identifier
stringlengths
11
32
pdf_url
stringlengths
17
4.62k
lang
stringclasses
120 values
error
stringclasses
1 value
title
stringlengths
2
500
source_name
stringlengths
1
435
publication_year
float64
1.9k
2.02k
license
stringclasses
3 values
word_count
int64
0
1.64M
text
stringlengths
1
9.75M
https://openalex.org/W3183662835
http://insight.jci.org/articles/view/147474/files/pdf
English
null
The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes
JCI insight
2,021
cc-by
16,272
The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes Shen Dong,1,2 Kamir J. Hiam-Galvez,3,4,5,6,7,8,9 Cody T. Mowery,10,11 Kevan C. Herold,12 Stephen E. Gitelman,2,13 Jonathan H. Esensten,14 Weihong Liu,1,2 Angela P. Lares,1,2 Ashley S. Leinbach,1,2 Michael Lee,1,2 Vinh Nguyen,1,2 Stanley J. Tamaki,15 Whitney Tamaki,2 Courtney M. Tamaki,2 Morvarid Mehdizadeh,1,2 Amy L. Putnam,1,2 Matthew H. Spitzer,3,4,6,7,8,9 Chun Jimmie Ye,10 Qizhi Tang,16 and Jeffrey A. Bluestone1,2,3 1Sean N. Parker Autoimmune Research Laboratory and 2Diabetes Center, UCSF, San Francisco, California, USA. 3Parker Institute for Cancer Immunotherapy, San Francisco, California, USA. 4Department of Microbiology and Immunology, 5Biomedical Sciences Graduate Program, 6Department of Otolaryngology, 7Department of Microbiology and Immunology, and 8Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA. 9Chan Zuckerberg Biohub, San Francisco, California, USA. 10Institute for Human Genetics and 11Medical Scientist Training Program, UCSF, San Francisco, California, USA. 12Department of Immunobiology and Internal Medicine, Yale University, New Haven, Connecticut, USA. 13Division of Pediatric Endocrinology, Department of Pediatrics; 14Department of Laboratory Medicine; 15Parnassus Flow Cytometry Core; and 16Transplantation Research Lab, Division of Transplant Surgery, Department of Surgery, UCSF, San Francisco, California, USA. Conflict of interest: JAB is a member of the scientific advisory boards of Arcus Biosciences, Solid Biosciences, and VIR Biotechnology and a member of the board of directors of both Gilead Sciences and Provention Bio. JAB is cofounder, president, and CEO of Sonoma Biotherapeutics, a company developing Treg-based cell therapies for the treatment of autoimmune diseases. JAB has a patent 62/667,981 licensed to Juno, a patent 62/744,058 pending, a patent 7,722,862 issued to Sonoma Biotherapeutics, a patent 9,012,134 issued to Sonoma Biotherapeutics, a patent 62/629,103 pending, and a patent 20060292142 issued to Provention Bio. KCH has consulted for Roche Pharmaceuticals. SEG has consulted for Biolojic, Caladrius Biosciences, Roche Pharmaceuticals, Avotres, Immunomolecular Therapeutics, and Tolerion. MHS receives research funding from Genentech (Roche), Bristol Myers Squibb, and Valitor and has been a paid consultant for Five Prime Therapeutics, and Ono Pharmaceutical, and January Inc. QT is a cofounder of Sonoma Biotherapeutics. BACKGROUND. A previous phase I study showed that the infusion of autologous Tregs expanded ex vivo into patients with recent-onset type 1 diabetes (T1D) had an excellent safety profile. However, the majority of the infused Tregs were undetectable in the peripheral blood 3 months postinfusion (Treg-T1D trial). The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes Therefore, we conducted a phase I study (TILT trial) combining polyclonal Tregs and low-dose IL-2, shown to enhance Treg survival and expansion, and assessed the impact over time on Treg populations and other immune cells. METHODS. Patients with T1D were treated with a single infusion of autologous polyclonal Tregs followed by one or two 5-day courses of recombinant human low-dose IL-2 (ld-IL-2). Flow cytometry, cytometry by time of flight, and 10x Genomics single-cell RNA-Seq were used to follow the distinct immune cell populations’ phenotypes over time. RESULTS. Multiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a substantial increase from baseline in a subset of activated NK, mucosal associated invariant T, and clonal CD8+ T cell populations. CONCLUSION. These data support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with preexisting active immunity. TRIAL REGISTRATION. ClinicalTrials.gov NCT01210664 (Treg-T1D trial), NCT02772679 (TILT trial). FUNDING. Sean N. Parker Autoimmune Research Laboratory Fund, National Center for Research Resources. Copyright: © 2021, Dong et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Submitted: January 12, 2021 Accepted: July 28, 2021 Published: September 22, 2021 Reference information: JCI Insight. 2021;6(18):e147474. https://doi.org/10.1172/jci. insight.147474. Copyright: © 2021, Dong et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E shown that the Tregs in the peripheral blood of patients with T1D can produce effector cytokines, such as IFN-γ (7, 8), and fail to regulate activated T effector cells (9, 10). shown that the Tregs in the peripheral blood of patients with T1D can produce effector cytokines, such as IFN-γ (7, 8), and fail to regulate activated T effector cells (9, 10). Adoptive transfer of Tregs has been shown to reverse T1D in a mouse model of spontaneous disease (11). Based on these results, we previously conducted a phase I trial (NCT01210664 — Treg-T1D) testing expanded autologous polyclonal Tregs as therapeutics aimed to restore tolerance in patients with recent- onset T1D (12). We demonstrated that Tregs could be efficiently isolated from peripheral blood of patients with T1D and expanded 300- to 2000-fold within a 2-week period. Moreover, the dysfunction that was associated with Tregs from patients with T1D was improved during culture. The expanded Tregs expressed higher levels of cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and latency-associated peptide, and a defect in IL-2– induced STAT5 signaling was reversed. Infusion of the Tregs in patients with recent-onset T1D had an excellent safety profile, and about 50% of recipients maintained their insulin production for 2 years. Moreover, we showed that the adoptively transferred Treg cells remained phenotypically stable in the blood for at least 1 year postin- fusion. However, there was a considerable and rapid decline in the percentage of infused Tregs in the peripheral blood, with, on average, over 75% of the infused Tregs absent from the circulation within 90 days. We hypothe- sized that the rapid decline might be attributed, at least in part, to a lack of IL-2 in patients with T1D, especially because GWAS have suggested a genetic link to IL-2 deficiency in this autoimmune setting (13). IL-2 is a survival and growth factor cytokine essential for Treg development and function, inducing effec- tive STAT5-mediated signaling to achieve full functionality and survival (14–17). In fact, genetic deficiency in the IL-2/IL-2R pathway leads to systemic autoimmunity (18). Tregs express the highest level of high-affinity α chain (CD25) as part of the trimeric receptor (α, β, γ) receptor complex, which make them highly responsive to even small amounts of the cytokine in the biological environment. In vivo administration of low doses of IL-2 can expand Tregs and ameliorate disease in multiple autoimmune disease models in mice. Introduction Submitted: January 12, 2021 Accepted: July 28, 2021 Published: September 22, 2021 Reference information: JCI Insight. 2021;6(18):e147474. https://doi.org/10.1172/jci. insight.147474. Type 1 diabetes mellitus (T1D) is an increasingly prevalent, yet still poorly understood, medical condition with devastating long-term complications, such as retinopathy, neuropathy, cardiovascular disease, and renal failure. The disease pathogenesis, which is influenced by genetic and environmental factors, is attributed to the loss of immune tolerance, which results in the development and inadequate control of pathogenic, autoreactive T cells that recognize and destroy islet β cells (1–3). Tregs play a predominant role in maintaining peripheral tolerance, and defects in their function have been described in patients with T1D (4–6). For instance, several studies have 1 C L I N I C A L M E D I C I N E For instance, low-dose IL-2 (ld-IL-2) prevents and reverses diabetes in the spontaneous NOD mouse model (19, 20). Ear- ly clinical studies suggested that ld-IL-2 blocks the progression of graft-versus-host disease, systemic lupus erythematosus, and autoimmune hepatitis (21–24). However, IL-2 is also a growth factor for conventional T, NK, and other potentially pathogenic cells due to the ubiquitous expression of the IL-2 receptor subunits in multiple activated immune cell populations. Hence, the dual activity of IL-2 put the drug at the crossroads of tolerance versus activation, and predicting its therapeutic effects remains uncertain (25). Thus, we designed a phase I study to determine if a combination of autologous Treg and ld-IL-2 therapies could demonstrate significant biologic activity based on multiparametric biomarker analyses of peripheral blood samples collected longitudinally from patients with T1D enrolled in the study. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Results Study design, patient population, and analysis of metabolic function levels. The phase I trial was designed as an open-label, dose-escalating study conducted at 2 sites, Yale University and the UCSF. Nine patients met the eligibility criteria. Seven were enrolled in the first cohort and 2 in the second cohort (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.147474DS1). A schematic representation of the Treg dose escalation plan and the approved plan for Proleukin (IL-2) dosing are illustrated in Supplemental Figure 2, A and B, respectively. Representation of an individual’s planned par- ticipation is shown in Supplemental Figure 2C. All the participants in the first cohort received the prescribed infusion of the expanded Tregs (3 × 106/kg) (Supplemental Table 1). There were 2 dosing cohorts planned, each consisting of 6–8 subjects treated with a single infusion of polyclonal Tregs and ld-IL-2 at the doses shown in Supplemental Table 1. Supplemental Table 1 and Supplemental Data File 3 show the demograph- ics and baseline hemoglobin A1c (HbA1c) levels. The mean age was 25.7 ± 4.7 years, and the mean disease duration was 46.1 ± 15.7 weeks at the time of screening. Metabolic assessment of all patients from cohort 1 and cohort 2 was followed up to 104 weeks and 78 weeks, respectively. Changes in β cell function over time were assessed by measuring the mixed meal tolerance test–stimulated (MMTT-stimulated) 4-hour C-peptide AUC, HbA1c, and insulin usage. The adverse events were limited to local events at the site of IL-2 injection (Supplemental Data File 1). The stimulated C-peptide levels decreased in all the patients within the first 30 days of treatment (Figure 1A). After 30 days, the stimulated C-peptide levels stabilized or increased slightly in 2 participants but continued to decline in the other 7 participants. Consistent with this, the insulin usage and HbA1c increased in 6/7 and 5/7 participants, respectively (Fig- ure 1A). Retrospective analyses, made available after the initiation of the TILT study, showed a similar C- peptide decay in the TILT patients when compared to the C-peptide responses in age-matched individuals JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 2 C L I N I C A L M E D I C I N E Figure 1. Metabolic assessments. (A) (Left column) C-peptide AUC is reported for fasting 4-hour mixed meal tolerance test (MMTT) without carbohydrate restriction for 3 days preceding testing. Results The target glucose level at the start of the test was between 70 and 200 mg/dL. Regular insulin or short-act- ing insulin analogs were allowed up to 6 and 2 hours before the test, respectively, to achieve the desired glucose level. The baseline blood samples (−10 minutes and 0 minutes) were drawn, and then patients drank Boost high protein nutritional energy drink (Nestle Nutrition) at 6 kcal/kg (1 kcal/mL) to a maximum of 360 mL. Blood was drawn at 15, 30, 60, 90, 120, 150, 180, 210, and 240 minutes following Boost dose. C-peptide AUC was calculated using the trapezoid rule. (Middle column) Hemoglobin A1c (HbA1c). (Right column) Insulin use. Insulin use for the 3 days immediately preceding the scheduled visit was self-reported. The average total insulin (long acting + short acting) use per day normalized to weight is reported. Table shows Treg and IL-2 dosage of each patient. MIU, million international units. (B) Percentage of relative C-peptide loss up to 104 and 78 weeks in patients from cohorts 1 and 2, respective- ly, of the TILT trial (2 left graphs) and from the placebo cohort of the AIDA and NT-14 trial (right graph). (C) Comparison of percentage of relative C-peptide loss at the indicated time point between the patients from TILT and placebo groups. Figure 1. Metabolic assessments. (A) (Left column) C-peptide AUC is reported for fasting 4-hour mixed meal tolerance test (MMTT) without carbohydrate restriction for 3 days preceding testing. The target glucose level at the start of the test was between 70 and 200 mg/dL. Regular insulin or short-act- ing insulin analogs were allowed up to 6 and 2 hours before the test, respectively, to achieve the desired glucose level. The baseline blood samples (−10 minutes and 0 minutes) were drawn, and then patients drank Boost high protein nutritional energy drink (Nestle Nutrition) at 6 kcal/kg (1 kcal/mL) to a maximum of 360 mL. Blood was drawn at 15, 30, 60, 90, 120, 150, 180, 210, and 240 minutes following Boost dose. C-peptide AUC was calculated using the trapezoid rule. (Middle column) Hemoglobin A1c (HbA1c). (Right column) Insulin use. Insulin use for the 3 days immediately preceding the scheduled visit was self-reported. The average total insulin (long acting + short acting) use per day normalized to weight is reported. Table shows Treg and IL-2 dosage of each patient. MIU, million international units. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Results (B) Percentage of relative C-peptide loss up to 104 and 78 weeks in patients from cohorts 1 and 2, respective- ly, of the TILT trial (2 left graphs) and from the placebo cohort of the AIDA and NT-14 trial (right graph). (C) Comparison of percentage of relative C-peptide loss at the indicated time point between the patients from TILT and placebo groups. who received placebo drugs in the anti-interleukin 1 inhibitor (Anakinra) AIDA and the anti-IL-1β Canaki- numab TrialNet TN-14 trials (26, 27). Thus, the TILT combination therapy did not improve islet metabolic function in any of the first 9 patients (Figure 1, B and C). These results suggested that the number of partici- pants planned for the trial would not be sufficient to demonstrate efficacy, which led to termination of the trial after the second patient in cohort 2 was treated. Longitudinal tracking of Treg postinfusion. In our previous studies, in vitro–expanded Tregs retained a stable functional phenotype after infusion but declined precipitously as a percentage of total T cells in the peripheral blood after 1 month. We had hypothesized that the ld-IL-2 treatment might increase the functional pheno- type and persistence of the adoptively transferred Treg population in vivo. To track the effect of ld-IL-2 on the infused Tregs’ persistence, Treg cell DNA was labeled with deuterium (2H) during the ex vivo expansion phase, and percentage of 2H-enriched DNA in PBMC sorted Tregs was measured by mass spectrometry. Fig- ure 2A shows detailed kinetics of 2H-enriched DNA of Tregs isolated from each patient at the indicated time point. In most patients, there was an increase in the percentage of 2H-labeled Tregs after each 5-day course of IL-2 (at day 7 and day 43, shown with dashed lines) (Figure 2A, small graphs). The increased percentage of 2H-labeled Tregs was transient and declined by 91 days. Depending on the participant, this occurred ~6–12 weeks after the last course of IL-2 treatment. Figure 2B shows the level of 2H enrichment normalized to the maximum value of each patient. Since the 2H labeling would be expected to dilute if the cells went into cycle, 3 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N Figure 2. Longitudinal tracking of in vitro–expanded Tregs postinfusion. Results (B) Graphs show the percentage of deuterated DNA enrichment normalized to the max- imum value in total PBMCs over time in each patient from the TILT trial. Light blue lines and gray areas show superimposition to normalized percentage of deuterated DNA enrichment of the T1D trial. Table shows Treg dosage. (C) Percentage of 2H level in postinfusion sorted non-Tregs versus Tregs in TILT trial patients. Paired 2-tailed t tests were performed in order to assess statistical significance. *P < 0.05. it seemed likely that the increase in the percentage of 2H label after IL-2 treatment reflected changes in Treg trafficking or survival rather than proliferation. A comparison of the kinetics of 2H enrichment in the periph- eral blood Tregs of TILT trial participants with analyses performed in the previous Treg-T1D trial in partici- pants treated with a similar number of infused Tregs (as described in Supplemental Table 1) showed that there was a longer term maintenance of the cells in the peripheral blood up to a year in the 6 patients who received both doses of IL-2 (Figure 2B), supporting the hypothesis that IL-2 therapy enhanced long-term Treg survival. To determine the stability of the adoptively transferred Tregs, at various time points, PBMCs were sorted into Treg (CD4+CD25+CD127lo/–) and non-Treg populations (CD4+CD25loCD127hi CD45RO+, CD45RO+C- D62Llo, CD45RO+CD62Lhi) and analyzed for the presence of 2H label. As seen in Figure 2C, all the 2H label- ing remained associated with the CD4+CD25+CD127lo/– Treg population over the first 28 days. Only back- ground levels of 2H label were observed in non-Treg subsets (including activated T effector/memory cells), suggesting that the adoptive cell transferred (ACT) Tregs retained a stable phenotype over time (Figure 2C). Phenotypic changes of infused and endogenous Treg population after ld-IL-2 treatment. The effects of ld-IL-2 on the Treg populations (both adoptively transferred and endogenous) in the 9 patients of the TILT trial were compared with those of 9 Treg-T1D study that had received a comparable or higher Treg dosage in the orig- inal phase I trial (NCT01210664-Treg-T1D trial) (Figure 3A legend table and Supplemental Table 1). A total of 76 frozen PBMC samples from the 18 patients, harvested at different time points (Supplemental Table 2), were analyzed by flow cytometry, cytometry by time of flight (CyTOF), single-cell RNA sequencing (RNA- Seq), and TCR sequencing (TCR-Seq). Results (A) Graphs show the percentage of DNA enrichment with deuterium (2H) in PBMC sorted Treg cells from TILT trial patients. Enlarged view of the 2H labeling kinetics up to 63 days is represented in the upper right of each graph. Black dashed lines indicate the fifth day of each IL-2 infusion course. Table shows Treg and IL-2 dosage of each patient. (B) Graphs show the percentage of deuterated DNA enrichment normalized to the max- imum value in total PBMCs over time in each patient from the TILT trial. Light blue lines and gray areas show superimposition to normalized percentage of deuterated DNA enrichment of the T1D trial. Table shows Treg dosage. (C) Percentage of 2H level in postinfusion sorted non-Tregs versus Tregs in TILT trial patients. Paired 2-tailed t tests were performed in order to assess statistical significance. *P < 0.05. Figure 2. Longitudinal tracking of in vitro–expanded Tregs postinfusion. (A) Graphs show the percentage of DNA enrichment with deuterium (2H) in PBMC sorted Treg cells from TILT trial patients. Enlarged view of the 2H labeling kinetics up to 63 days is represented in the upper right of each graph. Black dashed lines indicate the fifth day of each IL-2 infusion course. Table shows Treg and IL-2 dosage of each patient. (B) Graphs show the percentage of deuterated DNA enrichment normalized to the max- imum value in total PBMCs over time in each patient from the TILT trial. Light blue lines and gray areas show superimposition to normalized percentage of deuterated DNA enrichment of the T1D trial. Table shows Treg dosage. (C) Percentage of 2H level in postinfusion sorted non-Tregs versus Tregs in TILT trial patients. Paired 2-tailed t tests were performed in order to assess statistical significance. *P < 0.05. Figure 2. Longitudinal tracking of in vitro–expanded Tregs postinfusion. (A) Graphs show the Figure 2. Longitudinal tracking of in vitro–expanded Tregs postinfusion. (A) Graphs show the percentage of DNA enrichment with deuterium (2H) in PBMC sorted Treg cells from TILT trial patients. Enlarged view of the 2H labeling kinetics up to 63 days is represented in the upper right of each graph. Black dashed lines indicate the fifth day of each IL-2 infusion course. Table shows Treg and IL-2 dosage of each patient. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Results Data were normalized; cell populations were gated manually in CellEngine. Populations were then exported for analysis in R, and marker expression values were then arsinh-transformed with a cofactor of 5 and represented in dot plots. The results are plotted into 2 separated batches (batches 1 and 2) due to batch effect affecting the comparison of the samples within the same analysis pipeline (batches layout of the samples, Supplemental Table 3). Asterisks indicate significance relative to the control group determined by 1-way ANOVA. ***P < 0.001; ****P < 0.0001. CyTOF analysis showed an increase in the percentage and median expression of FOXP3 and also an increase in the median expression of activation markers such as CD27, CTLA-4, and HLA-DR in the Treg population after each course of ld-IL-2 treatment (day 7 and day 42) (Figure 3, B and C). The ld-IL-2–dependent increase of FOXP3+ Treg population was consistent with data shown in previous ld-IL-2 trials (28–30). To delve more deeply into phenotypic changes in immune subsets, CD45+ cells were isolated by FACS, combined into 30 pools, and sequenced in 3 distinct batches (Supplemental Table 4). The data from individual time points of each patient’s samples were then deconvoluted using the Demuxlet computational package (Supplemental Figure 3A) (31). The gene expression profile of more than 400,000 cells was integrated into a single uniform manifold approximation and projection (UMAP) plot clustering. The main immune cell pop- ulations were identified by known markers (Supplemental Figure 3B, including Tregs, B cells, NK cells, CD4 cells, CD8 cells, and dendritic cells/macrophages) as well as populations of granzyme B–activated (GZMB) and perforin-activated (PRF1) cells were illustrated using violin plots and UMAP plots (Supplemental Fig- ure 3, C and D). UMAP analysis showed that FOXP3 expression was localized to cluster 11 (Supplemen- tal Figure 3) in both the TILT and Treg-T1D samples (Figure 4A). Longitudinal analysis showed that each ld-IL-2 treatment (day 7 and day 42) resulted in an increased percentage of FOXP3+ T cells for all the patients from the TILT trial compared with Treg-T1D patients (Figure 4B). Expressed genes in the Treg cluster were compared between Treg-T1D and TILT samples. There were only a limited number of differences in gene expression between Treg-T1D and TILT Treg population before ld-IL-2 treatment (day 0) (Figure 4C, left volcano plot). In contrast, Tregs from TILT patients showed multiple upregulated mRNAs at day 7, after the first course of IL-2. Results Flow cytometric analyses of the whole PBMCs showed a significant increase in the percentage of the CD4+CD25+CD127lo/– population in all the TILT patients after each ld-IL-2 treatment (Figure 3A). Similarly, JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 4 C L I N I C A L M E D I C I N E Figure 3. Low-dose IL-2 induces activation phenotype in the Treg subset at a protein level. (A) Graphs represent the percentage of Tregs (left column) by flow cytometry at the indicated time points. TILT trial patient data are shown in upper graphs, and the Treg-T1D trial patients are represented in the lower graphs. Red asterisks indicate patients who received only 1 dose of IL-2. Tables indicate dosage of IL-2 and Tregs for each patient. Paired 2-tailed t tests were performed in order to assess statistical significance. (B and C) Percentage of FOXP3+ and median expression of FOXP3+ as well as median expression of CD27, CTLA-4, and HLA-DR was assessed by CyTOF. Data were normalized; cell populations were gated manually in CellEngine. Populations were then exported for analysis in R, and marker expression values were then arsinh-transformed with a cofactor of 5 and represented in dot plots. The results are plotted into 2 separated batches (batches 1 and 2) due to batch effect affecting the comparison of the samples within the same analysis pipeline (batches layout of the samples, Supplemental Table 3). Asterisks indicate significance relative to the control group determined by 1-way ANOVA. ***P < 0.001; ****P < 0.0001. C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E Figure 3. Low-dose IL-2 induces activation phenotype in the Treg subset at a protein level. (A) Graphs represent the percentage of Tregs (left column) by flow cytometry at the indicated time points. TILT trial patient data are shown in upper graphs, and the Treg-T1D trial patients are represented in the lower graphs. Red asterisks indicate patients who received only 1 dose of IL-2. Tables indicate dosage of IL-2 and Tregs for each patient. Paired 2-tailed t tests were performed in order to assess statistical significance. (B and C) Percentage of FOXP3+ and median expression of FOXP3+ as well as median expression of CD27, CTLA-4, and HLA-DR was assessed by CyTOF. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E 6 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Figure 4. Low-dose IL-2 induces activation phenotype in the Treg subset at the mRNA level. 10x Genomics single-cell RNA-Seq data were analyzed by Scanpy package. (A) UMAPs show expression of FOXP3 in cluster 11 from UMAP in Supplemental Figure 2 in the TILT patient samples and Treg-T1D patient samples. (B) Volcano plots represent differential gene expression analysis of the Treg cell compartment (Supplemental Figure 2, cluster 11) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2(fold change) (log2FC) with a P < 0.005. Gene expressions with P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. (C) Dot plot shows longitudinal changes over time of percentage of FOXP3+ cells in cluster 11 for the 2 trials. Asterisks indicate Figure 4. Low-dose IL-2 induces activation phenotype in the Treg subset at the mRNA level. 10x Genomics single-cell RNA-Seq data were analyzed by Scanpy package. (A) UMAPs show expression of FOXP3 in cluster 11 from UMAP in Supplemental Figure 2 in the TILT patient samples and Treg-T1D patient samples. (B) Volcano plots represent differential gene expression analysis of the Treg cell compartment (Supplemental Figure 2, cluster 11) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2(fold change) (log2FC) with a P < 0.005. Gene expressions with P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. (C) Dot plot shows longitudinal changes over time of percentage of FOXP3+ cells in cluster 11 for the 2 trials. Asterisks indicate Figure 4. Low-dose IL-2 induces activation phenotype in the Treg subset at the mRNA level. 10x Genomics single-cell RNA-Seq data were analyzed by Scanpy package. Results The upregulated gene expression profiles included both phenotypic and functional Treg 5 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E Sub- cluster 3 cells represented the gene expression profile of memory Treg population with high expression of CD25 (IL-2RA), BCL2, CTLA4, ICOS, and CD27 and low expression of CD62L (SELL) and Ki-67 (MKI67) while subcluster 4 cells expressed low levels of FOXP3, but high levels of ICOS and CTLA4, consistent with a population of conventional T cells that upregulated FOXP3. Subcluster 7 Tregs expressed markers similarly to subcluster 3 but high levels of the proliferation marker, MKI67 (Figure 4E, heatmap). Next, patient samples were examined for changes in Treg subclusters after Treg transfer and ld-IL-2 therapy. TILT patients showed a reduced number of activated Treg populations (subclusters 1 and 2) and an expanded percentage of memory and proliferating Treg cells (subclusters 3 and 7) (Figure 4E, stack bars chart). Interestingly, there was also an increase in subcluster 4, representing activated FOXP3lo conventional T cells, suggesting that ld-IL-2 treat- ment resulted in increased percentages of Tregs, and some conventional T cells, expressing activated/memory markers. However, TCR analysis of the TCR repertoire and Gini coefficient calculation suggested that there was no selective clonal expansion of the Treg TCR repertoire (see below in Gini index graph of the CD8 pop- ulation and Supplemental Table 5), consistent with the ld-IL-2 treatment broadly inducing the proliferation of activated Tregs and/or mobilized activated Tregs from the tissue into the circulation. Changes in innate cell subsets after Treg and ld-IL-2 treatment. Multiparameter longitudinal single-cell analysis allowed the identification of other immune populations affected by ld-IL-2 and Treg treatment. Previous clinical trials have shown that ld-IL-2 treatment altered the percentage of CD56+ NK populations in the cir- culation (23, 29, 33–35). In this study, single-cell analysis of differentially expressed genes in the GZMB+ NK cluster (cluster 4 in Supplemental Figure 3) between Treg-T1D and TILT samples showed limited differences at day 0 while a large set of phenotypic and activation markers of mature NK cells was altered in peripheral blood cells isolated from TILT patients at day 7 after the first course of IL-2 (Figure 5A). Longitudinal analy- sis showed that most of the patients in both trials presented a similar percentage of GZMB+ NK cells at day 0. This population was increased in 3 TILT trial participants, while 2 patients showed no change and 4 patients showed a decline in this population after each ld-IL-2 course (Figure 5B, upper graph). C L I N I C A L M E D I C I N E significance relative to the control group determined by 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Graphs represent mean mRNA expression of the indicated genes normalized to day 0 for the patients of each clinical trial group (TILT in upper graphs and Treg-T1D in lower graphs). Red asterisks indicate patients that received only 1 dose of IL-2. (E) UMAP and leiden clustering of the Treg cluster 11. Heatmap shows Treg markers’ and activation markers’ mean expression in the indicated clusters. Stacked bar chart shows the percentage of cells in each cluster in Treg-T1D versus TILT patients. significance relative to the control group determined by 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Graphs represent mean mRNA expression of the indicated genes normalized to day 0 for the patients of each clinical trial group (TILT in upper graphs and Treg-T1D in lower graphs). Red asterisks indicate patients that received only 1 dose of IL-2. (E) UMAP and leiden clustering of the Treg cluster 11. Heatmap shows Treg markers’ and activation markers’ mean expression in the indicated clusters. Stacked bar chart shows the percentage of cells in each cluster in Treg-T1D versus TILT patients. markers, including FOXP3, TNFRSF18 (GITR), LRRC32 (GARP), and IKZF1 (IKAROS); activation marker genes, including HLA-DRA and IL2RA; and IL-2 signaling genes, including CISH, SOCS2, BCL2, and PRDM1 (Figure 4C, right volcano plot). Moreover, corroborating CyTOF data (Figure 3, B and C), FOXP3, LRRC32, TNFRSF18, HLA-DR, and CD27 mean mRNA expression was increased in Treg populations in the majority of the participants after ld-IL-2 treatment at day 7 and day 42 (Figure 4D; upper graphs) when compared with samples from patients in the Treg-T1D trial (Figure 4D, lower graphs). Next, the cluster 11 was reanalyzed based on leiden clustering (UMAP, Figure 4E) to determine the heterogeneity of the Treg populations. A gene expression heatmap of the Treg population subclusters for all time points revealed increased CD44 and CCR7 expression in subclusters 1 and 2, matching the phenotype of an activated Treg population similar to activated Tregs seen in secondary lymphoid structures (32). C L I N I C A L M E D I C I N E (A) UMAPs show expression of FOXP3 in cluster 11 from UMAP in Supplemental Figure 2 in the TILT patient samples and Treg-T1D patient samples. (B) Volcano plots represent differential gene expression analysis of the Treg cell compartment (Supplemental Figure 2, cluster 11) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2(fold change) (log2FC) with a P < 0.005. Gene expressions with P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. (C) Dot plot shows longitudinal changes over time of percentage of FOXP3+ cells in cluster 11 for the 2 trials. Asterisks indicate JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 6 C L I N I C A L M E D I C I N E significance relative to the control group determined by 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Graphs represent mean mRNA expression of the indicated genes normalized to day 0 for the patients of each clinical trial group (TILT in upper graphs and Treg-T1D in lower graphs). Red asterisks indicate patients that received only 1 dose of IL-2. (E) UMAP and leiden clustering of the Treg cluster 11. Heatmap shows Treg markers’ and activation markers’ mean expression in the indicated clusters. Stacked bar chart shows the percentage of cells in each cluster in Treg-T1D versus TILT patients. C L I N I C A L M E D I C I N E In contrast, all the percentages of GZMB+ NK cells remained constant or decreased in samples from patients of the Treg-T1D trial (Figure 5B, lower graph). More importantly, at day 28, the percentage of GZMB+ NK cells significantly increased in all the TILT trial patients as compared with the patients in the Treg-T1D trial (Figure 5C). Inter- estingly, over time, the percentage of GZMB+ NK cells returned to baseline levels in all IL-2–treated patients. These data suggest that the administration of ld-IL-2 to patients with T1D led to changes in the phenotype and distribution of GZMB+ NK cells over time, likely a consequence of changes in expansion and trafficking in these patients. In fact, there was a positive correlation between changes in the Treg percentage and chang- es of the GZMB+ NK cells’ percentage after ld-IL-2 treatment (Figure 5D, upper graph). In contrast, those changes were not correlated in patients treated with Tregs only (Figure 5D, lower graph), suggesting that the ld-IL-2 treatment affected those 2 populations at the same time. Based on the above observations, we examined whether other innate cell subsets were altered by the Treg and ld-IL-2 treatment. Interestingly, TCR analysis of the cluster 6 cells showed that the TRAV2-1 gene was paired with TRAJ33/20/21 genes in a significant percentage of the cells (Supplemental Table 7). This TCR pairing was reminiscent of mucosal associated invariant T (MAIT) cells, a unique innate-like T cell subset, activated by conserved bacterial ligands presented by the invariant MHC MR1 molecule in host defense against JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 7 C L I N I C A L M E D I C I N E 8 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Figure 5. Low-dose IL-2 induces cytotoxic phenotype in the NK cell subset and mucosal invariant associated T cell subset. 10x Genomics single-cell RNA- Seq data were analyzed by Scanpy package. (A) Volcano plots represent differential gene expression analysis of the NK cell compartment (Supplemental Fig- ure 2, cluster 4) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. C L I N I C A L M E D I C I N E Those results suggest that ld-IL-2 selectively promoted expansion of activated NK and MAIT cell populations in the circulation of T1D patients treated with ld-IL-2. Changes of CD8 population after Treg and IL-2 treatment. Flow cytometry analysis of the PBMCs from Treg and ld-IL-2–treated patients showed that the overall percentage of CD3+CD8+ remained unchanged (Figure 6A), similar to what has been seen in other clinical studies. However, the percentage of CD3+CD8+CD25+ subset contracted after the first or second course of ld-IL-2. This was followed by a rapid recovery in the majority of the patients and, in some cases, exceeded the day 0 baseline when examined at day 63. These variations may be the consequence of the expansion of Treg population negatively regulating the expansion of the CD3+CD8+CD25+ subset or, alternatively, extravasation of this activated subset of cells from the peripheral blood compartment. Further analysis by single-cell RNA-Seq analysis supported the finding that selected activated CD8+ T cell subsets were altered dynamically by the ld-IL-2 plus Treg therapy. Specifically, there were significant changes in the gene expression profile of an activated CD8+ T cell subset expressing PRF1 and GZMB, 2 major cytotoxic proteins expressed by CD8+ T cells after activation (cluster 5, Supplemental Figure 3). Analysis of differentially expressed genes between Treg-T1D and TILT samples showed minor differences within the PRF1+GZMB+CD8+ T cells at day 0, while activation markers such as PRF1, CD69, and HLA- DRA were upregulated in the TILT patient samples by day 7, after the first course of IL-2 (Figure 6, B and C). Longitudinal analysis showed that PRF1 mean expression in cluster 5 was higher in TILT trial patients compared with Treg-T1D trial patients at day 7 after the first course of IL-2 (Figure 6D). These results suggest that the ld-IL-2 treatment expanded a preexisting population of PRF1+GZMB+CD8+ T cells in a subset of the patients. It is important to note that we have only assessed circulating cells. There may be activated cells in the inflamed tissue that may be mobilized as a consequence of the treatment, resulting in an increase in activated cells in the circulation. In order to assess whether the activated PRF1+GZMB+CD8+ T cells represented the expansion of a clonal population, we evaluated the sequences of TCRα and β chains. Analysis of the Gini coefficient of individual T cell subsets for the TCRα chains (left) and the TCRβ chain (right) was determined (Figure 7A). C L I N I C A L M E D I C I N E Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. C L I N I C A L M E D I C I N E plot represents percentage over time of GZMB+ cells in NK clusters in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. (D) Graphs show correlation of day 0 to day 28 changes in the percentage of GZMB+ NK and day 0 to day 7 changes in the percentage of FOXP3+ Treg cells in TILT patients (upper graph) and Treg-T1D patients (lower graph). (E) Volcano plots represent differential gene expression analysis of the MAIT cell compartment (Supplemental Figure 2, cluster 6) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. plot represents percentage over time of GZMB+ cells in NK clusters in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. (D) Graphs show correlation of day 0 to day 28 changes in the percentage of GZMB+ NK and day 0 to day 7 changes in the percentage of FOXP3+ Treg cells in TILT patients (upper graph) and Treg-T1D patients (lower graph). (E) Volcano plots represent differential gene expression analysis of the MAIT cell compartment (Supplemental Figure 2, cluster 6) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. bacterial and viral infections (36, 37) that can have a deleterious function in autoimmune diseases (38–40), especially when CD25 is upregulated on the cells (41). Although single-cell analysis of differentially expressed genes in the MAIT cell cluster (cluster 6 in Supplemental Figure 3B) showed limited differences between Treg- T1D and TILT samples at day 0, by day 7, after the first course of IL-2, activation markers such as PRF1, CD69, GZMA, CXCR4, and killer cell lectin like receptor B1 were upregulated in the MAIT population in the circulation of TILT patients (Figure 5E). C L I N I C A L M E D I C I N E Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. (B) Percentage of GZMB+ cells in the NK cluster (Supplemental Figure 2, cluster 4) were calculated and shown on upper graphs for TILT trial patients and lower graphs for Treg-T1D trial patients. Tables indicate dosage of IL-2 and Tregs for each patient. (C) Dot Figure 5. Low-dose IL-2 induces cytotoxic phenotype in the NK cell subset and mucosal invariant associated T cell subset. 10x Genomics single-cell RNA- Seq data were analyzed by Scanpy package. (A) Volcano plots represent differential gene expression analysis of the NK cell compartment (Supplemental Fig- ure 2, cluster 4) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate nonsignificant genes filtered out due to a P > 0.005. (B) Percentage of GZMB+ cells in the NK cluster (Supplemental Figure 2, cluster 4) were calculated and shown on upper graphs for TILT trial patients and lower graphs for Treg-T1D trial patients. Tables indicate dosage of IL-2 and Tregs for each patient. (C) Dot cytotoxic phenotype in the NK cell subset and mucosal invariant associated T cell subset. 10x Genomics single-cell RNA JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 8 C L I N I C A L M E D I C I N E plot represents percentage over time of GZMB+ cells in NK clusters in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. (D) Graphs show correlation of day 0 to day 28 changes in the percentage of GZMB+ NK and day 0 to day 7 changes in the percentage of FOXP3+ Treg cells in TILT patients (upper graph) and Treg-T1D patients (lower graph). (E) Volcano plots represent differential gene expression analysis of the MAIT cell compartment (Supplemental Figure 2, cluster 6) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E Volcano plots represent differential gene expression analysis of the PRF1+GZMB+CD8 T cell compartment (Supplemental Figure 2, cluster 5) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with a P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate non-significant genes filtered out due to a P > 0.005. (C) Table indicates the log2FC values of the indicated genes. Blue cells indicate non- significant genes filtered out due to a P > 0.005. (D) Dot plot shows PRF1 mRNA mean expression over time in PRF1+GZMB+CD8+ T cell cluster in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. C L I N I C A L M E D I C I N E Figure 6. Low-dose IL-2 treatment promotes a cytotoxic phenotype in the CD8+ T cell subset. (A) Flow cytometry analysis shows the percentage of total CD3+CD8+ T cells (left column) and CD8+CD25+ T cells (right column) at the collected time points. Upper graphs represent patients from the TILT trials, and lower graphs represent patients from the Treg-T1D trial. The table indicates the dosage of IL-2 and Tregs for each patient. (B) Single-cell RNA-Seq data were analyzed by Scanpy package. Volcano plots represent differential gene expression analysis of the PRF1+GZMB+CD8 T cell compartment (Supplemental Figure 2, cluster 5) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with a P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate non-significant genes filtered out due to a P > 0.005. (C) Table indicates the log2FC values of the indicated genes. Blue cells indicate non- significant genes filtered out due to a P > 0.005. C L I N I C A L M E D I C I N E (D) Dot plot shows PRF1 mRNA mean expression over time in PRF1+GZMB+CD8+ T cell cluster in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. Figure 6. Low-dose IL-2 treatment promotes a cytotoxic phenotype in the CD8+ T cell subset. (A) CD3+CD8+ T cells (left column) and CD8+CD25+ T cells (right column) at the collected time points. U lower graphs represent patients from the Treg-T1D trial. The table indicates the dosage of IL-2 and were analyzed by Scanpy package. Volcano plots represent differential gene expression analysis of Figure 2, cluster 5) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volc genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with a P values grea represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table in cells indicate non-significant genes filtered out due to a P > 0.005. (C) Table indicates the log2FC v significant genes filtered out due to a P > 0.005. (D) Dot plot shows PRF1 mRNA mean expression Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1 Figure 6. Low-dose IL-2 treatment promotes a cytotoxic phenotype in the CD8+ T cell subset. (A) Flow cytometry analysis shows the percentage of total CD3+CD8+ T cells (left column) and CD8+CD25+ T cells (right column) at the collected time points. Upper graphs represent patients from the TILT trials, and lower graphs represent patients from the Treg-T1D trial. The table indicates the dosage of IL-2 and Tregs for each patient. (B) Single-cell RNA-Seq data were analyzed by Scanpy package. Volcano plots represent differential gene expression analysis of the PRF1+GZMB+CD8 T cell compartment (Supplemental Figure 2, cluster 5) from TILT and Treg-T1D patients at day 0 (left volcano plot) and day 7 (right volcano plot). Downregulated (red dots) and upregulated genes (green dots) are indicated in log2FC with a P < 0.005. Gene expressions with a P values greater than 0.005 were filtered out. Vertical dashed lines represent thresholds of log2FC of –0.6 and 0.6 corresponding to a fold change of 1.5 times. Table indicates the log2FC values of the indicated genes. Blue cells indicate non-significant genes filtered out due to a P > 0.005. (C) Table indicates the log2FC values of the indicated genes. C L I N I C A L M E D I C I N E Blue cells indicate non- significant genes filtered out due to a P > 0.005. (D) Dot plot shows PRF1 mRNA mean expression over time in PRF1+GZMB+CD8+ T cell cluster in TILT and Treg-T1D trial patients. Asterisks indicate significance relative to the control group determined by 1-way ANOVA. *P < 0.05. C L I N I C A L M E D I C I N E The Gini coefficient is a measure of statistical dispersion intended to represent the distribution of the clonal diversity. As expected, the Gini index value was close to null in the Treg compartment for patients from the 2 trials, reflecting the high diversity of Treg TCRs with no clonal expansion. However, there were substantial changes in diversity of TCR usage in the PRF1+GZMB+CD8+ T cell compartment. Dot plots shown for both TCR chains demonstrated an increased Gini index in the Treg-T1D patients, indicating a preexistent clonal expansion of this T cell subset. Moreover, ld-IL-2 further enhanced the clonal expansion of this population, consistent with a role for IL-2 in the expansion of activated PRF1+GZMB+CD8+ T cells. In this regard, the Gini index calculation for TCRα and TCRβ for the whole CD3+ T cell population was unaltered after ld-IL-2 treatment or in samples from the Treg-T1D trials, suggesting that effects of ld-IL-2 treatment were manifested in the PRF1+GZMB+CD8+ T cell subset selectively (Figure 7A). The top CDR3 α and β sequences and frequencies from the PRF1+GZMB+CD8+ T cell population from the TILT patients are shown in the Supplemental Table 6. TCR sequences observed more than 30 times in the single-cell sequencing analyses mostly mapped within the PRF1+GZMB+CD8+ cluster 5 in both clinical studies (Figure 7B, Treg-T1D and TILT UMAPs). The cells from which these TCRs were derived (Figure 7C) exhibited very similar gene expression profiles for activation markers and cytotoxic genes in the TILT and Treg-T1D patients, confirming that a similar PRF1+GZMB+CD8+ T cell subset was present in both patient groups but selectively enriched in the ld-IL-2–treated TILT patients. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 9 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E Figure 6. Low-dose IL-2 treatment promotes a cytotoxic phenotype in the CD8+ T cell subset. (A) Flow cytometry analysis shows the percentage of total CD3+CD8+ T cells (left column) and CD8+CD25+ T cells (right column) at the collected time points. Upper graphs represent patients from the TILT trials, and lower graphs represent patients from the Treg-T1D trial. The table indicates the dosage of IL-2 and Tregs for each patient. (B) Single-cell RNA-Seq data were analyzed by Scanpy package. Discussion The conduct of the TILT trial was designed to test for the safety of the combination of Tregs and chang- es in molecular and cellular biomarkers that could be used to assess the clinical impact of T1D disease progression. There was no evidence that the combined therapy led to preservation of insulin production. Moreover, there were limited effects on the proportion of circulating adoptively transferred polyclonal Tregs 1 0 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E 7. Low-dose IL-2 increases clonal expansion of the PRF1+GZMB+CD8+ T cell compartment. (A) Clonal diversity from the Treg, PRF1+GZMB+CD8 T cell, al CD3+ populations was evaluated by the calculation of the Gini index. Dot plots show values of Gini index for each patient from the TILT and the D trial. Unpaired 2-tailed t tests were performed in order to assess statistical significance. (B) Left UMAP plot represents clusters of immune cells i ed in Supplemental Figure 2. Density plots on the right represent mapping of TCR clones expanded more than 30 times in each clinical trial group. tmaps represent cytotoxicity and activation markers gene expression (log normalized) of all the cells expressing expanded TCRs. Left y axis links ient to the depicted expanded TCRs. C L I N I C A L M E D I C I N E JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E activation rather than tolerance. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune disease. activation rather than tolerance. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune disease. Many studies of IL-2 and CD25 deficiency have demonstrated that IL-2 is critical for the maintenance of the survival and the function of Treg cells (20, 42–45). In adoptive Treg transfer therapies, the survival and function of infused Tregs is the key to their potential efficiency in the prevention of T1D progression. In a polyclonal Treg therapy trial, Marek-Trzonkowska et al. showed that infusing up to 2 doses of Tregs at 30 × 106/kg in young individuals led to a decrease of IL-2 in the serum of T1D patients after Treg infusion (46). In addition, in a previous Treg ACT trial, we showed a decrease of the adoptively transferred ex vivo expanded Treg populations 2 weeks after ACT, which may have reflected lack of access to IL-2 in the periphery (12). Therefore, we hypothesized that the combination of ld-IL-2 and Tregs might result in a more robust survival and function of the adoptively transferred polyclonal Tregs. In fact, multiple studies have examined the use of ld-IL-2 therapy to boost Treg cell survival and function for the treatment of autoimmunity and graft-versus- host disease (GvHD) (21, 30, 34, 47, 48). One of the earliest studies testing the effects of ld-IL-2 treatment was performed after hematopoietic stem cell transplantation. The investigators showed that treatment with ld-IL-2 increased early Treg expansion and suppressed acute and chronic GvHD (21, 22). Moreover, in 2011, Saadoun et al. showed that ld-IL-2 treatment could reduce hepatitis C virus–induced (HCV-induced) vasculitis in a majority of patients (34). Several T1D clinical trials using ld-IL-2 have been conducted, including a phase I/II trial IL-2 dose-defining study of 24 patients with T1D. In this study, patients were treated with 0.33 M to 3 M IU daily, and the treatment was shown to be well tolerated and led to an increase in the percentage of Tregs (28), although it should be pointed out that Treg percentages went back to pretreatment levels after discontin- uation of the therapy. C L I N I C A L M E D I C I N E Importantly, in a study that tested a higher dose of IL-2 combined with rapamycin, an increased and persistent ability of IL-2 to induce STAT5 phosphorylation, a defect found in endogenous Tregs isolated from patients with T1D, was observed (49). The gene expression profile of endogenous Tregs was assessed in order to understand whether ld-IL-2 could have affected the phenotype and function of the Treg population in patients with T1D. In all the TILT patients, CyTOF, flow cytometry, and single-cell RNA-Seq data showed that the whole population of Tregs exhibited upregulated activation markers, including CD25, GITR, CD27, HLA-DRA, and GARP, after ld-IL-2 treatment. Moreover, an increase in Treg memory phenotype and secondary lymph node homing marker expression in the whole Treg population was observed, suggesting that there was dynamic circulation of Tregs between tissues and the peripheral blood. Taken together those data corroborate previous studies showing that ld-IL-2 treatment resulted in an expansion of Tregs with an activated and memory phenotype (30, 35, 50). In parallel, the overall increase in the percentage of adoptively transferred Tregs in the peripheral blood, as shown by the 2H tracking, suggested that the ld-IL-2 treatment enhances survival and/or redistri- bution of Tregs in the circulation. In the latter case, the ld-IL-2 may have activated ACT Tregs, which then redistributed from the tissue where they had migrated after the transfer. Similar results were observed in the single dose of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in type 1 Diabetes trial (DILT1D), where Todd et al. showed the early altered trafficking of the Treg population (51). However, the limitation of using 2H labeling and subsequent mass spectrophotometry analyses is that the labeling method does not allow the determination of proliferative activity as the label dilutes during this process, allowing only a summation of total percentage of 2H-enriched DNA as a percentage of total Tregs. In summary, the impact of ld-IL-2 on the endogenous and infused Tregs populations in the TILT study was similar as compared with other clinical trials treating patients with T1D with ld-IL-2, and no evidence links those changes to a less functional Treg compartment. In the TILT study, we observed that some patients treated with the ld-IL-2 plus PolyTregs showed a tran- sient downregulation of both NK and CD25+CD8+ population followed by a recovery phase. C L I N I C A L M E D I C I N E Figure 7. Low-dose IL-2 increases clonal expansion of the PRF1+GZMB+CD8+ T cell compartment. (A) Clonal diversity from the Treg, PRF1+GZMB+CD8 T cell, and total CD3+ populations was evaluated by the calculation of the Gini index. Dot plots show values of Gini index for each patient from the TILT and the Treg-T1D trial. Unpaired 2-tailed t tests were performed in order to assess statistical significance. (B) Left UMAP plot represents clusters of immune cells identified in Supplemental Figure 2. Density plots on the right represent mapping of TCR clones expanded more than 30 times in each clinical trial group. (C) Heatmaps represent cytotoxicity and activation markers gene expression (log normalized) of all the cells expressing expanded TCRs. Left y axis links the patient to the depicted expanded TCRs. (PolyTregs), and we were unable to determine if there were functional changes in the cells, as the cells could not be analyzed on a per-cell level. However, the longitudinal study confirmed that the ld-IL-2 treatment increased the number of endogenous Tregs in the peripheral blood as previously reported (20, 42–45). More- over, there were transient changes in the distribution of the adoptively transferred Treg product, something underappreciated in previous studies of ld-IL-2 treatment, where individual Treg populations could not be tracked effectively. Importantly, the treatment led to the expansion of PRF1- and GZMB-expressing CD8+ T, MAIT, and NK cells and clonal expansion of a subset of the activated CD8+ T cells. This effect was likely due to expansion of preexisting activated immune cells, including a subset of activated CD8+ T cells express- ing high levels of the IL-2 receptor (CD25, CD122, CD132) complex cells in response to the exogenous- ly administered IL-2. These results support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also indicates how the same dose of ld-IL-2 can expand non-Tregs with increased expression of activation/cytotoxic genes in several immune cell subsets, potentially shifting the immune balance toward JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 1 1 C L I N I C A L M E D I C I N E Age and environmental exposure play a major role in shaping TCR repertoires in each individual; those factors may therefore play an important part in the response of the cytotoxic CD8 compartment to Proleukin. Taken together these data suggest that the individual’s TCR repertoire of cytotoxic/activated immune cell populations could be targeted by ld-IL-2 treatment. Profiling of the basal cytotoxic immune cells in young children, healthy individuals, or other autoimmune disease patients may bring more insight to cellular mechanisms involved in the onset and development of T1D, as well as the impact of Proleukin and other immunotherapies within diverse groups of individuals. There were limitations of our clinical study. Although we did not detect a significant improvement or even maintenance of insulin secretion in the participants studied when compared with placebo-treated patients from 2 other clinical trials, the number of patients that were studied was small and lacked clear statistical power to declare a correlation between IL-2–induced activation of cytotoxic cells and the inability of the combined therapy to maintain or improve C-peptide level. The concern about dramatic reduction in C-peptide production at 28 days led the Data and Safety Monitoring Board (DSMB) to recommend altering the IL-2 dose and ultimately led to termination of the study because of the low likelihood that clinical benefit could be achieved with the Treg plus ld-IL-2 combination. This decision was based on current information available to the DSMB and based on incomplete data on the impact of T1D on C-peptide production in short intervals of time. Only through retrospective comparisons with a placebo group identified through access to data in other clinical trials including patients who were age matched was it determined that many patients can experience dramatic reduction in C-peptide production in this limited time frame. However, even given these additional comparative data, it was clear that the combination therapy did not significantly preserve or improve C-peptide production in the treated individuals. In conclusion, the off-target effect of ld-IL-2 stresses the need to develop immunotherapy molecules targeting the Treg population more specifically. We and others have developed reagents such as antibodies, pegylated IL-2, engineered or even mutant IL-2 that can expand Tregs but not NK or CD8+ T cells (54–57). These studies show the versatility of IL-2 and lay the foundations for design of a more specific cytokine in the treatment of T1D. C L I N I C A L M E D I C I N E were similar to those reported (0.33 to 1 MIU/d for 5 consecutive days) (35). Most recently, the same group tested an ld-IL-2 in children with T1D within 3 months of diagnosis (52). The authors showed by immune cytometry no differences in the CD8 and NK population between IL-2–treated and placebo groups. However, the dosage used was significantly different from the TILT trial as 0.125, 0.250, or 0.500 MIU/m2 was given daily for a 5-day course and then every 2 weeks for 1 year. were similar to those reported (0.33 to 1 MIU/d for 5 consecutive days) (35). Most recently, the same group tested an ld-IL-2 in children with T1D within 3 months of diagnosis (52). The authors showed by immune cytometry no differences in the CD8 and NK population between IL-2–treated and placebo groups. However, the dosage used was significantly different from the TILT trial as 0.125, 0.250, or 0.500 MIU/m2 was given daily for a 5-day course and then every 2 weeks for 1 year. A number of factors, including the dosage scheme, amount of IL-2, regimen of ld-IL-2 administration, or length of administration, as well as disease-related factors such as time after diagnosis or age, might also affect the outcomes with IL-2 administration. Moreover, it has been shown that genetic variants of the enhancer region of IL-2RA could affect activation of the gene (53). In the present study, there was a positive correlation between IL-2–dependent NK/Treg proliferation, suggesting that the effectiveness of ld-IL-2 may depend on responsiveness and sensitivity of IL-2 activation/regulation pathways in the Treg and the cytotoxic cell popula- tions. In addition, differences in the basal level of percentage of GZMB+ NK Treg-T1D and TILT patients could account for the effects of IL-2 administration on the expansion of non-Treg cells. It is possible that the presence of preexisting populations of activated cells, either due to the remitting/relapsing nature of the disease or viral or other infections in the patients concurrent with ld-IL-2 administration, could impact effector cell expansion. In this regard, it should be noted that 2 patients in the Treg-T1D trial showed expanded activated Tregs. The characterization of specificities of the expanded PRF1+GZMB+CD8+ T cells for self-antigen and/or viruses such as CMV or EBV would be informative in order to determine if IL-2 targets a functional autoim- mune subset of the adaptive immune cell population. C L I N I C A L M E D I C I N E Furthermore, a better understanding of the endogenous cytotoxic profiles in patients with T1D and other autoimmune diseases would be also valuable in order to anticipate the effect of IL-2 treatment in future clinical trials. C L I N I C A L M E D I C I N E Other patients showed increased percentages of activated GZMB+ NK, PRF1+ MAIT, and PRF1+GZMB+CD8+ T cell sub- sets. Several studies in healthy donors and GvHD showed that ld-IL-2 induced proliferation of non-Treg pop- ulations such as NK and CD8+ T cells (29, 35). In T1D, Todd et al. reported in an IL-2 clinical trial that a single dose ranging from 0.04 to 1.5 MIU/m2 body surface area induced transient downregulation of NK cells’ percentage in circulation at 90 minutes followed by an expansion of the population above baseline in an IL-2 dose-dependent manner (51). These results and others (35, 52) suggest that ld-IL-2 treatment affects the homeostasis of NK and activated CD8+ T cells, raising a concern about the limited dosing window with this cytokine. In another dose-defining study in T1D, Rosenzwajg et al. reported a transient induction of CD8+ T cells at 3 MIU/d, and the NK gene expression signature was upregulated. The doses used in the TILT study JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 1 2 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E Participants were seen for follow-up assessments on days 1, 3, 7, 14, 28, 38, 42, 49, 63, and 91 after infusion, then every 13 weeks up to 1 year postinfusion, and then every 26 weeks up to 2 years postinfusion. Telephone monitoring for adverse events continued every 26 weeks up to 3 years after infusion. IL-2 administration. Each course (5 consecutive days) of IL-2 was prepared by, and dispensed from, the investigational pharmacy at each clinical site for administration by subcutaneous injection in the thigh at days 3–7 and 38–42 (if applicable). Clinical personnel at the research site administered the first injection of each 5-day course of IL-2. Self-administration by participants was allowed for subsequent IL-2 injections. An administration diary was provided to record the date, time, and injection site for each administration. The cell infusions were well tolerated based on safety criteria outlined in the protocol. There was a precipitous decline of C-peptide (<50% of the baseline C-peptide response at day 28) in the first patient (patient 002-003) enrolled in the trial. Thus, the second course of IL-2 was withheld based on a mandated stopping rule and DSMB review. The rapid loss of C-peptide production was observed in 2 additional participants (002-003 and 002-005), who were administered 1 × 106 and 0.33 × 106 U/d 5 times, respectively (Supplemental Table 1). These results led to a modification of the protocol with additional participants in the second cohort receiving 2 courses of IL-2 of 0.33 × 106 U/d × 5 each. Phenotypic analysis of expanded Treg populations and peripheral blood samples. Frozen PBMCs were thawed in 10 mL X-VIVO media, spun down, and resuspended in 5 mL of X-VIVO media. Each sample was counted and distributed into 3 aliquots and processed for flow cytometry analysis, CyTOF, and 10x Genomics anal- ysis. Phenotypic and functional analyses showed that the percentage and level of expression of FOXP3+ in expanded Tregs before infusion was similar in this trial as compared with the previous Treg-T1D study (Supplemental Figure 4). Moreover, the expanded population showed high levels of CD25 expression as well as a highly demethylated Treg-specific demethylated region (TSDR) locus consistent with a bona fide Treg population (Supplemental Figure 4). Flow cytometry. A total of 1 × 106 PBMCs were washed in FACS buffer (phosphate-buffered saline + 1% FBS) and distributed into 96-well plates. C L I N I C A L M E D I C I N E hemoglobin < 10.0 g/dL; leukocytes < 3000/mL; neutrophils < 1500/mL; lymphocytes < 800/mL; platelets < 100,000/mL; Tregs < 10/mL; evidence of active infection (HIV-1/HIV-2, hepatitis B, hepatitis C, EBV or CMV genomes, or positive purified protein derivative skin test); chronic use of systemic glucocorticoids or other immunosuppressive agents or biologic immunomodulators within 6 months before study entry; history of malignancy except adequately treated basal cell carcinoma; or any chronic illness or previous treatment that, in the opinion of the investigator, should preclude participation in the trial. Pregnant or breastfeeding women were excluded from the study, as well as any woman who was unwilling to use a reliable and effective form of contraception for 2 years after Treg dosing, and any man who was unwilling to use a reliable and effective form of contraception for 3 months after Treg dosing. hemoglobin < 10.0 g/dL; leukocytes < 3000/mL; neutrophils < 1500/mL; lymphocytes < 800/mL; platelets < 100,000/mL; Tregs < 10/mL; evidence of active infection (HIV-1/HIV-2, hepatitis B, hepatitis C, EBV or CMV genomes, or positive purified protein derivative skin test); chronic use of systemic glucocorticoids or other immunosuppressive agents or biologic immunomodulators within 6 months before study entry; history of malignancy except adequately treated basal cell carcinoma; or any chronic illness or previous treatment that, in the opinion of the investigator, should preclude participation in the trial. Pregnant or breastfeeding women were excluded from the study, as well as any woman who was unwilling to use a reliable and effective form of contraception for 2 years after Treg dosing, and any man who was unwilling to use a reliable and effective form of contraception for 3 months after Treg dosing. Administration and follow-up. Results of blood chemistries and hematology were reviewed, and a histo- ry of any recent illness or fever was obtained before infusion of the cells. Patients received premedication with acetaminophen and diphenhydramine. PolyTregs were manufactured under cGMP conditions from autol- ogous participant whole blood, as previously described (12). PolyTregs were infused fresh after a 14-day ex vivo expansion via a peripheral intravenous line over 10 to 30 minutes. Vital signs were taken before infusion and 15, 30, 60, 120, and 240 minutes postinfusion. Participants were discharged after being monitored at the clinical research unit for 4 hours postinfusion. Methods Participants. This study enrolled men and women diagnosed with T1D according to the American Diabetes Association standard criteria within 3 to 24 months of screening who were 18 to 45 years of age with peak C-peptide greater than 0.2 pmol/mL (0.6 ng/mL) during MMTT challenge and who were positive for at least 1 of the following antibodies: ICA 512-antibody (IA-2ic), glutamate decarboxylase (GAD), insulin, and zinc transporter 8 (ZnT8). Patients also had to have adequate venous access to support draw of 400 mL of whole blood and infusion of investigational therapy. They were determined to be ineligible if they had 1 3 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E ratios. After blocking, 50 μL of cell surface cocktail was added and incubated on ice for an additional 40 minutes. Antibodies were washed with CSM and pelleted. Cells were resuspended in residual volume after aspiration of CSM wash, and 200 μL of fix/perm buffer (eBioscience catalog 88-8824-00) was added to each sample and incubated for 30 minutes at RT. Subsequently, 1 mL of diluted eBioscience Perm Buffer (catalog 00-8333-56) was added and centrifuged at 400g for 7 minutes. These centrifuge conditions were used for all subsequent steps. The pellets were resuspended in 50 μL intracellular antibodies diluted with the Perm Buffer and incubated on a shaker for 1 hour at RT. One mL of diluted Perm Buffer was added to each tube and centrifuged. Cells were further washed with 14 mL CSM and pelleted. Pellet was resuspended in 1 mL of 3.2% paraformaldehyde, 0.02% Saponin (MilliporeSigma catalog 47036), and 100 nM Ir (iridium) used for identifying nucleated cells (Fluidigm catalog 201192B)/PBS and stored at 4oC. Prior to running the samples on the CyTOF machine, 10 μL of 20-plex Pd (palladium) (15 μM each isotope, UCSF flow core) barcode was added to each sample and incubated at RT for 20 minutes. A total of 14 mL of CSM was added to each tube to sequester the remaining free Pd isotopes and the tubes were centrifuged. Each barcoded sample was resuspended in 0.5 mL PBS and pooled into a single 15 mL conical tubes and centrifuged. Pooled cells were resuspended in MilliQ water (MilliporeSigma) and centrifuged at 400g for 10 minutes. Cells were resuspended in water containing 1:10 diluted Eq beads (Fluidigm catalog 2010778) and adjusted to a concentration of 1.2 × 106/mL and run on the Helios CyTOF at approximately 500 cells per second at the UCSF flow core. Anti- body panel is listed in Supplemental Data File 2. FCS files acquired on Helios CyTOF at approximately 500 cells per second at the UCSF flow core are available on request. Data normalization was performed as previously described (58). All mass cytometry files were normal- ized together using the mass cytometry data normalization algorithm (59), which uses the intensity values of a sliding window of these bead standards to correct for instrument fluctuations over time and between samples. After data collection, each condition was deconvoluted using a single-cell debarcoding algorithm (60). C L I N I C A L M E D I C I N E After normalization and debarcoding of files, singlets were gated by event length and DNA. Live cells were identified by cisplatin-negative cells. All positive and negative populations and antibody-staining concentrations were determined by titration on positive and negative control cell populations. After debarcoding and normaliza- tion of CyTOF data, cell populations were gated manually in CellEngine. Populations were then exported for analysis in R. Marker expression values were then arsinh-transformed with a cofactor of 5, and then expres- sion values from all cells from a trial and time point were plotted as a violin plot using ggplot2. The results are plotted into 2 separated batches (1 and 2) due to batch effect affecting the comparison of the samples within the same analysis pipeline (batches layout of the samples, Supplemental Table 3). 10x Genomics. For 10x Genomics single-cell RNA-Seq, we first used FACS to isolate living cells (CD45+ and LIVE/DEAD blue) and then implemented the Chromium Single Cell V(D)J Enriched and 5′ Gene Expression library generation and sequencing under the guidance of the official instruction manual. Cells were loaded onto a Chromium Next GEM chip G. Cells were lysed for reverse transcription and cDNA amplification in the Chromium Controller (10x Genomics). Then 76 samples were pooled into 5 samples, and 3 duplicate wells were run for each sample pool. Full-length cDNA along with cell barcode identifiers were PCR amplified, and sequencing libraries were prepared and normalized. The constructed library was sequenced on NovaSeq S4 flow cell (Illumina). Libraries’ preparation and sequencing were performed at the institute for human genetics at the Parnassus campus of UCSF. Cell Ranger Single-Cell Software Suite (version 3.1.0, 10x Genomics) was used for library demultiplexing, FASTQ file generation, read alignment, doublet filtering, barcode counting, unique molecular identifier (UMI) counting, and to generate feature- barcode matrices, determine clusters, and perform gene expression analysis. Droplet-based sequencing data were aligned and quantified against the GRCh38 human reference genome. Quality of cells in each demulti- plexed sample was then assessed based on 3 metrics: the number of total UMI counts per cell (library size), the number of detected genes per cell, and the proportion of mitochondrial gene counts. After quality control metric filtering, a total of 464,348 cells were retained for downstream analysis. C L I N I C A L M E D I C I N E Cell were stained in FACS buffer containing 1/500 of LIVE/DEAD blue reagent (Invitrogen) and incubated at room temperature for 30 minutes protected from light. After 1 wash with FACS buffer, cells were incubated with human Fc block reagent for 5 minutes, stained with cell surface fluorochrome-conjugated anti-human CD19, CD45RA, HLA-DR, CD8, CD127, CD27, CD4, CRO, CD40, CD56, CD38, CD3, CD14, and CD25 for 30 minutes on ice. After 2 washes in FACS buffer, cells were ana- lyzed by BD LSRII flow cytometer. All data analysis was performed using FlowJo software. Antibody panel is listed in Supplemental Data File 2. CyTOF. Cryopreserved cells were thawed and placed in 10 mL of 10% FBS-DMEM and pelleted, resus- pended in 2 mL of the same media, and counted, and 1 × 106 cells were removed for CyTOF staining. Cells were centrifuged and resuspended in 0.5 mL PBS in 15 mL conical tubes, and an equal volume of 1:5000 diluted cisplatin was added for a final concentration of 5 μM (Fluidigm catalog 201198) and incubated at room temperature (RT) for 5 minutes. Cisplatin reaction was quenched by adding 14 mL CSM (0.5% BSA, 2 mM EDTA in PBS) to each tube and pelleted. Cells were resuspended in 50 μL of FcBlock (Miltenyi Biotec catalog 130-059-901), diluted to manufacturer’s recommendations for 5 minutes. Mass cytometry antibod- ies were previously titrated using cryopreserved peripheral blood controls to achieve optimal signal-to-noise JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 1 4 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E the GRCh38 human VDJ reference genome. Resultant filtered annotated contigs were analyzed and concate- nated to the 10x single-cell gene expression data in python. the GRCh38 human VDJ reference genome. Resultant filtered annotated contigs were analyzed and concate- nated to the 10x single-cell gene expression data in python. Frequency of each clonotype was calculated in python and Gini coefficient was calculated using Olivia Guest, Gini, and Calculate the Gini coefficient of a numpy array, from GitHub repository, https://github. com/oliviaguest/gini. The Gini coefficient measures the inequality among values of a frequency distribution. A Gini score equals 0 when each element of the population has the same frequency. A high Gini score, closer to 1, suggests clonotype enrichment. Demuxlet pipeline analysis. Samples from each patient were genotyped by Infinium Omni Express Exome array (Illumina). Resulting VCF genotype files and single-cell RNA-Seq BAM files were used to run Demuxlet (31) with default parameters. Demultiplexing results were largely concordant with freemuxlet (https://github. com/statgen/popscle/). Subsequently, the multiplexed single-cell RNA-Seq and single-cell TCR-Seq data were filtered according to a singlet white list extracted from the demuxlet.best output file. TSDR methylation assay. Genomic DNA from 1 × 106 expanded Tregs was analyzed by Epiontis GmbH according to established protocol. Percentages of demethylated TSDR were calculated as follows: (mean copy numbers of unmethylated DNA/[mean copy numbers of unmethylated DNA + copy numbers of methylated DNA]) × 100. For Tregs from women, the percentages calculated above were multiplied by 2 to correct for X chromosome inactivation. Treg 2H tracking. During the 14-day clinical expansion period, the 2H2 label from [6,6-2H2]glucose in the X-VIVO 15 culture medium was incorporated into the DNA of replicating polyclonal CD4+CD127lo/−CD25+ Tregs as previously described (42). Initial qualifying experiments showed no differences in fold expansions, phenotype, or percentage TSDR demethylation between Tregs grown in X-VIVO 15 and Tregs grown in [2H2] glucose-containing X-VIVO 15. Functional suppression assay results showed similar inhibition between Tregs expanded in either type of medium, and cultures were free from bacteria, fungi, mycoplasma, or endotoxin contaminants. Mass spectrometry analyses showed that Tregs expanded in X-VIVO 15 with [6,6-2H2]glucose in the medium at 100% enrichment were approximately 60% enriched for 2H2 in the deoxyribose moiety of purine deoxyribonucleotides isolated from DNA, which is the theoretical maximum deuterium enrichment observed in deoxyribose in fully replaced cells that divided in the presence of [6,6-2H2]glucose (42, 43). C L I N I C A L M E D I C I N E This 60% enrich- ment level was consistently observed in all 7 preparations in this clinical study. After infusion of the labeled Tregs, peripheral blood was collected from the study participants, and PBMCs were sorted into the Treg population (CD4+CD25+CD127lo/–) and 3 non-Treg populations (CD4+CD25loC- D127hi CD45RO+, CD45RO+CD62Llo, CD45RO+CD62Lhi). Deuterium enrichments were compared between the Treg population and a mixture of the 3 non-Treg populations. Laboratory tests. Biochemical autoantibody titers were assayed at the Barbara Davis Center using radioim- munobinding assays, and ICA was measured at the University of Florida. C-peptide and HbA1c were mea- sured at the Northwest Lipid Research Laboratory. Viral loads for EBV and CMV were assessed by ViraCor Laboratories. Chemistries and hematology were performed at local clinical laboratories at UCSF and Yale. Mass spectrometry analysis was performed the Department of Nutritional Sciences & Toxicology at the Uni- versity of California, Berkeley. Data availability. The 10x Genomics single-cell sequencing data reported in this paper have been deposited in the Gene Expression Omnibus database (Study: GSE178991 and Series record: GSE178991). Data availability. The 10x Genomics single-cell sequencing data reported in this paper have been deposited in the Gene Expression Omnibus database (Study: GSE178991 and Series record: GSE178991). Statistics. Data analyses were performed using GraphPad Prism 6.0 software, and values at P < 0.05 were deemed significant. One-way ANOVA tests were performed in order to assess statistical significance. Study approval. All participants provided written informed consent before participating in any study proce- dures. UCSF Institutional Review Board, San Francisco, California, USA, approved the study. C L I N I C A L M E D I C I N E Using Scanpy (61), we performed BBKNN batch correction and principal components analysis on the gene expression matrix, and we then built a nearest neighbor graph to calculate a UMAP and find clusters via the louvain algorithm. Clusters were identified based on the expression of canonical marker genes. Differential gene expressions were generated using Scanpy, and volcano plots depicting gene expression changes in distinct immune cell subsets were generated using GraphPad Prism 6.0 software. Single-cell TCR-Seq data processing and RNA-Seq integration. Data from 10x Genomics single-cell VDJ sequencing were aligned and quantified using the Cell Ranger Single-Cell Software Suite (version 3.1.0) against JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 1 5 C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E experiment design and supervised Demuxlet analysis; and SD and JAB analyzed the results and wrote the article. All authors edited the report and approved the final manuscript. experiment design and supervised Demuxlet analysis; and SD and JAB analyzed the results and wrote the article. All authors edited the report and approved the final manuscript. Acknowledgments The authors would like to thank the members of the Bluestone and UCSF cGMP facility for their efforts to develop Treg therapies to treat human disease. Study funding was provided through the Sean N. Parker Autoim- mune Research Laboratory Fund and supported in part by the National Center for Research Resources through Clinical Translational Science Awards NIH/NCRR UCSF-CTSI grant number UL 1 TR000004. Study drug was provided by Prometheus Laboratories, Inc. Biomarker studies were performed in collaboration with the Immune Tolerance Network and supported by the National Institute of Allergy and Infectious Diseases of the NIH under award number UM1AI109565. Flow core funding was provided by RRID:SCR_018206 and DRC Center Grant NIH P30 DK063720, NIH S10 1S10OD018040-01, and NIH S10 1S10OD021822-01. The con- tent is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Address correspondence to: Jeffrey A. Bluestone, HSW 1112 Box 0540, 513 Parnassus Ave., San Francisco, California 94143, USA. Phone: 415.514.0417; Email: Jeff.Bluestone@ucsf.edu. 1. Bluestone JA, et al. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010;464(7293):1293–1300. 2. Menke A, et al. The prevalence of type 1 diabetes in the United States. Epidemiology. 2013;24(5):773–774. 3. Miller KM, et al. Current state of type 1 diabetes treatment in the U.S.: updated data from the T1D Exchange clinic registry. Diabetes Care. 2015;38(6):971–978. 4. Brusko TM, et al. Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes. 2005;54(5):1407–1414. 5. Lindley S, et al. Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes. 2005;54(1):92–99. 6 L SA l D f i IL 2R i li ib di i i h d i f FOXP3 i i CD4( )CD25( ) l 5. Lindley S, et al. Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes. 2005;54(1):92–99. 6. Long SA, et al. Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in CD4(+)CD25(+) regulatory Lindley S, et al. Defective suppressor function in CD4(+)CD25(+) T-c 5. Lindley S, et al. Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes. 2005;54(1):92–99. 6. Long SA, et al. Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in CD4(+)CD25(+) regulatory T-cells of type 1 diabetic subjects. Diabetes. 2010;59(2):407–415. T-cells of type 1 diabetic subjects. Diabetes. 2010;59(2):407–415. 7. Marwaha AK, et al. Acknowledgments Cutting edge: increased IL-17-secreting T cells in children with new-onset type 1 diabetes. J Immunol. 2010;185(7):3814–3818. 8. McClymont SA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol. 2011;186(7):3918–3926. 9. Schneider A, et al. The effector T cells of diabetic subjects are resistant to regulation via CD4+ FOXP3+ regulatory T cells. J Immunol. 2008;181(10):7350–7355. 10. Buckner JH. Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases. Nat Rev Immunol. 2010;10(12):849–859. 11. Tang Q, Bluestone JA. Regulatory T-cell therapy in transplantation: moving to the clinic. Cold Spring Harb Perspect Med. 2013;3(11):a015552. 12. Bluestone JA, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med. 2015;7(315):315ra189. 13. Webb GJ, Hirschfield GM. Using GWAS to identify genetic predisposition in hepatic autoimmunity. J Autoimmun. 2016;66:25–39. 14. Yu A, Malek TR. Selective availability of IL-2 is a major determinant controlling the production of CD4+CD25+Foxp3+ T regulatory cells. J Immunol. 2006;177(8):5115–5121. regulatory cells. J Immunol. 2006;177(8):5115–5121. 15. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol. 2012;12(3):180–190. 16. Weist BM, et al. Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat Immunol. 2015;16(6):635–641. competition. Nat Immunol. 2015;16(6):635–641. 17. Liao W, et al. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38(1):13–25. 17. Liao W, et al. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38(1):13–25. 18. Roth TL, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature. 2018 559(7714) 405 409 17. Liao W, et al. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38(1):13–25. 18 R th TL t l R i h T ll f ti d ifi it ith i l t ti N t 18. Roth TL, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature. 2018;559(7714):405–409. ; ( ) 19. Grinberg-Bleyer Y, et al. IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J Exp Med 2010;207(9):1871 1878 19. Grinberg-Bleyer Y, et al. IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J Exp Med. 2010;207(9):1871–1878. 20. Tang Q, et al. Author contributions JAB, SEG, KJHG, and QT were the principal investigators of the study, participated in its design, and ana- lyzed results. JHE supervised Treg production and study design and monitoring. SD performed flow and 10x Genomics experiments and analysis; KCM performed analysis of CyTOF data; CTM performed Demuxlet analysis; WL, SJT, CMT, and WT performed CyTOF experiments; APL, WL, VN, ML, and ALP generated Treg products and performed the quality control assays; ASL is the clinical coordinator and analyzed clinical data and metabolic responses; WL, MM, and SD participated in sample preparation for flow cytometry, CyTOF, and 10x Genomics experiments; MHS advised for CyTOF analysis; CJY advised 10x Genomics 1 6 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 2. Menke A, et al. The prevalence of type 1 diabetes in the United States. Epidemiology. 2013;24(5):773–774. 1. Bluestone JA, et al. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010;464(7293):1293–13 3. Miller KM, et al. Current state of type 1 diabetes treatment in the U.S.: updated data from the T1D Exchange clinic registry. Diabetes Care. 2015;38(6):971–978. C L I N I C A L M E D I C I N E 27. Moran A, et al. Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised, double-blind, placebo- controlled trials. Lancet. 2013;381(9881):1905–1915. 27. Moran A, et al. Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised, double-blind, placebo controlled trials. Lancet. 2013;381(9881):1905–1915. ; ( ) 28. Hartemann A, et al. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo- controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295–305. 28. Hartemann A, et al. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo- 28. Hartemann A, et al. Low-dose interleukin 2 in patients with typ controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295–305. 28. Hartemann A, et al. Low dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double blind, placebo controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295–305. controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295–305. 29. Hirakawa M, et al. Low-dose IL-2 selectively activates subsets of CD4+ Tregs and NK cells. JCI Insight. 2016;1(18):89278. 29. Hirakawa M, et al. Low-dose IL-2 selectively activates subsets o 30. Matsuoka K, et al. Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-h disease. Sci Transl Med. 2013;5(179):179ra43. g HM, et al. Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat Biotechnol. 2018;36(1):89–94. nblum MD et al Regulatory T cell memory Nat Rev Immunol 2016;16(2):90 101 31. Kang HM, et al. Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat Biotechnol. 2018;36(1):89–9 31. Kang HM, et al. Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat Biotechnol. 2018;36(1):89–94. 32. Rosenblum MD, et al. Regulatory T cell memory. Nat Rev Immunol. 2016;16(2):90–101. 31. Kang HM, et al. Multiplexed droplet single cell RNA sequencing using natural genetic variation. Nat Biotechnol. 2018;36(1):89 94. 32. Rosenblum MD, et al. Regulatory T cell memory. Nat Rev Immunol. 2016;16(2):90–101. 32. Rosenblum MD, et al. Regulatory T cell memory. Nat Rev Immunol. 2016;16(2):90–101. 33. Yu A, et al. A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells Immunity. 2009;30(2):204–217. 34. Saadoun D, et al. Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. N Engl J Med. 2011;365(22):2067–2077. ( ) 35. Rosenzwajg M, et al. Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. J Autoimmun 2015;58:48–58. 36. Carnero Contentti E, et al. C L I N I C A L M E D I C I N E Mucosal-associated invariant T cell features and TCR repertoire characteristics during the course of multiple sclerosis. Front Immunol. 2019;10:2690. 37. Reantragoon R, et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J Exp Med. 2013;210(11):2305–2320. 38. Gulden E, et al. MAIT cells: a link between gut integrity and type 1 diabetes. Cell Metab. 2017;26(6):813–815. 39. Rouxel O, et al. Cytotoxic and regulatory roles of mucosal-associated invariant T cells in type 1 diabetes. Nat Immunol. 2017;18(12):1321–1331. Toubal A, et al. Mucosal-associated invariant T cells and disease. N 41. Dias J, et al. Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity. J Leukoc Biol. 2016;100(1):233–240. 42. Fontenot JD, et al. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol. 2005;6(11):1142–1151. 43. Knoechel B, et al. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J Exp Med. 2005;202(10):1375–1386. 44. Barron L, et al. Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells. J Immunol. 2010;185(11):6426–6430. 45. Abbas AK, et al. Revisiting IL-2: biology and therapeutic prospects. Sci Immunol. 2018;3(25):eaat1482. 46. Marek-Trzonkowska N, et al. Factors affecting long-term efficacy of T regulatory cell-based therapy in type 1 diabetes. J Transl Med. 2016;14(1):332. 47. Koreth J, et al. Efficacy, durability, and response predictors of low-dose interleukin-2 therapy for chronic graft-versus-host disease. Blood. 2016;128(1):130–137. 48. Castela E, et al. Effects of low-dose recombinant interleukin 2 to promote T-regulatory cells in alopecia areata. JAMA Dermatol. 2014;150(7):748–751. 48. Castela E, et al. Effects of low-dose recombinant interleukin 2 to promote T-regulatory cells in alopecia areata. JAMA Dermatol. 2014;150(7):748–751. 49. Long SA, et al. Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function. Diabetes. 2012;61(9):2340–2348. β-cell function. Diabetes. 2012;61(9):2340–2348. 50. Yu A, et al. Selective IL-2 responsiveness of regulatory T cells th 50. Yu A, et al. Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes Diabetes 2015;64(6):2172–2183 50. Yu A, et al. Selective IL-2 responsiveness of regulatory T cells thro IL-2 therapy in type 1 diabetes. Diabetes. 2015;64(6):2172–2183. IL-2 therapy in type 1 diabetes. Diabetes. 2015;64(6):2172–2183 p p 51. Todd JA, et al. Regulatory T cell responses in participants with type 1 diabetes after a single dose of interleukin-2: a non-randomised, open label, adaptive dose-finding trial. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 Acknowledgments Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity. 2008;28(5):687–697. 21. Koreth J, et al. Interleukin-2 and regulatory T cells in graft-versus-host disease. N Engl J Med. 2011;365(22):2055–2066. 22. Matsuoka KI. Low-dose interleukin-2 as a modulator of Treg homeostasis after HSCT: current understanding and future perspectives. Int J Hematol. 2018;107(2):130–137. 23. Zhao C, et al. Low dose of IL-2 combined with rapamycin restores and maintains the long-term balance of Th17/Treg cells in refractory SLE patients. BMC Immunol. 2019;20(1):32. 24. He J, et al. Efficacy and safety of low-dose IL-2 in the treatment of systemic lupus erythematosus: a randomised, double-blin l b t ll d t i l A Rh Di 2020 79(1) 141 149 24. He J, et al. Efficacy and safety of low-dose IL-2 in the treatment of systemic lupus erythematosus: a randomised, double-blind, placebo-controlled trial. Ann Rheum Dis. 2020;79(1):141–149. placebo-controlled trial. Ann Rheum Dis. 2020;79(1):141–149. 25. Spolski R, et al. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol. 2018;18(10):648–659. 26. Cabrera SM, et al. Interleukin-1 antagonism moderates the inflammatory state associated with Type 1 diabetes during clinical trials conducted at disease onset. Eur J Immunol. 2016;46(4):1030–1046. JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 1 7 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E C L I N I C A L M E D I C I N E PLoS Med. 2016;13(10):e1002139. 51. Todd JA, et al. Regulatory T cell responses in participants with type 1 diabetes after a single dose of interleukin-2: a non-randomi 51. Todd JA, et al. Regulatory T cell responses in participants with 51. Todd JA, et al. Regulatory T cell responses in participants with type 1 diab open label, adaptive dose-finding trial. PLoS Med. 2016;13(10):e1002139. 51. Todd JA, et al. Regulatory T cell responses in participants with type 1 diab open label, adaptive dose-finding trial. PLoS Med. 2016;13(10):e1002139. open label, adaptive dose-finding trial. PLoS Med. 2016;13(10):e1002139. 52. Rosenzwajg M, et al. Low-dose IL-2 in children with recently diagnosed type placebo-controlled, dose-finding study. Diabetologia. 2020;63(9):1808–1821. 52. Rosenzwajg M, et al. Low-dose IL-2 in children with recently diagnosed type 1 diabetes: a phase I/II randomised, double-blind, placebo-controlled, dose-finding study. Diabetologia. 2020;63(9):1808–1821. 52. Rosenzwajg M, et al. Low dose IL 2 in children with recently diagnosed type 1 diabetes: a phase I/II randomised, double blind, placebo-controlled, dose-finding study. Diabetologia. 2020;63(9):1808–1821. placebo-controlled, dose-finding study. Diabetologia. 2020;63(9):1808–1821. 53. Simeonov DR, et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature. 2017;549(7670):111–115. 54. Trotta E, et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism. Nat Med. 2018;24(7):1005–1014. 55. Khoryati L, et al. An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice. Sci Immunol. 2020;5(50):eaba5264. 56. Charych DH, et al. NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models. Clin Cancer Res. 2016;22(3):680–690. efficacy in mouse tumor models. Clin Cancer Res. 2016;22(3):680–690. efficacy in mouse tumor models. Clin Cancer Res. 2016;22(3):680–690. 57. Sockolosky JT, et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes. Science. 2018;359(6379):1037–1042. 58. Spitzer MH, et al. Systemic immunity is required for effective cancer immunotherapy. Cell. 2017;168(3):487–502. 59. Finck R, et al. Normalization of mass cytometry data with bead standards. Cytometry A. 2013;83(5):483–494. 60. Zunder ER, et al. Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm Nat Protoc. 2015;10(2):316–333. 61. Wolf FA, et al. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018;19(1):15. 1 8 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474 JCI Insight 2021;6(18):e147474 https://doi.org/10.1172/jci.insight.147474
https://openalex.org/W3037587206
https://repository.ubn.ru.nl//bitstream/handle/2066/227352/227352.pdf
English
null
Gray matter covariations and core symptoms of autism. The EU-AIMS Longitudinal European Autism Project
bioRxiv (Cold Spring Harbor Laboratory)
2,020
cc-by
10,446
Gray matter covariations and core symptoms of autism: the EU-AIMS Longitudinal European Autism Project Mei, Ting; Llera, A.; Floris, D.L.; Forde, N.; Tillmann, J.; Durston, Sarah; Moessnang, C.; Rausch, A.; Beckmann, C.F.; Buitelaar, J.K. 2020, Article / Letter to editor (Molecular Autism, 11, 1, (2020), article 86) Doi link to publisher: https://doi.org/10.1186/s13229-020-00389-4 Version of the following full text: Publisher’s version Downloaded from: https://hdl.handle.net/2066/227352 Download date: 2024-10-24 Open Access Open Access Gray matter covariations and core symptoms of autism: the EU‑AIMS Longitudinal European Autism Project Ting Mei1*  , Alberto Llera1,2, Dorothea L. Floris1, Natalie J. Forde1, Julian Tillmann3, Sarah Durston4, Carolin Moessnang5, Tobias Banaschewski6, Rosemary J. Holt7, Simon Baron‑Cohen7, Annika Rausch1, Eva Loth8, Flavio Dell’Acqua8, Tony Charman3, Declan G. M. Murphy8, Christine Ecker8,9, Christian F. Beckmann1,10 and Jan K. Buitelaar1,2* on behalf of the EU-AIMS LEAP group © The Author(s) 2020. 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://creat​iveco​mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​ mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Mei et al. Molecular Autism (2020) 11:86 https://doi.org/10.1186/s13229-020-00389-4 Abstract Background:  Voxel-based morphometry (VBM) studies in autism spectrum disorder (autism) have yielded diverging results. This might partly be attributed to structural alterations being associating with the combined influence of several regions rather than with a single region. Further, these structural covariation differences may relate to continuous measures of autism rather than with categorical case–control contrasts. The current study aimed to identify structural covariation alterations in autism, and assessed canonical correlations between brain covariation patterns and core autism symptoms. Methods:  We studied 347 individuals with autism and 252 typically developing individuals, aged between 6 and 30 years, who have been deeply phenotyped in the Longitudinal European Autism Project. All participants’ VBM maps were decomposed into spatially independent components using independent component analysis. A generalized linear model (GLM) was used to examine case–control differences. Next, canonical correlation analysis (CCA) was performed to separately explore the integrated effects between all the brain sources of gray matter variation and two sets of core autism symptoms. Results:  GLM analyses showed significant case–control differences for two independent components. The first component was primarily associated with decreased density of bilateral insula, inferior frontal gyrus, orbitofrontal cortex, and increased density of caudate nucleus in the autism group relative to typically developing individuals. The second component was related to decreased densities of the bilateral amygdala, hippocampus, and parahippocampal gyrus in the autism group relative to typically developing individuals. The CCA results showed significant correlations between components that involved variation of thalamus, putamen, precentral gyrus, frontal, parietal, and occipital lobes, and the cerebellum, and repetitive, rigid and stereotyped behaviors and abnormal sensory behaviors in autism individuals. Limitations:  Only 55.9% of the participants with autism had complete questionnaire data on continuous parent- reported symptom measures. *Correspondence: t.mei@donders.ru.nl; Jan.Buitelaar@radboudumc.nl 1 Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Full list of author information is available at the end of the article *Correspondence: t.mei@donders.ru.nl; Jan.Buitelaar@radboudumc.nl 1 Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Full list of author information is available at the end of the article Background and incorporate an alternative that adheres more closely to the hypothesis of autism as a disconnection syndrome [13], thus providing greater sensitivity for between-group effects. For this purpose, we identify inter-regional sources of structural covariation using independent component analysis (ICA) [15], a data-driven unsupervised approach that allows the identification of interconnected brain regions across the whole brain. It has previously been applied successfully to identify covariance of brain morphometry in patients with psychiatric disorders [16–18]. Second, we move beyond the categorical autism case–control comparison towards exploring associations between brain structure and symptom dimensions or profiles of autism. Although former studies have used univariate approaches to explore the relationship between brain substrates and clinical phenotypes [6, 19], such associations are potentially the consequence of integrated effects across multiple symptoms dimensions and brain regions, rather than simple associations between a specific brain region and a specific symptom dimension. To study such multidimensional associations multivariate methods are effective [20, 21] and here we achieve this integration using Canonical Correlation Analysis (CCA) [22]. g Autism spectrum disorder (henceforth autism) is an early onset neurodevelopmental condition characterized by core deficits in social interaction and communication, along with restrictive interests and behavior, and sensory abnormalities [1]. Magnetic resonance imaging (MRI) studies have increased our understanding of the neuroanatomical underpinnings of autism and show that autism is associated, at the group level, with brain structural changes [2]. However, many results are not robust across different studies. For example, two studies using the same large-scale open access Autism Brain Imaging Data Exchange (ABIDE) dataset [3] came to different conclusions with regard to the volume of the pallidum [4, 5]. Also, across whole brain approaches investigating cortical (i.e., cortical thickness and surface area) and subcortical (i.e., volume) features have been inconsistent; two large-scale pooled estimate analytical studies observed diverging results of cortical changes in autism [6, 7]. Similarly, autism studies quantifying voxel- wise gray matter (GM) density also found divergent results of GM differences between autism diagnosed and control individuals; for instance, meta-analyses reported diverse changes of GM morphometry in autistic individuals on average, reporting either increased or decreased density of right inferior temporal gyrus in autism [8, 9]. Even when taking age into account, studies still observed different structural brain alterations in children and adolescents with autism [10, 11]. Background In summary, we investigate alterations in GM morphometric covariations in a deeply phenotyped large European autism case–control sample [23, 24] that allows us to improve our understanding of correlated structural brain alterations in autism. Subsequently, we focus on the covariation between the identified structural features and symptom behavior profiles among individuals with autism. A commonality to all these studies is their reliance on mass-univariate statistics. This approach identifies alterations in isolated regions or voxels but ignores possible relationships between them. The brain is a complex system of interconnected networks, and research into the neural basis of autism has moved away from focusing on local abnormalities into conceptualizing autism as a disorder of alterations in structural and functional brain connectivity [12]. This implies that structural brain alterations in autism likely reflect the combined influence of several regions and are not confined to one specific region [13, 14]. The present paper aims to advance prior work on brain structural neural correlates of autism in two ways. First, we aim to move away from the standard univariate approach © The Author(s) 2020. 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://creat​iveco​mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​ mmons​.org/publi​cdoma​in/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 (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism Conclusions:  Covaried areas associated with autism diagnosis and/or symptoms are scattered across the whole brain and include the limbic system, basal ganglia, thalamus, cerebellum, precentral gyrus, and parts of the frontal, parietal, and occipital lobes. Some of these areas potentially subserve social-communicative behavior, whereas others may underpin sensory processing and integration, and motor behavior. Keywords:  Autism, Magnetic resonance imaging, Voxel-based morphometry, Independent component analysis, Canonical correlation analysis GM density estimation Voxel-based morphometry (VBM) is a spatially unbiased whole-brain approach that extracts voxel-wise GM density (the amount of GM at a voxel) estimations. We performed VBM analyses using the CAT12 toolbox [30] in SPM12 (Wellcome Department of Imaging Neuroscience, London, UK). T1-weighted images were automatically segmented into GM, white matter, and cerebrospinal fluid and affine registered to the MNI template to improve segmentation. All resulting segmented GM maps were then used to generate a study-specific template and registered to MNI space via a high-dimensional, nonlinear diffeomorphic registration algorithm (DARTEL) [31]. A Jacobian modulation step was included using the flow fields to preserve voxel- wise information on local tissue volume. Images were smoothed with a 4  mm full-width half-max (FWHM) isotropic Gaussian kernel. MRI data acquisition ll All participants were scanned on 3  T MRI scanners (University of Cambridge: Siemens Verio; King’s College London: GE Medical Systems Discovery MR 750; Mannheim University: Siemens TimTrio; Radboud University: Siemens Skyra; Rome University: GE Medical Systems Sigma HDxTt; Utrecht University: Philips Medical Systems Achieva/Ingenia CX). High-resolution structural T1-weighted images were acquired with full head coverage, at 1.2  mm thickness with 1.1 × 1.1  mm in-plane resolution.For all other scanning parameters, please see Additional file 1: Subsection 1. In the present study, we selected participants with available structural MRI data. All images were inspected visually and participants were excluded in cases of brain injury or structural abnormalities (e.g., enlarged ventricles or cysts), excessive head motion, or preprocessing failure (n = 29). We excluded the participants from the Rome site due to low sample size (n = 1). We also excluded participants without full- scale intelligence quotient (FSIQ) data in the further statistical analyses (n = 5). This resulted in a sample of 599 participants from 5 sites, including 347 individuals with autism and 252 typically developing (TD) controls. Demographic and clinical information is shown in Table 1. Participantsh The participants were selected from the first wave of the European Autism Interventions—A Multicentre Study for Developing New Medications (EU-AIMS) Longitudinal European Autism Project (LEAP) dataset, which is a large multicenter study that aims to identify and validate biomarkers for autism [24]. In total, six centers are involved: Institute of Psychiatry, Psychology and Neuroscience, King’s College London, United Kingdom; Autism Research Centre, University of Cambridge, Page 3 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism United Kingdom; Radboud University Medical Centre, Nijmegen, the Netherlands; University Medical Centre Utrecht, the Netherlands; Central Institute of Mental Health, Mannheim, Germany; and University Campus Bio-Medico, Rome, Italy. Each participant underwent clinical, cognitive, and MRI assessment. Autism diagnoses were confirmed by clinicians according to the Diagnostic and Statistical Manual-IV (DSM-IV), International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10), or DSM-5. The study was approved by local ethical committees in each participating center, and written informed consent was provided by all participants and/or their legal guardians (for those < 18 years old). For further details on experimental design and clinical assessments, see [23, 24]. Disorder (ADHD), we included comorbidity with ADHD as an additional covariate in analyses. ADHD symptoms were assessed with the ADHD DSM-5 rating scale that includes symptom scales of inattention and hyperactivity/ impulsivity symptoms. The ADHD DSM-5 rating scale was based on either parent-report or self-report scores; self-report scores were only used when parent-reports were unavailable. The categorical output of the ADHD rating scale was used in this study. The summary for each of these clinical measures can be found in Table 1. Clinical measures W d h A We considered calibrated severity scores g  Parent report SRS scores were available for 284 participants with autism and 135 TD individuals h  RBS scores were available for 277 participants with autism and 133 TD individuals i  SSP scores were available for 201 participants with autism and 115 TD individuals g,h,i  In all questionnaires, the scores of the autism group only were used in our study, and they are all parent-rated Demographic Autism, n = 347 TD, n = 252 t/χ2 p value Mean SD Mean SD Age, ­yearsa 16.79 5.56 16.92 5.71 − 0.270 0.788 FSIQa,b 99.40 18.94 104.88 18.26 − 3.549 p < 0.001  FSIQ ≥ 75 104.29 14.95 109.02 13.07 − 3.883 p < 0.001  FSIQ < 75 66.61 5.44 63.69 9.20 1.399 0.172 n % n % Sex, male/femalec 253/94 72.9/27.1 163/89 64.7/35.3 4.658 0.031 ADHD rating ­scaled, with/ without ADHD 139/160 46.5/53.5 21/180 10.4/89.6 71.750 p < 0.001 Symptom profiles Mean SD Mean SD ADIe  Social interaction 16.80 6.66  Communication 13.50 5.62  RRB 4.32 2.67 ADOSf  Social affect 6.04 2.59  RRB 4.73 2.78 SRS T-scoreg 70.59 12.06 47.24 8.79 RBSh 15.76 13.42 2.20 8.28 SSPi 138.62 27.28 175.97 16.18 Table 1  Participant characteristics TD, typically developing; SD, standard deviation; FSIQ, full-scale intelligence quotient; ADHD, Attention Deficit Hyperactivity Disorder; ADI, Autism Diagnostic Interview-Revised; RRB restricted repetitive behaviors; ADOS Autism Diagnostic Observational Schedule 2; SRS Social Responsiveness Scale 2nd Edition; RBS Demographic Autism, n = 347 TD, n = 252 t/χ2 p value Mean SD Mean SD Age, ­yearsa 16.79 5.56 16.92 5.71 − 0.270 0.788 FSIQa,b 99.40 18.94 104.88 18.26 − 3.549 p < 0.001  FSIQ ≥ 75 104.29 14.95 109.02 13.07 − 3.883 p < 0.001  FSIQ < 75 66.61 5.44 63.69 9.20 1.399 0.172 n % n % Sex, male/femalec 253/94 72.9/27.1 163/89 64.7/35.3 4.658 0.031 ADHD rating ­scaled, with/ without ADHD 139/160 46.5/53.5 21/180 10.4/89.6 71.750 p < 0.001 Symptom profiles Mean SD Mean SD ADIe  Social interaction 16.80 6.66  Communication 13.50 5.62  RRB 4.32 2.67 ADOSf  Social affect 6.04 2.59  RRB 4.73 2.78 SRS T-scoreg 70.59 12.06 47.24 8.79 RBSh 15.76 13.42 2.20 8.28 SSPi 138.62 27.28 175.97 16.18 Table 1  Participant characteristics after preprocessing, whereas none of them were observed obvious artifacts, and they were consequently included in the final analyses. Clinical measures W d h A We used the Autism Diagnostic Interview-Revised (ADI) [25] and the Autism Diagnostic Observational Schedule 2 (ADOS) [26] to quantify past (ever and previous 4-to-5 years) and current autism symptoms of the social interaction, communication, and restricted repetitive behaviors (RRB) domains. We used T-scores (age- and sex-adjusted) of the Social Responsiveness Scale 2nd Edition (SRS) [27] in the autism group to assess severity of autistic traits/symptoms and the Repetitive Behavior Scale-Revised (RBS) [28] to measure repetitive and rigid behaviors associated with autism. Moreover, sensory processing abnormalities of autism were assessed with the Short Sensory Profile (SSP) [29]. To examine associations between clinical features in autism and brain measures, we created two sets of clinical measures: (1) the subscale scores of ADI-R and ADOS, both instruments were rated by qualified examiners, and (2) the total scores of SRS, RBS, and SSP, we included parent- rated reports only and limited the analyses to within the autism group. Further, concerning the potential effect of comorbidity with Attention Deficit Hyperactivity A full quality control report was generated by the CAT SPM pipeline for each participant that included visualizations of the segmentation, which were evaluated visually, and quantitative quality measures including mean correlation from sample homogeneity module and weighted overall image quality ratings that were additionally used to detect and exclude images of insufficient quality for inclusion in analysis. We visually checked the images with the mean correlation smaller than three standard deviations from the sample mean. Accordingly, 5 participants required visual inspection Mei et al. Molecular Autism (2020) 11:86 Page 4 of 13 Mei et al. Molecular Autism Table 1  Participant characteristics TD, typically developing; SD, standard deviation; FSIQ, full-scale intelligence quotient; ADHD, Attention Deficit Hyperactivity Disorder; ADI, Autism Diagnostic Interview-Revised; RRB, restricted, repetitive behaviors; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile a  Statistical differences were assessed by two-sample t test b  In Schedule A, B, and C (FSIQ ≥ 75), there are 302 participants with autism and 229 participants with TD. Schedule D (FSIQ < 75) comprised 45 participants with autism and 23 TD individuals c  Sex difference was examined by the chi-square test d  ADHD rating scores were available for 500 participants, including 299 individuals with autism and 201 TD individuals e  ADI scores were available for 332 participants f  ADOS scores were available for 339 participants. Clinical measures W d h A orders (i.e., number of components) tend to extract spatially sparser maps, and hence participant loading vectors variance would be explained by smaller subsets of participants. Therefore, 100 components were chosen to capture as much variation as possible while remaining statistically powered. However, the choice of model order is heuristic, we thereby examined the dependence on model order using different dimensionalities; more precisely, in addition to the 100 dimensional factorization, we also considered an automatic dimension estimation approach as implemented in MELODIC-ICA and a 50 dimensional independent component factorization. Statistical approach level. Canonical variates are generated independently for the brain and the behavioral datasets according to the product of the canonical coefficients (learned through CCA) and the original datasets. We referred to each pair of canonical variates as CCA mode [38]. The canonical coefficients separately represent weights for each brain variable and each symptom measure. For interpretation of the contribution of each independent source and each clinical measure to the CCA mode, their canonical coefficients were corrected as noted in [39]. In this work we focused on the first canonical mode since it provides the strongest associations and we denoted it as the main CCA mode. Furthermore, for each CCA analysis the statistical significance of the main CCA mode was determined by permutation testing (10,000 permutations, Bonferroni corrected p < 0.05/number of CCA modes). Here, we performed two separate CCA analyses to link the independent components participants’ contributions to subsets of behavioral measures; in the first CCA analyses (CCA​1) we included the subscales of ADI and ADOS as clinical measures and in the second (CCA​2) we used total scores of SRS, RBS, and SSP. The reliability of the CCA results presented as well as its dependence on the number of participants were tested using a leave- one-out cross-validation approach (Additional file  1: Subsection 4). For completeness we first performed a standard mass- univariate statistical analyses directly on the GM densities. To that end we used a generalized linear model (GLM) to detect group differences (autism vs. TD) on GM densities using the FMRIB Software Library v6.0 (FSL) [33]. Participants’ VBM maps were considered as the dependent variable and diagnostic group as the independent factor, with age, sex, FSIQ, and scan site as covariates. Significance was assessed using permutation testing (5000 permutations) and correction for multiple comparisons was achieved using Threshold-Free Cluster Enhancement (TFCE, two-tailed, threshold at p < 0.05) [34, 35]. Next, we considered the results of the ICA factorization of the VBM data. A GLM was used to examine differences between autistic and TD individuals, by using each participant’s loading to each component as a dependent variable, diagnostic group as independent variable, and age, sex, FSIQ, and scan site as regressors. To avoid the results of case–control differences being biased by IQ, we repeated the same procedure by excluding participants in Schedule D (FSIQ < 75, more details see Additional file 1: Subsection 2). Structural ICA decomposition All participants’ VBM data were simultaneously decomposed into 100 spatially independent sources of spatial variation using MELODIC-ICA [15]. Such ICA decomposition provides, at each independent component, a brain map reflecting a pattern of GM density covariation across participants, and a participant’s loading vector reflecting the contribution of each participant to each component. Higher model Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 Page 5 of 13 Mass‑univariate statisticsh The standard mass-univariate GLM analysis of the VBM data comparing cases and controls did not show significant group differences for voxel-wise GM volumes after multiple comparison correction. We provide the uncorrected statistical results (p < 0.05) in Additional file 1: Subsection 5. Statistical approach Considering that autism is highly comorbid with ADHD [36], we also controlled for comorbidity with ADHD by adding a dummy- coded variable (with/without ADHD) to the original GLM analyses (autism vs. TD) in the case–control ICA analysis. Additionally, we also conducted analyses that included a dimensional score (instead of categorical code) of the two ADHD subscales as an additional covariate in the group effect analyses (see Additional file 1: Subsection 3). Furthermore, we separately checked the effects of age squared, age-by-group, age squared- by-group, sex-by-group interactions, anxiety, depression comorbidity, medication use and image quality on case– control differences of structural covariance. The results can be found in Additional file 1: Subsection 3. Multiple comparison correction was implemented using false discovery rate (FDR) (p < 0.05) [37]. Group effect on ICA decompositionh The structural data ICA decomposition provided a set of 100 independent spatial sources, each of which is connected to a vector that depicts the degree of each participant’s contribution to the corresponding components. For clarity, we further refer to these vectors as the participant loadings. Post-hoc GLM analyses of these participant loadings showed case–control differences at nine independent components (ICs) (p < 0.05, i.e., IC10, IC13, IC14, IC15, IC23, IC28, IC31, IC48, and IC 99, see Additional file  1: Subsection  6). Of these, two components, IC10 ( β = −0.147 , p = 8.850 × 10–5, effect size [Cohen’s d] d = − 0.358) and IC14 ( β = −0.132 , p = 5.450 × 10–4, d = − 0.321), survived multiple comparison correction (FDR corrected, p < 8.072 × 10–4). These results were not driven by age, sex, or scan site. We also explored independently the relationships between each estimated brain component and subscales of ADI and ADOS, SRS, RBS, and SSP in the autism group using GLM analyses and again correction for multiple comparisons was implemented with the FDR method (p < 0.05). Then, to simultaneously explore the relationship between all the brain structural phenotypes estimated through ICA and all symptom phenotypes in the autism group, we used CCA. In the considered scenario, CCA is able simultaneously to learn linear projections (via canonical coefficients) of the brain structural sources and the behavioral measures that maximize the correlation between them at the participant Page 6 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 independent of the model order choice. For details, see Additional file 1: Subsection 7. In Fig. 1, we present summary images reflecting the brain areas involved in the structural variances occurring at these two components. The top row of Fig. 1 shows that IC10 primarily relates to structural variation in the bilateral insula, inferior frontal gyrus (IFG), orbitofrontal cortex (OFC), and caudate nuclei. Among these brain regions, the bilateral caudate exhibits alterations in the opposite direction to the others. Given the negative beta coefficient obtained from the GLM analysis between participant loadings at IC10 and the diagnosis group labels, individuals with autism demonstrate increased GM densities in the bilateral caudate and decreased densities in the bilateral insula, IFG, and OFC. The bottom row of Fig. Group effect on ICA decompositionh 1 shows that IC14 mainly involves variations in the bilateral amygdala, hippocampus, and parahippocampal gyrus (PHG). Similarly, according to the sign of the beta values obtained through the GLM, the autism group shows decreased densities in the areas involved in IC14. i To validate the ICA results not being biased by low IQ participants, an additional validation was performed by taking FSIQ into account to exclude the participants in Schedule D from the ICA factorization. This showed that a unique IC, corresponding to the original IC10, survived FDR correction (see Additional file  1: Subsection  3). Further, the effect of comorbidity with ADHD on brain structural variations was determined using data from 500 participants (for detailed demographic information, see Additional file 1: Subsection 3). This analysis showed that IC14 remained significantly associated with the autism group (FDR corrected, p = 9.669 × 10–4). However, IC10 was no longer associated with autism (p = 0.004). Further, post-hoc GLM analyses of the relationships between brain ICs and symptom ratings did not provide any significant associations (Additional file  1: Subsection 8). The robustness of the ICA results to the model order choice was evaluated by considering, in addition to the original 100-dimensional factorization, an automatic dimensionality estimation procedure resulting in a 91-dimensional factorization, and a 50-dimensional factorization. We observed that the main components reported (IC10 and IC14) are highly reproducible Relating gray matter spatial variation patterns to symptoms profilesi The regions involved in IC14 were mentioned above (canonical weight: − 0.312). Since higher scores of the ADI and ADOS reflect more severe autism symptoms, positive values of IC16 suggest that higher loading on this component is related to more severe symptoms in autism, and negative values of IC61, IC89, and IC14 meant that lower loadings on these three ICs are associated with more severe symptoms. In Fig. 2a, participants were color coded according to their ADOS- RRB scores to illustrate how the ADOS-RRB score drives the canonical correlation. In CCA​2 we linked SRS, RBS, and SSP scores to the brain measures of 194 individuals with autism, which is 55.9% of all participants with autism (lower number due to missing questionnaire data). We found a Bonferroni corrected (p = 0.05/3 = 0.017) significant relationship (Fig. 2b, r = 0.840, permutation p = 0.002, Fig. 2d). In this main CCA mode, IC82, IC99, and IC100 were the highest contributors to the correlation with behavior profiles, and SSP score loaded most on the association with the brain measures in the autism group (Fig. 3c, d). IC82 mainly Fig. 2  The first row shows the scatterplot of the main CCA mode of the brain structural covariations versus the symptom profiles for CCA​1 and CCA​ 2 respectively. One dot per participant in each graph is coded with gradient colors according to the scores of ADOS RRB (a) and SSP (b), respectively. The second row shows the histograms of the null distribution of correlation values obtained from the main CCA mode at 10,000 random participants’ permutations in the autism sample with ADI and ADOS scores (c), and with SRS, RBS, and SSP scores (d). The true r-value is marked by a red cross. ADI, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile; RRB, restricted and repetitive behaviors i i Fig. 2  The first row shows the scatterplot of the main CCA mode of the brain structural covariations versus the symptom profiles for CCA​1 and CCA​ 2 respectively. One dot per participant in each graph is coded with gradient colors according to the scores of ADOS RRB (a) and SSP (b), respectively. Relating gray matter spatial variation patterns to symptoms profilesi As a final step, we applied CCA to examine the associations between the 100 components and the two sets of clinical measures among the autism cases only. The CCA​1 (linking ADI and ADOS subscale scores to brain Fig. 1  The components showed significant case–control differences. The component maps were thresholded at 3 < |Z| < 5 . IC10, component number 10; IC14, component number 14 Fig. 1  The components showed significant case–control differences. The component maps were thresholded at 3 < |Z| < 5 . IC10, component number 10; IC14, component number 14 Page 7 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism measures), involved 325 autism participants and showed a Bonferroni corrected (p = 0.05/5 = 0.010) significant relationship (Fig. 2a,c, r = 0.701, permutation p = 0.008). In this main CCA mode, IC16, IC61, IC89, and IC14 were the highest contributors to the correlation with autism symptoms, and the ADOS RRB subscale loaded most on the association with the brain measures (Fig. 3a, b). Among the four components, IC16 mainly involved density variations in bilateral thalamus and putamen (canonical weight: 0.447), IC61 in right lateral occipital and left superior parietal lobe (canonical weight: − 0.366), and IC89 in the right precentral gyrus (canonical weight: − 0.333). For details, see Additional file 1: Subsection 9. Note that IC14 is among the components previously reported showing linear significant case–control group effects. The regions involved in IC14 were mentioned above (canonical weight: − 0.312). Since higher scores of the ADI and ADOS reflect more severe autism measures), involved 325 autism participants and showed a Bonferroni corrected (p = 0.05/5 = 0.010) significant relationship (Fig. 2a,c, r = 0.701, permutation p = 0.008). In this main CCA mode, IC16, IC61, IC89, and IC14 were the highest contributors to the correlation with autism symptoms, and the ADOS RRB subscale loaded most on the association with the brain measures (Fig. 3a, b). Among the four components, IC16 mainly involved density variations in bilateral thalamus and putamen (canonical weight: 0.447), IC61 in right lateral occipital and left superior parietal lobe (canonical weight: − 0.366), and IC89 in the right precentral gyrus (canonical weight: − 0.333). For details, see Additional file 1: Subsection 9. Note that IC14 is among the components previously reported showing linear significant case–control group effects. Relating gray matter spatial variation patterns to symptoms profilesi The second row shows the histograms of the null distribution of correlation values obtained from the main CCA mode at 10,000 random participants’ permutations in the autism sample with ADI and ADOS scores (c), and with SRS, RBS, and SSP scores (d). The true r-value is marked by a red cross. ADI, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile; RRB, restricted and repetitive behaviors Mei et al. Molecular Autism (2020) 11:86 Page 8 of 13 Mei et al. Molecular Autism Fig. 3  The top row shows the corrected canonical coefficients (weights) of the main CCA mode for the CCA​1 analyses (ADI&ADOS), and the bottom row for the CCA​2 analyses (SRS&RBS&SSP). a, c display the degree that each brain component contributed to the main CCA mode in each analysis. The two components with significant group effects are displayed in red. b, d display the degree that each symptom profile contributes to each analysis. CCA, canonical correlation analysis; ADI, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile Fig. 3  The top row shows the corrected canonical coefficients (weights) of the main CCA mode for the CCA​1 analyses (ADI&ADOS), and the bottom row for the CCA​2 analyses (SRS&RBS&SSP). a, c display the degree that each brain component contributed to the main CCA mode in each analysis. The two components with significant group effects are displayed in red. b, d display the degree that each symptom profile contributes to each analysis. CCA, canonical correlation analysis; ADI, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile Fig. 3  The top row shows the corrected canonical coefficients (weights) of the main CCA mode for the CCA​1 analyses (ADI&ADOS), and the bottom row for the CCA​2 analyses (SRS&RBS&SSP). a, c display the degree that each brain component contributed to the main CCA mode in each analysis. The two components with significant group effects are displayed in red. b, d display the degree that each symptom profile contributes to each analysis. Discussionh The present study used a reliable approach to quantify inter-individual differences in GM morphometry covariations in a deeply phenotyped large sample of individuals with and without autism. The standard, univariate VBM analysis did not show significant case–control differences. We then utilized an ICA decomposition of all participants GM density images, and similarly performed a case–control post-hoc statistical analyses. This analysis showed that autism was significantly associated with alterations in two independent sources of GM density covariations. These findings corroborated our hypothesis that alterations in brain morphometry in autism are associated with the combined influence of several regions rather than with a single region. In a following step, we applied CCA to explore multivariate associations between sets of continuous measures of core symptoms and sets of ICA-derived morphometry measures within the autism group, and were able to identify significant relationships between brain components and symptom profiles. Notably, one of the components which showed significant case–control differences was also among the highest loading components in the CCA. The brain regions with high loadings on either of these two components, i.e., insula, amygdala, hippocampus and PHG have lower densities in autism and have earlier been associated with autism [9, 43]. The opposite direction of the alteration of the caudate nucleus in autism has also previously been found [44]. This is not the case for the IFG and OFG, which showed lower densities in autism in our study, where prior studies found mixed results [45, 46]. Importantly, the brain regions identified by our analyses have earlier been implicated in the neurobiology and/or neurocognition of autism. In IC10, structural and/or functional alterations of the insula, IFG, and OFC have been associated with social and non-social cognitive impairments in autism [46–49]. A meta- analysis reported abnormal functional activations of the insula, IFG, and OFG during social cognition tasks in autism [50]. Additionally, variance of the caudate nucleus volume was found to correlate with the severity of RRB symptoms in autism [44]. Together with deviant structural and functional connectivity between frontal cortical areas and striatum in autism [47, 51, 52], structural covariation in striatum and frontal areas may underlie atypical functional fronto-striatal connectivity, and this has been associated with repetitive behavior and executive functioning impairments in autism [3, 45]. Relating gray matter spatial variation patterns to symptoms profilesi CCA, canonical correlation analysis; ADI, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observational Schedule 2; SRS, Social Responsiveness Scale 2nd Edition; RBS, Repetitive Behavior Scale-Revised; SSP, Short Sensory Profile interpretation (referring to uncorrected coefficients) of the CCA​2 weights ranks IC14 as the third strongest contributor to this canonical correlation (Additional file 1: Subsection 10).h involved variations in the bilateral cerebellum (canonical weight: 0.414), IC99 in the left lateral occipital and parietal lobe, and bilateral precentral gyrus (canonical weight: 0.277), and IC100 in the left inferior frontal gyrus and right middle frontal lobe (canonical weight: 0.262). For details, see Additional file 1: Subsection 9. Similarly, lower loadings on these three ICs were related to more severe symptoms. In Fig. 2b, each participant was color coded according to their SSP score, and it shows how SSP score drives the correlation. In this case, both IC10 and IC14 ranked outside the top 20 of the 100 components, suggesting that these two components with significant case–control difference have no strong contribution to the CCA​2 correlation. However, for completeness, direct i The CCA robustness analyses indicated that the main CCA modes of both CCA analyses were reliably estimated in a leave-one-subject out setting (Additional file 1: Subsection 4). In CCA​1, the weights of the main CCA mode of each leave-one-out analysis correlated on average above 0.94 with the weights of original main CCA mode in brain loadings and above 0.95 in behavior phenotypes when the sample was bigger than 122 participants. In CCA​2, the weights of the main CCA mode related on average above 0.92 in brain loadings Page 9 of 13 Mei et al. Molecular Autism (2020) 11:86 Page 9 of 13 Mei et al. Molecular Autism (2020) 11:86 brain areas. This is in line with a previous study that used a similar approach [40]. It is further in line with organizing principles of the brain that regions tend to be more interconnected when they are located close to each other [41, 42]. However, when we compared the regions loading on the two components, the covarying regions of each component distribute in different brain locations. This suggests that neuroanatomic alterations underlying autism are more widely distributed at the whole brain level. It is of note that, when accounting for ADHD comorbidity, IC14 remained significant but IC10 did not. Relating gray matter spatial variation patterns to symptoms profilesi This suggests that IC14 is more specifically related to autism associated structural variations, even after linearly accounting for ADHD effects, while IC10 might reflect variations associated with both autism and ADHD.h and above 0.96 in behavior profiles when the sample was bigger than 111 participants. Both CCA analyses are no reproducible for sample sizes smaller than (approximately) 100 participants. Discussionh In the present study, the density of caudate nuclei increase simultaneously with densities decreasing on other areas in autism, which fits with the results of a few functional studies that indicate inverse functional changes of these areas [53]. Particularly, the special pattern of GM densities changes in frontal and striatal areas might serve an important role in autism-related symptoms. We found nonsignificant case–control difference in univariate analysis in current study, which is inconsistent with previous studies (e.g., [8, 9]). In light of the substantial biological heterogeneity among autistic individuals, it is expected that findings differ across datasets, especially those of smaller size. Specifically, the current study included the individuals with a relative wide age and IQ range, and did not exclude co-occurring psychiatric symptoms. Additionally, integrating the findings from multivariate analyses in our study, the absence of diagnostic differences at single region/voxel underscores the detection sensitivity of group effect of a multivariate approach which evidently verified our hypothesis. Our findings showed two covarying sets of brain areas that structurally differed between cases and controls. While one source of GM density covariation, IC10, mainly related to the bilateral insula, IFG, OFG, and caudate, another source, IC14, primarily involved the bilateral amygdala, hippocampus, and PHG. The brain regions within each component are anatomically clustered and symmetrical, which indicates that the independent structural covariation alteration in the GM of individuals with autism is concentrated in nearby In IC14, we found decreased densities of amygdala and hippocampus, where the structural alterations have previously been related to social deficits in autism [6, 54]. The amygdala, hippocampus, and PHG subserve cognitive and emotional functions that were found abnormal in individuals with autism [50, Page 10 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 55, 56]. In addition to being involved in emotion and face processing, the three areas have been proposed as structures critical for working memory in autism [57]. Furthermore, these cognitive domains exert bidirectional effects on each other, with atypical social- emotional processing influencing memory performance in individuals with autism, and memory being involved in complex information processing and executive functioning, which in turn affects social cognition [57, 58]. Strengths and limitationsh The strengths of our study are the use of a large deeply phenotyped sample, bottom-up data-driven analyses, a multivariate approach for examining brain-symptom associations, and a large set of continuous measures of core autism symptoms. There are several limitations in our study. First, only 55.9% of the autism group had complete questionnaire data on continuous parent- reported symptom measures, which may have lowered statistical power for this analysis. Second, due to the clinical characteristic of autism, the participants showed significant differences in the proportion of sex distribution. Despite this we regressed out the sex effect in the group difference analyses, future studies may consider balancing the sex distributions in case and control groups; however this may be difficult to achieve. Third, albeit we ran a series of sensitivity and control analyses, the effect of other potential sources of variance, such as the complex effects of medication use that may influence our findings. Our multivariate correlation analyses moved from the case–control comparison to the use of continuous symptoms among individuals with autism and identified two prominent relationships between all structural brain covariances and symptoms in autism. Three of the four brain components that ranked top in this analysis did not show case–control differences, while there was one component (IC14) that differed between cases and controls and also significantly correlated with the severity of autism symptoms assessed by ADI and ADOS. The brain areas loading high on the brain components identified in the CCA are somewhat different from those implicated in the case–control analyses. These former brain areas are the thalamus, putamen, precentral gyrus, and parietal and occipital lobes in CCA​1, and the cerebellum, frontal lobe, and again precentral gyrus, and parietal and occipital lobes in CCA​2. These are foremost areas of the brain implicated in the processing and higher order integration of sensory information and motor functions. This makes sense since repetitive, rigid and stereotyped behaviors and abnormal sensory behaviors seem to drive the brain-behavior associations much more than the measures on social-communication symptoms. Note that variance within the different autism symptom domains (social-communication, repetitive behaviors and sensory abnormalities) was similar and cannot explain the differential symptom- brain associations. Supplementary information Supplementary information accompanies this paper at https​://doi. org/10.1186/s1322​9-020-00389​-4. Additional file 1: Subsection 1. Acquisition parameters. Subsection 2. Demographic information of each schedule. Subsection 3. Sensitivity analyses. Subsection 4. Leave-one-out (LOO) validation of CCA results. Subsection 5. Group differences at voxel-wise gray matter vol umes. Subsection 6. Independent components with case-control differences. Subsection 7. Robustness assessment of ICA model orders. Subsection 8. GLM results of the association between brain components and symptom profiles. Subsection 9. Components with highest loadings in CCA. Subsection 10. Uncorrected main CCA mode loadings of each component. Additional file 1: Subsection 1. Acquisition parameters. Subsection 2. Demographic information of each schedule. Subsection 3. Sensitivity analyses. Subsection 4. Leave-one-out (LOO) validation of CCA results. Subsection 5. Group differences at voxel-wise gray matter vol umes. Subsection 6. Independent components with case-control differences. Subsection 7. Robustness assessment of ICA model orders. Subsection 8. GLM results of the association between brain components and symptom profiles. Subsection 9. Components with highest loadings in CCA. Subsection 10. Uncorrected main CCA mode loadings of each component. Overall, the results of our multivariate analyses on case–control differences and on continuous measures of symptom severity among those with autism demonstrate the complexity of the brain morphometry correlates of autism. Brain areas involved are scattered across the whole brain and include the limbic system, basal ganglia, thalamus, cerebellum, precentral (motor) gyrus, and parts of the frontal, parietal, and occipital lobes. Discussionh In sum, given the potential functional interactions between these three brain areas, the structural covariance alterations of the amygdala, hippocampus, and PHG found in our study, may underlie or contribute to abnormal functional connections of these areas, and thus underlie poor performance on social cognition and memory tasks in individuals with autism. Conclusions We demonstrate brain morphometry differences between individuals with autism and typical controls in the inter-regional covariation of the insula, frontal area, caudate, amygdala, hippocampus, and PHG. Further, we highlight associations between covariation in density of the thalamus, putamen, precentral gyrus, frontal, parietal, and occipital lobes, and the cerebellum, and core autism symptoms, in particular repetitive behaviors and abnormal sensory behavior. Future studies may link our morphometry findings with data on brain function obtained from cognitive tests and/or functional and resting-state MRI, and with genomics data. Funding h k This work is primarily supported by the EU-AIMS consortium (European Autism Interventions), which receives support from Innovative Medicines Initiative Joint Undertaking Grant No.115300, the resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (Grant No. FP7/2007-2013), from the European Federation of Pharmaceutical Industries and Associations companies’ in-kind contributions; and by the AIMS-2-TRIALS consortium (Autism Innovative Medicine Studies-2-Trials), which has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement No. 777394, and this Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and AUTISM SPEAKS, Autistica, SFARI. TM is supported by a China Scholarship Council Grant (No 201806010408). This work has been further supported by the European Union Seventh Framework Programme Grant Nos. 602805 (AGGRESSOTYPE) (to JKB), 603016 (MATRICS) (to JKB), and 278948 (TACTICS) (to JKB); European Community’s Horizon 2020 Programme (H2020/2014–2020) Grant Nos. 643051 (MiND) (to JKB), 642996 (BRAINVIEW) (to JKB) and 847818 (CANDY) (to JKB and CFB); the Netherlands Organization for Scientific Research VICI Grant No. 2020/TTW/00836465 (to CFB); Wellcome Trust Collaborative Award Grant No. 215573/Z/19/Z (to CFB); the Autism Research Trust (to SBC). Received: 25 May 2020 Accepted: 5 October 2020 Consent for publication Independent component; ICA: Independent component analysis; ICD- 10: International Statistical Classification of Diseases and Related Health Problems 10th Revision; ID: Intellectual disability; IFG: Inferior frontal gyrus; OFC: Orbitofrontal cortex; MRI: Magnetic resonance imaging; PHG: Parahippocampal gyrus; RBS: Repetitive Behavior Scale-Revised; RRB: Restricted and repetitive behaviors; SA: Social affect; SD: Standard deviation; SRS: Social Responsiveness Scale 2nd Edition; SSP: Short Sensory Profile; TD: Typically developing; TFCE: Threshold-Free Cluster Enhancement; VBM: Voxel- based morphometry. Consent for publication was obtained from all participants prior to the study. Acknowledgements We thank all participants and their families for participating in this study. We gratefully acknowledge the contributions of all members of the EU-AIMS LEAP group: Jumana Ahmad, Sara Ambrosino, Bonnie Auyeung, Tobias Banaschewski, Simon Baron-Cohen, Sarah Baumeister, Christian F. Beckmann, Sven Bölte, Thomas Bourgeron, Carsten Bours, Michael Brammer, Daniel Brandeis, Claudia Brogna, Yvette de Bruijn, Jan K. Buitelaar, Bhismadev Chakrabarti, Tony Charman, Ineke Cornelissen, Daisy Crawley, Flavio Dell’Acqua, Guillaume Dumas, Sarah Durston, Christine Ecker, Jessica Faulkner, Vincent Frouin, Pilar Garcés, David Goyard, Lindsay Ham, Hannah Hayward, Joerg Hipp, Rosemary Holt, Mark H. Johnson, Emily J.H. Jones, Prantik Kundu, Meng-Chuan Lai, Xavier Liogier D’ardhuy, Michael V. Lombardo, Eva Loth, David J. Lythgoe, René Mandl, Andre Marquand, Luke Mason, Maarten Mennes, Andreas Meyer-Lindenberg, Carolin Moessnang, Nico Mueller, Declan G.M. Murphy, Bethany Oakley, Laurence O’Dwyer, Marianne Oldehinkel, Bob Oranje, Gahan Pandina, Antonio M. Persico, Annika Rausch, Barbara Ruggeri, Amber Ruigrok, Jessica Sabet, Roberto Sacco, Antonia San José Cáceres, Emily Simonoff, Will Spooren, Julian Tillmann, Roberto Toro, Heike Tost, Jack Waldman, Steve C.R. Williams, Caroline Wooldridge, and Marcel P. Zwiers. References 1. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed. Arlington: Author; 2013. 2. Ecker C, Bookheimer SY, Murphy DG. Neuroimaging in autism spectrum disorder: brain structure and function across the lifespan. Lancet Neurol. 2015;14(11):1121–34. 3. Di Martino A, Yan CG, Li Q, Denio E, Castellanos FX, Alaerts K, et al. The autism brain imaging data exchange: towards a large-scale evaluation of the intrinsic brain architecture in autism. Mol Psychiatry. 2014;19(6):659–67. 4. Turner AH, Greenspan KS, van Erp TGM. Pallidum and lateral ventricle volume enlargement in autism spectrum disorder. Psychiatry Res Neuroimaging. 2016;252:40–5. 5. Zhang W, Groen W, Mennes M, Greven C, Buitelaar J, Rommelse N. Revisiting subcortical brain volume correlates of autism in the ABIDE dataset: effects of age and sex. Psychol Med. 2018;48(4):654–68. Author details 1 Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. 2 Karakter Child and Adolescent Psychiatry University Centre, Nijmegen, The Netherlands. 3 Department of Psychology, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK. 4 Department of Psychiatry, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands. 5 Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany. 6 Department of Child and Adolescent Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany. 7 Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK. 8 Department of Forensic and Neurodevelopmental Sciences, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK. 9 Department of Child and Adolescent Psychiatry, University Hospital, Goethe University, Frankfurt am Main, Germany. 10 Centre for Functional MRI of the Brain, University of Oxford, Oxford, UK. Competing interests p g JKB has been a consultant to, advisory board member of, and a speaker for Janssen Cilag BV, Eli Lilly, Shire, Lundbeck, Roche, and Servier. He is not an employee of any of these companies, and not a stock shareholder of any of these companies. He has no other financial or material support, including expert testimony, patents or royalties. CFB is director and shareholder in SBGNeuro Ltd. TB served in an advisory or consultancy role for Lundbeck, Medice, Neurim Pharmaceuticals, Oberberg GmbH, Shire, and Infectopharm. He received conference support or speaker’s fee by Lilly, Medice, and Shire. He received royalties from Hogrefe, Kohlhammer, CIP Medien, and Oxford University Press. TC has received consultancy from Roche and received book royalties from Guildford Press and Sage. DGM has been a consultant to, and advisory board member, for Roche and Servier. He is not an employee of any of these companies, and not a stock shareholder of any of these companies. The present work is unrelated to the above grants and relationships. The other authors report no biomedical financial interests or potential conflicts of interest. Authors’ contributions JT, SD, CM, TB, RH, SB-C, AR, EL, TC, DGM, CE, CFB, JKB designed the EU-AIMS study, developed data acquisition and/or analysis protocols. TM ran analyses, visualized the findings, wrote the first draft, and revised the draft; AL conceptualized and supervised the analysis, and revised the manuscript; DLF ran the VBM analysis and revised the draft; JT & NJF revised the draft; CFB supervised and revised the manuscript; JKB supervised and revised the manuscript. All authors read and approved the final manuscript. Abbreviations ADHD: Attention Deficit Hyperactivity Disorder; ADI: Autism Diagnostic Interview-Revised; ADOS: Autism Diagnostic Observational Schedule 2; CCA​: Canonical correlation analysis; DSM-IV: Diagnostic and Statistical Manual-IV; EU-AIMS: European Autism Interventions—A Multicentre Study for Developing New Medications; FDR: False discovery rate; FSIQ: Full-scale intelligence quotient; GLM: Generalized linear model; GM: Gray matter; IC: Page 11 of 13 Page 11 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 Consent for publication Availability of data and materials Data collected in EU-AIMS LEAP are stored and curated at the central EU-AIMS database at the Pasteur Institute in Paris. The database is open to members of the wider scientific community upon request and submission of a paper and data analytic proposal. 6. van Rooij D, Anagnostou E, Arango C, Auzias G, Behrmann M, Busatto GF, et al. Cortical and subcortical brain morphometry differences between patients with autism spectrum disorder and healthy individuals across the lifespan: results from the ENIGMA ASD working group. Am J Psychiatry. 2018;175(4):359–69. Ethics approval and consent to participate The disrupted connectivity hypothesis of autism spectrum disorders: time for the next phase in research. Biol Psychiatry Cogn Neurosci Neuroimaging. 2016;1(3):245–52. 37. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B (Methodolo). 1995;57(1):289–300. y y g g g 13. Geschwind DH, Levitt P. Autism spectrum disorders: developmental disconnection syndromes. Curr Opin Neurobiol. 2007;17(1):103–11. 14. Picci G, Gotts SJ, Scherf KS. A theoretical rut: revisiting and critically evaluating the generalized under/over-connectivity hypothesis of autism. Dev Sci. 2016;19(4):524–49. 38. Smith SM, Nichols TE, Vidaurre D, Winkler AM, Behrens TE, Glasser MF, et al. A positive-negative mode of population covariation links brain connectivity, demographics and behavior. Nat Neurosci. 2015;18(11):1565–7. 15. Beckmann CF, Smith SM. Probabilistic independent component analysis for functional magnetic resonance imaging. IEEE Trans Med Imaging. 2004;23(2):137–52. 39. Haufe S, Meinecke F, Gorgen K, Dahne S, Haynes JD, Blankertz B, et al. On the interpretation of weight vectors of linear models in multivariate neuroimaging. Neuroimage. 2014;87:96–110. 16. Xu L, Groth KM, Pearlson G, Schretlen DJ, Calhoun VD. Source-based morphometry: the use of independent component analysis to identify gray matter differences with application to schizophrenia. Hum Brain Mapp. 2009;30(3):711–24. 40. Di X, Biswal BB. Similarly expanded bilateral temporal lobe volumes in female and male children with autism spectrum disorder. Biol Psychiatry Cogn Neurosci Neuroimaging. 2016;1(2):178–85. 41. Beul SF, Grant S, Hilgetag CC. A predictive model of the cat cortical connectome based on cytoarchitecture and distance. Brain Struct Funct. 2015;220(6):3167–84. 17. Hafkemeijer A, Moller C, Dopper EG, Jiskoot LC, van den Berg-Huysmans AA, van Swieten JC, et al. Differences in structural covariance brain networks between behavioral variant frontotemporal dementia and Alzheimer’s disease. Hum Brain Mapp. 2016;37(3):978–88. 42. Oligschlager S, Xu T, Baczkowski BM, Falkiewicz M, Falchier A, Linn G, et al. Gradients of connectivity distance in the cerebral cortex of the macaque monkey. Brain Struct Funct. 2019;224(2):925–35. 18. Vanasse TJ, Fox PM, Barron DS, Robertson M, Eickhoff SB, Lancaster JL, et al. BrainMap VBM: An environment for structural meta-analysis. Hum Brain Mapp. 2018;39(8):3308–25. 43. Nickl-Jockschat T, Habel U, Michel TM, Manning J, Laird AR, Fox PT, et al. Brain structure anomalies in autism spectrum disorder–a meta-analysis of VBM studies using anatomic likelihood estimation. Hum Brain Mapp. 2012;33(6):1470–89. 19. Ecker C, Suckling J, Deoni SC, Lombardo MV, Bullmore ET, Baron-Cohen S, et al. Ethics approval and consent to participate Ethical approval for this study was obtained through ethics committees at each site. Ethical approval for this study was obtained through ethics committees at each site. 7. Bedford SA, Park MTM, Devenyi GA, Tullo S, Germann J, Patel R, et al. Large-scale analyses of the relationship between sex, age and Page 12 of 13 Page 12 of 13 Mei et al. Molecular Autism (2020) 11:86 Mei et al. Molecular Autism (2020) 11:86 31. Ashburner J. A fast diffeomorphic image registration algorithm. Neuroimage. 2007;38(1):95–113. intelligence quotient heterogeneity and cortical morphometry in autism spectrum disorder. Mol Psychiatry. 2019;25(3):614–28. intelligence quotient heterogeneity and cortical morphometry in autism spectrum disorder. Mol Psychiatry. 2019;25(3):614–28. p y y 8. DeRamus TP, Kana RK. Anatomical likelihood estimation meta-analysis of grey and white matter anomalies in autism spectrum disorders. Neuroimage Clin. 2015;7:525–36. y y 8. DeRamus TP, Kana RK. Anatomical likelihood estimation meta-analysis of grey and white matter anomalies in autism spectrum disorders. Neuroimage Clin. 2015;7:525–36. 32. Groves AR, Smith SM, Fjell AM, Tamnes CK, Walhovd KB, Douaud G, et al. Benefits of multi-modal fusion analysis on a large-scale dataset: life-span patterns of inter-subject variability in cortical morphometry and white matter microstructure. Neuroimage. 2012;63(1):365–80. 9. Carlisi CO, Norman LJ, Lukito SS, Radua J, Mataix-Cols D, Rubia K. Comparative multimodal meta-analysis of structural and functional brain abnormalities in autism spectrum disorder and obsessive-compulsive disorder. Biol Psychiatry. 2017;82(2):83–102. 33. Jenkinson M, Beckmann CF, Behrens TE, Woolrich MW, Smith SM. Fsl Neuroimage. 2012;62(2):782–90. 34. Eklund A, Nichols TE, Knutsson H. Cluster failure: why fMRI inferences for spatial extent have inflated false-positive rates. Proc Natl Acad Sci USA. 2016;113(28):7900–5. 10. Liu J, Yao L, Zhang W, Xiao Y, Liu L, Gao X, et al. Gray matter abnormalities in pediatric autism spectrum disorder: a meta-analysis with signed differential mapping. Eur Child Adolesc Psychiatry. 2017;26(8):933–45. 35. Winkler AM, Ridgway GR, Douaud G, Nichols TE, Smith SM. Faster permutation inference in brain imaging. Neuroimage. 2016;141:502–16. f 11. Cai J, Hu X, Guo K, Yang P, Situ M, Huang Y. Increased left inferior temporal gyrus was found in both low function autism and high function autism. Front Psychiatry. 2018;9:542. 36. Simonoff E, Pickles A, Charman T, Chandler S, Loucas T, Baird G. Psychiatric disorders in children with autism spectrum disorders: prevalence, comorbidity, and associated factors in a population-derived sample. J Am Acad Child Adolesc Psychiatry. 2008;47(8):921–9. y y 12. Vasa RA, Mostofsky SH, Ewen JB. Ethics approval and consent to participate Brain anatomy and its relationship to behavior in adults with autism spectrum disorder: a multicenter magnetic resonance imaging study. Arch Gen Psychiatry. 2012;69(2):195–209. 44. Langen M, Bos D, Noordermeer SD, Nederveen H, van Engeland H, Durston S. Changes in the development of striatum are involved in repetitive behavior in autism. Biol Psychiatry. 2014;76(5):405–11. 20. Smith SM, Nichols TE. Statistical challenges in “big data” human neuroimaging. Neuron. 2018;97(2):263–8. 21. Mulders P, Llera A, Tendolkar I, van Eijndhoven P, Beckmann C. Personality profiles are associated with functional brain networks related to cognition and emotion. Sci Rep. 2018;8(1):13874. 45. Foster NE, Doyle-Thomas KA, Tryfon A, Ouimet T, Anagnostou E, Evans AC, et al. Structural gray matter differences during childhood development in autism spectrum disorder: a multimetric approach. Pediatr Neurol. 2015;53(4):350–9. 22. Hotelling H. Relations between two sets of variates. Biometrika. 1936;28:312–77. 46. Sato W, Kochiyama T, Uono S, Yoshimura S, Kubota Y, Sawada R, et al. Reduced gray matter volume in the social brain network in adults with autism spectrum disorder. Front Hum Neurosci. 2017;11:395. 23. Loth E, Charman T, Mason L, Tillmann J, Jones EJH, Wooldridge C, et al. The EU-AIMS Longitudinal European Autism Project (LEAP): design and methodologies to identify and validate stratification biomarkers for autism spectrum disorders. Mol Autism. 2017;8:24. 47. Kana RK, Keller TA, Minshew NJ, Just MA. Inhibitory control in high- functioning autism: decreased activation and underconnectivity in inhibition networks. Biol Psychiatry. 2007;62(3):198–206. 24. Charman T, Loth E, Tillmann J, Crawley D, Wooldridge C, Goyard D, et al. The EU-AIMS Longitudinal European Autism Project (LEAP): clinical characterisation. Mol Autism. 2017;8:27. y y 48. Wadsworth HM, Maximo JO, Donnelly RJ, Kana RK. Action simulation and mirroring in children with autism spectrum disorders. Behav Brain Res. 2018;341:1–8. 25. Rutter M, Le Couteur A, Lord C. Autism diagnostic interview-revised. Los Angeles: Western Psychological Services; 2003. 49. Nomi JS, Molnar-Szakacs I, Uddin LQ. Insular function in autism: Update and future directions in neuroimaging and interventions. Prog Neuropsychopharmacol Biol Psychiatry. 2019;89:412–26. 26. Lord C, Rutter M, DiLavore PC, Risi S, Gotham K, Bishop S. Autism diagnosticobservation schedule, Second Edition (ADOS-2) manual (part I): modules 1–4. Torrance: Western Psychological Services; 2012. 50. Patriquin MA, DeRamus T, Libero LE, Laird A, Kana RK. Neuroanatomical and neurofunctional markers of social cognition in autism spectrum disorder. Hum Brain Mapp. 2016;37(11):3957–78. y g 27. Constantino JN, Gruber CP. Social responsivenes Angeles: Western Psychological Services; 2012. y g 27. Ethics approval and consent to participate Constantino JN, Gruber CP. Social responsiveness scale. 2nd ed. Los Angeles: Western Psychological Services; 2012. Angeles: Western Psychological Services; 2012. 51. Langen M, Leemans A, Johnston P, Ecker C, Daly E, Murphy CM, et al. Fronto-striatal circuitry and inhibitory control in autism: findings from diffusion tensor imaging tractography. Cortex. 2012;48(2):183–93. 28. Bodfish JW, Symons FJ, Parker DE, Lewis MH. Varieties of repetitive behavior in autism: comparisons to mental retardation. J Autism Dev Disord. 2000;30(3):237–43. 29. Tomchek SD, Dunn W. Sensory processing in children with and without autism: a comparative study using the short sensory profile. Am J Occup Ther. 2007;61(2):190–200. 52. Di Martino A, Kelly C, Grzadzinski R, Zuo XN, Mennes M, Mairena MA, et al. Aberrant striatal functional connectivity in children with autism. Biol Psychiatry. 2011;69(9):847–56. 30. Gaser C, Dahnke R. https​://dbm.neuro​.uni-jena.de/cat/. Accessed 2 Sept 2018. 53. Clements CC, Zoltowski AR, Yankowitz LD, Yerys BE, Schultz RT, Herrington JD. Evaluation of the social motivation hypothesis of autism: a systematic review and meta-analysis. JAMA Psychiatry. 2018;75(8):797–808. Mei et al. Molecular Autism (2020) 11:86 Page 13 of 13 Page 13 of 13 56. Klapwijk ET, Aghajani M, Colins OF, Marijnissen GM, Popma A, van Lang ND, et al. Different brain responses during empathy in autism spectrum disorders versus conduct disorder and callous-unemotional traits. J Child Psychol Psychiatry. 2016;57(6):737–47. 57. Boucher J, Mayes A, Bigham S. Memory in autistic spectrum disorder. Psychol Bull. 2012;138(3):458–96. 58. Barendse EM, Hendriks MP, Jansen JF, Backes WH, Hofman PA, Thoonen G, et al. Working memory deficits in high-functioning adolescents with autism spectrum disorders: neuropsychological and neuroimaging correlates. J Neurodev Disord. 2013;5(1):14. 54. Baribeau DA, Dupuis A, Paton TA, Hammill C, Scherer SW, Schachar RJ, et al. Structural neuroimaging correlates of social deficits are similar in autism spectrum disorder and attention-deficit/hyperactivity disorder: analysis from the POND Network. Transl Psychiatry. 2019;9(1):72. 55. Aoki Y, Cortese S, Tansella M. Neural bases of atypical emotional face processing in autism: A meta-analysis of fMRI studies. World J Biol Psychiatry. 2015;16(5):291–300. 54. Baribeau DA, Dupuis A, Paton TA, Hammill C, Scherer SW, Schachar RJ, et al. Structural neuroimaging correlates of social deficits are similar in autism spectrum disorder and attention-deficit/hyperactivity disorder: analysis from the POND Network. Transl Psychiatry. 2019;9(1):72. 55. Aoki Y, Cortese S, Tansella M. Neural bases of atypical emotional face processing in autism: A meta-analysis of fMRI studies. World J Biol Psychiatry. 2015;16(5):291–300. 56. Klapwijk ET, Aghajani M, Colins OF, Marijnissen GM, Popma A, van Lang ND, et al. Different brain responses during empathy in autism spectrum disorders versus conduct disorder and callous-unemotional traits. J Child Psychol Psychiatry. 2016;57(6):737–47. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • 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: • 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:
https://openalex.org/W141199635
https://cfjournal.hse.ru/article/download/1697/2418
Russian
null
Взаимодействие реальных опционов на примере девелоперских проектов в России
Korporativnye finansy
2,010
cc-by
7,315
ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 40 Зубцов Н.Н.17, Пирогов Н.К.18 Несмотря на большое количество методологических и аналитических работ, посвященных методу реальных опционов, существует лишь несколько исследований, затрагивающих проблему анализа портфеля реальных опционов и их взаимодействий. Мы делаем попытку рассмотреть взаимодействие нескольких реальных опционов на примере инвестиционного проекта на бурно развивающемся российском рынке недвижимости. Информация по ряду проектов позволяет сформировать типичную структуру денежных потоков девелоперского проекта. Далее, с помощью построенной финансовой модели и используя метод симуляций, оценивается премия за наличие управленческой гибкости (реальных опционов) в типичном девелоперском проекте. Для построения модели используется подход, предложенный Тригеоргисом [Trigeorgis, 1993]. Интересно, что премия за наличие управленческой гибкости в девелоперских проектах коммерческой и жилой недвижимости не превышает 10% стоимости всего проекта. При этом подтверждается полученный ранее вывод об отсутствии свойства адитивности стоимостей отдельных реальных опционов в ситуации, когда опционы сосуществуют одновременно в рамках единого проекта. 17 Магистр программы «Стратегическое управление финансами фирмы» ГУ ВШЭ. 18 18 Старший преподаватель кафедры экономики и финансов фирмы ГУ ВШЭ. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, р р ф р ф ф р Выпуск #2(6) 2008 © Электронный журнал Корпоративные Фин 18 Старший преподаватель кафедры экономики и финансов фирмы ГУ ВШЭ. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 р р р р у р ф ф р 18 Старший преподаватель кафедры экономики и финансов фирмы ГУ ВШЭ. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Фин 18 Старший преподаватель кафедры экономики и финансов фирмы ГУ ВШЭ. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 17 Магистр программы «Стратегическое управление финансами фирмы» ГУ ВШЭ. 18 Старший преподаватель кафедры экономики и финансов фирмы ГУ ВШЭ. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 41 41 №2(6) 2008 Реальные опционы присутствуют в инвестиционных проектах во многих отраслях: в добыче полезных ископаемых [Dimitrakopoulos Roussos G., Sabry A. Abdel Sabour, 2007)], научно-исследовательских разработках [Trigeorgis L., 2002], недвижимости [Quigg, Laura, 1993]. Реальные опционы присутствуют в инвестиционных проектах во многих отраслях: в добыче полезных ископаемых [Dimitrakopoulos Roussos G., Sabry A. Abdel Sabour, 2007)], научно-исследовательских разработках [Trigeorgis L., 2002], недвижимости [Quigg, Laura, 1993] Целью данного исследования является исследование взаимодействия нескольких реальных опционов, в частности проверка свойства аддитивности на примере девелоперских проектов, а также численная оценка премии за реальные опционы в типичном девелоперском проекте. Методология исследования была заимствована в работах Тригеоргиса [1991, 1993]. В 2007 году объем инвестиций в недвижимость в России составил 5 млрд долл. США, продемонстрировав 16%-ный рост по сравнению с 2006 годом (данные Jones Lang LaSalle). Эти данные свидетельствуют о бурном развитии рынка недвижимости в России, поэтому имеет смысл рассмотреть реальные опционы в девелоперских проектах. Кроме того, как будет показано далее, существует лишь ограниченное число исследований реальных опционов в недвижимости. Введение Одним из самых распространенных методов для оценки привлекательности инвестиционных проектов является метод дисконтированных денежных потоков. Однако, несмотря на уже проверенную временем эффективность и относительную простоту применения, этот метод имеет ряд недостатков. Например, он не позволяет учитывать управленческую гибкость менеджмента, в результате чего зачастую происходит недооценка проектов, которые зависят от факторов с высокой степенью неопределенности, таких как цены на нефть, уголь, металлы, недвижимость. Метод реальных опционов позволяет количественно оценивать управленческую гибкость менеджмента в принятии решений. Подобно финансовому аналогу, реальный опцион предоставляет владельцу право, но не обязательство, реализации определенного условия. Отличие от финансовых опционов заключается в том, что базовым является реальный актив, а не ценная бумага [Myers, 1977]. Теория реальных опционов относительно молода и использует сложный математический аппарат, и в ней до сих пор присутствуют малоисследованные области. Например, на данный момент, существует очень мало исследований, посвященных оценке нескольких реальных опционов в совокупности в одном проекте и их взаимодействиям. Большинство работ фокусируется на оценке какого-то одного вида реальных опционов и при этом не учитывают, что в подавляющем большинстве инвестиционных проектов менеджмент имеет возможность несколькими способами влиять на реализацию проекта. Иначе говоря, в проекте может присутствовать несколько реальных опционов. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" © Электронный журнал Корпоративные Финансы, 2008 1. Обзор существующих исследований по тематике Темой данной работы является оценка инвестиционных проектов с множественными реальными опционами. Чаще всего в инвестиционном проекте присутствует не один реальный опцион. Типичный проект представляет собой набор реальных опционов, когда в некоторые моменты на протяжении срока жизни проекта у менеджмента возникает возможность на основании поступившей к этому моменту информации повлиять на развитие проекта. Менеджмент обладает так называемой управленческой гибкостью (managerial flexibility) и может отложить реализацию проекта или одного из его этапов, уменьшить или увеличить масштаб или вовсе отказаться от реализации. Для того чтобы получить справедливую оценку такой управленческой гибкости, необходимо провести оценку всего набора реальных опционов в совокупности, а не каждый опцион по отдельности, а затем складывать их стоимости. При наличии в одном проекте нескольких реальных опционов необходимо учитывать взаимодействие между ними, что часто приводит к нарушению свойства аддитивности. В большинстве случаев стоимость совокупности реальных опционов меньше суммы их отдельных стоимостей, хотя бывают и исключения. Прежде чем приступить непосредственно к исследованию, необходимо рассмотреть предыдущие работы, посвященные данной тематике. Само понятие реальных опционов появилось в финансовой теории еще в 70-х годах XX века в исследованиях Майерса и Пиндайка, которые ввели в рассмотрение понятие управленческой гибкости, а также подняли вопрос о её оценке. Прорыв в этой области произошел с появлением знаменитой работы Фишера Блэка и Майрона Шоулза [Black, Scholes, 1973], которая значительно развила математический аппарат для оценки опционов, а также дала аналитическое выражение для оценки финансовых опционов (формула Блэка-Шоулза), которое с успехом применяется для оценки отдельных реальных опционов. В отличие от темы оценки реальных опционов в отдельности, по которой существует довольно много как теоретических, так и эмпирических работ, оценке взаимодействующих реальных опционов посвящено не так много исследований. По сути их всего три. Первая из них дает математический аппарат, на основе которого оценивается совокупность реальных опционов, вторая описывает результаты, полученные численными методами, а третья дает практический пример применения данной методологии при оценке инфраструктурных проектов. Но есть еще ряд работ, в которых эта тематика затрагивается в той или иной степени. Самым первым исследованием, в котором предпринималась попытка оценить несколько опционов в совокупности, была работа Геске [Geske R., 1979], в которой впервые дается аналитическая формула для оценки сложных опционов (compound options – опционы Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 42 №2(6) 2008 на опцион). При получении этой формулы используется сложный математический аппарат стохастических дифференциальных уравнений, а сама формула довольно громоздка, поэтому на практике она используется редко. 1. Обзор существующих исследований по тематике Тем более что при оценке инвестиционных проектов с множественными реальными опционами мы сталкиваемся не со сложными опционами в прямом смысле этого слова, а скорее с неким портфелем реальных опционов, присутствующих в проекте. Методология, предложенная Геске, была развита в статье Аглиарди [Agliardi R., 2006], в которой автор получает аналитическое выражение для оценки двух последовательных реальных опционов на расширение, а также в работе Аглиарди [Agliardi R., 2007], которая дает аналитическое решение для оценки комбинации опционов на уменьшение и увеличение масштабов проекта, основываясь на том же математическом аппарате, с помощью которого была получена формула Блэка-Шоулза. Однако и здесь сложность используемого математического аппарата является препятствием для его практического применения. Самая ранняя статья, посвященная взаимодействующим реальным опционам, датируется 1991 годом [L. Trigeorgis, 1991]. В ней один из основоположников данной теории, Л. Тригеоргис, предлагает алгоритм для оценки взаимодействующих реальных опционов и сравнивает предложенный алгоритм с существующими методами оценки. Главным преимуществом предложенного метода является то, что он, в отличие от других способов (модель Блэка-Шоулза, биномиальная модель), позволяет оценивать совокупность реальных опционов, учитывая их влияние друг на друга. Также, как доказывает автор, предложенный метод является довольно эффективным благодаря скорости и точности расчетов.. Следующая статья, посвященная рассматриваемой тематике, — это более поздняя статья того же автора [L. Trigeorgis, 1993]. В ней с использованием предложенного ранее метода проводится более подробный анализ оценки инвестиционных проектов с множественными реальными опционами, а также более детально раскрываются причины их взаимодействия. В качестве примера берется инвестиционный проект, в котором одновременно присутствуют следующие реальные опционы: • опцион на отказ (option to abandon); • опцион на переключение (option to switch use); • опцион на задержку (option to defer); • опцион на уменьшение масштаба (option to contract); • опцион на увеличение масштаба (option to expand). Далее с помощью метода, предложенного в более ранней работе [Trigeorgis, 1991], анализируются численные результаты. Основной вывод, к которому приходит автор, — что стоимость реальных опционов вместе не равна сумме стоимостей реальных опционов по отдельности (чаще всего она меньше, но встречаются случаи, когда она может быть и больше). Основной причиной, по которой наблюдается различие в стоимости, является существование единого базового актива (приведенной стоимости денежных потоков от проекта) для всех реальных опционов в одном инвестиционном проекте. Из этого вытекает следующее. • Если рассматривать последовательность опционов, то стоимость реального опциона может изменяться по причине существования потенциального влияния следующего реального опциона (стоимость базового актива включает и стоимость следующих опционов). • На стоимость реального опциона (а следовательно, и на стоимость всего проекта) может влиять исполнение (неисполнение) предыдущих опционов. 1. Обзор существующих исследований по тематике • На стоимость реального опциона (а следовательно, и на стоимость всего проекта) может влиять исполнение (неисполнение) предыдущих опционов. Эти две причины являются основными для взаимодействия опционов и, следовательно, для нарушения свойства аддитивности стоимостей. Эти две причины являются основными для взаимодействия опционов и, следовательно, для нарушения свойства аддитивности стоимостей. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 Еще одной статьей на данную тему является работа Боуи и Динг Лан Ли [Bowe M., Ding Lun Lee, 2004]. В ней проводится тестирование модели, предложенной Тригеоргисом, Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 Еще одной статьей на данную тему является работа Боуи и Динг Лан Ли [Bowe M., Ding Lun Lee, 2004]. В ней проводится тестирование модели, предложенной Тригеоргисом, ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 43 на примере реального инфраструктурного проекта по строительству железнодорожной магистрали на Тайване. Основным преимуществом данной статьи является то, что авторы использовали реальные численные данные, а не проводили симуляции: это позволило протестировать метод оценки реальных опционов, предложенный Тригеоргисом, на практике. на примере реального инфраструктурного проекта по строительству железнодорожной магистрали на Тайване. Основным преимуществом данной статьи является то, что авторы использовали реальные численные данные, а не проводили симуляции: это позволило протестировать метод оценки реальных опционов, предложенный Тригеоргисом, на практике. Метод расчета стоимости проекта полностью аналогичен предложенному Тригеоргисом; при этом авторы дают пример расчета различных численных входных данных, таких как волатильность, ставка дисконтирования и т.д. Результаты проведенного исследования подтвердили выводы, сделанные Тригеоргисом, о том, что стоимости реальных опционов в одном проекте не являются аддитивными. Стоимость совокупности реальных опционов для данного примера составила 80,4% от суммы стоимостей реальных опционов в отдельности (наблюдается отрицательная синергия). А в целом стоимость реальных опционов составила довольно значительную долю (21,98%) от чистой приведенной стоимости проекта, что еще раз подтверждает вывод о значимости реальных опционов при оценке инвестиционных проектов. Также методология, предложенная Тригеоргисом для оценки реальных опционов, была развита им самим в статье «Evaluating Leases With Complex Operating Options» [L. Trigeorgis, 1996], где он делает попытку оценить реальные опционы в такой области, как лизинг (под реальными опционами здесь понимаются возможности для каждой из сторон, заложенные в контракте). Модель, используемая для оценки, аналогична логарифмической модели, за исключением незначительных модификаций. Выводы, к которым приходит автор, соответствуют полученным в предыдущих работах: при наличии нескольких реальных опционов в одном проекте нарушается свойство аддитивности их стоимостей. Метод оценки реальных опционов применяется для инвестиционных проектов в различных отраслях, таких как добыча полезных ископаемых [Dimitrakopolous Roussos G., Abdel Sabour, 2007], научно-исследовательские разработки [L. 1. Обзор существующих исследований по тематике Trigeorgis, 1990]), который и будет использоваться далее в данной работе. р • Существует ряд работ, в которых анализируются отдельные реальные опционы на примере сделок c недвижимостью и девелоперских проектов [Grenadier R. Steven, 1995; Rocha K., Salles L., Garcia F.A.A., Sardinha J.A., Texeira J.P., 2007; Quigg Laura, 1993]; но мы не нашли ни одной работы, которая бы оценивала совокупность реальных опционов в девелоперских проектах, тем более в условиях российского рынка. • Существует ряд работ, в которых анализируются отдельные реальные опционы на примере сделок c недвижимостью и девелоперских проектов [Grenadier R. Steven, 1995; Rocha K., Salles L., Garcia F.A.A., Sardinha J.A., Texeira J.P., 2007; Quigg Laura, 1993]; но мы не нашли ни одной работы, которая бы оценивала совокупность реальных опционов в девелоперских проектах, тем более в условиях российского рынка. Так как практически нет работ, оценивающих взаимодействующие реальные опционы в недвижимости, то эта тема дает возможность проведения новых научных исследований. В качестве исследуемого рынка берется российский рынок недвижимости. В качестве расчетной модели – логарифмическая модель Тригеоргиса, рассмотренная выше. 1. Обзор существующих исследований по тематике Trigeorgis, Han T.J. Smit], т.е. в тех отраслях, где есть значительная неопределенность относительно будущих денежных потоков. Что же касается реальных опционов в недвижимости, то здесь нам удалось найти лишь несколько работ, которые, впрочем, не затрагивают непосредственно взаимодействия реальных опционов. Например, исследование Куиг [Quigg Laura, 1993], которое является эмпирическим исследованием на выборке из 2700 сделок с недвижимостью в Сиэтле, США, за период с 1976-го по 1979 год. Ценность этого исследования заключается в том, что оно дает численную оценку стоимости реальных опционов в сделках с недвижимостью. С помощью статистических методов автор предпринял попытку оценить стоимость опциона на задержку в реализации проекта в реальных сделках с недвижимостью. Не вдаваясь в детали построения регрессий, было выяснено, что средняя премия за опцион на задержку составляет около 6%, варьируясь от 1 до 30%. По итогам анализа существующих исследований и литературы на эту тему можно сделать следующие выводы: • Методология анализа и оценки реальных опционов в отдельности является хорошо разработанной темой: существует большое количество научных работ, в которых решается данная проблема [Black, Scholes, 1973; Myers, 1977; McDonald R. & Siegel D., 1985; McDonald R. & Siegel D., 1986; Pindyck, 1988]. • Методология анализа и оценки реальных опционов в отдельности является хорошо разработанной темой: существует большое количество научных работ, в которых решается данная проблема [Black, Scholes, 1973; Myers, 1977; McDonald R. & Siegel D., 1985; McDonald R. & Siegel D., 1986; Pindyck, 1988]. • Существует ряд работ, в которых получены аналитические выражения для оценки сложных опционов и некоторых комбинаций реальных опционов, но данные формулы не находят практического применения из-за их сложности и громоздкости [Geske R., 1979; Agliardi R., 2006; Agliardi R., 2007; M.J. Brennan, E.S. Schwartz, 1985; Meng-Yu Lee, Fang-Bo Yeh, An-Pin Chen, 2008]. • Существует ряд работ, преимущественно одного автора [L. Trigeorgis, 1993; M. Bowe, Ding Lun Lee, 2004; L. Trigeorgis, 1996], посвященных анализу нескольких реальных опционов в совокупности. Также этот автор предлагает вычислительный метод для #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 © Электронный журнал Корпоративные Финансы, 2008 © Электронный журнал Корпоративные Финансы, 2008 Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 44 оценки реальных опционов в совокупности, учитывающий все их взаимодействия (логарифмическая модель Тригеоргиса [L. Trigeorgis, 1990]), который и будет использоваться далее в данной работе. оценки реальных опционов в совокупности, учитывающий все их взаимодействия (логарифмическая модель Тригеоргиса [L. Trigeorgis, 1990]), который и будет использоваться далее в данной работе. оценки реальных опционов в совокупности, учитывающий все их взаимодействия (логарифмическая модель Тригеоргиса [L. © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 45 №2(6) 2008 Теоретически существует возможность изменить назначение объекта на этапе подготовки проектной документации (например, с жилого комплекса на офисный), тем самым изменяя структуру денежных потоков проекта. Но на практике это практически не происходит, так как требует значительных денежных и временных затрат (дополнительные согласования и дополнительные денежные вложения на реконструкцию здания), поэтому данная возможность не рассматривалась. Четвертым опционом, который рассматривается в данном исследовании, является отказ от реализации проекта: мы предполагаем, что после окончания подготовки и согласования проектной документации девелопер может продать стороннему покупателю участок с готовой документацией, руководствуясь какими-либо соображениями (неблагоприятные рыночные условия, потребность в денежных средствах). Цена продажи определяется понесенными на предыдущих этапах капитальными затратами. Данный опцион позволяет минимизировать убытки, отказавшись от реализации «плохого» проекта. Рис. 1. Типы реальных опционов в девелоперском проекте 1 год 2 год 3 год Этап 2: подготовка проекта и согласование документации Этап 3: земляные работы Этап 4: строительно – монтажные работы Этап 1: покупка земельного участка Опцион на задержку в реализации проекта Опцион на увеличение/уменьшение масштабов проекта Опцион на отказ Этап 5: инженерные работы Продажа квартир / Сдача в аренду Рис. 1. Типы реальных опционов в девелоперском проекте Все рассмотренные выше опционы, в такой последовательности и с такими особенностями, характерны именно для инвестиционных проектов в области недвижимости, и само наличие вышеописанных возможностей повлиять на ход реализации проекта обеспечивает добавку к стоимости проекта, рассчитанной по методу дисконтированных денежных потоков. Величина премии за наличие управленческой гибкости определяется путем оценки реальных опционов с использованием моделей, рассмотренных выше. 2.1. Типы реальных опционов в девелоперских проектах Метод оценки реальных опционов применяется для инвестиционных проектов в различных отраслях, таких как добыча полезных ископаемых [Dimitrakopolous Roussos G., Abdel Sabour, 2007], научно-исследовательские разработки [L. Trigeorgis, Han T.J. Smit], т.е. в тех отраслях, где есть значительная неопределенность относительно будущих денежных потоков. Одной из таких отраслей является недвижимость, где главным фактором неопределенности выступает цена на недвижимость. При реализации девелоперского проекта менеджмент имеет относительную свободу действий, так как в определенные моменты времени он может принимать решения, влияющие на дальнейший ход реализации проекта. Эти моменты времени сосредоточены в основном до начала строительно-монтажных работ, так как после согласования всей проектной документации и начала работ основные характеристики проекта (площадь, класс здания, назначение и т.д.) практически невозможно поменять: они закреплены законодательно, а также требуют значительных капитальных вложений. Реальный опцион на задержку чаще всего встречается в проектах, требующих масштабных инвестиций. При покупке земельного участка или при долгосрочной аренде девелопер волен откладывать начало реализации проекта до тех пор, пока не сочтет рыночные условия благоприятными или не получит дополнительную информацию о рынке, которая позволит с большей точностью спрогнозировать денежные потоки. Если участок находится в собственности, то откладывать начало подготовки документации можно бесконечно долго. Данная возможность позволяет максимизировать стоимость здания с помощью выхода на рынок в оптимальный момент времени. Реальный опцион на увеличение (уменьшение) масштаба проекта существует на этапе разработки проектной документации. Согласно п. 2.3 «Разработка, согласование и утверждение архитектурно-градостроительного решения (архитектурного проекта)» Положения о едином порядке предпроектной и проектной подготовки строительства в городе Москве, для того, чтобы изменить технико-экономические показатели объекта, установленные в исходной разрешительной документации, более, чем на 10%, необходимо пройти ряд дополнительных согласований, которые требуют значительных временных и денежных затрат. Поэтому мы считаем, что после определения основных показателей проекта (в финансовой модели это площадь и назначение объекта) мы можем изменять их на 10%; таким образом, у нас есть опцион на увеличение (уменьшение) масштабов проекта на этапе подготовки и согласования проектной документации. Данный опцион позволяет увеличить прибыль или уменьшить убытки в зависимости от рыночных условий. © Электронный журнал Корпоративные Финансы, 2008 Выпуск #2(6), 2008 Выпуск #2(6), 2008 Выпуск #2(6), 2008 Методология исследования Активы на рынке недвижимости представляют особый класс активов, который отличается как от финансовых активов (акций, облигаций, финансовых инструментов и т.д.), так и от других реальных активов (драгоценных камней и металлов, лицензий, контрактов, нематериальных активов и т. п.). Ниже приведены основные характеристики активов в области недвижимости. • Высокая стоимость отдельных активов (значительные капитальные вложения, необходимые для строительства зданий). • Высокая стоимость отдельных активов (значительные капитальные вложения, необходимые для строительства зданий). Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 46 46 • Низкая ликвидность (значительные временные затраты при совершении сделок на рынке недвижимости). • Низкая ликвидность (значительные временные затраты при совершении сделок на рынке недвижимости). • Низкая ликвидность (значительные временные затраты при совершении сделок на рынке недвижимости). • Низкая ликвидность (значительные временные затраты при совершении рынке недвижимости). р ) • Неоднородность активов (каждый актив индивидуален, нет абсолютно одинаковых зданий и сооружений). • Неоднородность активов (каждый актив индивидуален, нет абсолютно одинаковых зданий и сооружений). • Недостаток информации (большинство компаний отрасли являются закрытыми, и даже публичные компании предоставляют только необходимый по законодательству минимум информации). • Недостаток информации (большинство компаний отрасли являются закрытыми, и даже публичные компании предоставляют только необходимый по законодательству минимум информации). Непрозрачность рынка недвижимости заставила отказаться от проведения эмпирических исследований статистическими методами и воспользоваться методами симуляций, так как для оценки инвестиционных проектов необходимо знать прогнозы денежных потоков, а также характеристики самого проекта, что, в принципе, является конфиденциальной информацией, которую невозможно найти в открытых источниках. ф ф р ру р Российский рынок недвижимости, как и рынок недвижимости в целом, можно сегментировать следующим образом: • жилая недвижимость; • коммерческая недвижимость: • офисная недвижимость, • складская недвижимость, • торговые центры (торговая недвижимость). • торговые центры (торговая недвижимость). Основным отличием активов жилой недвижимости от активов коммерческой является структура получения доходов, а именно то, что коммерческая недвижимость представляет собой стабильный поток относительно небольших доходов от аренды здания, и продажа проекта другому инвестору в конце прогнозного периода (здесь уместно сравнение с облигацией: поток купонных платежей и погашение номинала). В то время как жилая недвижимость подразумевает получение большего количества доходов во время осуществления строительства (продажа квартир в недостроенных домах) и по окончании строительства. Следует заметить, что каждый проект на рынке недвижимости является абсолютно индивидуальным (различные земельные участки, планировка задания, дизайн и т.д.), поэтому все закономерности на этом рынке являются средними. Следовательно, при рассмотрении любого конкретного проекта обязательно следует учитывать его индивидуальные особенности. Так как мы пользуемся методом симуляций, то нам необходимо выделить основные закономерности, характерные для проектов, в каждом сегменте рынка, а именно разбить по времени основные денежные потоки (как инвестиции, так и денежные поступления) и определить их масштаб (т.е. какую долю от общего объема инвестиций и на что необходимо затратить в каждый момент времени, а также какая доля от общего объема денежных поступлений от проекта поступает в каждый момент времени). Для этого мы разбиваем реализацию проекта на стадии, оцениваем их продолжительность, а также масштаб инвестиций и продаж для каждого этапа реализации проекта. © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 19 Средние сроки продолжительности отдельных стадий получены путем анализа завершенных проектов. 20 Затраты на приобретение земельного участка не следует рассматривать как фиксированные издержки, не зависящие от масштаба проекта, так как при приобретении земельного участка или прав аренды на него на покупателя накладываются некоторые ограничения в плане объема реализуемого проекта (этажности, площади). © Электронный журнал Корпоративные Финансы, 2008 Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 21 Цифры означают долю от общего объема инвестиций или продаж соответственно. Для коммерческой недвижимости картина выглядит таким же образом, за исключением того что доходы от реализации проекта представляют собой не разовые платежи, а одинаковые ежегодные выплаты и терминальный денежный поток в конце горизонта прогнозирования, соответствующий выходу из проекта. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" Основными стадиями типичного инвестиционного проекта в области недвижимости являются19: • оформление документов на право собственности земельного участка и совершение сделки по покупке (средний срок – 0,5 года)20; • оформление документов на право собственности земельного участка и совершение сделки по покупке (средний срок – 0,5 года)20; • подготовка проекта, исходно-разрешительной документации и её согласование в государственных органах (средний срок – 1 год); • подготовка проекта, исходно-разрешительной документации и её согласование в государственных органах (средний срок – 1 год); 19 Средние сроки продолжительности отдельных стадий получены путем анализа завершенных проектов. 20 Затраты на приобретение земельного участка не следует рассматривать как фиксированные издержки, не зависящие от масштаба проекта, так как при приобретении земельного участка или прав аренды на него на покупателя накладываются некоторые ограничения в плане объема реализуемого проекта (этажности, площади). © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" пуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 р р р , у у р ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 47 РНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 47 • земляные работы (средний срок — 0,5 года); • строительно-монтажные работы (средний срок – 1 год); • инженерные работы (средний срок – 1 год). Разбиение денежных потоков в зависимости от стадии инвестиционного проекта иведено на рисунке 2. -0.2 -0.05 -0.05 -0.05 -0.275 -0.275 -0.1 0.3 0.6 0.1 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Инвестиции Продажи Рис. 2. Структура денежных потоков для проектов жилой недвижимости21 Рис. 2. Структура денежных потоков для проектов жилой недвижимости21 Для расчета ставки дисконтирования для оценки девелоперских проектов теоретические модели (например CAPM) в полной мере неприменимыми, и необходимо учитывать экспертное мнение участников рынка. Согласно этому мнению, ставка варьируется от степени законченности проекта (имеется ли исходно-разрешительная документация, вышел ли проект на нулевой уровень, построено ли здание полностью); и ставка, по которой следует дисконтировать проекты в области жилой недвижимости с нуля (существует лишь проект, отсутствует проектная документация, здание не построено), лежит в диапазоне 20—30% (среднее значение 25%), а в области коммерческой — 15—25% (среднее значение – 20%). Именно эти ставки дисконтирования и использовались в качестве предпосылок в расчетной модели. Для проведения численных расчетов по оценке премии за наличие реальных опционов в девелоперских проектах была построена модель в программе Microsoft Excel. Как и большинство финансовых моделей, она состоит из блока входных данных, расчетного блока и вывода результатов. Краткая схема приведена на рисунке 3. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 48 ЖУРНАЛ КОРПОРАТИВНЫЕ ФИНАНСЫ №2(6) 2008 48 Рис. 3. Краткая схема модели Входные данные базируются на данных из открытых источников и экспертных оценок. В качестве входных данных использовались следующие параметры: • объем проекта (площадь здания); • распределение инвестиций по времени; • распределение продаж по времени; • ставка дисконтирования; • безрисковая ставка; • затраты на 1 кв. м; Рис. 3. Краткая схема модели Рис. 3. Краткая схема модели Рис. 3. Краткая схема модели Входные данные базируются на данных из открытых источников и экспертных оценок. В качестве входных данных использовались следующие параметры: Входные данные базируются на данных из открытых источников и экспертных оценок. В качестве входных данных использовались следующие параметры: Также использовалась временная структура и масштаб денежных потоков из предыдущего раздела. Численные значения входных данных приведены в таблице 1. Таблица 1. Основные входные данные для модели Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 Таблица 1. Основные входные данные для модели Жилая недвижимость Офисная недвижимость Торговая недвижимость Складская недвижимость Затраты на строительство, долл. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 49 №2(6) 2008 49 На основе входных данных с помощью алгоритма Тригеоргиса рассчитывалась стоимость совокупности реальных опционов. Горизонт прогнозирования составлял 5 лет с разбивкой по полугодиям. Расчет денежных потоков производился исходя из структуры, описанной выше: т.е. оценивалась общая сумма инвестиций и денежных поступлений в прогнозном периоде, исходя из затрат и цены/арендных ставок за 1 кв. м, а далее, в соответствии с заданным распределением, рассчитывались денежные потоки в каждом временном периоде. Помимо оценки стоимости проекта с учетом совокупности реальных опционов, проводился расчет «стандартной» чистой приведенной стоимости и стоимости реальных опционов в отдельности для оценки синергии. Для учета управленческой гибкости в модель были включены следующие четыре реальных опциона, рассмотренные выше: • опцион на задержку (Option to defer) – возможность отложить реализацию проекта на 5 лет (горизонт прогнозирования); • опцион на задержку (Option to defer) – возможность отложить реализацию проекта на 5 лет (горизонт прогнозирования); • опцион на увеличение масштаба (Option to expand) – возможность увеличить масштаб проекта (площадь) на 10% на этапе подготовки и согласования проектной документации (в соответствии с нормативными актами); • опцион на увеличение масштаба (Option to expand) – возможность увеличить масштаб проекта (площадь) на 10% на этапе подготовки и согласования проектной документации (в соответствии с нормативными актами); • опцион на уменьшение масштаба (Option to contract) – возможность уменьшить масштаб проекта (площадь) на 10% на этапе подготовки и согласования проектной документации (в соответствии с нормативными актами); • опцион на отказ (Option to abandon) – возможность продать в случае неблагоприятной конъюнктуры рынка земельный участок вместе с подготовленным проектом за стоимость, равную сумме вложенных инвестиций. • опцион на отказ (Option to abandon) – возможность продать в случае неблагоприятной конъюнктуры рынка земельный участок вместе с подготовленным проектом за стоимость, равную сумме вложенных инвестиций. Расчет проводился для различных значений цены/арендной ставки (для того чтобы оценить зависимость премии за реальные опционы при изменении рыночных условий) и волатильности (как величины, которая может быть рассчитана различными способами). Волатильность рассчитывалась тремя способами: • Дисперсия роста прогнозируемых денежных потоков ( ) (ln 1 t t CF CF Var + ). Стоит отметить, что при данном методе расчета для каждого значения цены (арендной ставки) получается свое значение волатильности, что влияет на итоговый результат. t CF отметить, что при данном методе расчета для каждого значения цены (арендной ставки) получается свое значение волатильности, что влияет на итоговый результат. отметить, что при данном методе расчета для каждого значения цены (арендной ставки) получается свое значение волатильности, что влияет на итоговый результат. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" • Историческая волатильность российского рынка жилой недвижимости (в качестве меры волатильности рынка использовалась волатильность индекса www.irn.ru). • Историческая волатильность российского рынка жилой недвижимости (в качестве меры волатильности рынка использовалась волатильность индекса www.irn.ru). • Историческая волатильность американского рынка недвижимости. В качестве меры волатильности использовалась волатильность индекса National Association of Real Estate Investment Trusts (NAREIT)22. Для расчетов брались месячные данные за 1971— 2008 годы. Оценка стоимостей реальных опционов по отдельности производилась с помощью формулы Блэка-Шоулза. Далее, для получения стоимости проекта с реальными опционами, но без учета их взаимодействия, эти стоимости добавлялись к «стандартной» чистой приведенной стоимости проекта. В итоге получалась стоимость проекта с реальными опционами, но без учета их взаимодействия. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 22 Real Estate Investment Trust (REIT) – инвестиционный трастовый фонд, специализирующийся на вложениях в недвижимость. Представляет собой инвестиционный фонд, который инвестирует в недвижимость посредством её приобретения. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" США/кв.м 1500 2500 2000 1000 Среднерыночная арендная ставка (цена), долл. США/кв. м 5500 1200 1200 150 Диапазон арендных ставок (цен), долл. США/кв. м 3500—7000 500—2500 500—2500 50—1000 Ставка дисконтирования, % 25 20 20 20 пуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 © Электронный журнал Корпоративные Финансы, 2008 2.1. Результаты В результате была выявлена зависимость стоимости проекта с учетом взаимодействующих реальных опционов (а также стоимости без учета реальных опционов и Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 22 Real Estate Investment Trust (REIT) – инвестиционный трастовый фонд, специализирующийся на вложениях в недвижимость. Представляет собой инвестиционный фонд, который инвестирует в недвижимость посредством её приобретения. © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 50 №2(6) 2008 с учетом реальных опционов, но без учета их взаимодействия) от цены для различных значений волатильности. Также были рассчитаны значения премий за наличие реальных опционов для различных значений цены и волатильности, а также различных сегментов рынка недвижимости. Учитывая, что вычисления проводились тремя способами расчета волатильности, полученные результаты можно разделить на три группы соответственно, что и сделано ниже. Для каждого способа расчета волатильности проводился анализ чувствительности к ценам (арендным ставкам) на недвижимость, а также вычислялись значения для текущей рыночной ситуации. Сначала была сделана попытка рассчитать волатильность, как отклонение денежных потоков. Данный способ расчета волатильности использовался в статье Боуи и Динг Дан Ли «Project evaluation in the presence of multiple embedded real options: evidence from the Taiwan High-Speed Rail Project». При данном методе расчета волатильности для жилой недвижимости чистая приведенная стоимость денежных потоков проекта, с учетом множественных реальных опционов, превосходит как чистую приведенную стоимость проекта без поправки на реальные опционы, так и чистую приведенную стоимость проекта с поправками на реальные опционы; причем с ростом цены увеличивается разница между оценками, т.е. наличие нескольких реальных опционов в одном проекте обеспечивает положительную синергию. Однако здесь стоит учитывать тот факт, что для каждого значения цены получается свое значение волатильности, так как изменяются денежные потоки, что влияет на значение расчетной волатильности. Таблица 2. Основные результаты вычислений (волатильность, рассчитанная как дисперсия денежных потоков) Среднерыночный уровень цен (арендных ставок), долл. США/кв. м Премия за множественн ые реальные опционы, % Диапазон цен (арендных ставок), долл. США/кв.м Диапазон премий за множественные реальные опционы, % Жилая недвижимость 5500 151 4500—6500 138—265 Офисная недвижимость 1500 41 800—2500 30—185 Торговая недвижимость 1500 36 800—2500 28—94 Складская недвижимость 130 58 100—250 31—112 Таблица 2. Основные результаты вычислений (волатильность, рассч дисперсия денежных потоков) ица 2. Основные результаты вычислений (волатильность, рассчитанная как денежных потоков) Что касается остальных видов недвижимости, то наблюдается обратная картина – премия за множественные реальные опционы меньше суммы стоимостей реальных опционов в отдельности, т.е. наличие множества реальных опционов обеспечивает негативную синергию. Единственной причиной этого могут являться различия в структуре денежных потоков. 2.1. Результаты Причиной такой большой премии является большое значение волатильности как одного из факторов стоимости опциона; также волатильность является причиной столь значительного отличия результатов для жилой недвижимости от результатов для коммерческой недвижимости. Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 Численное значение премии за реальные опционы получилось довольно значительным (для жилой недвижимости — около 150%), что позволяет сделать вывод о том, что данный метод расчета волатильности не является корректным, так как на рыночных данных приводит к результатам, не соответствующим здравому смыслу (стоимость возможностей не может превосходить чистую приведенную стоимость денежных потоков от реализации ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 51 №2(6) 2008 51 проекта; более того ни в одном исследовании премия за реальные опционы не превосходила даже 50%). © Электронный журнал Корпоративные Финансы, 2008 проекта; более того ни в одном исследовании премия за реальные опционы не превосходила даже 50%). проекта; более того ни в одном исследовании премия за реальные опционы не превосходила даже 50%) Далее для расчета волатильности использовались исторические цены российского рынка недвижимости. В качестве волатильности российского рынка недвижимости использовалась волатильность индекса цен агентства www.irn.ru. При подстановке данного фиксированного значения волатильности картина получается приблизительно одинаковой для всех типов недвижимости: начиная с некоторого значения цены наиболее высокой оценкой становится NPV и сумма стоимостей реальных опционов в отдельности; далее по убыванию следует NPV с поправкой на совокупность реальных опционов и затем — традиционный NPV (именно такие результаты получались при симуляциях, проводимых Тригеоргисом). Таблица 3. Основные результаты вычислений (волатильность как историческая волатильность российского рынка недвижимости) Среднерыночный уровень цен (арендных ставок), долл. США/кв. м Премия за множествен- ные реальные опционы, % Диапазон цен (арендных ставок), долл. США/кв.м Диапазон премий за множественные реальные опционы, % Жилая недвижимость 5500 8 4500—6500 6—26 Офисная недвижимость 1500 9 800—2500 10—20 Торговая недвижимость 1500 9 700—2500 10—18 Складская недвижимость 130 10 100 —250 9—13 ица 3. Основные результаты вычислений (волатильность как историческая ость российского рынка недвижимости) Таблица 3. Основные результаты вычислений (волатильность как и волатильность российского рынка недвижимости) Численное значение премии за множественные реальные опционы получилось приблизительно одинаковым для всех типов недвижимости и составляет около 9% от чистой приведенной стоимости, что представляется довольно разумным. Например, как уже было сказано выше, американские ученые в своих исследованиях получали оценку стоимости реальных опционов в размере 6%. Таким образом, на данный момент для учета управленческой гибкости в качестве оценки для стоимости реальных опционов можно брать 9% от чистой приведенной стоимости проекта. Хотя, конечно, эта оценка является довольно условной, так как каждый девелоперский проект является индивидуальным и для каждого проекта стоит рассчитывать свою премию. Но эта новая премия не будет значительно отклоняться от 9%, так как все основные закономерности уже учтены. В качестве третьей альтернативы была использована историческая волатильность американского рынка недвижимости. Такой подход может быть полезен для более поздних стадий развития рынка недвижимости, когда пройдет период бурного роста и российский рынок станет схож с американским или европейским. В качестве меры волатильности использовался месячные данные за 1971—2008 годы по американскому индексу National Association of Real Estate Investment Trusts (NAREIT). По результатам расчетов дисперсия доходностей за рассматриваемый промежуток времени составила 0,2%. Соответственно в качестве дисперсии базового актива мы принимали именно эту величину. Выпуск #2(6), 2008 Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 №2(6) 2008 52 Таблица 4. проекта; более того ни в одном исследовании премия за реальные опционы не превосходила даже 50%). Основные результаты вычислений (историческая волатильность американского рынка недвижимости) Среднерыночный уровень цен (арендных ставок), долл. США/кв. м Премия за множественны е реальные опционы, % Диапазон цен (арендных ставок), долл. США/кв.м Диапазон премий за множественные реальные опционы, % Жилая недвижимость 5500 1 4500—6500 3—18 Офисная недвижимость 1500 9 800—2500 6—10 Торговая недвижимость 1500 9 700—2500 6—10 Складская недвижимость 130 9 100—250 7—10 4. Основные результаты вычислений (историческая волатильность о рынка недвижимости) Здесь картина схожа с наблюдаемой при расчетах для волатильности российского рынка недвижимости. Стоит отметить, что с уменьшением волатильности уменьшается премия за реальные опционы, особенно это заметно для жилой недвижимости, для волатильности, соответствующей американскому рынку недвижимости, премия за множественные реальные опционы составляет всего лишь 1%. Таким образом, с увеличением степени развитости рынка падает премия за реальные опционы (хотя и незначительно), что вполне логично, так как на развитом рынке возможностей повлиять на ход проекта становится все меньше — ведь развитый рынок оставляет меньше простора для реализации возможностей. В целом можно сказать, что одним из факторов, определяющих премию за реальные опционы в девелоперских проектах, является способ расчета волатильности. На мой взгляд, наиболее разумно использовать историческую волатильность для исследуемого рынка, что подтверждается выполненными расчетами. Для российского рынка данная премия составляет 9%, с незначительными различиями между сегментами. Также важным фактором является ставка дисконтирования (к ней чувствительна чистая приведенная стоимость, независимо от того, используется при расчетах метод реальных опционов или нет). При выполнении данной работы были использованы текущие рыночные ставки дисконтирования; и поскольку довольно сложно спрогнозировать будущие ставки дисконтирования, тем более что на рынке недвижимости они в большинстве своем определяются с помощью экспертной оценки, анализ чувствительности по данному параметру не проводился — в связи с отсутствием его практической ценности. Полученные результаты показали, что нет ощутимой разницы между различными сегментами коммерческой недвижимости: проекты в этом секторе рынка имеют сходный масштаб и временную структуру денежных потоков (различия только в величине затрат и арендных ставок). Основные различия проявляются между проектами в сфере жилой и коммерческой недвижимости, так как в этих секторах рынка их структура довольно разнообразна. Также важно отметить, что относительная величина премии за наличие реальных опционов (в процентах) не зависит от масштаба проекта (площади). Играют роль только структура проекта и другие входные данные, упомянутые ранее (ставка дисконтирования, волатильность, величины затрат, арендных ставок и т.д.). Заключение Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 В настоящей работе мы исходили из предположения, что в ходе реализации проекта менеджмент может ее отложить (опцион на задержку), может изменять масштаб ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 53 №2(6) 2008 реализуемого проекта (опцион на увеличение/уменьшение масштабов проекта), а также принять решение об отказе от реализации проекта (опцион на отказ). Была сделана попытка оценить портфель реальных опционов в недвижимости. В качестве исходных данных использовались сведения о текущем состоянии рынка недвижимости, а также исследовалась зависимость стоимости проектов от значений цены и волатильности рынка. Взяв за основу численный метод. предложенный Л. Тригеоргисом в 1991 году и развитый им же в статье 1993 года, мы получили премии за наличие взаимодействующих реальных опционов в девелоперских проектах в различных сегментах рынка недвижимости (жилая, офисная, складская и торговая недвижимость). р ) Проведенное исследование показало, что значимое влияние на премию за наличие реальных опционов оказывает способ расчета волатильности. Существенные различия (более чем 100% для жилой недвижимости) были выявлены между результатами, использующими дисперсию денежных потоков и историческую волатильность. Так как при использовании денежных потоков получаются противоречащие здравому смыслу и предыдущим исследованиям результаты (премия за наличие реальных опционов в жилой недвижимости – 151%), то наиболее разумным представляется использовать историческую волатильность исследуемого рынка. Было выявлено незначительное отличие в результатах, полученных для различных сегментов коммерческой недвижимости (офисная, складская и торговая): премия за наличие реальных опционов отличается не более чем на 1%. Это объясняется сходством этих сегментов в плане структуры денежных потоков реализуемых проектов. Были выявлены незначительные отличия в размере премии в проектах жилой и коммерческой недвижимостью. В условиях текущей рыночной конъюнктуры премия за реальные опционы в проектах жилой недвижимости оставляет 8%, в секторе коммерческой недвижимости – 9%. Данная работа подтвердила отсутствие при наличии нескольких опционов в одном проекте свойства аддитивности стоимостей реальных опционов, которая была ранее сформулирована Тригеоргисом. С практической точки зрения данное исследование может быть интересно девелоперским компаниям и различным инвесторам, делающим вложения в активы на рынке недвижимости, так как оно дает в численной форме оценку премии за реальные опционы. Это может быть использовано для учета управленческой гибкости при оценке инвестиционной привлекательности девелоперского проекта. Результаты исследования позволили выявить возможные направления для следующих работ в рамках данной тематики. Во-первых, можно использовать предложенный подход для исследований стоимости проекта с несколькими реальными опционами в других отраслях, таких как добыча полезных ископаемых, научно-исследовательские разработки и т.д. Заключение Во-вторых, при наличии соответствующей информации можно протестировать метод Тригеоргиса на реальных данных по девелоперским проектам и выяснить, насколько отличается значение премии, полученное в результате симуляций, от значения премии, полученного в ходе статистических исследований. В-третьих, возможно включение в рассмотрение более сложных опционов, таких как возможность изменения назначения объекта, например перевод его из класса коммерческой недвижимости в жилую и наоборот, в ходе подготовки проектной документации. Список литературы 1. Agliardi R. Options to expand: Some remarks // Finance Research Letters, No. 3, 2006, р. 65–72. 1. Agliardi R. Options to expand: Some remarks // Finance Research Letters, No. 3, 2006, р. 65–72. 2. Agliardi R. Options to expand and to contract in combination // Applied Mathematical Letters, No. 20, 2007, р. 790—794. 2. Agliardi R. Options to expand and to contract in combination // Applied Mathematical Letters, No. 20, 2007, р. 790—794. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" 54 №2(6) 2008 3. Agliardi, E., Agliardi, R. A generalization of the Geske formula for compound options // Mathematical Social Sciences, No. 45, 2003, р. 75–82. р 4. Antikarov V., Copeland T. Real options: A Practioner’s Guide. TEXERE, LLC, 200 5. Black F., Scholes M. The pricing of options and corporate liabilities // Journal of Economy, No. 81, 1973, р. 637—654. 6. Bowe M., Ding Lun Lee. Project evaluation in the presence of multiple embedded real options: evidence from the Taiwan High-Speed Rail Project // Journal of Asian Economics, No. 15, 2004, p. 71–98. p 7. Brennan M., Schwarz E. Evaluating natural resource investments // Journal of Business, No. 58, 1985, p.135—157. 8. Dimitrakopoulos Roussos G., Abdel Sabour Sabry A. Evaluating mine plans under uncertainty: Can the real options make a difference? // Resources Policy, No. 32, 2007, p. 116–125. 9. Holland, Steven, Steven Ott and Tim Riddiough. The Role of Uncertainty in Investment: An Examination of Competing Investment Models Using Commercial Real Estate Data, Real Estate Economics, No. 28(1), 2001, p. 33—64/ 10. Geltner D.M., Norman M.G. Commercial Real Estate Analysis and Investments, South- Western Publishing, 2001. 11. Geske R. The Valuation of Compound Options // Journal of Financial Economics, No. 7, 1979, p. 63—81/ 12. Grenadier Steven R. Flexibility and Tenant Mix in Real Estate Projects // Journal of Urban Economics, No. 38, 1995, p. 357—378/ 13. McDonald R., Siegel D. The value of waiting to invest // Quarterly Journal of Economics, No. 101, 1986, p. 707—727. 14. McDonald R., Siegel D. Investment and the valuation of firms when there is an option to shut down // International Economic Review, No. 28, 1985, p. 331–349. 15. Meng-Yu Lee, Fang-Bo Yeh, An-Pin Chen. The generalized sequential compound options pricing and sensitivity analysis // Mathematical Social Sciences, No. 55, 2008, p. 38–54. 16. Myers S.C. Determinants of corporate borrowing // Journal of Financial Economics, No. 5, 1977, p. 147–175. 17. Ott Steven H. Real Options and Real Estate: A Review and Valuation Illustration, Belk College of Business Administration, University of North Carolina at Charlotte, NC 28223- 0001. 18. Ott Steven H. and Ha-Chin Yi. Real Options and Development: A Model of Regional Supply and Demand // Real Estate Finance, No. 18(1), 2001, p. 47—55. 19. Pindyck R. Irreversible Investment, Capacity Choice and Value of the Firm // The American Economic Review, No. 78, 1988, p. 969—985. 20. Quigg Laura. © Электронный журнал Корпоративные Финансы, 2008 Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" Empirical Testing of Real Option-Pricing Models // Journal of Finance, 1993, p. 621—640. 21. Rocha K., Salles L., Garcia F.A.A., Sardinha J.A., Texeira J.P. Real Estate and Real Options – A Case Study // Emerging Markets Review, No. 8, 2007, p. 67–79. 22. Trigeorgis L. A Log-Transformed Binomial Numerical Analysis Method for Valuing Complex Multi-Option Investments // The Journal of Financial and Quantitative Analysis, Vol. 26, No. 3. (Sep., 1991), p. 309—326. ( p ) p 23. Trigeorgis L. Evaluating Leases With Complex Operating Options // European Journal of Operational Research, No. 91, 1996, p. 315—329. 24. Trigeorgis L. The Nature of Option Interactions and the Valuation of Investments with Multiple Real Options // The Journal of Financial and Quantitative Analysis, Vol. 28, No. 1. (Mar., 1993), p. 1—20. ( ) p 25. Trigeorgis L., Martzoukos S.H. Real (investment) options with multiple sources of rare events // European Journal of Operational Research, No. 136. 2002, p. 696—706. © Электронный журнал Корпоративные Финансы, 2008 Выпуск #2(6), 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 55 26. Грибовский С.В., Иванова Е.Н., Львов Д.С., Медведева О.Е. Оценка стоимости недвижимости, М, Интерреклама, 2003. 27. Обзор рынка недвижимости, Россия, 2008. Colliers International. 28. Положение о едином порядке предпроектной и проектной подготовки строительства в г. Москве, 2-я редакция. © Электронный журнал Корпоративные Финансы, 2008 ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ" №2(6) 2008 55 26. Грибовский С.В., Иванова Е.Н., Львов Д.С., Медведева О.Е. Оценка стоимости недвижимости, М, Интерреклама, 2003. 27. Обзор рынка недвижимости, Россия, 2008. Colliers International. 28. Положение о едином порядке предпроектной и проектной подготовки строительства в г. Москве, 2-я редакция. ЖУРНАЛ "КОРПОРАТИВНЫЕ ФИНАНСЫ 55 55 27. Обзор рынка недвижимости, Россия, 2008. Colliers International. 28. Положение о едином порядке предпроектной и проектной подготовки строительства в г. Москве, 2-я редакция. 28. Положение о едином порядке предпроектной и проектной подготовки строительства в г. Москве, 2-я редакция. Выпуск #2(6), 2008 Выпуск #2(6), 2008 © Электронный журнал Корпоративные Финансы, 2008 © Электронный журнал Корпоративные Финансы, 2008
https://openalex.org/W4360771945
https://www.jmir.org/2023/1/e43359/PDF
English
null
Preliminary Attainability Assessment of Real-World Data for Answering Major Clinical Research Questions in Breast Cancer Brain Metastasis: Framework Development and Validation Study (Preprint)
null
2,022
cc-by
8,014
JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Original Paper Preliminary Attainability Assessment of Real-World Data for Answering Major Clinical Research Questions in Breast Cancer Brain Metastasis: Framework Development and Validation Study Min Jeong Kim1*, MSc; Hyo Jung Kim1,2*, PhD; Danbee Kang3,4, PhD; Hee Kyung Ahn5, MD; Soo-Yong Shin1,2,6, PhD; Seri Park7, BSN; Juhee Cho3,4,8, PhD; Yeon Hee Park1,9, MD, PhD PhD; Seri Park7, BSN; Juhee Cho3,4,8, PhD; Yeon Hee Park1,9, MD, PhD 1Department of Digital Health, Samsung Advanced Institute for Health Science & Technology, Sungkyunkwan University, Seoul, Republic of Korea 2Center for Research Resource Standardization, Research Institution for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 3Center for Clinical Epidemiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4Department of Clinical Research Design and Evaluation, Samsung Advanced Institute for Health Science & Technology, Sungkyunkwan University, Seoul, Republic of Korea 5Division of Medical Oncology, Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Republic of Korea 6Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University, Suwon, Republic of Korea 7Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea 8Department of Epidemiology and Medicine, The Welch Center for Prevention, Epidemiology, and Clinical Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States 9Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea *these authors contributed equally Corresponding Author: Yeon Hee Park, MD, PhD Division of Hematology-Oncology Department of Medicine, Samsung Medical Center Sungkyunkwan University School of Medicine 81, Ilwon-ro Gangnam-gu Seoul, 06351 Republic of Korea Phone: 82 10 9933 1780 Email: yhparkhmo@skku.edu 1Department of Digital Health, Samsung Advanced Institute for Health Science & Technology, Sungkyunkwan University, Seoul, Republic of Korea 2Center for Research Resource Standardization, Research Institution for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Clinical Epidemiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4Department of Clinical Research Design and Evaluation, Samsung Advanced Institute for Health Science & Technology, Sungkyunkwan University, Seoul, Republic of Korea 6Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University, Suwon, Republic of Korea 7Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea 8 9Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea * *these authors contributed equally Corresponding Author: Yeon Hee Park, MD, PhD Division of Hematology-Oncology Department of Medicine, Samsung Medical Center Sungkyunkwan University School of Medicine 81, Ilwon-ro Gangnam-gu Seoul, 06351 Republic of Korea Phone: 82 10 9933 1780 Email: yhparkhmo@skku.edu J Med Internet Res 2023 | vol. 25 | e43359 | p. 1 (page number not for citation purposes) Abstract Background: In recent decades, real-world evidence (RWE) in oncology has rapidly gained traction for its potential to answer clinical questions that cannot be directly addressed by randomized clinical trials. Integrating real-world data (RWD) into clinical research promises to contribute to more sustainable research designs, including extension, augmentation, enrichment, and pragmatic designs. Nevertheless, clinical research using RWD is still limited because of concerns regarding the shortage of best practices for extracting, harmonizing, and analyzing RWD. In particular, pragmatic screening methods to determine whether the content of a data source is sufficient to answer the research questions before conducting the research with RWD have not yet been established. Objective: We examined the PAR (Preliminary Attainability Assessment of Real-World Data) framework and assessed its utility in breast cancer brain metastasis (BCBM), which has an unmet medical need for data attainability screening at the preliminary step of observational studies that use RWD. Methods: The PAR framework was proposed to assess data attainability from a particular data source during the early research process. The PAR framework has four sequential stages, starting with clinical question clarification: (1) operational definition of variables, (2) data matching (structural/semantic), (3) data screening and extraction, and (4) data attainability diagramming. We identified 5 clinical questions to be used for PAR framework evaluation through interviews and validated them with a survey J Med Internet Res 2023 | vol. 25 | e43359 | p. 1 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 O XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Kim et al of breast cancer experts. We used the Samsung Medical Center Breast Cancer Registry, a hospital-based real-time registry implemented in March 2021, leveraging the institution’s anonymized and deidentified clinical data warehouse platform. The number of breast cancer patients in the registry was 45,129; it covered the period from June 1995 to December 2021. The registry consists of 24 base data marts that represent disease-specific breast cancer characteristics and care pathways. The outcomes included screening results of the clinical questions via the PAR framework and a procedural diagram of data attainability for each research question. Results: Data attainability was tested for study feasibility according to the PAR framework with 5 clinical questions for BCBM. We obtained data sets that were sufficient to conduct studies with 4 of 5 clinical questions. The research questions stratified into 3 types when we developed data fields for clearly defined research variables. KEYWORDS real-world data; preliminary attainability assessment; observational study; clinical data warehouse; PAR framework; brain metastasis; breast cancer However, pragmatic screening methods to determine whether the content of a data source is sufficient to answer the research questions before conducting research with RWD have not yet been established. Accurate but oversimplified instructions could lead to confusion among clinical researchers who seek to select the optimal RWD source for their hypothesis and research purpose [16-19]. In detail, specifying the research question and determining the appropriate level for understanding massive and complex RWD is still challenging for clinical researchers. The vagueness inherent in this complex process at an early stage of research is one cause of concern and controversy regarding the utility of RWD for generating scientific evidence [13,17]. Therefore, effective screening methods are needed to determine whether the contents of a data source are sufficient to answer the research questions before conducting studies using RWD [20,21]. Introduction Brain metastasis (BM) is a major cause of mortality in patients with breast cancer, and it increases the difficulty of treatment. Advancements in treatment and the development of brain imaging technology have increased the survival of patients with metastatic breast cancer, leading to an increased incidence of BM [1-4]. Nonetheless, the opportunity to participate in prospective randomized clinical trials (RCTs) is typically only available to a limited number of patients with breast cancer brain metastasis (BCBM). Design challenges, including heterogeneity of patients, varying definitions of clinical end points, and different methods to assess these end points, have led to excluding most patients with BCBM from RCTs [5,6]. Consequently, while the incidence and survival duration have expanded, clinical research methods for BCBM remain limited [7]. This study suggests a method to screen specific data sources for their ability to address a clinical research question at the preliminary step of research design and evaluate the method’s utility in assessing data attainability for BCBM. Meanwhile, real-world evidence (RWE) in oncology has rapidly gained traction in recent decades, with the potential to answer clinical questions that cannot be directly or completely addressed by RCTs [8,9]. Integrating real-world data (RWD) into clinical research promises to contribute to more sustainable research designs, including extension, augmentation, enrichment, and pragmatic design [10]. To support the need for RWE generation, medical institutions have provided RWD through the construction of clinical data warehouses (CDWs) based on electronic health records (EHRs) [11,12]. Nevertheless, clinical research using RWD is still limited because of concerns regarding confidence in nonrandomized RWD analyses and the shortage of best practices for extracting, harmonizing, and analyzing RWD—practices that improve transparency and reproducibility [13,14]. To address these concerns, researchers have emphasized the importance of comprehensively understanding data representation and data content while clarifying research questions [15]. Abstract In the first, only 1 question could be answered using direct data variables. In the second, the other 3 questions required surrogate definitions that combined data variables. In the third, the question turned out to be not feasible for conducting further analysis. Conclusions: The adoption of the PAR framework was associated with more efficient preliminary clinical research using RWD from BCBM. Furthermore, this framework helped accelerate RWE generation through clinical research by enhancing transparency and reproducibility and lowering the entry barrier for clinical researchers. (J Med Internet Res 2023;25:e43359) doi: 10.2196/43359 (J Med Internet Res 2023;25:e43359) doi: 10.2196/43359 J Med Internet Res 2023 | vol. 25 | e43359 | p. 2 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 Participants The Samsung Medical Center Breast Cancer Registry (SMC BCR) is a hospital-based real-time registry implemented in March 2021 that leverages the institution’s anonymized and deidentified CDW platform: the Data Analytics and Research Window for Integrated Knowledge (DARWIN)–C. The inclusion criteria for the SMC BCR comprise the intersection of three patient conditions: (1) visited at least one of the medical oncology, breast surgery, or radiation oncology departments, (2) diagnosed with breast cancer under codes C50 or D05 of the International Classification of Diseases codes, and (3) aged older than 15 years at enrollment [19]. The number of breast cancer patients in SMC BCR was 45,129, and the registry covered the period from June 1995 to December 2021. The SMC BCR consists of 24 base data marts that represent disease-specific breast cancer characteristics and care pathways. The registry provides clinical variables in the form of structured data, including demographics, diagnostic history, treatment information (operation, chemotherapy, and radiation therapy), laboratory test results, and featured data fields extracted from free-text records, such as pathology, radiology, or genomic laboratory reports [19]. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH BCBM data mart was evaluated using manual chart reviews performed by 2 clinical nurses with expertise in data management; data validity was greater than 98% via 10% random sampling. Data attainability was defined in this study as the availability of data for reconstruction and extraction required from a particular data source to conduct clinical research regarding sample size, data fields, and content. Working Group for Data Attainability Assessment A working group was formed for data attainability testing. It comprised people with clinical expertise and at least 10 years of experience in clinical research across various interdisciplinary areas, including medical informatics and epidemiology. Clinical question E Clinical question E Can any record of neurological symptoms described by patients be translated as a surrogate factor of brain metastasis? clinical question in the early research stages. The PAR framework has 4 sequential stages, starting with clarification of the clinical question (Figure 1). Clinicians Interviewed to Develop the Key Clinical Questions Seven breast cancer experts from 6 hospitals in the Republic of Korea participated in a survey. Two breast cancer experts were interviewed in advance to define clinical questions regarding BCBM. J Med Internet Res 2023 | vol. 25 | e43359 | p. 3 (page number not for citation purposes) Clinical Question Preparation and Validation We established clinical questions through 2 rounds of interviews with 2 breast cancer clinical experts in April and June 2021 (Textbox 1). From the interviews, we identified clinical questions that reflected the most recent and internationally relevant unmet clinical needs in BCBM [21-26]. We conducted a survey of experts’opinions to validate the clinical significance and the feasibility of these clinical questions. Seven breast cancer clinical experts participated in the survey from September 27 to October 5, 2021. In this survey, clinical experts scored clinical significance, research availability, data attainability, and method suitability using EHR data on a 5-point Likert scale (range 1-5). Multimedia Appendices 1 and 2 show the average scores and correlation analysis results. All data analyses and calculations were performed using Microsoft Excel (2016 version; Microsoft Corp). Graphs and plots were designed using R (version 4.0.2; R Foundation for Statistical Computing). For BCBM patient identification, we used a BCBM data mart in the SMC BCR. The BCBM data mart contains predefined and preprocessed data from patients (n=1443 up to December 2021) with at least one BM indicator after a breast cancer diagnosis. The indicators, defined by clinical experts, are as follows: craniotomy record (payment-based), whole-brain radiotherapy treatment (regions for radiation treatment are brain, whole brain, and partial), gamma knife (code for gamma knife), and intrathecal methotrexate treatment. The data quality of the Textbox 1. List of clinical questions developed from interviews with experts. Does systemic treatment for brain metastasis patients affect survival outcomes according to subtype? Clinical question C Does the timing of systemic treatment initiation affect survival outcomes when brain metastasis is accompanied by systemic metastasis? Clinical question D Overview In line with major clinical research questions on BCBM, this study evaluated screening performance using the Preliminary Attainability Assessment of Real-World Data (PAR) framework, which can assist clinicians in assessing data attainability from an RWD source at a preliminary stage of the study design. This study was divided into 2 phases. In the preparation phase, we first identified clinical questions related to current unmet needs according to the perspectives of clinical experts on BCBM for evaluation of the framework. In the evaluation phase, the working group assessed the preliminary data attainability of the listed clinical questions with a specific data source, using the PAR framework presented in the following section. https://www.jmir.org/2023/1/e43359 https://www.jmir.org/2023/1/e43359 J Med Internet Res 2023 | vol. 25 | e43359 | p. 2 (page number not for citation purposes) J Med Internet Res 2023 | vol. 25 | e43359 | p. 2 (page number not for citation purposes) XSL•FO RenderX Kim et al Clinical question D Is there a difference in the prediction of brain metastasis in patients who previously received trastuzumab alone for neoadjuvant the who received pertuzumab and trastuzumab for neoadjuvant therapy? Stage 4: Data Attainability Diagramming In stage 3, the actual data values identified during the previous stages were extracted. The interdisciplinary team, including clinical experts, continued to validate the contents of the extracted data sets. Through this cross-validation, we confirmed whether the data were logically aligned with previously well-known clinical evidence. Data sets for 4 of the 5 clinical questions proposed by the breast cancer experts from a clinical perspective were extracted. Finally, we draw a diagram showing the extracted sample size according to the respective clinical question and matching process from the sub–data sets acquired in stage 3. Stage 3: Data Screening and Extraction Here, the actual data values are extracted and screened from the data source for each data variable identified in the previous stages. Stage 2: Data Matching (Structural/Semantic) This stage involves matching the data variables with specific data fields in the data source selected as the research material. Data fields refer to stored data elements, such as the column name of the data table, that depend on the data source. By contrast, the data variable is a unified conceptual term. A one-to-one direct match has priority; however, a surrogate definition for the data variable is explored by combining the values from multiple data fields. In stage 2, the data fields were matched with the defined operational data variables. At this stage, some questions required variable replacement to match data in the SMC BCR. The variables “BM event time,” “death date,” “neoadjuvant regimen start date,” and “neoadjuvant regimen name” were respectively coupled with corresponding data fields in the SMC BCR. By contrast, for “systematic metastasis event time,” a one-to-one direct match was not possible for any data field within the data source. Hence, we used “palliative treatment start date” as a surrogate variable, following clinical expert opinion, which reflected institutional treatment protocols. PAR Framework The PAR framework was proposed to assess the data attainability of a data source from the perspective of a particular https://www.jmir.org/2023/1/e43359 XSL•FO RenderX Kim et al JOURNAL OF MEDICAL INTERNET RESEARCH Figure 1. Illustration of the Preliminary Attainability Assessment of Real-World Data (PAR) framework and study design. CQ: clinical question. Figure 1. Illustration of the Preliminary Attainability Assessment of Real-World Data (PAR) framework and study design. CQ: clinical question. informed consent as our study data were deidentified, and anonymized data were extracted from the CDW. This study followed the Standards for Quality Improvement Reporting Excellence (SQUIRE) guidelines [20]. Stage 1: Operational Definitions of Variables In this step, we identify variables inherent in the clinical question, describe them in natural language, and clarify operational definitions of the variables at 2 distinct levels. One level is the research-variable level and includes dependent or independent variables at the description level of the research hypothesis. The other level is a data-variable level that can be used as an atomic condition to declare the research variable. For example, “overall survival (time)” can be one of the research variables that the researcher intends to observe, and “first diagnosis date” and “death date” are the data variables that constitute the research variable. Results The working group assessed data attainability pertaining to the 5 clinical questions through stages 1 to 4. In stage 1, the research and data variables were defined based on the SMC BCR data structure (Figure 2). For clinical question E, the variable “neurological symptoms” did not match with any structured or semistructured fields in the data source. Furthermore, terminological code systems covering “neurological symptoms” had not been applied in the EHR prior to the SMC BCR. Therefore, we concluded that clinical question E was not properly answered using our data source. J Med Internet Res 2023 | vol. 25 | e43359 | p. 4 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al was 143 (28.7%). In the subgroup with no systemic treatment records, 54 patients (33%) were classified into the HR positive/HER2 negative group, 44 patients (26.8%) into the HR/HER2 positive group, and 66 patients (40.2%) into the TNBC group. At the final stage, only clinical question D had a data set obtained from a directly matched data variable (Figure 3D). We gathered data for 4292 patients from 2006 to 2021 from the BCR for trastuzumab treatment. Among these patients, 1382 patients received trastuzumab treatment as a neoadjuvant treatment, 832 patients received combined trastuzumab and pertuzumab neoadjuvant treatment, and 550 patients received trastuzumab neoadjuvant treatment alone. The remaining clinical questions required substitutional variables owing to a lack of matched variables in the CDW system. Therefore, we obtained appropriate data sets for the other clinical questions from the data source with a defined surrogate variable. In stage 2, the data-matching step, a surrogate data variable, “systemic treatment in a palliative setting,” was set to represent the presence of systemic metastasis. Among the 1443 BCBM patients, 956 patients were classified as having BM with systemic metastasis based on records of both BM-related and systemic treatment in a palliative setting. Of these 956 patients, 237 patients were classified as having BM that later progressed to systemic metastasis. Data attainability was verified for 2 clinical questions: “Is there a difference in survival outcomes between the brain as the primary metastasis site and brain metastasis accompanied by systemic metastasis?” and “Does the timing of systemic treatment initiation affect survival outcomes when brain metastasis is accompanied by systemic metastasis?” (Figure 3A and Figure 3C). To derive the data set for the clinical question “Does systemic treatment for brain metastasis patients affect survival outcomes according to subtype?” the BCBM mart and clinical subtyping information based on immunohistochemistry test results were used (Figure 3B). Clinical subtyping information was available for 662 patients who had test results for estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2 (HER2) items. HER2 results were curated based on supplementing the silver in situ hybridization or fluorescence in situ hybridization tests. During the process of clearly defining research variables in terms of data fields, the research questions stratified into 3 types. J Med Internet Res 2023 | vol. 25 | e43359 | p. 5 (page number not for citation purposes) Ethical Considerations The study protocol was approved by the Institutional Review Board of the SMC (2021-09-036), which waived the need for J Med Internet Res 2023 | vol. 25 | e43359 | p. 4 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 https://www.jmir.org/2023/1/e43359 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Figure 3. Data attainability for the clinical questions. Clinical questions were as follows. (A) Is there a difference in survival outcomes between the brain as the primary metastasis site and brain metastasis accompanied by systemic metastasis? (B) Does systemic treatment for brain metastasis patients affect survival outcomes according to subtype? (C) Does the timing of systemic treatment initiation affect survival outcomes when brain metastasis is accompanied by systemic metastasis? and (D) Is there a difference in the prediction of brain metastasis in patients who previously received trastuzumab alone for neoadjuvant therapy and in patients who received pertuzumab and trastuzumab for neoadjuvant therapy? BCBM: breast cancer brain metastasis; HER2: human epidermal growth factor receptor 2; HR: hormone receptor; TNBC: triple-negative breast cancer. Figure 4. Preliminary Attainability Assessment of Real-World Data framework evaluation in stages 1 to 4. CQ: clinical question; CDW: clinical data warehouse Attainability Assessment of Real-World Data framework evaluation in stages 1 to 4. CQ: clinical question; CDW: clinical da Figure 4. Preliminary Attainability Assessment of Real-World Data framework evaluation in stages 1 to 4. CQ: clinical question; CDW: clinical data warehouse. Figure 4. Preliminary Attainability Assessment of Real-World Data framework evaluation in stages 1 to 4. CQ: clinical qu h Discussion significance of the clinical questions. The mean score was 4.37 (range 3 57 5 00) We found that the correlation between scores Discussion Principal Findings In this study, we propose the PAR framework for data attainability screening at the preliminary step and evaluate its utility with clinical questions that reflect the most recent and internationally relevant unmet needs of clinicians in BCBM [21-26]. A survey was conducted to evaluate the clinical significance of the clinical questions. The mean score was 4.37 (range 3.57-5.00). We found that the correlation between scores given by experts was higher for the questions with higher average scores (Multimedia Appendices 1 and 2). RWE generation has received attention in the BCBM therapeutic area owing to limited clinical trial opportunities despite increasing clinical importance. However, incomplete gold standards for RWD study protocols and the unpredictable “hidden labor” of the secondary use of clinical data serve as barriers when clinical J Med Internet Res 2023 | vol. 25 | e43359 | p. 6 https://www.jmir.org/2023/1/e43359 (page number not for citation purposes) significance of the clinical questions. The mean score was 4.37 (range 3.57-5.00). JOURNAL OF MEDICAL INTERNET RESEARCH We found that the correlation between scores given by experts was higher for the questions with higher average scores (Multimedia Appendices 1 and 2). RWE generation has received attention in the BCBM therapeutic area owing to limited clinical trial opportunities despite increasing clinical importance. However, incomplete gold standards for RWD study protocols and the unpredictable “hidden labor” of the secondary use of clinical data serve as barriers when clinical https://www.jmir.org/2023/1/e43359 J Med Internet Res 2023 | vol. 25 | e43359 | p. 6 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH Among the 5 clinical questions suggested by clinical experts, we gathered data sets for 4 clinical questions using the stages suggested above. Only 1 clinical question could be answered using the matched direct data variables. However, the additional questions could be answered using surrogate variables (Figure 4). Systemic treatment records were only available for 498 patients. The number of patients with any hormone receptor (HR)/HER2 positive clinical subtype was 180 (36.2%), the number with triple-negative breast cancer (TNBC) was 175 (35.1%), and the number with an HR positive /HER2 negative clinical subtype Figure 2. Results at stages 1 and 2 with the breast cancer brain metastasis clinical questions. CQ: clinical question. Figure 2. Results at stages 1 and 2 with the breast cancer brain metastasis clinical questions. CQ: clinical question. https://www.jmir.org/2023/1/e43359 Principal Findings The methodology of this study can contribute to the acceleration of RWE generation by strengthening the transparency and reproducibility of the RWD research process and lowering the entry barrier for clinical researchers. CDWs contain clinical data from EHRs for retrospective analysis to enable clinical researchers to utilize RWD for research directly, and the scope of RWD use has significantly expanded over the past few decades [31-34]. However, we identified several challenges while conducting RWD studies using the SMC BCR with clinical questions. A deficiency in the exact data variables for the questions recognized by clinical researchers was detected in stage 1. Researchers need to reassess stages 1 and 2 when no directly matching data field exists for a research variable. A surrogate definition could be considered as a substitution. For example, we adopted an indirect variable to represent systemic metastasis using the data for systemic treatment and used the start date of the palliative treatment cycle as the date of metastasis. Since most metastatic breast cancer patients with systemic metastasis receive systemic treatments, data on these indirect variables were readily available from the SMC BCR. However, when using an indirect variable as a surrogate, the validity of the research outcome is lower than when using a direct variable, which could be a primary limitation of RWD studies. Alternatively, if the variables will be frequently used for the generation of RWE, construction of featured data marts based on raw data and local practice rules is recommended. Since the SMC CDW has a well-constructed BCR with a BM mart, it was relatively easy for the investigators to extract the necessary data variables. Clinical researchers with research questions derived from empirical insight in clinical practice have difficulties securing an understanding of accumulated RWD and solidifying their study designs. In contrast to conventional medical research methodologies, securing the reliability and validity of research variables for RWD studies is designed at a post– rather than pre–data collection stage. In addition, RWD is a conceptual collective term encompassing all data obtained through health care activities, and the content varies by data source [27]. Therefore, research using RWD requires significant and iterative effort prior to formal hypothesis testing, including selecting an appropriate data source, curating the data, repurposing it, and preprocessing it [28,29]. Principal Findings In this study, we propose the PAR framework for data attainability screening at the preliminary step and evaluate its utility with clinical questions that reflect the most recent and internationally relevant unmet needs of clinicians in BCBM [21-26]. A survey was conducted to evaluate the clinical https://www.jmir.org/2023/1/e43359 XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Kim et al reproducibility is further extended when applied to data sources that adopt not only institution-specific structures but also common data models, such as the Observational Medical Outcomes Partnership (OMOP) common data model [31] or the Informatics for Integrating Biology & the Bedside (i2b2) data model [32]. As specific data field conditions and query code levels with standardized data models and vocabularies can be reused, the accumulation of operational definitions of data levels can be considered an additional knowledge base. Furthermore, the accumulation of these definitions enhances consistency in the conduct of RWD research by enabling discussion regarding content validity in a more comparative manner. Accordingly, connecting formative efforts in the RWD research process, from data storage to processing, is a promising way to ensure reliability of research outcomes [33]. researchers attempt to design the most suitable methods to address their research questions [16,17]. We identified particular gaps when SMC built a site-specific CDW platform and developed a BCR as its first implementation case. After the release of the SMC BCR, clinical researchers attempted to generate RWE by using this registry, especially in areas with significant unmet needs, such as BCBM. Nevertheless, clinical research using RWD has not been as successful as expected in our experience despite the fact that well-known technical barriers are addressed; the CDW’s functional user interface provides clinicians with access to nonidentified and anonymous high-quality data sets. The greatest challenge faced by clinical researchers at the next step was securing a sufficient understanding of the data to avoid information distortion during the research data preparation stage. Therefore, we proposed the PAR framework to assess the feasibility of research at the preliminary stage and evaluated this framework with BCBM clinical questions with various entry points. To the best of our knowledge, a systematic framework to explore research feasibility at the conjunction between the data source and clinical questions has yet to be presented. Without such a framework, sample size, data fields, and content have not been properly accounted for. J Med Internet Res 2023 | vol. 25 | e43359 | p. 7 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 Multimedia Appendix 1 Comparison of expert assessments for each clinical question was conducted by surveying 7 experts' opinions from September 27th, 2021 to October 5th, 2021. [PNG File , 355 KB-Multimedia Appendix 1] JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Kim et al Data Availability The data used for this study were extracted from a real-time breast cancer registry and breast cancer brain metastasis data mart integrated into the Data Analytics and Research Window for Integrated Knowledge–C clinical data warehouse of Samsung Medical Center. The data presented in this study are available on reasonable request from the corresponding author. Conflicts of Interest YHP is a consultant or advisory board member for AstraZeneca, Pfizer, Lilly, MSD, Eisai, Roche, Daiichi-Sankyo, Menarini, Bixink, Everest, and Novartis Pharmaceuticals and received research funds from AstraZeneca, MSD, Pfizer, Novartis, and Roche. MJK is an employee of Roche Korea. HKA is a consultant for Daiichi Sankyo, Amgen, Yuhan, Novartis, Takeda, and Roche, and received payment for lectures from Bristol Myers Squibb, MSD, Eli Lilly, AstraZeneca, Boehringer Ingelheim, Menarini, Eisai, Pfizer, Boryung Pharmaceutical, Celltrion, Yuhan, and Pharmbio Korea. All other authors declare no conflicts of interest. Multimedia Appendix 2 The statistical analysis of the survey results utilized correlation analysis between the mean value of each score and the SD. [PNG File , 115 KB-Multimedia Appendix 2] Acknowledgments This study was supported by the Future Medicine 2030 Project of Samsung Medical Center (SMO1230451) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A1A01058250). We thank the clinical experts who contributed to this study: In Hae Park from Korea University Guro Hospital, Ji-Yeon Kim from Samsung Medical Center, Jae Ho Jeong from Asan Medical Center, Kyung-Hun Lee from Seoul National University Hospital, and Min Hwan Kim from Severance Hospital; they provided insight and expertise that greatly assisted preparation for our research into clinical researchers’ unmet needs regarding RWD. The clinical question assessment part of this study was previously presented as a poster at the San Antonio Breast Cancer Symposium Congress 2022 (December 6-10, 2022) as poster number P4-02-27. Principal Findings As a result, local information system expertise and deep content knowledge are often required to understand the idiosyncratic manner in which data sources are captured and stored [17,30]. Depending on the level of the data structure, data extraction frequently requires a high level of technical training as well. Through the process of matching research variables with data fields, the PAR framework illustrates how the process of clarifying research questions refines the range of data that should be analyzed before addressing a specific research question. Consequently, time and effort could be greatly reduced to ensure understanding of the selected data source. Additionally, it was not possible to extract key variables for clinical question E. This result aligned with the experts’survey scores for the least feasible and least suitable questions using EHR data. Symptoms described by patients were only recorded in EHRs as free text, and there was no specific template or location. It was not possible to preprocess this information using a rule-based semantic engine for the CDW. Despite capturing symptoms of patients’ complaints, integrating the information into the CDW was difficult, because the location and template of the data were not aligned across departments. Moreover, the terminology for neurological symptoms is not standardized and is subject to a relatively high level of cultural dependency. To address this, integrating other types of data, such as prospective cohort studies or patient-generated data from mobile or watch-type devices, can be considered. If investigators continue to track patient-reported outcomes through cohort studies and integrate this information with other variables in CDWs, the use of RWE based on CDW data can be increased. The advantage of the PAR framework is that the results of stages 1 and 2 are explicitly communicated and can be reused. For example, in clinical question D, the research variable “time from neoadjuvant to BM” can be measured as the time between “neoadjuvant regiment start date” and “BM event date.” This level of operational definition can be reviewed and reused by peers with clinical expertise regardless of the storage structure of the data source. Above all, this reusability-enhancing J Med Internet Res 2023 | vol. 25 | e43359 | p. 7 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 XSL•FO RenderX XSL•FO RenderX Limitations We proposed and evaluated a PAR framework to assess data attainability to answer major clinical research questions in BCBM. The adoption of the PAR framework is associated with improved efficiency in clinical research using RWD in the preliminary stage. This framework could contribute to improving the quality of RWD-based clinical research by enhancing its transparency and reproducibility. This study is limited in its generalizability. We assessed a single clinical domain, BCBM, and extracted data from a single data source, the SMC BCR. Further application of the PAR framework in different domains or with different types of RWD will be needed to develop the framework. However, the PAR framework and training case presented in this study could help guide clinical researchers in assessing preliminary attainability for future studies using RWD. Authors' Contributions MJK and HJK contributed equally to the development of the idea for the study, design of the study protocol, collection of patient data, data analysis (including underlying data verification and interpretation), development of the statistical analysis plan, and writing of the first draft of the manuscript. DK contributed to survey preparation and data analysis. HKA contributed to the conception and design of the study and interpretation of the data and provided breast cancer expertise. S-YS contributed to the design of the study, interpretation of the data, and provided advice regarding clinical data warehouse use. SP contributed to the conception and design of the study and the institutional review board process. YHP and JC equally contributed to the study and they evaluated the overall study process; contributed to conceptualization and design of the trial and data collection, analysis, and interpretation; and reviewed and edited the manuscript. All authors read and approved the final manuscript. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al 2. Morgan AJ, Giannoudis A, Palmieri C. The genomic landscape of breast cancer brain metastases: a systematic review. Lancet Oncol 2021 Jan;22(1):e7-e17. [doi: 10.1016/S1470-2045(20)30556-8] [Medline: 33387511] 3. Mills MN, Figura NB, Arrington JA, Yu HM, Etame AB, Vogelbaum MA, et al. Management of brain metastases in breast cancer: a review of current practices and emerging treatments. Breast Cancer Res Treat 2020 Apr;180(2):279-300. [doi: 10.1007/s10549-020-05552-2] [Medline: 32030570] 4. Ahn HK, Park YH, Lee SJ, Park S, Maeng CH, Park W, et al. Clinical implication of Time To Brain Metastasis (TTBM) according to breast cancer subtypes. Springerplus 2013 Dec;2(1):136 [FREE Full text] [doi: 10.1186/2193-1801-2-136] [Medline: 23667803] 5. Joe NS, Hodgdon C, Kraemer L, Redmond KJ, Stearns V, Gilkes DM. A common goal to CARE: Cancer Advocates, Researchers, and Clinicians Explore current treatments and clinical trials for breast cancer brain metastases. NPJ Breast Cancer 2021 Sep 14;7(1):121 [FREE Full text] [doi: 10.1038/s41523-021-00326-5] [Medline: 34521857] , p Cancer 2021 Sep 14;7(1):121 [FREE Full text] [doi: 10.1038/s41523-021-00326-5] [Medline: 345 6. Che W, Liu J, Fu T, Wang X, Lyu J. Recent trends in synchronous brain metastasis incidence and mortality in the United States: ten-year multicenter experience. Curr Oncol 2022 Nov 02;29(11):8374-8389 [FREE Full text] [doi: 10.3390/curroncol29110660] [Medline: 36354720] 7. Puhalla S, Elmquist W, Freyer D, Kleinberg L, Adkins C, Lockman P, et al. Unsanctifying the sanctuary: challenges and opportunities with brain metastases. Neuro Oncol 2015 May;17(5):639-651 [FREE Full text] [doi: 10.1093/neuonc/nov023] [Medline: 25846288] 8. Booth CM, Tannock IF. Randomised controlled trials and population-based observational research: partners in the evolution of medical evidence. Br J Cancer 2014 Feb 04;110(3):551-555 [FREE Full text] [doi: 10.1038/bjc.2013.725] [Medline: 24495873] ] 9. Di Maio M, Perrone F, Conte P. Real-world evidence in oncology: opportunities and limitations. Oncologist 2020 May;25(5):e746-e752 [FREE Full text] [doi: 10.1634/theoncologist.2019-0647] [Medline: 31872939] Maio M, Perrone F, Conte P. Real-world evidence in oncology: opportunities and limitations. Oncologist Di Maio M, Perrone F, Conte P. Real-world evidence in oncology: opportunities and limitations. Oncolog May;25(5):e746-e752 [FREE Full text] [doi: 10.1634/theoncologist.2019-0647] [Medline: 31872939] Di Maio M, Perrone F, Conte P. Real world evidence in oncology: opportunities and limitations. Oncolog May;25(5):e746-e752 [FREE Full text] [doi: 10.1634/theoncologist.2019-0647] [Medline: 31872939] 10. National Academies of Sciences, Engineering, and Medicine; Health and Medicine Division; Board on Health Sciences Policy; Forum on Drug Discovery, Development, and Translation. In: Shore C, Gee AW, Kahn B, Forstag EH, editors. JOURNAL OF MEDICAL INTERNET RESEARCH Examining the Impact of Real-World Evidence on Medical Product Development: Proceedings of a Workshop Series. Washington, DC: National Academies Press; 2019. 11. Pavlenko E, Strech D, Langhof H. Implementation of data access and use procedures in clinical data warehouses. A systematic review of literature and publicly available policies. BMC Med Inform Decis Mak 2020 Jul 11;20(1):157 [FREE Full text] [doi: 10.1186/s12911-020-01177-z] [Medline: 32652989] 12. Dhindsa K, Bhandari M, Sonnadara RR. What's holding up the big data revolution in healthcare? BMJ 2018 Dec 28;363:k5357. [doi: 10.1136/bmj.k5357] [Medline: 30593447] 12. Dhindsa K, Bhandari M, Sonnadara RR. What's holding up the big data revolution in healthcare? BMJ 2018 Dec 28;363:k5357. [doi: 10.1136/bmj.k5357] [Medline: 30593447] 13. Hong JC. Strategies to turn real-world data into real-world knowledge. JAMA Netw Open 2021 Oct 01;4(10):e2128045 [FREE Full text] [doi: 10.1001/jamanetworkopen.2021.28045] [Medline: 34618043] 14. Simon GE, Platt R, Watanabe JH, Bindman AB, John London A, Horberg M, et al. When can we rely on real-world evidence to evaluate new medical treatments? Clin Pharmacol Ther 2022 Jan;111(1):30-34 [FREE Full text] [doi: 10.1002/cpt.2253] [Medline: 33895994] 15. Penberthy LT, Rivera DR, Lund JL, Bruno MA, Meyer A. An overview of real-world data sources for oncology and considerations for research. CA Cancer J Clin 2022 May;72(3):287-300 [FREE Full text] [doi: 10.3322/caac.21714] [Medline: 34964981] 16. McVey L, Alvarado N, Greenhalgh J, Elshehaly M, Gale CP, Lake J, et al. Hidden labour: the skilful work of clinical audit data collection and its implications for secondary use of data via integrated health IT. BMC Health Serv Res 2021 Jul 16;21(1):702 [FREE Full text] [doi: 10.1186/s12913-021-06657-0] [Medline: 34271925] 17. Brown JS, Bastarache L, Weiner MG. Aggregating electronic health record data for COVID-19 research-caveat emptor. JAMA Netw Open 2021 Jul 01;4(7):e2117175 [FREE Full text] [doi: 10.1001/jamanetworkopen.2021.17175] [Medline: 34255055] 18. Miksad RA, Abernethy AP. Harnessing the power of real-world evidence (RWE): a checklist to ensure regulatory-grade data quality. Clin Pharmacol Ther 2018 Feb;103(2):202-205 [FREE Full text] [doi: 10.1002/cpt.946] [Medline: 29214638] q y ; ( ) [ ] [ p ] [ ] 19. Kim H, Kim HJ, Kim H, Kim HR, Jo H, Hong J, et al. Real-world data from a refractory triple-negative breast cancer cohort selected using a clinical data warehouse approach. Cancers (Basel) 2021 Nov 21;13(22):5835 [FREE Full text] [doi: 10.3390/cancers13225835] [Medline: 34830990] 19. Kim H, Kim HJ, Kim H, Kim HR, Jo H, Hong J, et al. References 1. Pellerino A, Internò V, Mo F, Franchino F, Soffietti R, Rudà R. Management of brain and leptomeningeal metastases from breast cancer. Int J Mol Sci 2020 Nov 12;21(22):8534 [FREE Full text] [doi: 10.3390/ijms21228534] [Medline: 33198331] J Med Internet Res 2023 | vol. 25 | e43359 | p. 8 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al 23. Che W, Wang Y, Wang X, Lyu J. Association between age and the presence and mortality of breast cancer synchronous brain metastases in the United States: A neglected SEER analysis. Front Public Health 2022;10:1000415 [FREE Full text] [doi: 10.3389/fpubh.2022.1000415] [Medline: 36211679] 24. Nayak L, DeAngelis LM, Brandes AA, Peereboom DM, Galanis E, Lin NU, et al. The Neurologic Assessment in Neuro-Oncology (NANO) scale: a tool to assess neurologic function for integration into the Response Assessment in Neuro-Oncology (RANO) criteria. Neuro Oncol 2017 May 01;19(5):625-635 [FREE Full text] [doi: 10.1093/neuonc/nox029] [Medline: 28453751] 25. Cardoso F, Paluch-Shimon S, Senkus E, Curigliano G, Aapro M, André F, et al. 5th ESO-ESMO international consensus guidelines for advanced breast cancer (ABC 5). Ann Oncol 2020 Dec;31(12):1623-1649 [FREE Full text] [doi: 10.1016/j.annonc.2020.09.010] [Medline: 32979513] j 26. Cardoso F, Kyriakides S, Ohno S, Penault-Llorca F, Poortmans P, Rubio I, et al. Early breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2019 Aug 01;30(8):1194-1220 [FREE Full text] [doi: 10.1093/annonc/mdz173] [Medline: 31161190] 27. Makady A, de Boer A, Hillege H, Klungel O, Goettsch W, (on behalf of GetReal Work Package 1). What Is real-world data? a review of definitions based on literature and stakeholder interviews. Value Health 2017;20(7):858-865 [FREE Full text] [doi: 10.1016/j.jval.2017.03.008] [Medline: 28712614] j j 28. Meystre SM, Lovis C, Bürkle T, Tognola G, Budrionis A, Lehmann CU. Clinical data reuse or secondary use: current status and potential future progress. Yearb Med Inform 2017 Sep 11;26(01):38-52. [doi: 10.15265/iy-2017-007] 29. Campion JT, Craven C, Dorr D, Knosp B. Understanding enterprise data warehouses to support clinical and translational research. J Am Med Inform Assoc 2020 Jul 01;27(9):1352-1358 [FREE Full text] [doi: 10.1093/jamia/ocaa089] [Medline: 32679585] 30. Hersh W, Weiner M, Embi P, Logan J, Payne P, Bernstam E, et al. Caveats for the use of operational electronic health record data in comparative effectiveness research. Med Care 2013 Aug;51(8 Suppl 3):S30-S37 [FREE Full text] [doi: 10.1097/MLR.0b013e31829b1dbd] [Medline: 23774517] 30. Hersh W, Weiner M, Embi P, Logan J, Payne P, Bernstam E, et al. Caveats for the use of operational electronic health record data in comparative effectiveness research. Med Care 2013 Aug;51(8 Suppl 3):S30-S37 [FREE Full text] [doi: 10.1097/MLR.0b013e31829b1dbd] [Medline: 23774517] 31. Voss EA, Makadia R, Matcho A, Ma Q, Knoll C, Schuemie M, et al. Feasibility and utility of applications of the common data model to multiple, disparate observational health databases. JOURNAL OF MEDICAL INTERNET RESEARCH J Am Med Inform Assoc 2015 May;22(3):553-564 [FREE Full text] [doi: 10.1093/jamia/ocu023] [Medline: 25670757] 31. Voss EA, Makadia R, Matcho A, Ma Q, Knoll C, Schuemie M, et al. Feasibility and utility of applications of the common data model to multiple, disparate observational health databases. J Am Med Inform Assoc 2015 May;22(3):553-564 [FREE Full text] [doi: 10.1093/jamia/ocu023] [Medline: 25670757] 32. Murphy SN, Weber G, Mendis M, Gainer V, Chueh HC, Churchill S, et al. Serving the enterprise and beyond with informatics for integrating biology and the bedside (i2b2). J Am Med Inform Assoc 2010;17(2):124-130 [FREE Full text] [doi: 10.1136/jamia.2009.000893] [Medline: 20190053] 32. Murphy SN, Weber G, Mendis M, Gainer V, Chueh HC, Churchill S, et al. Serving the enterprise and beyond with informatics for integrating biology and the bedside (i2b2). J Am Med Inform Assoc 2010;17(2):124-130 [FREE Full text] [doi: 10.1136/jamia.2009.000893] [Medline: 20190053] j ] [ ] 33. Evans RS, Lloyd JF, Pierce LA. Clinical use of an enterprise data warehouse. AMIA Annu Symp Proc 2012;2012:189-198 [FREE Full text] [Medline: 23304288] 33. Evans RS, Lloyd JF, Pierce LA. Clinical use of an enterprise data warehouse. AMIA Annu Symp Proc 2012;2012:189-198 [FREE Full text] [Medline: 23304288] 34. Shin S, Kim WS, Lee J. Characteristics desired in clinical data warehouse for biomedical research. Healthc Inform Res 2014 Apr;20(2):109-116 [FREE Full text] [doi: 10.4258/hir.2014.20.2.109] [Medline: 24872909] 34. Shin S, Kim WS, Lee J. Characteristics desired in clinical data warehouse for biomedical research. Healthc Inform Res 2014 Apr;20(2):109-116 [FREE Full text] [doi: 10.4258/hir.2014.20.2.109] [Medline: 24872909] Abbreviations BCBM: breast cancer brain metastasis BCR: breast cancer registry BM: brain metastasis CDW: clinical data warehouse DARWIN: Data Analytics and Research Window for Integrated Knowledge EHR: electronic health records HER2: human epidermal growth factor receptor 2 HR: hormone receptor PAR: Preliminary Attainability Assessment of Real-world Data RCT: randomized clinical trial RWD: real-world data RWE: real-world evidence SMC BCR: Samsung Medical Center Breast Cancer Registry TNBC: triple-negative breast cancer JOURNAL OF MEDICAL INTERNET RESEARCH Real-world data from a refractory triple-negative breast cancer cohort selected using a clinical data warehouse approach. Cancers (Basel) 2021 Nov 21;13(22):5835 [FREE Full text] [doi: 10.3390/cancers13225835] [Medline: 34830990] 20. Ogrinc G, Davies L, Goodman D, Batalden P, Davidoff F, Stevens D. Squire 2.0 (Standards for Quality Improvement Reporting Excellence): revised publication guidelines from a detailed consensus process. Am J Crit Care 2015 Nov 31;24(6):466-473. [doi: 10.4037/ajcc2015455] [Medline: 26523003] 20. Ogrinc G, Davies L, Goodman D, Batalden P, Davidoff F, Stevens D. Squire 2.0 (Standards for Quality Improvement Reporting Excellence): revised publication guidelines from a detailed consensus process. Am J Crit Care 2015 Nov 31;24(6):466-473. [doi: 10.4037/ajcc2015455] [Medline: 26523003] 21. Kim M, Kizilbash SH, Laramy JK, Gampa G, Parrish KE, Sarkaria JN, et al. Barriers to effective drug treatment for brain metastases: a multifactorial problem in the delivery of precision medicine. Pharm Res 2018 Jul 12;35(9):177 [FREE Full text] [doi: 10.1007/s11095-018-2455-9] [Medline: 30003344] 21. Kim M, Kizilbash SH, Laramy JK, Gampa G, Parrish KE, Sarkaria JN, et al. Barriers to effective drug treatment for brain metastases: a multifactorial problem in the delivery of precision medicine. Pharm Res 2018 Jul 12;35(9):177 [FREE Full text] [doi: 10.1007/s11095-018-2455-9] [Medline: 30003344] 22. Liu X, Fang Y, Li Y, Li Y, Qi L, Wang X. Pertuzumab combined with trastuzumab compared to trastuzumab in the treatment of HER2-positive breast cancer: A systematic review and meta-analysis of randomized controlled trials. Front Oncol 2022;12:894861 [FREE Full text] [doi: 10.3389/fonc.2022.894861] [Medline: 36249045] 22. Liu X, Fang Y, Li Y, Li Y, Qi L, Wang X. Pertuzumab combined with trastuzumab compared to trastuzumab in the treatment of HER2-positive breast cancer: A systematic review and meta-analysis of randomized controlled trials. Front Oncol 2022;12:894861 [FREE Full text] [doi: 10.3389/fonc.2022.894861] [Medline: 36249045] J Med Internet Res 2023 | vol. 25 | e43359 | p. 9 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Please cite as: Kim MJ, Kim HJ, Kang D, Ahn HK, Shin SY, Park S, Cho J, Park YH Preliminary Attainability Assessment of Real-World Data for Answering Major Clinical Research Questions in Breast Cancer Brain Metastasis: Framework Development and Validation Study J Med Internet Res 2023;25:e43359 URL: https://www.jmir.org/2023/1/e43359 doi: 10.2196/43359 PMID: J Med Internet Res 2023 | vol. 25 | e43359 | p. 11 (page number not for citation purposes) Abbreviations J Med Internet Res 2023 | vol. 25 | e43359 | p. 10 (page number not for citation purposes) J Med Internet Res 2023 | vol. 25 | e43359 | p. 10 (page number not for citation purposes) https://www.jmir.org/2023/1/e43359 https://www.jmir.org/2023/1/e43359 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Kim et al Edited by A Mavragani; submitted 10.10.22; peer-reviewed by AS Ibrahim, K Wac, R Bellazzi; comments to author 07. version received 02.01.23; accepted 03.01.23; published 23.03.23 Please cite as: Kim MJ, Kim HJ, Kang D, Ahn HK, Shin SY, Park S, Cho J, Park YH Preliminary Attainability Assessment of Real-World Data for Answering Major Clinical Research Questions in Breast Cancer Brain Metastasis: Framework Development and Validation Study J Med Internet Res 2023;25:e43359 URL: https://www.jmir.org/2023/1/e43359 doi: 10.2196/43359 PMID: ©Min Jeong Kim, Hyo Jung Kim, Danbee Kang, Hee Kyung Ahn, Soo-Yong Shin, Seri Park, Juhee Cho, Yeon Hee Park. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 23.03.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2023/1/e43359 XSL•FO RenderX
https://openalex.org/W2002379724
https://europepmc.org/articles/pmc4274539?pdf=render
English
null
Magnetic structure and Magnetic transport Properties of Graphene Nanoribbons With Sawtooth Zigzag Edges
Scientific reports
2,014
cc-by
6,556
OPEN SUBJECT AREAS: MAGNETIC DEVICES ELECTRONIC DEVICES ELECTRONIC PROPERTIES AND DEVICES D. Wang1, Z. Zhang1, Z. Zhu1 & B. Liang1,2 Received 7 August 2014 Accepted 25 November 2014 Published 23 December 2014 1Institute of Nanomaterial & Nanostructure, Changsha University of Science and Technology, Changsha 410114, China, 2School of Automotive & Mechanical Engineering, Changsha University of Science and Technology, Changsha 410114, China. The magnetic structure and magnetic transport properties of hydrogen-passivated sawtooth zigzag-edge graphene nanoribbons (STGNRs) are investigated theoretically. It is found that all-sized ground-state STGNRs are ferromagnetic and always feature magnetic semiconductor properties, whose spin splitting energy gap Eg changes periodically with the width of STGNRs. More importantly, for the STGNR based device, the dual spin-filtering effect with the perfect (100%) spin polarization and high-performance dual spin diode effect with a rectification ratio about 1010 can be predicted. Particularly, a highly effective spin-valve device is likely to be realized, which displays a giant magnetoresistace (MR) approaching 1010%, which is three orders magnitude higher than the value predicted based on the zigzag graphene nanoribbons and six orders magnitude higher than previously reported experimental values for the MgO tunnel junction. Our findings suggest that STGNRs might hold a significant promise for developing spintronic devices. Correspondence and requests for materials should be addressed to Z.Zg. (lgzzhang@ sohu.com) S pintronic devices (STDs), using spin instead of charge as an information carrier, have recently attracted tremendous attention within the scientific community. Due to unique electronics as well as magneto- electronics properties, such as a weak spin-orbital coupling and long spin correlation length for electrons, which are some key features for developing STDs1–3, the two-dimensional planar graphene and the corresponding quasi-unidimensional graphene nanoribbons (GNRs) have been extensively studied1–7. Especially for zigzag graphene nanoribbons (ZGNRs), one predicts that they will play an important role in spintronic applications. Ground-state ZGNRs exhibit ferromagnetically (FM) ordered states at each edge individually due to the unsat- urated p electron existing for each edge carbon atom but antiferromagnetic (AFM) coupling between opposite edges, leading to a spin-polarized semiconducting behavior with zero net spin4, thus their applications in STDs are severely limited. Heretofore, many effective approaches, such as edge modifications8, doping9, introducing topologic defects10, and applying an external electrical field4 or magnetic field5, have been proposed to break the spin degeneracy and stabilize ferromagnetic (FM) state in ZGNRs, achieving metallic or half- metallic features accordingly. OPEN Based on these ways, some important phenomena are found and promising STDs, such as giant magnetoresistance devices and conductance switchers5, spin-filtering devices6, bipolar spin diodes6,7, spin-polar- ized current amplifiers7, and bipolar field-effect spin-filtering devices11, are designed theoretically. S However, in fact, the realistic applications of the ZGNR magnetism and the above methods to obtain a favorable magnetic ordering might not be feasible. Generally, a very large electrical field is required to split the spin-degenerated band structures and achieve a half-metallicity experimentally. The theoretical predicted mag- netic moment for edge carbon atoms is not very strong12, so that the spin-polarized states of the ZGNRs become unstable to be transformed to the spin-unpolarized state in the presence of ballistic current through the ZGNRs13,14 or at finite temperatures15. It was estimated that the magnetic states can only be stabilized at tem- peratures T , 10K16,17, and become paramagnetic(PM) behaviors as T increases, which has been confirmed experimentally18. And also, as reported in previous works5–7, STD effects based on ZGNRs essentially occur for geometrically symmetrical ZGNRs with respect to the axis due to the intrinsic transmission selection rule of the wave function of spin subbands near the Fermi level, but not for geometrically asymmetrical ZGNRs. Particularly, realizing the specific magnetic device features needs the externally magnetic field simultaneously applied on left and right ZGNR electrodes to create the [1,-1] magnetic configurations5–7, however, it is difficult to limit two SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 1 cycle for calculations and takes into account the interface coupling and screening layer effects. magnetic fields with the opposite direction in two local nano-scale regions, which might exceed those available experimentally19. Additionally, heteroatom doping would influence the mobility and spin correlation length of carriers, even resulting in a spin flip, and edge modification might weaken the geometrical structure stability. Therefore, designing graphene-based systems to have a large ground- state magnetic moment and become experimentally feasible STDs remains a challenge. g y We firstly investigate electronic and magnetic structures of STGNRs with STGNR(5,3) as example. The isosurface plots for the spin polarized density (=r 5 ra 2 rb) of the FM and AFM states are shown in Fig. 2(a) and (b), respectively, where ra and rb denote the electron density of majority spin (a) (red) and minority spin (b) (blue), respectively. For the FM state as shown in Fig. OPEN For the FM state as shown in Fig. 2(a), all the carbon atoms display spin-polarized states, specially, there exists a spin parallel coupling between both edges. While for the AFM state, as shown in Fig. 2(b), STGNR displays FM ordering at each edge carbon atoms individually, but AFM coupling between both edges, similarly to ground-state ZGNRs. Our calculations show that the FM state is a ground state, its total energy is found to be 31 meV per unit cell lower than the AFM state. These results are easy to be under- stood. For graphene, the spin of the A and B sublattices is antiparallel. In a STGNR, the edge carbon atoms always belong to the same sublattice, A sublattice, except for carbon atoms with the armchair bond at corners. The polarization of carbon atoms in the A sublattice, especially at the edges, is much stronger than that in the B sublattice, which is actually due to the unsaturated bond of those atoms, even after they are hydrogenated. Upon the strong interactions between the carbon atoms, the inside atoms are also spin-polarized. For a STGNR (m, n), the number of A-type carbon atoms and B-type z1 In this paper, we present investigations on magnetic structure and magnetic transport properties for the graphene nanoribbons (GNRs) with sawtooth (ST) zigzag edges (STGNRs)20 and passivated by mono- hydrogen atom21, which is related to modified-edge GNRs22,23. It is found that at a FM ground state they are all a magnetic semi- conductor. In particular, unique band overlap pattern for two elec- trodes and its particular sensitivity to a switching magnetic field lead to the dual spin-filtering effect with the perfect (100%) spin polar- ization and high-performance dual spin diode effect with a rectifica- tion ratio about 1010, as well as a highly effective spin-valve device feature with a giant magnetoresistace (GMR) value approaching 1010% can be observed. Results ll Fig. 1 illustrates the schematic structure of STGNRs (m, n), and the rectangle box drawn with a dotted line denotes a unit cell. STGNRs can be view as the armchair graphene nanoribbons (AGNRs) being tailored or introducing defects at their edge to form zigzag edges with a large sawtooth periodically. Current nano-size lithographic tech- niques have provided the possibility for cutting or patterning gra- phene into well-defined geometric structures with atom precision, for example, the controlled formation of sharp zigzag edges in GNRs has been recently fabricated experimentally24. The dangling bond on each edge carbon atom for STGNRs (m, n) is all saturated by single hydrogen (H) atom in order to retain the sp2 hybridization of carbon atoms. Here, two integers, m and n, are used to represent the unit-cell size of STGNRs, which are the number of hexagonal rings along the m and n directions, respectively, as shown in Fig. 1, corresponding to the width of nanoribbon and the length of a large sawtooth at edge. It is important to note that to form a periodic nanoribbon structure, n must be an odd number and equal to or bigger than 3, i.e., n $ 3, and both m and n satisfy m 2 n $ 1 to assure STGNRs being the same width for the whole nanoribbon. Further, we construct device models by the STGNRs to investigate magnetic transport behaviors, where a device is divided into three regions: left electrode, right electrode, and the scattering region (the device region). Each electrode is repre- sented by a unit cell of STGNR along the axis and descried by self- energies, and the scattering region is involved in the self-consistent STGNR (m, n), the number of A type carbon atoms and B type carbon atoms is of unbalanced, they are NA~ nz1 2 (2m{nz2)z (n{1) and NB~ nz1 2 (2m{nz2) per unit cell, respectively. Thus the difference of the number for two types of carbon atoms is NA-NB 5 n-1, independent of m. According to Lieb’s theorem, total net spin magnetization of a graphene structure is M~ 1 2 NA{NB j jgmB, where g < 2 for the electron, mB is the Bohr magneton. Applying this rule, we can obtain that the magnetic moment is 2mB for STGNR(5, 3). OPEN 2(a), all the carbon atoms display spin-polarized states, specially, there exists a spin parallel coupling between both edges. While for the AFM state, as shown in Fig. 2(b), STGNR displays FM ordering at each edge carbon atoms individually, but AFM coupling between both edges, similarly to ground-state ZGNRs. Our calculations show that the FM state is a ground state, its total energy is found to be 31 meV per unit cell lower than the AFM state. These results are easy to be under- stood. For graphene, the spin of the A and B sublattices is antiparallel. In a STGNR, the edge carbon atoms always belong to the same sublattice, A sublattice, except for carbon atoms with the armchair bond at corners. The polarization of carbon atoms in the A sublattice, especially at the edges, is much stronger than that in the B sublattice, which is actually due to the unsaturated bond of those atoms, even after they are hydrogenated. Upon the strong interactions between the carbon atoms, the inside atoms are also spin-polarized. For a STGNR (m, n), the number of A-type carbon atoms and B-type carbon atoms is of unbalanced, they are NA~ nz1 2 (2m{nz2)z (n{1) and NB~ nz1 2 (2m{nz2) per unit cell, respectively. Thus the difference of the number for two types of carbon atoms is NA-NB 5 n-1, independent of m. According to Lieb’s theorem, total net spin magnetization of a graphene structure is M~ 1 2 NA{NB j jgmB, where g < 2 for the electron, mB is the Bohr magneton. Applying this rule, we can obtain that the magnetic moment is 2mB for STGNR(5, 3). Our calculations on Mulliken Population involving the spin polar- ization show that the magnetic moment for STGNR(5, 3) is about 1.85mB per unit cell, which basically agrees with Lieb’s theorem. This slight smaller value is because the interactions between edge carbon atoms and H atoms induce a rearrangement of electron spins of carbon atoms25. h l d b d ( ) h d f ( ) g y We firstly investigate electronic and magnetic structures of STGNRs with STGNR(5,3) as example. The isosurface plots for the spin polarized density (=r 5 ra 2 rb) of the FM and AFM states are shown in Fig. 2(a) and (b), respectively, where ra and rb denote the electron density of majority spin (a) (red) and minority spin (b) (blue), respectively. Results ll This indicates that STGNR(5, 3) features the properties of bipolar magnetic semiconductors (BMS)11 with a well-defined spin splitting energy gap Eg, ,75 meV. The BMS is an important material for realizing bipolar spin-filtering (BSF) devices11. The middle and right panels for the PDOS suggest that the DOS around the Fermi level is mainly derived from p (p) orbital (the s- and d-orbital PDOS is negligible small) of C atoms(at both edges and others). ond-lowest energy state (GS11), and the nonmagnetic state pos- sesses a highest energy (GS12)(not exhibited here). Generally, the thermal stability of materials is regarded to be related to DEmag. As can be seen, DEmag decreases with increasing m, but arises monoton- ically with increasing n, this is easy to be understood. When the value of m increases to form a wider ribbon, the edge magnetic coupling (exchange interaction) is weakened accordingly to display pristine AGNR properties partially. Conversely, increasing n means increas- ing the length of zigzag edges and in turn decreasing their spacing, so that the two-edge magnetic coupling is strengthen unambiguously. The thermal stability is usually quantified by the Curie temperature Tc based on the mean-field theory and Heisenberg model27, Tc 5 2D/ 3kB, whereD is the energy required to flip one spin to the GS11 state. We can obtain D 5 12.24 meV for STGNR (4, 3), leading to Tc , 95 K. It is almost 10 times as large as ZGNRs by the experiment value Tc , 10K16,17, but still much below room temperature. To find the size effects, the band structure and spin splitting energy gap as a function of geometrical parameters, m and n, corresponding to the width of nanoribbon and the length of a large sawtooth at edge for STGNRs, are shown in Fig. 3(a–c). It is clear that the magnetic semiconducting behaviors for all STGNRs are always preserved regardless of the value of m and n. Interestingly, the spin splitting energy gap Eg changes periodically with the value of m by 3 as a period, and satisfies E3iz1 g wE3i g wE3i{1 g (i is a positive integer), simi- larly to the case of AGNRs for the gap alteration with width. With increasing the value of n, the spin splitting energy gap Eg drops at n55 then rises sharply. Results ll Our calculations on Mulliken Population involving the spin polar- ization show that the magnetic moment for STGNR(5, 3) is about 1.85mB per unit cell, which basically agrees with Lieb’s theorem. This slight smaller value is because the interactions between edge carbon atoms and H atoms induce a rearrangement of electron spins of carbon atoms25. The spin-resolved band structure (BS), the density of states (DOS), and the projected density of states (PDOS) for STGNR(5, 3) at the FM ground state are exhibited in Fig. 2 (c). The middle and right panels display the atom-PDOS and orbital-PDOS, respectively. As can be seen, a- and b-spin subbands are prominently split, but no subband (or DOS) crosses (or touches) the Fermi level, and the conduction and valance bands nearest to the Fermi level belong to Figure 1 | The schematic diagram of geometrical structures and the constructed device model for STGNRs(m, n), the dashed box indicates the unit cell, and (m, n) denotes the size of a unit cell. Figure 1 | The schematic diagram of geometrical structures and the constructed device model for STGNRs(m, n), the dashed box indicates the unit cell, and (m, n) denotes the size of a unit cell. of geometrical structures and the constructed device model for STGNRs(m, n), the dashed box indicates the unit cell, nit cell. SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 2 www.nature.com/scientificreports Figure 2 | Electronic and magnetic structures of STGNR(5,3). Isosurface plots of the spin density (=r 5 ra 2 rb) of the FM ground state (a) and AFM state (b) for an optimized H-STGNR(5,3). Values for red (a-spin) and blue (b-spin) isosurfaces are 60.01 |e |/A˚ 3, respectively. (c) The band structure (BS), density of states (DOS), and projected density of states (PDOS) for STGNR(5,3). PDOS includes the atom-PDOS and orbital- PDOS. Figure 2 | Electronic and magnetic structures of STGNR(5,3). Isosurface plots of the spin density (=r 5 ra 2 rb) of the FM ground state (a) and AFM state (b) for an optimized H-STGNR(5,3). Values for red (a-spin) and blue (b-spin) isosurfaces are 60.01 |e |/A˚ 3, respectively. (c) The band structure (BS), density of states (DOS), and projected density of states (PDOS) for STGNR(5,3). PDOS includes the atom-PDOS and orbital- PDOS. b- and a-spin states, respectively. Results ll p To develop future STDs based on STGNRs, it is highly desirable to fully understand their intrinsic magnetic transport properties under bias. The constructed device model is shown in Fig. 1, as stated above. Here, two types of magnetic configurations are considered, respect- ively : (1) P configuration, namely, whole device is taken as a FM ground state, and the spin ordering for two electrodes in parallel points to the same direction. a- and b-spin states are major and minor spin components, respectively. (2) AP configuration, i.e., an externally switching magnetic field is applied perpendicular to the plane of the right electrode to switch its spin ordering antiparallel to the left electrode, namely, the left electrode is still a-spin polarized but the right one is turned to be b-spin polarized, in order to dem- onstrate the response of STGNRs to an application of the magnetic field. Furthermore, the spins across the magnetic domain wall in the scattering region is set in a collinear case for calculations. We still For realistic applications of magnetic materials, the magnetic stability is an important aspect that needs to be considered. As we know, the paramagnetic response of a system is described by the Pauli susceptibility which depends only on the electronic density of states at the Fermi level (Stoner instability)26. STGNRs have almost zero DOS at the Fermi level at the FM ground-state as shown in Figs. 2 (c), thus giving rise to a higher ground-state magnetic stability for STGNRs than that for the FM state of ZGNRs with finite DOS at the Fermi level5. To further investigate a thermal stability, the energy difference DEmag between the AFM and FM states as a function of m and n is drawn in Fig. 3(d). As mentioned above, the FM is the ground state (GS) with a lowest energy, while the AFM is the sec- SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 3 Figure 3 | Size effects for STGNRs(m,n). (a), (b), (c), and (d) show the change of the band structures, spin splitting energy gap, and energy difference DEmag between the AFM state and the FM state with geometrical parameters m and n, respectively Figure 3 | Size effects for STGNRs(m,n). Results ll (a), (b), (c), and (d) show the change of the band structures, spin splitting energy gap, and energy difference DEmag between the AFM state and the FM state with geometrical parameters m and n, respectively Figure 3 | Size effects for STGNRs(m,n). (a), (b), (c), and (d) show the change of the band structures, spin splitting DEmag between the AFM state and the FM state with geometrical parameters m and n, respectively take STGNR(5,3) as example, and for the corresponding device, the spin-resolved I-V characteristics in P and AP configurations are manifested in Figs. 4 (a) and (b), respectively. Obviously, several important features can be visible: (1) In P configuration, the electron tunneling channels for both a- and b-spin states are almost blocked off completely, the negligible small currents (, 5 3 1027 mA)in a region of interest can be observed. (2) In AP configuration, the unidirectional nature of the spin-polarized current shows up, namely, the a-spin channel is only opened under positive bias but suppressed fully under negative bias, while for the b-spin channel, it is just opposite. These mean that the STGNRs can act as a dual spin filter and a dual spin diode in AP configuration. (3) Making a com- parison on currents between P and AP configurations, one can find that there is a much larger current (several mA) in AP configuration than that (, 5 3 1027 mA)in P configuration, which implies that a switching magnetic field features a tremendous tuning effects on the spin transport and thus the giant magnetoresistance (MR) effect can be expected reasonably. the overlap of b- and a- spin bands in both electrodes above and below the Fermi level leads to two transmission peaks, respectively, but they do not contribute to the realistic electron transmission due to this transmission gap occurring around the Fermi level. When this device is negatively (positively) biased, the bands of the left and right electrodes are driven to move downward (upward) and upward (downward), respectively. Until - 0.2 (10.2) V, as shown in Fig. 5(a) ((c)), no spin band overlap appears for the same spin state of two electrodes within the BW, resulting in the BW always lying in an transmission gap. And from then onwards, the trans- mission gap will increases monotonically with bias and the BW remains in between. SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 Results ll Therefore, almost zero current arises in a device for P configuration regardless of the bias polarity and spin compo- nents. But for AP configurations, the situation is greatly changed. The applied switching magnetic field turns the role of a- and b-spin states in the right electrode to be exchanged each other, namely, minor and major spin components for a- and b-spin states, respect- ively. At zero bias, as shown in Fig. 5(e), no spin band overlap can be detected, and thereby no transmission peak occurs. However, when a negative bias is applied, the b-spin band in two electrodes approaches gradually and final overlapping, for example, at -0.2 V, as shown in Fig. 5(d), both of them have already a large overlap in the BW and generate a broaden and high transmission peak, thus a large b-spin current can emerge, while a-spin band in two electrodes goes far away gradually with bias, leading to a neg- ligible small a-spin current. Under positive bias, behaviors for a (b)- spin state are similar to those for b(a)-spin state under negative bias, for example, at 0.2 V, as shown in Fig. 5(f), only a-spin band over- lap occurs in the BW and creates a broaden and high transmission peak, and thus only a large a-spin current under positive bias is p y To understand the origin of distinctive transport behaviors, the relation of the transmission spectrum and electrode band structures under several typical biases, 0.0 and 60.2 V, are displayed in Fig. 5(a) – (f), in which figures (a) – (c) and (d) – (f) correspond the P con- figuration and AP configuration, respectively. In each figure, the left, middle, and right panels show the band structure of the left electrode, the transmission spectrum of the device, and the band structure of the right electrode, respectively. To obtain a spin-dependent current, the band overlap is necessary, namely, the bands for the same spin state in two electrodes form an overlapping region in the bias window (BW) and make the electronic band-to-band tunneling available for carrying current. In P configuration, at zero bias, as shown in Fig. 5(b), SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 4 Figure 4 | The spin-resolved I-V characteristics in P (a) and AP (b) configurations for STGNR(5,3). Results ll As can be seen, an unexpectedly high rectification ratio, up to 1010, can be reached in AP configuration, as shown in Fig. 6(a), which is a forward rectification for a-spin state and an inverse rectification for b-spin state. This rectification ratio is a much larger value as compared to that for a ZGNR diode (,105)6 and macroscopic p-n junction diodes (105 , 107), this means that the STGNR can act as an excellent dual spin diode. And also, the perfect (100%) spin polarization is achieved under a slightly higher bias To quantify the STD characteristics of STGNRs, we define the spin-independent rectification ratio as RRs~ Is(+V) j j Is(+V) j j (s 5 a, b), spin polarization as SPs~ Is Itotal |100% (s 5 a, b), and magne- toresistace as MR~ IAp{Ip Ip |100%. The calculated results are Results ll In P configuration, the electron tunneling channels for both a- and b-spin states are almost blocked off completely, but in AP configuration, the bias-polarity-dependent spin-polarized current shows up. Figure 4 | The spin-resolved I-V characteristics in P (a) and AP (b) configurations for STGNR(5,3). In P configuration, the electron tunneling channels for both a- and b-spin states are almost blocked off completely, but in AP configuration, the bias-polarity-dependent spin-polarized current shows up. derived. Remarkably, these obtained results are entirely consistent with those in Fig. 4. ($0.1 V) in AP configuration regardless of a bias being positive or negative, as shown in Fig. 6(b), serving as a dual spin-filtering device, namely, by the selection of bias polarity, we can obtain an electron flow with different spin directions. More importantly, one can see that, by a switching magnetic field applied on STGNRs, a highly effective spin-valve device is likely to be realized, which displays a giant magnetoresistace (GMR) approaching 1010%, which is three orders magnitude higher than that predicted based on the ZGNRs5 and six orders magnitude higher than previously reported experi- mental values for the MgO tunnel junction28–30. To quantify the STD characteristics of STGNRs, we define the spin-independent rectification ratio as RRs~ Is(+V) j j Is(+V) j j (s 5 a, b), spin polarization as SPs~ Is Itotal |100% (s 5 a, b), and magne- toresistace as MR~ IAp{Ip Ip |100%. The calculated results are shown in Fig. 6. As can be seen, an unexpectedly high rectification ratio, up to 1010, can be reached in AP configuration, as shown in Fig. 6(a), which is a forward rectification for a-spin state and an inverse rectification for b-spin state. This rectification ratio is a much larger value as compared to that for a ZGNR diode (,105)6 and macroscopic p-n junction diodes (105 , 107), this means that the STGNR can act as an excellent dual spin diode. And also, the perfect (100%) spin polarization is achieved under a slightly higher bias To quantify the STD characteristics of STGNRs, we define the spin-independent rectification ratio as RRs~ Is(+V) j j Is(+V) j j (s 5 a, b), spin polarization as SPs~ Is Itotal |100% (s 5 a, b), and magne- toresistace as MR~ IAp{Ip Ip |100%. The calculated results are shown in Fig. 6. Discussion The k-point sampling is 1, 1, and 150 in the x, y, and z directions, respectively, where the z is the period direction of nanoribbon, and the cut off energy is set to 200 Ry. For all models studied, a 15A˚ vacuum slab is used to eliminate interaction between the models, and all calculation was performed after the geometries are optimized until all residual forces on each atom are smaller than 0.05 eV/A˚. Once the convergence in self-consistency calcula- tions is achieved, the spin-polarized current through a device is computed by the Landauer-like formula36. In our calculations, the average Fermi level, an average value of the chemical potential of the left and right electrodes, is set as zero. effect with the perfect (100%) spin polarization and high-perform- ance dual spin diode effect with a rectification ratio about 1010 can be achieved, which is a much larger value as compared to that for a ZGNR diode (,105) and macroscopic p-n junction diodes (105 , 107). Particularly, a highly effective spin-valve device is likely to be realized, which displays a giant magnetoresistace (GMR) approach- ing 1010% that is three orders magnitude higher than the value pre- dicted based on the ZGNRs and six orders magnitude higher than previously reported experimental values for the MgO tunnel junc- tion. These distinctive features can be attributed to their unique band overlap pattern for two electrodes and particular sensitivity to a switching magnetic field. Our findings suggest that STGNRs have a promising performance for developing STDs. 1. Yazyev, O. V. Emergence of magnetism in graphene materials and nanostructures. Rep. Prog. Phys. 73, 056501 (2010). 2. Pesin, D. & MacDonald, A. H. Spintronics and pseudospintronics in graphene and topological insulators. Nat. Mater. 11, 409–416 (2012). 2. Pesin, D. & MacDonald, A. H. Spintronics and pseudospintronics in graphene and topological insulators. Nat. Mater. 11, 409–416 (2012). 3. Dlubak, B. et al. Highly efficient spin transport in epitaxial graphene on SiC. Nat. Phys. 8, 557–561 (2012). 3. Dlubak, B. et al. Highly efficient spin transport in epitaxial graphene on SiC. Nat. Phys. 8, 557–561 (2012). p g p p g As we know, there exist two types of hybridizations, sp2- and sp3- hybridizations, for edge carbon atoms of GNRs. Under a lower hydrogen-concentration, the usual edge structure is generally regarded to be the sp2 hybridized mono-hydrogen termination. Discussion Therefore, mono-hydrogen terminated structures for GNRs are pre- sented and investigated in most of literatures. In this present work, we also research on this type of termination for STGNRs. In fact, edge structure of GNRs is very diversified, such as dihydrogen termina- tion31, edge reconstruction32, Z211 passivated edge32, edge defects32, and other chemical modifications, which might have an impact on magnetism. These complicated cases will be studied in our further works. Additionally, it might be difficult in achieving the required atomic precision for fabricating STGNRs in the current experiments, but theoretical modeling to understand the magnetic structure and magnetic transport properties of ideal STGNRs is very necessary. 4. Young-Woo Son, M. L. C. & Steven, G. Louie. Half-Metallic Graphene Nanoribbons. Nature 444, 347–349 (2006). 5. Kim, W. Y. & Kim, K. S. Prediction of very large values of magnetoresistance in a graphene nanoribbon device. Nat. Nanotechnol. 3, 408–412 (2008). 6. Zeng, J., Chen, K.-Q., He, J., Zhang, X.-J. & Sun, C. Q. Edge Hydrogenation- Induced Spin-Filtering and Rectifying Behaviors in the Graphene Nanoribbon Heterojunctions. J. Phys. Chem.C 115, 25072–25076 (2011). 7. Zeng, M., Shen, L., Zhou, M., Zhang, C. & Feng, Y. Graphene-based bipolar spin diode and spin transistor: Rectification and amplification of spin-polarized current. Phys. Rev. B 83, 115427 (2011). y ( ) 8. Kan, E.-J., Li, Z., Yang, J. & Hou, J. Half-metallicity in edge-modified zigzag graphene nanoribbons. J. Am. Chem. Soc. 130, 4224–4225 (2008). g p 9. Dutta, S., Manna, A. K. & Pati, S. K. Intrinsic half-metallicity in modified graphene nanoribbons. Phys. Rev. Lett. 102, 096601 (2009). 10. Lin, X. & Ni, J. Half-metallicity in graphene nanoribbons with topological line defects. Phys. Rev. B 84, 075461 (2011). 11. Li, X., Wu, X., Li, Z., Yang, J. & Hou, J. Bipolar magnetic semiconductors: a new class of spintronics materials. Nanoscale 4, 5680–5685 (2012). Finally, we would like to point out that exchange -correlation functionals for DFT used in our work might underestimate the band gap of AGNRs compared with other algorithms, such as the GW method33. Therefore, a larger spin splitting energy gap Eg might occur if using other more exact methods for calculations. In our work, the spin polarization and spin diode effect as well as the spin-valve device effect are all closely related to the spin splitting energy gap, which would lead to need a higher threshold voltage to start spintronic device effects. 12. Discussion Spintronics and magnetic device properties of STGNRs are investi- gated theoretically. It is found that at a FM ground state the STGNRs always feature the typical properties of bipolar magnetic semi- conductors. And their Curie temperature Tc is much higher as com- pared with that for ZGNRs. More importantly, the dual spin-filtering Figure 5 | The relation of the transmission spectrum and electrode band structures for STGNR(5,3) under several typical biases, 0.0 and 60.2 V. (a) – (c) For P configuration. (d) – (f) For AP configuration. The region highlighted with green represents the bias window. Figure 5 | The relation of the transmission spectrum and electrode band structures for STGNR(5,3) under several typical biases, 0.0 and 60.2 V. (a) – (c) For P configuration. (d) – (f) For AP configuration. The region highlighted with green represents the bias window. SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 5 Figure 6 | The spin-independent rectification ratio (a) and spin polarization (b) in AP configuration as well as magnetoresistace (c) for STGNR(5,3). The high-performance dual spin diode effect with a rectification ratio about 1010 and the dual spin-filtering effect with the perfect (100%) spin polarization, as well as a giant magnetoresistace (GMR) value approaching 1010%, can be achieved. Figure 6 | The spin-independent rectification ratio (a) and spin polarization (b) in AP configuration as well as magnetoresistace (c) for STGNR(5,3). The high-performance dual spin diode effect with a rectification ratio about 1010 and the dual spin-filtering effect with the perfect (100%) spin polarization, as well as a giant magnetoresistace (GMR) value approaching 1010%, can be achieved. double-zeta plus polarization (DZP) basis for other atoms. The k-point sampling is 1, 1, and 150 in the x, y, and z directions, respectively, where the z is the period direction of nanoribbon, and the cut off energy is set to 200 Ry. For all models studied, a 15A˚ vacuum slab is used to eliminate interaction between the models, and all calculation was performed after the geometries are optimized until all residual forces on each atom are smaller than 0.05 eV/A˚. Once the convergence in self-consistency calcula- tions is achieved, the spin-polarized current through a device is computed by the Landauer-like formula36. In our calculations, the average Fermi level, an average value of the chemical potential of the left and right electrodes, is set as zero. double-zeta plus polarization (DZP) basis for other atoms. Discussion Pisani, L., Chan, J., Montanari, B. & Harrison, N. Electronic structure and magnetic properties of graphitic ribbons. Phys. Rev. B 75, 064418 (2007). 13. Mermin, N. D. & Wagner, H. Absence of ferromagnetism or antiferromagnetism in one-or two-dimensional isotropic Heisenberg models. Phys. Rev. Lett. 17, 1133–1136 (1966). 14. Areshkin, D. A. & White, C. T. Building blocks for integrated graphene circuits. Nano Lett. 7, 3253–3259 (2007). 15. Li, Z., Qian, H., Wu, J., Gu, B.-L. & Duan, W. Role of Symmetry in the Transport Properties of Graphene Nanoribbons under Bias. Phys. Rev. Lett. 100, 206802 (2008). 16. Kunstmann, J., O¨ zdog˘an, C., Quandt, A. & Fehske, H. Stability of edge states and edge magnetism in graphene nanoribbons. Phys. Rev. B 83, 045414 (2011). 17. Yazyev, O. V. & Katsnelson, M. Magnetic correlations at graphene edges: basis for novel spintronics devices. Phys. Rev. Lett. 100, 047209 (2008). 1. Yazyev, O. V. Emergence of magnetism in graphene materials and nanostructures. Rep. Prog. Phys. 73, 056501 (2010). 2. Pesin, D. & MacDonald, A. H. Spintronics and pseudospintronics in graphene and topological insulators. Nat. Mater. 11, 409–416 (2012). Acknowledgments g This work was supported by the National Natural Science Foundation of China (Grant Nos. 61371065, 61101009, 61201080, and 51302022), the Construct Program of the Key Discipline in Hunan Province, Aid Program for Science and Technology Innovative Research Team in Higher Educational Instituions of Hunan Province, and the Scientific Research Innovation Fund for Postgraduate of Changsha University of Science and Technology. ( ) 24. Jia, X. et al. Controlled formation of sharp zigzag and armchair edges in graphitic nanoribbons. Science 323, 1701–1705 (2009). 25. Zhang, J., Zhang, Z., Tang, G., Deng, X. & Fan, Z. Modulating magnetic ordering of the zigzag-edge trigonal graphene by functionalizations. Org. Electron. 15, 1338–1346 (2014). 26. Narozhny, B., Aleiner, I. & Larkin, A. Magnetic fluctuations in two-dimensional metals close to the Stoner instability. Phys. Rev. B 62, 14898 (2000). Author contributions y y 27. Hynninen, T., Raebiger, H. & von Boehm, J. Structural and magnetic properties of (Ga,Mn)N from first principles. Phys. Rev. B 75, 125208 (2007). Device design and theoretical analysis were performed by Z. Zg. Calculations for Electronic and magnetic structures, transmission spectra, and the I-V characteristics were performed mainly by D. W. and Secondarily by Z. Zu and B. L. All the authors discussed the results and wrote the manuscript. 28. Parkin, S. S. et al. Giant tunnelling magnetoresistance at room temperature with MgO (100) tunnel barriers. Nat. Mater. 3, 862–867 (2004). 29. Yuasa, S., Nagahama, T., Fukushima, A., Suzuki, Y. & Ando, K. Giant room- temperature magnetoresistance in single-crystal Fe/MgO/Fe magnetic tunnel junctions. Nat. Mater. 3, 868–871 (2004). Methods The geometric optimization as well as calculations of the electronic structure and transport are performed by using the spin-polarized DFT combined with the non- equilibrium Green’s function(NEGF) method34,35. We employ Troullier-Martins norm-conserving pseudopotentials to represent the atom core and linear combina- tions of local atomic orbitals to expand the valence states of electrons. The spin- dependent generalized gradient approximation (SGGA) is used as the exchange– correlation functional is used as the exchange–correlation functional. The wave function is expanded by a single-zeta plus polarization (SZP) basis for H atoms, and 18. Sepioni, M. et al. Limits on intrinsic magnetism in graphene. Phys. Rev. Lett. 105, 207205 (2010). 19. Saha, K. K., Nikolic´, B. K., Meunier, V., Lu, W. & Bernholc, J. Quantum- interference- controlled three-terminal molecular transistors based on a single ring-shaped molecule connected to graphene nanoribbon electrodes. Phys. Rev. Lett. 105, 236803 (2010). 20. Wang, Z. F., Jin, S. & Liu, F. Spatially separated spin carriers in spin- Semiconducting graphene nanoribbons. Phys. Rev. Lett. 111, 096803 (2013). SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 6 36. Landauer, R. Electrical resistance of disordered one-dimensional lattices. Philos. Mag. 21, 863–867 (1970). 21. Yu, D., Lupton, E. M., Gao, H. J., Zhang, C. & Liu, F. A unified geometric rule for designing nanomagnetism in graphene. Nano Res. 1, 497–501 (2008). g g g g p 22. Girao, E. C., Cruz-Silva, E. & Meunier, V. Electronic transport properties of g g g g p 22. Girao, E. C., Cruz-Silva, E. & Meunier, V. Electronic transport properties of assembled carbon nanoribbons. ACS nano 6, 6483–6491 (2012). 23. Costa Gira˜o, E., Liang, L., Cruz-Silva, E., Filho, A. G. S. & Meunier, V. Emergence of Atypical Properties in Assembled Graphene Nanoribbons. Phys. Rev. Lett. 107, 135501 (2011). SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 Additional information j 30. Chopra, H. D., Sullivan, M. R., Armstrong, J. N. & Hua, S. Z. The quantum spin- valve in cobalt atomic point contacts. Nat. Mater. 4, 832–837 (2005). Competing financial interests: The authors declare no competing financial interests. 31. Sun, Y. Y. et al. Phase diagram of graphene nanoribbons and band-gap bifurcation of Dirac fermions under quantum confinement. Phys. Rev. B 85, 195464 (2002). How to cite this article: Wang, D., Zhang, Z., Zhu, Z. & Liang, B. Magnetic structure and Magnetic transport Properties of Graphene Nanoribbons With Sawtooth Zigzag Edges. Sci. Rep. 4, 7587; DOI:10.1038/srep07587 (2014). 32. Kunstmann, J., Ozdoˇgan, C., Quandt, A. & Fehske, H. Stability of edge states and edge magnetism in graphene nanoribbons. Phys. Rev. B 83, 045414 (2011). 33. Yang, L., Park, C.-H., Son, Y.-W., Cohen, M. L. & Louie, S. G. Quasiparticle energies and band gaps in graphene nanoribbons. Phys. Rev. Lett. 99, 186801 (2007). 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 permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ g energies and band gaps in graphene nanoribbons. Phys. Rev. Lett. 99, 186801 (2007). 34. Brandbyge, M., Mozos, J.-L., Ordejo´n, P., Taylor, J. & Stokbro, K. Density- functional method for nonequilibrium electron transport. Phys. Rev. B 65, 165401 (2002). 35. Pan, J., Zhang, Z., Deng, X., Qiu, M. & Guo, C. The transport properties of D-s-A molecules: A strikingly opposite directional rectification. Appl. Phys. Lett. 98, 013503 (2011). SCIENTIFIC REPORTS | 4 : 7587 | DOI: 10.1038/srep07587 7
https://openalex.org/W2600584577
https://hal.archives-ouvertes.fr/hal-01605881/file/2017_Rigault_Frontiers%20in%20Plant%20Science_1.pdf
English
null
Quantitative Methods to Assess Differential Susceptibility of Arabidopsis thaliana Natural Accessions to Dickeya dadantii
Frontiers in plant science
2,017
cc-by
8,279
Quantitative methods to assess differential susceptibility of arabidopsis thaliana natural accessions to dickeya dadantii Martine Rigault, Amélie Buellet, Céline Masclaux-Daubresse, Mathilde Fagard, Fabien Chardon, Alia Dellagi To cite this version: Martine Rigault, Amélie Buellet, Céline Masclaux-Daubresse, Mathilde Fagard, Fabien Chardon, et al.. Quantitative methods to assess differential susceptibility of arabidopsis thaliana natural accessions to dickeya dadantii. Frontiers in Plant Science, 2017, 8, pp.1-10. ￿10.3389/fpls.2017.00394￿. ￿hal- 01605881￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01605881 https://hal.science/hal-01605881v1 Submitted on 25 May 2020 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. Distributed under a Creative Commons Attribution 4.0 International License ORIGINAL RESEARCH published: 28 March 2017 doi: 10.3389/fpls.2017.00394 Quantitative Methods to Assess Differential Susceptibility of Arabidopsis thaliana Natural Accessions to Dickeya dadantii Martine Rigault, Amélie Buellet, Céline Masclaux-Daubresse, Mathilde Fagard, Fabien Chardon and Alia Dellagi* Institut Jean-Pierre Bourgin, UMR INRA- AgroParisTech 1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France Among the most devastating bacterial diseases of plants, soft rot provoked by Dickeya spp. cause crop yield losses on a large range of species with potato being the most economically important. The use of antibiotics being prohibited in most countries in the field, identifying tolerance genes is expected to be one of the most effective alternate disease control approaches. A prerequisite for the identification of tolerance genes is to develop robust disease quantification methods and to identify tolerant plant genotypes. In this work, we investigate the feasibility of the exploitation of Arabidopsis thaliana natural variation to find tolerant genotypes and to develop robust quantification methods. We compared different quantification methods that score either symptom development or bacterial populations in planta. An easy to set up and reliable bacterial quantification method based on qPCR amplification of bacterial DNA was validated. This study demonstrates that it is possible to conduct a robust phenotyping of soft rot disease, and that Arabidopsis natural accessions are a relevant source of tolerance genes. INTRODUCTION Specialty section: This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science Plants are exposed to biotic and abiotic stresses that lead to important crop yield losses. Considerable advances have been made recently that allow a better understanding of the mechanisms underlying plant–pathogen interactions which determine disease severity at the end. On the one hand, pathogens deploy various virulence systems to invade the plant tissues either by killing cells or by suppressing plant defenses. On the other hand, plants use different defense strategies to counteract this invasion (Dangl et al., 2013). Characterizing the main processes involved in virulence on the pathogen side and involved in defense on the plant side requires a reliable system to score disease severity parameters (Brouwer et al., 2003; Trontin et al., 2011). Indeed, new sequencing technologies and phenotyping open the way to investigate plant genetic determinants of resistance/tolerance to pathogens, but strictly depend on robust quantification methods (Mutka and Bart, 2015). Quantification of disease can rely on different parameters that vary according to the pathogen considered. In most cases, symptom severity is considered as a good indicator of the pathogen’s impact on its host during disease. Another parameter that can be considered, is the in planta pathogen growth or pathogen burden (Brouwer et al., 2003). Received: 05 December 2016 Accepted: 07 March 2017 Published: 28 March 2017 Edited by: Edited by: Richard Berthomé, Centre Toulouse Midi-Pyrénées (INRA), France Reviewed by: Reviewed by: Javier Plasencia, Universidad Nacional Autónoma de México (UNAM), Mexico Oswaldo Valdes-Lopez, Universidad Nacional Autónoma de México (UNAM), Mexico Reviewed by: Javier Plasencia, Universidad Nacional Autónoma de México (UNAM), Mexico Oswaldo Valdes-Lopez, Universidad Nacional Autónoma de México (UNAM), Mexico *Correspondence: Alia Dellagi dellagi@agroparistech.fr *Correspondence: Alia Dellagi dellagi@agroparistech.fr Keywords: natural variation, Arabidopsis thaliana, Dickeya dadantii, quantification, qPCR Keywords: natural variation, Arabidopsis thaliana, Dickeya dadantii, quantification, qPCR Citation: Citation: Rigault M, Buellet A, Masclaux-Daubresse C, Fagard M, Chardon F and Dellagi A (2017) Quantitative Methods to Assess Differential Susceptibility of Arabidopsis thaliana Natural Accessions to Dickeya dadantii. Front. Plant Sci. 8:394. doi: 10.3389/fpls.2017.00394 March 2017 | Volume 8 | Article 394 1 Frontiers in Plant Science | www.frontiersin.org Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. Monitoring the pathogen burden allows a better understanding of the mechanisms controlling disease impact on host. Indeed, plants can be tolerant or resistant to a certain pathogen. Resistance consists of reduced disease severity due to pathogen growth restriction, and relies on specific recognition of pathogen strains via the product of a resistance (R) gene. In the majority of the cases, R genes contain two conserved domains: nucleotide binding domain (NB) and a leucine rich repeat domain (LRR) (St Clair, 2010). Tolerance consists in limiting detrimental effects of disease without a strong reduction of pathogen burden. The underlying mechanisms of tolerance are not specific and mobilize different immunity processes. The outcomes of tolerance or resistance on pathogen populations are different. Resistance can lead to pathogen eradication, but increases the risks of emergence of new hyper-virulent strains. In contrast, tolerance does not reduce pathogen populations and does not favor arms race between hosts and pathogens. Thus, combining quantification of disease severity and pathogen populations in planta is crucial. to set up genetic screens, it is necessary to find differentially susceptible plant genotypes by scoring robust quantitative traits. The use of Arabidopsis as a model plant to accelerate discovery of tolerance or resistance genes in crops proved to be efficient in several instances (Piquerez et al., 2014). The rationale of using the model plant Arabidopsis rather than potato is the availability of genetic tools such as fully sequenced genotypes, mutant libraries allowing a rapid identification of candidate genes. In this work, we investigated the feasibility of the exploitation of natural variation in Arabidopsis thaliana L. (Arabidopsis) to highlight differential susceptibilities by comparing different quantitative traits. In addition, Arabidopsis is a host for D. dadantii displaying a compatible interaction with spreading symptoms (Fagard et al., 2007). In potato, candidate genes involved in tolerance QTLs to Phytophthora infestans were found to be involved in tolerance to the oomycete after expression in Arabidopsis (Pajerowska- Mukhtar et al., 2008) indicating that common mechanisms of tolerance do exist in Arabidopsis and potato. Citation: Bacterial pathogens have the peculiarity of being difficult to control. Indeed, antibiotic use in agriculture is prohibited in most European countries, due to the risk of resistance emergence in the field that could be transferred to clinically important bacteria isolates (Williams-Nguyen et al., 2016). Among the most devastating bacterial diseases, soft rot causes crop yield losses in a large range of species with potato being the most economically important (Czajkowski et al., 2011). Soft rot can be caused by Dickeya species or Pectobacterium species (Toth et al., 2003; Toth et al., 2011). Bacteria survive in water or in plant organs like potato tubers and infect growing plants in the field (Reverchon et al., 2016). Bacteria multiply on the plant surface then enter apoplastic spaces where they multiply. Typical symptoms are maceration and rotting of infected tissues mainly caused by the massive production of plant cell wall degrading enzymes (PCWDE) that lead to tissue disorganization and then death. Cell wall degradation liberates sugars that are used as carbon source for bacterial cells (Reverchon and Nasser, 2013). The process by which Dickeya species infect their hosts corresponds to what is generally described as a necrotrophic lifestyle (van Kan, 2006). Production of these enzymes is tightly controlled at the transcriptional and post transcriptional level in response to environmental factors. Because control of soft rot species is complex, it is a prerequisite to develop a robust and reliable system to score the disease parameters (Czajkowski et al., 2011). For instance, breeding for resistance is hampered by the difficulty to score symptoms and then to correlate this with the bacterial population. The most studied disease quantitative trait in genetic studies is the visible symptom. Another important trait, is the in planta pathogen burden. In planta bacterial population estimation by classical counting of bacterial colonies is tedious and time- consuming. To improve bacterial population estimation in planta, we designed primers to PCR-amplify bacterial DNA from infected tissues. We have determined the correlation between several disease parameters in different plant genotypes harboring different tolerance levels to D. dadantii. Frontiers in Plant Science | www.frontiersin.org Plant and Pathogen Material Plant and Pathogen Material Wild-type accessions of Arabidopsis Bur-0, Can-0, Col-0, Cvi-0, Edi-0, Ge-0, Oy-0 and Sakata were obtained from the Versailles Arabidopsis Stock Center (INRA Versailles France1). Arabidopsis seeds were stratified by incubation for 2 days at 4◦C in 0.01% agarose in water (w/v) in the dark, then, were sown in sand. Homogeneous germination occurred 2 days after sowing. Three times per week, the pots were watered (by immersion of their base) in a mineral solution containing 2 mM nitrate or 10 mM nitrate (Supplementary Table 1). The pH of the watering solutions remained between 5.1 and 5.5 (Lemaitre et al., 2008). During the first 2 weeks, they were watered with the 2 mM nitrate containing solution to avoid intoxication with excess nitrate. The four following weeks, they were watered with a solution containing 10 mM nitrate (Supplementary Table 1). Plants were grown under short days (8-h light/16-h dark) with 21◦C temperature and 70% relative humidity. Six- week-old plants were heavily watered and covered with a transparent plastic 16 h before inoculation. The cover was kept in place throughout the assay to maintain high-humidity conditions. Very few data about plant defense against Dickeya dadantii are available (Reverchon et al., 2016). Accumulation of reactive oxygen species is involved in reducing D. dadantii infection on Saintpaulia ionantha and Arabidopsis (Santos et al., 2001; Reverchon et al., 2002; Fagard et al., 2007). Jasmonic acid is involved in bacterial attraction and defense (Fagard et al., 2007; Antunez-Lamas et al., 2009). Iron homeostasis plays a pivotal role in Arabidopsis defense against D. dadantii (Aznar et al., 2014, 2015; Nam Phuong et al., 2012). In order to gain insight into the plant defense arsenal effective against Dickeya spp. and to identify tolerance genes, genetic screens can be very powerful. To be able Bacterial Strain and Inoculation Method 1http://publiclines.versailles.inra.fr/ Bacterial Strain and Inoculation Method The experiments were performed with the D. dadantii 3937 strain constitutively expressing a gfp fusion and resistant to gentamycin 1http://publiclines.versailles.inra.fr/ March 2017 | Volume 8 | Article 394 2 Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. (Asselbergh et al., 2008; Chapelle et al., 2015). Bacteria were grown in Luria-Bertani medium supplemented with 10 µg/mL gentamycin (Sigma). For plant inoculations, a small hole was made with a needle in the leaf, and then, 5 µL of a bacterial suspension at a density of 1 × 108 Colony Forming Unit/mL (CFU) made up in 50 mM potassium phosphate buffer (pH 7) was spotted on the top of the hole. In each experiment, six plants were inoculated for each genotype and three leaves by plant were inoculated. In order to avoid bias linked to symptom severity, leaves were randomly chosen to be processed by each method of bacterial quantification (CFU counting or qPCR). then DNA was precipitated by adding 300 µL of isopropanol. After centrifuging 15 min at 7000 rpm, the pellet was recovered and washed with 70% ethanol then DNA was dissolved in 30 µL of TE buffer. (Asselbergh et al., 2008; Chapelle et al., 2015). Bacteria were grown in Luria-Bertani medium supplemented with 10 µg/mL gentamycin (Sigma). For plant inoculations, a small hole was made with a needle in the leaf, and then, 5 µL of a bacterial suspension at a density of 1 × 108 Colony Forming Unit/mL (CFU) made up in 50 mM potassium phosphate buffer (pH 7) was spotted on the top of the hole. In each experiment, six plants were inoculated for each genotype and three leaves by plant were inoculated. In order to avoid bias linked to symptom severity, leaves were randomly chosen to be processed by each method of bacterial quantification (CFU counting or qPCR). Symptom Quantification on a Collection of Arabidopsis Natural Accessions Visual symptom scoring is usually performed based on a disease severity scale or based on surface measurement. We compared these two methods for the quantification of visible symptoms caused by D. dadantii on different Arabidopsis accessions. The first method consists in attributing a disease severity index (DSI) to each symptom based on the scale indicated in Figure 1A as previously described (Fagard et al., 2007). The second method consists in measuring the surface of the lesion hereafter designated as “lesion surface” (LS). Surface Measurement Photographs of infected plants were taken for each individual leaf. The surface of the lesions were analyzed using the open source software ImageJ https://imagej.nih.gov/ij/. Quantitative PCR Amplification of Bacterial DNA DNA from D. dadantii bacterial cultures was used as a standard. A standard curve was obtained by a fivefold serial dilution starting from a concentration of 5 ng/µL. Amplifications of DNA from infected leaves was performed on a 1/50th of the purified DNA (see section above). Primers used are: PelA-Forward: 5′-CCGCAACGTCTACATCCAAA-3′; PelA- Reverse: 5′- CGTCGCCTTTTTCGTAATGC-3′; RpoB-Forward: 5′- AATCGAAGGTTCCGGGATTC -3′; RpoB-Reverse: 5′- GCCGTTACGGATATCGATGAG -3′. Two and a half microliters of the 1/50th diluted DNA was subjected to real time qPCR using SYBR Green PCR Mastermix (Eurogentec) and gene-specific primers. Absolute DNA quantities were obtained based on the standard curve of pure bacterial DNA. The DNA content in each leaf was converted into Bacterial cell EqDNA according to Supplementary Table 2 formulas. Count of Bacterial Colonies To monitor in planta viable bacterial populations, infected leaves were individually harvested in 500 µL 0.9% NaCl. After grinding, the suspension was diluted following a 10-fold dilution series up- to 10−6 or 10−7 fold. Ten microliters droplets were then plated out on LB medium containing 10 µg/mL gentamycin following the drop-plate method (Herigstad et al., 2001). For each dilution in the series, two 10-µL droplets were placed on LB plates and incubated at 28◦C for 48 h. CFUs were counted on plated droplets that contained between 4 and 40 colonies and the number of CFU per leaf was calculated. Bacterial DNA Extraction Bacterial DNA was extracted from pure D. dadantii liquid cultures. An overnight LB grown bacterial culture was pelleted by centrifugation. The pellet was suspended in TE(Tris-EDTA) buffer Proteinase K and SDS were added to a final concentration of 100 µg/mL and 0.5%, respectively. The extract was thoroughly mixed and incubated at 37◦C for 1 h, then NaCl was added to a final concentration of 0.83 M. To precipitate cell debris, cetyltrimethylammonium bromide (CTAB)/NaCl (10% CTAB, 0.7 M NaCl) was added to reach the final proportion of 8/70th of the volume, then incubated 10 min at 65◦C. An equal volume of chloroform/isoamylic alcohol was added, followed by mixing and spinning for 5 min. The supernatant was recovered and subjected to a phenol/chloroform/isoamylic alchohol extraction followed by a precipitation in isopropanol. To minimize environmental effects potentially due to soil composition, seedlings were grown on sand and watered with a mineral solution as indicated. Leaves of 6-week-old seedlings were inoculated by making a hole with a needle followed by the application of 5 µL of a bacterial suspension to ensure that each leaf was inoculated with the same number of bacteria. D. dadantii infection causes tissue maceration visible 24–48 h post infection (24–48 h p.i.). The maceration spreads starting from the hole. Symptoms are scored during the 1st days (here 3 days) using the 0–5 DSI scale (Figure 1A). To compare these two procedures, photographs were taken at different time points after inoculation, areas of maceration were measured by computer-assisted analysis of the images. In order to compare the two methods, we chose Arabidopsis accessions displaying different sensitivities to infection based on symptom scoring (data not shown). These accessions are Bur-0 displaying a reduced sensitivity and Cvi-0 displaying a high sensitivity. DNA Extraction from Infected Leaves After harvesting, infected leaves were individually frozen in liquid nitrogen then ground. DNA extraction was performed using the CTAB extraction method. In each tube containing one ground leaf, 400 µL of CTAB buffer (2% cetyltrimethyl-ammonium bromide, 1% polyvinylpyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, 0.2% β-mercapto-ethanol) was added. The samples were then incubated 30 min at 60◦C. Four hundred microliters of chloroform: isoamylic alcohol (24:1) were then added. The samples were centrifuged for 10 min at 7000 rpm in a micro-centrifuge and 300 µL of the supernatant were harvested, The progression curves of LS and DSI indicated that the relative susceptibilities of the two accessions were the same for March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 3 igault et al. Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii FIGURE 1 | Correlation between macerated surface and disease severity on Arabidopsis leaves infected with Dickeya dadantii. Leaves were inoculated with 5 µL of bacterial suspension. (A) Image representing stages of the disease severity index scale. (B) Leaf symptoms were scored according to the scale of 0–5 (Fagard et al., 2007). (C) Photographs were taken at indicated times after inoculation. Diseased areas were measured using the computer program ImageJ. (D) Correlation between DSI and LS 48 h p.i. Experiments were repeated at least three times. Data from one representative experiment are shown. Error bars: standard deviation. Mean were compared using Student’s T-test. n = 10–18. ∗p-value < 0.05, ∗∗p-value < 0.001. Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. FIGURE 1 | Correlation between macerated surface and disease severity on Arabidopsis leaves infected with Dickeya dadantii. Leaves were inoculated with 5 µL of bacterial suspension. (A) Image representing stages of the disease severity index scale. (B) Leaf symptoms were scored according to the scale of 0–5 (Fagard et al., 2007). (C) Photographs were taken at indicated times after inoculation. Diseased areas were measured using the computer program ImageJ. (D) Correlation between DSI and LS 48 h p.i. Experiments were repeated at least three times. Data from one representative experiment are shown. Error bars: standard deviation. Mean were compared using Student’s T-test. n = 10–18. ∗p-value < 0.05, ∗∗p-value < 0.001. quantification methods (DSI and LS). Statistically significant linear correlation was obtained between LS and DSI with a Pearson correlation coefficient of 0.92 (Figure 1D). DNA Extraction from Infected Leaves Thus, DSI and LS provide similar estimations of the Arabidopsis genotypes susceptibility to D. dadantii. both symptom-scoring methods, Bur-0 being the most tolerant, Cvi-0 being the most susceptible. However, significant differences were observed between DSI and LS after 24 and 48 h following infection. Indeed, the DSI progression curve shows a regular increase over 3 days (Figure 1B) while the LS started to diminish in Cvi-0 at 48 h p.i. (Figure 1C). This reduction could be attributed to the fact that severe maceration causes shrinking and folding of the leaf tissue. Thus, at later infection stages, LS may not correctly reflect the severity of symptoms. We concluded that the best time to highlight different sensitivity levels is at 48 h p.i. We determined the degree of correlation between both To determine whether the quantification of disease based on symptom severity can be used to discriminate between Arabidopsis genotypes, we studied D. dadantii infection on a panel of eight accessions chosen for their different origins (data not shown). For this purpose, bacterial infection was performed on the accessions Bur-0, Can-0, Col-0, Cvi-0, Edi-0, Ge-0, and March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 4 Rigault et al. Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii FIGURE 2 | Quantification of disease on a collection of Arabidopsis accessions. Disease severity was quantified by LS on the indicated Arabidopsis genotypes 48 h p.i. Experiment was repeated three times with similar results. Representative data are shown. Means with different letters are significantly different at p < 0.05 as determined using XLSTAT ANOVA Tukey HSD test. FIGURE 2 | Quantification of disease on a collection of Arabidopsis accessions. Disease severity was quantified by LS on the indicated Arabidopsis genotypes 48 h p.i. Experiment was repeated three times with similar results. Representative data are shown. Means with different letters are significantly different at p < 0.05 as determined using XLSTAT ANOVA Tukey HSD test. efficiencies with D. solani genomic DNA were out of acceptable range thus confirming the specificity of our primers (data not shown). Sequence alignments of pelA and rpoB primers with the corresponding genes from D. solani show several mismatches (Supplementary Figure 1). To quantify D. dadantii DNA, a standard curve was obtained using a serial dilution of genomic bacterial DNA from D. dadantii 3937. Bacterial Quantification In planta by qPCR Amplification of Bacterial DNA To perform large scale phenotyping of plants, robust and simple methods are needed. We were interested in phenotyping bacterial burden in planta, which is a complementary trait to the visible symptom. To investigate in planta bacterial populations, serial dilutions of samples followed by plating and colony counting are usually performed (Tornero and Dangl, 2001). However, this method is time-consuming and requires the use of a bacterial strain that harbors a resistance to an antibiotic. We wanted to develop a system to quantify bacteria by qPCR that could be easier to perform on a larger scale. For this purpose, before quantifying unknown bacterial titers, we checked whether DNA amplification allowed us to correctly determine a known bacterial titer in planta. To obtain leaves with a known bacterial titer, Arabidopsis leaves were inoculated with different known amounts of bacteria, then were immediately frozen. Total DNA was extracted as described, and two bacterial genes were amplified (pelA and rpoB) to avoid artifacts related to primer sequence. The pelA gene encodes a pectate lyase present in pectinolytic bacteria (Hugouvieux-Cotte-Pattat et al., 2014). The rpoB gene encodes an RNA polymerase which is a powerful tool used for bacterial identification (Mollet et al., 1997). These primers were used in a previous study (Nam Phuong et al., 2012), and their specificity was checked using as a template DNA of the closely related D. solani (Pedron et al., 2014). Amplification DNA Extraction from Infected Leaves Data on Figure 3 show that bacterial DNA estimated by qPCR is in agreement with the expected DNA in the inoculum (Supplementary Table 2) based on the fact that a D. dadantii bacterial suspension of 0D600 = 0.1 contains approximately 5 × 108 CFU/mL (Antunez-Lamas et al., 2009) and that a bacterial cell contains approximately 5 fg of DNA (Hutchison and Venter, 2006). Bacterial DNA amounts estimated via pelA and rpoB amplification were similar indicating that DNA quantification does not depend on the primers’ sequence. A highly statistically significant correlation was observed between experimentally quantified bacterial DNA via qPCR and theoretically expected bacterial DNA (Figure 3B). These data indicate that our qPCR-based quantification method is a reliable tool to estimate bacterial populations in planta at the initial stage of infection. Bacterial populations estimated by qPCR will be designated hereafter as “Bacterial cell EqDNA.” Oy-0. Disease severity estimated by LS at 48 h p.i. indicates that three statistically distinct groups could be defined. According to the LS criterion, the most tolerant accessions are Oy-0, Edi- 0, and Bur-0. The most susceptible ones are Ge-0, Sakata, and Can-0 (Figure 2). These data indicate that natural accessions of Arabidopsis display different levels of tolerance to D. dadantii suggesting that tolerance associated loci might be found in this plant species. Frontiers in Plant Science | www.frontiersin.org Individual Sample Analyses of Bacterial Population and Symptom Severity As expected, bacterial populations at 0 h p.i., quantified by both methods corresponded to the inoculum, i.e., 25,000 bacteria/leaf (data not shown). Then, at 24 and 48 h. p.i., the bacterial titers in each inoculated leaf were determined, using either the DNA quantification method or the serial dilution method. By this means, we were able to attribute to each infected leaf 3 parameters: (1) DSI, (2) LS, (3) bacterial titer/leaf either estimated via CFU or via Bacterial cell EqDNA. show, an important variability at the early stages of the infection, which may explain why it is quite complex in some cases to evaluate disease severity with this bacterium (Czajkowski et al., 2011). We note that the estimated bacterial populations by qPCR were in general higher than the bacterial populations estimated by counting of CFU although immediately after the inoculations, the initial inocula were identical. One possible explanation for this difference is that qPCR-based estimation of bacterial cells includes a part of the bacterial populations that died during the infection process and thereby over-estimates the amount of bacteria. Conversely, grinding the infected tissue then diluting and counting growing colonies as CFU probably kills a proportion of the bacterial cells and from the plant defense system. y To compare CFU/leaf and Bacterial Cell EqDNA/leaf, it was not technically possible to have two different estimations of the bacterial populations for the same leaf. To circumvent this problem, we compared the CFU/leaf and Bacterial Cell EqDNA/leaf for leaves displaying the same DSI. Indeed, this parameter could be scored for every leaf. To be more precise in our procedure, we divided the symptom stages as follows: Stage 0, Stage 0.5, Stage 1, Stage 1.5, Stage 2, Stage 2.5, Stage 3, Stage 3.5, Stage 4 (In our data, there was no Stage 5 because we used data for the 24 and 48 h p.i. time-points, when the symptoms rarely reach stage 5). Thus, for each stage, we could determine a mean bacterial population quantified as CFU/leaf and a mean bacterial population as Bacterial Cell EqDNA/leaf. This allowed us to check whether there is a correlation between these two parameters. Figure 4C shows that the Pearson correlation coefficient between the two datasets is 0.814 with statistically significant correlation indicating that the two bacterial quantification methods are equivalent. Individual Sample Analyses of Bacterial Population and Symptom Severity Quantifying disease based on symptoms or based on bacterial populations provides different biological information. Indeed, it is conceivable that some plants may harbor a high inoculum without displaying symptoms. For instance, Dickeya species are known to survive in a latent state in plant tissues without causing symptoms (Toth et al., 2003; Czajkowski et al., 2011, 2015). Thus, we investigated possible correlations between bacterial populations and visible symptoms in individual leaves. For this purpose, we quantified bacteria in planta using two different methods. The first method is the classical count March 2017 | Volume 8 | Article 394 5 Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. FIGURE 3 | Bacterial population estimation by qPCR in samples with known bacterial titer. (A) Leaves were inoculated with 5 µL of a bacterial suspension at increasing concentrations. Leaves were immediately frozen and total DNA extracted then qPCR was performed with primers amplifying RPOB or PELA. Bacterial DNA quantities were determined based on a standard curve obtained with pure bacterial DNA. (B) Estimated bacterial DNA was plotted against expected values to check reliability of the quantification method. FIGURE 3 | Bacterial population estimation by qPCR in samples with known bacterial titer. (A) Leaves were inoculated with 5 µL of a bacterial suspension at increasing concentrations. Leaves were immediately frozen and total DNA extracted then qPCR was performed with primers amplifying RPOB or PELA. Bacterial DNA quantities were determined based on a standard curve obtained with pure bacterial DNA. (B) Estimated bacterial DNA was plotted against expected values to check reliability of the quantification method. of bacterial colonies after tissue grinding and serial dilutions followed by plating and counting the CFUs which is widely used in the literature (Tornero and Dangl, 2001; Lebeau et al., 2008; Antunez-Lamas et al., 2009). The second method is based on qPCR quantification of bacterial DNA in inoculated plants. We therefore compared these two methods, using plants at the extremes of the gradient of sensitivity of our panel of differentially susceptible accessions to investigate whether symptom severity is related to bacterial populations when disease progression has occurred (24 and 48 h p.i.). Leaves of Cvi-0 and Bur-0 plants were inoculated with 5 µL of a bacterial suspension at 5 × 108 CFU/mL. Leaves displaying different intensities of DSI and of LS were harvested individually. Frontiers in Plant Science | www.frontiersin.org DISCUSSION Identifying novel genes involved in defense processes against the necrotrophic pathogen D. dadantii is very challenging. Indeed, this bacterium causes major economic losses but very few data are available about potential tolerance genes. Taking advantage of the available novel genomics tools and QTL mapping populations in the model plant Arabidopsis opens new perspectives concerning the identification of tolerance genes to this bacterium. The feasibility of such approaches with D. dadantii is hampered by the lack of availability of reliable disease quantification methods. We addressed this issue by monitoring different disease parameters in a detailed experimental design. This experimental design was aimed at validating and comparing the different quantification methods in order to allow the choice for their use at larger scales (Table 1). We first supported our previously published scoring system by a quantitative method based on measurements of the macerated area. We then showed that quantifying bacterial populations by qPCR in planta is a reliable method to phenotype plant susceptibility to D. dadantii. FIGURE 4 | Correlation between lesion surface and bacterial population estimated by CFU or DNA in Arabidopsis leaves. (A) Bacterial population estimated by CFU was plotted against LS. (B) Bacterial population estimated by qPCR was plotted against LS. (C) Bacterial population estimated by qPCR was plotted against bacterial populations estimated by CFU in each class of symptoms (S0 = Stage 0, S0.5 = Stage 0.5, etc). Experiment was repeated twice with similar results. Representative data are shown. Determined coefficients are indicated (Spearman for A and B, Pearson for C). FIGURE 4 | Correlation between lesion surface and bacterial population estimated by CFU or DNA in Arabidopsis leaves. FIGURE 4 | Correlation between lesion surface and bacterial population estimated by CFU or DNA in Arabidopsis leaves. (A) Bacterial population estimated by CFU was plotted against LS. (B) Bacterial population estimated by qPCR was plotted against LS. (C) Bacterial population estimated by qPCR was plotted against bacterial populations estimated by CFU in each class of symptoms (S0 = Stage 0, S0.5 = Stage 0.5, etc). Experiment was repeated twice with similar results. Representative data are shown. Determined coefficients are indicated (Spearman for A and B, Pearson for C). Our data and others’ shows that the infection process by D. dadantii is quite complex and may result in asymptomatic lesions containing large amounts of bacteria in some cases, and important lesion development with limited bacterial populations in other cases. DISCUSSION This asymptomatic phase led to the proposition that soft rot causing bacteria may be considered as hemi- biotrophs (Kraepiel and Barny, 2016). The asymptomatic phase ends at the onset of pectinase production that is under the control of a complex regulatory network in Dickeya spp. Signaling networks that sense environmental cues are activated via transcription factors including KdgR, PecS, Fur, and PecT responding to pectin fragments, immune signals, iron and temperature, respectively (Reverchon et al., 2016). In D. dadantii, a quorum sensing signal produced via a gene cluster named Vfm; coordinates the production of PCWDE at high bacterial density (Nasser et al., 2013). Notwithstanding the occurrence of asymptomatic infections, we show here that it is possible to obtain reliable estimations of bacterial populations by qPCR that are equivalent to estimations monitored by the classical CFU Individual Sample Analyses of Bacterial Population and Symptom Severity Although CFU and Bacterial cell EqDNA do not derive from the same individuals, this comparison provides an additional indication that both methods are consistent. To determine whether visible symptoms were correlated with bacterial titers, we plotted LS against bacterial titers estimated by CFU or by Bacterial cell EqDNA. Figures 4A,B indicate that there was a statistically significant linear correlation between bacterial populations either estimated as CFU or as Bacterial Cell EqDNA and symptom severity (LS). Interestingly, measured bacterial populations were most variable at the beginning of the infection process, corresponding to LS below 0.2 cm2 (stages 1–2). Furthermore, we observed that in some cases bacterial populations reached higher levels in leaves with low LS compared to leaves with higher LS, indicating that in some cases bacteria can survive in leaves under a latent state. Taken together, our data show that bacterial populations are positively correlated to disease symptoms. However, bacterial populations in planta March 2017 | Volume 8 | Article 394 6 Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. FIGURE 4 | Correlation between lesion surface and bacterial population estimated by CFU or DNA in Arabidopsis leaves. (A) Bacterial population estimated by CFU was plotted against LS. (B) Bacterial population estimated by qPCR was plotted against LS. (C) Bacterial population estimated by qPCR was plotted against bacterial populations estimated by CFU in each class of symptoms (S0 = Stage 0, S0.5 = Stage 0.5, etc). Experiment was repeated twice with similar results. Representative data are shown. Determined coefficients are indicated (Spearman for A and B, Pearson for C). displaying the highest bacterial population estimated by bacterial cell EqDNA, are Ge-0 and Cvi-0 which are also found in the most susceptible group according to the LS criterion. Disease symptoms as LS were plotted against bacterial populations for each accession to further determine the level of correlation between these two criteria. We found that the LS and bacterial DNA were significantly correlated with a correlation coefficient of 0.62 indicating again that there is an overall consistency between bacterial populations and visible disease symptoms although LS is more discriminant in this experiment. Estimation of bacterial populations provides complementary biological information about the mechanisms underlying the interaction. Taken together, these data show that although, this pathosystem presents a difficulty due to the presence of latent bacterial populations during infection, Arabidopsis is a good model to study genetic diversity of tolerance. Quantification of Bacterial Populations on a Collection of Arabidopsis Natural Accessions To assess whether differential symptom severity on the eight accessions analyzed in Figure 2 was associated with a differential pathogen growth, bacterial populations were quantified by qPCR in these accessions. This allowed the definition of two statistically distinct groups (Figure 5). According to the bacterial population estimated by Bacterial cell EqDNA, the most tolerant accessions are Bur-0 and Edi-0 which are also classified as the most tolerant according to the LS criterion (Figure 2). The accessions March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 7 Rigault et al. Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii FIGURE 5 | Quantification of disease and bacterial populations on a collection of Arabidopsis accessions. (A) Bacterial population estimated by qPCR on the indicated Arabidopsis genotypes 48 h p.i. (B) Bacterial population estimated by qPCR was plotted against LS for each genotype. Experiment was repeated at least twice with similar results. Representative data are shown. Means with different letters are significantly different at p < 0.05 as determined using XLSTAT ANOVA Tukey HSD test. FIGURE 5 | Quantification of disease and bacterial populations on a collection of Arabidopsis accessions. (A) Bacterial population estimated by qPCR on the indicated Arabidopsis genotypes 48 h p.i. (B) Bacterial population estimated by qPCR was plotted against LS for each genotype. Experiment was repeated at least twice with similar results. Representative data are shown. Means with different letters are significantly different at p < 0.05 as determined using XLSTAT ANOVA Tukey HSD test. TABLE 1 | Comparative analysis of the different methods to quantify Arabidopsis susceptibility levels against Dickeya dadantii. TABLE 1 | Comparative analysis of the different methods to quantify Arabidopsis susceptibility levels against Dickeya dadantii. Methods to score susceptibility Advantages Drawbacks DSI – Simple and quick - Limited precision LS – Precise – Time consuming – Not informative at later infection stages CFU/leaf – Informative about bacterial titer – Tedious and time consuming – Cheap – Underestimates bacterial populations – Precise – Requires the use of a strain resistant to an antibiotic – Requires immediate processing Bacterial Cell EqDNA/leaf – Informative about bacterial titer – Costly – Simple and quick – Frozen tissue can be processed after sampling – Precise omparative analysis of the different methods to quantify Arabidopsis susceptibility levels against Dickeya dadantii. Quantification of Bacterial Populations on a Collection of Arabidopsis Natural Accessions TABLE 1 | Comparative analysis of the different methods to quantify Arabidopsis susceptibility levels against Dickeya counting, and that this quantification method is a good indicator of the overall plant susceptibility (based on measurement of symptoms). This is consistent with a recent study showing that Pseudomonas sp. quantification in planta by qPCR is counting, and that this quantification method is a good indicator of the overall plant susceptibility (based on measurement of symptoms). This is consistent with a recent study showing that Pseudomonas sp. quantification in planta by qPCR is correlated with counting of bacterial colonies estimated by CFU in previously characterized susceptible Arabidopsis mutants (Ross and Somssich, 2016). In our work, instead of validating the quantification methods on previously characterized susceptible March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 8 Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. mutants, we were able to uncover significantly different susceptibility groups within a collection of Arabidopsis natural accessions uncharacterized previously following D. dadantii infection. be involved in tolerance to bacterial wilt in Medicago truncatula (Vailleau et al., 2007). By the present work, we provide a tool simple to set up in order to phenotype sensitivity of Arabidopsis plant genotypes to the bacterial pathogen D. dadantii. This study demonstrates that it is possible to conduct a robust phenotyping of soft rot disease despite the occurrence of latent bacteria, and that Arabidopsis natural accessions are a relevant source of tolerance genes. Thus, it would be of great interest to use our scoring methods to identify tolerance genes either using QTL mapping in a recombinant segregating population or by performing a genome wide association study. Quantifying DNA as a phenotyping method is a well-known method for fungal pathogens (Klosterman, 2012). Estimation of pathogenic bacteria by qPCR is performed to determine the level of natural contaminations in the field (Czajkowski et al., 2015), but it is not routinely used for pathogenic bacteria in an experimental setting. A previous work showed that it was possible to use qPCR to study bacterial populations in planta using Pseudomonas syringae and Pectobacterium carotovorum as examples (Brouwer et al., 2003), but here we go further to demonstrate the correlation with CFU and disease severity, a prerequisite for the use of this technique in experimental plant pathology. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00394/ full#supplementary-material ACKNOWLEDGMENT We are grateful to C. R. Tinsley for critical reading of the manuscript. AUTHOR CONTRIBUTIONS AD: Designed experiments, analyzed the data, and wrote the paper. MR and AB: performed experiments and analyzed the data. AD, FC, MF, and CM-D: contributed writing the paper. Quantification of Bacterial Populations on a Collection of Arabidopsis Natural Accessions Although quantifying bacterial populations in planta based on DNA includes dead bacteria, we were able to show that this does not hamper the significant correlations between CFU bacterial counting and Bacterial Cell EqDNA. Quantifying bacterial RNA by RT-qPCR may allow the quantification of living bacteria. However, this would require the use of a constitutively expressed bacterial gene in planta, and would require an additional expensive step, which is the reverse transcription. FUNDING This work was supported by the grant INRA NITROPATH BAP2014_63. For most necrotrophs and hemi-biotrophs infecting a large range of hosts, the genetic determinants of plant immunity rely on tolerance rather than specific resistance (Lai and Mengiste, 2013; Roux et al., 2014). Thus, it is likely that the plant immunity against D. dadantii relies on tolerance processes. Because tolerance is a quantitative trait, the availability of powerful quantification methods is crucial. Tolerance is usually controlled by multiple loci and resistance is usually controlled by a single locus encoding a resistance (R) gene (St Clair, 2010). However, some exceptions do exist especially with the example of the bacterial wilt Ralstonia solanacearum (Huet, 2014). Tolerance can be conferred by a single R gene like it is the case for the R gene RRS-1 that confers tolerance in Arabidopsis accession Kil-0 (Van der Linden et al., 2013). Conversely, multiple R genes can REFERENCES (2008). The GacA global regulator is required for the appropriate expression of Erwinia chrysanthemi 3937 pathogenicity genes during plant infection. Environ. Microbiol. 10, 545–559. doi: 10.1111/j.1462-2920.2007. 01473.x Tornero, P., and Dangl, J. L. (2001). A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana. Plant J. 28, 475–481. doi: 10.1046/j.1365-313X.2001.01136.x Toth, I. K., Bell, K. S., Holeva, M. C., and Birch, P. R. J. (2003). Soft rot erwiniae: from genes to genomes. Mol. Plant Pathol. 4, 17–30. doi: 10.1046/j.1364-3703. 2003.00149.x Lemaitre, T., Gaufichon, L., Boutet-Mercey, S., Christ, A., and Masclaux- Daubresse, C. (2008). Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession. Plant Cell Physiol. 49, 1056–1065. doi: 10.1093/pcp/pcn081 Toth, I. K., van der Wolf, J. M., Saddler, G., Lojkowska, E., Helias, V., Pirhonen, M., et al. (2011). Dickeya species: an emerging problem for potato production in Europe. Plant Pathol. 60, 385–399. doi: 10.1111/j.1365-3059.2011.02427.x Trontin, C., Tisne, S., Bach, L., and Loudet, O. (2011). What does Arabidopsis natural variation teach us (and does not teach us) about adaptation in plants? Curr. Opin. Plant Biol. 14, 225–231. doi: 10.1016/j.pbi.2011.03.024 Mollet, C., Drancourt, M., and Raoult, D. (1997). rpoB sequence analysis as a novel basis for bacterial identification. Mol. Microbiol. 26, 1005–1011. doi: 10.1046/j. 1365-2958.1997.6382009.x Mutka, A. M., and Bart, R. S. (2015). Image-based phenotyping of plant disease symptoms. Front. Plant Sci. 5:734. doi: 10.3389/fpls.2014.00734 Vailleau, F., Sartorel, E., Jardinaud, M. F., Chardon, F., Genin, S., Huguet, T., et al. (2007). Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula. Mol. Plant Microbe Interact. 20, 159–167. doi: 10.1094/mpmi-20-20-0159 Nam Phuong, K., Aznar, A., Segond, D., Rigault, M., Simond-Cote, E., Kunz, C., et al. (2012). Iron deficiency affects plant defence responses and confers resistance to Dickeya dadantii and Botrytis cinerea. Mol. Plant Pathol. 13, 816–827. doi: 10.1111/j.1364-3703.2012.00790.x Van der Linden, L., Bredenkamp, J., Naidoo, S., Fouche-Weich, J., Denby, K. J., Genin, S., et al. (2013). Gene-for-gene tolerance to bacterial wilt in Arabidopsis. Mol. Plant Microbe Interact. 26, 398–406. doi: 10.1094/mpmi-07-12-0188-r Nasser, W., Dorel, C., Wawrzyniak, J., Van Gijsegem, F., Groleau, M. C., Deziel, E., et al. (2013). Vfm a new quorum sensing system controls the virulence of Dickeya dadantii. Environ. Microbiol. 15, 865–880. doi: 10.1111/1462-2920. 12049 van Kan, J. A. L. (2006). Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci. 11, 247–253. REFERENCES Dickeya ecology, environment sensing and regulation of virulence programme. Environ. Microbiol. Rep. 5, 622–636. doi: 10.1111/1758-2229.12073 Erwinia chrysanthemi. Mol. Plant Microbe Interact. 20, 794–805. doi: 10.1094/ mpmi-20-7-0794 Herigstad, B., Hamilton, M., and Heersink, J. (2001). How to optimize the drop plate method for enumerating bacteria. J. Microbiol. Methods 44, 121–129. doi: 10.1016/s0167-7012(00)00241-4 Reverchon, S., Rouanet, C., Expert, D., and Nasser, W. (2002). Characterization of indigoidine Biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity. J. Bacteriol. 184, 654–665. doi: 10.1128/JB.184.3.654- 665.2002 Huet, G. (2014). Breeding for resistances to Ralstonia solanacearum. Front. Plant Sci. 5:715. doi: 10.3389/fpls.2014.00715 Ross, A., and Somssich, I. E. (2016). A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae Hugouvieux-Cotte-Pattat, N., Condemine, G., and Shevchik, V. E. (2014). Bacterial pectate lyases, structural and functional diversity. Environ. Microbiol. Rep. 6, 427–440. doi: 10.1111/1758-2229.12166 Ross, A., and Somssich, I. E. (2016). A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana. Plant Methods 12:48. doi: 10.1186/s13007-016-0149-z robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana. Plant Methods 12:48. doi: 10.1186/s13007-016-0149-z Hutchison, C. A., and Venter, J. C. (2006). Single-cell genomics. Nat. Biotechnol. 24, 657–658. doi: 10.1038/nbt0606-657 Roux, F., Voisin, D., Badet, T., Balague, C., Barlet, X., Huard-Chauveau, C., et al. (2014). Resistance to phytopathogens e tutti quanti: placing plant quantitative disease resistance on the map. Mol. Plant Pathol. 15, 427–432. doi: 10.1111/ mpp.12138 Klosterman, S. J. (2012). “Real-time PCR for the quantification of fungi in planta,” in Plant Fungal Pathogens: Methods and Protocols, Vol. 835, eds M. D. Bolton and B. P. H. J. Thomma 121–132. doi: 10.1007/978-1-61779-501-5_8 Santos, R., Franza, T., Laporte, M. L., Sauvage, C., Touati, D., and Expert, D. (2001). Essential role of superoxide dismutase on the pathogenicity of Erwinia chrysanthemi strain 3937. Mol. Plant Microbe Interact. 14, 758–767. doi: 10. 1094/MPMI.2001.14.6.758 Kraepiel, Y., and Barny, M. A. (2016). Gram-negative phytopathogenic bacteria, all hemibiotrophs after all? Mol. Plant Pathol. 17, 313–316. doi: 10.1111/mpp. 12345 St Clair, D. A. (2010). Quantitative disease resistance and quantitative resistance loci in breeding. Annu. Rev. Phytopathol. 48, 247–268. doi: 10.1146/annurev- phyto-080508-081904 Lai, Z. B., and Mengiste, T. (2013). Genetic and cellular mechanisms regulating plant responses to necrotrophic pathogens. Curr. Opin. Plant Biol. 16, 505–512. doi: 10.1016/j.pbi.2013.06.014 Lebeau, A., Reverchon, S., Gaubert, S., Kraepiel, Y., Simond-Cote, E., Nasser, W., et al. REFERENCES fluorescence PCR. FEMS Microbiol. Lett. 228, 241–248. doi: 10.1016/s0378- 1097(03)00759-6 Antunez-Lamas, M., Cabrera-Ordonez, E., Lopez-Solanilla, E., Raposo, R., Trelles- Salazar, O., Rodriguez-Moreno, A., et al. (2009). Role of motility and chemotaxis in the pathogenesis of Dickeya dadantii 3937 (ex Erwinia chrysanthemi 3937). Microbiology 155, 434–442. doi: 10.1099/mic.0.022244-0 Chapelle, E., Alunni, B., Malfatti, P., Solier, L., Pedron, J., Kraepiel, Y., et al. (2015). A straightforward and reliable method for bacterial in planta transcriptomics: application to the Dickeya dadantii/Arabidopsis thaliana pathosystem. Plant J. 82, 352–362. doi: 10.1111/tpj.12812 Czajkowski, R., Perombelon, M. C. M., Jafra, S., Lojkowska, E., Potrykus, M., van der Wolf, J. M., et al. (2015). Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Ann. Appl. Biol. 166, 18–38. doi: 10.1111/aab. 12166 Asselbergh, B., Achuo, A. E., Hofte, M., and Van Gijsegem, F. (2008). Abscisic acid deficiency leads to rapid activation of tomato defence responses upon infection with Erwinia chrysanthemi. Mol. Plant Pathol. 9, 11–24. doi: 10.1111/j.1364- 3703.2007.00437.x Aznar, A., Chen, N. W. G., Rigault, M., Riache, N., Joseph, D., Desmaele, D., et al. (2014). Scavenging iron: a novel mechanism of plant immunity activation by microbial siderophores. Plant Physiol. 164, 2167–2183. doi: 10.1104/pp.113. 233585 Czajkowski, R., Perombelon, M. C. M., van Veen, J. A., and van der Wolf, J. M. (2011). Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant Pathol. 60, 999–1013. doi: 10.1111/j.1365-3059.2011.02470.x Aznar, A., Chen, N. W. G., Thomine, S., and Dellagi, A. (2015). Immunity to plant pathogens and iron homeostasis. Plant Sci. 240, 90–97. doi: 10.1016/j.plantsci. 2015.08.022 Dangl, J. L., Horvath, D. M., and Staskawicz, B. J. (2013). Pivoting the plant immune system from dissection to deployment. Science 341, 746–751. doi: 10.1126/science.1236011 Brouwer, M., Lievens, B., Van Hemelrijck, W., Van den Ackerveken, G., Cammue, B. P. A., and Thomma, B. (2003). Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time Fagard, M., Dellagi, A., Roux, C., Perino, C., Rigault, M., Boucher, V., et al. (2007). Arabidopsis thaliana expresses multiple lines of defense to counterattack March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 9 Natural Variation of Arabidopsis Susceptibility to Dickeya dadantii Rigault et al. Erwinia chrysanthemi. Mol. Plant Microbe Interact. 20, 794–805. doi: 10.1094/ mpmi-20-7-0794 Reverchon, S., and Nasser, W. (2013). Frontiers in Plant Science | www.frontiersin.org REFERENCES doi: 10.1016/j.tplants.2006.03.005 Williams-Nguyen, J., Sallach, J. B., Bartelt-Hunt, S., Boxall, A. B., Durso, L. M., McLain, J. E., et al. (2016). Antibiotics and antibiotic resistance in agroecosystems: state of the science. J. Environ. Qual. 45, 394–406. doi: 10.2134/ jeq2015.07.0336 Pajerowska-Mukhtar, K. M., Mukhtar, M. S., Guex, N., Halim, V. A., Rosahl, S., Somssich, I. E., et al. (2008). Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis. Planta 228, 293–306. doi: 10.1007/s00425-008-0737-x Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Pedron, J., Mondy, S., des Essarts, Y. R., Van Gijsegem, F., and Faure, D. (2014). Genomic and metabolic comparison with Dickeya dadantii 3937 reveals the emerging Dickeya solani potato pathogen to display distinctive metabolic activities and T5SS/T6SS-related toxin repertoire. BMC Genomics 15:283. doi: 10.1186/1471-2164-15-283 Copyright © 2017 Rigault, Buellet, Masclaux-Daubresse, Fagard, Chardon and Dellagi. 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) or licensor 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. Piquerez, S. J. M., Harvey, S. E., Beynon, J. L., and Ntoukakis, V. (2014). Improving crop disease resistance: lessons from research on Arabidopsis and tomato. Front. Plant Sci. 5:671. doi: 10.3389/fpls.2014.00671 Reverchon, S., Muskhelisvili, G., and Nasser, W. (2016). Virulence program of a bacterial plant pathogen: the Dickeya model. Prog. Mol. Biol. Transl. Sci. 142, 51–92. doi: 10.1016/bs.pmbts.2016.05.005 March 2017 | Volume 8 | Article 394 Frontiers in Plant Science | www.frontiersin.org 10
https://openalex.org/W4389339192
https://press.nordicopenaccess.no/index.php/noasp/catalog/download/205/1127/9565
Norwegian
null
PISA 2022. Norske elevers kompetanse i matematikk, naturfag og lesing
null
2,023
cc-by
18,978
Matematikk I PISA 2022 presterer norske elever i gjennomsnitt lavere i matematikk enn de har gjort noen gang tidligere i PISA-undersøkelsen. Resultatet er på samme nivå som OECD-gjennomsnittet, og det var det også forrige gang matematikk var hovedområde, i 2012. Både i 2018 og 2015 presterte norske elever over OECD-gjennomsnittet. ISBN: 978-82-02-83047-2 ISBN: 978-82-02-83047-2 ISBN PDF: 978-82-02-83046-5 ISBN EPUB: 978-82-02-83283-4 ISBN HTML: 978-82-02-83284-1 ISBN XML: 978-82-02-83285-8 DOI: https://doi.org/10.23865/noasp.205 ISBN PDF: 978-82-02-83046-5 ISBN EPUB: 978-82-02-83283-4 ISBN HTML: 978-82-02-83284-1 ISBN XML: 978-82-02-83285-8 DOI: https://doi.org/10.23865/noasp.205 ISBN PDF: 978-82-02-83046-5 ISBN EPUB: 978-82-02-83283-4 ISBN HTML: 978-82-02-83284-1 ISBN XML: 978-82-02-83285-8 DOI: https://doi.org/10.23865/noasp.205 DOI: https://doi.org/10.23865/noasp.205 ©Fredrik Jensen, Andreas Pettersen, Tove Stjern Frønes, Anna Eriksen, Maria Løvgren og Eva K. Narvhus Formgiving: Cappelen Damm AS Foto: Side 3: skif - stock.adobe.com, side 7: Getty Images, side 8: Getty Images/Maskot, side 22: Getty Images/iStockphoto, side 24: Andrey Popov – stock.adobe.com, side 30: Getty Images Bilder er ikke omfattet av rapportens lisens og kan ikke gjenbrukes Trykk: AIT Grafisk AS Font: Minion Pro 9,5/13 pkt. Formgiving: Cappelen Damm AS Materialet i denne publikasjonen er utgitt som Open Access / åpen tilgang og er omfattet av åndsverkslovens bestemmelser og Creative Commons-lisens CC-BY 4.0 Dette verket omfattes av bestemmelsene i Lov om opphavsretten til åndsverk m.v. av 1961. Verket utgis Open Access under betingelsene i Creative Commons-lisensen CC-BY 4.0. Denne tillater tredjepart å kopiere, distribuere og spre verket i hvilket som helst medium eller format, og å remixe, endre, og bygge videre på materialet til et hvilket som helst formål, inkludert kommersielle, under betingelse av at korrekt kreditering og en lenke til lisensen er oppgitt, og at man indikerer om endringer er blitt gjort. Tredjepart kan gjøre dette på enhver rimelig måte, men uten at det kan forstås slik at lisensgiver bifaller tredjepart eller tredjeparts bruk av verket. Lisensvilkår: http://creativecommons.org/licenses/by/4.0/ Vil du sitere denne kortrapporten? Jensen, F., Pettersen, A., Frønes, T. S., Eriksen, A., Løvgren, M. & Narvhus, E. K. (2023). PISA 2022. Norske elevers kompetanse i matematikk, naturfag og lesing. Cappelen Damm Akademisk. https://doi.org/10.23865/noasp.205 Vil du sitere denne kortrapporten? Jensen, F., Pettersen, A., Frønes, T. S., Eriksen, A., Løvgren, M. & Narvhus, E. K. (2023). g PISA 2022. Norske elevers kompetanse i matematikk, naturfag og lesing. Cappelen Damm Akadem https://doi.org/10.23865/noasp.205 Betydelig flere lavtpresterende elever I PISA 2022 er det en langt større andel norske elever som presterer på lavt nivå i alle de tre fagområdene enn ved tidligere undersøkelser. Fra 2018 til 2022 har andelen lavtpresterende elever økt fra 19 til 31 prosent i matematikk, fra 21 til 28 prosent i naturfag og fra 19 til 27 prosent i lesing. Pandemi siden forrige PISA-måling i PISA-undersøkelsen i Norge. Prestasjonene er på samme nivå som OECD-gjennomsnittet. Til sammenlikning presterte norske elever over OECD-gjennomsnittet i 2018 og 2009, som var de to forrige gangene lesing var hovedområde. Elevene som deltok i PISA-undersøkelsen 2022, gikk på ungdoms­ skolen i en periode preget av pandemirestriksjoner. Vi antar at pandemien har hatt betydning for flere av endringene i resultatene fra PISA 2018 til 2022. Samtidig kan vi ikke si sikkert hvor store endringer som kan tilskrives pandemien og hvor mye som skyldes andre faktorer. De fleste opplever tilhørighet til skolen og god arbeidsro De fleste opplever tilhørighet til skolen og god arbeidsro Andelen elever som opplever tilhørighet til skolen, er omtrent like stor i PISA 2022 som i 2015 og 2018. Norske elever rapporterer i større grad om et godt forhold til skolen enn elever i andre nordiske land og OECD. På samme måte oppgir de fleste norske elever at matematikk­ timene er preget av arbeidsro, men en del elever opplever at de blir distrahert av egen eller andres bruk av digitale ressurser i disse timene. Større forskjeller mellom elevgrupper Som i tidligere undersøkelser er elevenes hjemmebakgrunn av stor betydning for prestasjonene. Men som tidligere betyr hjemmebakgrunn mindre i Norge enn i de fleste andre land. Samtidig ser vi i et lengre perspektiv, fra PISA 2012, at det er økende prestasjonsforskjeller mellom elever med høy og lav sosioøkonomisk status (SØS). Den generelle faglige tilbakegangen for de lavtpresterende elevene, og at de er mange flere enn før, er også en bekymring for likeverdig opplæringstilbud. Det samme er de fortsatt store kjønnsforskjellene i leseprestasjoner, samt prestasjonsforskjellen mellom minoritets- og majoritetselevene. Sammendrag Pandemi siden forrige PISA-måling Naturfag I PISA 2022 er det norske gjennomsnittsresultatet i naturfag under OECD-gjennomsnittet og på samme lave nivå som i 2006. Til sammen­ likning presterte norske elever på OECD-gjennomsnittet både i PISA 2018 og forrige gang naturfag var hovedområde, i 2015. Tilbakegang i prestasjoner i alle tre fagområder De norske resultatene for PISA 2022 viser en tilbakegang i prestasjoner i alle de tre fagområdene sammenliknet med 2018. Tilbakegangen er stor i matematikk (33 poeng) og lesing (23 poeng), og noe mindre i naturfag (12 poeng). OECD-gjennomsnittet har også gått ned i alle fagområdene, men det er stor variasjon mellom landene. Lesing I PISA 2022 er de norske elevenes gjennomsnittlige prestasjoner i lesing på samme nivå som i 2006 – det laveste nivået som er målt for lesing PISA 2022 1 Matematikk PISAs rammeverk for matematikk definerer matematisk kompe­ tanse (mathematical literacy) som den enkeltes evne til å resonnere matematisk og å formulere, bruke og tolke matematikk for å løse problemer i mange forskjellige virkelighetsnære situasjoner (OECD, 2023a). Oppgavene legger opp til at elevene skal bruke kunnskaper og ferdigheter i matematikk til å løse problemer innenfor person­ lige, yrkesrelaterte og samfunnsmessige kontekster. Dette er knyttet til problemløsing og matematisk modellering, som alltid har vært en sentral del av matematikk i PISA. En viktig endring i PISA 2022 Om PISA-undersøkelsen 2022 PISA (Programme for International Student Assessment) er en internasjonal undersøkelse av 15-åringers kompetanse i matematikk, naturfag og lesing. I undersøkelsen svarer elevene på oppgaver innenfor de tre fagområdene samt et spørreskjema med spørsmål om hjemmebakgrunn, interesser, holdninger og hvilke meninger de har om undervisningen og læringsmiljøet ved skolen de går på. er at matematisk resonnering har fått en mer framtredende rolle enn tidligere. Matematisk resonnering handler blant annet om å trekke slutninger, både induktivt og deduktivt, som er en viktig del av problemløsing og modellering. Men matematisk resonnering er også noe mer enn det å løse problemer, for eksempel inngår også det å vurdere og legge fram argumenter, samt vurdere tolkninger og slutninger knyttet til påstander og matematiske resultater. Dette betyr at matematisk resonnering også er en viktig del av det å tenke kritisk. I PISA 2022 er matematikk hovedområdet i undersøkelsen. Det vil si at prøven og spørreskjemaet inneholdt henholdsvis flere oppgaver og spørsmål knyttet til matematikk enn de andre fagområdene. I 2022 ble PISA-undersøkelsen gjennomført for åttende gang. Under­ søkelsen har vært gjennomført hvert tredje år siden 2000, men ble utsatt fra 2021 til 2022 på grunn av koronapandemien. I PISA 2022 deltok rundt 690 000 elever fra 81 land. I Norge deltok omtrent 8 500 elever fra 260 skoler. PISA-undersøkelsen administreres av Orga­ nisation for Economic Co-operation and Development (OECD). I Norge er det Institutt for lærerutdanning og skoleforskning (ILS) ved Universitetet i Oslo som gjennomfører undersøkelsen på oppdrag fra Utdanningsdirektoratet. Naturfagi Scientific literacy i PISA er evnen og viljen til å engasjere seg i natur­ fagsrelaterte temaer. Dette innebærer følgende tre kompetanser: forklare fenomener på en naturvitenskapelig måte, vurdere og plan­ legge naturvitenskapelige undersøkelser og tolke data og evidens på en naturvitenskapelig måte. Disse kompetansene avhenger av fagkunnskap og holdninger til faget. Rammeverket er detaljert beskrevet av Kjærnsli og Jensen (2016), og kapittelet kan lastes ned fra pisa.no. Dette rammeverket har blitt oppdatert og videre­ utviklet til PISA 2025, som er neste gang naturfag er hovedområde (OECD, 2023b). Elevene som deltar i PISA-undersøkelsen, får to timer til å svare på en faglig prøve som består av oppgaver i matematikk, naturfag og lesing. I PISA 2022 fikk noen elever også oppgaver i økonomiforståelse. Oppgavene som inngår i PISA-prøven, er utviklet på bakgrunn av rammeverk for de ulike fagområdene. Rammeverkene er utviklet av internasjonale ekspertgrupper bestående av skolefolk og forskere og beskriver hva slags kompetanse undersøkelsen måler i matematikk, naturfag og lesing. Oppgavene i prøven legger vekt på at elevene skal bruke sin kunnskap og kompetanse for å løse problemer og håndtere utfordringer som hovedsakelig er knyttet til virkelighetsnære situa­ sjoner eller autentiske tekster. Nedenfor gis det en kort beskrivelse av hvilken type kompetanse som måles i de tre fagområdene. Lesing Lesekompetanse (reading literacy) er i PISA definert som at elever skal forstå, bruke, evaluere, reflektere over og engasjere seg i tekster, for å kunne nå sine mål, utvikle sine kunnskaper og evner, og delta i samfunnet (Weyergang & Magnusson, 2020). Å lese med forståelse innebærer tre kognitive prosesser med nær forbindelse: å finne fram til informasjon, å tolke informasjon og å vurdere og reflektere over informasjon. Rammeverket er beskrevet og diskutert i Weyergang og Magnusson (2020) og er åpent tilgjengelig på pisa.no. Hvorfor deltar Norge i PISA-undersøkelsen? Den overordnede hensikten med PISA-undersøkelsen er å vurdere hvor godt elever på slutten av den obligatoriske skolegangen er forberedt på å bruke sine ferdigheter og kunnskaper i de tre fag­områdene i videre studier, arbeidsliv og privatliv. Utformingen av undersøkelsen gjør det mulig å se på endringer i prestasjoner over tid (trend). I tillegg kan resultatene sammenliknes på tvers av land, noe som gir informasjon om styrker og svakheter ved norsk skole. Fra et norsk perspektiv er det særlig interessant å sammenlikne norske resultater mellom år og å se på likheter og forskjeller mellom de nordiske landene. Dataene fra PISA-undersøkelsene blir lagt ut fritt tilgjengelig på OECD sine nettsider og brukes av ulike miljøer til videre forskning. Utvalgsusikkerhet i PISA 2022: merkede land Utvalgsusikkerhet i PISA 2022: merkede land PISA-undersøkelsen gir ikke informasjon om årsakssammenhenger, noe som betyr at det ikke er mulig å trekke konklusjoner om hvilke endringer som skyldes pandemien, og hva som kan forklares med andre forhold. Se side 28 for nærmere beskrivelse. I PISA 2022 var det flere land som opplevde utfordringer med å innfri de tekniske kravene knyttet til deltakelse, delvis på grunn av pandemien. Blant OECD-landene som omtales i denne rapporten, knytter avvikene seg til to ulike fenomener: andelen ekskluderte elever og responsrate (svarprosent) for elever og skoler. Land hvor andelen ekskluderte elever er betydelig høyere enn 5 prosent, eller hvor det har vært en stor økning i eksklusjon siden 2018, er merket med 1 i denne rapporten. Danmark er ett av landene dette gjelder. En del land har ikke oppfylt kriteriene for svarprosent, enten blant skolene eller elevene som er trukket ut, og disse landene er merket med 2. På elevnivå kan dette skyldes at de var fraværende på prøvedagen. Se side 29 for oversikt over hvilke land dette gjelder. I Norge er andelen deltakende skoler og elever innenfor de tekniske kravene, og andelen ekskluderte elever har gått noe ned fra PISA 2018. Hvorfor deltar Norge i PISA-undersøkelsen? Den overordnede hensikten med PISA-undersøkelsen er å vurdere hvor godt elever på slutten av den obligatoriske skolegangen er forberedt på å bruke sine ferdigheter og kunnskaper i de tre fag­områdene i videre studier, arbeidsliv og privatliv. Utformingen av undersøkelsen gjør det mulig å se på endringer i prestasjoner over tid (trend). I tillegg kan resultatene sammenliknes på tvers av land, noe som gir informasjon om styrker og svakheter ved norsk skole. Fra et norsk perspektiv er det særlig interessant å sammenlikne norske resultater mellom år og å se på likheter og forskjeller mellom de nordiske landene. Dataene fra PISA-undersøkelsene blir lagt ut fritt tilgjengelig på OECD sine nettsider og brukes av ulike miljøer til videre forskning. Resultatene fra den faglige prøven sees også i sammenheng med informasjon fra spørreskjemaet. Dette gjør det mulig å for eksempel undersøke i hvilken grad elever med ulik hjemmebakgrunn og forut­ setninger har like muligheter til å lykkes på skolen. Det er også en rekke spørsmål knyttet til læring i fagene, trivsel og andre skolefaktorer. PISA-undersøkelsen er en del av kvalitetsvurderingssystemet. Det betyr at data og resultater fra PISA inngår som en av en rekke informasjonskilder for å vurdere kvaliteten i norsk skole. Økonomiforståelse I PISA 2022 deltok elevene i en tilleggsundersøkelse om økonomi­ forståelse med oppgaver innen blant annet personlig økonomi. Resultatene fra denne undersøkelsen kommer i juni 2024. Personlig økonomi er også vektlagt i LK20, både i det tverrfaglige temaet folke­ helse og livsmestring og i matematikkfaget. 2 PISA 2022 PISA 2022 PISA 2022 Utvalgsusikkerhet i PISA 2022: merkede landl Poengskala i PISA-undersøkelsen Poengskala i PISA-undersøkelsen Resultatene fra PISA rapporteres på en skala hvor gjennomsnitts­ resultatet er satt til 500 poeng, og standardavviket er satt til 100 poeng. Denne skalaen er basert på gjennomsnittsresultatet for OECD-landene første gangen et fag var hovedområdet, det vil si år 2000 for lesing, 2003 for matematikk og 2006 for naturfag. Et standardavvik på 100 betyr at resultatene for to tredjedeler av elevene i OECD-landene var mellom 400 og 600 poeng det året skalaen ble satt. Poengskalaen i PISA gjør det mulig å undersøke endringer i prestasjoner over tid. Tilbakegang i matematikk, naturfag og lesing Resultatene for de norske elevene viser en tilbakegang i prestasjoner fra PISA 2018 til 2022 i alle de tre fagområdene (figur 1). Tilbake­ gangen er størst i matematikk og lesing, men den er også relativt stor i naturfag sammenliknet med tidligere undersøkelser. I matematikk er det norske gjennomsnittsresultatet lavere enn noen gang tidligere, og i naturfag og lesing må vi tilbake til 2006 for å finne et tilsvarende lavt gjennomsnittsresultatet som i 2022. PISA 2022 ble gjennomført rett etter pandemien Resultatene fra den faglige prøven sees også i sammenheng med informasjon fra spørreskjemaet. Dette gjør det mulig å for eksempel undersøke i hvilken grad elever med ulik hjemmebakgrunn og forut­ setninger har like muligheter til å lykkes på skolen. Det er også en rekke spørsmål knyttet til læring i fagene, trivsel og andre skolefaktorer. Resultatene fra denne undersøkelsen baserer seg på data som er samlet inn rett i etterkant av at smitteverntiltakene i Norge ble opphevet og samfunnet åpnet igjen etter koronapandemien. PISA 2022 gir derfor et innblikk i norske 15-åringers kompetanse etter det som for mange elever var to år med svært spesielle omstendigheter og betingelser for læring. Samtidig er det viktig å huske at ikke alle resultater eller endringer siden PISA 2018 nødvendigvis kan forklares med pandemien. PISA-undersøkelsen er en del av kvalitetsvurderingssystemet. Det betyr at data og resultater fra PISA inngår som en av en rekke informasjonskilder for å vurdere kvaliteten i norsk skole. 3 PISA 2022 3 PISA 2022 Prestasjoner i matematikk, naturfag og lesing Her presenterer vi hovedresultatene fra PISA 2022. Resultatene for de norske elevene blir sammenliknet med tidligere år og med de andre nordiske landene. PISA 2022 gir et innblikk i norske elevers kompetanse i matematikk, naturfag og lesing rett etter at samfunnet gjenåpnet etter covid-19-pandemien. Tilbakegang i lesing og matematikk for OECD-gjennomsnittet Det er også en tilbakegang for OECD-gjennomsnittet i alle de tre fagområdene. Resultatene fra PISA 2022 viser også et mønster på tvers av land, der tilbakegangen er størst i matematikk og lesing og noe mindre i naturfag. Hovedtrekk ved den norske tilbakegangenl I PISA 2022 er det langt flere norske elever på de laveste mestrings­ nivåene i alle de tre fagområdene enn tidligere. I matematikk presterer nesten en av tre norske elever (31 prosent) under mestringsnivå 2, mens tilsvarende tall er 28 prosent for naturfag og 27 prosent for lesing. Tilbakegangen er noe ulik for fagområdene. For lesing og naturfag skyldes nedgangen først og fremst at elevene med lavest kompetanse presterer lavere enn tidligere. I matematikk er det en tilbakegang i prestasjoner for elever langs hele skalaen. Spredningen har med andre ord økt i lesing og naturfag siden PISA 2018. I PISA 2022 er det langt flere norske elever på de laveste mestrings­ nivåene i alle de tre fagområdene enn tidligere. I matematikk presterer nesten en av tre norske elever (31 prosent) under mestringsnivå 2, mens tilsvarende tall er 28 prosent for naturfag og 27 prosent for lesing. I PISA 2022 presterer de norske elevene på samme nivå som OECD-gjennomsnittet i matematikk og lesing, mens de i naturfag presterer under OECD-gjennomsnittet. Dette er en endring fra PISA 2018, da de norske elevene presterte over OECD-gjennomsnittet i matematikk og lesing, og på samme nivå som OECD-gjennomsnittet i naturfag (se også tabell 13, 14 og 15). Tilbakegangen er noe ulik for fagområdene. For lesing og naturfag skyldes nedgangen først og fremst at elevene med lavest kompetanse presterer lavere enn tidligere. I matematikk er det en tilbakegang i prestasjoner for elever langs hele skalaen. Spredningen har med andre ord økt i lesing og naturfag siden PISA 2018. Figur 1. Norske elevers prestasjoner i matematikk, naturfag og lesing, fra det året de ulike fagene var hovedområde første gang: matematikk i 2003, naturfag i 2006 og lesing i 2000. Usikkerheten rundt hvert målepunkt er omtrent 5 poeng i hver retning. Figur 1. Norske elevers prestasjoner i matematikk, naturfag og lesing, fra det året de ulike fagene var hovedområde første gang: matematikk i 2003, naturfag i 2006 og lesing i 2000. Usikkerheten rundt hvert målepunkt er omtrent 5 poeng i hver retning. 4 PISA 2022 Matematikk Figur 2. Gjennomsnittsresultater i matematikk, naturfag og lesing for norsk elever, fra det året de ulike fagene var hovedområde første gang: matematik 2003, naturfag i 2006 og lesing i 2000. De norske resultatene i PISA 2022 e signifikant forskjellig fra tidligere år, med unntak av naturfag og lesing i 2006 De norske gjennomsnittsresultatene i matematikk i PISA 2022 er 468 poeng, 33 poeng mindre enn i 2018. Til sammenlikning har resultatene i matematikk variert mellom 489 og 502 poeng for norske elever i PISA-undersøkelsene fra 2003 til 2018 (figur 2). Naturfag I naturfag er gjennomsnittsresultatet for de norske elevene 478 poeng, noe som er en tilbakegang på 12 poeng fra PISA 2018. Dette er også en tilbakegang fra tidligere undersøkelser, men likt som i 2006 (figur 2). Tilbakegangen i naturfag er likevel mindre enn tilbakegangen i mate­ matikk og lesing. Tilbakegang i Norden, men store forskjeller mellom landene Også i de andre nordiske landene er det en tilbakegang i prestasjoner i alle de tre fagområdene fra PISA 2018 til 2022, med unntak av naturfagresultatene for svenske og danske elever, som er på samme nivå som i 2018. Sett under ett er det størst tilbakegang for islandske elever. I Norge og Finland er det også en markant tilbakegang i alle fagområdene (se tabell 13, 14 og 15). Både i Danmark, Finland og Sverige presterer elevene over OECD-gjennomsnittet i alle de tre fagområdene i PISA 2022, mens elevene på Island presterer under OECD-gjennomsnittet i alle de tre fagområdene. Lesing I lesing er gjennomsnittsresultatet for de norske elevene 477 poeng. Dette er en tilbakegang på 23 poeng fra PISA 2018, og resultatet er på det laveste nivået i hele perioden fra første gjennomføring i 2000, men likt som i 2006. Fra 2015 til 2022 er tilbakegangen på 37 poeng. Resultatene i lesing har variert mellom 484 og 513 poeng (figur 2). Hvordan forklarer vi disse endringene? Det er ingen tvil om at pandemien har hatt betydning for den til dels store tilbakegangen vi ser på tvers av land. Samtidig vet vi ikke hvor stor del av tilbakegangen blant de norske elevene som skyldes pandemien og begrensningene som fulgte med denne, og hvilke andre faktorer som eventuelt kan ligge bak tilbakegangen. Figur 2. Gjennomsnittsresultater i matematikk, naturfag og lesing for norske elever, fra det året de ulike fagene var hovedområde første gang: matematikk i 2003, naturfag i 2006 og lesing i 2000. De norske resultatene i PISA 2022 er signifikant forskjellig fra tidligere år, med unntak av naturfag og lesing i 2006. 22 5 5 PISA 2022 Matematikk Det er en betydelig tilbakegang i gjennomsnittsresultatene i matematikk for norske elever i PISA 2022 sammenliknet med 2018. Det er det også i de andre nordiske landene og i de aller fleste andre europeiske land. Mens norske elever presterte over OECD-gjennomsnittet i 2018 og 2015, presterer de i 2022 på samme nivå som OECD-gjennomsnittet. I PISA 2022 er det norske gjennomsnittsresultatet i matematikk lavere enn noen gang tidligere. Dette gjelder for alle de nordiske landene, med unntak av Sverige, hvor resultatene i 2022 er på samme nivå som i PISA 2012 (figur 4). Betydelig tilbakegang i Norge, Norden og i OECD-landene Resultatene for PISA 2022 viser en tilbakegang i matematikkpresta­ sjoner i alle de nordiske landene (figur 3). Samtidig viser resultatene at det er relativt store forskjeller i hvor stor denne tilbakegangen er. Størst tilbakegang er det blant islandske og norske elever, der tilbakegangen er markant større enn for gjennomsnittet blant OECD- landene. For danske og svenske elever er tilbakegangen omtrent som gjennomsnittet i OECD. Figur 5. Gjennomsnittsresultater i matematikk i PISA 2022 for de nordiske landene i poeng over og under OECD-gjennomsnittet (472 poeng) som er satt til 0 i figuren. Det norske resultatet er ikke signifikant forskjellige fra OECD-gjennomsnittet, noe som er vist med grå farge på søylen. Figur 3. Endring i gjennomsnittsresultater i matematikk fra PISA 2018 til 2022 for de nordiske landene og OECD. Figur 5. Gjennomsnittsresultater i matematikk i PISA 2022 for de nordiske landene i poeng over og under OECD-gjennomsnittet (472 poeng) som er satt til 0 i figuren. Det norske resultatet er ikke signifikant forskjellige fra OECD-gjennomsnittet, noe som er vist med grå farge på søylen. Figur 3. Endring i gjennomsnittsresultater i matematikk fra PISA 2018 til 2022 for de nordiske landene og OECD. Figur 4. Gjennomsnittsresultater i matematikk for de nordiske landene og OECD i PISA 2003–2022. Norske elever presterer som OECD-gjennomsnittet I PISA 2022 presterer de norske elevene på samme nivå som gjennomsnittet for OECD-landene i matematikk, mens islandske elever presterer under (figur 5). Danske, finske og svenske elever presterer fortsatt over OECD-gjennomsnittet. Til sammenlikning presterte elever i alle de nordiske landene over OECD-gjennomsnittet i PISA 2018. Betydelig tilbakegang i Norge, Norden og i OECD-landene Stor økning i andelen norske elever på lavt mestringsnivå Poengskalaen som PISA-resultatene måles etter, er delt inn i mest­ ringsnivåer, fra nivå 1 til 6. Nivåene beskriver hva som kjennetegner elevenes matematiske kompetanse på ulike steder på skalaen. Elever med resultater på nivå 5 og 6 betegnes som høytpresterende, mens elever under nivå 2 betegnes som lavtpresterende. Nivå 2 vurderes som et minimum for å være godt forberedt til videre utdanning og arbeidsliv. I PISA 2022 presterer nesten én av tre norske elever (31 prosent) under mestringsnivå 2 i matematikk. Dette er en stor økning fra tidligere PISA-undersøkelser, hvor denne andelen ikke har vært over 22 prosent (figur 6). I 2018 var den tilsvarende andelen omtrent én av fem elever (19 prosent). Figur 4. Gjennomsnittsresultater i matematikk for de nordiske landene og OECD i PISA 2003–2022. 6 PIS 6 PISA 2022 Videre viser resultatene fra PISA 2022 at kun 7 prosent av norske elever presterer på de to høyeste mestringsnivåene (figur 8). Dette er en lavere andel enn ved tidligere undersøkelser og nesten en halvering siden PISA 2018. Blant de nordiske landene er andelen elever på høyt mestringsnivå mellom 5 prosent (Island) og 10 prosent (Sverige) i PISA 2022. Figur 6. Prosentandel norske elever som presterer på lavt nivå (under nivå 2) i matematikk. Resultatene for PISA 2022 er signifikant forskjellig fra 2003, 2012 og 2018. Figur 8. Prosentandel norske elever som presterer på høyt nivå (nivå 5 og 6) i matematikk. Resultatene for PISA 2022 er signifikant forskjellig fra 2003, 2012 og 2018. Figur 6. Prosentandel norske elever som presterer på lavt nivå (under nivå 2) i matematikk. Resultatene for PISA 2022 er signifikant forskjellig fra 2003, 2012 og 2018. Andelen lavtpresterende elever i Norge er lik som for gjennomsnittet i OECD-landene, men høyere enn i Danmark, Finland og Sverige (figur 7). Samtidig har det også i de andre nordiske landene vært en økning i andelen lavtpresterende elever siden PISA 2018. Figur 8. Prosentandel norske elever som presterer på høyt nivå (nivå 5 og 6) i matematikk. Resultatene for PISA 2022 er signifikant forskjellig fra 2003, 2012 og 2018. Figur 7. Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i matematikk i PISA 2022. Stor økning i andelen norske elever på lavt mestringsnivå Ved å undersøke spredningen i resultatene i matematikk finner vi at tilbakegangen blant norske elever fra PISA 2018 til 2022 er omtrent like stor blant elever som presterer høyt og lavt (10- og 90-prosentilene har omtrent like stor tilbakegang). Hvis vi sammenlikner resultatene fra 2012 til 2022, finner vi derimot at tilbakegangen er størst blant de lavest presterende elevene, og dermed at spredningen i resultatene har økt i denne perioden. Figur 7. Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i matematikk i PISA 2022. PISA 2022 7 PISA 2022 7 Flere gutter enn jenter både på høyt og lavt nivå Hvis vi sammenlikner spredningen i resultatene og andelen elever på ulike mestringsnivåer, finner vi derimot noen forskjeller i resultatene for norske gutter og jenter. I PISA 2022 er både andelen høytpresterende og lavtpresterende elever høyere blant gutter enn jenter (figur 10). Dette mønsteret finner vi igjen også i spredningen i gjennomsnittsresultatene. Standardavviket for gutter er 100, mens standardavviket for jenter er 87. I PISA 2022 er det ingen kjønnsforskjeller blant norske elever i gjennomsnittsresultatene i matematikk. Det betyr at vi ikke finner kjønnsforskjellen som var til stede i 2018. Også i de andre nordiske landene og for gjennomsnittet i OECD er det relativt små forskjeller i gjennomsnittsresultatene for gutter og jenter (figur 9). Guttene presterer noe bedre enn jentene i Danmark og i OECD-landene sett under ett, mens i Finland presterer jentene noe bedre enn guttene. I PISA 2022 er det ingen kjønnsforskjeller blant norske elever i gjennomsnittsresultatene i matematikk. Det betyr at vi ikke finner kjønnsforskjellen som var til stede i 2018. Også i de andre nordiske landene og for gjennomsnittet i OECD er det relativt små forskjeller i gjennomsnittsresultatene for gutter og jenter (figur 9). Guttene presterer noe bedre enn jentene i Danmark og i OECD-landene sett under ett, mens i Finland presterer jentene noe bedre enn guttene. Figur 9. Forskjell i gjennomsnittsresultat i matematikk mellom jenter og gutter i de nordiske landene og OECD i PISA 2022. Danmark, Finland og OECD-gjennom­ snittet har statistisk signifikante kjønnsforskjeller. Figur 10. Prosentandelen norske gutter og jenter på lavt mestringsnivå (under nivå 2) og høyt mestringsnivå (nivå 5 og 6) i matematikk i PISA 2022. Kjønnsforskjellene er signifikante. Figur 9. Forskjell i gjennomsnittsresultat i matematikk mellom jenter og gutter i de nordiske landene og OECD i PISA 2022. Stor økning i andelen norske elever på lavt mestringsnivå Danmark, Finland og OECD-gjennom­ snittet har statistisk signifikante kjønnsforskjeller. Figur 10. Prosentandelen norske gutter og jenter på lavt mestringsnivå (under nivå 2) og høyt mestringsnivå (nivå 5 og 6) i matematikk i PISA 2022. Kjønnsforskjellene er signifikante. Figur 9. Forskjell i gjennomsnittsresultat i matematikk mellom jenter og gutter i de nordiske landene og OECD i PISA 2022. Danmark, Finland og OECD-gjennom­ snittet har statistisk signifikante kjønnsforskjeller. Figur 10. Prosentandelen norske gutter og jenter på lavt mestringsnivå (under nivå 2) og høyt mestringsnivå (nivå 5 og 6) i matematikk i PISA 2022. Kjønnsforskjellene er signifikante. 8 PISA 2022 PISA 2022 PISA 2022 r enn 20 poeng over OECD and 510 (2,0) its 508 (2,1) erland1/2 493 (3,8) 0 poeng over OECD nd2 492 (2,0) gia 489 (2,2) mark1 489 (1,9) rbritannia2 489 (2,2) en 489 (2,3) errike 487 (2,3) ekkia 487 (2,1) venia 485 (1,2) and 484 (1,9) via1 483 (2,0) rige 482 (2,1) OECD-gjennomsnittet uen 475 (1,8) kland 475 (3,1) nkrike 474 (2,5) nia 473 (1,5) arn 473 (2,5) CD-gjennomsnitt 472 (0,4) tugal 472 (2,4) a 471 (3,1) ge 468 (2,1) 0 poeng under OECD ta 466 (1,6) vakia 464 (2,9) atia 463 (2,4) nd 459 (1,6) kia 453 (1,6) 50 poeng under OECD aina* 441 (4,1) bia 440 (3,0) as 430 (2,3) mania 428 (4,0) r enn 50 poeng under OECD ros 418 (1,2) garia 417 (3,3) dova 414 (2,3) ntenegro 406 (1,1) rgia 390 (2,4) d-Makedonia 389 (0,9) ania 368 (2,1) ovo 355 (1,0) Figur 11. Oversikt over resultater i matematikk for europeiske land som deltok i PISA 2022. Tabellen viser gjennomsnitt resultatene for de europeiske landene som deltok i PISA 2022 med standardfeil i parentes. Intervallene for poenggrensen er valgt med OECD-gjennomsnittet som utgangspunkt. Grensene mellom de øvrige gruppene er ikke statistisk begrunne men er valgt for å gi en relativt jevn fordeling av land og for å illustrere små og store forskjeller fra OECD-gjennomsnitte Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. * I Ukraina ble PISA gjennomført i 18 av 27 regioner. Figur 11. Oversikt over resultater i matematikk for europeiske land som deltok i PISA 2022. Tabellen viser gjennomsnitts­ resultatene for de europeiske landene som deltok i PISA 2022 med standardfeil i parentes. Intervallene for poenggrensene er valgt med OECD-gjennomsnittet som utgangspunkt. Grensene mellom de øvrige gruppene er ikke statistisk begrunnet, men er valgt for å gi en relativt jevn fordeling av land og for å illustrere små og store forskjeller fra OECD-gjennomsnittet. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. * I Ukraina ble PISA gjennomført i 18 av 27 regioner. 9 PISA 2022 Naturfag I PISA 2022 er det norske gjennomsnittsresultatet i naturfag på samme lave nivå som i 2006. Gjennomsnittsresultatene i PISA 2006 var åtte poeng høyere, men ikke signifikant forskjellig fra 2022. PISA 2022 viser en tilbakegang i norske elevers gjennomsnitts­ resultat i naturfag sammenliknet med 2018. Selv om det også er tilbakegang i flere andre land, ser det ut til å være relativt stabile prestasjoner i naturfag internasjonalt. De norske resultatene er preget av at mange flere elever enn tidligere presterer på lavt mestringsnivå. Norske elevers gjennomsnittsresultat i naturfag (478 poeng) er like under OECD-gjennomsnittet på 485 poeng, mens danske og svenske elever presterer over OECD-gjennomsnittet (figur 13). Til tross for en jevn tilbakegang siden 2006, presterer fortsatt finske elever på høyt nivå i naturfag. Finland er det eneste nordiske landet der gjennomsnittsresultatet er over 500 poeng i noen av fagområdene. Islandske elever presterer betydelig under OECD-gjennomsnittet. Tilbakegang i naturfag, men mindre enn i matematikk Det er mindre tilbakegang i prestasjonene i naturfag enn det er for matematikk. Dette gjelder både i Norge og i mange andre land. I Norden er det Island som har størst tilbakegang i naturfag, men det er også en tilbakegang i resultatene i Norge og Finland. I Danmark og Sverige er resultatene på samme nivå som i 2018. For OECD-gjennom­ snittet er det en liten tilbakegang på 4 poeng (figur 12). Mange flere elever på lavt mestringsnivå Resultatene i naturfag er delt inn i seks mestringsnivåer som beskriver i hvilken grad elever behersker ulike kunnskaper og kompetanser i naturfag. Nivå 2 ansees som et minimum for å være godt forberedt til videre utdanning og arbeidsliv, og elever som presterer under dette nivået, betegnes som lavtpresterende elever. I naturfag i PISA 2022 gjelder dette 28 prosent av de norske elevene (figur 14), som er en stor økning i andelen lavtpresterende elever fra alle de tidligere undersøkelsene. 7 prosent av de norske elevene presterer på de høyeste nivåene (nivå 5 og 6), og denne andelen har holdt seg stabil sammenliknet med tidligere år. Figur 12. Endring i gjennomsnittsresultater i naturfag fra PISA 2018 til 2022 for de nordiske landene og OECD. Figur 14. Prosentandel norske elever som presterer på lavt mestringsnivå (under nivå 2) i naturfag. Resultatene for PISA 2022 er signifikant forskjellig fra 2006, 2015 og 2018. Figur 12. Endring i gjennomsnittsresultater i naturfag fra PISA 2018 til 2022 for de nordiske landene og OECD. Figur 13. Gjennomsnittsresultater i naturfag for de nordiske landene og OECD i PISA 2006–2022. Figur 14. Prosentandel norske elever som presterer på lavt mestringsnivå (under nivå 2) i naturfag. Resultatene for PISA 2022 er signifikant forskjellig fra 2006, 2015 og 2018. Andelen norske elever på de laveste nivåene er høyere enn i de fleste andre nordiske landene (figur 15) og blant de høyeste i OECD. Sverige har en lik andel som OECD-gjennomsnittet, mens Danmark og Finland har en lavere andel. Island har den høyeste andelen i Norden, med 36 prosent lavtpresterende elever. Figur 13. Gjennomsnittsresultater i naturfag for de nordiske landene og OECD i PISA 2006–2022. 10 PISA 2022 10 PISA 10 PISA 2022 Figur 15. Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i naturfag i PISA 2022. Figur 16. Forskjell i gjennomsnittsresultat i naturfag mellom gutter og jenter i de nordiske landene og OECD i PISA 2022. Figur 15. Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i naturfag i PISA 2022. Figur 15. Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i naturfag i PISA 2022. Figur 16. Forskjell i gjennomsnittsresultat i naturfag mellom gutter og jenter i de nordiske landene og OECD i PISA 2022. Figur 15. Økende avstand mellom elever Vi kan også undersøke tilbakegangen i prestasjoner ved å se på endringen i resultater for elevene som presterer høyest og lavest på PISA-prøven. Poenggrensen som skiller de 10 prosent lavest presterende elevene fra resten av utvalget, er definert som 10-prosentilen, mens poenggrensen som skiller de 10 prosent høyest presterende elevene fra resten av utvalget, er definert som 90-prosentilen. Resultatene viser at 10-prosentilen gikk ned med 19 poeng fra PISA 2018 (357 poeng) til 2022 (338 poeng). Til sammenlikning var det ingen endring for 90-prosentilen, som vil si at de høyest presterende elevene presterte på samme nivå i 2022 som i 2018. Dette betyr at gapet i prestasjoner mellom de som presterer høyest og lavest, har økt siden 2018. Sammenlikner vi med PISA 2015, finner vi en enda større forskjell i prestasjoner mellom de lavest og høyest presterende elevene. Fra 2015 til 2022 er det en nedgang på 32 poeng for de lavest presterende elevene (10-prosentilen), mens det er en liten, ikke-signifikant nedgang (7 poeng) for de høyest presterende elevene (90-prosentilen). Dette ser vi også hvis vi sammenlikner andelen lavt- og høyt­ presterende gutter og jenter (se figur 17). Mens det er omtrent like mange gutter og jenter som presterer på de øverste mestringsnivåene, er det 7 prosentpoeng flere lavtpresterende gutter enn jenter. Kjønns­ forskjellen i naturfag blant norske elever i PISA 2022 handler altså hovedsakelig om at det er flere lavtpresterende gutter enn jenter. Figur 17. Prosentandelen norske gutter og jenter på lavt mestringsnivå (under nivå 2) og høyt mestringsnivå (nivå 5 og 6) i naturfag i PISA 2022. Kjønnsfor­ skjellene er signifikant forskjellig for lavtpresterende elever, men ikke-signifikant forskjellig for høytpresterende elever. Mange flere elever på lavt mestringsnivå Prosentandel elever i de nordiske landene og OECD som presterer på lavt mestringsnivå (under nivå 2) i naturfag i PISA 2022. Figur 16. Forskjell i gjennomsnittsresultat i naturfag mellom gutter og jenter i de nordiske landene og OECD i PISA 2022. Når det gjelder høytpresterende elever, er denne andelen lik i Norge og Danmark (7 prosent). Andelen høytpresterende elever er størst i Finland (13 prosent) og Sverige (10 prosent), og lavest på Island (2 prosent). Kjønnsforskjellen i naturfag blant norske elever er svært ulikt fordelt utover prestasjonsskalaen. Hvis vi sammenlikner resultatene for de høyest presterende guttene og jentene (90-prosentilen), finner vi ingen signifikante forskjeller. Sammenlikner vi resultatene for de lavest presterende guttene og jentene (10-prosentilen), finner vi derimot at poenggrensen er 29 poeng lavere for guttene. Mens de 10 prosent svakeste jentene presterer fra 354 poeng og ned, er den tilsvarende poenggrensen for de 10 prosent svakeste guttene på 325 poeng. Tilbakegang for elever i Norden og internasjonalt Resultatene for PISA 2022 viser en betydelig tilbakegang i lesepresta­ sjoner i alle de nordiske landene (figur 18). Tilbakegangen i antall poeng er spesielt stor for islandske og finske elever, men også for norske og svenske elever er tilbakegangen stor. Tilbakegangen for de danske elevene er omtrent lik tilbakegangen i hele OECD-området. Figur 18. Endring i gjennomsnittsresultater i lesing fra PISA 2018 til 2022 for de nordiske landene og OECD. Lesing For de norske elevene er resultatene i lesing i PISA 2022 betydelig lavere enn ved alle tidligere gjennomføringer, med unntak av i 2006. Mens Norge i de fleste gjennomføringer har hatt resultater rundt det opprinnelige OECD-gjennomsnittet på 500 poeng, skiller resultatet i PISA 2022 seg ut ved å være på et langt lavere nivå (figur 19). For finske og islandske elever er resultatene i lesing i 2022 også betydelig lavere enn ved alle tidligere PISA-undersøkelser. For svenske elever er resultatene i lesing i 2022 på samme nivå som i 2009 og 2012, men lavere enn i alle de andre tidligere undersøkelsene. I Danmark er det en tilbakegang sammenliknet med 2018 og 2015, men resultatene fra PISA 2022 er på samme nivå som i 2012 og i de tidligere undersøkelsene. I PISA 2022 er gjennomsnittsresultatet for norske elever (477 poeng) på samme nivå som gjennomsnittet for OECD-landene (476 poeng). Finske, danske og svenske elever presterer over OECD-gjennomsnittet, mens islandske elever presterer betydelig under. PISA 2022 viser en stor tilbakegang i norske elevers gjennom­ snittsresultat i lesing sammenliknet med PISA 2018. Det norske gjennomsnittsresultatet er tilbake på samme nivå som i 2006, som er det laveste nivået for hele perioden undersøkelsen har vært gjennomført i Norge. Mens norske elever presterte over OECD-gjennomsnittet i lesing både i 2015 og 2018, er gjennom­ snittsresultatet for norske elever nå på samme nivå som for OECD. PISA 2022 viser en stor tilbakegang i norske elevers gjennom­ snittsresultat i lesing sammenliknet med PISA 2018. Det norske gjennomsnittsresultatet er tilbake på samme nivå som i 2006, som er det laveste nivået for hele perioden undersøkelsen har vært gjennomført i Norge. Mens norske elever presterte over OECD-gjennomsnittet i lesing både i 2015 og 2018, er gjennom­ snittsresultatet for norske elever nå på samme nivå som for OECD. Fortsatt kjønnsforskjeller i jentenes favør Figur 17. Prosentandelen norske gutter og jenter på lavt mestringsnivå (under nivå 2) og høyt mestringsnivå (nivå 5 og 6) i naturfag i PISA 2022. Kjønnsfor­ skjellene er signifikant forskjellig for lavtpresterende elever, men ikke-signifikant forskjellig for høytpresterende elever. Norske jenter presterer i gjennomsnitt 13 poeng bedre enn norske gutter i naturfag i PISA 2022 (figur 16). Det var en tilsvarende kjønns­ forskjell i 2018 (11 poeng), mens i 2015 presterte norske gutter og jenter på samme nivå. Blant de nordiske landene har Finland størst kjønnsforskjell i jentenes favør, mens Danmark er det eneste nordiske landet med kjønnsforskjell i guttenes favør. PISA 2022 11 Økende avstand mellom eleveri Over tid finner vi at tilbakegangen i lesing er mye større blant de lavest presterende enn blant de høyest presterende elevene. Poenggrensen som skiller de 10 prosent lavest presterende elevene fra resten av utvalget (10-prosentilen), har gått ned med 33 poeng fra PISA 2018 (356 poeng) til 2022 (323 poeng). Poenggrensen som skiller de 10 prosent høyest presterende elevene (90-prosentilen), har gått ned med 13 poeng i samme periode. Dette betyr at forskjellene i prestasjoner i lesing mellom de høyest og lavest presterende har økt fra 2018 til 2022. Undervisning i kritisk lesing i norskfaget Undervisning i kritisk lesing i norskfaget I tillegg til store forskjeller i gjennomsnittsresultater er det også langt flere gutter enn jenter som presterer på lavt nivå. I PISA 2022 presterer 34 prosent av de norske guttene under nivå 2, det vil si mer enn hver tredje gutt. Til sammenlikning presterer 20 prosent av norske jenter under nivå 2. Elevene ble spurt om hva læreren ba dem gjøre med tekster i norsk­ faget, og hvor ofte læreren oppfordret til ulike praksiser. Svaralter­ nativene var «I alle timene», «I de fleste timene», «I noen timer» og «Aldri eller nesten aldri». De fleste blir oppmuntret til å ta i bruk kritiske, undersøkende strategier, men bare noe over halvparten oppgir at dette skjer i de fleste eller alle timene de leser tekster (tabell 1). Det er store forskjeller i andelen lavtpresterende elever mellom de nordiske landene. Island har den høyeste andelen (40 prosent), mens Norge (27 prosent) og Sverige (24 prosent) er nær OECD-gjennom­ snittet (26 prosent). Danmark (19 prosent) og Finland (21 prosent) har den laveste andelen lavtpresterende elever i Norden. Tabell 1. Prosentandelen elever som svarer «I alle timene» eller «I de fleste timene» på følgende spørsmål: «Når du leser tekster i norsktimene, hvor ofte blir du oppfordret av læreren til å gjøre det følgende?» Elevers strategier for utforsking og dybdelæring Det er fortsatt store prestasjonsforskjeller i lesing i jentenes favør i alle de nordiske landene (figur 21), og forskjellen er langt større i Norge og Finland enn i de fleste andre land. Elevers strategier for utforsking og dybdelæring I LK20 legges det også vekt på en utforskende tilnærming i lærings­ arbeidet, og elevene ble derfor spurt hvor ofte de møtte skolearbeidet med ulike tilnærminger. Svaralternativene var «Aldri eller nesten aldri», «Omtrent en eller to ganger i året», «Omtrent en eller to ganger i måneden», «Omtrent en eller to ganger i uka» eller «Hver dag eller nesten hver dag». En lav andel elever oppgir at de ulike strategiene er i deres faste repertoar. Kun halvparten stiller regelmessig spørsmål ved teksters meningsinnhold, leter etter evidens som støtter påstander og reflekterer for å danne seg egne meninger (tabell 2). Elevers strategier for utforsking og dybdelæring I LK20 legges det også vekt på en utforskende tilnærming i lærings­ arbeidet, og elevene ble derfor spurt hvor ofte de møtte skolearbeidet med ulike tilnærminger. Svaralternativene var «Aldri eller nesten aldri», «Omtrent en eller to ganger i året», «Omtrent en eller to ganger i måneden», «Omtrent en eller to ganger i uka» eller «Hver dag eller nesten hver dag». En lav andel elever oppgir at de ulike strategiene er i deres faste repertoar. Kun halvparten stiller regelmessig spørsmål ved teksters meningsinnhold, leter etter evidens som støtter påstander og reflekterer for å danne seg egne meninger (tabell 2). Figur 21. Antall poeng forskjell i gjennomsnittsresultat i lesing mellom jenter og gutter i de nordiske landene og OECD i PISA 2022. Tabell 2. Prosentandelen elever som svarer «Omtrent en eller to ganger i uka» eller «Hver dag eller nesten hver dag» på følgende spørsmål: «Hvor ofte gjør du det følgende i skoletimene eller når du gjør lekser?» Figur 21. Antall poeng forskjell i gjennomsnittsresultat i lesing mellom jenter og gutter i de nordiske landene og OECD i PISA 2022. Stor økning i andelen elever på lavt nivå I lesing er mestringsnivå 2 vurdert som en nedre grense for hva elever bør ha av kompetanse for videre skolegang og arbeidsliv. Elever som presterer under dette nivået, betegnes som lavtpresterende. I PISA 2022 presterer mer enn én av fire norske elever under nivå 2 (figur 20). Dette er 8 prosentpoeng flere enn i 2018 og 12 prosentpoeng flere enn i 2009. Andelen høytpresterende elever (nivå 5 og 6) er 9 prosent i 2022, en nedgang fra 11 prosent i 2018 og på nivå med PISA 2009 (8 prosent). Figur 18. Endring i gjennomsnittsresultater i lesing fra PISA 2018 til 2022 for de nordiske landene og OECD. Figur 19. Gjennomsnittsresultater i lesing for de nordiske landene og OECD i PISA 2000–2022. Figur 20. Prosentandel norske elever som presterer på lavt mestringsnivå (under nivå 2) i lesing. Resultatene for PISA 2022 er signifikant forskjellig fra 2000, 2009 og 2018. Figur 20. Prosentandel norske elever som presterer på lavt mestringsnivå (under nivå 2) i lesing. Resultatene for PISA 2022 er signifikant forskjellig fra 2000, 2009 og 2018. Figur 19. Gjennomsnittsresultater i lesing for de nordiske landene og OECD i PISA 2000–2022. Figur 20. Prosentandel norske elever som presterer på lavt mestringsnivå (under nivå 2) i lesing. Resultatene for PISA 2022 er signifikant forskjellig fra 2000, 2009 og 2018. Figur 19. Gjennomsnittsresultater i lesing for de nordiske landene og OECD i PISA 2000–2022. 12 PISA 2022 12 PISA 2022 PISA 2022 Problemløsing og matematisk modellering Problemløsing og matematisk modellering Formulere, bruke og tolke viser til ulike steg elevene må arbeide seg gjennom når de bruker matematikk for å løse problemer i ulike kontekster. Denne typen problemløsing kalles matematisk model­ lering. Det første steget handler om å gjenkjenne muligheter til å bruke matematikk i den gitte situasjonen og å kunne omforme problemet til et matematisk problem (formulere). Etter at det opprinnelige problemet er omformet til noe som kan løses matematisk, er det neste steget å løse det gjennom for eksempel utregninger, operasjoner og algoritmer, for å komme fram til en matematisk løsning (bruke). I det siste steget skal den matematiske løsningen tolkes i lys av den opprinnelige konteksten (tolke). Dette kan innebære å vurdere hvor gyldig eller relevant løsningen er, eller forklare hvorfor et matematisk resultat er – eller ikke er – en fornuftig løsning på det opprinnelige problemet. Denne typen matematisk kompetanse innebærer ikke bare god forståelse av en rekke matematiske begreper og prosedyrer og hvordan de brukes, men også å kunne vurdere når det er hensikts­ messig å bruke ulike begreper og prosedyrer. Holdninger til lesing og lesevaner I PISA 2022 var det ingen spørsmål knyttet til elevers lesing på fritida eller på skolen, siden lesing ikke var undersøkelsens hovedområde. De norske elevene fikk imidlertid to spørsmål som har til hensikt å belyse sentrale elementer i LK20. Spørsmålene handlet om hvilken undervisning elevene rapporterer å få innen kritisk lesing, samt hvilke strategier de bruker for utforsking og dybdelæring. PISA 2022 13 Hva er matematisk kompetanse? Det har lenge vært en utbredt oppfatning at det å lære matematikk handler om å opparbeide en slags verktøykasse med matematiske begreper, algoritmer og regler. De siste tiårene har det blitt lagt fram flere beskrivelser av matematisk kompetanse som bygger på en bredere forståelse av hva det vil si å mestre matematikk (Kilpatrick, 2020). Disse beskrivelsene handler blant annet om at elevene skal tenke matematisk, identifisere matematikk i ulike sammenhenger, lage matematiske modeller og vurdere og tolke løsninger. En slik bred forståelse av matematisk kompetanse finner vi også i læreplanen (LK20), der kjerneelementene i matematikkfaget blant annet inkluderer problemløsing, modellering, resonnering og kommunikasjon. Denne brede forståelsen av hva det vil si å mestre matematikk ligger også til grunn for matematisk kompetanse i PISA. Matematikk i og utenfor skolen Matematisk kompetanse i PISA I PISA brukes begrepet mathematical literacy om det å være matematisk forberedt til å håndtere ulike utfordringer vi kan møte på i ulike situasjoner i livet. Mathematical literacy er vanskelig å oversette direkte, men vi har valgt å omtale det som matematisk kompetanse. I PISA 2022 er matematisk kompetanse definert som den enkeltes evne til å resonnere matematisk og å formulere, bruke og tolke matematikk for å løse problemer i mange forskjellige virkelighetsnære kontekster (OECD, 2023a). Og det er nettopp de matematiske prosessene resonnere, formulere, bruke og tolke elevene må ta i bruk for å løse oppgavene som i PISA er gitt i personlige, yrkesrettede, samfunnsmessige og vitenskapelige kontekster. Både i den norske læreplanen og i PISA 2022 legges det vekt på at matematikk spiller en viktig rolle i å forberede barn og unge til å være aktive deltakere i samfunnet og arbeidslivet, mestre utfordringer og ta veloverveide valg i eget liv. Men hva må egentlig elevene lære i matematikk for å være rustet til dette? Hva innebærer matematisk kompetanse for en 15-åring? Både i den norske læreplanen og i PISA 2022 legges det vekt på at matematikk spiller en viktig rolle i å forberede barn og unge til å være aktive deltakere i samfunnet og arbeidslivet, mestre utfordringer og ta veloverveide valg i eget liv. Men hva må egentlig elevene lære i matematikk for å være rustet til dette? Hva innebærer matematisk kompetanse for en 15-åring? Matematikk i samfunnet Viktigheten av matematisk kompetanse blir stadig løftet fram i stor­ tingsmeldinger, nasjonale strategier og andre fora. Samtidig er bruken av matematikk i stor grad skjult i hverdagen, yrkeslivet og teknologien. Vi får presentert værmeldinger som bygger på matematiske modeller og beregninger. Det samme gjelder apper og programvare vi bruker, og alle sikkerhetssystemene som er nødvendige for å holde oss trygge i vår digitale verden. Sykepleiere, elektrikere, ingeniører, økonomer og andre arbeidstakere innenfor mange ulike yrkesgrupper bruker matematikk uten at de nødvendigvis tenker over at det de gjør krever matematiske kunnskaper og ferdigheter. I privatlivet trenger vi mate­ matikk for å gjøre beregninger og vurderinger rundt økonomiske spørsmål, utdanning og helse. I tillegg er det å kunne tenke matematisk viktig for å gjøre kritiske vurderinger, for eksempel når vi skal vurdere utsagn og påstander som bygger på statistikk, tabeller og figurer. Matematisk resonnering og kritisk tenkningi Definisjonen av matematisk kompetanse i PISA 2022 legger også vekt på at elevene skal kunne resonnere matematisk. Dette handler blant annet om å trekke slutninger og inngår i alle modellerings­stegene når man løser virkelighetsnære problemer. Men matematisk reson­ 4 PISA 2022 14 utvalg av disse oppgavene. Oppgavene er organisert i oppgave­enheter, hvor oppgavene innenfor hver enhet er knyttet til samme kontekst. Trekantet mønster er en av de frigitte oppgaveenhetene fra PISA 2022. Her ser vi det typiske oppsettet for oppgavene i PISA, med innle­ dende tekst og figur til høyre, og spørsmål til venstre. Prøven består av både åpne oppgaveformater, hvor elevene skal formulere et svar med egne ord, og ulike typer flervalgformater. Noen oppgaver krever at elevene skal gjøre simuleringer eller bruke regneark som en del av løsningen. Hver av oppgavene måler en liten del av rammeverket, og prøven som helhet måler matematisk kompetanse slik det er definert i rammeverket. nering handler også om noe mer enn det å løse problemer, for eksempel vil det å vurdere og legge fram argumenter, vurdere tolkninger og slutninger knyttet til påstander og matematiske resultater involvere matematisk resonnering. I læreplanen knyttes denne typen matematisk kompetanse til kritisk tenkning, som er nødvendig når elevene skal ta stilling til viktige spørsmål i samfunnet og ta egne valg i livet (Utdanningsdirektoratet, 2020). Matematikkoppgavene i PISA 2022 Matematikkprøven i PISA 2022 består av 234 oppgaver. Av disse var 160 oppgaver nye i 2022, mens 74 er brukt i tidligere undersøkelser slik at det er mulig å måle endringer over tid. Hver elev får tildelt et PISA 2022 15 Matematisk kompetanse på ulike nivåer Noen typiske kjennetegn ved elever på mestringsnivå 1a er at de kan • svare på tydelig definerte spørsmål i en enkel kontekst hvor all nødvendig informasjon er gitt • hente ut relevant informasjon fra to kilder når disse er presentert i et enkelt format • utføre enkle, innøvde prosedyrer som er tydelig gitt i oppgaven og som hovedsakelig involverer heltall Noen typiske kjennetegn ved elever på mestringsnivå 3 er at de kan • utarbeide egne løsningsstrategier som involverer flere steg • bruke enkel algoritmisk tenkning • utføre flere forskjellige innøvde prosedyrer som ikke er gitt i problemstillingen • tolke og bruke ulike representasjoner og informasjonskilder og gjøre resonnementer ut fra dem Noen typiske kjennetegn ved elever på mestringsnivå 3 er at de kan • utarbeide egne løsningsstrategier som involverer flere steg • bruke enkel algoritmisk tenkning • utføre flere forskjellige innøvde prosedyrer som ikke er gitt i problemstillingen • tolke og bruke ulike representasjoner og informasjonskilder og gjøre resonnementer ut fra dem • hente ut relevant informasjon fra to kilder når disse er presentert i et enkelt format • tolke og bruke ulike representasjoner og informasjonskilder og gjøre resonnementer ut fra dem • utføre enkle, innøvde prosedyrer som er tydelig gitt i oppgaven og som hovedsakelig involverer heltall Nivå 1 under 420 poeng Nivå 1 under 420 poeng Nivå 3 482–545 poeng Nivå 3 482–545 poeng Nivå 2 420–482 poeng Informasjonen i denne oppgaven er gitt i et enkelt format og med små heltall (seks blå og ti røde trekanter). Både spørsmålet og svaralterna­ tivene gjør det tydelig at oppgaven omhandler prosentregning, men elevene må selv finne ut hvilken løsningsmetode de skal benytte. Noen elever vil se at det er færre blå enn røde trekanter og at svaret derfor må være mindre enn 50 %, altså 37,5 som er riktig. 68 prosent av de norske elevene svarte korrekt på denne oppgaven. Nivå 1 under 420 poeng 4 under 420 poeng 4 Informasjonen i denne oppgaven er gitt i et enkelt format og med små heltall (seks blå og ti røde trekanter). Både spørsmålet og svaralterna­ tivene gjør det tydelig at oppgaven omhandler prosentregning, men elevene må selv finne ut hvilken løsningsmetode de skal benytte. Noen elever vil se at det er færre blå enn røde trekanter og at svaret derfor må være mindre enn 50 %, altså 37,5 som er riktig. Matematisk kompetanse på ulike nivåer På disse to sidene er det gitt eksempler på fire oppgaver som var med i PISA 2022. De fire oppgavene er typiske oppgaver som elevene på mestringsnivå 1a, 3, 5 og 6 får til. Det er også gitt en kort beskrivelse av nivåene oppgavene er knyttet til. Prosentandelen elever på ulike nivåer varierer for de nordiske landene (figur 22). For andelene under nivå 2 er det tydelige forskjeller på tvers av landene. For de norske elevene er fordelingen på de ulike nivåene omtrent lik som for OECD-landene. I PISA blir elevenes resultater knyttet til mestringsnivåer. Det er utviklet beskrivelser av hva som kjennetegner kompetansen til elever på hvert nivå. I PISA 2022 brukes mestringsnivåene 1 til 6, hvor nivå 1 er delt inn i 1a, 1b og 1c. Kompetanse på nivå 2 er vurdert til å være minimums­ kompetansen elever trenger for å kunne delta aktivt i samfunnet. I den tekniske rapporten for PISA 2022 gis det utdypende beskrivelser av alle nivåene og hvordan de er utviklet (OECD, 2023c). Figur 22. Prosentandel elever på mestrings­ nivåer i matematikk i de nordiske landene og for OECD-gjennomsnittet i PISA 2022. Nedre grense for nivå 2 er markert med en vertikal svart linje. Matematisk kompetanse på ulike nivåer 68 prosent av de norske elevene svarte korrekt på denne oppgaven. Informasjonen i denne oppgaven er gitt i et enkelt format og med små heltall (seks blå og ti røde trekanter). Både spørsmålet og svaralterna­ tivene gjør det tydelig at oppgaven omhandler prosentregning, men elevene må selv finne ut hvilken løsningsmetode de skal benytte. Noen elever vil se at det er færre blå enn røde trekanter og at svaret derfor må være mindre enn 50 %, altså 37,5 som er riktig. 68 prosent av de norske elevene svarte korrekt på denne oppgaven. Informasjonen i denne oppgaven er gitt i et enkelt format og med små heltall (seks blå og ti røde trekanter). Både spørsmålet og svaralterna­ tivene gjør det tydelig at oppgaven omhandler prosentregning, men elevene må selv finne ut hvilken løsningsmetode de skal benytte. Noen elever vil se at det er færre blå enn røde trekanter og at svaret derfor må være mindre enn 50 %, altså 37,5 som er riktig. 68 prosent av de norske elevene svarte korrekt på denne oppgaven. I denne oppgaven må elevene tolke en modell og skjønne at tallene viser differansen mellom de gjennomsnittlige avstandene fra sola, som de finner i tabellen. Oppgaven må løses i flere steg, og elevene må bruke flere representasjoner for å plassere planetene riktig. Regneoperasjonen er enkel (subtraksjon), men prosedyren er ikke tydelig gitt i oppgaven. Riktig svar er, fra venstre til høyre, Jupiter, Saturn, Uranus. 47 prosent av de norske elevene svarte korrekt på denne oppgaven. 16 PISA 2022 Figur 22. Prosentandel elever på mestrings­ nivåer i matematikk i de nordiske landene og for OECD-gjennomsnittet i PISA 2022. Nedre grense for nivå 2 er markert med en vertikal svart linje. Figur 22. Prosentandel elever på mestrings­ nivåer i matematikk i de nordiske landene og for OECD-gjennomsnittet i PISA 2022. Nedre grense for nivå 2 er markert med en vertikal svart linje. • løse abstrakte problemer ved hjelp av fleksibel tenkning og kreative løsningsstrategieri • gjenkjenne når en prosedyre som ikke er spesifisert i en oppgave kan brukes i ukjent kontekst • bruke simuleringer og regneark på en hensiktsmessig måte • tenke kritisk og fremstille resonnementer på en tydelig måte p g ppg • vurdere matematiske resultater opp mot den virkelige konteksten Nivå 6 669 poeng og over Nivå 4 545–607 poeng I denne oppgaven må elevene gjøre en kritisk vurdering av Helenes påstand. Matematikkangst Matematikkangst handler om negative følelser som oppstår i møte med matematikk, for eksempel stress, uro eller frykt (Ashcraft, 2002). «Angst» må her ikke misforstås som en psykisk lidelse eller en diagnose, men handler om en stressreaksjon som kan ha store konsekvenser for læring. De negative følelsene vi kaller matematikkangst, kan oppstå i formelle sammenhenger, for eksempel i en prøvesituasjon, men følelsene kan også oppstå i mer hverdagslige situasjoner, for eksempel når man skal finne ut hvor mye hver enkelt skal betale når man deler en regning. I spørreskjemaet i PISA 2022 blir elevene bedt om å ta stilling til seks utsagn om tanker og følelser knyttet til matematikk (se figur 23), hvor svaralternativene er «Svært enig», «Enig», «Uenig» og «Svært uenig». Resultatene viser at omtrent en av tre norske elever rapporterer at de blir veldig nervøse når de arbeider med matematikkoppgaver, og at omtrent halvparten av norske elever rapporterer at de blir veldig stresset når de må gjøre lekser i matematikk. Omtrent to av tre elever rapporterer å være redd for å få dårlige karakterer i matematikk. De fleste av disse utsagnene var også med i PISA 2003 og 2012. Sammenlikner vi resultatene fra PISA 2022 med tidligere år, ser vi en økning i andelen elever som svarer «Svært enig» eller «Enig» for alle utsagnene Figur 23. Prosentandel av de norske elevene i PISA 2022, 2012 og 2003 som svarer «Svært enig» eller «Enig» for de seks utsagnene knyttet til matematikkangst. Tabell 3. Verdier for konstruktet matematikkangst i PISA 2022 og 2012. Høyere verdi betyr høyere grad av matematikkangst. Skalaen er standardisert, med 0 som gjennomsnitt og 1 som standardavvik for OECD-landene det første året skalaen ble laget. Statistisk signifikante endringer er uthevet. Tabell 3. Verdier for konstruktet matematikkangst i PISA 2022 og 2012. Høyere verdi betyr høyere grad av matematikkangst. Skalaen er standardisert, med 0 som gjennomsnitt og 1 som standardavvik for OECD-landene det første året skalaen ble laget. Statistisk signifikante endringer er uthevet. Konstruktverdi 2022 Konstruktverdi 2012 Endring Norge 0,17 0,02 0,15 Danmark -0,12 -0,37 0,25 Finland -0,29 -0,33 0,04 Island 0,06 -0,33 0,39 Sverige -0,07 -0,35 0,27 OECD 0,17 0,00 0,17 Konstruktverdi 2022 Konstruktverdi 2012 Endring Resultatene for de seks utsagnene danner til sammen en verdi for samlevariabelen matematikkangst (se side 30 for nærmere forklaring av begrepet samlevariabel), der en høyere verdi betyr høyere grad av matematikkangst. Matematisk kompetanse på ulike nivåer Oppgaven er på mestringsnivå 6 og noe mer abstrakt enn de tidligere oppgavene i enheten ved at elevene må vurdere hva tallene representerer. Det riktige svaret er «Nei», med en tilhørende begrunnelse som påpeker at regnearket kun viser andelen skogareal, eller at det ikke viser det totale landarealet. Kun 9 prosent av norske elever fikk riktig svar, og i PISA 2022 ser vi at oppgavene som krever en begrunnelse, er vanskelige for elevene. I denne oppgaven skal elevene bruke informasjonen i tabellen til å finne ut hvilke land som har størst, ingen og minst endring i skogareal mellom 2005 og 2015. Her har elevene mulighet til å gjøre utregninger og sorteringer i regnearket for å komme frem til riktig svar. 23 prosent av de norske elevene valgte riktig land på alle de tre spørsmålene. I denne oppgaven skal elevene bruke informasjonen i tabellen til å finne ut hvilke land som har størst, ingen og minst endring i skogareal mellom 2005 og 2015. Her har elevene mulighet til å gjøre utregninger og sorteringer i regnearket for å komme frem til riktig svar. 23 prosent av de norske elevene valgte riktig land på alle de tre spørsmålene. I denne oppgaven skal elevene bruke informasjonen i tabellen til å finne ut hvilke land som har størst, ingen og minst endring i skogareal mellom 2005 og 2015. Her har elevene mulighet til å gjøre utregninger og sorteringer i regnearket for å komme frem til riktig svar. 23 prosent av de norske elevene valgte riktig land på alle de tre spørsmålene. I denne oppgaven må elevene gjøre en kritisk vurdering av Helenes påstand. Oppgaven er på mestringsnivå 6 og noe mer abstrakt enn de tidligere oppgavene i enheten ved at elevene må vurdere hva tallene representerer. Det riktige svaret er «Nei», med en tilhørende begrunnelse som påpeker at regnearket kun viser andelen skogareal, eller at det ikke viser det totale landarealet. Kun 9 prosent av norske elever fikk riktig svar, og i PISA 2022 ser vi at oppgavene som krever en begrunnelse, er vanskelige for elevene. PISA 2022 17 PISA 2022 17 Matematikkangst og tankesett Resultatene fra elevspørreskjemaet i PISA 2022 gir innsikt i elevenes interesse for og holdninger til matematikkfaget, deres mestringsforventninger i faget og deres tanker om læring. Her presenterer vi resultater knyttet til to av temaene i spørreskjemaet, matematikkangst og tankesett. Figur 23. Prosentandel av de norske elevene i PISA 2022, 2012 og 2003 som svarer «Svært enig» eller «Enig» for de seks utsagnene knyttet til matematikkangst. Lærende tankesett – tro på innsats og hardt arbeid Figur 24. Gjennomsnittsresultater i matematikk i PISA 2022 for norske elever med lærende tankesett og låst tankesett. Andelen elever i hver kategori er vist i parentes. Hvilke oppfatninger og tanker du har knyttet til matematikk og det å lære matematikk, har noe å si for hva du selv tror du har mulighet til å lære. Noen tror for eksempel at ikke alle kan lære matematikk, og at noen er født med et «mattegen» eller en «mattehjerne». Det skilles gjerne mellom å ha et lærende tankesett (growth mindset) og et låst eller fiksert tankesett (fixed mindset) (Dweck & Yeager, 2019). Et lærende tankesett vil si å ha en oppfatning av at evner og intelligens er noe som kan utvikles med innsats og trening. Et slikt tankesett innebærer en tro på at utholdenhet, hardt arbeid og motgang fostrer læring og motiverer til å ikke gi opp. Et låst tankesett derimot vil si å ha en oppfatning av at evner og intelligens er et talent man er født med og som i liten grad kan endres. Med et låst tankesett vil det å oppleve motgang oppleves som nederlag og en bekreftelse på manglende evner, og dermed undergrave troen på at innsats og hardt arbeid har en verdi. En persons tankesett er ikke bundet til å være lærende eller låst, men påvirkes av erfaringer og læringssituasjoner. For eksempel kan ros og tilbakemeldinger fra voksne som legger vekt på innsats og arbeidsprosess («nå har du jobbet hardt»), påvirke barns tankesett til å bli mer lærende, mens ros og tilbakemeldinger som går på barnas intelligens eller dyktighet («du er smart», «du er flink i matematikk»), kan føre til et mer låst tankesett (Gunderson et al., 2013). Figur 24. Gjennomsnittsresultater i matematikk i PISA 2022 for norske elever med lærende tankesett og låst tankesett. Andelen elever i hver kategori er vist i parentes. Figur 25 viser gjennomsnittsresultater i matematikk for fire grupper: elever med lav matematikkangst og lærende tankesett (536 poeng), elever med lav matematikkangst og låst tankesett (504 poeng), elever med høy matematikkangst og lærende tankesett (449 poeng) og elever med høy matematikkangst og låst tankesett (440 poeng). Elever med lav og høy matematikkangst vil si de 25 prosent av elevene med lavest og høyest verdi på samlevariabelen for mate­matikkangst. Resultatene i figur 25 viser betydningen av samspillet mellom følelser knyttet til matematikk, tankesett og prestasjoner. Matematikkangst De norske elevene i PISA 2022 rapporterer om omtrent samme grad av matematikkangst som gjennomsnittet for elever i OECD-landene (tabell 3). Samtidig viser resultatene at norske elever har høyest grad av matematikkangst i Norden, og at de rapporterer om høyere grad av angst for matematikk i PISA 2022 enn i 2012, noe som er i tråd med det som vises i figur 23. Også i de andre nordiske landene, bortsett fra Finland, og for gjennomsnittet i OECD-landene uttrykker elevene større grad av matematikkangst i 2022 enn i 2012. Det å ha negative tanker og følelser for matematikk er krevende i seg selv, men har også en sammenheng med svake matematikkpresta­ sjoner. Forskning tyder på at effekten går begge veier (Carey et al., 2016); på den ene siden er matematikkangst en slags barriere, eller sperre, som hindrer læring og faglig utvikling, og på den andre siden fører svake prestasjoner i matematikk til mer angst. Resultatene fra PISA 2022 viser en tydelig sammenheng mellom matematikkangst og 18 PISA 2022 hvor forskjellen i gjennomsnittsresultatet for elever med et lærende og låst tankesett varierer mellom 18 poeng (Finland) og 33 poeng (Danmark). Resultatene viser også en sammenheng mellom tankesett og matematikkangst, hvor elever med låst tankesett uttrykker mer engstelse for matematikk enn elever med lærende tankesett (i gjennomsnitt 0,26 høyere verdi for samlevariabelen for matematikkangst). Dette stemmer overens med forskning som viser at et låst tankesett kan bidra til negative følelser knyttet til matematikk, for eksempel å føle hjelpeløshet hvis det er noe man ikke mestrer. prestasjoner i matematikk. Gjennomsnittsresultatene for de 25 prosent norske elevene med høyest verdi på samlevariabelen matematikkangst, er 77 poeng lavere enn for de 25 prosent med lavest verdi. Sammen­ likner vi resultatene for gutter og jenter i PISA 2022, finner vi store forskjeller i alle de nordiske landene. Jentene uttrykker større grad av negative følelser knyttet til matematikk enn guttene, noe de også gjorde i PISA 2012. I PISA 2022 er denne kjønnsforskjellen 0,63 poeng på samlevariabelen for matematikkangst. Lærende tankesett – tro på innsats og hardt arbeid Vi finner et tilsvarende mønster blant elever i alle de nordiske landene. I PISA 2022 ble elevene spurt om hvor enige eller uenige de er i utsagnet «Intelligens er noe ved deg som du ikke kan endre så mye». Elever som rapporterte at de er «Svært enig» eller «Enig» i dette utsagnet, ansees å ha et låst tankesett, mens elever som rapporterer «Uenig» eller «Svært uenig», ansees å ha et lærende tankesett. Det tas forbehold om at resultatene som rapporteres her, er basert på elevenes svar på dette ene utsagnet. En samlevariabel basert på flere utsagn, ville gitt et mer reliabelt mål på elevenes tankesett. Figur 25. Gjennomsnittsresultater i matematikk i PISA 2022 for elever med henholdsvis lav og høy grad av matematikkangst og lærende/låst tankesett. Resultatforskjellene er signifikante. Resultatene fra PISA 2022 viser at 59 prosent av norske elever har holdninger som forbindes med et lærende tankesett, mens 41 prosent ser ut til å ha et låst tankesett (figur 24). Videre viser analysene at gjennomsnittsresultatene til elever med et lærende tankesett er 29  poeng høyere enn elever med et låst tankesett. Tilsvarende forskjeller finner vi også i de andre nordiske landene, Figur 25. Gjennomsnittsresultater i matematikk i PISA 2022 for elever med henholdsvis lav og høy grad av matematikkangst og lærende/låst tankesett. Resultatforskjellene er signifikante. PISA 2022 19 Læringsmiljø timene i PISA 2022, er omtrent lik som i 2012 og betydelig mindre enn i 2003. Resultatene for denne samlevariabelen viser at svenske og finske elever rapporterer om litt dårligere arbeidsro enn norske elever. Elevene ble også spurt om arbeidsro i PISA 2018, men den gangen var utsagnene knyttet til norsktimene. For de fleste utsagnene rapporterte elevene ganske likt for arbeidsro i norsktimene i 2018 som for matematikktimene i 2022. For utsagnene «Elevene begynner ikke å arbeide før lenge etter at timen har begynt» og «Elevene får ikke arbeidet ordentlig» var det derimot noe bedre resultater i 2018. I Norge har alle elever rett til et trygt og godt læringsmiljø som fremmer helse, trivsel og læring, og dette er i 2023 på nytt stadfestet i opplæringsloven. Et godt læringsmiljø avhenger av at elevene opplever at de føler seg trygge, både fysisk og psykisk, føler tilhørighet og støtte, og at de utfordres faglig og intellektuelt. I PISA får elevene spørsmål om ulike sider ved læringsmiljøet, blant annet forholdet til skolen og lærerne, mobbing, fravær og arbeidsro og klasseledelse i undervisningstimene. Arbeidsro i matematikktimene I PISA 2022 ble elevene også spurt om de ble distrahert av digitale ressurser i klasserommet. Svaralternativene er «Alle timene», «De fleste timene», «Noen timer» og «Aldri eller nesten aldri». Resultatene I PISA 2022 er arbeidsro i matematikktimene undersøkt ved at elevene tar stilling til fem utsagn. Svaralternativene er «Alle timene», «De fleste timene», «Noen timer» og «Aldri eller nesten aldri». De samme utsagnene var også med i 2003 og 2012, da matematikk også var hovedområde, slik at det er mulig å undersøke hvordan elevenes opplevelse av arbeidsro har endret seg over tid. Figur 27. Prosentandel elever i de nordiske landene som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikk­ timene?» i PISA 2022. Figur 26 viser de fem utsagnene som danner en samlevariabel for arbeidsro. Andelen norske elever som rapporterer om uro i Figur 26. Prosentandel norske elever som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikktimene?» i PISA 2022, 2012 og 2003. Figur 26. Prosentandel norske elever som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikktimene?» i PISA 2022, 2012 og 2003. Figur 27. Prosentandel elever i de nordiske landene som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikk­ timene?» i PISA 2022. Figur 27. Prosentandel elever i de nordiske landene som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikk­ timene?» i PISA 2022. Figur 26. Prosentandel norske elever som svarer «Alle timene» eller «De fleste timene» på spørsmålet «Hvor ofte skjer dette i matematikktimene?» i PISA 2022, 2012 og 2003. 20 PISA 2022 20 PISA 2022 viser at omtrent en av tre elever rapporterer at de blir distrahert av egen bruk, mens en av fire elever blir distrahert av andre elever som bruker digitale ressurser i alle eller i de fleste timene (figur 27). Dette er på samme nivå, eller lavere, enn for elever i de andre nordiske landene. Det er viktig å ta hensyn til at disse resultatene er basert på elevenes selvrapportering om hvor ofte de opplever å bli distrahert, og at det kan være vanskelig å vurdere i ettertid om noe har vært distraherende eller ikke. Uansett er det grunn til å tro at mange elever opplever bruk av digitale ressurser som et distraherende element i matematikktimene. Arbeidsro i matematikktimene «Uenig» og «Svært uenig». Resultatene fra 2022 viser at de fleste norske elever føler tilhørighet til skolen, og at graden av tilhørighet er på omtrent samme nivå som i 2018 og 2015. Hvis vi ser på samlevariabelen basert på elevenes svar om de seks utsagnene, er det likevel en liten nedgang i skoletilhørighet fra PISA 2018. Det er verdt å merke seg at elevene som deltok i PISA-undersøkelsene i 2003 rapporterte om høyere grad av tilhørighet til skolen. Resultatene fra PISA 2022 viser også at norske elever rapporterer om høyere grad av tilhørighet enn elever i de andre nordiske landene og gjennomsnittet for OECD-landene. Samtidig tyder resultatene på at det er en stor andel norske elever som ikke trives på skolen, for eksempel at de føler de ikke hører til på skolen (23 prosent), at de føler seg ensomme (14 prosent) og at de føler seg annerledes og ikke passer inn (19 prosent). Omtrent lik grad av skoletilhørighet Å føle tilhørighet til skolen er viktig for et godt psykososialt miljø og har stor betydning for elevenes læring og mentale helse. I PISA 2022 blir elevenes følelse av tilhørighet til skolen undersøkt gjennom seks utsagn de skal ta stilling til – tre utsagn som er negativt formulert, og tre utsagn som er positivt formulert (se figur 28). Disse utsagnene har også vært med tidligere. Svaralternativene er «Svært enig», «Enig», At de fleste norske elever har et godt forhold til skolen, vises også i resultatene fra Elevundersøkelsen i 2022, hvor 85 prosent av elevene svarte at de trives godt eller svært godt på skolen. Resultatene fra Elevundersøkelsen viser også at elevenes trivsel har vært relativt stabil i perioden 2014 til 2022. I PISA 2022 er det en positiv sammenheng mellom norske elevers følelse av tilhørighet til skolen og matematikkprestasjoner (tabell 4). Denne sammenhengen var like sterk i 2012. Figur 28. Prosentandel elever i Norge og OECD som svarer «Svært enig» eller «Enig» på spørsmålet «Tenk på skolen din: Hvor enig er du i disse utsagnene», i PISA 2022, 2018 og 2003. Elevenes følelse av tilhørighet til skolen varierer på tvers av elev­ grupper og skoler. I Norge rapporterer gutter om høyere grad av tilhørighet enn jenter, og elever med høy sosioøkonomisk status (25 prosent høyeste) rapporterer om høyere grad av tilhørighet enn elever med lav sosioøkonomisk status (25 prosent laveste). Resultatene viser også at elever som går på skoler preget av høy SØS blant elevene (25 prosent høyeste), rapporterer om høyere grad av tilhørighet enn elever som går på skoler med tilsvarende lav SØS. Tabell 4. Poengforskjell i matematikkresultater mellom elever som svarer «Svært enig» eller «Enig» sammenliknet med elever som svarer «Uenig» eller «Svært uenig» på spørsmålet «Tenk på skolen din: Hvor enig er du i disse utsagnene?». Statistisk signifikante forskjeller er uthevet. Utsagn Poengforskjell Jeg føler meg annerledes og at jeg ikke passer inn på skolen. -24 Jeg føler meg ensom på skolen. -25 Jeg føler at jeg blir holdt utenfor på skolen. -45 Det virker som om andre elever liker meg. 30 Jeg får lett venner på skolen. 2 Jeg føler at jeg hører til på skolen. 27 Figur 28. Prosentandel elever i Norge og OECD som svarer «Svært enig» eller «Enig» på spørsmålet «Tenk på skolen din: Hvor enig er du i disse utsagnene», i PISA 2022, 2018 og 2003. PISA 2022 21 Likeverd Norge er det landet i PISA-undersøkelsen med høyest gjennom­ snittlig sosioøkonomisk status (SØS) og blant landene med minst forskjeller i SØS blant elevene. Tidligere PISA-undersøkelser har vist at Norge har vært blant landene hvor hjemmebakgrunn har minst betydning for elevers prestasjoner, sett i et internasjonalt perspektiv. Dette gjelder også for PISA 2022. Tabell 5 viser at mellom 8,3 og 9,6 prosent av variasjonen i prestasjoner blant norske elever kan forklares av SØS. Dette er lave tall både i nordisk og internasjonal sammenheng. For gjennomsnittet i OECD-landene er de tilsvarende tallene mellom 12,6 og 15,5 prosent. Likeverd er et viktig prinsipp i norsk skole. Alle barn og unge skal ha lik rett til skole og utdanning. Samtidig vet vi at faktorer som hjemmebakgrunn og kjønn kan ha stor betydning for om elever lykkes på skolen. I dette kapittelet bruker vi resultatene fra PISA 2022 til å undersøke spørsmålet om likeverd fra ulike sider. Rettferdighet og inkludering som mål for utdanningssystemer I PISA 2022 defineres to dimensjoner av likeverd i utdanning (equity): rettferdighet og inkludering. Rettferdighet vil si at alle elever skal ha mulighet til å nå sitt fulle læringspotensial, uavhengig av bakgrunn, mens inkludering innebærer at alle skal ha tilgang til god utdanning. Likeverd blir definert som en kombinasjon av disse to, og utdannings­ systemer som kombinerer høy rettferdighet og høy inkludering, blir vurdert til å ha høy grad av likeverd. l Det er verdt å merke seg at Sverige skiller seg ut negativt i Norden og er på samme nivå som OECD-gjennomsnittet når det kommer til betydningen av hjemmebakgrunn for elevenes prestasjoner. Tabell 5. Prosentandel av variansen i prestasjoner i de tre fagområdene som kan forklares av elevenes SØS. Resultatene er gitt for de nordiske landene og for gjennomsnittet i OECD. Tabell 5. Prosentandel av variansen i prestasjoner i de tre fagområdene som kan forklares av elevenes SØS. Resultatene er gitt for de nordiske landene og for gjennomsnittet i OECD. Resultatene fra PISA 2022 peker på flere utfordringer med å gi norske elever like muligheter til å lykkes. I de tidligere kapitlene har vi sett at det er store andeler av de norske elevene som presterer under nivå 2 i de ulike fagområdene. Dette kan knyttes til begrepet om inkludering. Vi har også sett at det fortsatt er store kjønnsforskjeller i elevenes gjennomsnittsresultater i lesing. Likeverd I dette kapittelet skal vi se videre på betydningen av elevenes hjemmebakgrunn og prestasjons­ forskjeller mellom minoritets- og majoritetselever. Varians i prestasjoner forklart av SØS Matematikk Naturfag Lesing Norge 9,6 % 9,4 % 8,3 % Danmark 12,2 % 10,7 % 9,8 % Finland 12,4 % 10,5 % 9,9 % Island 9,3 % 10,8 % 8,6 % Sverige 15,0 % 15,8 % 14,1 % OECD 15,5 % 14,2 % 12,6 % Mobbing forskjellen er liten. Resultatene viser også at mobbing henger sammen med elevenes hjemmebakgrunn; elever med lavere SØS rapporterer om mer mobbing enn elever med høyere SØS. Mobbing kan defineres som negative handlinger som utøves mot en part som ikke så lett kan forsvare seg, og det kan ha store konsekvenser for alle involverte. I PISA 2022 tok elevene stilling til flere utsagn om mobbing med svaralternativene «Aldri eller nesten aldri», «Noen få ganger i året», «Noen få ganger i måneden» eller «En gang i uka eller mer». Andelen norske elever som rapporterer at de jevnlig har opplevd mobbing det siste året («En gang i uka eller mer» og «Noen få ganger i måneden»), er ganske lik i 2022 som i 2018 (figur 29). I Elevundersøkelsen var det en økning i andelen elever som rapporterte at de blir mobbet av medelever i 2022 sammenliknet med tidligere år. Figur 29. Prosentandel elever i Norge og OECD i PISA 2022 og 2018 som svarer «Noen få ganger i måneden» eller «En gang i uka eller mer» på spørsmålet: «Hvor ofte har du opplevd noe av dette på skolen i løpet av de siste 12 månedene?» Resultatene fra TIMSS-undersøkelsen viser økt rapportert mobbing fra 2015 til 2019 på 9. trinn. Andelen elever som rapporterer om mobbing er på samme nivå i Norge, Sverige og gjennomsnittet for OECD-landene, mens den er noe høyere i Danmark og noe lavere i Finland og på Island. At elever opplever mobbing, bryter med opplæringsloven og elevers rett til et trygt og godt skolemiljø og den lovfestede nulltoleransen mot mobbing. Mobbing kan føre til alvorlige og langsiktige fysiske og psykiske problemer. Det kan også gå ut over elevenes faglige utvikling. I  PISA 2022 er det en tydelig sammenheng mellom elevenes rapportering om mobbing og faglige prestasjoner. Norske jenter rapporterer i større grad enn gutter at de blir mobbet, selv om Figur 29. Prosentandel elever i Norge og OECD i PISA 2022 og 2018 som svarer «Noen få ganger i måneden» eller «En gang i uka eller mer» på spørsmålet: «Hvor ofte har du opplevd noe av dette på skolen i løpet av de siste 12 månedene?» 22 22 PISA 2022 2 PISA 2022 PISA 2022 Betydningen av hjemmebakgrunn I naturfag og lesing finner vi en betydelig økning i forskjeller også fra PISA 2018 til 2022. I nordisk sammenheng skiller Sverige seg ut med betydelig større forskjeller i prestasjoner mellom elevgrupper enn de andre nordiske landene. Disse forskjellene har også økt betydelig siden 2018 i alle de tre fagområdene. Figur 30. Gjennomsnittsresultat i matematikk i Norge for fire grupper av elever med ulik sosioøkonomisk status: de 25 prosent elevene med lavest, nest lavest, nest høyest og høyest verdi på samlevariabelen for SØS. Selv om betydningen av hjemmebakgrunn for prestasjoner er mindre i Norge enn i de aller fleste andre land som deltar i PISA, er det relativt store forskjeller i prestasjoner hvis vi sammenlikner elever med ulik SØS. En måte å undersøke denne forskjellen på er å sammenlikne gjennomsnittsresultatene for de 25 prosent elevene med lavest SØS (gruppen lengst til venstre i figur 30) med de 25 prosent elevene med høyest SØS (gruppen lengst til høyre i figur 30). Tabell 6 viser Selv om betydningen av hjemmebakgrunn for prestasjoner er mindre i Norge enn i de aller fleste andre land som deltar i PISA, er det relativt store forskjeller i prestasjoner hvis vi sammenlikner elever med ulik SØS. En måte å undersøke denne forskjellen på er å sammenlikne gjennomsnittsresultatene for de 25 prosent elevene med lavest SØS (gruppen lengst til venstre i figur 30) med de 25 prosent elevene med høyest SØS (gruppen lengst til høyre i figur 30). Tabell 6 viser Betydningen av hjemmebakgrunn Mye forskning viser at hjemmebakgrunn er en av de viktigste fakto­ rene for skoleprestasjoner. Andelen variasjon i prestasjoner som kan forklares av SØS, kan brukes som et mål på i hvor stor grad skolen lykkes med å utjevne sosiale forskjeller. I land hvor en større andel av variasjonen i prestasjoner kan forklares av SØS, betyr det at elevenes hjemmebakgrunn i større grad kan brukes til å forutsi hvorvidt de vil lykkes på skolen eller ikke. Samtidig er det vanskelig å si noe om hvor liten betydningen av hjemmebakgrunn bør være for at man skal kunne si at utdanningssystemet har «lykkes» med å utjevne sosiale forskjeller. Om måling av elevenes sosioøkonomiske status i PISA Begrepet hjemmebakgrunn brukes i denne rapporten om sosio­­­­­ økonomisk status (SØS). I PISA måles elevenes SØS ut fra spørre­ skjemaspørsmål knyttet til foreldrenes utdanningsnivå og yrkesstatus, og ressurser i hjemmet. Dette kombineres til en samlevariabel for SØS, der gjennomsnittet for elever i OECD-landene er satt til 0 med et standardavvik på 1. I PISA 2022 er Norge og Island de to landene med høyest gjennomsnittlig SØS og minst forskjell i SØS blant elevene. Se Jensen med kollegaer (2020) for en utfyllende beskrivelse av SØS-målet i PISA-undersøkelsen. PISA 2022 23 23 PISA 2022 23 PISA 2022 2 Tabell 6. Forskjeller i gjennomsnittsresultater mellom 25 prosent av de norske elevene med høyest og lavest SØS. Forskjeller som er signifikant forskjellig fra PISA 2022, er uthevet. Figur 30. Gjennomsnittsresultat i matematikk i Norge for fire grupper av elever med ulik sosioøkonomisk status: de 25 prosent elevene med lavest, nest lavest, nest høyest og høyest verdi på samlevariabelen for SØS. Forskjeller i gjennomsnittsresultater mellom elever med høy og lav SØS PISA 2012 PISA 2015 PISA 2018 PISA 2022 Matematikk 62 68 68 81 Naturfag 65 73 75 90 Lesing 60 63 72 89 at siden PISA 2012 har forskjellen i gjennomsnittsresultater mellom elevgruppene med høy og lav SØS økt i alle de tre fagområdene. I naturfag og lesing finner vi en betydelig økning i forskjeller også fra PISA 2018 til 2022. I nordisk sammenheng skiller Sverige seg ut med betydelig større forskjeller i prestasjoner mellom elevgrupper enn de andre nordiske landene. Disse forskjellene har også økt betydelig siden 2018 i alle de tre fagområdene. at siden PISA 2012 har forskjellen i gjennomsnittsresultater mellom elevgruppene med høy og lav SØS økt i alle de tre fagområdene. Inkludering I PISA 2022 blir inkludering undersøkt på to måter. Dekningsgraden gir et anslag på hvor stor andel av 15-åringene i et land som går på skole og ikke er ekskludert fra PISA-undersøkelsen (se side 26 for mer informasjon om dekningsgrad). Norge er blant landene med høy dekningsgrad. I tillegg blir det undersøkt hvor mange elever som oppnår det som ansees som minimumskompetanse for å være forberedt til videre skolegang og arbeidsliv (mestringsnivå 2 eller høyere) i alle de tre fagområdene. Norske minoritetselever presterer i gjennomsnitt 36 poeng lavere i matematikk enn majoritetselever i PISA 2022. I 2012, forrige gang matematikk var hovedområde, var denne forskjellen på 47 poeng (prosentandelen minoritetselever var da 9,5). Når vi kontrollerer for elevenes sosioøkonomiske status i PISA 2022, reduseres forskjellen betraktelig. Forskjeller i elevenes hjemmebakgrunn forklarer dermed mye av forskjellen i gjennomsnittsresultater mellom minoritets- og majoritetselever. Figur 31 viser at i Norge presterer 59 prosent av elevene på nivå 2 eller høyere i matematikk, naturfag og lesing. Det betyr at 41 prosent av elevene presterer under nivå 2 i minst ett av fagområdene. Figuren viser også at blant norske elever er andelen som presterer på nivå 2 eller høyere i alle fagområdene på samme nivå som blant elevene i OECD-landene, men betydelig lavere enn blant elevene i Danmark, Finland og Sverige. Sammenlikner vi resultatene på mestringsnivåer, finner vi at andelen majoritetselever under nivå 2 er 27 prosent, og den tilsvarende andelen for minoritetselever er 43 prosent. Dette er høye tall for begge grupper og en betydelig forskjell mellom gruppene. Det er vanskelig å sammenlikne resultater for grupper av minoritetselever på tvers av land. Minoritetselever er en mangfoldig elevgruppe som består av elever med svært ulik bakgrunn, og sammen­ setningen av denne elevgruppen kan variere mye. I tillegg består denne gruppen av relativt få elever, noe som gjør at det blir større usikkerhet rundt disse resultatene. Figur 31. Prosentandel elever i de nordiske landene og OECD som presterer på mestringsnivå 2 eller høyere i alle de tre fagområdene (matematikk, naturfag og lesing) i PISA 2022. Det er betydelige forskjeller i matematikkprestasjonene mellom majoritets- og minoritetselever også i de andre nordiske landene. Til tross for relativt stor usikkerhet i resultatene for disse gruppene er forskjellen i poeng mellom majoritets- og minoritetselever signifikant i alle de nordiske landene (tabell 7). Det samme gjelder også for OECD-gjennomsnittet, selv om forskjellen der er mindre (30 poeng). Resultater for minoritets- og majoritetselever I spørreskjemaet i PISA får elevene spørsmål om i hvilket land de og foreldrene er født. Elever blir definert som minoritetselever hvis de oppgir at begge foreldrene er født i et annet land. I Norge er det 24 PISA 2022 PISA 2022 Tabell 7. Prosentandel minoritetselever og gjennomsnittsresultater i matematikk for majoritetselever og minoritetselever i de nordiske landene og OECD i PISA 2022. Forskjellene er gitt før og etter at det er kontrollert for sosioøkonomisk status (SØS). Signifikante forskjeller er uthevet. Prosentandel minoritetselever Majoritetselever gjennomsnitts­poeng (S.E.) Minoritetselever gjennomsnittspoeng (S.E.) Forskjell mellom minoritetselever og majoritetselever Forskjell kontrollert for SØS Norge 15,9 % 479 (2,2) 443 (4,0) 36 9 Danmark¹ 10,7 % 497 (2,1) 442 (4,0) 54 28 Finland 6,8 % 491 (1,9) 425 (3,3) 65 42 Island 7,4 % 464 (1,7) 427 (5,3) 37 15 Sverige 21,3 % 499 (2,0) 436 (3,5) 63 34 OECD 12,9 % 479 (0,4) 447 (1,2) 30 15 nordiske landene og i OECD reduseres forskjellene i prestasjoner når det tas hensyn til elevenes hjemmebakgrunn, men de forsvinner ikke. nordiske landene og i OECD reduseres forskjellene i prestasjoner når det tas hensyn til elevenes hjemmebakgrunn, men de forsvinner ikke. 15,9 prosent minoritetselever i PISA 2022 (tabell 7). Dette er noe høyere enn i 2018, da andelen var 12,4. Island og Finland har lavest andel minori­tetselever i Norden, og Sverige har høyest. I Norge er andelen med lav SØS 54 prosent blant minoritetselever og 19 prosent blant majoritetselever. Inkludering Den lavere gjennomsnittsforskjellen mellom majoritets- og minoritets­ elever i OECD-landene skyldes flere faktorer, blant annet at i noen få land, som for eksempel Canada, Storbritannia og Australia, presterer minoritetselever like godt som eller bedre enn majoritetselever. I de Figur 31. Prosentandel elever i de nordiske landene og OECD som presterer på mestringsnivå 2 eller høyere i alle de tre fagområdene (matematikk, naturfag og lesing) i PISA 2022. PISA 2022 25 PISA 2022 25 Fortsatt høy dekningsgrad i Norge PISA-undersøkelsen skal være så inkluderende som mulig, og eksklusjon av skoler eller enkeltelever kan bare gjøres i tråd med internasjonale kriterier. Kriteriene for eksklusjon av elever er beskrevet i tekstboksen. Begrepet dekningsgrad betegner i hvor stor grad PISA-undersøkelsen dekker alle 15-åringer i et land. Det regnes ut ved at antall elever (vektet) som deltok på PISA-undersøkelsen, deles på antall 15-åringer i landet. Både andelen elever som er ekskludert fra prøven og andelen 15-åringer som ikke går på skole, har betydning for hvor stor dekningsgrad et land har. I Norge er det hovedsakelig eksklusjon som påvirker dekningsgraden. Hvor nøyaktig estimatet for antall 15-åringer i et land er, vil variere. Tallet kan for eksempel være basert på folketellinger som ikke blir gjort hvert år. Dette forklarer hvorfor et par land i tabell 13, 14 og 15 har dekningsgrad på mer enn 100 prosent. Noe lavere eksklusjon enn tidligere I Norge var det en stigning i andelen ekskluderte elever fra PISA 2000 til 2018, mens det i 2022 er en liten nedgang (se tabell 8). I 2022 er eksklusjonen på 1,5 prosent på skolenivå og 5,8 prosent på elevnivå. Dette gir en samlet eksklusjon på 7,3 prosent (tabell 8). I 2015 og 2018 var den samlede eksklusjonen på henholdsvis 6,7 og 7,9 prosent. For å møte standardene i undersøkelsen er grensen for samlet eksklusjon satt til 5 prosent. Selv om samlet eksklusjon i Norge har vært over denne grensen siden 2009, har andelen endret seg lite etter dette og har derfor liten betydning for trendmålinger i denne I Norge var det en stigning i andelen ekskluderte elever fra PISA 2000 til 2018, mens det i 2022 er en liten nedgang (se tabell 8). I 2022 er eksklusjonen på 1,5 prosent på skolenivå og 5,8 prosent på elevnivå. Dette gir en samlet eksklusjon på 7,3 prosent (tabell 8). I 2015 og 2018 var den samlede eksklusjonen på henholdsvis 6,7 og 7,9 prosent. Hvem deltar ikke i PISA-undersøkelsen? • Elever med fysisk funksjonsnedsettelse. Dette gjelder bare elever med en type funksjonsnedsettelse som kan hindre dem i å gjennomføre prøven. • Elever med kognitive, psykiske og/eller emosjonelle vansker. Dette er elever som PP-tjenesten, BUP eller andre faginstanser har vurdert, og som ikke er i stand til å forstå og følge instruksjonen i undersøkelsen. Elever skal ikke utelukkes bare fordi de presterer dårlig på skolen eller har disiplinproblemer. I PISA 2022 er dekningsgraden for norske 15-åringer 91 prosent. Til sammenlikning er gjennomsnittlig dekningsgrad for OECD-landene 89 prosent. Denne statistikken er rapportert sammen med beskrivel­ sene av andel elever på ulike prestasjonsnivåer i den internasjonale rapporten (OECD, 2023d) og i tabellverket i denne rapporten. • Elever med begrensede norskkunnskaper. Dette er elever som må oppfylle alle tre følgende kriterier: (1) ikke har norsk som morsmål, (2) har begrensede norskkunnskaper og (3) har hatt mindre enn ett års undervisning i norsk. Det norske datasettet har blitt vurdert til å ha god nok kvalitet til å bli inkludert i det internasjonale datasettet i PISA 2022, og til å kunne sammenliknes på tvers av land og over tid (OECD, 2023c). Deltakelse i PISA 2022 I PISA 2022 blir datakvaliteten i flere land utfordret av enten lav dekningsgrad, lav responsrate eller andre mulige kilder til skjevhet i utvalgets representativitet. I Norge er andelen deltakende skoler og elever innenfor de tekniske kravene, og andelen ekskluderte elever har gått noe ned fra PISA 2018. perioden. Internasjonalt har man beregnet at 1 prosentpoeng økning i eksklusjon tilsvarer omtrent 0,6–1 poeng lavere gjennomsnittsresultat (OECD, 2017), dersom man antar at elevene som er ekskludert, ville prestert lavere enn elevene som deltok på undersøkelsen. Tabell 8. Prosentandel norske elever ekskludert fra PISA-undersøkelsen i 2000–2022. Tabell 8. Prosentandel norske elever ekskludert fra PISA-undersøkelsen i 2000–2022. Tabell 8. Prosentandel norske elever ekskludert fra PISA-undersøkelsen i 2000–2022. Eksklusjon 2000 2003 2006 2009 2012 2015 2018 2022 Elevnivå 1,9 2,9 2,9 3,8 5,0 5,5 6,6 5,8 Skolenivå 0,8 0,5 0,7 2,2 1,2 1,3 1,4 1,5 Samlet 2,7 3,4 3,5 5,9 6,1 6,7 7,9 7,3 I Norge ble PISA-undersøkelsen gjennomført fra 14. mars til 6. mai 2022. Målgruppen var elever født i 2006. Det innebærer at omtrent 99 prosent av deltakerne gikk på 10. trinn, mens i underkant av 1 prosent gikk enten på 9. trinn eller vg1 da de tok prøven. Til sammen deltok ca. 8 500 elever fra 260 skoler. Deltakerprosenten var 87 på elevnivå og 99 på skolenivå. Eksklusjon 2000 2003 2006 2009 2012 2015 2018 2022 Elevnivå 1,9 2,9 2,9 3,8 5,0 5,5 6,6 5,8 Skolenivå 0,8 0,5 0,7 2,2 1,2 1,3 1,4 1,5 Samlet 2,7 3,4 3,5 5,9 6,1 6,7 7,9 7,3 PISA 2022 Noe lavere testmotivasjon enn i PISA 2018l I PISA 2022 var det inkludert flere mål på elevenes innsats og utholdenhet da de gjennomførte undersøkelsen. Rett etter at elevene hadde fullført prøven, vurderte de sin egen innsats på en skala fra 1 til 10, og tilsvarende for hvor stor innsats de ville ha lagt ned dersom prøven hadde hatt betydning for karakterene. Tabell 9 viser at gjennomsnittet har gått litt ned på begge disse spørsmålene om innsats for nordiske land og OECD-landene samlet. For å møte standardene i undersøkelsen er grensen for samlet eksklusjon satt til 5 prosent. Selv om samlet eksklusjon i Norge har vært over denne grensen siden 2009, har andelen endret seg lite etter dette og har derfor liten betydning for trendmålinger i denne 26 26 PISA 2022 26 PISA 2022 PISA 2022 motivasjon, er lavere i PISA 2022 enn tidligere år (tabell 11). To av tre elever rapporterer at de gjorde en god innsats på prøven, mens rundt halvparten av elevene rapporterer at de gjorde sitt beste og at de var motivert for å gjøre sitt beste. Det er en forholdsvis lav andel elever som rapporterer at det betød mye og at det var viktig for dem å gjøre det bra på PISA-prøven. Dette er ikke overraskende med tanke på at prøven ikke teller på elevenes karakterer og at ingen får kjennskap til prøveresultatet. Samtidig er det vanskelig å vite hvorfor det er en nedgang for disse og de andre utsagnene sammenliknet med de tidligere PISA-undersøkelsene. En mulighet er at denne nedgangen i motivasjon gjelder for skole og læring generelt, og at det er knyttet til turbulensen mange elever har opplevd gjennom ungdomsskolen som følge av pandemien. Tabell 9. Selvrapportert innsats på PISA-prøven for elever i de nordiske landene og OECD i PISA 2022 og 2018. Verdiene viser gjennomsnittlig innsats på PISA- prøven på en skala fra 1 til 10, der 10 er høyeste mulige innsats. Signifikante endringer fra 2018 til 2022 er uthevet. Gjennomsnittlig innsats på PISA-prøven Gjennomsnittlig innsats dersom elevene hadde fått karakter på PISA-prøven 2022 2018 Endring 2022 2018 Endring Norge 6,9 7,4 -0,5 9,0 9,2 -0,2 Danmark 7,3 7,5 -0,2 9,3 9,4 -0,1 Finland 7,7 8,0 -0,3 9,1 9,3 -0,2 Island 7,1 7,7 -0,5 8,8 9,1 -0,2 Sverige 7,2 7,4 -0,2 9,3 9,4 -0,1 OECD 7,5 7,7 -0,2 9,0 9,1 -0,1 Det ble også beregnet gjennomsnittsresultat for den første timen av prøven og for den andre timen av prøven. Noe lavere testmotivasjon enn i PISA 2018l Forskjellen i prestasjoner på den første og andre timen kan gi en indikasjon på elevenes utholden­­het i prøvesituasjonen. Tabell 10 viser at den gjennomsnittlige prestasjonen går ned fra den første til den andre prøvetimen for de fleste nordiske land og for OECD-landene samlet. Man kan forvente at utholdenheten blir lavere desto lenger man sitter med prøven. Endringen fra den første til den andre timen er ikke større for norske elever i PISA 2022 enn den var i PISA 2018. 2009 2012 2015 2018 2022 Jeg gjorde en god innsats på PISA-prøven 86 81 85 76 67 Jeg gjorde mitt beste på PISA-prøven 80 69 82 68 58 Jeg var motivert for å gjøre mitt beste på PISA-prøven 75 69 78 66 52 Jeg arbeidet med oppgavene uten å gi opp selv om noen av dem var vanskelige 70 60 72 64 55 Det betydde mye for meg å gjøre det bra på PISA-prøven 43 36 46 41 29 Det var viktig for meg å gjøre det bra på PISA-prøven 54 47 58 52 35 Tabell 10. Forskjell i gjennomsnittlig poengsum i de tre fagområdene fra den første til den andre prøvetimen. Signifikante endringer mellom den første og andre timen er uthevet med fet skrift. Prestasjonsforskjell fra den første til den andre prøvetimen Matematikk Naturfag Lesing 2022 2018 2022 2018 2022 2018 Norge -10 -9 -10 -19 -20 -24 Danmark -6 -9 -15 -12 -5 -18 Finland -3 -8 -9 0 -12 -16 Island -2 -12 -14 -14 -26 -20 Sverige -6 -10 -14 -14 -9 -21 OECD -4 -8 -8 -9 -14 -19 Tekniske utfordringer på enkelte skoler i Norge og Island g p g g I Norge og på Island opplevde noen elever som gjennomførte prøven på Chromebook, tekniske problemer. Det viste seg å skyldes kapasitetsproblemer på internasjonale servere, og feilen ble rettet tre uker inn i gjennomføringsperioden. I disse tre ukene var det 16 skoler i Norge som rapporterte om tekniske problemer. På disse skolene var det i overkant av 400 elever som deltok i PISA, det vil si 6–7 prosent av det totale utvalget. Analyser av dataene tyder på at responstid og responsrate er annerledes blant elevene på skoler med tekniske problemer, men at det ikke er merkbare forskjeller i prestasjoner eller for statistisk profil på oppgavene (item fit) for disse elevene sammenliknet med resten av utvalget. Den tekniske ekspertgruppen i PISA har likevel konkludert med at det er umulig å utelukke at de tekniske problemene kan ha påvirket elevenes engasjement og motivasjon, og at det kan ha hatt en negativ innvirkning på elevenes resultat på prøven. Til slutt i spørreskjemaet som elevene svarte på etter prøven, fikk de norske elevene på nytt spørsmål om innsatsen på PISA-prøven, og de tok stilling til påstander hvor svaralternativene var «Svært enig», «Enig», «Uenig» og «Svært uenig» (tabell 11). Konteksten for å svare på disse spørsmålene er dermed ulik det foregående spørsmålet (tabell 9), siden elevene hadde brukt enda mer tid på undersøkelsen. Her ser vi at andelen som er svært enig eller enig i de ulike utsagnene om Til slutt i spørreskjemaet som elevene svarte på etter prøven, fikk de norske elevene på nytt spørsmål om innsatsen på PISA-prøven, og de tok stilling til påstander hvor svaralternativene var «Svært enig», «Enig», «Uenig» og «Svært uenig» (tabell 11). Konteksten for å svare på disse spørsmålene er dermed ulik det foregående spørsmålet (tabell 9), siden elevene hadde brukt enda mer tid på undersøkelsen. Her ser vi at andelen som er svært enig eller enig i de ulike utsagnene om PISA 2022 27 Bakteppe for PISA 2022 Pandemien i et internasjonalt lys Elevene som deltok i PISA 2022, hadde pandemirestriksjoner alle årene de gikk på ungdomsskolen. Våren 2020, da pandemien brøt ut og skolene stengte, gikk disse elevene på 8. trinn. Figur 32 viser en tidslinje over ungdomsskoletida for populasjonen av elever som deltok i PISA 2022 og sammenfall med smittevernrestriksjoner i samme periode. j y Elever i alle deltakerland i PISA var selvsagt berørt av pandemien, men det er store variasjoner i hvor lenge skolene stengte fysisk og hvor god overgangen til digital undervisning ble (OECD, 2023e). I de fleste landene opplevde minst tre av fire elever skolestengning i en viss tid, og halvparten av elevene var på skoler som var stengt i mer enn tre måneder på grunn av pandemien. I gjennomsnitt opplevde 16 prosent av elevene i OECD-landene ingen skolestengning. Den landsdekkende skolestengningen våren 2020 var relativt kortvarig, men helt fram til februar 2022 ble det gjennomført lokale eller regionale skolestengninger på grunn av smittefare. For eksempel var 25 prosent av barne- og ungdomsskolene helt eller delvis stengt minst en gang vinteren 2021, i tråd med den såkalte trafikklys­ modellen (Utdanningsdirektoratet, 2021b). Under pandemien ble de yngste elevene prioritert for fysisk tilstedeværelse på skolen, mens ungdomstrinnet hadde en større andel digital hjemmeskole deler av tida (Utdanningsdirektoratet, 2021b). Konsekvenser for gjennomføringen av PISA-undersøkelsenl Konsekvenser for gjennomføringen av PISA-undersøkelsen På grunn av koronapandemien ble PISA-undersøkelsen flyttet fra 2021 til 2022, men også i 2022 var det krevende for mange land å gjennomføre en storskala skoleundersøkelse. Usikkerheten og ustabilitet knyttet til utviklingen i pandemien og smitteverntiltak på skoler og i samfunnet generelt bød på ekstra utfordringer med å planlegge og gjennomføre PISA-undersøkelsen. Mens det i de fleste land, inkludert Norge, ser ut til at undersøkelsen har blitt gjennomført som normalt, er det noen land som har hatt problemer med å innfri kriteriene for deltakelse blant utvalgte skoler og elever. Dette er nærmere beskrevet på side 29. Vi kan slå fast at det var stor variasjon blant PISA 2022-elevene med hensyn til hvordan skolehverdagen artet seg i løpet av ungdoms­ skolen. Oslo og nærområdet og en del av de andre store byene hadde gjentatte perioder med store begrensninger, mens mange mindre norske kommuner kun opplevde kortvarige smittebølger etter den første skolestengningen. Figur 32. Tidslinje over sammenfall mellom smittevernrestriksjoner på grunn av covid-19-pandemien og ungdomsskoletida for populasjonen som deltok i PISA 2022. 99 prosent av elever født i 2006 gikk i 8. klasse i 2020, mens 1 prosent fulgte undervisningen enten med årskullet over eller under. Figur 32. Tidslinje over sammenfall mellom smittevernrestriksjoner på grunn av covid-19-pandemien og ungdomsskoletida for populasjonen som deltok i PISA 2022. 99 prosent av elever født i 2006 gikk i 8. klasse i 2020, mens 1 prosent fulgte undervisningen enten med årskullet over eller under. 28 PISA 2022 28 PISA 2022 Tolkning av resultatendringer For PISA 2022 bruker OECD et enkelt, avrundet tall på 20 poeng som anslag for ett års gjennomsnittlig faglig progresjon. Dette er basert på studier av elevers utvikling i 30 land i PISA 2018 (Avvisati & Givord, 2023). Norske elever var ikke en del av denne studien, og det tas forbehold om at årlig læring ser ut til å variere mellom land og også mellom fag. Økt usikkerhet i PISA 2022 for merkede land For å sikre kvaliteten på dataene og lik gjennomføring av prøven i alle land, er PISA-undersøkelsen underlagt en rekke retningslinjer og tekniske standarder. Det stilles strenge krav til blant annet utvalg av elever og deltakelse, og det blir gjennomført en kvalitetskontroll Måleusikkerhet og utvalgsusikkerhet av dataene for hvert av deltakerlandene, hvor eventuelle avvik fra kriteriene rapporteres. PISAs tekniske ekspertgruppe, som har ansvar for å overvåke kvaliteten på dataene, har konkludert med at avvikene kan ha betydning for resultatene i noen land og for validiteten i slutningene som trekkes. I denne rapporten og i alle internasjonale publikasjoner er land med slike avvik merket for å signalisere at det er større usikkerhet knyttet til resultatene for disse landene enn for andre land. Måleusikkerhet og utvalgsusikkerhet I PISA-undersøkelsen er måleusikkerhet og utvalgsusikkerhet de to viktigste formene for usikkerhet. Måleusikkerhet har sammenheng med at hver elev bare får et utvalg av oppgaver, mens utvalgsusikkerhet er knyttet til at det er et utvalg av elever, og ikke hele populasjonen av 15-åringer, som deltar. I tillegg er det en ekstra usikkerhet, en såkalt lenkefeil, knyttet til det å sammenlikne resultater fra ulike PISA-gjennomføringer, for eksempel resultatene fra PISA 2022 med PISA 2018. I tabell 13, 14 og 15 vil noen land bli merket (se tabell 12). For noen land anslås usikkerheten til å være 5–10 poeng, mens for andre land er det ikke gjort et slikt anslag. Usikkerheten kan tallfestes for hvert resultat. For eksempel er gjennomsnittsresultatet i matematikk for norske elever 468 poeng i PISA 2022, og usikkerheten er på 4,1 poeng i hver retning (for et 95 prosent konfidensintervall). Det betyr at vi forventer at gjennom­ snittsresultatet til hele populasjonen befinner seg et sted mellom 464 og 472 poeng. Statistisk signifikans Når vi sammenlikner gjennomsnittsverdiene til to elevgrupper, er det viktig å være sikker på at forskjeller vi finner ikke skyldes tilfeldig­ heter i målingene eller i utvalget av elever som deltar. Ved å ta hensyn til usikkerheten knyttet til gjennomsnittsverdiene, er det mulig å beregne hvor stor sannsynlighet det er for at en forskjell mellom to elevgrupper skyldes slike tilfeldigheter. Hvis det er mindre enn 5 prosent sannsynlighet for at en forskjell skyldes tilfeldigheter, sier vi at det er et statistisk signifikant resultat. Når vi har et statistisk signifikant resultat, er det stor sannsynlighet for at resultatet ikke bare eksisterer i datasettet, men også i populasjonen dataene representerer. Tabell 12. Oversikt over land som er merket på grunn av forbehold om utvalgsusikkerhet i PISA 2022. Usikkerheten knyttes til eksklusjon, responsrate eller begge. eller begge. PISA 2022: Økt usikkerhet knyttet til eksklusjon1 responsrate2 Danmark X Latvia X New Zealand X Canada X Australia X Irland X Storbritannia X Nederland X X USA X gg PISA 2022: Økt usikkerhet knyttet til eksklusjon1 responsrate2 Danmark X Latvia X New Zealand X Canada X Australia X Irland X Storbritannia X Nederland X X USA X Når vi noen steder i denne rapporten skriver at det ikke er noen forskjell mellom to elevgruppers resultat, selv om man kan se en forskjell i tilhørende tabell eller figur, er det fordi denne forskjellen ikke er statistisk signifikant. PISA 2022 29 29 PISA 2022 29 Forklaringer til tabellverket Tabell 13, 14 og 15 oppsummerer hovedresultatene for OECD-landene i henholdsvis matematikk, naturfag og lesing. For hvert land er det oppgitt gjennomsnittlig resultat, med standardfeil i parentes, og spredningen i resultatene i form av standardavvik. Videre sammenliknes resultatene fra PISA 2022 med 2018 og med tidligere gjennomføringer da faget var hovedområde. Endringer som er signifikant forskjellig, er merket med stjerne (*). Det er også oppgitt dekningsgrad for hvert av landene (se mer om dette på side 26) og fordeling av elever på mestringsnivåer. Endring i OECD- gjennomsnittet over tid er basert på de OECD-landene som deltok det gjeldende året. Samlevariabler 10 prosent av elevene presterer under. For norske elever i PISA 2022 er 10-prosentilen 345 poeng i matematikk. Dette betyr at 10 prosent av norske elever presterer under 345 poeng i matematikk, mens 90 prosent av elevene presterer over 345 poeng. Tilsvarende er 90-prosentilen 589 poeng, som betyr at 90 prosent av norske elever presterer under 589 poeng, mens 10 prosent presterer over 589 poeng. I kapitlene om matematikkangst, lærende tankesett, likeverd og læringsmiljø presenterer vi resultater basert på samlevariabler. En samlevariabel er et mål som er satt sammen av flere spørsmål eller utsagn knyttet til et bestemt tema eller begrep. For eksempel er samle­ variabelen «Arbeidsro» basert på elevenes svar til utsagnene «Elevene hører ikke etter hva læreren sier», «Det er bråk og uro», «Læreren må vente lenge før elevene roer seg», «Elevene får ikke arbeidet ordentlig» og «Elevene begynner ikke å arbeide før lenge etter at timen har begynt». Skalaen for samlevariablene er standardiserte og basert på resultatene for OECD-landene, hvor gjennomsnittet er satt til 0 og standardavviket til 1. Prosentiler I noen tilfeller bruker vi prosentiler til å sammenlikne resultater for ulike elevgrupper. Prosentil viser i denne rapporten til et punkt på poengskalaen (en poengsum) som skiller ut en bestemt prosentandel av elevene, når elevene er sortert fra lavest til høyest poengsum. For eksempel viser 10-prosentilen til den poenggrensen som nøyaktig 30 30 PISA 2022 PISA 2022 Tabell 13. Resultater i matematikk for OECD-landene i PISA 2022. Land med et gjennomsnittsresultat som ikke er signifikant forskjellig fra det norske resultatet, er markert med grønn bakgrunn. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. Tabell 13. Resultater i matematikk for OECD-landene i PISA 2022. Land med et gjennomsnittsresultat som ikke er signifikant forskjellig fra det norske resultatet, er markert med grønn bakgrunn. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. 31 PISA 2022 31 PISA 2022 31 Tabell 14. Resultater i naturfag for OECD-landene i PISA 2022. Land med et gjennomsnittsresultat som ikke er signifikant forskjellig fra det norske resultatet, er markert med grønn bakgrunn. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. det norske resultatet, er markert med grønn bakgrunn. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. 32 PISA 2022 Tabell 15. Resultater i lesing for OECD-landene i PISA 2022. Land med et gjennomsnittsresultat som ikke er signifikant forskjellig fra det norske resultatet, er markert med grønn bakgrunn. Land med utfordringer med datakvalitet er merket (1, 2), se side 29 for forklaring. ( ) PISA 2022 33 33 PISA 2022 33 PISA 2022 33 PISA 2022 Deltakerland i PISA 2022 Tabell 16. Oversikt over land og regioner som deltar i PISA 2022. Land som har gjennomført prøven i PISA 2022 på papir, er merket *. og regioner som deltar i PISA 2022. Land som har gjennomført prøven i PISA 2022 på papir, er merket * Tabell 16. Oversikt over land og regioner som deltar i PISA 2022. Land som har gjennomført prøven i OECD-land Australia Belgia Canada Chile Colombia Costa Rica Danmark Estland Finland Frankrike Hellas Irland Island Israel Italia Japan Latvia Litauen Mexico Nederland New Zealand Norge Polen Portugal Slovakia Slovenia Spania Storbritannia Sveits Sverige Sør-Korea Tsjekkia Tyrkia Tyskland Ungarn USA Østerrike OECD-land Australia Belgia Canada Chile Colombia Costa Rica Danmark Estland Finland Frankrike Hellas Irland Island Israel Italia Japan Latvia Litauen Mexico Nederland New Zealand Norge Polen Portugal Slovakia Slovenia Spania Storbritannia Sveits Sverige Sør-Korea Tsjekkia Tyrkia Tyskland Ungarn USA Østerrike Land eller regioner som ikke er medlem av OECD Albania Argentina Baku (Aserbajdsjan) Brasil Brunei Bulgaria De forente arabiske emirater Den dominikanske republikk El Salvador Filippinene Georgia Guatemala* Hongkong (Kina) Indonesia Jamaica Jordan Kambodsja* Kasakhstan Kina Kosovo Kroatia Kypros Macao (Kina) Malaysia Malta Marokko Moldova Mongolia Montenegro Nord-Makedonia Palestina Panama Paraguay* Peru Qatar Romania Saudi-Arabia Serbia Singapore Taipei (Taiwan) Thailand Ukraina (18 av 27 regioner) Uruguay Usbekistan Vietnam* Land eller regioner som ikke er medlem av OECD Land eller regioner som ikke er medlem av OECD Singapore Taipei (Taiwan) Thailand Ukraina (18 av 27 regioner) Uruguay Usbekistan Vietnam* Kypros Mongolia Peru Qatar Romania 34 PISA 2022 4 PISA 2022 Litteratur Kilpatrick, J., Swafford, J., Findell, B., Mathematics Learning Study Committee & National Research Council Center for Education. (2001). Adding it up: Helping children learn mathematics. National Academy Press. Denne rapporten er basert på resultater som er gitt i de to første bindene av den internasjonale rapporten, som blant annet inne­ holder tabeller med detaljerte resultater for alle deltakerlandene. Kjærnsli, M. & Jensen, F. (2016). Naturfag i PISA: Definisjon og oppgaver. I M. Kjærnsli & F. Jensen (Red.), Stø kurs. Norske elevers kompetanse i naturfag, matematikk og lesing i PISA 2015 (s. 32–49). Universitetsforlaget. Ashcraft, M. H. (2002). Math anxiety: Personal, educational, and cognitive consequences. Current Directions in Psychological Science, 11(5), 181–185. https://doi.org/10.1111/1467-8721.00196 Kunnskapsdepartementet. (2021). Skolen etter koronapandemien. Et løft for trivsel og læring (Rapport fra arbeidsgruppen for tapt læring). https://www. regjeringen.no/no/dokumenter/skolen-etter-koronapandemien/id2861088/ Avvisati, F. & Givord, P. (2023). The learning gain over one school year among 15-year-olds: An international comparison based on PISA. Labour Economics, 84. https://doi.org/10.1016/j.labeco.2023.102365 Mælan, E. N., Gustavsen, A. M., Stranger-Johannessen, E. & Nordahl, T. (2021). Norwegian students experiences of home schooling during the COVID-19 pandemic. European Journal of Special Needs Education, 36(1), 5–19. https:// doi.org/10.1080/08856257.2021.1872843 Carey, E., Hill, F., Devine, A. & Szücs, D. (2016). The chicken or the egg? The direction of the relationship between mathematics anxiety and mathe­ matics performance. Frontiers in Psychology, 6. https://doi.org/10.3389/ fpsyg.2015.01987 Niss, M. & Højgaard, T. (2019). Mathematical competencies revisited. Educa­ tion Studies in Mathematics, 102, 9–28. https://doi.org/10.1007/s10649-019- 09903-9 Caspersen, J., Hermstad, I. H., Hybertsen, I. D., Lynnebakke, B., Vika, K. S., Smedsrud, J., Wendelborg, C. & Federici, R. A. (2021). Koronapandemien i grunnskolen – håndtering og konsekvenser (NIFU-rapport). Nordisk insti­ tutt for studier av innovasjon, forskning og utdanning. https://hdl.handle. net/11250/2733034 OECD. (2017). PISA 2015 results: Vol. 3. Students’ well-being. OECD Publis­ hing. https://doi.org/10.1787/9789264273856-en OECD. (2023a). PISA 2022 assessment and analytical framework. PISA, OECD Publishing. https://doi.org/10.1787/dfe0bf9c-en Cimpian, A., Arce, H.-M. C., Markman, E. M. & Dweck, C. S. (2007). Subtle linguistic cues affect children’s motivation. Psychological Science, 18(4), 314–316. https://doi.org/10.1111/j.1467-9280.2007.01896.x OECD. (2023b). PISA 2025 science framework draft. OECD Publishing. https://pisa-framework.oecd.org/science-2025/ Dowker, A., Sarkar, A. & Looi, C. Y. (2016). Mathematics anxiety: What have we learned in 60 years?. Frontiers in Psychology, 7. https://doi.org/10.3389/ fpsyg.2016.00508 OECD. (2023c). PISA 2022 technical report. OECD Publishing. Dweck, C. S. & Yeager, D. S. (2019). Mindsets: A view from two eras. Perspectives on Psychological Science, 14(3), 481–496. https://doi. org/10.1177/1745691618804166 OECD. (2023d). PISA results 2022: Vol. Litteratur Prosjekt­ gruppen vil takke skolene og elevene som deltok i PISA 2022, både ved utprøvingene i 2020 og 2021 og i hovedgjennomføringen i 2022. Denne rapporten bygger på tidligere publikasjoner som finnes på pisa.no. Litteratur 1. The state of learning and equity in education. OECD Publishing. OECD. (2023e). PISA 2022 results – examining resilience. Learning during – and from – disruption. OECD Publishing. Gunderson, E. A., Gripshover, S. J., Romero, C., Dweck, C. S., Goldin‐Meadow, S. & Levine, S. C. (2013). Parent praise to 1‐ to 3‐year‐olds predicts children’s motivational frameworks 5 years later. Child Development, 84(5), 1526–1541. https://doi.org/10.1111/cdev.12064 Utdanningsdirektoratet. (2020). Læreplan i matematikk 1.–10. trinn (MAT01‑05). https://www.udir.no/lk20/mat01-05 Jensen, F., Kjærnsli, M., Björnsson, J. K. & Pettersen, A. (2020). Gir norsk skole alle elever like muligheter til å bli gode lesere? I T. S. Frønes & F. Jensen (Red.), Like muligheter til god leseforståelse? 20 år med lesing i PISA (s. 222–241). Universitetsforlaget. https://doi.org/10.18261/9788215040066-2020-09 Kilpatrick, J. (2020). Competency frameworks in mathematics education. I S. Lerman (Red.), Encyclopedia of mathematics education (s. 110–113). Springer. https://doi.org/10.1007/978-3-030-15789-0_27 Utdanningsdirektoratet. (2021). Utdanningsspeilet 2021. https://www.udir.no/ tall-og-forskning/publikasjoner/utdanningsspeilet/utdanningsspeilet-2021/ Weyergang, C. & Magnusson, C. G. (2020). Hva er relevant lesekompetanse i dagens samfunn, og hvordan måles lesing i PISA 2018? I T. S. Frønes & F. Jensen (Red.), Like muligheter til god leseforståelse? 20 år med lesing i PISA (s. 46–78). Universitetsforlaget. https://doi.org/10.18261/9788215040066-2020-03 Jensen, F., Kjærnsli, M., Björnsson, J. K. & Pettersen, A. (2020). Gir norsk skole alle elever like muligheter til å bli gode lesere? I T. S. Frønes & F. Jensen (Red.), Like muligheter til god leseforståelse? 20 år med lesing i PISA (s. 222–241). Universitetsforlaget. https://doi.org/10.18261/9788215040066-2020-09 Utdanningsdirektoratet. (2021). Utdanningsspeilet 2021. https://www.udir.no/ tall-og-forskning/publikasjoner/utdanningsspeilet/utdanningsspeilet-2021/ Weyergang, C. & Magnusson, C. G. (2020). Hva er relevant lesekompetanse i dagens samfunn, og hvordan måles lesing i PISA 2018? I T. S. Frønes & F. Jensen (Red.), Like muligheter til god leseforståelse? 20 år med lesing i PISA (s. 46–78). Universitetsforlaget. https://doi.org/10.18261/9788215040066-2020-03 Weyergang, C. & Magnusson, C. G. (2020). Hva er relevant lesekompetanse i dagens samfunn, og hvordan måles lesing i PISA 2018? I T. S. Frønes & F. Jensen (Red.), Like muligheter til god leseforståelse? 20 år med lesing i PISA (s. 46–78). Universitetsforlaget. https://doi.org/10.18261/9788215040066-2020-03 Kilpatrick, J. (2020). Competency frameworks in mathematics education. I S. Lerman (Red.), Encyclopedia of mathematics education (s. 110–113). Springer. https://doi.org/10.1007/978-3-030-15789-0_27 PISA 2022 35 PISA 2022 35 35 PISA 2022 Denne kortrapporten presenterer de norske resultatene fra PISA 2022. Undersøkelsen er finansiert av Utdanningsdirektoratet og er gjennomført av Institutt for lærerutdanning og skoleforskning ved Universitetet i Oslo med Fredrik Jensen som prosjektleder. Flere norske PISA-resultater høsten 2024 Det vil publiseres en egen antologi om matematikk i PISA- undersøkelsene 2003–2022 i løpet av høsten 2024. Antologien skrives av en større forfattergruppe med bakgrunn i matematikkforskning og vil bestå av primær- og sekundæranalyser av norske resultater i matematikk, med stor vekt på nordiske sammenlikninger. I flere av kapitlene suppleres analyser av PISA-materialet med mindre, kvalitative studier. Temaer i antologien vil blant annet være matematisk modellering, resonnering og argumentasjon, matematikkundervisning og hvor like muligheter elever har til å lære matematikk. Mer informasjon om PISA og hvordan vi måler elevers kompetanse i matematikk, naturfag og lesing finnes på pisa.no og oecd.org/pisa.
https://openalex.org/W2075999382
https://europepmc.org/articles/pmc3644241?pdf=render
English
null
Development of a green binder system for paper products
BMC biotechnology
2,013
cc-by
10,212
RESEARCH ARTICLE Open Access © 2013 Flory 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. Development of a green binder system for paper products Ashley R Flory1,2, Deborah Vicuna Requesens1, Shivakumar P Devaiah1, Keat Thomas Teoh1,3, Shawn D Mansfield4 and Elizabeth E Hood1,3* Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Abstract Background: It is important for industries to find green chemistries for manufacturing their products that have utility, are cost-effective and that protect the environment. The paper industry is no exception. Renewable resources derived from plant components could be an excellent substitute for the chemicals that are currently used as paper binders. Air laid pressed paper products that are typically used in wet wipes must be bound together so they can resist mechanical tearing during storage and use. The binders must be strong but cost-effective. Although chemical binders are approved by the Environmental Protection Agency, the public is demanding products with lower carbon footprints and that are derived from renewable sources. Results: In this project, carbohydrates, proteins and phenolic compounds were applied to air laid, pressed paper products in order to identify potential renewable green binders that are as strong as the current commercial binders, while being organic and renewable. Each potential green binder was applied to several filter paper strips and tested for strength in the direction perpendicular to the cellulose fibril orientation. Out of the twenty binders surveyed, soy protein, gelatin, zein protein, pectin and Salix lignin provided comparable strength results to a currently employed chemical binder. Conclusions: These organic and renewable binders can be purchased in large quantities at low cost, require minimal reaction time and do not form viscous solutions that would clog sprayers, characteristics that make them attractive to the non-woven paper industry. As with any new process, a large-scale trial must be conducted along with an economic analysis of the procedure. However, because multiple examples of “green” binders were found that showed strong cross-linking activity, a candidate for commercial application will likely be found. Keywords: Paper industry, Binders, Enzymes, Plant-produced proteins, Green chemistry Polymerized plant cell wall components result in a re- markably strong structure in nature. Since paper is made primarily of one of those polymers, cellulose, it is possible to simulate a cell wall assembly by applying and cross- linking other cell wall constituents with enzymes, such as laccase and peroxidases, which are responsible for this cross-linking in vivo. The ensuing interwoven network of substrates may increase paper tensile strength and provide an alternative to chemical binders. In this work, proteins, carbohydrates, and phenolic compounds were applied to paper with and without enzyme activation to determine if an increase in tensile strength could be achieved. * Correspondence: ehood@astate.edu 1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72467, USA 3College of Agriculture and Technology, Arkansas State University, Jonesboro AR 72467, USA Full list of author information is available at the end of the article Background These compounds can cross-link to themselves, to each other, or to a growing lignin polymer with the use of laccase [2]. Black liquor is a by-product from the pulp and paper in- dustry generated when wood pulp is chemically digested and bleached to remove lignin. Bleaching not only results in the removal of lignin, but also ferulic acid, cellulose, and hemicelluloses [3]. Because black liquor is a by- product generally used for heat energy, but rich in poten- tially valuable chemicals, it provides an inexpensive source of cell wall substrates. Since many of the substrates found in black liquor are phenolic compounds, this project sought to employ black liquor as a cross-linking agent in paper manufacture. However, black liquor obtained from different sources can have varying properties, depending on feedstock stream and pulping process employed at the pulp mill, and consequently is compositionally different. Another protein of interest was hydroxyproline-rich gly- coproteins (HRGP), which are highly glycosylated proteins found in the cell wall that contain repeated sequences of serine and proline, as well as an abundance of the amino acid tyrosine [6]. Peroxidase, which requires hydrogen per- oxide to facilitate its reactions, is responsible for moving electrons so that tyrosine can attach to other tyrosine resi- dues resulting in cross-linked HRGP in the cell wall [7]. Since this protein has been shown to cross-link in a peroxide-mediated fashion, it was also a suitable candidate as a paper binder. Elegir et al. [3] previously described an experiment that attempted to use phenolic compounds as paper binders. This group isolated lignin from black liquor and cross-linked it with the use of laccase and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in an attempt to increase the tensile strength of hot pressed pulp sheets. The authors found that the cross-linking of lignin provided more than a two-fold increase in wet tensile strength of the pulp sheet. However, dry tensile strength was negatively affected. As an alternative ap- proach, Mansfield [4] demonstrated that directly impreg- nating radiate pine with laccase during mechanical pulp production could not only reduce the refining energy re- quired to attain a target freeness, but also had enhanced paper strength properties. This strategy takes advantage of the cross-linking capacity of laccase to enhance the inter-fiber bonding using the native wood fiber lignin remaining on the pulp fibers. Background Globally, paper companies apply chemical binders during the paper-making process to attain target tensile strength of paper. Some components of these binders are acryl- amide, acetaldehyde, urea-formaldehyde and vinyl acetate. Alternatives to synthetic paper binders have been investi- gated, but a renewable binder that can equal the strength of the traditional binders has yet to be identified. Plant- based products would be an ideal alternative to chemicals currently used in the paper industry. * Correspondence: ehood@astate.edu 1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72467, USA 3College of Agriculture and Technology, Arkansas State University, Jonesboro, AR 72467, USA Full list of author information is available at the end of the article Phenolic compounds are the first and largest category of the three main plant-derived substrates that could poten- tially be utilized to create a strong binder for paper. They are made up of aromatic carbon rings with hydroxyl Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 2 of 14 from soybean and wheat as binders in cellulosic paper composites. In their experiments, they used the total pro- tein found in soybeans and wheat protein (gluten) to make a slurry with water, urea, sodium hydroxide, and acryl- amide. They found that wheat gluten increased paper strength as much as 60%, while total soy protein increased the paper strength by 46% compared to paper with no binder added. The authors concluded that although gluten was more effective at increasing tear strength of paper, soy protein would be better suited as a bulk binder because it is less expensive than gluten. While these proteins did not provide enough increase in tensile strength to replace the current chemical binders, it is possible to decrease the amount of synthetic binder used by supplementing the in- dustrial binder with this plant-derived protein binder. In the current project, we isolated soy proteins from defatted soybean meal, which were applied to paper, with and with- out enzyme, to determine if a significant increase in tensile strength would be possible. groups attached, and are a major component of the sec- ondary cell wall. Lignin is a complex phenolic polymer whose composition can change depending on what pre- cursors are involved in its assembly [1]. A few of the pre- cursors to lignin are ferulic acid, and sinapyl and coniferyl alcohol. Background Gel electrophoresis results with HRP showed that the increase in HRGP’s mo- lecular weight occurred most efficiently at 50°C after 1 conformation that will allow it to lay flat against the cellu- lose of paper. While this protein may provide adequate tensile strength, there may be problems implementing it in a commercial setting because of ethical and health con- cerns about using animal products. The last group of potential binders is carbohydrates. Carbohydrates are the most abundant class of organic compounds found in living organisms, vegetal and animal indistinctively. In this work, keeping within the scope of developing a green binder, we chose to test carbohydrates derived from plant sources, algae or fruit. Since these po- tential binders are made up of polysaccharides, laccase and peroxidase will not effectively cross-link these sub- strates. However, the experiment performed by Fahmy et al. [5] with soy protein and wheat gluten suggests that enzymatic cross-linking of substrates may not be neces- sary to produce a strong binder. Table 1 Summary of all substrates tested as binder Substrate category Substrate Source Protein Soy Protein Defatted soybean meal (Arkansas State University) HRGP Corn silk (Arkansas State University) Gelatin Knox Gelatin (Kraft Foods, Glenview, IL) JELL-O (Kraft Foods, Glenview, IL) Bovine & Porcine (Great Lakes Gelatin, Grayslake, IL) Zein Acros Organics, Geel, Belgium Carbohydrates Agar Seng Huad Limited Partnership (Bangkok, Thailand) Agarose Genetic Analysis (Fisher Scientific, Pittsburg, PA) Analytical Grade (Promega, Madison, WI) Pectin Sure-Jell (Kraft Foods, Glenview, IL) Ball (Jarden Home Brands, Daleville, IN) Apple (Sigma Chemical Company, St. Louis, MO) Grapefruit (Source Naturals, Inc., Scotts Valley, CA) Gum Arabic Sigma Chemical Company, St. Louis, MO Xanthan Gum Kountry Kupboard, Jonesboro, AR Locust Bean Gum Sigma Chemical Company, St. Louis, MO Carrageen Sigma Chemical Company, St. Louis, MO Kelp Powder Now Foods, Bloomingdale, IL Flaxseed Now Foods, Bloomingdale, IL Lignin/ Black Liquor Buckeye Technologies Inc., Memphis, TN Phenolic Compounds Lignin Low Sulfonate Sigma Chemical Company, St. Louis, MO Sodium Lignin Sulfonate MP Biomedicals, LLC, Solon, OH Salix Vertichem, Toronto, Canada Marasperse Lignotech, Rothschild, WI Ferulic Acid Sigma Chemical Company, St. Louis, MO Coniferyl Alcohol Sigma Chemical Company, St. Louis, MO Table 1 Summary of all substrates tested as binder We aimed to produce a renewable binder to replace chemical binders currently used by the paper industry in order to increase tensile strength of non-woven papers. Background Zein is an alcohol-soluble storage protein found in maize seed that is known as an excellent film-maker be- cause of its ability to cross-link. Kim et al. [8] report that reagents such as glutaraldehyde, epicholorhydrin and citric acid, induce cross-linking between zein molecules. An in- crease in tensile strength was reported in zein films after cross-linking occurred. These properties of zein protein suggested it could be an organic, renewable alternative for a paper binder. Although plant components are excellent choices for paper binders, some animal proteins may also serve the same purpose. Gelatin is a denatured form of collagen that is derived mainly from skin and bone of bovine and porcine sources (http://www.rousselot.com/en/rousselot- gelatine/gelatine-characteristics/definitions/gelatine-bloom/). Gelatin is tested and graded according to its bloom strength. Bloom strength is defined as the force, expressed in grams, necessary to depress a 6.67% gel (kept 17 hours at 10°C) by 4 mm with a standard plunger. Bloom strength generally ranges between 50 to 300 g and a higher bloom means stronger, usually more expensive gelatin. Gelatin is similar to HRGP in that it is inherently rich in hydroxy- proline residues, thus it has side chains available for cross-linking with peroxidase, and possesses a native Some cross-linking experiments have been performed simply to understand mechanisms by which cell wall en- zymes and substrates interact. Iiyama et al. [2] describe mechanisms for covalent cross-linking of polysaccharides, proteins, phenolic compounds, and hydroxycinnamic acids in cell walls, but any applications of these results was not discussed. Given that the mechanisms and compounds involved in these chemical reactions are understood, these processes may be replicated in vitro and then applied in the paper industry as a renewable, green paper binder. The second type of substrate predicted to be a strong binder in these experiments was protein. Many types of protein have already been utilized for making paper binders. Fahmy et al. [5] describe the use of plant proteins Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 3 of 14 glycoprotein, or HRGP extracted from maize silk, is ap- proximately 75 kDa. As heat and reaction time were in- creased, the band corresponding to HRGP at 75 kDa lessens in intensity and the bands at 160 kDa and those above 220 kDa become more intense. Background We studied the potential of oxidoreductase enzymes to cross-link substrates and produce an interwoven network of substrates within the cellulose of paper, thereby increas- ing tensile strength. Alternatively, these molecules may cross-link without added enzymes. In order to replace the chemical binder with an organic binder, the strength of paper had to be at least equal to that of the paper with a chemical binder. We found several potential binder com- pounds that produced adequate strength. Results O Our approach was to test a variety of binders and condi- tions on 1 × 0.5 inch strips of Whatman #1 filter paper. Binders were applied by submersion into solution, the paper was dried 10 minutes at high temperature and the strength was tested in the direction perpendicular to the orientation of the cellulose fibers, as this is the weakest dir- ection and the standard in the industry. In some cases en- zymes were used. To determine the utility of the enzymes, they were first tested with substrates without the paper strips to determine optimal reaction conditions. A list of all potential binders tested is found in Table 1. Tear weights for all groups of green binders were statistically tested for differences from the commercial binder control (Table 2). We were interested in binders that were as strong or stron- ger than the commercial binder. Enzyme reactions T Two enzymes were tested in this study, horseradish peroxidase (HRP) and laccase. Protein gels were used to determine HRP’s optimum conditions in cross-linking proteins. Figure 1A shows reactions incubated for 10, 30 and 60 minutes at 22, 28 and 37°C. HRP is approximately 50 kDa is size, whereas the substrate, hydroxyproline-rich Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 4 of 14 Table 2 Binder differences from the commercial binder Binder Equal to commercial binder1 Higher than commercial binder Proteins HRGP + 300 ug HRP No 3% Zein Yes 5% Zein Yes TSP (Pellet Resuspended) Yes TSP/ 250 μg HRP (10 min) No 9% Knox Gelatin Yes 9% Knox Gelatin + 200 ug HRP No Yes 9% JELL-O Yes Porcine 300 Bloom Yes Bovine 250 Bloom Yes Carbohydrates 0.8% Agar No 3% Agar (Analytical) No 1% Agar (Genetic Analysis) No 11% Sure-Jell (low sugar) No 9% Sure- Jell No 1% Gum Arabic No 5% GA + 250 ug HRP No Kelp No Flax Seed No Xanthan Gum No Locust Bean Gum No 1% Carrageen No Pectins 5% Ball Pectin Yes 7% Ball Pectin Yes 9% Ball Pectin Yes 5% Apple No 5% Apple + 1N HCl No 5% Apple+CA (0.50 g) Yes 3% Grapefruit No 3% Grapefruit + CA (0.23 g) Yes Lignins and Phenolics Black Liquor (Heat) No Black Liquor + HCl No Lignin Low Sulfonate (Heat) No Lignin Low Sulfonate + HCl No 5% Salix (Heat) Yes Lignin Sulfonate (Heat) No Table 2 Binder differences from the commercial binder (Continued) Lignin Sulfonate + HCl No Marasperse (Heat) No Marasperse + HCl No Ferulic Acid (2 mg/ml) No Coniferyl Alcohol (2 mg/ml) No The goal of these experiments was to find a green binder that was not significantly lower than the currently used commercial binder. All binders that were not significantly different at the 95% confidence level are marked “Yes”. Only the Knox gelatin binders were significantly higher than the commercial binder. 1Samples that are not significantly different are those that perform as well as the commercial binder. All samples that are significantly different have lower values except for the gelatin, which was significantly higher. See Methods for statistical treatments. Table 2 Binder differences from the commercial binder (Continued) minute of reaction using water as a buffer (pH approxi- mately 7; Figure 1B). Enzyme reactions T Figure 1B shows reactions that were incubated at 37, 42 and 50°C for 1, 5 and 10 minutes. The same concentration of substrate and enzyme was loaded on both gels shown in Figure 1A and B. The band correspond- ing to HRGP at 75 kDa is clearly seen at room temperature and 28°C in Figure 1A. This band is fainter and eventually disappears as the temperature increases to 50°C. In order to determine the efficacy of HRP on phenolic compounds, samples were analyzed by gel permeation chromatography. Low and high concentrations of HRP (15 μg and 150 μg) were combined separately with black liquor, lignin and ferulic acid before chromatographic analysis. Figure 2 shows a dramatic change in molecular weight when HRP is added to ferulic acid compared to when ferulic acid was analyzed alone. Only slight in- creases in molecular weight were observed in black li- quor and lignin when HRP was added to these samples. In order to test for activity of laccase on phenolic com- pounds, this enzyme was combined separately with black liquor, low sulfonate lignin and ferulic acid. It was then an- alyzed for molecular weight changes by gel permeation chromatography. Figure 3 shows that when lignin and lac- case were combined, lignin eluted earlier than when lignin was run by itself, demonstrating an increase in molecular weight of the lignin stemming from laccase cross-linking. These results also indicate there was no change in mo- lecular weight when laccase was combined with black li- quor and only a slight change when this enzyme was combined with ferulic acid under the conditions tested. The peaks of ferulic acid in Figures 2 and 3 vary because conditions for GPC detection were adjusted for the second compound in the mix rather than the ferulic acid and are thus are not quantitative. Protein binders l Several proteins were tested as binders using small paper strips (Figure 4). The units on the y-axis show the weight (in grams) necessary to tear the paper strips. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 5 of 14 Figure 1 SDS-PAGE of HRGP and HRP reactions, A. RT to 37°C; B. 37°C to 50°C. C to 50°C. Control strips of paper were received from a paper manufacturer with their vinyl acetate binder already ap- plied. The column labeled “Commercial Binder” shows the weight needed to tear paper with the industrial binder applied. The column labeled “No Binder” refers to the average weight needed to tear three pieces of wet paper in the cross direction with no binder applied. For each binder several concentrations of enzyme and sub- strate were tested, as well as different incubation times with the various enzyme preparations. Strength values for selected experiments are shown. to application to the paper strips. Enzymes were only used when working with gum arabic since this polysaccharide also contains some gylcoprotein [9]. Strength results from agar and two different grades of agarose (analytical grade and genetic analysis grade – see Table 1) are shown in Figure 5. These binders were able to withstand between 337 and 458 g of weight. Ball pectin’s average tear weight (745 g) was slightly higher than that of the commercial binder but not significantly different. Regular Sure-JellW pectin was the weakest of the three pectins evaluated holding only 495 g, while low sugar Sure-Jell was intermediate, withstanding 584 g of weight. Xanthan gum and locust bean gum binders resulted in no extra strength added to the paper. Carrageen also served as a very weak binder only holding 243 g of weight com- pared to the commercial binder. Brown flax seed was ground with liquid nitrogen, then mixed with water and applied to paper, and was able to hold 548 g of weight. These carbohydrate binders were all significantly lower than the commercial binder. Knox gelatin with HRP was able to withstand 925 grams of weight before the paper tore, a significantly higher tear weight (95% confidence level) compared to the commercial binder which tore at 720 grams weight. Even without enzyme, the Knox gelatin binder was able to withstand 819 g before tearing which was not differ- ent from the commercial binder. Carbohydrate binders Several carbohydrates were also used as substrates in this study (Figure 5). Results show the concentration of sub- strate that produced the strongest binding strength. Each vertical bar is an average of the weight it took to tear three individual pieces of wet paper in the cross direction. All carbohydrate binders were dissolved in boiling water prior Protein binders l Textured soy protein held slightly less weight than gelatin (790 g), but still outperformed the commercial binder. Zein protein served as an average binder withstanding 574 grams of weight. Zein and TSP were not significantly different from the commercial binder. The weakest protein binder was HRGP, which was only able to hold about 77 g be- fore the paper tore. As shown in Figure 6, pure pectin (apple and grapefruit) applied to paper resulted in weak binding, only holding between 230 and 360 g of weight. However, when an acidic component such as citric acid was added, the binder was capable of withstanding between 680 and 740 g, re- spectively, allowing pectin to equal that of the commercial binder. Pectin concentration was also shown to affect the binder strength, e.g., 5% pectin holds about 100 g less weight than 7 or 9% pectin, although this amount is still not significantly lower than the commercial binder. Baby wipe solution test y In order to determine the efficacy of our experiments for commercial applications, we tested our bound paper soaked in baby wipe solution over a 3 month period. Baby wipe so- lution tests were performed on paper treated with the ball pectin binder exclusively since this was the strongest green binder from a non-animal source tested in previous experi- ments. These values were compared to strength values pro- duced by a vinyl acetate binder currently in use. We observed no significant differences between the chemical binder and the Ball pectin binder used in this experiment, before application of the baby wipe solution or after 3 months soaking in the solution (data not shown). Phenolic binders Finally, lignin and phenolic compounds were tested as po- tential binders. For each type of lignin applied to the paper Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 6 of 14 Figure 2 Gel permeation chromatography of various substrates with HRP. strips, two types of experiments were carried out: lignin combined with hydrochloric acid or lignin alone, dried at high temperatures, between 100°C and 200°C. Figure 7 shows that lignin sulfonate and marasperse were only able to hold 103 g weight when high heat was applied. How- ever, black liquor and lignin low sulfonate were able to withstand 337 and 456 g, respectively, when they were dried at 200°C. When 1N HCl was sprayed onto lignin coated paper strips, lignin sulfonate and marasperse were able to hold 123 and 198 g, respectively. The black liquor binder held 290 g and lignin low sulfonate 282 g after be- ing sprayed with HCl. Neither coniferyl alcohol nor ferulic acid was capable of holding much weight, tearing at 65 g. Salix lignin when dried at 200°C was the strongest lignin/ phenolic compound binder, holding 714 g, the only phen- olic binder that was equal to the commercial binder. The Salix lignin comprises an alcohol soluble fraction from ground wood that contains complex polymerized forms of lignin (USP# 8,053,566). Discussion Several proteins, carbohydrates, and lignins were tested as paper binders to explore the possibility of replacing chemical binders currently used by manufacturers of non-woven paper products with an alternative green, re- newable compound. Horseradish peroxidase and laccase were combined with green binders in order to initiate cross-linking between these molecules. We tested envir- onmentally friendly binders that could be implemented into a commercial setting while still providing the same strength as the commonly used vinyl acetate binder. In order to determine optimum conditions for the en- zymes, cross-linking tests were performed by using molecu- lar weight determination of cross-linked proteins with SDS-PAGE, or gel permeation chromatography for cross- linked lignins. Protein gels showed that HRP cross-linked HRGP, increasing its molecular weight. Otte and Barz [10] conducted a similar experiment to investigate if cross- linking was occurring between an extensin-like protein and a proline-rich protein extracted from chickpea cell walls. Figure 2 Gel permeation chromatography of various substrates with HRP. Figure 2 Gel permeation chromatography of various substrates with HRP. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 7 of 14 Page 7 of 14 Figure 3 Gel permeation chromatography of various substrates with laccase. The authors performed SDS-PAGE analysis to demonstrate the formation of one large band above 205 kDa from two protein bands of 60 and 190 kDa when treated with HRP and H2O2, similar to the results shown in the present work. Therefore, it can be concluded that cross-linking of HRGP is occurring, as described by Deepak et al. [11]. Gel permeation chromatography (GPC) was used to es- tablish conditions for reactions using phenolic compounds. GPC is commonly used to distinguish differences in mo- lecular weight when trying to determine if cross-linking is occurring using oxidoreductase enzymes and phenolic compounds as substrates. Felby et al. [12] described an ex- periment that used laccase to cross-link lignin in order to create stronger fiberboards. The authors used GPC to compare molecular weight of lignin extracted from pulp and fiberboards to lignin from each source treated with laccase. In a similar experiment, Felby et al. [13] showed an increase in strength properties of beech wood fiber- boards when laccase was used compared to boards that did not have the enzyme treatment. Discussion In the present work, GPC results showed little to no change in molecular weight when enzymes were added to substrates, with the exception of ferulic acid and HRP which showed a dramatic increase in molecular weight. Because laccase was shown to have such a minimal effect on its substrates, its use was minimized in binder strength tests on paper. Also, because of the GPC results, HRP was primarily used with proteins and not with lignin or other phenolic compounds. Lignin samples were analyzed with GPC and prelimin- ary results led us to predict changes could be made to improve efficiency of laccase reaction conditions. Even though pH 5 sodium acetate buffer was used in all reac- tions as suggested by Bailey et al. [14], this buffer was not able to establish an ideal pH for the reaction to occur, as the lignin is extremely basic in nature. Black li- quor has an especially high pH of 10–12 and the sodium acetate was only able to maintain the reaction conditions to approximately pH 8. Madzak et al. [15] showed that laccase has an activity range of pH 2 to 6. Also, we predict supplementing the reaction with oxy- gen would improve the efficacy of the laccase reaction. Laccase works by removing electrons from oxygen, transferring them to its substrates, thereby creating free radicals or reactive species that will cross-link with each other [16]. All reactions analyzed by GPC were carried out in closed 1.5 ml Eppendorf tubes. Although these tubes were shaken during the reaction, fresh oxygen ex- change could not occur. Mattinen et al. [17] showed the importance of oxygen in the reaction by mixing laccase and lignin together and measuring the amount of oxygen consumed over a 3 hour period with an oxygen elec- trode. After these solutions were shaken for 3 hours in a sealed glass flask, the oxygen concentration remaining in Figure 3 Gel permeation chromatography of various substrates with laccase. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 8 of 14 Figure 4 Summary of average tensile strength of protein binders. Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. HRP: Horseradish peroxidase. HRGP: Hydroxyproline-rich glycoprotein. TSP: Textured soy protein. Figure 4 Summary of average tensile strength of protein binders. Discussion Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. HRP: Horseradish peroxidase. HRGP: Hydroxyproline-rich glycoprotein. TSP: Textured soy protein. the flask was very low for those reactions that performed efficiently. To resolve this problem of depleted oxygen, Elegir et al. [3] supplemented more oxygen than was re- quired by the reaction, ensuring oxygen would not be the limiting factor. smoothness. A Sheffield test is commonly used to test the quality of paper sizing, where the longer it takes for paper to absorb a liquid, the better. Gelatin applied to paper not only improves the surface properties, but also minimizes the effect of aging and photooxidation [19]. However, it is not clear if the use of gelatin in this way has ever been shown to increase the tear strength of paper. Mediators such as 2,2'-azino-bis(3-ethylbenzthiazoline- 6-sulphonic acid) (ABTS) have also been employed to induce a conformational change in laccase that results in greater access to the enzyme’s active site. If the active site is more accessible, the enzyme should work more ef- ficiently to cross-link substrates [2]. However, enzymes and mediators are expensive and not likely to be used in manufacturing unless substantial benefit can be gained in product quality or process efficiency. Gelatin could be easily implemented into the paper in- dustry process. It is organic, renewable, readily available at low price, needs little reaction time to produce high tear strength, and produces a non-viscous solution that will not clog sprayers. Textured soy protein solution has all of these advantages as well. However, if using the resuspended pellet of soy protein, the solution may be too viscous and could clog sprayers. Gelatin is a substance that is commonly used in paper sizing [18]. Paper sizing is defined as a substance applied to paper in order to provide a glossy finish that will de- crease liquid absorption and improve printability and In the current investigation, gelatin and textured soy protein showed the best tear strength properties of all the protein binders assessed when applied to paper, providing Figure 5 Summary of average tensile strength of carbohydrate binders. Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. GA: Gum arabic. HRP: Horseradish peroxidase. Figure 5 Summary of average tensile strength of carbohydrate binders. Discussion Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. GA: Gum arabic. HRP: Horseradish peroxidase. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 9 of 14 Figure 6 Average tensile strength of pectin binders. Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. CA: Citric Acid. Figure 6 Average tensile strength of pectin binders. Tear weight is the average of the weight required to tear three strips of paper individually. Error bars represent standard deviation. CA: Citric Acid. between a 12 and 14-fold increase in wet paper tear strength when compared to paper with no binder applied. Furthermore, the strength provided by the gelatin binder gradually increases as the percentage of gelatin increases, and is the only green binder tested that was superior to the commercial binder strength. Also, the strength pro- vided by gelatin increases slightly when HRP is added to the reaction mixture. Gelatin from both porcine and bo- vine sources was tested along with a high and low bloom for each source. Neither the source of the gelatin nor the bloom strength seemed to affect the tear strength pro- duced by these binders. resuspended in water, the wet tear strength of paper in- creased more than 50%, offering a product that was slightly stronger than the commercial binder product. In theory, HRGP is an excellent substrate to be used as a paper binder. It has side chains available for cross- linking with peroxidase, which will form strong covalent bonds, and its conformation will allow it to lay flat against the cellulose of paper. There have been no previous sug- gestions in the literature for industrial applications of HRGP unlike the plethora of other substrates that have been evaluated as binders. However, it is known that the cross-linking of HRGP, as well as lignin and cellulose are crucial in order to provide structure and strength to the plant cell wall [2]. Therefore, if larger quantities of this protein were employed, it is reasonable to assume that an HRGP binder could provide acceptable tear strength to The supernatant of the textured soy protein provided approximately 50% less strength than gelatin and the use of HRP with soy protein did not seem to change this strength value. Discussion However, when the remaining pellet was Figure 7 Average tensile strength of lignin/phenolic compound binders. Error bars represent standard deviation. HCl: Hydrochloric acid HRP: Horseradish peroxidase. Figure 7 Average tensile strength of lignin/phenolic compound binders. Error bars represent standard deviation. HCl: Hydrochloric acid HRP: Horseradish peroxidase. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 10 of 14 Page 10 of 14 paper products. Nonetheless, in the present study, HRGP did not provide any extra strength to wet paper tear strength. This could very likely be due to the very low quantities of protein applied. Therefore, three treatments were performed on all lignin samples: addition of laccase, spraying with HCl, and ap- plication of high heat when drying. No increase in strength was apparent in any of the lignin samples when laccase was added. Slight increases in strength were ob- served in marasperse, black liquor, and low sulfonate lig- nin binders when HCl was sprayed on lignin coated paper. HCl spraying had the most effect on black liquor and low sulfonate lignin binders. High heat obviously had the greatest effect (Figure 7). Zein protein from corn kernels is also a good candi- date, as when zein is dissolved in ethanol and heated, it is not viscous and would not clog sprayers. Also, zein protein provided a 9-fold increase in tear strength when compared to paper with no binder applied. Because the development of low-cost manufacturing methods is needed, this protein could fit this need as it is available in large amounts. It is 40 to 50% of the protein in corn as reported by Shukla & Cheryan [20]. Results from zein, gelatin, pectin, soy and lignin binders show that application of heat dramatically increased the strength of paper. The possible reasons for this are as fol- lows. First, the application of heat may simply be dehydrat- ing the binder mix and forming a strong film around the cellulose of paper that would not be soluble in water. The literature shows that films are formed from several of the binders used in this work such as zein, pectin and gelatin [20,25,26]. These authors reported remarkable strength produced by each of these films and proposed their use in industrial applications to replace the use of petroleum products as adhesives. Discussion Another possibility is that chemical or physical changes may be occurring as a consequence of the high heat treatment, changing the flow properties of the molecules. Therefore, the strength provided by heat could be occurring because of dehydration, melting of com- pounds or a combination thereof. Carbohydrates have already been used as wood and paper/cardboard adhesives [21,22]. For example, pectin from orange peels serves as an excellent binder for drug tablets [23]. These authors also suggest that this binder would be more appealing than synthetic binders because of its availability and low cost. Coffin & Fishman [24] in- vestigated the tensile strength and other physical and mechanical properties produced by citrus pectin films and compared those properties to films produced by sugar beet and almond pectin. Determining the differ- ences in the properties of films made by different pectins will help to understand what type of pectin would make the best binder. Pectin provided the highest tear strength out of all carbohydrate binders, equaling the strength produced by the commercial binder. However, there are several differ- ent types of pectin and all of them are slightly different in terms of solubility and strength provided as a binder. While all food grade pectins already contained an acidic component to assist in gelling, usually citric acid or so- dium citrate, these components must be added to pure pectin. Without this added acidic component, pure pectin produced low strength values. We clearly demonstrate how adding citric acid to the pure pectin increased strength values, by up to 1.5-fold. Unfortunately, in this work, all carbohydrate binders, other than pectin either produced average to low strength results and/or their so- lutions were too viscous to be used in manufacturing. The phenolic compounds used did not produce compar- able strength values to the lignin binders. After application of ferulic acid and coniferyl alcohol, the paper tore with the same weight as did paper with no binder applied. The GPC results showed that ferulic acid had a dramatic increase in molecular weight when HRP was added, suggesting that ferulic acid could cross-link with cellulose fibers and serve as a strong binder. Unfortunately, only small amounts of ferulic acid and coniferyl alcohol could be used when apply- ing solutions to paper because of their low solubility in water. Using ethanol as a solvent would resolve solubility, but is incompatible with enzymes. Discussion The Salix binder provided the same amount of strength as the commercial vinyl acetate binder. After being dissolved in ethanol and heated, this binder forms a non- viscous solution that would not likely clog sprayers. Con- sequently, this binder is the most practical out of all other lignin/phenolic compound binders tested. Since lignin is the second most abundant polymer in nature, it can be obtained in large quantities and there- fore would be suitable for a commercial setting. It was observed that when HCl was added to lignin, a sticky, gel-like mixture formed. This observation implies some form of cross-linking is occurring between the lignin molecules. Thus, we hypothesized that applying lignin to paper and then spraying that paper with HCl may have a positive effect on the strength of paper. Also, after ob- serving an increase in strength after heat was applied to gelatin and pectin samples, it suggested that heat may have the same effect on lignin samples, as was previously reported by Mansfield [4] with mechanical pulp. If black liquor provided adequate strength, it would have been the most ideal binder. Paper manufacturers produce between 250 and 400 gallons of black liquor per ton of pulp (AF&PA, http://www.afandpa.org/). Most of this is used as an energy source to avoid the use of fossil fuels, but much is excess. If paper manufacturers could use their by- product not only to fuel their mill, but also as the binder in their manufactured products, this could drastically lower production costs. As shown in the strength results for Page 11 of 14 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 11 of 14 necessary to determine the true potential of these enzymes in binder systems. This effort clearly shows that there is po- tential for renewable resources, such as plant and animal by-products, to substitute the current level of petroleum- derived chemicals used as binders in the paper industry. Gelatin at 9% concentration combined with HRP provided significantly higher tear strength than the current commer- cial binder. different lignin binders, slight changes in lignin compos- ition or source of lignin can dramatically affect the strength of the binder. Therefore, in future experiments, it would be worthwhile to survey black liquor from different paper mills to determine if any samples could be implemented as a strong binder for paper products, and evaluate other reac- tion conditions. Testing method A small-scale version of a column-testing instrument (Instron, Norwood, MA) was made in order to test the strength of paper in the laboratory setting. A die cutter was used to ensure all pieces of paper were cut exactly the same in the cross-direction. To test paper strength, the top portion of a small piece of Whatman #1 filter paper (1.5 in × 0.5 in) was attached to a holder and weight was added to the opposite end of the strip. We calculated the exact amount of weight required to tear that individual piece of paper. This process was repeated three times for each binder applied. To test all the potential green binders, approximately 2 ml of each enzyme/substrate mixture described below was dispensed into incubator trays and the strips of paper were allowed to incubate in this solution. All reac- tions were incubated at 50°C and the pH used depended on the results of the enzyme tests previously performed. Reactions containing enzymes were allowed to proceed for 1 to 15 minutes. The binders developed in this project could be applied to different commercial products, such as wet wipe appli- cators. Since binders produced adequate strength without the use of enzymes, covalent bonds were not formed, so it was important to determine if binders could retain their strength in commercial solutions. Therefore, paper with binder applied was soaked in baby wipe solution for up to three months before tensile strength was tested. The plant binder retained its strength just as well as the vinyl acetate binder after soaking in baby wipe solution for these pe- riods of time. After incubating in binder solution, the strips were dried at approximately 200°C for 10 min, submerged in water, and the strength of each wet paper strip was tested in the cross-direction. Three paper strips (i.e., 3 replicates) were used for each treatment. The mean weight needed to tear each set of three strips of paper for each treatment was recorded. These measurements were compared to two control treatments: paper with no binder applied and paper with the vinyl acetate binder applied. Conclusions h This project aimed to identify potential renewable and green replacements for the currently used synthetic paper binders. A selection of twenty binders was surveyed, and among them, soy protein, gelatin, zein protein, pectin, and Salix lignin provided wet tear strength equivalent to that of the vinyl acetate binder currently used in the manufactur- ing process. All of these binders are organic, renewable, available in large quantities at reasonable prices, require very short reaction time and do not form viscous solutions that would clog sprayers. For these reasons, all binders chosen in this work could easily be implemented in an in- dustrial process. Oxidoreductase enzymes were combined with binder substrates to determine if enzymatic cross- linking could provide an increase in tear strength. Gelatin showed a slight increase in strength when horseradish per- oxidase was added, but more research in this area is Discussion Depending on the type of trees used and differences in the pulping process between facilities, the constituents of black liquor may be altered which may in turn have an effect on its properties as a binder [1]. In this work, lignin from hardwood trees showed strengths 2-fold higher than the black liquor from softwood trees. The lignins were also extracted by very different processes, perhaps contributing to the differences in binding activ- ity. Many studies have been done to determine the struc- tural and chemical differences between hardwood and softwood lignin. For example, Mohan et al. [27] found that the molecular weight of softwood pyrolysis lignin is larger than that of hardwood pyrolysis lignin. They also reported that softwood lignin which consists solely of guaiacyl-derived monomers results from high amounts of polymerized phenylpropane units, while hardwood lignin is made up of guaiacyl-syringyl lignin and results in mixed polymers of lower molecular mass. Protein binders ( Gelatin (Great Lakes Gelatin Co., Grayslake, IL), hydro xyproline-rich glycoprotein (HRGP, isolated from maize silk), textured soy protein (TSP, residue post oil extrac- tion) and zein (Acros Organics, New Jersey, USA) were tested as protein binders. JELL-OW (Kraft Foods, Glenview, IL) and KnoxW unflavored gelatin (Kraft Foods, Glenview, IL) were tested. Several different types of pectin were utilized through- out this project. Two types of Sure-JellW pectin and BallW pectin were obtained from local grocers. The first pectin had less sugar added than the second, regular pectin. These two pectins also contained different acidic compo- nents that assist in gelling. Low sugar pectin contained fu- maric acid and sodium citrate whereas the regular pectin only contained citric acid. BallW pectin’s ingredients in- cluded citric acid, sodium citrate and potassium sorbate. Boiling water was not necessary to dissolve BallW pectin. Sure-JellW pectins were dissolved in boiling water. KnoxW gelatin concentrations of 5, 7 and 9% were dissolved in boiling water then applied to Whatman #1 filter paper. For each percentage of KnoxW gelatin used, 0, 150, 200, and 250 μg of HRP was added. All reactions were incubated for 1 minute at 50°C. Gelatin from por- cine sources (blooms 100 and 300) and bovine sources (blooms 100 and 250) were obtained from Great Lakes Gelatin Company (Grayslake, IL). The highest and low- est bloom strength available from each source was pur- chased. For each source and for each bloom strength, 5, 7 and 9% gelatin solutions were made as above and ap- plied to paper. The 9% JELL-OW was applied to paper with and without HRP. To determine the effectiveness of pure pectins as binders, apple pectin was purchased from Sigma Chem- ical Co. (St. Louis, MO) and grapefruit pectin from a local health food store. For pure apple and grapefruit pectin, solutions of 1-5% were dissolved in boiling water and were applied to paper with and without citric acid. The amount of citric acid added was half the amount by weight of pectin added in each solution. HRGP was extracted from the cell walls of maize silk with calcium chloride and sodium metabisulfite as de- scribed by Hood et al. [6]. Concentration of HRGP was determined by absorbance at 280 nm using a microplate reader (Synergy HT, Bio-Tek). Substrate cross-linking H di h id Horseradish peroxidase (HRP) was obtained from Sigma Chemical Co. (P6782 St. Louis, MO). Gel electrophoresis of reaction products was used to determine the extent the substrate gained molecular weight, or was cross-linked, after reacting with the enzyme under certain conditions. Thermo Scientific gradient gels (4-20% acrylamide) were used with HEPES buffer. Approximately 6 μg of HRGP, 5 μg of HRP, and 6 μg of 8 mM hydrogen peroxide were used, as described by Devaiah & Shetty [28], and incubated at room temperature for one hour, then analyzed by SDS-PAGE. The same Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 12 of 14 water, ground meal, HRP, and H2O2 was analyzed by protein electrophoresis to determine if cross-linking oc- curred with this substrate. The ground soy slurry was centrifuged for 5 minutes at 1,860 × g. The supernatant was then applied to paper strips with 250 μg of HRP and 8 mM H2O2 and was carried out for 1 and 10 minutes at 50°C. The supernatant was also applied to paper with no enzyme present. Finally, the pellet was resuspended in water and applied to paper with no enzyme present. concentrations were used in reaction mixtures that were in- cubated at 22, 28, 37, 42 and 50°C, for 1, 5, 10, 30 and 60 minutes before being stopped with 0.1% SDS and mercaptoethanol and separated on a gel. The same experi- ment was per-formed at different pH (2, 3, 4, 5 and 6). HRP was also combined with lignin, black liquor and ferulic acid, individually. These samples were tested with gel permeation chromatography, as described by Mansfield et al. [29], to determine if this enzyme caused any molecu- lar weight changes in these phenolic compounds. Gel per- meation chromatography columns, series 60 and 300, were utilized (YMC Co., Ltd. Kyoto, Japan). The column packing material was silica derivatized with 1,2-dihydroxypropane. Zein protein was obtained from Sigma Chemical Co. (St. Louis, MO). A cross-linking test for this substrate was done by gel electrophoresis. A 3% solution of zein in 50% ethanol was applied to paper with and without HRP. Lignin, black liquor and ferulic acid were each combined with laccase and gel permeation chromatography was performed. Reactions were incubated in sodium acetate buffer, pH 5, at 50°C as reported by Bailey et al. [14]. Substrate cross-linking H di h id Sam- ples were allowed to incubate for 15 and 60 minute time periods to ensure that cross-linking occurred. For each time period, a high and low concentration of 150 μg and 15 μg were chosen for the reaction mixture. Protein binders ( The concentration of HRGP applied to paper was approximately 1mg/ml and the reaction with HRP and H2O2 added was allowed to proceed at 50°C for 15 minutes before the paper was dried and the strength was tested. Carbohydrate binders A variety of carbohydrates that are common substitutes for gelatin were tested. A range of 1 to 15% of each carbo- hydrate was dissolved in boiling water. Since gum Arabic contains both polysaccharides and glycoproteins, a 5% so- lution was made with boiling water and applied to paper with 250 μg of HRP and 8mM H2O2 and without enzyme. Flax seeds were ground with liquid nitrogen and 10 ml of water was added to every gram of ground flaxseed before application to paper. Abbreviations ABTS 2 2' i Abbreviations ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); DMSO: Dimethyl sulfoxide; FA: Ferulic acid; GPC: Gel permeation chromatography; HCl: Hydrochloric acid; HRGP: Hydroxyproline-rich glycoprotein; HRP: Horseradish peroxidase; kDa: KiloDaltons; PEO: Poly (ethylene) oxide; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TSP: Textured soy protein. Three types of tests were carried out with this binder. First, black liquor was applied to Whatman #1 filter paper with and without enzymes. Three solutions were made; black liquor with 250 μg of laccase, black liquor with 250 μg of HRP and 8mM H2O2, and black liquor with no en- zyme. After applying these solutions to paper, they were dried at 100°C for 10 minutes. Acknowledgements ld l k k We would like to acknowledge Buckeye Technologies Inc. (Perry, FL) for providing us with samples of black liquor and Vertichem (Toronto, Canada) for supplying Salix lignin. References 1. Lora JH, Glasser WG: Recent industrial applications of Lignin: a sustainable alternative to nonrenewable materials. J Polym Environ 2002, 10(1):39–48. 1. Lora JH, Glasser WG: Recent industrial applications of Lignin: a sustainable alternative to nonrenewable materials. J Polym Environ 2002, 10(1):39–48. 2. Iiyama K, Lam TT, Stone BA: Covalent cross-links in the cell wall. Plant Physiol 1994, 104:315–320. 3. Elegir G, Bussini D, Antonsson S, Lindström M, Zoia L: Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor. Appl Microbiol Biotechnol 2007, 77(4):809–817. Finally, 0.05 g/ml and 0.10 g/ml of Salix lignin (Vertichem, Toronto, Canada) were dissolved in 60% ethanol. This solu- tion was heated to approximately 55°C in the microwave to dissolve any insoluble fractions. The solution was then ap- plied to paper, allowed to dry at room temperature for 5 mi- nutes then dried at 200°C for 10 minutes before rewetting and testing strength in the cross-direction. 4. Mansfield SD: Laccase impregnation during mechanical pulp processing: improved refining efficiency and sheet strength, Volume 55. Carlton, Australia: Appita; 2002. pp 5. Fahmy Y, El-Wakil NA, El-Gendy AA, Abou-Zeid RE, Youssef MA: Plant proteins as binders in cellulosic paper composites. Int J Biol Macromol 2010, 47(1):82–85. 6. Hood EE, Shen QX, Varner JE: A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls. Plant Physiol 1988, 87:138–142. 7. Ringli C: The hydroxyproline-rich glycoprotein domain of the Arabidopsis LRX1 requires Tyr for function but not for insolubilization in the cell wall. Plant J 2010, 63(4):662–669. Received: 23 August 2012 Accepted: 18 March 2013 Published: 26 March 2013 Received: 23 August 2012 Accepted: 18 March 2013 Published: 26 March 2013 Ferulic acid and coniferyl alcohol were obtained from Sigma Chemical Co. (St. Louis, MO). Two mg of ferulic acid were first dissolved in 100 μl of dimethyl sulfoxide (DMSO), then water was added to equal 1 ml. Two mg of coniferyl alcohol were dissolved in 1ml of water. Both substrates were mixed with 250 μg of HRP and 8mM H2O2 for application on to Whatman #1 filter paper. Authors’ information h k d h EEH has worked in the area of cell wall structure and function for 25 years. This project represents an application of that knowledge. SPD is an enzyme biochemist and advised the enzyme reactions. SDM is a lignin chemist and advised the lignin trials and performed the GPC. Lignin low sulfonate (Sigma Chemical Co. St. Louis, MO), sodium lignin sulfonate, (MP Biomedicals, LLC. Solon, OH) and marasperse (Lignotech Rothschild, WI) were dissolved in water to make 0.05, 0.1 and 0.15 g/ml solutions for application to paper. Each concentration of each type of lignin was applied to paper and sprayed with HCl as previously described for black liquor. HCl was added to lignin samples in order to reduce the pH of the solution [14]. These paper strips were dried at 100°C for 10 minutes. Lastly, all concentrations of each lignin were applied to paper and dried at temperatures ranging from 100°C to 200°C for 10 minutes before ten- sile strength was tested. Competing interests Competing interests The authors declare that they have no competing interests. Lignin/Phenolic compound binders Black liquor, lignin low sulfonate, sodium lignin sulfon- ate, marasperse, ferulic acid, coniferyl alcohol and Salix lignin were the potential substrates used in these sets of experiments. Samples of black liquor were obtained from Buckeye Technologies (Perry, FL). Ultra filtration of black liquor, using nitrogen gas and a regenerated cellulose membrane with a 1,000 Dalton molecular weight cutoff, was used to remove unwanted sulfur salts and to concentrate the Defatted soybean meal was ground in a coffee grinder and then mixed with water at 1:10 (w/v). The mixture of Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Page 13 of 14 commercial binder) that had also been soaked in solution for the same amount of time. sample. Approximately 5 ml of black liquor was mixed with 100 ml of water in order to ensure adequate filtration was achieved and to lessen the chance of the black liquor clogging the filter. Authors’ contributions Second, paper strips were dipped in black liquor, hung up with binder clips in the fume hood and 1N HCl was sprayed on both sides of the strips with a thin-layer chro- matography sprayer. The strips were allowed to air dry for 5 minutes then dried completely at 100°C for 10 minutes. AF carried out the experiments with binders and enzyme testing. DVR coordinated, drafted and edited the manuscript. SD participated in the design of the study and performed some enzyme reactivity assays. SM performed the gel permeation chromatography. KT served as advisor throughout the project and performed the initial lignin-acid precipitation experiments. EH conceived the study, participated in its coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Third, high heat was applied to black liquor treated paper. After liquor application on to paper, the paper strips were dried at 200°C for 10 minutes before being rewet and tested for strength in the cross-direction. Author details 1A k B 1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72467, USA. 2Department of Biology, Arkansas State University, Jonesboro, AR 72467, USA. 3College of Agriculture and Technology, Arkansas State University, Jonesboro, AR 72467, USA. 4Department of Wood Science, 1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72467, USA. 2Department of Biology, Arkansas State University, Jonesboro, AR 72467, USA. 3College of Agriculture and Technology, Arkansas State University, Jonesboro, AR 72467, USA. 4Department of Wood Science, University of British Columbia, 4030-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada. University of British Columbia, 4030-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada. University of British Columbia, 4030-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada. Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 10. Otte O, Barz W: Characterization and oxidative in vitro cross-linking of an extension-like protein and proline-rich protein purified from chickpea cell walls. Phytochemistry 2000, 53:1–5. 11. Deepak S, Shailasree S, Kini RK, Muck A, Mithöfer A, Shetty SH 11. Deepak S, Shailasree S, Kini RK, Muck A, Mithöfer A, Shetty SH: Hydroxyproline-rich Glycoproteins and Plant Defence. J Phytopathol 2010, 158(9):585–593. 12. Felby C, Hassingboe J, Lund M: Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin. Enzyme Microb Technol 2002, 31(6):736–741. 13. Felby C, Thygesen LG, Sanadi A, Barsberg S: Native lignin for bonding of fiber boards—evaluation of bonding mechanisms in boards made from laccase-treated fibers of beech (Fagus sylvatica). 6th International Lignin Institute conference 2004, 20(2):181–189. 14. Bailey MR, Woodard SL, Callaway E, Beifuss K, Magallanes-Lundback M, Lane JR, Horn ME, Mallubhotla H, Delaney DD, Ward M, et al: Improved recovery of active recombinant laccase from maize seed. Appl Microbiol Biotechnol 2004, 63(4):390–397. 15. Madzak C, Mimmi MC, Caminade E, Brault A, Baumberger S, Briozzo P, Mougin C, Jolivalt C: Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis. Protein Eng Des Sel 2006, 19(2):77–84. Protein Eng Des Sel 2006, 19(2):77–84. g 16. Thurston CF: The structure and function of fungal laccases. Microbiology 1994, 140(1):19–26. 17. Mattinen M, Suortti T, Gosselink R, Argyropoulos DS, Evtuguin D, Suurnakki A, Jong E, Tamminena T: Polymerization of different lignins by laccase. BioResources 2008, 3:549–565. 18. Bryce R: Method of sizing paper. 1944, US Patent No. 2,354,662 19. Basta Altaf H, Fadl Naim A: Effects of Grammage and Gelatin Additive on the Durability of Paper. Restaurator 2003, 24:253. 20. Shukla R, Cheryan M: Zein: the industrial protein from corn. Ind Crop Prod 2001, 13(3):171–192. 21. Pizzi A: Recent developments in eco-efficient bio-based adhesives for wood bonding: opportunities and issues. J Adhes Sci Technol 2006, 20(8): 829–846. 22. Emengo FN, Chukwu SER, Mozie J: Tack and bonding strength of carbohydrate-based adhesives from different botanical sources. Int J Adhes Adhes 2002, 22(2):93–100. 23. Srivastava P, Malviya R, Kulkarni GT: Formulation and evaluation of Paracetamol tablets to assess binding property of orange peel pectin. Int J Pharm Sci 2010, 3:30–34. 24. Baby wipe solution tests Small strips of Whatman filter paper with binder of inter- est applied were soaked in baby wipe solution, at room temperature for up to 3 months. The strength was tested and compared to the factory’s binder (referred to as 8. Kim S, Sessa DJ, Lawton JW: Characterization of zein modified with a mild cross-linking agent. Ind Crop Prod 2004, 20(3):291–300. 9. Qi W, Fong C, Lamport DTA: Gum arabic glycoprotein is a twisted hairy rope: A new model based on O-galactosylhydroxyproline as the polysaccharide attachment site. Plant Physiol 1991, 96:848–855. 9. Qi W, Fong C, Lamport DTA: Gum arabic glycoprotein is a twisted hairy rope: A new model based on O-galactosylhydroxyproline as the polysaccharide attachment site. Plant Physiol 1991, 96:848–855. Page 14 of 14 Flory et al. BMC Biotechnology 2013, 13:28 http://www.biomedcentral.com/1472-6750/13/28 Coffin DR, Fishman ML: Physical and mechanical properties of highly plasticized pectin/starch films. J Appl Polymer Sci 1994, 54(9):1311–1320. 25. Hoagland P, Parris N: Chitosan/pectin laminated films. J Agric Food Chem 1996, 44:1915–1919. 26. Matsuda S, Iwata H, Se N, Ikada Y: Bioadhesion of gelatin films crosslinked with glutaraldehyde. J Biomed Mater Res 1999, 45(1):20–27. 27. Mohan D, Pittman CU, Steele PH: Pyrolysis of Wood/Biomass for Bio-oil: A Critical Review. Energy Fuel 2006, 20(3):848–889. 28. Devaiah SP, Shetty HS: Purification of an infection-related acidic peroxidase from pearl millet seedlings. Pestic Biochem Phys 2009, 94(2– 3):119–126. 28. Devaiah SP, Shetty HS: Purification of an infection-related acidic peroxidase from pearl millet seedlings. Pestic Biochem Phys 2009, 94(2– 3):119–126. 29. Mansfield SD, De Jong E, Saddler JN: Appl Environ Microbiol 1997, 63(10):3804–3809. 29. Mansfield SD, De Jong E, Saddler JN: Appl Environ Microbiol 1997, 63(10):3804–3809. doi:10.1186/1472-6750-13-28 Cite this article as: Flory et al.: Development of a green binder system for paper products. BMC Biotechnology 2013 13:28. 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: • 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: • 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: • Convenient online submission
https://openalex.org/W2032367481
https://hal.science/hal-03476538/file/m069p025.pdf
English
null
Medium-term acclimation of feeding and of digestive and metabolic enzyme activity in the neritic copepod Acartia clausi. I. Evidence from laboratory experiments
Marine ecology. Progress series
1,991
cc-by
13,225
MARINE ECOLOGY PROGRESS SERIES Mar. Ecol. Prog. Ser. MARINE ECOLOGY PROGRESS SERIES Mar. Ecol. Prog. Ser. Vol. 69: 25-40, 1991 Published January 10 Published January 10 Medium-term acclimation of feeding and of digestive and metabolic enzyme activity in the neritic copepod Acartia clausi. I. Evidence from laboratory experiments INRS-Oceanologie, 310 Allee des Ursulines. Rimouski, Quebec, Canada G5L 3A1 Department of Oceanography, Texas A and M University, College Station, Texas 77843, USA ABSTRACT: The time-relationship between food supply, intermediate metabolism (ETS, GDH activities) and nutritional processes (ingestion, digestive enzyme activities) was established experimen- tally for the small neritic copepod Acartia clausi. In the first 3 experiments, food-limiting conditions (suboptimal growth) were imposed and the food supply simulated: (1) different concentrations of a single pulse of phytoplankton; (2) different declining phytoplankton concentrations; or (3) a double pulse of phytoplankton. In all 3 cases positive acclimation of ingestion rates and digestive enzyme activities (amylase, laminarinase and trypsin) was observed. Trypsin appeared to have a low threshold of sensitivity to food changes suggesting that the copepods may enhance their potential for protein digestion at low food concentrations. In the fourth experiment we confirmed that under saturating food concentrations, copepods showed increased food intake and intermediate metabolism but no acclima- tion of their digestive enzyme systems. Except during starvation, day-to-day patterns of ETS and GDH activities did not covary with food quantity, although ETS varied with food quality and food type. Contradictions between previously published and present results are discussed and confirm that the time acclimation hypothesis of Mayzaud & Poulet (1978; Limnol. Oceanogr. 23: 1144-1154) applies only for food-limiting conditions. Under the present experimental conditions, A, clausi appeared to require 48 h to significantly acclimate to changes in food ration exceeding a threshold of 1.5 to 2 bgC ind.-'. The time acclimation hypothesis should be revised to emphasize that nutrition is likely controlled by feed- back mechanisms which operate to meet the metabolic requirements of individuals and that acclimation of the different aspects of nutrition (ingestion and digestion) take place over different time scales. O Inter-Research/Printed in Germany INTRODUCTION authors to other groups of copepods. Linear relation- ships between ingestion rates and food concentration were described in several studies (Reeve & Walter 1977, Conover 1978, Koeller et al. 1979, Huntley 1981, Uye & Kasahara 1983, Conover & Mayzaud 1984) but not found in recent reports by Paffenhofer & Van Sant (1985) and Ayukai (1987). Similarly, positive acclima- tion of digestive enzymes (i.e. increase in enzyme activity in agreement with the food ingested) was observed in several studies (cf. review by Mayzaud 1986), confirmed experimentally (Boucher et al. 1976, Cox 1981, Hirche 1981, Cox & Willasson 1981) and recently disputed by Hassett & Landry (1983, 1988), Landry & Hassett (1985), Harris et al. (1986) and Bbm- stedt (1988). The concept of time acclimation of the nutritional processes in zooplankton was introduced by Mayzaud & Poulet (1978) in order to explain the apparent sea- sonal covariation of the particulate potential food sup- ply with the ingestion rate and the digestive enzyme activity of small neritic copepods. They proposed that under natural, food-limiting conditions (i.e. suboptimal growth), ingestion rates should follow more or less the changes in food standing stock and that digestion rate should positively track changes in food quantity as long as the qualitative characteristics of the food-intake remained constant. Although primarily defined for small neritic copepods, the concept was extended by various The overall contradiction among published results Mar. Ecol. Prog. Ser. 69: 2540, 1991 26 seems to have originated from: (1) the failure by vari- ous authors to reproduce in the laboratory the field conditions in which time acclimation occurs and (2) attempts to compare experimental results which are not strictly comparable. Moreover, most experimental studies have not recognized that the ultimate control of nutritional acclimation is not food supply but the organ- ism's energy requirements. seems to have originated from: (1) the failure by vari- ous authors to reproduce in the laboratory the field conditions in which time acclimation occurs and (2) attempts to compare experimental results which are not strictly comparable. Moreover, most experimental studies have not recognized that the ultimate control of nutritional acclimation is not food supply but the organ- ism's energy requirements. were subsampled daily for 3 d after their capture on 18 March 1985; those from Group B for 3 d after their capture on 19 March; and those from Group C for 4 d after their capture on 21 March. MATERIALS AND METHODS Adults and stage V copepodites of the copepod Acar- tia clausi were collected from the Bay of Villefranche, France, using a 333 pm mesh size net. Water from tow depth was also collected using a Niskin sampler. The copepods were returned to the laboratory within half an hour and sorted immediately according to develop- mental stages under a binocular microscope. The specimens (females with some CV) were generally acclimated to laboratory conditions overnight in natural sea water at in situ temperature. The diatom Skeletonema costatum was generally used as food, except in Expt 4 where the chrysophyte Hymenomonas elongata was used in combination with the diatom. Samples of adult Acartia clausi were deep-frozen (-70 "C) immediately after capture to determine field level of digestive enzymes (Day 0 condition). In Expt 2, copepods collected in spring (13 May), were initially acclimated to laboratory conditions in natural seawater for 24 h before being divided into 3 groups. To simulate an acclimation to decreasing food concentrations, the individuals in the first group were starved throughout the experiment, while the 2 other groups were subjected to 2 different and decreasing concentrations of diatom food initially set at 2 high levels: i.e. 70 000 cells mlpl (5600 pgC I-') and 14 000 cells ml-' (1100 pgC 1-l). The culture of Skeletonema costatum used was harvested at the same stage of growth as in the previous experiment. The time dependency of feeding rates, digestive en- zyme levels, and ETS and GDH activities in relation to food variation was investigated through initial starva- tion experiments in filtered seawater, plus 4 different sets of feeding experiments (Expts 1 to 4) during April, May and June 1985. In designing the feeding experi- ment protocol, care was taken to meet 3 criteria in order to confirm the nature of digestive acclimation: (1) the copepod should be subjected to 2 or more differ- ent levels of the same food for a period of several days; (2) as in nature, the food supply should be limiting and variable over time; and (3) the chemical and biochem- ical composition of the food should be appropriate to the enzyme system examined. Expt 3 attempted to simulate the influence of a dou- ble pulse of phytoplankton. The first pulse lasted for 24 h and was separated from the second by a period of 24 h. INTRODUCTION Skeletonema costatum or Hymenomonas elongata were grown under a 12 : 12 h light-dark cycle in ES Provasoli medium. In all 4 experiments, algal batch cultures were initiated every other day and each batch was used as food for 2 consecutive days to provide the copepods with roughly similar-age algae. The experi- mental food media were prepared by adding known concentrations of phytoplankton batch culture to GF/C filtered seawater. For practical reasons, the seawater was filtered immediately after collection and kept at natural temperature in a cold room until use the next day. In the present study, we have tried to: (1) clarify the nature of the ingestive and digestive response to medium term (days) food changes of small neritic copepods; (2) ascertain experimentally the reality of time acclimation of digestive processes; and (3) relate these nutritional changes to those of the intermediary metabolism as expressed by measurements of the elec- tron transport system (ETS) and glutamate dehydro- genase (GDH) activity. In Expt 1, a set of copepods collected on 1 April (late winter conditions) was divided into 3 groups. Immedi- ately after capture, each group was fed on 1 of 3 different concentrations of Skeletonema costatum: 'High', 14 000 cells ml-l (2.9 ppm by volume or 1400 pgC 1-l); 'Medium', 7000 cells ml-' (1.5 ppm by volume or 700 pgC 1-l); or 'Low', 1500 cells ml-l (0.7 ppm by volume or 350 pgC I-'). The experiment attempted to simulate the influence of a single pulse of phytoplankton over 4 d. The culture of S. costatum used was in the exponential growth phase and was harvested 5 d after inoculation (105 cells ml-l). MATERIALS AND METHODS Copepod mortality, at any rate, remained well below 5 % dC1 in all feeding experiments. On each sampling day, one jar at each food level was randomly picked and, after removal of dead or moribund copepods, the remainder were counted and then deep-frozen (-0°C) for subsequent enzyme ana- lyses. The remaining jars were checked for dead indi- viduals and their food medium replaced to the desired concentration. Aliquot samples of phytoplankton cul- tures were filtered on precombusted Whatman GF/C glass-fibre filters for subsequent analyses (in triplicate) of carbon, nitrogen, protein, total carbohydrates and soluble carbohydrate content per cell. Microscope enumeration of the algal stock-culture density was also carried out to check the growth status of the culture. Amylase, laminarinase, trypsin and alkaline protease activities were measured on 3 subsamples of 80 and 120 individuals each after homogenization in Tris-HC1 buffer (pH: 7.0; 0.05 M) and centrifugation at 10 000 X g and 2°C. Optimum conditions for measur- ing amylase and laminarinase activity were previously established with crude extracts and purified enzymes (Mayzaud & Mayzaud 1981, Roche-Mayzaud 1985, Roche-Mayzaud & Mayzaud 1987) while optimum conditions for trypsin and alkaline protease activities were established only with crude extracts. Amylase, laminarinase and trypsin activities were measured according to the methods described by Mayzaud (1980) and Mayzaud et al. (1984). Alkaline protease activity was estimated from the hydrolysis of casein-yellow (Calbiochem.) according to the procedure described by Van Wormhoudt (1973). Protein measurements were carried out according to Lowry et al. (1951) using bovine albumin as a standard. Enzyme activity was expressed on a per individual basis because soluble protein concentrations per individual varied seasonally in the Bay of ViLlefranche, and also generally increased with increasing food supply in our experiments. In all experiments, copepod mean density ranged from 300 to 400 Acartia clausi which were placed in 1 1 jars and attached to a rotating wheel in a constant temperature room under dim light at 18°C. Concen- trations of algal food in each jar were monitored and adjusted daily using a Coulter counter (model TA) during Expts 1 and 2. During Expt 2, a major failure of the Coulter counter led us to use an alternate method. Thus daily monitoring in the subsequent Expts 3 and 4 was done fluorometrically following the standard method introduced by Yentsch & Menzel (1963) using an Aminco fluorometer. MATERIALS AND METHODS As previously, copepods collected in late spring (20 May) were acclimated for 24 h in unfiltered sea- water before being divided into 3 groups. One group was starved throughout the 6 d period and the other 2 supplied on Day 1 with the same 2 concentrations of Skeletonema costatum as in Expt 2. Early stationary growth phase diatom culture was used (i.e. 10 d after inoculation) in order to potentially optimize the laminarinase response. For our initial work, we tracked the daily enzyme activity of 3 groups of copepods that we collected in late winter and held in the laboratory for several days in 0.45 pm filtered seawater. Individuals from Group A Expt 4 was designed to evaluate the influence of alternating limiting/saturating/limiting food conditions on the ingestion-digestion response. After being held Roche-Mayzaud et al.: Acclimation of ft ?eding and digestive enzyme activity 27 in the laboratory for 24 h in natural seawater, copepods collected in early summer (3 June) were fed a limiting diet of Skeletonema costatum for 4 d at a concentration of 20 000 cells ml-'(1600 ygC 1-l). On the fifth day, one group of individuals was maintained on the same diet while another group was fed on saturating concen- trations (40 mgC 1-l) of Hymenomonas elongata each day for 3 d. After this latter change of food regime, this group was then returned to the initial diet of S, cos- tatum diet for an additional 3 d. The use of 2 different phytoplankters was made necessary because of the impossibility of achieving sufficient volume of batch culture of S. costatum to maintain saturating and limit- ing food conditions at the same time. By the use of a larger-size, better-accepted species of phytoplankton, saturating food concentrations could be obtained with moderate volume of culture without reduced ingestion rates. As we will see later, because digestive enzyme acclimation is governed by concentration rather than nature of food (Head & Conover 1983, Harris et al. 1986), such an experimental strategy is not likely to confound the interpretation by added variability. dependent. Although concentrations in particles or chlorophyll remaining after each 20 to 24 h sampling were on average 45 % of the initial values, indicating that this worst case seldom occurred, we of course recognize that a significant fraction of the particulate chlorophyll left could originate from the grazing activ- ity of the copepods (O'Connors et al. 1976). MATERIALS AND METHODS Because of this failure, vol- umes estimated in Expts 1 and 2 should be considered only on a relative basis. Indeed relationships between cell number, cell carbon and volume suggested a con- sistent overestimation of the particulate volume by a factor of 5. Copepod feeding rates were calculated from changes in food concentrations between sampling times (usu- ally 20 to 24 h). Algal growth rates (i.e. grazing con- trols) were determined from changes in cell densities in samples of food without copepods incubated under similar conditions. Calculations of ingestion were made according to the equation of Frost (1972). Available food supply was expressed on a per individual basis rather than per unit volume to correct for variations (+ 20 %) in the initial number of live experimental individuals per jar without corresponding changes in total volume. Indeed, considering the normal filtering rate of adult Acartia clausi and the relatively high copepod densities, a worst case possibility existed that the entire experimental volume would be swept clear making food availability strongly time- and density- Electron transport system (ETS) and glutamate dehydrogenase (GDH) activities were estimated on a fourth subsample of 80 to 120 copepods after homogenization in Tris-acetate buffer (pH: 8.6; 0.1 M; 0.2 % Triton X-100) according to the methodologies described by Bidigare et al. (1982). However, because of the small sample sizes available (only 300 to 500 km soluble protein per 100 individuals), both ETS and GDH were modified for small-volume analyses (0.25 m1 homogenate and 1.5 m1 total reaction volume for ETS; Mar. Ecol. Prog. Ser. 69: 25-40, 1991 28 1.4 m1 total volume of reaction mixture for GDH, con- taining 5.4 pmol ADP, 3.2 pm01 NAD, 48 pmol gluta- mate and 100 p1 enzyme homogenate). Table 1. Acartia clausi. Protein content of adult females during the course of the 4 experiments of digestion acclimation. Data are featured from sampling dates on which the maximum range of food concentrations were offered. Starv.: starvation; SD: standard deviation Carbon and nitrogen contents of the experimental phytoplankton were measured with a Perkin-Elmer CHN analyzer. Total protein and total carbohydrate concentrations were estimated by the methods of Lowry et al. (1951) and Dubois et al. (1956) respec- tively. Soluble carbohydrate content was measured according to Mayzaud et al. (1985) using a double extraction in 0.05 N HC1 at 50 "C. MATERIALS AND METHODS Expt Sampling Food level Protein concentration date (pg ind.T1) f SD 1 D4 Low 1.88 f 0.08 Medium 2.15 + 0.19 High 3.31 f 0.13 2 Dz Stam. 1.63 f 0.14 Low 1.99 f 0.07 High 2.91 f 0.14 3 D5 Starv. 1.86 f 0.12 Low 2.28 + 0.01 High 3.09 f 0.18 4 D6 Sk. cost. 2.28 f 0.14 Hymen. 3.24 + 0.10 D? Sk. cost. Hymen. 3.22 -t 0.10 D8 Sk. cost. 2.70 f 0.15 Hymen. 3.28 f 0.21 Statistical analyses were performed using SPSS and SYSTAT software packages. Significance of time changes was tested using the non-parametric Kruskal- Wallis test while differences between treatments were compared using both the non-parametric Wilcoxon 2-sample test and the multiple comparison test. Both tests yield identical results. Starvation Following Harris et al. (1986), food saturation can be defined, at least to a first approximation, as the concentration of food which sustains maximum body protein content. Although such a definition is some- what different from that of optimal growth, it follows that under food saturation, body protein content should be high and more or less constant. By corol- lary, body protein content should change with food concentrations under food-limiting conditions. The data presented in Table 1 summarize how individual protein content varied on dates when the ranges in food supply within each experiment was greatest. As expected for food-limited individual, mean per indi- vidual protein content followed the food availability for all 4 experiments with Skeletonema costatum. By contrast, those specimens fed Hymenornonas elon- gata exhibited nearly identical body protein concen- trations confirming that H. elongata was a food- saturating ration. Our initial experiment demonstrated that field levels of GDH and ETS activity declined rapidly when copepods were held without food in laboratory aquaria. Fig. lA, B shows that copepods starved over 2 to 4 d had GDH and ETS activities less than half those of 'wild' individuals freshly collected from the field. These activities appeared to approach an asymptote of about 5 f. 2 nmol ind.-' h-' for GDH and 10 + 2 nmol ind.-' h-' for ETS (n = 6). Throughout the starvation time- course, however, the ratio ETS/GDH activity remained more or less constant (2.1 * 0.6). Feeding In our series of 4 feeding experiments, we also observed that GDH and ETS activities were generally A B week of 18 March 85 I i v mean Fig. 1. Acartia clausi. Decrease in ETS and GDH 2 4 2 activity for individuals held for 1 to 4 d without DAYS OF STARVATION DAYS OF STARVATION feeding in laboratory aquaria A I i 2 4 DAYS OF STARVATION B week of 18 March 85 v mean 2 DAYS OF STARVATION B Roche-Mayzaud et al.: Acclirnation of feeding and digestive enzyme activity 29 16- 12. 4 0- Fig. 2. Acartia clausi. Changes in (A) food ration and (B) ingestion rates for 3 different food levels of the diatom Skeletonema costatum during Expt l. 'High' food level = initially 14 000 cells ml-l or 2.9 mm3 1-l; 'Medium' food level = initially 7000 cells ml-l or 1.5 mm3 I-'; 'Low' food level = initially 1500 cells ml-l or 0.7 mm3 1-l. Vertical bars in- dicate standard deviation. D_, stands for the 24 h delay be- tween food adjustment and ingestion measurement on D, FOOD RATION 10'3mm3 ind.-l A A - HIGH FOOD LEVEL ---- MEDIUM FOOD LEVEL LOW FOOD LEVEL SD(0.2 #.' *.. '*## .A..., ' f - $.-*-- ...' N. #-.- .-.-r 'I I D-2 D D-4 6- 5. 4 3' 2- l 0- DO D( D2 D3 Dq TIME (DAYS) INGESTION 103mm3 ind.-l d-l B - - - l 16- 12. 4 0- FOOD RATION 10'3mm3 ind.-l A A - HIGH FOOD LEVEL ---- MEDIUM FOOD LEVEL LOW FOOD LEVEL SD(0.2 #.' *.. '*## .A..., ' f - $.-*-- ...' N. #-.- .-.-r 'I I D-2 D D-4 lower in copepods held in the laboratory than those freshly collected from the field, although not as low as in those individuals starved for several days. In contrast, as we will describe below, activities of the 3 digestive enzymes usually recovered when the experimental copepods were fed, and in some instances experimental digestive enzyme activity per individual exceeded those of specimens freshly collected from the field. A A- Expt 1 elemental C : N ratio (5.5 to 6.5) and in protein : carbo- hydrate : lipid ratio (5 : 3 : 1 to 6 : 4 : 1). The diatom concentrations in our first experiment simulated conditions that copepods might encounter in a spring bloom when they would be exposed for several days to a single pulse of phytoplankton. The 'High', 'Medium' and 'Low' regimes allowed us to contrast 3 different intensities of this 'bloom'. As indicated in Fig. 2A, each level of concentration was increased by 30 % over the first 2 d and brought back to the initial value on the last day. Because it was growing in log-phase when harvested for Days 1 and 3, the different batches of culture of Skeletonema costatum used throughout the first experiment did not exhibit large changes in either chemical or biochemical composition. However, by Day 4 the last batch of culture had apparently reached early stationary phase as evidenced by an increase in protein and total carbohydrate content per cell (Table 2). Throughout the experiment, however, the cells of S. costatum were characterized by a narrow range in - INGESTION 103mm3 ind.-l d-l 6- 5. 4 3' 2- l 0- DO D( D2 D3 Dq TIME (DAYS) INGESTION 103mm3 ind.-l d-l B - - - l B Fig. 2. Acartia clausi. Changes in (A) food ration and (B) ingestion rates for 3 different food levels of the diatom Skeletonema costatum during Expt l. 'High' food level = initially 14 000 cells ml-l or 2.9 mm3 1-l; 'Medium' food level = initially 7000 cells ml-l or 1.5 mm3 I-'; 'Low' food level = initially 1500 cells ml-l or 0.7 mm3 1-l. Vertical bars in- dicate standard deviation. D_, stands for the 24 h delay be- tween food adjustment and ingestion measurement on D, The experimental copepods were subjected to the different food concentrations immediately after capture on 1 April and, except for the first 24 h, displayed a positive functional relationship between ingestion rate and phytoplankton changes (Fig. 2B). A Wilcoxon 2- sample test indicated that the responses were signifi- cantly different (p < 0.01) for all 3 food levels and could be ranked accordingly: High > Medium > Low. How- ever, the time changes were only significant for the highest food levels (Kruskal-Wallis test; p < 0.03), sug- gesting some threshold which compounded the accli- mation with the experimental variability. Table 2. Skeletonema costatum. Chemical and biochemical composition of different batches of the log-phase culture used in Expt 1. C: carbon; N: nitrogen; Chl: chlorophyll; Prot.: total proteins; T. Carb.: total carbohydrates; S. Carb.: soluble carbohydra- tes; Lip.: total lipids. Values in parentheses: standard deviation. The negative time scale indicates that the food was added to the experiment vessels 24 h before the ingestion and enzyme activity measurements Expt 1 Vertical bars indicate standard deviation zn [ GDH Experiment I (week of I April 85) 2 4 6 DAY 0 - 0 - "0 DI D2 D3 D4 Do Dl D2 D3 D4 TRYPSIN p M p nitmniline mm l i n d l C 0 - - "0 DI D2 D3 D4 3[ Experiment I (weekof I Aoril 85) ob ' I 2 4 6 DAY C TRYPSIN p M p nitmniline mm-l i n d l TRYPSIN 80[ p M p nitmniline mm-l i n d l Do DI D2 D3 D4 TIME (DAYS) 80[ p M p nitmniline mm l i n d l Do DI D2 D3 D4 TIME (DAYS) -HIGH ----MEDIUM m-.-, LOW Fig. 4. Acartia clausi. Changes in electron transport system (ETS; ng-at. O2 ind.-' h-') and glutamate dehydrogenase (GDH; ng-at. N ind.-' h-') specific activities during Expt 1 Fig. 3. Acartia clausi. Medium-term acclimation of the diges- tive enzymes (A) amylase, (B) laminarinase (LAMNASE), and (C) trypsin, subjected to the food changes defined in Expt 1. 'High' = initially 14 000 cells ml-' or 2.9 mm3 I-'; 'Medium' = initially 7000 cells mlpl or 1.5 mm3 I-'; 'Low' = initially ' 1500 cells ml-' or 0.7 mm3 lpl. Vertical bars indicate standard deviation Expt 1 The changes in amylase, laminarinase and trypsin Table 2. Skeletonema costatum. Chemical and biochemical composition of different batches of the log-phase culture used in Expt 1. C: carbon; N: nitrogen; Chl: chlorophyll; Prot.: total proteins; T. Carb.: total carbohydrates; S. Carb.: soluble carbohydra- tes; Lip.: total lipids. Values in parentheses: standard deviation. The negative time scale indicates that the food was added to the experiment vessels 24 h before the ingestion and enzyme activity measurements Sampling Conc. C N Chl Prot. T. Carb. S . Carb. Lip. C:N C:Chl N:Chl date (106 cells ml-l) (10-6 pg cell-') Day -1 to -3 3.3 72 13 1.5 6 1 38 38 12 5.5 48 8.7 (2) (0.1) (0.5) (1) (1) (2) (0.2) Day -4 5.4 85 13 1.3 108 59 37 10 6.5 65 10.0 (0.4) (0.1) (0.5) (1) (1) (5) (0.3) Mar. Ecol. Prog. Ser. 69: 25-40, 1991 30 zn [ GDH Experiment I (week of I April 85) 2 4 6 DAY 3[ Experiment I (weekof I Aoril 85) ob ' I 2 4 6 DAY Fig. 4. Acartia clausi. Changes in electron transport system (ETS; ng-at. O2 ind.-' h-') and glutamate dehydrogenase (GDH; ng-at. N ind.-' h-') specific activities during Expt 1 0 - 0 - "0 DI D2 D3 D4 Do Dl D2 D3 D4 TRYPSIN 80[ p M p nitmniline mm-l i n d l C Do DI D2 D3 D4 TIME (DAYS) -HIGH ----MEDIUM m-.-, LOW Fig. 3. Acartia clausi. Medium-term acclimation of the diges- tive enzymes (A) amylase, (B) laminarinase (LAMNASE), and (C) trypsin, subjected to the food changes defined in Expt 1. 'High' = initially 14 000 cells ml-' or 2.9 mm3 I-'; 'Medium' = initially 7000 cells mlpl or 1.5 mm3 I-'; 'Low' = initially ' 1500 cells ml-' or 0.7 mm3 lpl. Expt 2 The design of the first experiment was essentially concerned with the influence of increasing food supply and left one question unanswered: do the copepods acclimate to medium-term (days) decreasing food con- centrations in a way similar to increasing food concen- tration or is there an influence of the previous trophic history? In Expt 2, after 24 h acclimation to laboratory conditions in natural seawater, the copepods were introduced to 2 different high food levels of Skeleto- nema costatum. After 24 h, each of these 2 food con- centrations was sharply decreased (by 50 % the first day and by 10 % the remaining 2 d; Fig. 5A). A third group of individuals was maintained without food in fil- tered seawater as a control. activities recorded during the same experiment dis- played a sharp decrease during the first day of labora- tory confinement followed by a pattern of changes which differed somewhat with the individual enzyme (Fig. 3). Laminarinase and trypsin activities showed respectively complete and partial recovery, while amyl- ase activity diminished throughout the experiment. Those copepods exposed to 'High' food concentration demonstrated a positive acclimation for all enzyme activity after the first 24 h, with significantly higher activities per individual than for those held at 'Medium' and 'Low' levels of food (Wilcoxon 2-samples test; p <0.05). The amylase and laminarinase activities recorded for the 2 lower food concentrations were not significantly different (Wilcoxon 2-sample test) whereas trypsin activity was ranked according to the positive acclimation hypothesis, i.e. highest at highest concen- tration and lowest at the lowest (Wilcoxon 2-sample test; p < 0.02). The influence of the variable day-to-day pattern of ingestion was strongly reflected by laminarin- ase activity but not evident for the other 2 enzymes. The characteristics of Skeletonema costatum used in the second experiment are summarized in Table 3. As previously, the cell composition was protein-dominated at all times but both biochemical and chemical compos- itions showed some variations throughout the experi- ment. The protein : carbohydrate : lipid ratio varied from 5:4:1onDays2and3to3:2:1onDay4and10:6:1on Day 5 when the stock culture had reached stationary phase. Throughout the 5 d experiment, the diatom's C:N ratio remained more or less constant (5.3 + 0.4). GDH and ETS activities (Fig. 4) did not vary markedly among the 3 food concentration groups tested. Expt 2 'High' = initially 70 000 cells ml-' or 19 mm3 1-l; 'Low' = initially 14 000 cells ml-' or 3.5 mm3 1-l; 'Starved' = starved control cope- pods. Vertical bars indicate standard deviation Table 3. Skeletonema costatum. Chemical and biochemical composition of different batches of the log-phase culture used in Expt 2. (For key to symbols, see Table 2) Table 3. Skeletonema costatum. Chemical and biochemical composition of different batches of the log-phase culture used in Expt 2. (For key to symbols, see Table 2) p ( y y , ) Sampling Conc. C N Chl Prot. T. Carb. S. Carb. Lip. C:N C:Chl N:Chl date (106 cells ml-l) (lop" ~g cell-') Day -2 -3 2.3 75 13 1.1 54 45 43 11 5.8 68 12 (1) (0.1) (0.1) (1) (2) (3) (1.5) Day -4 1.0 74 15 0.8 55 42 37 21 4.9 93 19 (5) (0.2) (0.1) (1) (0.5) (2) (1) Day -5 5.4 85 15 1.0 98 59 37 10 5.7 85 13 (0.4) (0.1) (0.1) (5) (1) (5) (0.3) the available food supply. In contrast to the previous experiment where field-collected copepods had high activities of all 3 enzymes, all the copepods captured on 13 May displayed very low trypsin activity coupled with relatively high amylase and laminarinase activities (compare Do conditions Fig. 5B to D). Ex- posure to different levels of food supply for a short time (24 h) did not seem to influence the enzyme levels (D2) which, except for amylase, remained similar to the activity exhibited by the starved control individuals. However, positive acclimation became evident during the next 3 d, even though the food available was dras- tically decreasing (Fig. 5A), suggesting a strong mem- ory effect. A Wilcoxon 2-sample test confirmed that the enzyme responses were significantly different (p < 0.05) and could again be ranked according to the food supply (D2 for trypsin excepted). Over time, amyl- ase and laminarinase of fed copepods displayed signifi- cant variations (Kruskal-Wallis test; 0.01 < p <0.05) with an initial decrease over the first 2 d, followed by either a stabilization or slow increase in activity (Fig. 5B, C). Trypsin also showed significant variations (Kruskal-Wallis test; p < 0.01) with a maximum activity occurring 24 h after the initial pulse of food supply, followed by a decrease which agreed with that of the food concentrations. Expt 2 Both activities decreased sharply from the values observed in the field (Do) and thenleveled off at about 20 % more than the activity levels seen in copepods starved for 4 d. GDH and ETS activities (Fig. 4) did not vary markedly among the 3 food concentration groups tested. Both activities decreased sharply from the values observed in the field (Do) and thenleveled off at about 20 % more than the activity levels seen in copepods starved for 4 d. A failure of the electronic particle counter halfway through the second experiment prevented us from pre- cisely estimating day-to-day changes in ingestion rate changes. Nevertheless, the few data obtained sug- gested that ingestion generally tracked the decrease in Roche-Mayzaud et al.: Acclimation of feeding and digestive enzyme activity 3 1 Fig. 5. Acartia clausi. Changes in (A) food ration, and related medium-term acclimation of the di- gestive enzymes; (B) laminarinase (LAMNASE); (C) amylase and (D) trypsin, for 2 different food levels of the diatom Skeletonema costatum du- ring Expt 2. 'High' = initially 70 000 cells ml-' or 19 mm3 1-l; 'Low' = initially 14 000 cells ml-' or 3.5 mm3 1-l; 'Starved' = starved control cope- pods. Vertical bars indicate standard deviation l ~ ~ r n r n ~ ind.-l A LAMNASE B p g glucose mm-l ind.-l -HIGH 4 0 *---- LOW AMYLASE C TRYPSIN '1 rrq mylose rnin-1 ind.-1 16[ p M pn~troanil~ne rn~n-l ind.-1 D TIME (DAYS) TIME (DAYS) Table 3. Skeletonema costatum. Chemical and biochemical composition of different batches of the log-phase culture used in Expt 2. (For key to symbols, see Table 2) l ~ ~ r n r n ~ ind.-l A LAMNASE p g glucose mm-l ind.-l -HIGH 4 0 *---- LOW AMYLASE C TRYPSIN '1 rrq mylose rnin-1 ind.-1 16[ p M pn~troanil~ne rn~n-l in TIME (DAYS) TIME (DAYS) l ~ ~ r n r n ~ ind.-l A -HIGH 4 0 *---- LOW AMYLASE C '1 rrq mylose rnin-1 ind.-1 16[ TIME (DAYS) B C D TIME (DAYS) TIME (DAYS) Fig. 5. Acartia clausi. Changes in (A) food ration, and related medium-term acclimation of the di- gestive enzymes; (B) laminarinase (LAMNASE); (C) amylase and (D) trypsin, for 2 different food levels of the diatom Skeletonema costatum du- ring Expt 2. Expt 2 Thus, the data suggest that the reactivity of the enzyme system varies with the enzyme considered and that under the present experimental conditions trypsin activity is the only one to both show an acclimation to food concentration and reflect the decreasing characteristic of the food availability. Once again, the changes in ETS and GDH activities Mar. Ecol. Prog. Ser. 69: 25-40, 1991 32 FOOD RATION A ( ng Chl o ind:l 1 0 - '32 '33 D4 0-5 '36 TlME (DAYS) o l ' ~ " " D2 03 04 05 D6 01 TlME (DAYS) 5 4. 3- 2- I. Fig. 7. Acartia clausi. Medium-term changes in (A) food ration, and (B) ingestion rates, for 2 different food levels of the diatom Skeletonema costatum during Expt 3. Initial concentrations were similar to those of Expt 2 (see Fig. 5). Vertical bars indicate standard deviation. For time axis legend see Fig. 1 INGESTION B ng Chl o ind.-l h-' - UPPER LEVEL LEVEL - High - Low + Starved ob I 2 4 6 DAY 70 ETS Experiment 2(weekof 13 May 601 - High 4 0 - LOW - Starved Ob 2 - 4 6 I DAY Fig. 6. Acartia clausi. Changes in electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for the 2 food levels and starving individuals defined in Expt 2. 'High' = 70 000 cells ml-l or 19 mm3 1-l; 'Low' = 14 000 cells ml-' or 3.5 mm3 l-l; 'Starved' = starved control copepods - High - Low + Starved ob I 2 4 6 DAY FOOD RATION A ( ng Chl o ind:l 1 0 - '32 '33 D4 0-5 '36 TlME (DAYS) o l ' ~ " " D2 03 04 05 D6 01 TlME (DAYS) 5 4. 3- 2- I. INGESTION B ng Chl o ind.-l h-' - UPPER LEVEL LEVEL B Fig. 6. Acartia clausi. Changes in electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for the 2 food levels and starving individuals defined in Expt 2. 'High' = 70 000 cells ml-l or 19 mm3 1-l; 'Low' = 14 000 cells ml-' or 3.5 mm3 l-l; 'Starved' = starved control copepods Fig. 7. Acartia clausi. Medium-term changes in (A) food ration, and (B) ingestion rates, for 2 different food levels of the diatom Skeletonema costatum during Expt 3. Expt 2 Initial concentrations were similar to those of Expt 2 (see Fig. 5). Vertical bars indicate standard deviation. For time axis legend see Fig. 1 failed to show a positive functional response to either of the 2 food levels. Fig. 6 demonstrates that the activities of individuals exposed to both food regimes decreased with time, ultimately reaching ETS and GDH levels similar to those exhibited by starving late-winter col- lected specimens. jected to 2 successive pulses of Skeletonema costatum over 4 d (Fig. 7, top). The 2 food levels used were identical to those used in Expt 2 and as previously a group of non- fed copepods was used as control. The diatom cells, which were harvested during the stationary growth phase, except on Day 2, contained a considerably higher concentration of carbohydrates than cells from Expt 2. Soluble polysaccharides made up 76 to 96 % of the total carbohydrates, while the protein : carbohydrate : lipid ratio displayed some important variations with values of 4:7:1onDays2,5and6,9:8:1onDay3and6:7:1on Day 4 (Table 4). However, the C: N ratio showed less variation, with most values ranging between 7.1 and 8.1. Expt 3 Vertical bars indicate standard deviation; 'starved' = starved control copepods The general pattern of changes in ingestion rate 301 Experiment 3 (weekof 20 May 85) --C Upper -A--- Lower - Starved ~ b " " " 2 4 6 DAY 70 Experiment 3(weekof 20 May 85) r:, 50 Upper Lower --c Starved 30 C - 10 2 4 6 M Y Fig. 9. Acartia clausi. Changes in the electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for the 2 food levels and starving individuals during Expt 3. 'Starved' = starved control copepods; initial food levels similar to those of Expt 2 (see Fig. 5) 'los3 LAMNASE rngornylose min-l ind.-l ,.~g glucose min-l ind.-l Do 01 "2 D3 D4 D5 '% Do D2 D3 D4 4 '% X I O ' ~ TRYPSIN c p M Tyr mind tnd.-l - Upper level 4 Lower level Starved TIME (DAYS) 'los3 LAMNASE ,.~g glucose min-l ind.-l Do D2 D3 D4 4 '% N c 301 Experiment 3 (weekof 20 May 85) --C Upper -A--- Lower - Starved ~ b " " " 2 4 6 DAY rngornylose min-l ind.-l Do 01 "2 D3 D4 D5 '% X I O ' ~ c X I O ' ~ TRYPSIN c p M Tyr mind tnd.-l - Upper level 4 Lower level Starved TIME (DAYS) 70 Experiment 3(weekof 20 May 85) r:, 50 Upper Lower --c Starved 30 C - 10 2 4 6 M Y TIME (DAYS) Fig. 8. Acartia clausi. Medium-term acclimation of the diges- tive enzymes (A) amylase, (B) laminarinase (LAMNASE), and (C) trypsin, for the 2 different food levels of the diatom Skeletonema costatum during Expt 3. Initial concentrations were similar to those of Expt 2 (see Fig. 5). Vertical bars indicate standard deviation; 'starved' = starved control copepods Fig. 9. Acartia clausi. Changes in the electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for the 2 food levels and starving individuals during Expt 3. 'Starved' = starved control copepods; initial food levels similar to those of Expt 2 (see Fig. 5) The general pattern of changes in ingestion rate closely followed the oscillatory nature of the food availability, with differences in ingestion at the upper and lower level most pronounced when differences in food ration were most marked (Days 3, 5 and 6; Fig. 7). Expt 3 The significance of the general trend was tested using the non-parametric Kruskal-Wallis test (p < 0.03), while that of the acclirnation to food level was verified by a Wilcoxon 2-sample test (p < 0.02). ated, although it tested significant (Kruskal-Wallis test, p < 0.05) when compared to the uniformly low trypsin activity of starved control specimens. The time course of variation in GDH and ETS activities showed some differences between upper and lowedstarved conditions, although neither exhibited a clear relationship with the variation in food supply (Fig. 9). As already described for the digestive enzymes, the overall trend corresponded to an initial decrease in activity, but this was followed by an almost complete recovery of GDH and ETS activity at the upper food levels to oscillate about mean activity some 50% higher than that of unfed copepods. As was the case in the previous experiments, all 3 digestive enzyme activities (i.e. amylase, laminarinase, trypsin) showed a marked initial decrease related to the laboratory acclimation (D1) which was not fully reversed after the first 24 h of feeding (D2) (Fig. BA to C). Signifi- cant positive acclirnation to the food concentration had set in by Day 3 (Wilcoxon 2-sample test; p < 0.01), even though oscillations in ingestion rates seem attenuated in terms of enzyme changes and shifted rhythmicity was apparent for trypsin activity. The amylase andlaminarin- ase activities at high food concentration demonstrated almost complete recovery to their initial 20 May field levels, which might reflect the dominance of carbohy- drates in the diet. The same significant trend (Kruskal- Wallis test; p < 0.02) was apparent for the low food level carbohydrase activities, although good discrimination between feeding and non-feeding groups was only established after the second day (Fig. 8). Recovery of trypsin activity was more limited and observed only for the higher food concentration. The general variation in trypsin activity at lower food level was more attenu- Expt 3 The time-averaging phenomenon which seemed to characterize the enzyme response in the previous 2 experiments cannot be fully identified using a single short pulse type experiment. Consequently, for the third experiment the copepods were acclimated for 24 h in natural seawater to laboratory conditions and then sub- Table 4. Skeletonema costatum. Chemical and biochemical composition of different batches of the stationary phase culture used in Expt 3. (For key to symbols, see Table 2) Sampling Conc. C N Chl Prot. T. Carb. S. Carb. Lip. C:N C:Chl N:Chl date (106 cells ml-l) ( 1 0 ~ ~ pg cell-') Day -2 -5 -6 6.2 134 19 1.3 92 150 144 23 7.1 103 15 (0.3) (0.1) (0.1) (0.3) (19) (12) (2) Day -3 5.8 113 14 1.3 108 96 73 12 8.1 87 11 (0.1) (0.03) (0.01) (0.1) (5) (7) ('3.4) Day -4 5.9 124 16 1.3 115 123 109 18 7.8 95 12 (0.1) (0.1) (0.1) (0.3) (9) (8) (1) emical and biochemical composition of different batches of the stationary phase culture used in Expt 3. (For key to symbols, see Table 2) Roche-Mayzaud et al.: Acclimation of feeding and digestive enzyme activity 33 301 Experiment 3 (weekof 20 May 85) --C Upper -A--- Lower - Starved ~ b " " " 2 4 6 DAY 70 Experiment 3(weekof 20 May 85) r:, 50 Upper Lower --c Starved 30 C - 10 2 4 6 M Y Fig. 9. Acartia clausi. Changes in the electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for the 2 food levels and starving individuals during Expt 3. 'Starved' = starved control copepods; initial food levels similar to those of Expt 2 (see Fig. 5) 'los3 LAMNASE rngornylose min-l ind.-l ,.~g glucose min-l ind.-l Do 01 "2 D3 D4 D5 '% Do D2 D3 D4 4 '% X I O ' ~ TRYPSIN c p M Tyr mind tnd.-l - Upper level 4 Lower level Starved TIME (DAYS) Fig. 8. Acartia clausi. Medium-term acclimation of the diges- tive enzymes (A) amylase, (B) laminarinase (LAMNASE), and (C) trypsin, for the 2 different food levels of the diatom Skeletonema costatum during Expt 3. Initial concentrations were similar to those of Expt 2 (see Fig. 5). Expt 4 From a theoretical point of view, positive acclimation of the digestive system should be related to previous or actual food limitation. Once fully acclimated in the laboratory under non-limiting conditions (1 to 2 mgC I-'), current theory predicts that further pulses in food supply should not result in large changes in digestive enzyme activity. Consequently, for our final experi- ment, the copepods were acclimated for 5 d under laboratory conditions with Skeletonema costatum as food source, then subjected to a pulse of the larger Hymenornonas elongata for 3 d before being returned to the original diet of S. costatum for another 3 d. Mar. Ecol. Prog. Ser. 69: 2540, 1991 34 Table 5. Skeletonema costatum and Hymenomonas elongata. Chemical and biochemical composition of different batches of the stationary phase cultures used in Expt 4. (For key to symbols, see Table 2) g Day -6 -7 (200) (16) (0.4) ( Day -8 1.23 2090 170 28.0 9 (40) (1) (0.3) ( 700- O L ~ , . . ~ . . . . : . ~ D2 D4 D6 D8 DIO (TIME DAYS) 600. oL l D-2 D-4 D - 6 ~ - 8 ' "-10 Fig. 10. Acartia clausi. Long-term changes (10 d) in (A) food ration and (B) ingestion rates for 1 food level (10 to 16 yg chl 1-l) of the diatom Skeletonema costatum and for a FOOD RATION ng Chlg ind.-l 5- 4. h - HYMENOMONAS EUINGATA 1 - SKELETONEMA COSTATUM INGESTION ng Chlg ~nd.-l h-l B 700- 600. oL l D-2 D-4 D - 6 ~ - 8 ' "-10 FOOD RATION ng Chlg ind.-l h - HYMENOMONAS EUINGATA 1 - SKELETONEMA COSTATUM is a 'good' maintenance food source for the culture of Acartia-type copepods. The H. elongata culture used was also harvested during the early sationary phase and displayed similar biochemical characteristics. The protein : carbohydrate : lipid ratio varied between 4 : 7 : 1 for S. costatum and 2 : 5 : 1 for H, elongata. Values for the C: N ratio of both cultures varied between 7.2 and 12.3, while their soluble polysaccharide contents ranged from 68 to 96 % of the total carbohydrates. As expected, the ingestion rate tracked the available food ration, although the amount of Hyrnenomonas elongata actually ingested was not strictly proportional to the 20-fold increase in available chlorophyll that we added in this 3 d pulse (Fig. DISCUSSION ase and protease activities in Calanus hyperboreus fed different phytoplankters but observed a temperature- dependent time course of 8 to 16 d. More than likely, the use of late diapausing copepods which did not feed until 5 din the laboratory explains both the long time scale and the non-specificity observed. As we will see later, a time course of 24 to 48 h for medium-term digestive acclima- tion seems more representative of actively feeding copepods. Other studies by Hassett & Landry (1983, 1988) on C. pacificusand Harris et al. (1986) on Calanus helgolandicus led to negative relationships between potential food supply and digestive enzyme activity but these authors reached somewhat differing conclusions. Hassett & Landry (1983) suggested that the maintenance of high levels of digestive enzymes could be in anticipa- tion of future feeding opportunity, while Harris et al. (1986) proposed that such results indicated a compensa- tory mechanism to maintain assimilation rate under non- limiting food situations. The acclimation hypothesis, first proposed by May- zaud & Poulet (1978) and completed by Mayzaud & Mayzaud (1985) and Mayzaud (1986), suggested that small neritic copepods under food-limiting conditions acclimated behaviourly and biochemically to time-aver- aged food concentrations. Experimental demonstration of digestive acclimation has been limited to a small number of studies with contradictory conclusions. Cox (1981) and Cox & Willasson (1981) demonstrated a specific positive acclimation of laminarinase in Euphausiapacifica and Calanuspacificusfeeding on the diatom Skeletonema costatum or particulate laminarin and found a time course of 1 d. Head & Conover (1983) also described a positive response of amylase, laminarin- 141 Experiment 4 (week of 3 June 85) 126 4 b . . s s . . . . . , . l ~ 4 2 4 6 8 10 12 DAY Fig. 12. Acartia clausi. Changes in electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for individuals fed limiting levels of the diatom Skeletonerna costatum and subjected to a pulse of saturating concentrations of the chrysophysae Hymenomonas elongata on Days 6 to 8. Open symbols: controls fed on S, costatum during the pulse of H. elongata 141 Experiment 4 (week of 3 June 85) 126 4 b . . s s . . . . . , . Expt 4 10). O L ~ , . . ~ . . . . : D2 D4 D6 D8 DIO (TIME DAYS) 5- 4. INGESTION ng Chlg ~nd.-l h-l B The digestive enzyme activities oscillated about mean activity which did not strikingly increase when the pulse of Hymenomonas elongata was introduced (Fig. 11). In fact, the controls fed on Skeletonema cos- tatum generally had equivalent or higher digestive enzyme activities than the copepods fed on H, elon- gata. The specific patterns of changes observed fell into 2 categories: (1) an overall decrease in activity followed by a partial recovery to between 50 and 75 % of the initial activity (e.g. amylase), or (2) an initial decrease followed by a more or less complete recovery back to the initial field activity (e.g. the other 3 enzymes). As expected, laminarinase, trypsin and alkaline protease activities were significantly intercorrelated (0.05 < p < 0.01) with maximum activities either during or 2 d after the pulse of H. elongata (Fig. 11). As we will see later, this increase apparently reflected a general increase in intermediate metabolism. B D4 D6 D (TIME DAYS) D4 D6 D (TIME DAYS) Fig. 10. Acartia clausi. Long-term changes (10 d) in (A) food ration and (B) ingestion rates for 1 food level (10 to 16 yg chl 1-l) of the diatom Skeletonema costatum and for a short saturating pulse (3 d) of the chrysophycae Hymeno- monas elongata during Expt 4. Vertical bars indicate standard deviation ETS and GDH activity covaried for the first 5 d of the experiment but diverged after Hymenomonas elongata was presented (Fig. 12). The ETS activity clearly increased in response to H. elongata as food supply to reach activity levels comparable to those in the field at the time and twice those of the corresponding controls fed on Skeletonema costatum. A correlation analysis confirmed the similarities among changes of ETS, laminarinase and proteases (0.05 < p < 0.01; r > 0.64, df = 9). The food level and culture of Skeletonema costatum used were similar to the low concentration of the previ- ous experiment. The diatom culture was again har- vested during the stationary phase and was carbohy- drate-dominated (Table 5). Expt 4 We introduced the chryso- phyte Hymenomonas elongata because laboratory rearing studies (Yassen 1985) have demonstrated that it Roche-Mayzaud et al.: Acclimation of feeding and digestive enzyme activity 35 XIO-~ AMYLASE LAMNASE { rng amylose mm-l i n d l A l2;p3 W glucose min-l ind.' B , TRYPSIN i- y- C) c- 8 p- n Sk. costaturn fed t- o- - Hym. elongata fed al 0 . . . . , , . . , 0 a , m n . . * * l % D4 D6 D8 '% D2 D4 D6 D8 D10 TIME (DAYS) TIME (DAYS) XIO-~ AMYLASE LAMNASE { rng amylose mm-l i n d l A l2;p3 W glucose min-l ind.' B , A TRYPSIN 8 Sk. costaturn fed - Hym. elongata fed 0 . . . . , , . . , 0 a , m n . . * * l % D4 D6 D8 '% D2 D4 D6 D8 D10 TIME (DAYS) TIME (DAYS) Fig. 11. Acartia clausi. Long-term accli- mation of the digestive enzymes (A) amy- lase, (B) laminarinase (LAMNASE), (C) trypsin, and (D) alkaline proteases ac- tivities to change in the limiting food sup- ply of the diatom Skeletonema costaturn and short-term acclimation to a saturat- ing pulse of the chrysophysae Hymeno- monas elongata during Expt4. Vertical bars indicate standard deviation Fig. 11. Acartia clausi. Long-term accli- mation of the digestive enzymes (A) amy- lase, (B) laminarinase (LAMNASE), (C) trypsin, and (D) alkaline proteases ac- tivities to change in the limiting food sup- ply of the diatom Skeletonema costaturn and short-term acclimation to a saturat- ing pulse of the chrysophysae Hymeno- monas elongata during Expt4. Vertical bars indicate standard deviation DISCUSSION l ~ 4 2 4 6 8 10 12 DAY The contradictions between these different studies and our results should be analyzed both from a methodological and theoretical point of view. As indi- cated by Mayzaud (1986), changes in digestive enzyme activities are likely to be representative of the ability to adapt to variations in food supply in order to achieve through assimilation the best possible fulfilment of metabolic requirements. Such dual control by the food concentration and the metabolic needs imposes on any experimental design: (1) a choice of enzyme represen- tative of the digestive processes, (2) a continued exist- ence of a food-limiting environment (i.e. suboptimal growth conditions), and (3) the persistence of similar metabolic requirements. Fig. 12. Acartia clausi. Changes in electron transport system (ETS) and glutamate dehydrogenase (GDH) specific activities for individuals fed limiting levels of the diatom Skeletonerna costatum and subjected to a pulse of saturating concentrations of the chrysophysae Hymenomonas elongata on Days 6 to 8. Open symbols: controls fed on S, costatum during the pulse of H. elongata Mar. Ecol. Prog. Ser. 69: 2 5 4 0 , 1991 36 From these considerations and the arguments developed by Samain et al. (1981) for Arternia and by Mayzaud & Mayzaud (1985) for neritic copepods, we suggest that the apparent contradictions between vari- ous laboratory experiments can be reconciled by con- sidering the experimental trophic situations. Harris et al. (1986) designed their experiments to achieve saturating conditions, where maximal growth and/or egg production could be achieved. Under such condi- tions, the copepods they used had no need to acclimate, as any increase in food supply was surfeit and digestive enzyme levels varied according to a compensatory mechanism between the digestive enzyme activity and the substrate ingested, i.e. the copepods adjusted their digestion and assimilation rates to their metabolic needs. Hassett & Landry (1983), on the other hand, experimented with both non-saturating conditions and saturating conditions, and tracked what, in our opinion, were enzymes not expected to be fully coupled to extracellular digestive processes. Indeed, endocellular enzymes hydrolyze those molecules resulting from the action of the extracellular digestion and are not directly related to the molecules ingested. The higher enzyme activity they reported for the 2 lower, food-limiting regimes would have been expected from the experi- mental conditions they imposed as positive acclimation only occurred for these 2 food concentrations. DISCUSSION In sum- mary both of these studies illustrated very specific trophic situations which were not central to the May- zaud & Poulet (1978) acclimation hypothesis. Considering the key role of protein nutrition (Paffenhofer & Van Sant 1985, Libourel-Houde & Roman 1987, Hassett & Landry 1988, Cowles et al. 1988), a balanced description of digestive system activ- ity should monitor proteases as well as those glycosid- ases involved in the extracellular digestion. In our opinion, the choice by Hassett & Landry (1983, 1988) to focus exclusively on glycosidases was unfortunate, for it left unknown the possibility of a differential response in the proteolytic activities like that documented in the present study, and more recently for an Antarctic, lipid- rich copepod (Drepanopus pectinatus; Mayzaud et al. unpubl.). Moreover, Hassett & Landry's selection of maltase and cellobiase as concurrent test enzymes was problematical as both are involved in the intracellular phase of digestion and are not likely to be directly related to food intake. Proper characterization of the experimental food conditions has been an important problem in most studies for it should be related to the nutritional status of the animals. Harris et al. (1986) used different food items at concentrations varying between 969 and 2149 ygC 1-l; such concentrations represented a non-limit- ing trophic environment as attested by the stability of the body protein level throughout the experiment, irrespective of the food source or concentration. In contrast, although Hassett & Landry (1983) used food concentrations ranging from 200 to 1452 pgC I-', the recorded changes in dry weight suggested that max- imum copepod growth (non-limitation) was achieved only at their 2 highest experimental food concen- trations (4200 cells mlpl = 924 ygC 1-I and 660 cells ml-I = 1452 ygC 1-l) while limitation seemed to occur at the first 2 concentrations (1000 cells ml-l = 220 ygC 1-I and 2000 cells mlpl = 440 pgC 1-l) although to a lesser degree than in the natural environment. In the present experiments, even though Skeletonerna cos- taturn concentrations were increased an order of mag- nitude, from 360 to 5600 ygC 1-' (1.5 to 18 pgC per Acartia clausi) during the course of our laboratory evaluation, this range of food ration appeared to be generally limiting. DISCUSSION Positive responses were recorded over a range of 2 to 12 pgC ind.-l but were more sustained for discontinu- ous food stimuli. These results agree with the early findings by Cox (1981) and Cox & Willasson (1981) and explain the lack of short-term experimental changes (4 to 6 h) described by Hassett & Landry (1988). However, they do not support the various field reports of die1 changes in enzyme activity (Boucher & Samain 1974, Tande & Slagstad 1982, Bdmstedt 1984, 1988, Mayzaud et al. 1984). The validity of a direct comparison between short- term and medium-term acclimation must be considered carefully. We might anticipate that short-term changes in digestive processes would be associated with the cycles of active feeding (6 to 8 h) and controlled by the sequence of gut processes. Most of the digestion takes place in the midgut, and 4 main cell-types are usually associated with that sequence: (1) R-cells (equivalent to the absorptive cells of decapods), (2) F-cells (site of synthesis of the digestive enzymes involved in extracellular digestion), (3) E-cells (undifferentiated which can develop into either R or F cells, and (4) B- cells (responsible for intracellular digestion) which must disintegrate to release their digestive products before assimilation by the R-cells (Amaud et al. 1978, 1980, 1983, 1984a, b). In a recent study, Nott et al. (1985) showed that at the onset of feeding, Calanus helgolandicus F-cells were still in their developmental stage while B-cells were small, reduced in number and showed little sign of bursting into the gut lumen. In contrast, l h after feeding some B-cells had disinte- grated and this process was almost complete at the end of 7 h. Since B-cells cannot be replaced continuously, a rebuilding period is required to reset the digestive structure. In Expt 4, longer-term acclimation at medium and more or less constant food ration (3 to 4 pgC ind.-l) showed that over an 8 d period, the day-to-day fluctua- tion in ingestion rates had no marked influence on the corresponding enzyme activity. Thus, variation in excess of 1 and 2 ygC ind.-l is probably required for the digestive enzyme system activity to fluctuate in response. It is worth noting that introducing a pulse of saturating food ration resulted in a stabilization (or a small decrease) in 3 of the 4 enzyme activities moni- tored. DISCUSSION The results we obtained in the first 3 experiments of the present study clearly confirm that Acartia-type copepods exposed to different food-limiting levels will positively adjust both their feeding pattern and their digestive enzyme activities, although these adjust- ments do not necessarily occur on identical time scales. For example, while changes in ingestion closely tracked day-to-day variations in food supply, changes in digestive enzyme activities were functionally linked to the medium-term, time-averaged food concentration and the medium-term, time-averaged ingestion. In all our experiments, in situ enzyme activities (Do) could be not be maintained under laboratory conditions and a transition period of 1 to 3 d was manifest before the new 'food regime(s)' could be correlated signifi- cantly with digestive enzymes. Note that this 1 to 3 d transition was necessary, irrespective of whether the copepods were previously acclimated (Expts 2 and 3) or not (Expt 1) to our laboratory environment. Starvation consistently resulted in a decrease of activity but, as already observed by Boucher & Samain (1974) and by Mayzaud & Mayzaud (1985), such a decrease in diges- tive enzyme activity generally had a lag time of 1 to 2 d. Unchanging levels of metabolic requirements are extremely difficult to achieve as they would require laboratory conditions which perfectly simulate the natural ones. Nevertheless, probable changes should be kept in mind during the interpretation of acclimation experiments. As indicated by Donaghay (1988), changes in growth response or egg production are not only related to food availability but also to previous history or precon- ditioning of the animals. Intuitively, one would expect that switching an organism from a turbulent, patchy food environment to laboratory conditions would result in changes in the energy and metabolic requirements associated with the new life environment. The lack of significant differences between amylase (or laminarinase) activities between low and medium Roche-Mayzaud et al.: Acclimation )f feeding and digestive enzyme activity 37 food levels (350 and 700 pgC 1-l) in Expt 1 was surpris- ing especially when compared to the reactivity of the trypsin system. Such differences among enzyme sys- tems suggest there may be a differential threshold of sensitivity which may functionally enhance the poten- tial for protein assimilation at low food concentration. DISCUSSION We suggest that such data underline the importance of protein assimilation for meeting the metabolic require- ments of copepods, as already emphasized by Paffenhofer & Van Sant (1985), Libourel-Houde & Roman (1987), Hassett & Landry (1988) and Cowles et al. (1988). In Expt 2, decreasing the food ration failed to initiate parallel decrease of any glycosidase activities but it strongly induced a drop in trypsin activity with a 24 h delay. Interestingly, the lack of a sharp decrease in glycosidase activities was still associated with a posi- tive acclimation to the time-averaged food concen- tration, suggesting the existence of 2 different levels of time regulation processes that are diversely affected by the 'memory effect' from the initial food conditions. The acclimation to the difference in food levels was unaf- fected by the direction of the food concentration changes which was only reflected by those enzyme with low initial activity level, i.e. s m d 'memory effect'. We believe it probable that the initial high food level (D2) increased the duration of the memory effect associ- ated with already high natural glycosidase activities. In Expt 3, successive pulses of phytoplankton were not recorded by either amylase or laminarinase but induced a 24 h shifted rhythmicity of trypsin at high food level. The dominance of carbohydrates over pro- teins in the food ration may explain the more limited induction of trypsin at the lower food concentration. tiate such adaptation. Under the present experimental conditions, Acartia clausi appeared to require 48 h to demonstrate a significant acclimation with changes in food ration exceeding a threshold of 1.5 to 2 pgC ind.-l. Positive responses were recorded over a range of 2 to 12 pgC ind.-l but were more sustained for discontinu- ous food stimuli. These results agree with the early findings by Cox (1981) and Cox & Willasson (1981) and explain the lack of short-term experimental changes (4 to 6 h) described by Hassett & Landry (1988). However, they do not support the various field reports of die1 changes in enzyme activity (Boucher & Samain 1974, Tande & Slagstad 1982, Bdmstedt 1984, 1988, Mayzaud et al. 1984). tiate such adaptation. Under the present experimental conditions, Acartia clausi appeared to require 48 h to demonstrate a significant acclimation with changes in food ration exceeding a threshold of 1.5 to 2 pgC ind.-l. DISCUSSION Stress situations such as copper poisoning (Bdmstedt 1980) or high noise (vibrations) levels (Regnault & Lagardere 1983) also resulted in an increase in either metabolic (respiration, ammonia excretion) or ETS levels. except through allosteric changes in the rate of enzyme hydrolysis or through differential use of the available F and B cells. However, on longer time scales (>48 h), acclimation could conceivably result from either an increase in the number of F- and B-cells, or an increase in the concentration of enzyme per cell, or both. Thus short- and medium-term acclirnation are likely to pro- ceed at the cellular level via different sets of regulatory factors (Mayzaud 1986) and the two are not directly comparable. Experiments to test the validity of ETS activity as an estimator of respiratory rates carried out by Bhmstedt (1980) and Ikeda & Skjoldal (1980) both showed a fair correspondence between the 2 measurements despite some differences in the time scales for acclimation. Fast decreases in ETS activity and respiration rates for either fed or starved Acartia were reported in the same 2 studies, with strong indications that the effect of feeding conditions was additive to another unknown underlying effect. The results of the present study confirm these observations and suggest a laboratory-effect-related food limitation which was only released (Expt 4) when feeding at saturation occurred. In contrast, GDH activity seemed more influenced by such an effect as it never recovered to a level close to the natural one. As recently noted by Donaghay (1988), it would be useful to review the definition of time acclimation given by Mayzaud & Poulet (1978) in order to emphasize: (1) that nutrition in copepods is controlled by feed-back mechanisms which operate to meet the metabolic requirements of individuals, and (2) that acclimation of the different aspects of nutrition (inges- tion, digestion, assimilation), when necessary, may operate on different time scales. The influences of food quality, feeding history or food selectivity on ingestion rates are all evidence of feed-back controls. Whether they occur systematically with digestive acclimation is doubtful but both processes are part of a general adap- tive strategy to a fluctuating trophic environment. DISCUSSION The limited increase in alkaline protease activ- ity, when compared with the sharp increase in inges- tion rate from Days 6 to 8, probably reflects a general increase in intermediary metabolism, evident from the ETS data. In summary, our final feeding experiment confirmed that medium-term acclirnation of digestive enzyme activity is best demonstrated and is likely restricted to situations of food limitation. Detailed data on the cycle of F-cells are lacking for copepods but the time courses of the processes are probably analogous to those that occur in decapod crustacea (Mohanna-A1 et al. 1985): (1) for about 30 min after feeding F-cells contain numerous zymo- gen-like granules; (2) these granules decrease in fre- quency after 1 or 2 h; (3) when the more proximal F- cells have released most of their enzyme content, they differentiate into early stage B-cells. If similar proces- ses occur in most copepods, individuals fed at satura- tion level will see their enzyme production limited by the availability of F-cells and B-cells, while individuals feeding at lower concentrations will be able to extend their feeding and digestive period. Note that in both cases, the copepods themselves have no possibility for short-term modification of their digestive capabilities, In an earlier study, Mayzaud & Poulet (1978) sug- gested that between 24 h and 6 days was required for the enzyme system to acclimate, but could not define the minimum size of the food ration which might ini- Mar. Ecol. Prog. Ser. 69: 2540, 1991 38 except through allosteric changes in the rate of enzyme hydrolysis or through differential use of the available F and B cells. However, on longer time scales (>48 h), acclimation could conceivably result from either an increase in the number of F- and B-cells, or an increase in the concentration of enzyme per cell, or both. Thus short- and medium-term acclirnation are likely to pro- ceed at the cellular level via different sets of regulatory factors (Mayzaud 1986) and the two are not directly comparable. The overall time stability of the both ETS and GDH in most our experiments is also an index of normal phy- siological behaviour, despite high food concentrations and high animal densities. Indeed, Bhmstedt (1980) found no influence of Acartia tonsa densities between 15 and 800 ind. 1-I and observed an increase in ETS activity from 1200 to 4000 ind. 1-l. DISCUSSION Correspondence between intermediary metabolism (respiration and/or nitrogen excretion) and digestive enzyme activity has been proposed from comparisons of different copepod species living in the same environ- ment (Gaudy & Boucher 1983), and postulated from observations of variations in ETS activity during studies of die1 changes (Harris & Malej 1986, Bdmstedt 1988) Except during starvation, day-to-day patterns of ETS and GDH activities in the present experiments failed to covary with food quantity, although ETS activity appeared to be influenced by food quality (protein versus carbohyrate dominated) and food type (chryso- phyte versus diatom). Under our experimental condi- tions, such independence from food concentration was to be expected, for several recent studies have indi- cated saturation of respiration rates may occur above food levels of 500 pgC I-', in species as different as the copepod Acartia tonsa (Kinrboe et al. 1985) and the cladoceran Daphnia magna (Lampert 1986). Because of this, the increase in ETS activity which occurred in our Expt 4 when A. clausi were fed on an extremely strong pulse of Hymenomonas elongata was surprising, although obviously tied to the increase in specific dynamic action associated to the increase in ingestion rate. Moreover, why we observe a 'memory effect' after these copepods were transferred back to lower food levels is unclear, but could be related to the same regulatory processes that maintained high glycosidase activities under decreasing food concentrations in Expt 2. Acknowledgements. The authors thank Dr R. J. Conover for his helpful criticisms and comments of the manuscript. This research was supported by ERA-CNRS 228, NSERC grant #3667 and FCAR team grant #3578 to P. M. D.C. Biggs' participation was made possible by a Faculty Academic Study Leave Award from Texas A & M University, supplemented by a TAMU minigrant for International Enhancement. We thank Mrs Laura Cassiani who performed most of the measurements on the biochemical composition of the phytoplankton cultures. Acknowledgements. The authors thank Dr R. J. Conover for his helpful criticisms and comments of the manuscript. This research was supported by ERA-CNRS 228, NSERC grant #3667 and FCAR team grant #3578 to P. M. D.C. Biggs' participation was made possible by a Faculty Academic Study Leave Award from Texas A & M University, supplemented by a TAMU minigrant for International Enhancement. We thank Mrs Laura Cassiani who performed most of the measurements on the biochemical composition of the phytoplankton cultures. LITERATURE CITED Arnaud, J., Brunet, M., Mazza, J. (1978). Stuhes on the midgut of Centropages typicus (copepod, calanoid). Cell Tissue Res. 187: 333-353 Arnaud, J., Brunet, M., Mazza, J. (1980). Structure et ultra- structure comparee de l'intestin chez plusieurs especes de copepodes calanoides. Zoomorphologie 95: 213-333 Arnaud, J., Brunet, M., Mazza, J. (1983). Detection d'une activite phosphatasique acide dans les cellules B de l'intes- tin moyen de Centropages typicus (Copepode, calanoide) C. R. Acad. Sci. Paris 296: 727-730 Arnaud, J., Brunet, M., Mazza, J. (1984a). Detection d'une activite arylsulfatasique dans les cellules B de l'intestin moyen de Centropages typicus (Copepode, calanoide). C. R. Acad. Sci. Paris 298: 499-502 Arnaud, J., Brunet, M., Mazza, J. (198413). Cytochemical detection of phosphatase and arylsulfatase in the midgut of Centropages typicus (Copepod, calanoid). Bas. appl. Histochem. 28: 399412 Ayukai, T. (1987). Feeding by the planktonic calanoid copepod Acartia clausi, Giesbrecht, on natural suspended particulate matter of varying quantity and quality. J. exp. mar. Biol. Ecol. 106: 137-149 Roche-Mayzaud et al.: Acclimation of feeding and digestive enzyme activity 39 Bbmstedt, U. (1980). ETS activity as an estimator of respiratory rate of zooplankton populations. The significance of varia- tions in environmental factors. J, exp. mar. Biol. Ecol. 42: 267-283 adults of Calanus finmarchicus and Calanus helgolandicus in relation to particulate matter. Kieler Meeresforsch. Sonderh. 5: 174-185 adults of Calanus finmarchicus and Calanus helgolandicus in relation to particulate matter. Kieler Meeresforsch. Sonderh. 5: 174-185 Huntley, M. (1981). Non-selective, non-saturating feeding by three calanoid copepod species of the Labrador Sea. Limnol. Oceanogr. 26: 831-842 Bimstedt, U. (1984). Diel variations in the nutritional physiol- ogy of Calanus glacialis from lat. 78"N in the summer. Mar. Biol. 79: 257-267 Ikeda, T., Skjoldal, H. R. (1980). The effect of laboratory conditions on the extrapolation of experimental measure- ments to the ecology of marine plankton. VI. Changes in physiological activities and biochemical components of Acetes sibogae and Acartia australis after capture. Mar. Biol. 58: 285-293 Bhstedt, U. (1988). Interspecific, seasonal and die1 variations in zooplankton trypsin and amylase activity in Kosterfjor- den, western Sweden. Mar. Ecol. Prog. Ser. 44: 15-24 Bihgare, R. R., King, F. D., Biggs, D. C. (1982). Glutamate dehydrogenase (GDH) and respiratory electron-transport system (ETS) activities in Gulf of Mexico zooplankton. J. Plankton Res. 4: 895-91 1 Kimboe, T., M~lenberg, F., Hamburger, K. (1985). LITERATURE CITED Digestive enzymes and their relation to nutrition. In: Corner, E. D. S., O'Hara, S. C. M. (eds.) The biological chemistry of marine copepods. Clarendon Press, Oxford, p. 165-225 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. (1956). Colorimetric method for the determination of sugars and related substances. Analyt. Chem. 28: 350-356 Mayzaud, P,, Farber-Lorda, J., Corre, M. C. (1985). Aspects of the nutritional metabolism of two Antarctic euphausiids Euphausia superba and Tysanoessa macrura. In: Siegfried, W. R., Condy, P. R., Laws, R. M. (eds.) Antarctic nutrient cycles and food webs. Springer-Verlag, Berlin, p. 330-338 Frost, B. W. (1972). Effect of size and concentration of food particles on the feeding behavior of the marine planktonic copepod Calanus pacificus. Limnol. Oceanogr. 17: 805-815 Mayzaud, P., Mayzaud, 0. (1981). Kinetic properties of diges- tive carbohydrases and proteases of zooplankton. Can. J. Fish. Aquat. Sci. 38: 535-543 Gaudy, R., Boucher, J. (1983). Relation between respiration, excretion (ammonia and inorganic phosphorus) and activ- ity of amylase and trypsin in different species of pelagic copepods from an Indian ocean equatorial area. Mar. Biol. 75: 37-45 Mayzaud, P,, Mayzaud, 0. (1985). The influence of food limi- tation on the nutritional adaptation of marine zooplankton. Arch. Hydrobiol. Beih. Ergebn. Limnol. 21: 223-233 Arch. Hydrobiol. Beih. Ergebn. Limnol. 21: 223-23 Harris, R. P,, Malej, A. (1986). Diel patterns of ammonium excretion and grazing rhythms in Calanus helgolandicus in surface stratified waters. Mar. Ecol. Prog. Ser. 31: 75-85 Mayzaud, O., Mayzaud, P., de la Bigne, C., Grohan, P,, Con- over, R. J. (1984). Diel changes in the particulate environ- ment, feeding activity and digestive enzyme concentration in neritic zooplankton. J. exp. mar. Biol. Ecol. 84: 15-35 Hanis, R. P,, Samain, J.-F., Moal, J., Martin-Jezequel, V., Poulet, S. A. (1986). Effect of algal diet on digestive enzyme activity in Calanus helgolandicus. Mar. Biol. 90: 353-361 eritic zooplankton. J. exp. mar. Biol. Ecol. 84: 15-3 Mohanna-Al, S. Y., Nott, J. A., Lane, D. J. W. (1985). Mitotic E- and secretary F-cells in the hepatopancreas of the shrimp Penaeus semisulcatus (Crustacea, decapoda). J. mar. biol. Ass. U.K. 65: 901-910 Hassett, R. P,, Landry, M. R. (1983). Effect of food level acclimation on digestive enzyme activities and feeding behavior of Calanus pacificus. Mar. Biol. 75: 47-55 Nott, J. A., Corner, E. D. S., Marin, L. J., O'Hara, S. C. M. (1985). LITERATURE CITED Bioenerge- tics of the planktonic copepod Acartia tonsa: relation between feeding, egg production and respiration, and composition of specific dynamic action. Mar. Ecol. Prog. Ser. 26: 85-97 Boucher, J., Samain, J.-F. (1974). L'activite amylasique indice de la nutrition du zooplancton; mise en evidence d'un rythme quotiden en zone d'upwelling. Tethys 6: 179-188 Boucher, J., Laurec, A., Samain, J.-F., Smith, S. L. (1976) etude de la nutrition du zooplancton en zone d'upwelling par la mesure des activites enzymatiques digestives. In: Personne, G., Jaspers, H. (eds.) Proc. 10th Eur. Mar. Biol. Symp., Vol. 11. University Press, Wetteren, p. 85-110 Koeller, P. A., Barwell-Clarke, J. E., Whitney, F., Takahashi, M. (1979). Winter conditions of marine zooplankton popu- lation in Saanich inlet, B.C., Canada. 3. Mesozooplankton. J. exp. mar. Biol. Ecol. 37: 161-174 Lampert, W. (1986). Response of the respiratory rate of Daph- nia magna to changing food conditions. Oecologia (Berl.) 70: 495-501 Conover, R. J. (1978). Transformation of organic matter. In: Kinne, 0. (ed.) Marine ecology, Vol. IV, Dynamics. Wiley, Chichester, p. 221-499 Landry, M. R., Hassett, R. P. (1985). Time scales in behavioral biochemical and energetic adaptations to food-limiting conditions by a marine copepod. Arch. Hydrobiol. Beih. Ergebn. Limnol. 21: 209-221 Conover, R. J., Mayzaud, P. (1984). Utilization of phytoplank- ton by zooplankton during the spring bloom in a Nova Scotia inlet. Can. J. Fish. Aquat. Sci. 41: 232-244 Cowles, T. J., Olson, R. J., Chisholm, S. W. (1988). Food selection by copepods: discrimination on the basis of food quality. Mar. Biol. 100: 41-49 Libourel-Houde, S. E., Roman, M. R. (1987). Effects of food quality on the functional ingestion response of the copepod Acartia tonsa. Mar. Ecol. Prog. Ser. 40: 60-77 Cox, J. L. (1981). Laminarinase induction in marine zooplank- ton and its variability in zooplankton samples. J. Plankton Res. 3: 345-356 Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J. (1951). Protein measurement with the Folin-phenol rea- gent. J. biol. chem. 193: 265-275 Cox, J. L., Willasson, S, W. (1981). Larninarinase induction in Calanuspacificus. Mar. Biol. Lett. 2: 307-311 Mayzaud, P. (1980). Some sources of variability in the deter- mination of digestive enzyme activity in zooplankton. Can. J. Fish. Aquat. Sci. 37: 1426-1432 Donaghay, P. L. (1988). The role of temporal scales of acclima- tion, food quality and trophic dominance in controlling the evolution of copepod feeding behavior. Bull. mar. Sci. 43: 469-485 Mayzaud, P. (1986). This article was presented by Dr R. J. Conover, Dartmouth, N.S., Canada LITERATURE CITED Cyclical contributions of the digestive epithelium to fecal pellet formation by the copepod Calanus helgoland- icus. Mar. Biol. 83: 271-280 Hassett, R. P,, Landry, M. R. (1988). Short term changes in feeding and digestion by the copepod Calanus pacificus. Mar. Biol. 99: 63-74 Head, E. J. H., Conover, R. J. (1983). Induction of digestive en- zyme in Calanus hyperboreus. Mar. Biol. Lett. 4: 219-231 Hirche, H. J. (1981). Digestive enzymes of copepodites and O'Connors, H. B., Small, L. F., Donaghay, P. L. (1976). Particle- size modification by two size classes of the estuarine copepod Acartia clausi. Limnol. Oceanogr. 21: 300-308 Mar. Ecol. Prog. Ser. 69: 2540, 1991 40 Paffenhofer, G. A., Van Sant, K. B. (1985). The feeding response of a marine planktonic copepod to quantity and quality of particles. Mar. Ecol. Prog. Ser. 27: 55-65 Possible processes of nutritive adaptations for zooplank- ton: a demonstration on Artemia. Kieler Meeresforsch. Sonderh. 5: 218-228 Possible processes of nutritive adaptations for zooplank- ton: a demonstration on Artemia. Kieler Meeresforsch. Sonderh. 5: 218-228 Reeve, M. R., Walter, M. A. (1977). Observations on the existence of lower threshold and upper critical food con- centrations for the copepod Acartia clausi. J. exp, mar. Biol. Ecol. 29: 211-221 Tande, K. S., Slagstadt, D. (1982). Ecological investigation on the zooplankton community of Balsfjorden, northern Nor- way. Seasonal and short-time variations in enzyme activity in copepodite Stage V and V1 male and female of Calanus finmarchicus. Sarsia 67: 63-68 Regnault, M,, Lagardere, J. P. (1983). Effects of ambient noise on the metabolic level of Crangon crangon (Decapoda, natantia). Mar. Ecol. Prog. Ser. 11: 71-78 Uye, S., Kasahara, S. (1983). Grazing of various developmen- tal sages of Pseudodiaptomus marinus (Copepode, calanoida) on naturally occurring particles. Bull. Plankton Soc. Japan 30: 147-158 Roche-Mayzaud, 0. (1985). Purification and kinetic properties of the a-amylase from the copepod Acartia clausi (Gies- brecht). Comp. Biochem. Physiol. 82B: 725-730 Van Wormhoudt, A. (1973). Activite des proteases, des amyl- ases et des proteines solubles au cours du developpement larvaire chez Palaemon serratus. Mar. Biol. 19: 245-258 Roche-Mayzaud, O., Mayzaud, P. (1987). Purification of end0 and exolaminarinase and partial characterization of the exoacting form from the copepod Acartia clausi (Gies- brecht). Comp. Biochem. Physiol. 88B: 105-110 Yentsch, C. S., Menzel, D. (1963). A method for the determina- tion of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep Sea Res. 10: 221-231 Samain, J.-F., Moal, J., Daniel, J. Samain, J.-F., Moal, J., Daniel, J. Y., Le Coz, .l. R. (1981). LITERATURE CITED Y., Le Coz, .l. R. (1981). Manuscript first received: January 8, 1990 Manuscript first received: January 8, 1990 Revised version accepted: October 10, 1990
https://openalex.org/W4313839430
https://egusphere.copernicus.org/preprints/2022/egusphere-2022-437/egusphere-2022-437.pdf
English
null
Reply on RC1 After Revision
null
2,023
cc-by
21,395
ERROR: type should be string, got "https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Sensitivities of subgrid-scale physics schemes, meteorological forcing, \nand \ntopographic \nradiation \nin \natmosphere-through-bedrock \nintegrated process models: A case study in the Upper Colorado River \nBasin Zexuan Xu1, Erica R. Siirila-Woodburn1, Alan M. Rhoades1, Daniel Feldman1 \n5 \n1 Earth and Environmental Sciences Area, Lawrence Berkeley National Laboratory \nCorrespondence to: Zexuan Xu (zexuanxu@lbl.gov) Abstract. Mountain hydrology is controlled by interacting processes extending from the atmosphere through the b Abstract. Mountain hydrology is controlled by interacting processes extending from Abstract. Mountain hydrology is controlled by interacting processes extending from the atmosphere through the bedrock. Integrated process models (IPM), one of the main tools needed to interpret observations and refine conceptual models of the \n10 \nmountainous water cycle, require meteorological forcing that simulates the atmospheric process to predict hydroclimate then \nsubsequently impacts surface-subsurface hydrology. Complex terrain and extreme spatial heterogeneity in mountainous \nenvironments drive uncertainty in several key considerations in IPM configurations, and require further quantification and \nsensitivity analyses. Here, we present an IPM using the Weather Research and Forecasting (WRF) model coupled with an \nintegrated hydrologic model, ParFlow-CLM, implemented over a domain centered over the East River Watershed (ERW), \n15 \nlocated in the Upper Colorado River Basin (UCRB). The ERW is a heavily-instrumented 300 km2 region in the headwaters \nof the UCRB near Crested Butte, CO, with a growing atmosphere-through-bedrock observation network. Through a series of \nexperiments in water year 2019 (WY19), we use four meteorological forcings derived from commonly used reanalysis \ndatasets, three subgrid-scale physics scheme configurations, and two terrain shading options within WRF to test the relative \nimportance of these experimental design choices on key hydrometeorological metrics including precipitation, snowpack, as \n20 \nwell as evapotranspiration, groundwater storage, and discharge simulated by the ParFlow-CLM. Results reveal that sub-grid \nscale physics configuration contributes to larger spatiotemporal variance in simulated hydrometeorological conditions, \nwhereas variance across meteorological forcing with common sub-grid scale physics configurations is more spatiotemporally \nconstrained. For example, simulated discharge shows greater variance in response to the WRF simulations across subgrid-\nscale physics schemes (26%) rather than meteorological forcing (6%). Topographic radiation option has minor effects on the \n25 \nwatershed-average hydrometeorological processes, but adds profound spatial heterogeneity to local energy budgets (+/-30 \nW/m2 in shortwave radiation and 1 K air temperature differences in late summer). The findings from this study provide \nguidance on an IPM setup that most accurately represents atmospheric-through-bedrock hydrometeorological processes and \ncan be used to guide future modeling and fieldwork in mountainous watersheds. Abstract. Mountain hydrology is controlled by interacting processes extending from the atmosphere through the bedrock. Sensitivities of subgrid-scale physics schemes, meteorological forcing, \nand \ntopographic \nradiation \nin \natmosphere-through-bedrock \nintegrated process models: A case study in the Upper Colorado River \nBasin Integrated process models (IPM), one of the main tools needed to interpret observations and refine conceptual models of the \n10 \nmountainous water cycle, require meteorological forcing that simulates the atmospheric process to predict hydroclimate then \nsubsequently impacts surface-subsurface hydrology. Complex terrain and extreme spatial heterogeneity in mountainous \nenvironments drive uncertainty in several key considerations in IPM configurations, and require further quantification and \nsensitivity analyses. Here, we present an IPM using the Weather Research and Forecasting (WRF) model coupled with an y\ny\n,\np\ng\ng (\n)\np\nintegrated hydrologic model, ParFlow-CLM, implemented over a domain centered over the East River Watershed (ERW), \n15 \nlocated in the Upper Colorado River Basin (UCRB). The ERW is a heavily-instrumented 300 km2 region in the headwaters \nof the UCRB near Crested Butte, CO, with a growing atmosphere-through-bedrock observation network. Through a series of \nexperiments in water year 2019 (WY19), we use four meteorological forcings derived from commonly used reanalysis \ndatasets, three subgrid-scale physics scheme configurations, and two terrain shading options within WRF to test the relative integrated hydrologic model, ParFlow-CLM, implemented over a domain centered over the East River Watershed (ERW), \n15 \nlocated in the Upper Colorado River Basin (UCRB). The ERW is a heavily-instrumented 300 km2 region in the headwaters \nof the UCRB near Crested Butte, CO, with a growing atmosphere-through-bedrock observation network. Through a series of \nexperiments in water year 2019 (WY19), we use four meteorological forcings derived from commonly used reanalysis \ndatasets, three subgrid-scale physics scheme configurations, and two terrain shading options within WRF to test the relative importance of these experimental design choices on key hydrometeorological metrics including precipitation, snowpack, as \n20 \nwell as evapotranspiration, groundwater storage, and discharge simulated by the ParFlow-CLM. Results reveal that sub-grid \nscale physics configuration contributes to larger spatiotemporal variance in simulated hydrometeorological conditions, \nwhereas variance across meteorological forcing with common sub-grid scale physics configurations is more spatiotemporally \nconstrained. For example, simulated discharge shows greater variance in response to the WRF simulations across subgrid- importance of these experimental design choices on key hydrometeorological metrics including precipitation, snowpack, as \n20 \nwell as evapotranspiration, groundwater storage, and discharge simulated by the ParFlow-CLM. Results reveal that sub-grid \nscale physics configuration contributes to larger spatiotemporal variance in simulated hydrometeorological conditions, \nwhereas variance across meteorological forcing with common sub-grid scale physics configurations is more spatiotemporally \nconstrained. 1. Introduction \n30 An improved predictive understanding of watershed dynamics and response to perturbations is particularly important for \nmountainous watersheds due to the multitude of natural services they provide even while those services are highly \nvulnerable to anthropogenic and natural environmental change (Hubbard et al., 2018; Siirila-Woodburn et al., 2021). The \nUpper Colorado River Basin (UCRB), which supports 40 million people and ecosystems that has experienced major \nhydrological change in recent decades (James et al., 2014). Discharge may have decreased by ~9.3% per degree Celsius of \n35 \nwarming, due to processes extending from the atmosphere through the subsurface (Milly and Dunne, 2020). Drought is \ncommon to the region, however, the current multi-decade drought is unprecedented in at least the last 1200 years (Williams \net al., 2022). To better estimate how aridification of the UCRB might continue, processes that shape the water cycle in this \nregion must be considered holistically, including atmospheric processes such as large-scale vapor transport, precipitation and \nradiation, land surface processes such as evapotranspiration and snowpack metamorphosis, and surface-through-subsurface \n40 \nhydrological processes. Atmospheric and land surface processes all interact and influence river discharge through riverine \nprocesses, infiltration, and subsurface flow and storage, but their impact varies depending on the temporal and spatial scales \nof analysis (Siirila-Woodburn et al., 2021). Unfortunately, there is a dearth of observational data to constrain these processes \nat their relevant scales, leading to persistent biases in the predictive understanding of the mountainous hydrologic cycle with \ndirect implications for water resource management (Sturm et al., 2017; Rhoades et al., 2018a,b,c; Xu et al., 2019). A recent \n45 \nstudy by Lundquist et al. (2019) highlighted that calibrated models, which themselves have numerous deficiencies, have \nlikely outpaced the skill of observationally-based gridded products in advancing the understanding of the integrated \nmountainous hydrologic cycle. Fortunately, observational campaigns, combined with coordinated modeling activities, \nrepresent a potential path forward towards enhancing our predictive understanding of the hydrologic cycle in complex terrain \n(Feldman et al., 2021). 50 An improved predictive understanding of watershed dynamics and response to perturbations is particularly important for \nmountainous watersheds due to the multitude of natural services they provide even while those services are highly \nvulnerable to anthropogenic and natural environmental change (Hubbard et al., 2018; Siirila-Woodburn et al., 2021). https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Sensitivities of subgrid-scale physics schemes, meteorological forcing, \nand \ntopographic \nradiation \nin \natmosphere-through-bedrock \nintegrated process models: A case study in the Upper Colorado River \nBasin For example, simulated discharge shows greater variance in response to the WRF simulations across subgrid- scale physics schemes (26%) rather than meteorological forcing (6%). Topographic radiation option has minor effects on the \n25 \nwatershed-average hydrometeorological processes, but adds profound spatial heterogeneity to local energy budgets (+/-30 \nW/m2 in shortwave radiation and 1 K air temperature differences in late summer). The findings from this study provide \nguidance on an IPM setup that most accurately represents atmospheric-through-bedrock hydrometeorological processes and \ncan be used to guide future modeling and fieldwork in mountainous watersheds. scale physics schemes (26%) rather than meteorological forcing (6%). Topographic radiation option has minor effects on the \n25 \nwatershed-average hydrometeorological processes, but adds profound spatial heterogeneity to local energy budgets (+/-30 \nW/m2 in shortwave radiation and 1 K air temperature differences in late summer). The findings from this study provide \nguidance on an IPM setup that most accurately represents atmospheric-through-bedrock hydrometeorological processes and \ncan be used to guide future modeling and fieldwork in mountainous watersheds. 1 1 1. Introduction \n30 Unfortunately, there is a dearth of observational data to constrain these processes \nat their relevant scales, leading to persistent biases in the predictive understanding of the mountainous hydrologic cycle with radiation, land surface processes such as evapotranspiration and snowpack metamorphosis, and surface-through-subsurface \n40 \nhydrological processes. Atmospheric and land surface processes all interact and influence river discharge through riverine \nprocesses, infiltration, and subsurface flow and storage, but their impact varies depending on the temporal and spatial scales \nof analysis (Siirila-Woodburn et al., 2021). Unfortunately, there is a dearth of observational data to constrain these processes \nat their relevant scales, leading to persistent biases in the predictive understanding of the mountainous hydrologic cycle with direct implications for water resource management (Sturm et al., 2017; Rhoades et al., 2018a,b,c; Xu et al., 2019). A recent \n45 \nstudy by Lundquist et al. (2019) highlighted that calibrated models, which themselves have numerous deficiencies, have \nlikely outpaced the skill of observationally-based gridded products in advancing the understanding of the integrated \nmountainous hydrologic cycle. Fortunately, observational campaigns, combined with coordinated modeling activities, \nrepresent a potential path forward towards enhancing our predictive understanding of the hydrologic cycle in complex terrain Here, we explore how modeling activities can best support that path forward. Process models provide an essential tool for \nquantifying linear and non-linear interacting processes across spatiotemporal scales that arise in mountains and can help to \nfill observational gaps. However, the processes that are represented in these process models are a mixture of fundamental Here, we explore how modeling activities can best support that path forward. Process models provide an essential tool for \nquantifying linear and non-linear interacting processes across spatiotemporal scales that arise in mountains and can help to \nfill observational gaps. However, the processes that are represented in these process models are a mixture of fundamental fill observational gaps. However, the processes that are represented in these process models are a mixture of fundamental \nphysics and subgrid-scale parameterizations, many of which were not developed with mountainous hydrologic cycle \n55 \nprocesses in theory, and/or are based on decades-old field and laboratory data. To further compound those issues, cross-scale \ninteractions in complex terrain are challenging to resolve at their native scales with currently available advanced computing \nresources (Siirila-Woodburn et al., 2021). 1. Introduction \n30 The \nUpper Colorado River Basin (UCRB), which supports 40 million people and ecosystems that has experienced major An improved predictive understanding of watershed dynamics and response to perturbations is particularly important for \nmountainous watersheds due to the multitude of natural services they provide even while those services are highly \nvulnerable to anthropogenic and natural environmental change (Hubbard et al., 2018; Siirila-Woodburn et al., 2021). The \nUpper Colorado River Basin (UCRB), which supports 40 million people and ecosystems that has experienced major hydrological change in recent decades (James et al., 2014). Discharge may have decreased by ~9.3% per degree Celsius of \n35 \nwarming, due to processes extending from the atmosphere through the subsurface (Milly and Dunne, 2020). Drought is \ncommon to the region, however, the current multi-decade drought is unprecedented in at least the last 1200 years (Williams \net al., 2022). To better estimate how aridification of the UCRB might continue, processes that shape the water cycle in this \nregion must be considered holistically, including atmospheric processes such as large-scale vapor transport, precipitation and hydrological change in recent decades (James et al., 2014). Discharge may have decreased by ~9.3% per degree Celsius of \n35 \nwarming, due to processes extending from the atmosphere through the subsurface (Milly and Dunne, 2020). Drought is \ncommon to the region, however, the current multi-decade drought is unprecedented in at least the last 1200 years (Williams \net al., 2022). To better estimate how aridification of the UCRB might continue, processes that shape the water cycle in this \nregion must be considered holistically, including atmospheric processes such as large-scale vapor transport, precipitation and radiation, land surface processes such as evapotranspiration and snowpack metamorphosis, and surface-through-subsurface \n40 \nhydrological processes. Atmospheric and land surface processes all interact and influence river discharge through riverine \nprocesses, infiltration, and subsurface flow and storage, but their impact varies depending on the temporal and spatial scales \nof analysis (Siirila-Woodburn et al., 2021). Unfortunately, there is a dearth of observational data to constrain these processes \nat their relevant scales, leading to persistent biases in the predictive understanding of the mountainous hydrologic cycle with radiation, land surface processes such as evapotranspiration and snowpack metamorphosis, and surface-through-subsurface \n40 \nhydrological processes. Atmospheric and land surface processes all interact and influence river discharge through riverine \nprocesses, infiltration, and subsurface flow and storage, but their impact varies depending on the temporal and spatial scales \nof analysis (Siirila-Woodburn et al., 2021). 1. Introduction \n30 While discipline-specific process models, such as those used to explore and \npredict atmospheric or subsurface processes have advanced scientific understanding in a myriad of ways through sustained \nengagement with extensive user communities (Gutowski et al., 2020), Integrated Process Models (IPMs), in which these \n60 \ndiscipline-specific process models are coupled, are relatively novel and are still being vetted for various scientific \napplications in complex terrain. For example, Maina et al. (2020) explored how the horizontal resolution of atmospheric physics and subgrid-scale parameterizations, many of which were not developed with mountainous hydrologic cycle \n55 \nprocesses in theory, and/or are based on decades-old field and laboratory data. To further compound those issues, cross-scale \ninteractions in complex terrain are challenging to resolve at their native scales with currently available advanced computing \nresources (Siirila-Woodburn et al., 2021). While discipline-specific process models, such as those used to explore and \npredict atmospheric or subsurface processes have advanced scientific understanding in a myriad of ways through sustained 60 engagement with extensive user communities (Gutowski et al., 2020), Integrated Process Models (IPMs), in which these \n60 \ndiscipline-specific process models are coupled, are relatively novel and are still being vetted for various scientific \napplications in complex terrain. For example, Maina et al. (2020) explored how the horizontal resolution of atmospheric 2 2 Additionally, Mallard et al. (2017) evaluated that the sensitivity of near-surface \n80 \ntemperatures and precipitation to changes in land use representation is smaller than the model error for those fields, while \nRudisill et al., (2021) found that the details of snow cover in the initial conditions of a WRF simulation in complex terrain \nare key to ensuring the skill of that simulation, not just in 2-meter air temperature but also in the surface energy budget. Meanwhile, Rahimi et al (2022) found minimal sensitivity of SWE in WRF simulations across the entire western United \nStates to microphysics schemes, but found large effects due to model resolution. On the other hand, the effects of \n85 \nmeteorological forcing as the lateral boundary conditions of WRF simulations have also been recognized. For instance, Xu et \nal. (2018) identified that the simulations of hydroclimate in California using WRF are largely driven by large-scale forcing \ndatasets. Taken together, the published literature suggests a one-size-fits-all WRF model configuration for hydrological \nstudies in complex terrain may not be possible. In other words, the WRF configuration is likely case- and region-specific, \nand could depend either on the representation of processes within the WRF simulation domain or the boundary conditions of \n90 \nWRF forced by the large-scale meteorological forcing. The options of subgrid-scale physics schemes and large-scale \nmeteorological forcing datasets need to be fully tested to understand their sensitivities to atmospheric and hydrological \nprocesses in the ERW. physics configuration on precipitation and snowpack processes in the UCRB (Rasmussen et al., 2011; Liu et al., 2011; Liu et \nal., 2017; Rasmussen et al, 2020). Outside of the UCRB, Orr et al. (2017) found cloud microphysics schemes have \nsignificant impacts on monsoon precipitation simulation in the complex-terrain Himalayan regions, with the Morrison \n75 \nmicrophysics scheme producing the best agreement with observations. Conversely, Comin et al. (2018) found that the \nMorrison microphysics scheme produced excessive snowfall and exhibited poor performance when evaluated in the Andes, \nwhile the Goddard (WDM6) scheme exhibited the best performance with respect to observed snowfall. In terms of land \nsurface process, Jin et al. (2010) explored that land surface model complexity improves temperature simulation, but has a significant impacts on monsoon precipitation simulation in the complex-terrain Himalayan regions, with the Morrison \n75 \nmicrophysics scheme producing the best agreement with observations. Conversely, Comin et al. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. forcings (40km to 0.5 km) in the Cosumnes River watershed, California, simulated by a widely-used regional climate model \n(Weather Research and Forecasting (WRF; Powers et al., 2017), result in differences in surface and subsurface hydrologic \nmetrics when used to force the integrated hydrologic model (ParFlow-CLM; Maxwell et al., 2015). We expand that \n65 \nsensitivity analysis in this study, including model meteorological forcing and subgrid-scale physics configuration choice, and \ntheir influence on the surface-through-subsurface response of the integrated hydrologic model. The goal of this work is to \nprovide the mountain hydrology research community with a properly-configured IPM that can inform ongoing and future \nfield campaigns and their process-modeling needs in the UCRB. forcings (40km to 0.5 km) in the Cosumnes River watershed, California, simulated by a widely-used regional climate model \n(Weather Research and Forecasting (WRF; Powers et al., 2017), result in differences in surface and subsurface hydrologic \nmetrics when used to force the integrated hydrologic model (ParFlow-CLM; Maxwell et al., 2015). We expand that \n65 \nsensitivity analysis in this study, including model meteorological forcing and subgrid-scale physics configuration choice, and \ntheir influence on the surface-through-subsurface response of the integrated hydrologic model. The goal of this work is to \nprovide the mountain hydrology research community with a properly-configured IPM that can inform ongoing and future \nfield campaigns and their process-modeling needs in the UCRB. 65 70 Standalone WRF simulations have been widely investigated in complex terrain, and provide context for the unfilled gaps in \nIPM investigation and development in complex terrain. For example, several papers detailed the role of subgrid-scale \nphysics configuration on precipitation and snowpack processes in the UCRB (Rasmussen et al., 2011; Liu et al., 2011; Liu et \nal., 2017; Rasmussen et al, 2020). Outside of the UCRB, Orr et al. (2017) found cloud microphysics schemes have \nsignificant impacts on monsoon precipitation simulation in the complex-terrain Himalayan regions, with the Morrison \n75 \nmicrophysics scheme producing the best agreement with observations. Conversely, Comin et al. (2018) found that the \nMorrison microphysics scheme produced excessive snowfall and exhibited poor performance when evaluated in the Andes, \nwhile the Goddard (WDM6) scheme exhibited the best performance with respect to observed snowfall. In terms of land \nsurface process, Jin et al. (2010) explored that land surface model complexity improves temperature simulation, but has a \nminimal impact on simulated precipitation. (2018) found that the \nMorrison microphysics scheme produced excessive snowfall and exhibited poor performance when evaluated in the Andes, \nwhile the Goddard (WDM6) scheme exhibited the best performance with respect to observed snowfall. In terms of land \nsurface process, Jin et al. (2010) explored that land surface model complexity improves temperature simulation, but has a significant impacts on monsoon precipitation simulation in the complex-terrain Himalayan regions, with the Morrison \n75 \nmicrophysics scheme producing the best agreement with observations. Conversely, Comin et al. (2018) found that the \nMorrison microphysics scheme produced excessive snowfall and exhibited poor performance when evaluated in the Andes, \nwhile the Goddard (WDM6) scheme exhibited the best performance with respect to observed snowfall. In terms of land \nsurface process, Jin et al. (2010) explored that land surface model complexity improves temperature simulation, but has a minimal impact on simulated precipitation. Additionally, Mallard et al. (2017) evaluated that the sensitivity of near-surface \n80 \ntemperatures and precipitation to changes in land use representation is smaller than the model error for those fields, while \nRudisill et al., (2021) found that the details of snow cover in the initial conditions of a WRF simulation in complex terrain \nare key to ensuring the skill of that simulation, not just in 2-meter air temperature but also in the surface energy budget. Meanwhile, Rahimi et al (2022) found minimal sensitivity of SWE in WRF simulations across the entire western United minimal impact on simulated precipitation. Additionally, Mallard et al. (2017) evaluated that the sensitivity of near-surface \n80 \ntemperatures and precipitation to changes in land use representation is smaller than the model error for those fields, while \nRudisill et al., (2021) found that the details of snow cover in the initial conditions of a WRF simulation in complex terrain \nare key to ensuring the skill of that simulation, not just in 2-meter air temperature but also in the surface energy budget. Meanwhile, Rahimi et al (2022) found minimal sensitivity of SWE in WRF simulations across the entire western United minimal impact on simulated precipitation. Additionally, Mallard et al. (2017) evaluated that the sensitivity of near-surface \n80 \ntemperatures and precipitation to changes in land use representation is smaller than the model error for those fields, while \nRudisill et al., (2021) found that the details of snow cover in the initial conditions of a WRF simulation in complex terrain \nare key to ensuring the skill of that simulation, not just in 2-meter air temperature but also in the surface energy budget. Meanwhile, Rahimi et al (2022) found minimal sensitivity of SWE in WRF simulations across the entire western United States to microphysics schemes, but found large effects due to model resolution. On the other hand, the effects of \n85 \nmeteorological forcing as the lateral boundary conditions of WRF simulations have also been recognized. For instance, Xu et \nal. (2018) identified that the simulations of hydroclimate in California using WRF are largely driven by large-scale forcing \ndatasets. Taken together, the published literature suggests a one-size-fits-all WRF model configuration for hydrological \nstudies in complex terrain may not be possible. In other words, the WRF configuration is likely case- and region-specific, and could depend either on the representation of processes within the WRF simulation domain or the boundary conditions of \n90 \nWRF forced by the large-scale meteorological forcing. The options of subgrid-scale physics schemes and large-scale \nmeteorological forcing datasets need to be fully tested to understand their sensitivities to atmospheric and hydrological \nprocesses in the ERW. and could depend either on the representation of processes within the WRF simulation domain or the boundary conditions of \n90 \nWRF forced by the large-scale meteorological forcing. The options of subgrid-scale physics schemes and large-scale \nmeteorological forcing datasets need to be fully tested to understand their sensitivities to atmospheric and hydrological \nprocesses in the ERW. Furthermore, few studies have assessed how these choices impact the subsequent simulation of surface-through-subsurface \n95 \nhydrologic processes. These types of analysis are needed because the WRF model can be configured in myriad ways for a Furthermore, few studies have assessed how these choices impact the subsequent simulation of surface-through-subsurface \n95 \nhydrologic processes. These types of analysis are needed because the WRF model can be configured in myriad ways for a 3 Using an IPM, we address an outstanding question: does synoptic-scale meteorological forcing or meso-to-micro scale atmospheric processes have a more direct effect on surface and subsurface hydrologic \n110 \nprocesses in a mountainous watershed? forcing or meso-to-micro scale atmospheric processes have a more direct effect on surface and subsurface hydrologic \n110 \nprocesses in a mountainous watershed? In order to answer this question, we undertake a series of experiments with different synoptic-scale meteorological forcings, \nand different, plausible choices for meso-to-micro scale parameterizations in the IPM. This is informed by prior standalone \nWRF studies that have utilized different shortwave and longwave radiation, microphysical, and surface and planetary \n115 \nboundary layer schemes (Skamarock et al., 2019). Additionally, topographical shortwave shading effects are tested to \nunderstand how spatial heterogeneity in the surface radiation budget influences evapotranspiration and snowpack \naccumulation and ablation processes (Arthur et al., 2018). Then we explore how the surface and subsurface hydrology fields \nrespond to these various experimental setup choices, especially discharge in the ERW of the UCRB (described below). With \na discrete set of simulations, we establish the relative importance of these choices. We can also establish the relative \n120 \nimportance of subgrid-scale parameterizations that affect water and energy budgets. Our hypothesis is that if synoptic-scale \nforcings produce a much larger spread in surface and subsurface hydrology fields than subgrid-scale physics scheme choice, \nthen predictive hydrology in the UCRB should prioritize improving large-scale weather products and analyses. Conversely, \nif model subgrid-scale physics scheme choice produces more variability in hydrologic response, then smaller scale \natmospheric and hydrological processes affected by surface heterogeneity in the ERW should be prioritized for model \n125 \ndevelopment. Finally, we can establish if there are spatial and/or temporal patterns to differences between models and \nobservations that point to model configuration choices and thereby motivate further, directed model development and \nsensitivity studies. In order to answer this question, we undertake a series of experiments with different synoptic-scale meteorological forcings, \nand different, plausible choices for meso-to-micro scale parameterizations in the IPM. This is informed by prior standalone \nWRF studies that have utilized different shortwave and longwave radiation, microphysical, and surface and planetary \n115 \nboundary layer schemes (Skamarock et al., 2019). Additionally, topographical shortwave shading effects are tested to \nunderstand how spatial heterogeneity in the surface radiation budget influences evapotranspiration and snowpack \naccumulation and ablation processes (Arthur et al., 2018). https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. given domain, and feedbacks to the surface and subsurface hydrology can yield a potentially large range of results. The \naforementioned IPM study by Maina et al. (2020) showed that biases of 5-10% in basin-average surface water storage can \nresult from forcing resolution differences in WRF alone, with localized differences in groundwater head by several meters. given domain, and feedbacks to the surface and subsurface hydrology can yield a potentially large range of results. The \naforementioned IPM study by Maina et al. (2020) showed that biases of 5-10% in basin-average surface water storage can \nresult from forcing resolution differences in WRF alone, with localized differences in groundwater head by several meters. Schreiner-McGraw and Ajami (2020) show that water partitioning across four commonly used meteorological forcings \n100 \ndiffers substantially within a Sierra Nevada watershed, and that the combination of precipitation uncertainty, soil \nparameterization, and topographic position all impact the severity to which these differences in forcing exert on the \nhydrology. Schreiner-McGraw and Ajami (2020) show that water partitioning across four commonly used meteorological forcings \n100 \ndiffers substantially within a Sierra Nevada watershed, and that the combination of precipitation uncertainty, soil \nparameterization, and topographic position all impact the severity to which these differences in forcing exert on the \nhydrology. 100 In spite of the range of WRF sensitivity investigations, the connections between uncertainty in a WRF configuration and its \n105 \ninfluence on surface-through-subsurface hydrology is underexplored and therefore the focus of this work. It should be noted \nthat our investigation is not to explore general principles behind IPM uncertainty quantification and error propagation, but \nrather to present a concrete use-case to guide the advancement of atmosphere-through-bedrock modeling and its connections \nto mountainous hydrological science. Using an IPM, we address an outstanding question: does synoptic-scale meteorological In spite of the range of WRF sensitivity investigations, the connections between uncertainty in a WRF configuration and its \n105 \ninfluence on surface-through-subsurface hydrology is underexplored and therefore the focus of this work. It should be noted \nthat our investigation is not to explore general principles behind IPM uncertainty quantification and error propagation, but \nrather to present a concrete use-case to guide the advancement of atmosphere-through-bedrock modeling and its connections \nto mountainous hydrological science. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Therefore, this article is organized as follows: first, we present details of study site and hydroclimate in the water year, as \n130 \nwell as the IPM including the coupling between WRF and ParFlow-CLM and the justifications for using WRF and ParFlow-\nCLM as the atmospheric and surface-through-subsurface process models in the IPM, respectively. Then, we describe the \nWRF experiments that we performed to test the relative importance of synoptic-scale boundary forcing and meso-to-micro \nscale model subgrid-scale physics schemes for driving ERW integrated hydrological simulations. Next, we present the Therefore, this article is organized as follows: first, we present details of study site and hydroclimate in the water year, as \n130 \nwell as the IPM including the coupling between WRF and ParFlow-CLM and the justifications for using WRF and ParFlow-\nCLM as the atmospheric and surface-through-subsurface process models in the IPM, respectively. Then, we describe the \nWRF experiments that we performed to test the relative importance of synoptic-scale boundary forcing and meso-to-micro \nscale model subgrid-scale physics schemes for driving ERW integrated hydrological simulations. Next, we present the simulated discharge, evapotranspiration and groundwater storage using ParFlow-CLM, to quantify the responses to changing \n135 \nWRF configurations. We conclude by contextualizing these results in light of the ongoing field campaign activities in the \nERW. simulated discharge, evapotranspiration and groundwater storage using ParFlow-CLM, to quantify the responses to changing \n135 \nWRF configurations. We conclude by contextualizing these results in light of the ongoing field campaign activities in the \nERW. simulated discharge, evapotranspiration and groundwater storage using ParFlow-CLM, to quantify the responses to changing \n135 \nWRF configurations. We conclude by contextualizing these results in light of the ongoing field campaign activities in the \nERW. Then we explore how the surface and subsurface hydrology fields \nrespond to these various experimental setup choices, especially discharge in the ERW of the UCRB (described below). With WRF studies that have utilized different shortwave and longwave radiation, microphysical, and surface and planetary \n115 \nboundary layer schemes (Skamarock et al., 2019). Additionally, topographical shortwave shading effects are tested to \nunderstand how spatial heterogeneity in the surface radiation budget influences evapotranspiration and snowpack \naccumulation and ablation processes (Arthur et al., 2018). Then we explore how the surface and subsurface hydrology fields \nrespond to these various experimental setup choices, especially discharge in the ERW of the UCRB (described below). With a discrete set of simulations, we establish the relative importance of these choices. We can also establish the relative \n120 \nimportance of subgrid-scale parameterizations that affect water and energy budgets. Our hypothesis is that if synoptic-scale \nforcings produce a much larger spread in surface and subsurface hydrology fields than subgrid-scale physics scheme choice, \nthen predictive hydrology in the UCRB should prioritize improving large-scale weather products and analyses. Conversely, \nif model subgrid-scale physics scheme choice produces more variability in hydrologic response, then smaller scale a discrete set of simulations, we establish the relative importance of these choices. We can also establish the relative \n120 \nimportance of subgrid-scale parameterizations that affect water and energy budgets. Our hypothesis is that if synoptic-scale \nforcings produce a much larger spread in surface and subsurface hydrology fields than subgrid-scale physics scheme choice, \nthen predictive hydrology in the UCRB should prioritize improving large-scale weather products and analyses. Conversely, \nif model subgrid-scale physics scheme choice produces more variability in hydrologic response, then smaller scale atmospheric and hydrological processes affected by surface heterogeneity in the ERW should be prioritized for model \n125 \ndevelopment. Finally, we can establish if there are spatial and/or temporal patterns to differences between models and \nobservations that point to model configuration choices and thereby motivate further, directed model development and \nsensitivity studies. atmospheric and hydrological processes affected by surface heterogeneity in the ERW should be prioritized for model \n125 \ndevelopment. Finally, we can establish if there are spatial and/or temporal patterns to differences between models and \nobservations that point to model configuration choices and thereby motivate further, directed model development and \nsensitivity studies. 4 4 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. The Weather Research & Forecasting (WRF) model version 4.0 is used in this study (Powers et al., 2017). WRF was chosen \nbecause of its widespread use in the investigation of atmospheric and land processes, and contextualizing observations in \ncomplex terrain (Rasmussen et al., 2011; Rasmussen et al., 2014). The WRF model is comprised of a fully coupled \n160 \natmospheric and land surface model with a range of user-specific options for subgrid-scale physics schemes. WRF is a \nregional climate model that requires boundary and initial conditions provided by either global climate model (GCM) outputs \nor atmospheric reanalyses datasets. Our configuration of the WRF model is designed with three nested domains, with an \nouter, middle and inner domains at grid resolution of 4.5 km, 1.5 km and 0.5 km, respectively, centered around Crested \nB tt\nC l\nd\nh\nth E\nt Ri\nt\nh d i l\nt d (Fi\n1)\n165 While the stand-alone WRF model has been used extensively to advance the understanding of atmospheric processes, it has \nlower fidelity and applicability to investigate surface-through-subsurface hydrologic processes, and therefore is limited as an \nassessment tool for understanding integrated mountainous hydrologic cycle. Therefore, to provide an estimate of the entire \nhydrologic budget, we use a one-way coupling between WRF and an integrated hydrologic model, ParFlow-CLM (Maxwell \n170 While the stand-alone WRF model has been used extensively to advance the understanding of atmospheric processes, it has \nlower fidelity and applicability to investigate surface-through-subsurface hydrologic processes, and therefore is limited as an \nassessment tool for understanding integrated mountainous hydrologic cycle. Therefore, to provide an estimate of the entire \nhydrologic budget, we use a one-way coupling between WRF and an integrated hydrologic model, ParFlow-CLM (Maxwell \n170 \net al., 2015, described in further detail below), in order to simulate the hydrological response of key variables not otherwise \nquantifiable in standalone WRF, such as discharge and groundwater storage. While the stand-alone WRF model has been used extensively to advance the understanding of atmospheric processes, it has \nlower fidelity and applicability to investigate surface-through-subsurface hydrologic processes, and therefore is limited as an \nassessment tool for understanding integrated mountainous hydrologic cycle. Therefore, to provide an estimate of the entire assessment tool for understanding integrated mountainous hydrologic cycle. 2. Study Site This investigation focused principally on modeling and analysis of the ERW, a representative mountainous headwater \ncatchment of the UCRB near Gothic, Colorado (Hubbard et al., 2018). This 300 km2 watershed of the Upper Colorado River \n140 \nBasin is at a high-level, representative of the UCRB that it has very large gradients in precipitation (e.g., a factor of 2 range \nin precipitation between the northern and southern boundary of the ERW) and surface-through-subsurface hydrology. The \nERW has a continental, subarctic climate with long, cold winters and short, cool summers. At an average elevation of 3266 \nmeters above sea level, the watershed has a mean annual temperature of 0℃, and distinct winter and growing seasons that influence hydrologic and biogeochemical cycles. River discharges are driven primarily by snowmelt in late spring to early \n145 \nsummer, with mid- to late-summer monsoonal rainfall inducing rapid but punctuated increases in streamflow. The ERW \nreceives ~1200 mm/yr of precipitation and we focus here on Water Year 2019 (Oct 1, 2018 - Sep 30, 2019). The ERW has become a mountainous community testbed for improving predictive understanding of multi-scale atmosphere-\nthrough-bedrock system dynamics and is the centerpiece of such focused activities because it is one of two major tributaries \n150 \nthat form the Gunnison River, which in turn accounts for near half of the Colorado River’s discharge at the Colorado–Utah \nborder. In the past decade, several synthesis research efforts have been established in this region, including a wide range of \nfieldwork and modeling activities (Hubbard et al., 2018). The ERW has become one of the most heavily-instrumented \nmountainous watersheds in the world, which makes it an ideal focus for this research given the potentially large number of \nobservational constraints available for the IPM efforts presented here. 155 3. Methods \n3.1. WRF models 3. Methods \n3.1. WRF models 5 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. cells or 100 by 100 km extent) and their associated elevations (left). The Global Multi-resolution Terrain Elevation Data \n2010 (GMTED2010) elevation data in meters above mean sea-level is used in the WRF simulation. Right: the innermost \nParFlow-CLM domain and spatial extent of the East River Watershed (white line) and associated land cover type derived \nfrom the National Land Cover Dataset (NLCD) (Homer et al, 2020) and upscaled to 100 m (right). 180 180 A major experimental design decision when simulating the integrated mountainous hydrologic cycle is computational cost \nassociated with the throughput of the simulations (e.g., simulated years per actual day) that are determined by model \nhorizontal, vertical, and timestep resolutions and subgrid scale physics parameterization complexity. Computational \nexpenses for exploring the sensitivities of WRF configuration choice in this study were significant: one simulated year \n185 \nrequires approximately 100,000 CPU hours on LBNL’s Lawrencium lr6 supercomputing system. As such, it was highly \nimpractical to simulate the entire configuration space of meteorological forcing and subgrid-scale parameterization choice. A \ndiscrete sub-sample of configurations, as presented here, is used to isolate and systematically determine which combination \nof subgrid scale parameterization choice is superior for a given domain such as the ERW. We therefore adopted a \nparsimonious approach to explore the space of possible WRF configurations, described below. 190 Therefore, to provide an estimate of the entire \nhydrologic budget, we use a one-way coupling between WRF and an integrated hydrologic model, ParFlow-CLM (Maxwell \n170 \net al., 2015, described in further detail below), in order to simulate the hydrological response of key variables not otherwise \nquantifiable in standalone WRF, such as discharge and groundwater storage. hydrologic budget, we use a one-way coupling between WRF and an integrated hydrologic model, ParFlow-CLM (Maxwell \n170 \net al., 2015, described in further detail below), in order to simulate the hydrological response of key variables not otherwise \nquantifiable in standalone WRF, such as discharge and groundwater storage. Figure 1. Left: Three nested WRF domains D01 (4.5 km grid resolution, 201 by 201 grid cells or 900 by 900 km extent), D01 \n175 \n(1.5 km grid resolution, 201 by 201 grid cells or 300 by 300 km extent), and D03 (0.5 km grid resolution, 201 by 201 grid Figure 1. Left: Three nested WRF domains D01 (4.5 km grid resolution, 201 by 201 grid cells or 900 by 900 km extent), D01 \n175 \n(1.5 km grid resolution, 201 by 201 grid cells or 300 by 300 km extent), and D03 (0.5 km grid resolution, 201 by 201 grid 6 3.1.1. Subgrid-scale physics schemes In addition, the \nNCEP FNL (NCEP, 2000) operational global analysis and forecast data are on a 0.25-degree grid resolution from the Global \nData Assimilation System (GDAS) (Kleist et al, 2009). All meteorological forcing datasets are processed at 6-hourly by the \nWRF Preprocessing System (WPS)\n220 atmosphere-land-ocean-sea ice coupling, assimilates satellite radiances. MERRA2 is another atmospheric reanalysis based \n215 \non data assimilation (Gelaro et al., 2017), which is the first long-term global reanalysis to assimilate space-based \nobservations of aerosols and represent their interactions with other physical processes in the climate system. In addition, the \nNCEP FNL (NCEP, 2000) operational global analysis and forecast data are on a 0.25-degree grid resolution from the Global \nData Assimilation System (GDAS) (Kleist et al, 2009). All meteorological forcing datasets are processed at 6-hourly by the 3.1.1. Subgrid-scale physics schemes Three well-established suites of subgrid scale physics schemes for WRF are evaluated in this study (Table 1). One scheme \nwas developed by NCAR and is used for a wide range of simulations over domains extending across the entire Conterminous \nUnited States (CONUS) (Liu et al., 2017). Another scheme that we consider here has been used for decadal-length \nhydroclimate simulation over California (Huang et al., 2017; Xu et al., 2018; Ullrich et al., 2018), and since it was initially \n195 \ndeveloped by researchers at the University of California, Davis, it is denoted as UCD here. More recently, Flores et al. (2016) and Rudisill et al. (2021) implemented a WRF configuration that focused on exploring land-atmosphere interactions \nin complex terrain. This configuration was developed by researchers at Boise State University, and is referred to as BSU \nhere. 200 \n \nTable 1: Microphysics, radiation, land surface model, surface layer, and planetary boundary layer schemes used for the \nthree different WRF configurations of the IPM tested here. Subgrid-scale \nphysics \nschemes \nNCAR (CONUS) \nBSU \nUCD \nMicrophysics \nThompson \nThompson \nWSM6 \nShortwave radiation \nRRTMG \nRRTM \nRRTMG Three well-established suites of subgrid scale physics schemes for WRF are evaluated in this study (Table 1). One scheme \nwas developed by NCAR and is used for a wide range of simulations over domains extending across the entire Conterminous \nUnited States (CONUS) (Liu et al., 2017). Another scheme that we consider here has been used for decadal-length \nhydroclimate simulation over California (Huang et al., 2017; Xu et al., 2018; Ullrich et al., 2018), and since it was initially \n195 \ndeveloped by researchers at the University of California, Davis, it is denoted as UCD here. More recently, Flores et al. (2016) and Rudisill et al. (2021) implemented a WRF configuration that focused on exploring land-atmosphere interactions \nin complex terrain. This configuration was developed by researchers at Boise State University, and is referred to as BSU \nhere. 200 Table 1: Microphysics, radiation, land surface model, surface layer, and planetary boundary layer schemes used for the \nthree different WRF configurations of the IPM tested here. Subgrid-scale \nphysics \nschemes \nNCAR (CONUS) \nBSU \nUCD \nMicrophysics \nThompson \nThompson \nWSM6 \nShortwave radiation \nRRTMG \nRRTM \nRRTMG 7 7 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Longwave radiation \nRRTMG \nRRTM \nRRTMG \nLand surface model \nNoah \nNoah-MP \nNoah \nSurface layer \nEta similarity \nMonin-Obukhov \nRevised MM5 \nPlanetary \nBoundary \nlayer \nMellor-Yamada-Janjic \nscheme \nMellor-Yamada-Janjic \nscheme \nUW (Brethert\nPark) \n3.1.2. 3.1.1. Subgrid-scale physics schemes Meteorological forcing RRTMG \nNoah \nRevised MM5 RRTMG \nNoah \nRevised MM5 \nUW (Bretherton and \nPark) RRTMG \nNoah \nRevised MM5 UW (Bretherton and \nPark) Mellor-Yamada-Janjic \nscheme Each of these WRF configurations must specify a set of initial and lateral boundary conditions at the synoptic scale and, at \n205 \nleast in the outer domain, are typically derived from high-resolution atmospheric reanalyses. The reanalysis from the \nNational Centers for Environmental Prediction (NCEP), Climate Forecast System Reanalysis version 2 (CFSR2), The \nModern-Era Retrospective analysis for Research and Applications - Version 2 (MERRA2), European Centre for Medium-\nRange Weather Forecasting Reanalysis version 5 (ERA5) were used in this study. 210 \nERA5 is the fifth generation ECMWF atmospheric reanalysis of the global climate on a 30 km grid resolution (Hersbach et \nal, 2020), and combines model data with observations from across the world into a globally complete and consistent dataset. The CFSR2 is also global and is designed to provide an operational product for forecasting and analysis purposes at 0.3 \ndegree grid resolution (Saha et al, 2010). The CFSR2 data were generated by an advanced assimilation schemes, 210 \nERA5 is the fifth generation ECMWF atmospheric reanalysis of the global climate on a 30 km grid resolution (Hersbach et \nal, 2020), and combines model data with observations from across the world into a globally complete and consistent dataset. The CFSR2 is also global and is designed to provide an operational product for forecasting and analysis purposes at 0.3 \ndegree grid resolution (Saha et al, 2010). The CFSR2 data were generated by an advanced assimilation schemes, ERA5 is the fifth generation ECMWF atmospheric reanalysis of the global climate on a 30 km grid resolution (Hersbach et \nal, 2020), and combines model data with observations from across the world into a globally complete and consistent dataset. The CFSR2 is also global and is designed to provide an operational product for forecasting and analysis purposes at 0.3 \ndegree grid resolution (Saha et al, 2010). The CFSR2 data were generated by an advanced assimilation schemes, atmosphere-land-ocean-sea ice coupling, assimilates satellite radiances. MERRA2 is another atmospheric reanalysis based \n215 \non data assimilation (Gelaro et al., 2017), which is the first long-term global reanalysis to assimilate space-based \nobservations of aerosols and represent their interactions with other physical processes in the climate system. 3.1.3 Topographic radiation Topographic effects for shortwave radiation flux calculations in complex terrain are evaluated (Arthur et al., 2018). One is \nthe “slope_rad” namelist option, which modifies surface solar radiation flux according to terrain slope by correcting it based \non the solar zenith angle relative to the local surface normal vector. This adjustment ensures that the solar radiation received \nat the surface in WRF is consistent with the geometric projection of incoming sunlight onto local, non-flat surfaces. The \n225 \nother namelist option, “topo_shading”, allows for shadowing of neighboring grid cells. When “topo_shading” is active, WRF \ndetermines if any topography intersects a line drawn between a given grid point and the location of the sun at the time step Topographic effects for shortwave radiation flux calculations in complex terrain are evaluated (Arthur et al., 2018). One is \nthe “slope_rad” namelist option, which modifies surface solar radiation flux according to terrain slope by correcting it based \non the solar zenith angle relative to the local surface normal vector. This adjustment ensures that the solar radiation received the “slope_rad” namelist option, which modifies surface solar radiation flux according to terrain slope by correcting it based \non the solar zenith angle relative to the local surface normal vector. This adjustment ensures that the solar radiation received \nat the surface in WRF is consistent with the geometric projection of incoming sunlight onto local, non-flat surfaces. The \n225 \nother namelist option, “topo_shading”, allows for shadowing of neighboring grid cells. When “topo_shading” is active, WRF \ndetermines if any topography intersects a line drawn between a given grid point and the location of the sun at the time-step \nof the WRF run. If so, a topographic shadow is cast on that grid point and the direct component of the incoming solar at the surface in WRF is consistent with the geometric projection of incoming sunlight onto local, non-flat surfaces. The \n225 \nother namelist option, “topo_shading”, allows for shadowing of neighboring grid cells. When “topo_shading” is active, WRF \ndetermines if any topography intersects a line drawn between a given grid point and the location of the sun at the time-step \nof the WRF run. If so, a topographic shadow is cast on that grid point and the direct component of the incoming solar 225 8 ParFlow is coupled to the land \nsurface model, the Common Land Model (CLM), which calculates a coupled water energy balance at every surface cell of surface model, the Common Land Model (CLM), which calculates a coupled water energy balance at every surface cell of \nthe domain (Dai et al., 2003) and incorporates spatially distributed vegetative processes by including specified land use types \n245 \nparameterized by the International Geosphere-Biosphere Program standard database. Hourly meteorological forcing derived \nfrom WRF drives ParFlow-CLM, and includes the following eight variables: precipitation, two-meter surface air \ntemperature, longwave radiation, shortwave radiation, 10-meter east-west and south-north wind speeds, atmospheric \npressure, and specific humidity. Computational expenses for ParFlow-CLM are also less substantial than that of WRF for \nthis model configuration, but still require high performance computing. Excluding the time for a multi-year initial condition \n250 \ni\ni\nl\nt\nf th P Fl\nCLM i\nl ti\n64\nth NERSC’ C\ni\nti\nt\ni the domain (Dai et al., 2003) and incorporates spatially distributed vegetative processes by including specified land use types \n245 \nparameterized by the International Geosphere-Biosphere Program standard database. Hourly meteorological forcing derived \nfrom WRF drives ParFlow-CLM, and includes the following eight variables: precipitation, two-meter surface air \ntemperature, longwave radiation, shortwave radiation, 10-meter east-west and south-north wind speeds, atmospheric \npressure, and specific humidity. Computational expenses for ParFlow-CLM are also less substantial than that of WRF for this model configuration, but still require high performance computing. Excluding the time for a multi-year initial condition \n250 \nspinup, a single water year of the ParFlow-CLM simulations on 64 cores on the NERSC’s Cori supercomputing system is \napproximately 1,000 CPU hours. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. radiation is set to 0. In this study, simulations in which “slope_rad” and “topo_shading” are jointly enabled are termed \n“3DRad” and when jointly disabled are termed “no3DRad”, in the inner domain of the WRF simulation. 230 230 Figure 2. Conceptual framework for developing a set of different WRF configurations of the IPM to evaluate the sensitivities \nof subgrid-scale physics parameterization choice, meteorological forcing, and radiation scheme in the representation of \nmountain water and energy budgets. 235 Figure 2. Conceptual framework for developing a set of different WRF configurations of the IPM to evaluate the sensitivities \nof subgrid-scale physics parameterization choice, meteorological forcing, and radiation scheme in the representation of \nmountain water and energy budgets. 235 Figure 2. Conceptual framework for developing a set of different WRF configurations of the IPM to evaluate the sensitivities \nof subgrid-scale physics parameterization choice, meteorological forcing, and radiation scheme in the representation of \nmountain water and energy budgets. 235 235 Table 2: East River Watershed WRF experiment configurations. Three subgrid-scale physics schemes, four meteorological \nforcings, and the topographic radiation options were assessed. Subgrid-scale physics \nschemes \nMeteorological \nforcing \nTopographic \nradiation \nBSU \nCFSR2 \n3DRad_inner \n \n \nno3DRad_inner \n \nERA5 \n3DRad_inner \n \n \nno3DRad_inner \n \nMERRA2 \n3DRad_inner \n \nNCEP \n3DRad_inner able 2: East River Watershed WRF experiment configurations. Three subgrid-scale physics schem\nrcings, and the topographic radiation options were assessed. 9 UCD \nCFSR2 \n3DRad_inner \n \nERA5 \n3DRad_inner \nNCAR \nCFSR2 \n3DRad_inner \n \nERA5 \n3DRad_inner \n3.2 ParFlow-CLM description \nhttps://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. UCD \nCFSR2 \n3DRad_inner \n \nERA5 \n3DRad_inner \nNCAR \nCFSR2 \n3DRad_inner \n \nERA5 \n3DRad_inner ParFlow is a physically based surface-subsurface hydrologic model that solves the coupled flow of saturated and variability-\n240 \nsaturated groundwater and overland surface water (Ashby and Falgout, 1996; Jones and Woodward, 2001; Maxwell, 2013). The three-dimensional form of Richards equation is used to solve for lateral and vertical groundwater flow in the subsurface \nand the kinematic wave approximation is used to solve two-dimensional overland flow. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. the year. On the other hand, the Schofield Pass station is located upstream of the ERW and, on average, receives 1.2 m of \n265 \nprecipitation and reaches 0.9 m in maximum snow water equivalent. In addition, we use the snow water equivalent product \nof the Airborne Snow Observatory (ASO; Painter et al, 2016) on 04/07/2019 to evaluate the spatial pattern skill of the \nsnowpack simulation across WRF configurations (Figure S-6). Notably, ASO SWE estimates are lower than SNOTEL SWE \nmeasurements (ASO: 389 mm at Butte, 938 mm at Schofield Pass; SNOTEL: 490 mm at Butte, 1260 mm at Schofield Pass). In addition to SNOTEL station data, stream gauges measurement of discharge at the pumphouse, the outlet of the ERW, is \n270 \nused to evaluate the ParFlow-CLM simulation results. the year. On the other hand, the Schofield Pass station is located upstream of the ERW and, on average, receives 1.2 m of \n265 \nprecipitation and reaches 0.9 m in maximum snow water equivalent. In addition, we use the snow water equivalent product \nof the Airborne Snow Observatory (ASO; Painter et al, 2016) on 04/07/2019 to evaluate the spatial pattern skill of the \nsnowpack simulation across WRF configurations (Figure S-6). Notably, ASO SWE estimates are lower than SNOTEL SWE \nmeasurements (ASO: 389 mm at Butte, 938 mm at Schofield Pass; SNOTEL: 490 mm at Butte, 1260 mm at Schofield Pass). In addition to SNOTEL station data, stream gauges measurement of discharge at the pumphouse, the outlet of the ERW, is \n270 \nused to evaluate the ParFlow-CLM simulation results. In addition to SNOTEL station data, stream gauges measurement of discharge at the pumphouse, the outlet of the ERW, is \n270 \nused to evaluate the ParFlow-CLM simulation results. 3.3 Reference Datasets The Parameter-elevation Relationships on Independent Slopes Model (PRISM) dataset (Daly et al., 2008) was used as the \nreference dataset to assess model performance of precipitation and temperature in this study. PRISM uses observations from \n255 \nquality-controlled meteorological stations along with a topographic correction method against elevation based on empirical \nregression to create daily gridded 800-meter total precipitation, and daily average, minimum and maximum two-meter \nsurface temperature. Snowpack Telemetry (SNOTEL) data have been widely used in snowpack assessment (Serreze et al, 1999; Fassnacht et al, \n260 \n2003), and we use three SNOTEL stations (Butte, Schofield Pass, Upper Taylor) within the WRF inner domain to assess the \nsnowpack simulation skill of each IPM configuration. Significant heterogeneity is sampled by the three SNOTEL stations \n(within or near the ERW) due to the complex topography. For example, the Butte station is located downstream of the ERW \nand, on average, receives approximately 0.8 m of precipitation, and reaches 0.4 m in maximum snow water equivalent over 10 4. Results We start by presenting a number of time-series of spatial averages over the ERW for WY19. They indicate the gross \nperformance of the IPM across the water year, and whether a configuration produces generally reasonable estimates relative \nto observational products. Figure 3 shows cumulative precipitation, two-meter surface air temperature, and snow water \n275 \nequivalent (SWE). For cumulative precipitation, each configuration produces amounts higher than PRISM (cumulative \nprecipitation of 1201 mm), and the UCD simulates the highest cumulative precipitation. For surface air temperature, the \nseasonal cycle and daily variability are captured by all configurations, however, exhibit systematic cold biases relative to \nPRISM (annual average two-meter surface air temperature of 0.6 degrees Celsius). In terms of SWE, all model \nconfigurations concur in their representation of the snowpack accumulation season and melt season in late spring and into \n280 \nsummer, except UCD which simulates an earlier peak timing of SWE. configurations concur in their representation of the snowpack accumulation season and melt season in late spring and into \n280 \nsummer, except UCD which simulates an earlier peak timing of SWE. The spread in cumulative precipitation when comparing across different meteorological forcing dataset is apparent (Figure \n3). Although UCD and NCAR configurations show greater difference in precipitation forced by ERA5 and CFSR2, the 3). Although UCD and NCAR configurations show greater difference in precipitation forced by ERA5 and CFSR2, the \nconsistency across BSU configurations is notable, which also shows the closest agreement with PRISM. When comparing \n285 \nthe relative roles of subgrid-scale physics scheme choice to meteorological forcing, the percent difference of cumulative \nprecipitation, calculated with (maximum - minimum)/minimum*100, across BSU-CSFR2, UCD-CSFR2 and NCAR-CSFR2 \nschemes is nearly 34% of the mininum cumulative precipitation simulated by BSU-CFSR2, compared to the 4.6% of the \nsimulations across BSU configurations with different meteorological forcing (CSFR2, ERA5, MERRA2 and NCEP). consistency across BSU configurations is notable, which also shows the closest agreement with PRISM. When comparing \n285 \nthe relative roles of subgrid-scale physics scheme choice to meteorological forcing, the percent difference of cumulative \nprecipitation, calculated with (maximum - minimum)/minimum*100, across BSU-CSFR2, UCD-CSFR2 and NCAR-CSFR2 \nschemes is nearly 34% of the mininum cumulative precipitation simulated by BSU-CFSR2, compared to the 4.6% of the \nsimulations across BSU configurations with different meteorological forcing (CSFR2, ERA5, MERRA2 and NCEP). consistency across BSU configurations is notable, which also shows the closest agreement with PRISM. 4. Results When comparing \n285 \nthe relative roles of subgrid-scale physics scheme choice to meteorological forcing, the percent difference of cumulative \nprecipitation, calculated with (maximum - minimum)/minimum*100, across BSU-CSFR2, UCD-CSFR2 and NCAR-CSFR2 \nschemes is nearly 34% of the mininum cumulative precipitation simulated by BSU-CFSR2, compared to the 4.6% of the \nsimulations across BSU configurations with different meteorological forcing (CSFR2, ERA5, MERRA2 and NCEP). 290 \nBSU simulations are generally in agreement with PRISM. However, the UCD simulations are outliers relative to the other \nsimulations, with cumulative precipitation of 1706 mm, or 42% higher at the end of the water year, with the most notable \ndifferences occurring in March through September. NCAR simulations show general agreement with PRISM and BSU \nthroughout the water year, save for June through September. The two-meter surface air temperature time-series reveals that \nthe UCD simulation is systematically colder throughout the winter and spring regardless of which meteorological forcing\n295 290 \nBSU simulations are generally in agreement with PRISM. However, the UCD simulations are outliers relative to the other \nsimulations, with cumulative precipitation of 1706 mm, or 42% higher at the end of the water year, with the most notable \ndifferences occurring in March through September. NCAR simulations show general agreement with PRISM and BSU \nthroughout the water year, save for June through September. The two-meter surface air temperature time-series reveals that \nthe UCD simulation is systematically colder throughout the winter and spring, regardless of which meteorological forcing \n295 the UCD simulation is systematically colder throughout the winter and spring, regardless of which meteorological forcing \n295 \ndataset is used. The persistent cold bias simulated by the UCD, NCAR and BSU schemes has been found in previous WRF 11 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. In addition to the domain-averages, spatial heterogeneity due to land-surface cover and topographic effects are shown in \n310 \nFigure 4. The systematic cold bias simulated throughout the water year appears to be an elevation-dependent phenomena \nwith higher-elevations exhibiting an enhanced cold bias compared with PRISM. However, the river valley and relatively \nlower-elevation areas at the southern edge of the ERW, which includes Crested Butte Mountain, stands out as these regions \nare warmer than the PRISM dataset. Figure 4b shows precipitation in BSU-CFSR2 is wetter in the western regions, and drier In addition to the domain-averages, spatial heterogeneity due to land-surface cover and topographic effects are shown in \n310 \nFigure 4. The systematic cold bias simulated throughout the water year appears to be an elevation-dependent phenomena \nwith higher-elevations exhibiting an enhanced cold bias compared with PRISM. However, the river valley and relatively \nlower-elevation areas at the southern edge of the ERW, which includes Crested Butte Mountain, stands out as these regions \nare warmer than the PRISM dataset. Figure 4b shows precipitation in BSU-CFSR2 is wetter in the western regions, and drier in the eastern, of the ERW in comparison against PRISM. Figure S-3 and S-4 show comparisons between PRISM and the \n315 \nIPM configurations and indicates no biases that are persistent across seasons. During summer, the BSU-CFSR2 simulation \nconsistently produces more precipitation than PRISM. in the eastern, of the ERW in comparison against PRISM. Figure S-3 and S-4 show comparisons between PRISM and the \n315 \nIPM configurations and indicates no biases that are persistent across seasons. During summer, the BSU-CFSR2 simulation \nconsistently produces more precipitation than PRISM. in the eastern, of the ERW in comparison against PRISM. Figure S-3 and S-4 show comparisons between PRISM and the \n315 \nIPM configurations and indicates no biases that are persistent across seasons. During summer, the BSU-CFSR2 simulation \nconsistently produces more precipitation than PRISM. Although the two-meter surface air temperature bias is evident, it doesn't vary significantly across either subgrid-scale \nphysics scheme or meteorological forcing, subsequent exploration will be predominantly focused on precipitation. The \n320 \nbottom row in Figure 4 shows the grid-cell standard deviation of monthly precipitation across subgrid-scale physics schemes \n(i.e., UCD, NCAR and BSU simulations with CFSR2 meteorological forcing – bottom left) and BSU simulation driven by \ndifferent meteorological forcing datasets (ERA5, CFSR2, MERR2 and NCEP – bottom right). https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. studies within western US mountain regions (Xu et al., 2018, Rudisill et al. 2021). The SWE time-series again shows a \nsimilar relationship with precipitation, with the outlier being UCD-ERA5, in terms of the seasonal timing of when snowpack \npeaks and melts (Figure S-4). 300 Figure 3. Cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) simulated within \nthe ERW using an IPM with different subgrid-scale physics schemes and meteorological forcings. The cumulative \nprecipitation and temperature results are compared relative to PRISM. 10-day moving averages of daily temperature are \n5 \nshown in b). The percent difference in cumulative precipitation across subgrid-scale physics schemes (black brackets) and \nmeteorological forcing (green brackets), calculated by (maximum - minimum)/minimum*100, are provided on the right y-\naxis. Figure 3. Cumulative precipitation, two-meter surface air temperature and snow water e Figure 3. Cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) simulated within \nthe ERW using an IPM with different subgrid-scale physics schemes and meteorological forcings. The cumulative \nprecipitation and temperature results are compared relative to PRISM. 10-day moving averages of daily temperature are \n5 \nshown in b). The percent difference in cumulative precipitation across subgrid-scale physics schemes (black brackets) and \nmeteorological forcing (green brackets), calculated by (maximum - minimum)/minimum*100, are provided on the right y-\naxis. Figure 3. Cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) simulated within \nthe ERW using an IPM with different subgrid-scale physics schemes and meteorological forcings. The cumulative \nprecipitation and temperature results are compared relative to PRISM. 10-day moving averages of daily temperature are \n05 \nshown in b). The percent difference in cumulative precipitation across subgrid-scale physics schemes (black brackets) and \nmeteorological forcing (green brackets), calculated by (maximum - minimum)/minimum*100, are provided on the right y-\naxis. 305 12 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. standard deviation of annual cumulative precipitation is plotted for subgrid-scale physics schemes (c) and meteorological \nforcings (d). The values in the parentheses are the domain average differences over the water year. standard deviation of annual cumulative precipitation is plotted for subgrid-scale physics schemes (c) and meteorological \nforcings (d). The values in the parentheses are the domain average differences over the water year. standard deviation of annual cumulative precipitation is plotted for subgrid-scale physics schemes (c) and meteorological \nforcings (d). The values in the parentheses are the domain average differences over the water year. Based on the assessment of simulated precipitation and two-meter surface air temperature compared with PRISM, the BSU-\n335 \nCFSR2 configuration is selected as a baseline to further explore the influence of topographic radiation scheme effects. Figure \n5 shows daily ERW spatial average time series over the water year for the major mountainous water and energy budget \nvariables. By isolating the impacts of subgrid-scale physics schemes and meteorological forcings across IPM simulations, it \nis easier to to systematically intercompare cause-and-effect across different topographic radiation options. Consistent with \ni\nfi di\nll\nfi\nti\ntill\nti\nt\nl ti\ni it ti\nd\nt\nld\nl ti\nt PRISM\n340 Based on the assessment of simulated precipitation and two-meter surface air temperature compared with PRISM, the BSU-\n335 \nCFSR2 configuration is selected as a baseline to further explore the influence of topographic radiation scheme effects. Figure \n5 shows daily ERW spatial average time series over the water year for the major mountainous water and energy budget \nvariables. By isolating the impacts of subgrid-scale physics schemes and meteorological forcings across IPM simulations, it \nis easier to to systematically intercompare cause-and-effect across different topographic radiation options. Consistent with \nprevious findings, all configurations still overestimate cumulative precipitation and are too cold relative to PRISM. 340 previous findings, all configurations still overestimate cumulative precipitation and are too cold relative to PRISM. 340 Figure 5. Spatial-average cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) \n(first row), shortwave and longwave radiation, and latent and sensible heat (second row) over the ERW as simulated by the \nIPM configurations with and without realistic topographic radiation effects, along with, where available, estimates from \n345 \nPRISM. 3DRad indicates a simulation with topo_shading and slope_rad turned on in the WRF inner domain but not the \nouter WRF domains, no3DRad indicates a simulation with top_shading and slope_rad turned off in both the inner and outer \nWRF domains. 10-day moving averages are shown in b) temperature, and radiation variables (d, e, f, g). Figure 5. Spatial-average cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) \n(first row), shortwave and longwave radiation, and latent and sensible heat (second row) over the ERW as simulated by the Figure 5. Spatial-average cumulative precipitation, two-meter surface air temperature a\n(first row), shortwave and longwave radiation, and latent and sensible heat (second row) Figure 5. Spatial-average cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) \n(first row), shortwave and longwave radiation, and latent and sensible heat (second row) over the ERW as simulated by the \nIPM configurations with and without realistic topographic radiation effects, along with, where available, estimates from \n345 \nPRISM. 3DRad indicates a simulation with topo_shading and slope_rad turned on in the WRF inner domain but not the \nouter WRF domains, no3DRad indicates a simulation with top_shading and slope_rad turned off in both the inner and outer \nWRF domains. 10-day moving averages are shown in b) temperature, and radiation variables (d, e, f, g). Figure 5. Spatial-average cumulative precipitation, two-meter surface air temperature and snow water equivalent (SWE) \n(first row), shortwave and longwave radiation, and latent and sensible heat (second row) over the ERW as simulated by the \nIPM configurations with and without realistic topographic radiation effects, along with, where available, estimates from \n345 \nPRISM. 3DRad indicates a simulation with topo_shading and slope_rad turned on in the WRF inner domain but not the \nouter WRF domains, no3DRad indicates a simulation with top_shading and slope_rad turned off in both the inner and outer \nWRF domains. 10-day moving averages are shown in b) temperature, and radiation variables (d, e, f, g). IPM configurations with and without realistic topographic radiation effects, along with, where available, estimates from \n345 \nPRISM. 3DRad indicates a simulation with topo_shading and slope_rad turned on in the WRF inner domain but not the \nouter WRF domains, no3DRad indicates a simulation with top_shading and slope_rad turned off in both the inner and outer \nWRF domains. 10-day moving averages are shown in b) temperature, and radiation variables (d, e, f, g). Similar to the conclusions \ndrawn from Figure 3, Supplementary Figure S-4 also shows the monthly spatial standard deviations across subgrid-scale physics schemes are generally greater than meteorological forcing, particularly in regions of higher-elevation during the \n325 \nwinter season. 13 13 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Figure 4. Upper row: Differences in spatial distributions of annual average two-meter surface air temperature (a\n330 \ncumulative precipitation (b) between the BSU-CFSR2 WRF configuration and PRISM. Lower row: For all scheme Figure 4. Upper row: Differences in spatial distributions of annual average two-meter surface air temperature (a) and \n330 \ncumulative precipitation (b) between the BSU-CFSR2 WRF configuration and PRISM. Lower row: For all schemes, the Figure 4. Upper row: Differences in spatial distributions of annual average two-meter surface air temperature (a) and \n330 \ncumulative precipitation (b) between the BSU-CFSR2 WRF configuration and PRISM. Lower row: For all schemes, the 14 Figure 6 shows the seasonally-resolved shortwave radiation, two-meter surface air temperature, latent heat flux and SWE for \n350 \ndifferent configurations of shortwave radiation in the simulation with and without topo_shaing and slope_rad options in the \ninner domain. While no3DRad does not adjust the SWdown, 3DRad simulation recalculates the SWdown based on the Figure 6 shows the seasonally-resolved shortwave radiation, two-meter surface air temperature, latent heat flux and SWE for \n350 \ndifferent configurations of shortwave radiation in the simulation with and without topo_shaing and slope_rad options in the \ninner domain. While no3DRad does not adjust the SWdown, 3DRad simulation recalculates the SWdown based on the Figure 6 shows the seasonally-resolved shortwave radiation, two-meter surface air temperature, latent heat flux and SWE for \n350 \ndifferent configurations of shortwave radiation in the simulation with and without topo_shaing and slope_rad options in the \ninner domain. While no3DRad does not adjust the SWdown, 3DRad simulation recalculates the SWdown based on the 15 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. shadows cast by nearby topography. Spatial differences in IPM-simulated shortwave radiation (Figure 6b) are seen in the \nnortheast and western portions of the ERW, when topographic effect of shortwave radiation is included. As a result, a \ncorresponding change in the spatial pattern of simulated two-meter surface air temperature and latent heat flux are seen, \n355 \nwhich are driven by the change in downwelling shortwave radiation with topographic shading (Figures 6a and 6d). The \nresulting pattern change in SWE (Figure 6c) shows that the northern and northeastern sections of the ERW, where snowpack \nare concentrated, are sensitive to shortwave radiation. This is expected and consistent with previous findings that included \ntopographic effects in shortwave radiation and found distinct spatial patterns of hydrologic variable sensitivity due to both \nshadows and surface reflection that produce time-varying effects on net surface radiation (Lee et al., 2015; Palazzi et al., \n360 \n2019; Gu et al., 2020; Hao et al., 2021). 355 shadows and surface reflection that produce time-varying effects on net surface radiation (Lee et al., 2015; Palazzi et al., \n360 \n2019; Gu et al., 2020; Hao et al., 2021). Although Figure 5 shows that realistic shortwave radiation produces small effects on the seasonal cycle of the surface energy \nand mass budgets when averaged over the entire watershed, including annual average SWE (Figure 5c), Figure 6c shows that Although Figure 5 shows that realistic shortwave radiation produces small effects on the seasonal cycle of the surface energy \nand mass budgets when averaged over the entire watershed, including annual average SWE (Figure 5c), Figure 6c shows that \nmountains and valleys have different amounts of SWE. Furthermore, seasonal patterns show simulated latent heat is \n365 \ndiminished at lower elevations from March to May, when snowmelt occurs in the valley, and the remaining snowpack in the \nmountains and late snowmelt in 3DRad simulation causes lower latent heat flux shown in July (Figure S-5). The 3DRad \nsimulation has less SWE in the valleys during the accumulation season but more SWE at higher elevations during the melt \nseason, which is a direct result of the differences in shortwave radiation redistribution. Figure S-5 also shows that the latent mountains and valleys have different amounts of SWE. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. To understand how the aforementioned WRF configurations and forcings impact the integrated water budget, Figure 7 shows \n380 \nthe simulated hydrologic output from the ParFlow-CLM model for watershed outlet discharge (top row) and watershed-\naverage groundwater storage (bottom row). Discharge at the watershed outlet (see exact location on Figure 1) shows general \nagreement across the different WRF subgrid-scale physics scheme configurations and meteorological forcings, where the \ndaily averaged time series (left) shows only minor differences through time. However, cumulative discharge by year-end \nreveals substantial differences (right), especially after peak snowmelt where estimates of cumulative discharge begin to \n385 \ndiverge. Differences across the WRF configurations are especially large; the difference across the three subgrid-scale physics \nscheme configurations with ERA5 (UCD, NCAR, and BSU) varies by 26% by year-end. Differences across meteorological \nforcing (using the BSU physics configuration as a control, shown in green) are also noteworthy, although smaller, \napproximately 6%. These results are consistent with variation of simulated precipitation in WRF described earlier, \nconfirming that for this basin, meteorological forcing drives less variance on hydrologic response than subgrid-scale physics \n390 \nscheme \nconfiguration. To understand how the aforementioned WRF configurations and forcings impact the integrated water budget, Figure 7 shows \n380 \nthe simulated hydrologic output from the ParFlow-CLM model for watershed outlet discharge (top row) and watershed-\naverage groundwater storage (bottom row). Discharge at the watershed outlet (see exact location on Figure 1) shows general \nagreement across the different WRF subgrid-scale physics scheme configurations and meteorological forcings, where the \ndaily averaged time series (left) shows only minor differences through time. However, cumulative discharge by year-end \nreveals substantial differences (right), especially after peak snowmelt where estimates of cumulative discharge begin to \n385 \ndiverge. Differences across the WRF configurations are especially large; the difference across the three subgrid-scale physics \nscheme configurations with ERA5 (UCD, NCAR, and BSU) varies by 26% by year-end. Differences across meteorological To understand how the aforementioned WRF configurations and forcings impact the integrated water budget, Figure 7 shows \n380 \nthe simulated hydrologic output from the ParFlow-CLM model for watershed outlet discharge (top row) and watershed-\naverage groundwater storage (bottom row). Furthermore, seasonal patterns show simulated latent heat is \n365 \ndiminished at lower elevations from March to May, when snowmelt occurs in the valley, and the remaining snowpack in the \nmountains and late snowmelt in 3DRad simulation causes lower latent heat flux shown in July (Figure S-5). The 3DRad \nsimulation has less SWE in the valleys during the accumulation season but more SWE at higher elevations during the melt \nseason, which is a direct result of the differences in shortwave radiation redistribution. Figure S-5 also shows that the latent heat differences in north-facing and south-facing sides are most apparent in the snowmelt and warm seasons. This is \n370 \nconsistent with previous findings (Lee et al., 2015; Palazzi et al., 2019; Gu et al., 2020; Hao et al., 2021), that a more \nrealistic treatment of shortwave radiation, which includes shadows and projected insolation on sloped surfaces, results in \nlower shortwave insolation on the surface at this time of year. The lower shortwave radiation should, in turn, decrease the \nenergy available for the IPM to produce snowmelt. 16 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. 5 \nFigure 6. Topographic radiation differences (3dRad minus no3dRad) annual average two-meter surface air tempera\nshortwave (SW) and latent heat flux, and snow water equivalent (SWE) over the ERW. The values in the parentheses ar\nERW average differences over the water year, which are small and consistent with Figure 5. 5 375 Figure 6. Topographic radiation differences (3dRad minus no3dRad) annual average two-meter surface air temperature, \nshortwave (SW) and latent heat flux, and snow water equivalent (SWE) over the ERW. The values in the parentheses are the \nERW average differences over the water year, which are small and consistent with Figure 5. 17 Discharge at the watershed outlet (see exact location on Figure 1) shows general \nagreement across the different WRF subgrid-scale physics scheme configurations and meteorological forcings, where the \ndaily averaged time series (left) shows only minor differences through time. However, cumulative discharge by year-end \nl\nb t\nti l diff\n( i ht)\ni ll\nft\nk\nlt\nh\nti\nt\nf\nl ti\ndi\nh\nb\ni\nt\n385 reveals substantial differences (right), especially after peak snowmelt where estimates of cumulative discharge begin to \n385 \ndiverge. Differences across the WRF configurations are especially large; the difference across the three subgrid-scale physics \nscheme configurations with ERA5 (UCD, NCAR, and BSU) varies by 26% by year-end. Differences across meteorological \nforcing (using the BSU physics configuration as a control, shown in green) are also noteworthy, although smaller, \napproximately 6%. These results are consistent with variation of simulated precipitation in WRF described earlier, \nconfirming that for this basin, meteorological forcing drives less variance on hydrologic response than subgrid-scale physics \n390 \nscheme \nconfiguration. 18 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Figure 7. Time-series of ParFlow-CLM simulations of discharge rate (a), cumulative discharge (b), groundwater storage \nper unit area of the watershed (c), and cumulative average unsaturated groundwater storage per area of the watershed (d) \nfor the IPM configurations described in Table 2. The brackets on the far-right indicate the percent difference of cumulative \ndischarge and unsaturated groundwater storage per area (b and d, respectively) for WRF simulations across different \nmeteorological forcings (green) and subgrid-scale physics schemes (black). Figure 7. Time-series of ParFlow-CLM simulations of discharge rate (a), cumulative di Figure 7. Time-series of ParFlow-CLM simulations of discharge rate (a), cumulative discharge (b), groundwater storage \nper unit area of the watershed (c), and cumulative average unsaturated groundwater storage per area of the watershed (d) \n395 \nfor the IPM configurations described in Table 2. The brackets on the far-right indicate the percent difference of cumulative \ndischarge and unsaturated groundwater storage per area (b and d, respectively) for WRF simulations across different \nmeteorological forcings (green) and subgrid-scale physics schemes (black). Figure 7. Time-series of ParFlow-CLM simulations of discharge rate (a), cumulative discharge (b), groundwater storage \nper unit area of the watershed (c), and cumulative average unsaturated groundwater storage per area of the watershed (d) \n395 \nfor the IPM configurations described in Table 2. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. ParFlow-CLM which is consistent with the discussion surrounding Figure 3 in relationship to PRISM. An early-fall peak in \nsimulated discharge is also seen in all WRF simulations, and not in observed discharge, although a significant increase in simulated discharge is also seen in all WRF simulations, and not in observed discharge, although a significant increase in \nSNOTEL precipitation was measured in October of that year (see Figure S-1, S-2). This further supports a temperature bias, \n405 \nalbeit opposite that of the cold-bias discussed previously, where precipitation around that October storm-event falling as rain \n(as opposed to snow) leads to a sharp increase in discharge. A sensitivity analysis of the BSU-ERA5 model run for a lower \nprecipitation year (water year 2018, which was nearly half the precipitation of 2019), showed better agreement with \nobserved discharge, which suggests the bias in timing may be a function of accumulated precipitation and/or snowmelt, and \nis reserved for future studies (not shown). 410 SNOTEL precipitation was measured in October of that year (see Figure S-1, S-2). This further supports a temperature bias, \n405 \nalbeit opposite that of the cold-bias discussed previously, where precipitation around that October storm-event falling as rain \n(as opposed to snow) leads to a sharp increase in discharge. A sensitivity analysis of the BSU-ERA5 model run for a lower \nprecipitation year (water year 2018, which was nearly half the precipitation of 2019), showed better agreement with \nobserved discharge, which suggests the bias in timing may be a function of accumulated precipitation and/or snowmelt, and \ni\nd f\nf\ndi\n(\nh\n)\n410 SNOTEL precipitation was measured in October of that year (see Figure S-1, S-2). This further supports a temperature bias, \n405 \nalbeit opposite that of the cold-bias discussed previously, where precipitation around that October storm-event falling as rain \n(as opposed to snow) leads to a sharp increase in discharge. The brackets on the far-right indicate the percent difference of cumulative \ndischarge and unsaturated groundwater storage per area (b and d, respectively) for WRF simulations across different \nmeteorological forcings (green) and subgrid-scale physics schemes (black). A comparison to observed discharge is also shown on Figure 7, which for all scenarios suggest a delayed snowmelt response \n400 \nin the IPM. While the objective of this study is not to replicate the observations, but rather determine sensitivity across IPM \nconfiguration choice, the mismatch in streamflow response suggests a systematic cold-bias from the WRF input into A comparison to observed discharge is also shown on Figure 7, which for all scenarios suggest a delayed snowmelt response \n400 \nin the IPM. While the objective of this study is not to replicate the observations, but rather determine sensitivity across IPM \nconfiguration choice, the mismatch in streamflow response suggests a systematic cold-bias from the WRF input into 19 A sensitivity analysis of the BSU-ERA5 model run for a lower \nprecipitation year (water year 2018, which was nearly half the precipitation of 2019), showed better agreement with \nobserved discharge, which suggests the bias in timing may be a function of accumulated precipitation and/or snowmelt, and \nis reserved for future studies (not shown)\n410 Basin-average groundwater storage, shown in Figure 7c in area-normalized units, shows a strong annual signal for all WRF \nconfigurations with notable, but minimal differences across IPM configurations. Here all groundwater, inclusive of saturated \nor unsaturated storage, is considered. The cumulative, area-normalized annual groundwater storage, when accounting for Basin-average groundwater storage, shown in Figure 7c in area-normalized units, shows a strong annual signal for all WRF \nconfigurations with notable, but minimal differences across IPM configurations. Here all groundwater, inclusive of saturated \nor unsaturated storage, is considered. The cumulative, area-normalized annual groundwater storage, when accounting for \nonly vadose zone storage (Figure 7d), which is most responsive to sub-annual differences in precipitation inputs, is \n415 \nmeaningful in this context because it relates a cumulative impact on near-surface groundwater storage due to IPM \nconfiguration. Similar to year-end cumulative discharge, year-end departures in vadose zone groundwater storage across the \ndifferent simulations are evident. Differences across the IPM configurations of subgrid-scale physics schemes are slightly \nlarger than the difference across the forcing simulations (4% versus 2%, respectively). While the differences in groundwater \nsignals are not as pronounced as the discharge signals, the slower, more muted-nature of infiltration and impacts on deeper \n420 \naquifer reserves relative to discharge would likely be more notable for multi-year simulations and/or in more water-limited only vadose zone storage (Figure 7d), which is most responsive to sub-annual differences in precipitation inputs, is \n415 \nmeaningful in this context because it relates a cumulative impact on near-surface groundwater storage due to IPM \nconfiguration. Similar to year-end cumulative discharge, year-end departures in vadose zone groundwater storage across the \ndifferent simulations are evident. Differences across the IPM configurations of subgrid-scale physics schemes are slightly \nlarger than the difference across the forcing simulations (4% versus 2%, respectively). While the differences in groundwater signals are not as pronounced as the discharge signals, the slower, more muted-nature of infiltration and impacts on deeper \n420 \naquifer reserves relative to discharge would likely be more notable for multi-year simulations and/or in more water-limited \nenvironments (or water years) where plant-water use demands are higher. signals are not as pronounced as the discharge signals, the slower, more muted-nature of infiltration and impacts on deeper \n420 \naquifer reserves relative to discharge would likely be more notable for multi-year simulations and/or in more water-limited \nenvironments (or water years) where plant-water use demands are higher. signals are not as pronounced as the discharge signals, the slower, more muted-nature of infiltration and impacts on deeper \n420 \naquifer reserves relative to discharge would likely be more notable for multi-year simulations and/or in more water-limited \nenvironments (or water years) where plant-water use demands are higher. Figure 8 shows maps of standard deviations in annual total evapotranspiration (ET) simulated by ParFlow-CLM across IPM \nconfigurations (top row), as well as the cell-binned relationship of those standard deviations of annual ET with land use and \n425 \ncover type, as well as elevation (bottom). Consistent with variations shown in the simulated discharge and groundwater \nstorage, Parflow-CLM simulates greater variations of ET under WRF configurations driven by different subgrid-scale \nphysics schemes (Figure 8a), compared to the simulations conducted with different meteorological forcings (Figure 8b). These results suggest that locations populated by high-water demanding vegetation (namely evergreen and deciduous forests) at mid-elevations result in the highest ET variability across IPM configurations. Conversely, low-water demanding \n430 \nvegetation (barren/sparsely vegetated land and grasses), which reside across a range of elevations in the study domain, result \nin the lowest variability in annual ET across IPM configurations. These differences in water demand essentially magnify any \ndifferences in atmospheric conditions. forests) at mid-elevations result in the highest ET variability across IPM configurations. Conversely, low-water demanding \n430 \nvegetation (barren/sparsely vegetated land and grasses), which reside across a range of elevations in the study domain, result \nin the lowest variability in annual ET across IPM configurations. These differences in water demand essentially magnify any \ndifferences in atmospheric conditions. forests) at mid-elevations result in the highest ET variability across IPM configurations. Conversely, low-water demanding \n430 \nvegetation (barren/sparsely vegetated land and grasses), which reside across a range of elevations in the study domain, result \nin the lowest variability in annual ET across IPM configurations. These differences in water demand essentially magnify any \ndifferences in atmospheric conditions. 20 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Figure 8. Pixel-level standard deviation in annual total evapotranspiration (ET) over the ParFlow-CLM domain from WR\nwith different subgrid-scale physics schemes (a,c) or meteorological forcing (b,d). https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. 5. Discussion and Conclusions \n440 In spite of previous efforts to characterize the sensitivity of WRF simulations to model configuration choices, the mountain \nclimate and hydrology scientific community has not sufficiently explored the implications of those choices for surface and \nsubsurface hydrology in high-altitude complex terrain. Here, we used an IPM produced by coupling WRF and ParFlow-\nCLM to assess the hydrometeorology of the ERW which is characterized by strong hydrological gradients indicative of \nmountain environments of the UCRB. 445 In this paper, we present a number of numerical experiment results that are informative for the scientific community to better \nunderstand atmosphere-through-bedrock process interactions, with an eye towards how to represent those interactions in \nclimate and hydrological models. First, the uncertainties associated with meteorological forcing choice are less important In this paper, we present a number of numerical experiment results that are informative for the scientific community to better \nunderstand atmosphere-through-bedrock process interactions, with an eye towards how to represent those interactions in \nclimate and hydrological models. First, the uncertainties associated with meteorological forcing choice are less important \nthan subgrid-scale physics scheme choice, at least in the ERW. This finding has important implications for IPM in complex \n450 \nterrain, since it reveals that the differences in reanalysis products are less consequential for initializing and forcing IPMs than \natmospheric configurations, and that efforts to advance IPMs such as collecting observations and using them to evaluate \nphysical process parameterizations at the sub-HUC-8 scale could help to better constrain model performance. This result also \nshows that the boundary conditions of the IPM simulation are less important in driving the magnitude and spatial variability \nf k\nh d\nl\ni\nl\ni bl\nh\nh d\nil i\nh\ni\nd\ni i i\nh i\nb\nid\nl\nh\ni\nh In this paper, we present a number of numerical experiment results that are informative for the scientific community to better \nunderstand atmosphere-through-bedrock process interactions, with an eye towards how to represent those interactions in \nclimate and hydrological models. First, the uncertainties associated with meteorological forcing choice are less important \nthan subgrid-scale physics scheme choice, at least in the ERW. 5. Discussion and Conclusions \n440 This finding has important implications for IPM in complex \n450 \nterrain, since it reveals that the differences in reanalysis products are less consequential for initializing and forcing IPMs than \natmospheric configurations, and that efforts to advance IPMs such as collecting observations and using them to evaluate \nphysical process parameterizations at the sub-HUC-8 scale could help to better constrain model performance. This result also \nshows that the boundary conditions of the IPM simulation are less important in driving the magnitude and spatial variability \nof key hydrometeorological variables than the details in choosing and optimizing atmospheric subgrid-scale physics schemes \n455 \n(e.g., microphysics or boundary layer turbulence). Ultimately, we found that the BSU-CFSR2 configuration produced the \nmost accurate recreation of WY2019 in the ERW which allows researchers, in this case, to prioritize process studies and the \ndevelopment of associated observational constraints within the ERW. However, further investigation is need to evaluate the \nsystemic cold bias across IPM configurations, particularly at higher elevations, and the consequence of delayed snowmelt climate and hydrological models. First, the uncertainties associated with meteorological forcing choice are less important \nthan subgrid-scale physics scheme choice, at least in the ERW. This finding has important implications for IPM in complex \n450 \nterrain, since it reveals that the differences in reanalysis products are less consequential for initializing and forcing IPMs than \natmospheric configurations, and that efforts to advance IPMs such as collecting observations and using them to evaluate \nphysical process parameterizations at the sub-HUC-8 scale could help to better constrain model performance. This result also \nshows that the boundary conditions of the IPM simulation are less important in driving the magnitude and spatial variability than subgrid-scale physics scheme choice, at least in the ERW. This finding has important implications for IPM in complex \n450 \nterrain, since it reveals that the differences in reanalysis products are less consequential for initializing and forcing IPMs than \natmospheric configurations, and that efforts to advance IPMs such as collecting observations and using them to evaluate \nphysical process parameterizations at the sub-HUC-8 scale could help to better constrain model performance. This result also \nshows that the boundary conditions of the IPM simulation are less important in driving the magnitude and spatial variability of key hydrometeorological variables than the details in choosing and optimizing atmospheric subgrid-scale physics schemes \n455 \n(e.g., microphysics or boundary layer turbulence). 5. Discussion and Conclusions \n440 This is because the spatial redistribution of shortwave radiation leads to In the investigation of topographical and slope gradient effects on shortwave radiation, our study shows those considerations \nin WRF are essential in redistributing radiation flux over regions of complex terrain, even though the differences in spatial-\naverage performance over ERW is minimal. This is because the spatial redistribution of shortwave radiation leads to in WRF are essential in redistributing radiation flux over regions of complex terrain, even though the differences in spatial-\naverage performance over ERW is minimal. This is because the spatial redistribution of shortwave radiation leads to \napproximately +/- 30 W/m2 difference in the east/west facing slopes that lead to +/- 1 K difference in two-meter surface air \n465 \ntemperature in August and September (when snowpack is nonexistent). Throughout most of the water year when snowpack \nexists, the spatial heterogeneity of temperature differences are less apparent than for shortwave radiation. Latent heat is \nposited to buffer differences in the shortwave radiation contribution to the radiation budget, and causes early snowmelt in the \nhigh elevation mountains in those simulations with topographical and slope gradient shortwave radiation effects turned on. approximately +/- 30 W/m2 difference in the east/west facing slopes that lead to +/- 1 K difference in two-meter surface air \n465 \ntemperature in August and September (when snowpack is nonexistent). Throughout most of the water year when snowpack \nexists, the spatial heterogeneity of temperature differences are less apparent than for shortwave radiation. Latent heat is \nposited to buffer differences in the shortwave radiation contribution to the radiation budget, and causes early snowmelt in the \nhigh elevation mountains in those simulations with topographical and slope gradient shortwave radiation effects turned on. At the same time, this finding is potentially indicative of challenges in extrapolating findings from one mountainous \n470 \nwatershed to another. If atmospheric process details are significant for surface and subsurface hydrological modeling and if \nthe findings regarding atmospheric processes in one study area are marginally or completely irrelevant to other mountainous At the same time, this finding is potentially indicative of challenges in extrapolating findings from one mountainous \n470 \nwatershed to another. The ERW outline is overlain in black \nthe upper row, a-b. The relationship between annual ET, elevation, and land cover type are shown as scatter plots on t\nbottom row, c-d. See Figure 1 for maps of land cover types. 435 Figure 8. Pixel-level standard deviation in annual total evapotranspiration (ET) over the ParFlow-CLM domain from WRF \nwith different subgrid-scale physics schemes (a,c) or meteorological forcing (b,d). The ERW outline is overlain in black in \nthe upper row, a-b. The relationship between annual ET, elevation, and land cover type are shown as scatter plots on the \nbottom row, c-d. See Figure 1 for maps of land cover types. 21 5. Discussion and Conclusions \n440 Ultimately, we found that the BSU-CFSR2 configuration produced the \nmost accurate recreation of WY2019 in the ERW which allows researchers, in this case, to prioritize process studies and the \ndevelopment of associated observational constraints within the ERW. However, further investigation is need to evaluate the \nsystemic cold bias across IPM configurations, particularly at higher elevations, and the consequence of delayed snowmelt \nand timing of discharge peaks. 460 of key hydrometeorological variables than the details in choosing and optimizing atmospheric subgrid-scale physics schemes \n455 \n(e.g., microphysics or boundary layer turbulence). Ultimately, we found that the BSU-CFSR2 configuration produced the \nmost accurate recreation of WY2019 in the ERW which allows researchers, in this case, to prioritize process studies and the \ndevelopment of associated observational constraints within the ERW. However, further investigation is need to evaluate the \nsystemic cold bias across IPM configurations, particularly at higher elevations, and the consequence of delayed snowmelt \nand timing of discharge peaks. 460 of key hydrometeorological variables than the details in choosing and optimizing atmospheric subgrid-scale physics schemes \n455 \n(e.g., microphysics or boundary layer turbulence). Ultimately, we found that the BSU-CFSR2 configuration produced the \nmost accurate recreation of WY2019 in the ERW which allows researchers, in this case, to prioritize process studies and the \ndevelopment of associated observational constraints within the ERW. However, further investigation is need to evaluate the \nsystemic cold bias across IPM configurations, particularly at higher elevations, and the consequence of delayed snowmelt \nand timing of discharge peaks. 460 In the investigation of topographical and slope gradient effects on shortwave radiation, our study shows those considerations \nin WRF are essential in redistributing radiation flux over regions of complex terrain, even though the differences in spatial-\naverage performance over ERW is minimal. This is because the spatial redistribution of shortwave radiation leads to \napproximately +/- 30 W/m2 difference in the east/west facing slopes that lead to +/- 1 K difference in two-meter surface air \n465 \ntemperature in August and September (when snowpack is nonexistent). Throughout most of the water year when snowpack \nexists, the spatial heterogeneity of temperature differences are less apparent than for shortwave radiation. Latent heat is In the investigation of topographical and slope gradient effects on shortwave radiation, our study shows those considerations \nin WRF are essential in redistributing radiation flux over regions of complex terrain, even though the differences in spatial-\naverage performance over ERW is minimal. Another methodological constraint is that our WRF and Parflow-CLM \n480 \nexperiments were only one-way coupled instead of two-way coupled, which ignores potentially important feedbacks from \nthe subsurface hydrology to the atmosphere via ET and the radiation budget. For example, Givati et al. (2016) reported that \nsimulated precipitation was improved with two-way coupling in WRF-Hydro compared to WRF-only and Forrester et al. (2018) showed that boundary layer dynamics were impacted in IPM simulations in regions where shallow water tables exist. 485 \nFuture work will include integration of data, either indirectly through IPM benchmarking or directly through data \nassimilation into the IPM, from a recently deployed atmospheric observatory in the ERW as part of the Surface Atmosphere \nIntegrated Field Laboratory (SAIL) Campaign, which will run from September, 2021 to June, 2023. SAIL is collecting a \nwide-array of observations with the intent to advance understanding of precipitation, snow, aerosol, aerosol-cloud Future work will include integration of data, either indirectly through IPM benchmarking or directly through data \nassimilation into the IPM, from a recently deployed atmospheric observatory in the ERW as part of the Surface Atmosphere \nIntegrated Field Laboratory (SAIL) Campaign, which will run from September, 2021 to June, 2023. SAIL is collecting a \nwide-array of observations with the intent to advance understanding of precipitation, snow, aerosol, aerosol-cloud \ninteraction, and radiation processes in complex terrain and establish the minimum-but-sufficient level of process \n490 \nunderstanding to develop a robust predictive understanding of seasonal surface water and energy budgets in the ERW \n(Feldman et al., 2021). SAIL is working in conjunction with the Watershed Function Scientific Focus Area (WF-SFA) and \npartners including the National Oceanic and Atmospheric Administration (NOAA)’s Study for Precipitation, the Lower \nAtmosphere, and Surface for Hydrometeorology (SPLASH), the United States Geological Survey’s Next Generation Water \nObserving System (NGWOS), the National Science Foundation’s Sublimation of Snow (SOS) project, and numerous state \n495 \nand local agencies and organizations, including the Rocky Mountain Biological Laboratory, to develop a wide range of \nhydrometeorological datasets to constrain atmosphere, surface, and subsurface processes simultaneously. Together, these \nresources are contributing to the establishment of a highly-instrumented and studied UCRB watershed. Our study highlights \nthat the benchmarking provided by these data collections will be critical in addressing the systemic IPM cold bias by \nproviding a more constrained estimate of radiation budgets in complex terrain that ultimately shape snowmelt and discharge. 5. Discussion and Conclusions \n440 If atmospheric process details are significant for surface and subsurface hydrological modeling and if \nthe findings regarding atmospheric processes in one study area are marginally or completely irrelevant to other mountainous At the same time, this finding is potentially indicative of challenges in extrapolating findings from one mountainous \n470 \nwatershed to another. If atmospheric process details are significant for surface and subsurface hydrological modeling and if \nthe findings regarding atmospheric processes in one study area are marginally or completely irrelevant to other mountainous 22 500 wide-array of observations with the intent to advance understanding of precipitation, snow, aerosol, aerosol-cloud \ninteraction, and radiation processes in complex terrain and establish the minimum-but-sufficient level of process \n490 \nunderstanding to develop a robust predictive understanding of seasonal surface water and energy budgets in the ERW \n(Feldman et al., 2021). SAIL is working in conjunction with the Watershed Function Scientific Focus Area (WF-SFA) and \npartners including the National Oceanic and Atmospheric Administration (NOAA)’s Study for Precipitation, the Lower \nAtmosphere, and Surface for Hydrometeorology (SPLASH), the United States Geological Survey’s Next Generation Water interaction, and radiation processes in complex terrain and establish the minimum-but-sufficient level of process \n490 \nunderstanding to develop a robust predictive understanding of seasonal surface water and energy budgets in the ERW \n(Feldman et al., 2021). SAIL is working in conjunction with the Watershed Function Scientific Focus Area (WF-SFA) and \npartners including the National Oceanic and Atmospheric Administration (NOAA)’s Study for Precipitation, the Lower \nAtmosphere, and Surface for Hydrometeorology (SPLASH), the United States Geological Survey’s Next Generation Water Observing System (NGWOS), the National Science Foundation’s Sublimation of Snow (SOS) project, and numerous state \n495 \nand local agencies and organizations, including the Rocky Mountain Biological Laboratory, to develop a wide range of \nhydrometeorological datasets to constrain atmosphere, surface, and subsurface processes simultaneously. Together, these \nresources are contributing to the establishment of a highly-instrumented and studied UCRB watershed. Our study highlights \nthat the benchmarking provided by these data collections will be critical in addressing the systemic IPM cold bias by Observing System (NGWOS), the National Science Foundation’s Sublimation of Snow (SOS) project, and numerous state \n495 \nand local agencies and organizations, including the Rocky Mountain Biological Laboratory, to develop a wide range of \nhydrometeorological datasets to constrain atmosphere, surface, and subsurface processes simultaneously. Together, these \nresources are contributing to the establishment of a highly-instrumented and studied UCRB watershed. Our study highlights \nthat the benchmarking provided by these data collections will be critical in addressing the systemic IPM cold bias by providing a more constrained estimate of radiation budgets in complex terrain that ultimately shape snowmelt and discharge. 500 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. watersheds, then additional field work would be needed in mountainous hydrology research to address this issue, given that \nthe extrapolation of fieldwork results remains a central challenge for field-based research and modeling activities. 475 A limitation of our study, given the computational constraints of running IPMs, is that it was infeasible to explore the full \nparameter spaces of WRF and ParFlow-CLM exhaustively; thus, our conclusions are limited to the selected subgrid-scale \nphysics schemes and meteorological forcing datasets analyzed. Additional work is needed to improve the systemic cold bias \nin two-meter surface air temperature throughout all experiments as this may have been the major driver in the delayed \nsnowmelt and peak discharge simulated by the IPM. Another methodological constraint is that our WRF and Parflow-CLM \n480 A limitation of our study, given the computational constraints of running IPMs, is that it was infeasible to explore the full \nparameter spaces of WRF and ParFlow-CLM exhaustively; thus, our conclusions are limited to the selected subgrid-scale \nphysics schemes and meteorological forcing datasets analyzed. Additional work is needed to improve the systemic cold bias \nin two-meter surface air temperature throughout all experiments as this may have been the major driver in the delayed \nsnowmelt and peak discharge simulated by the IPM. Another methodological constraint is that our WRF and Parflow-CLM \n480 \nexperiments were only one-way coupled instead of two-way coupled, which ignores potentially important feedbacks from \nthe subsurface hydrology to the atmosphere via ET and the radiation budget. For example, Givati et al. (2016) reported that \nsimulated precipitation was improved with two-way coupling in WRF-Hydro compared to WRF-only and Forrester et al. (2018) showed that boundary layer dynamics were impacted in IPM simulations in regions where shallow water tables exist. snowmelt and peak discharge simulated by the IPM. Another methodological constraint is that our WRF and Parflow-CLM \n480 \nexperiments were only one-way coupled instead of two-way coupled, which ignores potentially important feedbacks from \nthe subsurface hydrology to the atmosphere via ET and the radiation budget. For example, Givati et al. (2016) reported that \nsimulated precipitation was improved with two-way coupling in WRF-Hydro compared to WRF-only and Forrester et al. (2018) showed that boundary layer dynamics were impacted in IPM simulations in regions where shallow water tables exist. snowmelt and peak discharge simulated by the IPM. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Author contributions: ZX, ESW, AMR and DF designed the study together. ZX performed the WRF simulations and \nanalyzed the results. ESW performed the ParFlow-CLM simulations. All authors contributed to the writing and approve of \nthis manuscript. Author contributions: ZX, ESW, AMR and DF designed the study together. ZX performed the WRF simulations and \nanalyzed the results. ESW performed the ParFlow-CLM simulations. All authors contributed to the writing and approve of \nthis manuscript. Competing interests: The contact author has declared that neither they nor their co-authors have any competing interests. Competing interests: The contact author has declared that neither they nor their co-authors have any competing interests. 0 Competing interests: The contact author has declared that neither they nor their co-authors have any competing interests. Competing interests: The contact author has declared that neither they nor their co-authors have any competing interests. 510 \n \nAcknowledgements: This work was supported by the Laboratory Directed Research and Development Program of Lawrence \nBerkeley National Laboratory under U.S. Department of Energy Contract No. DE-AC02-05CH11231. This research used \nresources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported Acknowledgements: This work was supported by the Laboratory Directed Research and Development Program of Lawrence \nBerkeley National Laboratory under U.S. Department of Energy Contract No. DE-AC02-05CH11231. This research used \nresources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported Acknowledgements: This work was supported by the Laboratory Directed Research and Development Program of Lawrence \nBerkeley National Laboratory under U.S. Department of Energy Contract No. DE-AC02-05CH11231. This research used \nresources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported Acknowledgements: This work was supported by the Laboratory Directed Research and Development Program of Lawrence \nBerkeley National Laboratory under U.S. Department of Energy Contract No. DE-AC02-05CH11231. This research used \nresources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy under that same contract. This research also used the Lawrencium \n515 \ncomputational cluster resource provided by the IT Division at the Lawrence Berkeley National Laboratory (supported by the \nDirector, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under contract no. DE-\nAC02-05CH11231). Data Availability: All WRF model output files can be found at 505 23 Ashby, S. F. and Falgout, R. D.: A parallel multigrid preconditioned conjugate gradient algorithm for groundwater flow \nsimulations, Nuclear science and engineering, 124, 145–159, https://doi.org/10.13182/NSE96-A24230, 1996. Department of Energy Regional \nand Global Climate Modeling (RGCM) Program through the Calibrated and Systematic Characterization, Attribution and \nD\ni\nf E\n(CASCADE) S i\nF\nA\nd h\nI\nd E\nl\ni\nf h\nSi\nl\nd H d\nli\n530 Detection of Extremes (CASCADE) Science Focus Area and the Integrated Evaluation of the Simulated Hydroclimate \n530 \nSystem of the Continental US project also under the same contract. Arthur, R. S., Lundquist, K. A., Mirocha, J. D., and Chow, F. K.: Topographic effects on radiation in the WRF Model with \nthe immersed boundary method: Implementation, validation, and application to complex terrain, Monthly Weather \nR\ni\n146 3277 3292 htt\n//d i\n/10 1175/MWR D 18 0108 1 2018\n35 The authors acknowledge the helpful guidance provided by Professor Lejo Flores and Dr. Will Rudisill \nof Boise State University regarding the WRF configurations conducted in this study over the ERW. by the Office of Science of the U.S. Department of Energy under that same contract. This research also used the Lawrencium \n515 \ncomputational cluster resource provided by the IT Division at the Lawrence Berkeley National Laboratory (supported by the \nDirector, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under contract no. DE-\nAC02-05CH11231). The authors acknowledge the helpful guidance provided by Professor Lejo Flores and Dr. Will Rudisill \nof Boise State University regarding the WRF configurations conducted in this study over the ERW. y\ng\ng\ng\ny\n \n520 \nFinancial support: First author Xu's material pertaining to WRF simulations was supported by the Laboratory Directed \nResearch and Development Program of Lawrence Berkeley National Laboratory under U.S. Department of Energy Contract \nNo. DE-AC02-05CH11231, and his material pertaining to the analysis using reference and observation datasets was \nsupported by the Lawrence Berkeley National Laboratory’s Watershed Function Science Focus Area of the U.S. Department Financial support: First author Xu's material pertaining to WRF simulations was supported by the Laboratory Directed \nResearch and Development Program of Lawrence Berkeley National Laboratory under U.S. Department of Energy Contract \nNo. DE-AC02-05CH11231, and his material pertaining to the analysis using reference and observation datasets was \nsupported by the Lawrence Berkeley National Laboratory’s Watershed Function Science Focus Area of the U.S. Department \nof Energy’s Environmental System Science (ESS) Program also under the same contract\n525 of Energy’s Environmental System Science (ESS) Program, also under the same contract. 525 \nCo-authors Feldman and Siirila-Woodburn's material was supported by the Laboratory Directed Research and Development \nProgram of Lawrence Berkeley National Laboratory under the same contract. Co-author Rhoades was funded by the \nDirector, Office of Science, Office of Biological and Environmental Research of the U.S. Department of Energy Regional \nand Global Climate Modeling (RGCM) Program through the Calibrated and Systematic Characterization, Attribution and of Energy’s Environmental System Science (ESS) Program, also under the same contract. 525 \nCo-authors Feldman and Siirila-Woodburn's material was supported by the Laboratory Directed Research and Development \nProgram of Lawrence Berkeley National Laboratory under the same contract. Co-author Rhoades was funded by the \nDirector, Office of Science, Office of Biological and Environmental Research of the U.S. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Comin, A. N., Schumacher, V., Justino, F., and Fernandez, A.: Impact of different microphysical parameterizations on \nextreme \nsnowfall \nevents \nin \nthe \nSouthern \nAndes, \nWeather \nand \nclimate \nextremes, \n21, \n65–75, \nhttps://doi.org/10.1016/j.wace.2018.07.001, 2018. 540 Dai, Y., Zeng, X., Dickinson, R. E., Baker, I., Bonan, G. B., Bosilovich, M. G., Denning, A. S., Dirmeyer, P. A., Houser, P. R., Niu, G., Oleson, K. W., Schlosser, C. A., & Yang, Z.: The Common Land Model, Bulletin of the American \nMeteorological Society, 84(8), 1013-1024, doi:10.1175/BAMS-84-8-1013, 2003. Daly, C., Halbleib, M., Smith, J. I., Gibson, W. P., Doggett, M. K., Taylor, G. H., Curtis, J., and Pasteris, P. P.: Physiographically sensitive mapping of climatological temperature and precipitation across the conterminous United \n545 \nStates, International Journal of Climatology: a Journal of the Royal Meteorological Society, 28, 2031–2064, \nhttps://doi.org/10.1002/joc.1688, 2008. Deems, J. S., Painter, T. H., and Finnegan, D. C.: Lidar measurement of snow depth: a review, Journal of Glaciology, 59, \n467–479, https://doi.org/10.3189/2013JoG12J154, 2013. ,\ngy\ny\ng\ny,\n,\n,\nhttps://doi.org/10.1002/joc.1688, 2008. Deems, J. S., Painter, T. H., and Finnegan, D. C.: Lidar measurement of snow depth: a review, Journal of Glaciology, 59, \n467–479, https://doi.org/10.3189/2013JoG12J154, 2013. Deems, J. S., Painter, T. H., and Finnegan, D. C.: Lidar measurement of snow depth: a review, Journal of Glaciology, 59, \n467–479, https://doi.org/10.3189/2013JoG12J154, 2013. Fassnacht, S., Dressler, K., and Bales, R.: Snow water equivalent interpolation for the Colorado River Basin from snow \n550 \ntelemetry (SNOTEL) data, Water Resources Research, 39, https://doi.org/10.1029/2002WR001512, 2003. Feldman, D.R. et al.: Surface Atmosphere Integrated Field Laboratory (SAIL)
 Science Plan, Ed. by Robert Stafford, ARM \nuser facility. DOE/SC-ARM-21-004, 2021. Forrester, M. M., Maxwell, R. M., Bearup, L. A., and Gochis, D. J.: Forest disturbance feedbacks from bedrock to \natmosphere using coupled hydrometeorological simulations over the Rocky Mountain headwaters, Journal of \n555 \nGeophysical Research: Atmospheres, 123, 9026–9046, https://doi.org/10.1029/2018JD028380, 2018. Gelaro, R., McCarty, W., Suárez, M. J., Todling, R., Molod, A., Takacs, L., Randles, C. A., Darmenov, A., Bosilovich, M. G., Reichle, R., et al.: The modern-era retrospective analysis for research and applications, version 2 (MERRA-2), \nJournal of climate, 30, 5419–5454, https://doi.org/10.1175/JCLI-D-16-0758.1, 2017. References: Review, 146, 3277–3292, https://doi.org/10.1175/MWR-D-18-0108.1, 2018. Ashby, S. F. and Falgout, R. D.: A parallel multigrid preconditioned conjugate gradient algorithm for groundwater flow \nsimulations, Nuclear science and engineering, 124, 145–159, https://doi.org/10.13182/NSE96-A24230, 1996. 24 Fassnacht, S., Dressler, K., and Bales, R.: Snow water equivalent interpolation for the Colorado River Basin from snow \n550 \ntelemetry (SNOTEL) data, Water Resources Research, 39, https://doi.org/10.1029/2002WR001512, 2003. Feldman, D.R. et al.: Surface Atmosphere Integrated Field Laboratory (SAIL)
 Science Plan, Ed. by Robert Stafford, ARM \nuser facility. DOE/SC-ARM-21-004, 2021. Forrester, M. M., Maxwell, R. M., Bearup, L. A., and Gochis, D. J.: Forest disturbance feedbacks from bedrock to \natmosphere using coupled hydrometeorological simulations over the Rocky Mountain headwaters, Journal of \n555 \nGeophysical Research: Atmospheres, 123, 9026–9046, https://doi.org/10.1029/2018JD028380, 2018. Gelaro, R., McCarty, W., Suárez, M. J., Todling, R., Molod, A., Takacs, L., Randles, C. A., Darmenov, A., Bosilovich, M. G., Reichle, R., et al.: The modern-era retrospective analysis for research and applications, version 2 (MERRA-2), \nJournal of climate, 30, 5419–5454, https://doi.org/10.1175/JCLI-D-16-0758.1, 2017. Forrester, M. M., Maxwell, R. M., Bearup, L. A., and Gochis, D. J.: Forest disturbance feedbacks from bedrock to \natmosphere using coupled hydrometeorological simulations over the Rocky Mountain headwaters, Journal of \n555 \nGeophysical Research: Atmospheres, 123, 9026–9046, https://doi.org/10.1029/2018JD028380, 2018. Gelaro, R., McCarty, W., Suárez, M. J., Todling, R., Molod, A., Takacs, L., Randles, C. A., Darmenov, A., Bosilovich, M. G., Reichle, R., et al.: The modern-era retrospective analysis for research and applications, version 2 (MERRA-2), \nJournal of climate, 30, 5419–5454, https://doi.org/10.1175/JCLI-D-16-0758.1, 2017. Givati, A., Gochis, D., Rummler, T., and Kunstmann, H.: Comparing one-way and two-way coupled hydrometeorological \n560 \nforecasting \nsystems \nfor \nflood \nforecasting \nin \nthe \nMediterranean \nregion, \nHydrology, \n3, \n19, \nhttps://doi.org/10.3390/hydrology3020019, 2016. G\nC\nH\nA\nW\nY\nY\nB\nM\nX\nZh\nX\nd C i S\nEff\nt\nf\nb\nid t\ni\ndi ti\nf\ni\nth Givati, A., Gochis, D., Rummler, T., and Kunstmann, H.: Comparing one-way and two-way coupled hydrometeorological \n560 \nforecasting \nsystems \nfor \nflood \nforecasting \nin \nthe \nMediterranean \nregion, \nHydrology, \n3, \n19, \nhttps://doi.org/10.3390/hydrology3020019, 2016. Gu, C., Huang, A., Wu, Y., Yang, B., Mu, X., Zhang, X., and Cai, S.: Effects of subgrid terrain radiative forcing on the \nability of RegCM4. 1 in the simulation of summer precipitation over China, Journal of Geophysical Research: Gu, C., Huang, A., Wu, Y., Yang, B., Mu, X., Zhang, X., and Cai, S.: Effects of subgrid terrain radiative forcing on the \nability of RegCM4. 1 in the simulation of summer precipitation over China, Journal of Geophysical Research: Gu, C., Huang, A., Wu, Y., Yang, B., Mu, X., Zhang, X., and Cai, S.: Effects of subgrid terrain radiative forcing on the \nability of RegCM4. 1 in the simulation of summer precipitation over China, Journal of Geophysical Research: \nAtmospheres, 125, e2019JD032 215, https://doi.org/10.1029/2019JD032215, 2020. 565 Atmospheres, 125, e2019JD032 215, https://doi.org/10.1029/2019JD032215, 2020. 565 \nGutowski, W. J., Jr, Ullrich, P. A., Hall, A., Leung, L. R., O’Brien, T. A., Patricola, C. M., Arritt, R. W., Bukovsky, M. S., \nCalvin, K. V., Feng, Z., Jones, A. D., Kooperman, G. J., Monier, E., Pritchard, M. S., Pryor, S. C., Qian, Y., Rhoades, \nA. M., Roberts, A. F., Sakaguchi, K., Urban, N., and Zarzycki, C.: The Ongoing Need for High-Resolution Regional \nClimate Models: Process Understanding and Stakeholder Information, Bulletin of the American Meteorological Society, 570 25 Lee, W.-L., Gu, Y., Liou, K., Leung, L., and Hsu, H.-H.: A global model simulation for 3-D radiative transfer impact on \n600 \nsurface hydrology over the Sierra Nevada and Rocky Mountains, Atmospheric Chemistry and Physics, 15, 5405–5413, \nhttps://doi.org/10.5194/acp-15-5405-2015, 2015. Orr, A., Listowski, C., Couttet, M., Collier, E., Immerzeel, W., Deb, P., and Bannister, D.: Sensitivity of simulated summer \nmonsoonal precipitation in Langtang Valley, Himalaya, to cloud microphysics schemes in WRF, Journal of Geophysical \n625 \nResearch: Atmospheres, 122, 6298–6318, 2017. Painter, T. H., Berisford, D. F., Boardman, J. W., Bormann, K. J., Deems, J. S., Gehrke, F., Hedrick, A., Joyce, M., Laidlaw, \nR., Marks, D., et al.: The Airborne Snow Observatory: Fusion of scanning lidar, imaging spectrometer, and physically-\nbased modeling for mapping snow water equivalent and snow albedo, Remote Sensing of Environment, 184, 139–152, \nhttps://doi.org/10.1016/j.rse.2016.06.018, 2016. 630 \nPalazzi, E., Mortarini, L., Terzago, S., and Von Hardenberg, J.: Elevation-dependent warming in global climate model \nsimulations at high spatial resolution, Climate Dynamics, 52, 2685–2702, https://doi.org/10.1007/s00382-018-4287-z, \n2019. Powers, J. G., Klemp, J. B., Skamarock, W. C., Davis, C. A., Dudhia, J., Gill, D. O., Coen, J. L., Gochis, D. J., Ahmadov, R., \nPeckham, S. E., et al.: The weather research and forecasting model: Overview, system efforts, and future directions, \n635 \nBulletin of the American Meteorological Society, 98, 1717–1737, https://doi.org/10.1175/BAMS-D-15-00308.1., 2017. Milly, P. C. and Dunne, K. A.: Colorado River flow dwindles as warming-driven loss of reflective snow energizes \nevaporation, Science, 367, 1252–1255, https://doi.org/10.1126/science.aay9187, 2020. Orr, A., Listowski, C., Couttet, M., Collier, E., Immerzeel, W., Deb, P., and Bannister, D.: Sensitivity of simulated summer \nmonsoonal precipitation in Langtang Valley, Himalaya, to cloud microphysics schemes in WRF, Journal of Geophysical \n625 \nResearch: Atmospheres, 122, 6298–6318, 2017. 625 Painter, T. H., Berisford, D. F., Boardman, J. W., Bormann, K. J., Deems, J. S., Gehrke, F., Hedrick, A., Joyce, M., Laidlaw, \nR., Marks, D., et al.: The Airborne Snow Observatory: Fusion of scanning lidar, imaging spectrometer, and physically-\nbased modeling for mapping snow water equivalent and snow albedo, Remote Sensing of Environment, 184, 139–152, \nhttps://doi.org/10.1016/j.rse.2016.06.018, 2016. 30 630 Palazzi, E., Mortarini, L., Terzago, S., and Von Hardenberg, J.: Elevation-dependent warming in global climate model \nsimulations at high spatial resolution, Climate Dynamics, 52, 2685–2702, https://doi.org/10.1007/s00382-018-4287-z, \n2019. Powers, J. G., Klemp, J. B., Skamarock, W. C., Davis, C. A., Dudhia, J., Gill, D. O., Coen, J. L., Gochis, D. J., Ahmadov, R., Powers, J. G., Klemp, J. B., Skamarock, W. C., Davis, C. A., Dudhia, J., Gill, D. O., Coen, J. L Powers, J. G., Klemp, J. B., Skamarock, W. C., Davis, C. A., Dudhia, J., Gill, D. O., Coen, J. L., Gochis, D. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Hao, D., Bisht, G., Gu, Y., Lee, W.-L., Liou, K.-N., and Leung, L. R.: A parameterization of sub-grid topographical effects \non solar radiation in the E3SM Land Model (version 1.0): implementation and evaluation over the Tibetan Plateau, \nGeoscientific Model Development, 14, 6273–6289, https://doi.org/10.5194/gmd-14-6273-2021, 2021. Hersbach, H, Bell, B, Berrisford, P, et al.: The ERA5 global reanalysis. Quarterly Journal of the Royal Meteorological \nSociety, 146, 1999– 2049, doi:10.1002/qj.3803, 2020. 575 Homer, C., et al.: Conterminous United States land cover change patterns 2001–2016 from the 2016 National Land Cover \nDatabase, ISPRS Journal of Photogrammetry and Remote Sensing, 162, 184-199, doi:10.1016/j.isprsjprs.2020.02.019, \n2020. Huang, X., Rhoades, A. M., Ullrich, P. A., and Zarzycki, C. M.: An evaluation of the variable-resolution CESM for \nmodeling \nCalifornia’s \nclimate, \nJournal \nof \nAdvances \nin \nModeling \nEarth \nSystems, \n8, \n345–369, \n0 \nhttps://doi.org/10.1002/2015MS000559, 2016. 580 Hubbard, S. S., Williams, K. H., Agarwal, D., Banfield, J., Beller, H., Bouskill, N., Brodie, E., Carroll, R., Dafflon, B., \nDwivedi, D., et al.: The East River, Colorado, Watershed: A mountainous community testbed for improving predictive \nunderstanding \nof \nmultiscale \nhydrological–biogeochemical \ndynamics, \nVadose \nZone \nJournal, \n17, \n1–25, \nhttps://doi.org/10.2136/vzj2018.03.0061, 2018. 585 585 Ikeda, K., Rasmussen, R., Liu, C., Gochis, D., Yates, D., Chen, F., Tewari, M., Barlage, M., Dudhia, J., Miller, K., et al.: \nSimulation \nof \n60 \nseasonal \nsnowfall \nover \nColorado, \nAtmospheric \nResearch, \n97, \n462–477, \nhttps://doi.org/10.1016/j.atmosres.2010.04.010, 2010. James, T, et al.: The economic importance of the Colorado River to the basin region. Final Rep., L. William Seidman \nResearch Institute, Arizona State University, 54, https://businessforwater.org/wp-content/uploads/2016/12/PTF-Final-\n590 \n121814.pdf, 2014. Jin, J., Miller, N. L., and Schlegel, N.: Sensitivity study of four land surface schemes in the WRF model, Advances in \nMeteorology, 2010, https://doi.org/10.1155/2010/167436, 2010. Lee, W.-L., Gu, Y., Liou, K., Leung, L., and Hsu, H.-H.: A global model simulation for 3-D radiative transfer impact on \n600 \nsurface hydrology over the Sierra Nevada and Rocky Mountains, Atmospheric Chemistry and Physics, 15, 5405–5413, \nhttps://doi.org/10.5194/acp-15-5405-2015, 2015. 26 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Liu, C., Ikeda, K., Thompson, G., Rasmussen, R., and Dudhia, J.: High-resolution simulations of wintertime precipitation in \nthe Colorado Headwaters region: Sensitivity to physics parameterizations, Monthly Weather Review, 139, 3533–3553, \nhttps://doi.org/10.1175/MWR-D-11-00009.1, 2011. 605 Liu, C., Ikeda, K., Rasmussen, R., Barlage, M., Newman, A. J., Prein, A. F., Chen, F., Chen, L., Clark, M., Dai, A., et al.: \nContinental-scale convection-permitting modeling of the current and future climate of North America, Climate \nDynamics, 49, 71–95, https://doi.org/10.1007/s00382-016-3327-9, 2017. Lundquist, J., Hughes, M., Gutmann, E., and Kapnick, S.: Our skill in modeling mountain rain and snow is bypassing the \nskill of our observational networks, Bulletin of the American Meteorological Society, 100, 2473–2490, \nhttps://doi.org/10.1175/BAMS-D-19-0001.1, 2019. 610 Maina, F. Z., Siirila-Woodburn, E. R., and Vahmani, P.: Sensitivity of meteorological-forcing resolution on hydrologic \nvariables, Hydrology and Earth System Sciences, 24, 3451–3474, https://doi.org/10.5194/hess-24-3451-2020, 2020. Mallard, M. S., Spero, T. L., and Taylor, S. M.: Examining WRF’s sensitivity to contemporary Mallard, M. S., Spero, T. L., and Taylor, S. M.: Examining WRF’s sensitivity to contemporary land-use datasets across the \ncontiguous United States Using Dynamical Downscaling, Journal of applied meteorology and climatology, 57, 2561–\n615 \n2583, https://doi.org/10.1175/JAMC-D-17-0328.1, 2018. Mallard, M. S., Spero, T. L., and Taylor, S. M.: Examining WRF’s sensitivity to contemporary land-use datasets across the 615 contiguous United States Using Dynamical Downscaling, Journal of applied meteorology and climatology, 57, 2561–\n615 \n2583, https://doi.org/10.1175/JAMC-D-17-0328.1, 2018. Maxwell, R., Condon, L., and Kollet, S.: A high-resolution simulation of groundwater and surface water over most of the\ncontinental US with the integrated hydrologic model ParFlow v3, Geoscientific model development, 8, 923–937, contiguous United States Using Dynamical Downscaling, Journal of applied meteorology and climatology, 57, 2561–\n615 \n2583, https://doi.org/10.1175/JAMC-D-17-0328.1, 2018. Maxwell, R., Condon, L., and Kollet, S.: A high-resolution simulation of groundwater and surface water over most of the \ncontinental US with the integrated hydrologic model ParFlow v3, Geoscientific model development, 8, 923–937, \nhttps://doi.org/10.5194/gmd-8-923-2015, 2015. Maxwell, R. M.: A terrain-following grid transform and preconditioner for parallel, large-scale, integrated hydrologic \n620 \nmodeling, Advances in Water Resources, 53, 109–117, https://doi.org/10.1016/j.advwatres.2012.10.001, 2013. Maxwell, R. M.: A terrain-following grid transform and preconditioner for parallel, large-scale, integrated hydrologic \n620 \nmodeling, Advances in Water Resources, 53, 109–117, https://doi.org/10.1016/j.advwatres.2012.10.001, 2013. Milly, P. C. and Dunne, K. A.: Colorado River flow dwindles as warming-driven loss of reflective snow energizes \nevaporation, Science, 367, 1252–1255, https://doi.org/10.1126/science.aay9187, 2020. J., Ahmadov, R., \nPeckham, S. E., et al.: The weather research and forecasting model: Overview, system efforts, and future directions, \n5 \nBulletin of the American Meteorological Society, 98, 1717–1737, https://doi.org/10.1175/BAMS-D-15-00308.1., 2017. 635 Peckham, S. E., et al.: The weather research and forecasting model: Overview, system efforts, and future directions, \n635 \nBulletin of the American Meteorological Society, 98, 1717–1737, https://doi.org/10.1175/BAMS-D-15-00308.1., 2017. 27 645 \nRasmussen, R., Ikeda, K., Liu, C., Gochis, D., Clark, M., Dai, A., Gutmann, E., Dudhia, J., Chen, F., Barlage, M., et al.: \nClimate change impacts on the water balance of the Colorado headwaters: high-resolution regional climate model \nsimulations, Journal of Hydrometeorology, 15, 1091–1116, https://doi.org/10.1175/JHM-D-13-0118.1, 2014. 645 warmer climate, Journal of Climate, 24, 3015 3048, https://doi.org/10.1175/2010JCLI3985.1, 2011. 645 \nRasmussen, R., Ikeda, K., Liu, C., Gochis, D., Clark, M., Dai, A., Gutmann, E., Dudhia, J., Chen, F., Barlage, M., et al.: \nClimate change impacts on the water balance of the Colorado headwaters: high-resolution regional climate model \nsimulations, Journal of Hydrometeorology, 15, 1091–1116, https://doi.org/10.1175/JHM-D-13-0118.1, 2014. Rhoades, A. M., Ullrich, P. A., and Zarzycki, C. M.: Projecting 21st century snowpack trends in western USA mountains \nusing variable-resolution CESM, Climate Dynamics, 50, 261–288, https://doi.org/10.1007/s00382-017-3606-0, 2018a. 50 650 Rhoades, A. M., Ullrich, P. A., Zarzycki, C. M., Johansen, H., Margulis, S. A., Morrison, H., Xu, Z., and Collins, W. D.: \nSensitivity of mountain hydroclimate simulations in variable-resolution CESM to microphysics and horizontal \nresolution, Journal of Advances in Modeling Earth Systems, 10, 1357–1380, https://doi.org/10.1029/2018MS001326, \n2018b. Rhoades, A. M., Jones, A. D., and Ullrich, P. A.: Assessing mountains as natural reservoirs with a multimetric framework. 655 \nEarth's Future, 6, 1221– 1241. https://doi.org/10.1002/2017EF000789, 2018c. Rudisill, W., Flores, A., and McNamara, J.: The Impact of Initial Snow Conditions on the Numerical Weather Simulation of \na Northern Rockies Atmospheric River, Journal of Hydrometeorology, 22, 155–167, 2021. Skamarock, W.C., Klemp, J.B., Dudhia, J., Gill, D.O., Liu, Z., Berner, J., Wang, W., Powers, J.G., Duda, M.G., Barker, Rhoades, A. M., Jones, A. D., and Ullrich, P. A.: Assessing mountains as natural reservoirs with a multimetric framework. 655 \nEarth's Future, 6, 1221– 1241. https://doi.org/10.1002/2017EF000789, 2018c. Earth's Future, 6, 1221– 1241. https://doi.org/10.1002/2017EF000789, 2018c. Rudisill, W., Flores, A., and McNamara, J.: The Impact of Initial Snow Conditions on the Numerical Weather Simulation of \na Northern Rockies Atmospheric River, Journal of Hydrometeorology, 22, 155–167, 2021. Rudisill, W., Flores, A., and McNamara, J.: The Impact of Initial Snow Conditions on the Numerical Weather Simulation of \na Northern Rockies Atmospheric River, Journal of Hydrometeorology, 22, 155–167, 2021. a Northern Rockies Atmospheric River, Journal of Hydrometeorology, 22, 155–167, 2021. https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Rahimi, S., Krantz, W., Lin, Y.-H., Bass, B., Goldenson, N., Hall, A., Lebo, Z. J., and Norris, J.: Evaluation of a Reanalysis-\nDriven Configuration of WRF4 Over the Western United States From 1980 to 2020, Journal of Geophysical Research: \nAtmospheres, 127, e2021JD035 699, https://doi.org/10.1029/2021JD035699, 2022. Rasmussen, K. L., Prein, A. F., Rasmussen, R. M., Ikeda, K., and Liu, C.: Changes in the convective population and \n40 \nthermodynamic environments in convection-permitting regional climate simulations over the United States, Climate \nDynamics, 55, 383–408, https://doi.org/10.1007/s00382-017-4000-7, 2020. Rasmussen, K. L., Prein, A. F., Rasmussen, R. M., Ikeda, K., and Liu, C.: Changes in the convective population and \n640 \nthermodynamic environments in convection-permitting regional climate simulations over the United States, Climate \nDynamics, 55, 383–408, https://doi.org/10.1007/s00382-017-4000-7, 2020. Rasmussen, R., Liu, C., Ikeda, K., Gochis, D., Yates, D., Chen, F., Tewari, M., Barlage, M., Dudhia, J., Yu, W., et al.: High-\nresolution coupled climate runoff simulations of seasonal snowfall over Colorado: A process study of current and \nwarmer climate, Journal of Climate, 24, 3015–3048, https://doi.org/10.1175/2010JCLI3985.1, 2011. 645 \nRasmussen, R., Ikeda, K., Liu, C., Gochis, D., Clark, M., Dai, A., Gutmann, E., Dudhia, J., Chen, F., Barlage, M., et al.: \nClimate change impacts on the water balance of the Colorado headwaters: high-resolution regional climate model \nsimulations, Journal of Hydrometeorology, 15, 1091–1116, https://doi.org/10.1175/JHM-D-13-0118.1, 2014. Rhoades, A. M., Ullrich, P. A., and Zarzycki, C. M.: Projecting 21st century snowpack trends in western USA mountains \nusing variable-resolution CESM, Climate Dynamics, 50, 261–288, https://doi.org/10.1007/s00382-017-3606-0, 2018a. 650 \nRhoades, A. M., Ullrich, P. A., Zarzycki, C. M., Johansen, H., Margulis, S. A., Morrison, H., Xu, Z., and Collins, W. D.: \nSensitivity of mountain hydroclimate simulations in variable-resolution CESM to microphysics and horizontal \nresolution, Journal of Advances in Modeling Earth Systems, 10, 1357–1380, https://doi.org/10.1029/2018MS001326, \n2018b. Dynamics, 55, 383–408, https://doi.org/10.1007/s00382-017-4000-7, 2020. Rasmussen, R., Liu, C., Ikeda, K., Gochis, D., Yates, D., Chen, F., Tewari, M., Barlage, M., Dudhia, J., Yu, W., et al.: High-\nresolution coupled climate runoff simulations of seasonal snowfall over Colorado: A process study of current and \nwarmer climate, Journal of Climate, 24, 3015–3048, https://doi.org/10.1175/2010JCLI3985.1, 2011. Skamarock, W.C., Klemp, J.B., Dudhia, J., Gill, D.O., Liu, Z., Berner, J., Wang, W., Powers, J.G., Duda, M.G., Barker, Skamarock, W.C., Klemp, J.B., Dudhia, J., Gill, D.O., Liu, Z., Berner, J., Wang, W., Powers Skamarock, W.C., Klemp, J.B., Dudhia, J., Gill, D.O., Liu, Z., Berner, J., Wang, W., Powers, J.G., Duda, M.G., Barker, \nD.M. and Huang, X.Y.: A description of the advanced research WRF model version 4. National Center for Atmospheric \n660 \nResearch: Boulder, CO, USA, 145, p.145, 2019. D.M. and Huang, X.Y.: A description of the advanced research WRF model version 4. National Center for Atmospheric \n660 \nResearch: Boulder, CO, USA, 145, p.145, 2019. Saha, S., Moorthi, S., Pan, H.-L., Wu, X., Wang, J., Nadiga, S., Tripp, P., Kistler, R., Woollen, J., Behringer, D., et al.: The \nNCEP climate forecast system reanalysis, Bulletin of the American Meteorological Society, 91, 1015–1058, \nhttps://doi.org/10.1175/2010BAMS3001.1, 2010. Saha, S., Moorthi, S., Pan, H.-L., Wu, X., Wang, J., Nadiga, S., Tripp, P., Kistler, R., Woollen, J., Behringer, D., et al.: The \nNCEP climate forecast system reanalysis, Bulletin of the American Meteorological Society, 91, 1015–1058, \nhttps://doi.org/10.1175/2010BAMS3001.1, 2010. Schreiner-McGraw, A. P. and Ajami, H.: Impact of uncertainty in precipitation forcing data sets on the hydrologic budget of \n665 \nan integrated hydrologic model in mountainous terrain, Water Resources Research, 56, e2020WR027 639, \nhttps://doi.org/10.1029/2020WR027639, 2020. Serreze, M. C., Clark, M. P., Armstrong, R. L., McGinnis, D. A., and Pulwarty, R. S.: Characteristics of the western United \nStates snowpack from snowpack telemetry (SNOTEL) data, Water Resources Research, 35, 2145–2160, \nhttps://doi.org/10.1029/1999WR900090, 1999. 670 g\ny\ng\n,\n,\n,\n,\nhttps://doi.org/10.1029/2020WR027639, 2020. Serreze, M. C., Clark, M. P., Armstrong, R. L., McGinnis, D. A., and Pulwarty, R. S.: Characteristics of the western United \nStates snowpack from snowpack telemetry (SNOTEL) data, Water Resources Research, 35, 2145–2160, \nhttps://doi.org/10.1029/1999WR900090, 1999. 670 Serreze, M. C., Clark, M. P., Armstrong, R. L., McGinnis, D. A., and Pulwarty, R. S.: Characteristics of the western United \nStates snowpack from snowpack telemetry (SNOTEL) data, Water Resources Research, 35, 2145–2160, \nhttps://doi.org/10.1029/1999WR900090, 1999. 670 670 28 https://doi.org/10.5194/egusphere-2022-437\nPreprint. Discussion started: 27 June 2022\nc⃝Author(s) 2022. CC BY 4.0 License. Siirila-Woodburn, E. R., Rhoades, A. M., Hatchett, B. J., Huning, L. S., Szinai, J., Tague, C., Nico, P. S., Feldman, D. R., Siirila-Woodburn, E. R., Rhoades, A. M., Hatchett, B. J., Huning, L. S., Szinai, J., Tague, C., Nico, P. S., Feldman, D. R., \nJones, A. D., Collins, W. D., et al.: A low-to-no snow future and its impacts on water resources in the western United \nStates, Nature Reviews Earth & Environment, 2, 800–819, https://doi.org/10.1038/s43017-021-00219-y, 2021. Rhoades, A. M., Hatchett, B. J., Huning, L. S., Szinai, J., Tague, C., Nico, P. S., Feldman, D. R Siirila-Woodburn, E. R., Rhoades, A. M., Hatchett, B. J., Huning, L. S., Szinai, J., Tague, C., Nico, P. S., Feldman, D. R., \nJones, A. D., Collins, W. D., et al.: A low-to-no snow future and its impacts on water resources in the western United \nStates, Nature Reviews Earth & Environment, 2, 800–819, https://doi.org/10.1038/s43017-021-00219-y, 2021. Sturm, M., Goldstein, M. A., and Parr, C.: Water and life from snow: A trillion dollar science question, Water Resources \nh\n3 3 34 3 44 h\n//d i\n/0 1002/201\n020840 201 Jones, A. D., Collins, W. D., et al.: A low-to-no snow future and its impacts on water resources in the western United \nStates, Nature Reviews Earth & Environment, 2, 800–819, https://doi.org/10.1038/s43017-021-00219-y, 2021. Sturm, M., Goldstein, M. A., and Parr, C.: Water and life from snow: A trillion dollar science question, Water Resources \nResearch, 53, 3534–3544, https://doi.org/0.1002/2017WR020840, 2017. 675 Ullrich, P., Xu, Z., Rhoades, A., Dettinger, M., Mount, J., Jones, A., and Vahmani, P.: California’s drought of the future: A \nmidcentury \nrecreation \nof \nthe \nexceptional \nconditions \nof \n2012–2017, \nEarth’s \nfuture, \n6, \n1568–1587, \nhttps://doi.org/10.1029/2018EF001007, 2018 Williams, A. P., Cook, B. I., and Smerdon, J. E.: Rapid intensification of the emerging southwestern North American \nmegadrought in 2020–2021, Nature Climate Change, 12, 232–234, https://doi.org/10.1038/s41558-022-01290-z, 2022. 680 Xu, Y., Jones, A. and Rhoades, A.: A quantitative method to decompose SWE differences between regional climate models \nand reanalysis datasets. Sci Rep 9, 16520. https://doi.org/10.1038/s41598-019-52880-5, 2019. Xu, Z., Rhoades, A. M., Johansen, H., Ullrich, P. A., and Collins, W. D.: An intercomparison of GCM and RCM dynamical \ndownscaling for characterizing the hydroclimatology of California and Nevada, Journal of Hydrometeorology, 19, \n1485–1506, https://doi.org/10.1175/JHM-D-17-0181.1, 2018. 685 Xu, Z., Rhoades, A. M., Johansen, H., Ullrich, P. A., and Collins, W. D.: An intercomparison of GCM and RCM dynamical \ndownscaling for characterizing the hydroclimatology of California and Nevada, Journal of Hydrometeorology, 19, 1485–1506, https://doi.org/10.1175/JHM-D-17-0181.1, 2018. 685 1485–1506, https://doi.org/10.1175/JHM-D-17-0181.1, 2018. 685 685 29 29"
https://openalex.org/W2346730333
https://www.nature.com/articles/srep25557.pdf
English
null
Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device
Scientific reports
2,016
cc-by
5,358
Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device received: 08 January 2016 accepted: 18 April 2016 Published: 06 May 2016 Kin Fong Lei1,2,3, Hsueh-Peng Tseng1, Chia-Yi Lee4 & Ngan-Ming Tsang3,5 Cell invasion is the first step of cancer metastasis that is the primary cause of death for cancer patients and defined as cell movement through extracellular matrix (ECM). Investigation of the correlation between cell invasive and extracellular stimulation is critical for the inhabitation of metastatic dissemination. Conventional cell invasion assay is based on Boyden chamber assay, which has a number of limitations. In this work, a microfluidic device incorporating with impedance measurement technique was developed for quantitative investigation of cell invasion process. The device consisted of 2 reservoirs connecting with a microchannel filled with hydrogel. Malignant cells invaded along the microchannel and impedance measurement was concurrently conducted by measuring across electrodes located at the bottom of the microchannel. Therefore, cell invasion process could be monitored in real-time and non-invasive manner. Also, cell invasion rate was then calculated to study the correlation between cell invasion and extracellular stimulation, i.e., IL-6 cytokine. Results showed that cell invasion rate was directly proportional to the IL-6 concentration. The microfluidic device provides a reliable and convenient platform for cell-based assays to facilitate more quantitative assessments in cancer research. Cancer metastasis is a cancer that has spread from one part of the body (primary site) to another not directly con- nected with it. It represents the major problem in the cancer treatment and has dramatic effects on the survival of patients. To achieve metastasis, malignant tumor cells should penetrate tissue barriers, such as the basement membrane, migrate through blood or lymph vessels, and rise of distant colonies1. Cell invasion is the first step of metastasis and defined as cell movement through extracellular matrix (ECM) which requires adhesion, pro- teolysis of ECM, and migration2. Therefore, investigation of the basic principles and molecular pathways of cell invasion is critical to inhibit metastatic dissemination. For example, increase of interleukin-6 (IL-6) cytokine leads to increase the rate of malignancy and promote cancer metastasis3–6. When IL-6 engages the receptor of cells, a number of cellular phosphorylation and signaling pathways are triggered. It results a wide range of cellu- lar processes including cell proliferation, oncogenesis, and cancer metastasis7. Understanding of the correlation between cell invasion and extracellular stimulation, e.g., cytokine, is essential to study cell metastasis and thus develop effective therapeutic strategies for controlling invasive malignant tumor cells. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device pf p g g g Currently, most of cell invasion assays are based on Boyden chamber assay in biological laboratory. A tran- swell coating with a layer of ECM on membrane is used and cells move through the ECM to study the cell inva- sion process. Hence, invaded cells can be stained and quantified on the membrane under microscope. The Boyden chamber assay is widely used but also has inherent limitations. The pore size of the membrane highly influences the number of invaded cells. Also, since cells move from the upper transwell to the lower culture chamber, cell invasion may be induced by gravity. Moreover, this assay is an end-point assay and the quantification of invaded cells is subjective. These are the major concerns of the Boyden chamber assay and development of alternative methods for cell invasion assay becomes necessary. 1Graduate Institute of Medical Mechatronics, Chang Gung University, Taoyuan, Taiwan. 2Department of Mechanical Engineering, Chang Gung University, Taoyuan, Taiwan. 3Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou, Taiwan. 4Ph.D. Program in Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan. 5School of Traditional Chinese Medicine, Chang Gung University, Taoyuan, Taiwan. Correspondence and requests for materials should be addressed to K.F.L. (email: kflei@mail.cgu.edu.tw) Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 1 www.nature.com/scientificreports/ Figure 1. Microfluidic device for impedimetric monitoring of cell invasion process. (a) Photograph of the microfluidic device. (b) Schematic illustration of the principle of monitoring the cell invasion process. Figure 1. Microfluidic device for impedimetric monitoring of cell invasion process. (a) Photograph of the microfluidic device. (b) Schematic illustration of the principle of monitoring the cell invasion process. In the past decades, development of microfluidic technology becomes mature and a lot of biomedical applica- tions have been demonstrated on microfluidic systems8–10. For cell-based assays in microfluidic systems, one of the important advantages is to provide a well-controlled environment for precise study of cellular activities11–14. By designing special microchannels, cell invasion could be observed in the microfluidic systems15–18. For example, a microfluidic device was developed for monitoring cell migration across ECM-coated microgaps15. Migration of invasive MDA-MB-231 cells was tracked by real-time light microscopy. Alternatively, transendothelial invasion of tumor aggregates was successfully observed in a microfluidic system18. Adenoid cystic carcinoma cell aggregates transmigrated across the endothelium under the stimulation of chemokine CXCL12 and the invasion was inhib- ited by CXCR4 antagonist. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device This in vitro device allowed for detailed study of the attachment and transendothelial invasion of tumor aggregates. In the above excellent demonstrations, observation of cell invasion activity was based on imaging under optical microscope. Results were achieved by capturing and comparing the images at the beginning and at regular intervals during cell invasion process. However, quantitative correlation between cell invasion and extracellular stimulation is challenging to be evaluated by optical imaging. In order to achieve quantitative study of cell invasion under extracellular stimulation, in this work, a microfluidic device incorpo- rating with impedance measurement technique was developed to provide real-time, non-invasive, and objective investigation of cellular activities.l g A microfluidic device with 2 reservoirs connecting with a microchannel was developed. Malignant cells (cell line: NPC-BM1) were seeded in one reservoir and invaded to another reservoir through the microchannel filled with methyl cellulose (MC) hydrogel. The MC hydrogel was used to mimic the basement membrane in vivo. On the bottom surface of the microchannel, eight electrodes were embedded for impedance measurement. Cells moving along the microchannel could be monitored in a real-time and non-invasive manner. Hence, cell invasion rate could be calculated by the impedance change of the electrodes. Influence of cell invasion rate under stimu- lation of cytokine, i.e., IL-6, was demonstrated to quantitatively study the correlation between cell invasion and extracellular stimulation. As a whole, the current microfluidic device provides a platform to study cell invasion process without influences of pore size of the membrane and gravity that are the major concerns in Boyden cham- ber assay. Moreover, quantitative monitoring of cell invasion process was successfully achieved by impedance measurement technique. This microfluidic device is proposed to be a convenient and quantitative tool for cell invasion assay. Results and Discussionl Design of the microfluidic device for monitoring of cell invasion process. A microfluidic device incorporating with impedance measurement technique was developed to quantitatively study of cell invasion process under the stimulation of cytokine. The device was composed of a glass substrate embedded with 8 straight electrodes and a polydimethylsiloxane (PDMS) layer with 2 reservoirs connecting with a microchannel. Photograph of the microfluidic device and the principle of monitoring the cell invasion process are shown in Fig. 1. The electrodes were aligned vertically with the microchannel for the impedance measurement. The exper- imental procedures of the cell invasion assay in the microfluidic device is shown in Fig. 2. Before cell invasion experiment, the microchannel was filled with MC hydrogel to mimic the basement membrane in vivo. Cells were seeded in one reservoir and invaded to another reservoir through the microchannel. During the process, cells moved along the microchannel and the total impedance across each pair of electrodes increased to determine the presence of cells. Because the distance of electrodes was known, the impedance change of each pair of electrodes Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 2 www.nature.com/scientificreports/ Figure 2. Experimental procedures of the cell invasion assay in the microfluidic device. (a) Assembly of the microfluidic device. (b) Loading and solidification of MC hydrogel in the microchannel. (c) Loading of cells in serum-free medium in one reservoir and medium with 20% FBS in another reservoir. The microfluidic device was then transferred in an incubator. Impedance measurement was conducted by measuring each pair of electrodes using an impedance analyzer. Figure 2. Experimental procedures of the cell invasion assay in the microfluidic device. (a) Assembly of the microfluidic device. (b) Loading and solidification of MC hydrogel in the microchannel. (c) Loading of cells in serum-free medium in one reservoir and medium with 20% FBS in another reservoir. The microfluidic device was then transferred in an incubator. Impedance measurement was conducted by measuring each pair of electrodes using an impedance analyzer. Figure 3. Correlation between impedance magnitude and cell density under different measuring frequencies of 5, 10, 20, and 50 kHz. Error bars represent the standard errors of 3 repeated experiments. The solid lines represent the results of linear regressive approximation. Figure 3. Correlation between impedance magnitude and cell density under different measuring frequencies of 5, 10, 20, and 50 kHz. Error bars represent the standard errors of 3 repeated experiments. Results and Discussionl The solid lines represent the results of linear regressive approximation. represented the location and number of cells. Hence, cell invasion rate could be calculated by continuous imped- ance measurement of each pair of electrodes at different fixed distances. Optimization of the impedance measurement conditions. Because cell membrane is modeled as an equivalent electrical circuit of capacitance and resistance in series, the impedance magnitude of cells attaching on the electrode surface is varied by the measuring frequency19. When the frequency is sufficient low or high, sensitivity of the measurement of cellular behaviors becomes extremely low. Also, the frequency may be varied by different culture environments and measuring electrodes. Therefore, optimization of the measuring frequency was investigated in order to figure out the highest sensitivity and linearity between impedance magnitude and cell density in the microfluidic device. Cells at different densities of 103, 104, 105, 106, and 107 cells/mL were respec- tively introduced to the microchannel blocked by 2 obstructed plates at its ends. After 8 hr for cell attachment, MC hydrogel was applied to the microchannel. Hence, impedance magnitude across the electrodes located at the bottom of the microchannel was measured and analyzed. Correlation between impedance magnitude and cell density under different measuring frequencies of 5, 10, 20, and 50 kHz is shown in Fig. 3. Generally, cell density was found to be proportional to the impedance magnitude at all measuring frequencies. The sensitivity values, i.e., slope of the correlation, were respectively 111.7, 91.4, 95.0, and 60.8, and the linearity values, i.e., R-squared values, were respectively 0.7281, 0.7233, 0.8850, and 0.8463. Measurement at 20 kHz was found to have the high- est linear correlation with reasonable sensitivity. Hence, the measuring frequency of 20 kHz was identified and used in the following experiments in this study. Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 3 www.nature.com/scientificreports/ Figure 4. Successive microscopic images of cell invasion process. The red lines represents the leading edges of cells at the corresponding time points. Figure 4. Successive microscopic images of cell invasion process. The red lines represents the leading edges of cells at the corresponding time points. Figure 5. Impedance measurements across the electrodes at the distances of 200, 400, 600, and 800 μm at different successive time points. Error bars represent the standard errors of 3 repeated experiments. Materials and Methods Cell culture. An epithelial cell line (NPC-BM1) derived from a bone marrow biopsy of a female Taiwanese patient with nasopharyngeal carcinoma (NPC) was kindly provided by Dr. Jenny Liu, Chang Gung University. Culture medium was Dulbecco’s modified eagle medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, USA) and antibiotic/antimycotic (100 U/mL of penicillin G sodium, 100 mg/mL of streptomycin, and 0.25 mg/mL of amphotericin B; Gibco-BRL Life Technologies, USA). Cells were amplified by standard cell culture technique and trypsinized using 0.05% trypsin (Gibco-BRL Life Technologies, USA) for 3 min, centrifuged at 1200 rpm for 5 min, and re-suspended in the medium for further experiments. Fabrication of the microfluidic device. The microfluidic device consisted of a glass substrate and a PDMS (Model: Sylgard® 184; Dow Corning, USA) layer. Eight straight Cr/Au (50/500 Å) electrodes were fabricated on the glass substrate by standard microfabrication processes including thermal evaporation and lift-off. The elec- trodes were 100 μ m in width and the gap between electrodes was 100 μ m. The PDMS layer was fabricated by soft lithography and the fabrication process is briefly described. A poly(methyl methacrylate) (PMMA) mold with negative pattern of reservoirs and microchannel was machined by CNC engraving machine (Model: EGX-400; Roland, USA). Then, PDMS pre-polymer and curing agent in (w/w) 10:1 were mixed and poured to the PMMA mold. The PDMS was solidified at 70 °C for 1hr and then peeled off from the mold. Finally, the microfluidic device was completed by bonding the glass substrate and the PDMS layer using oxygen plasma (Model: PDC-32 G; Harrick Plasma, USA). Before conducting experiment, the microfluidic device was sterilized using 70% (w/v) ethanol for 6 h, washed by phosphate-buffered saline (PBS; 50 mM phosphate, 150 mM NaCl, and 10 mM EDTA; pH 7.6), and kept under ultraviolet light overnight. Cell invasion assay in the microfluidic device. The microfluidic device provides a platform for the inves- tigation of cell invasion without the use of membrane and the influence of gravity. In order to form a barrier to mimic basement membrane in vivo, MC hydrogel was used in this study. It was prepared by dispersing MC powder (Methocel®  MC; Sigma, USA) in PBS at a concentration of 3% (w/v) at room temperature and was sterilized by using autoclave at 121 °C under 100 kPa for 20 min for further use. Results and Discussionl The dash line represents the impedance change of 32 Ω, i.e., a threshold of certain cells reaching the electrode surface. Figure 5. Impedance measurements across the electrodes at the distances of 200, 400, 600, and 800 μm at different successive time points. Error bars represent the standard errors of 3 repeated experiments. The dash line represents the impedance change of 32 Ω, i.e., a threshold of certain cells reaching the electrode surface. Real-time and non-invasive impedimetric monitoring of cell invasion process. In order to con- firm the applicability of the microfluidic device for cell invasion assay, experiment of cell invasion was performed and results were observed under microscope. Noted that the microchannel was not embedded with the electrodes in order to have clear microscopic images. During the process, cells invaded from one reservoir to another res- ervoir through the connecting microchannel filled with MC hydrogel. Microscopic images of the microchannel were captured every 6 hr and is shown in Fig. 4. The leading edges of cells at different successive time points were manually identified from the images. Based on linear regressive approximation, cell invasion rate was estimated to be 12.6 μ m/h. Results showed that the microfluidic device is feasible to perform cell invasion assay with the advantages of without influences of pore size of the membrane and gravity that are the major concerns in Boyden chamber assay. The quantification based on microscopic observation was direct and convenient; but it was sub- jective. Therefore, to provide an objective approach, cell invasion process was then monitored by impedance measurement across 8 straight electrodes embedded on the bottom of the microchannel. Results of impedance measurement at different successive time points are shown in Fig. 5. Microscopic images correlating to the exper- imental data shown in Fig. 5 were included in Fig. S1 in the supplementary materials. Because cells reached the distance of 800 μ m in the experiment within 48 hr, only impedance changes of the electrodes located at the Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 4 www.nature.com/scientificreports/ distances of 200, 400, 600, and 800 μ m were shown in the figure. Increase of impedance change represented cell attachment on the electrode surface. Results showed that cells were progressively invaded from the reservoir until the distance of 800 μ m. Cell invasion process was quantitatively monitored in the microfluidic device. Conclusionl A microfluidic device incorporating with impedance measurement technique was developed for the investiga- tion of cell invasion process. Cells invaded along the microchannel filled with hydrogel and the process could be monitored in real-time and non-invasive manner. Moreover, quantitative study of the correlation between cell invasion rate and extracellular stimulation was achieved. Results showed that cell invasion rate was directly pro- portional to the IL-6 concentration. In conclusion, reliable assessment of cellular activity is very important for the development of effective therapeutic strategies. Design of the microfluidic device was to eliminate the influences of membrane and gravity that are the major concerns in Boyden chamber assay. Also, the microfluidic device provides a promising tool to quantitatively study cell invasion process under extracellular stimulation. This tech- nology has potential to be developed to a reliable and convenient analytical platform for facilitating quantitative assessments in cancer research. Results and Discussionl In order to calculate the cell invasion rate, we defined the impedance change of 32 Ω as a threshold of certain cells reaching the electrode surface. Because environmental noise may influence the impedance measurement, we found that impedance change over 32 Ω significantly indicated cell attachment on the electrode surface (data not shown). Based on linear regressive approximation, cell invasion rate was calculated to be 10.0 μ m/h. g pp In this work, results shown in Fig. 4 (microscopic observation) and Fig. 5 (impedance measurement) were 2 independent experiments. In cell biological study, experimental data are only compared in the same experiment under same condition. The most differences of different experiments are cell conditions, e.g., generation, cell cycle, and source, which cannot be controlled. Therefore, cell invasion rates calculated from 2 approaches could not be directly compared but they still reasonably matched. Cell invasion under the stimulation of cytokine. Understanding of the correlation between cell inva- sion rate and extracellular stimulation, e.g., cytokine, is essential to study cell metastasis for the development of effective therapeutic strategies. Cytokine is one of the interesting topics of influencing cancer pathogenesis20–22. It was reported that cells infected by Epstein-Barr virus lead to the oncogenic transformation23,24 and induce the production of cytokines such as IL-6 and IL-1025,26. The production of IL-6 from virus-transformed cells affects the neighboring cells to increase the rate of malignancy and promote cancer metastasis3–6. Such that, cell invasion rate could be promoted by the stimulation of cytokine. To quantitatively study this correlation, cells were respec- tively stimulated by IL-6 at different concentrations and the corresponding cell invasion rate was then calculated. Results of the impedance measurements are shown in Fig. 6. Analysis of percentage of invaded cells was included in Fig. S2 in the supplementary materials. Cell invasion rates under IL-6 concentrations of 2, 5, and 10 ng/mL were calculated to be 10.3, 12.2, and 20.3 μ m/h, respectively. Generally, cell invasion rate was directly proportional to the IL-6 concentration. It was reported that higher activation level of transcription factors is triggered by higher concentration of IL-627. Results confirmed that the microfluidic device could quantitatively monitor the entire cell invasion process. Materials and Methods The experimental procedures of the cell invasion assay in the microfluidic device is shown in Fig. 2. Two obstructed polycarbonate plates were inserted at 2 ends of the microchannel. Then, the MC hydrogel was loaded in the microchannel and a gel-like barrier was formed after solidification. Next, cells at cell density of 3 ×  105 cells/mL in serum-free medium were Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 5 ntificreports/ Figure 6. Impedimetric monitoring of cell invasion process under the stimulation of IL-6 cytokine at the concentrations of (a) 2, (b) 5, and (c) 10 ng/mL. Error bars represent the standard errors of 3 repeated experiments. The dash line represents the impedance change of 32 Ω, i.e., a threshold of certain cells reaching the electrode surface. www.nature.com/scientificreports/ applied to one reservoir. Culture medium with 20% FBS was a sion. The microfluidic device was then transferred in a 37 °C Figure 6. Impedimetric monitoring of cell invasion process u the concentrations of (a) 2, (b) 5, and (c) 10 ng/mL. Error bars experiments. The dash line represents the impedance change o the electrode surface. Figure 6. Impedimetric monitoring of cell invasion process un the concentrations of (a) 2, (b) 5, and (c) 10 ng/mL. Error bars experiments. The dash line represents the impedance change o the electrode surface. Figure 6. Impedimetric monitoring of cell invasion process under the stimulation of IL-6 cytokine at the concentrations of (a) 2, (b) 5, and (c) 10 ng/mL. Error bars represent the standard errors of 3 repeated experiments. The dash line represents the impedance change of 32 Ω, i.e., a threshold of certain cells reaching the electrode surface. applied to one reservoir. Culture medium with 20% FBS was applied to another reservoir for inducing cell inva- sion. The microfluidic device was then transferred in a 37 °C and 5% CO2 humidified incubator (Model: 370; Thermoscientific, USA). After 6 hr for cell seeding and spreading, the obstructed plates could be removed and the experiment was defined to be started (0 hr). Investigation of cell invasion without extracellular stimulation was first carried out up to 2 days. Result was a control to compare to the results with the stimulation of cytokine, i.e., IL-6 (Invitrogen, USA) in different concentrations. During these cell invasion experiments, impedance meas- urement across each pair of electrodes was concurrently conducted by using an impedance analyzer (Model: VersaSTAT 4; Princeton Applied Research, USA). Materials and Methods Hence, real-time and non-invasive monitoring of cell invasion activity was demonstrated in the microfluidic device. Impedance measurement of cell invasion in the microfluidic device. A pioneer work of using impedance measurement technique for the cellular monitoring was reported in 198428. Later, it has been widely demonstrated on the quantification of various cellular activities, i.e., proliferation, viability, physiological state of cells29–34. These demonstrations were based on using a pair of coplanar electrodes on a solid surface for the electrical transducer. Because cell membrane is lipid bilayer structure and has capacitance of 0.5–1.3 μ F/cm2 and resistance of 102–105 Ω/cm2 35, cells spreading on the electrode surface can induce the impedance change across Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 6 www.nature.com/scientificreports/ the electrodes. When cells attach onto the electrode surface, the total impedance across the electrodes can be modeled as capacitances and resistances in series, which reflects the biophysical properties of the insulating cell membrane. When the frequency is sufficient low or high, sensitivity of the cell impedance measurement becomes extremely low. It was suggested that the most appropriate frequency for cell impedance measurement is 20 kHz at which the sensor will be at the highest sensitivity19. Although the optimized frequency may vary from differ- ent device parameters, cell impedance measurement at medium frequencies (5–100 kHz) is a universal criteria. Moreover, it was reported that there was no detectable electrical influence on cells and the measurement can be used for long-term continuous monitoring36. Hence, the impedance measurement is generally believed that it can provide a convenient and reliable measurement for monitoring of cellular activities. p g In this study, during the 2 day cell invasion experiment, impedance measurement was performed every 6 hr. Potential of 0.1 Vrms was applied across the electrodes and the impedance was measured from 1 to 100 kHz. Eight electrodes were respectively located at the distances of 200, 400, 600, 800, 1000, 1200, 1400, and 1600 μ m. Seven measurements were respectively conducted by 7 pairs of electrodes located at 200 and 400 μ m, 400 and 600 μ m, 600 and 800 μ m, 600 and 1000 μ m, 1000 and 1200 μ m, 1200 and 1400 μ m, and 1400 and 1600 μ m. Therefore, seven impedance values were collected for each measurement at successive time point. Materials and Methods In order to eliminate the variations between electrodes, background impedance value of each pair of electrodes was measured before load- ing of cells. Impedance change was defined as the subtraction of the impedance value at the successive time point and the background impedance value. References Particle separation and sorting in microfluidic devices: a review. Microfluid. Nanofluid. 17, 1–52 (2014).l 14. Sajeesh, P. & Sen, A. K. Particle separation and sorting in micro lll 15. Chaw, K. C., Manimaran, M. & Tay, F. E. H. Matrigel coated polydimethylsiloxane based microfluidic devices for studying meta and non-metastatic cancer cell invasion and migration. Biomed. Microdevices 9, 597–602 (2007).l 6. Sung, K. E. et al. Transition to invasion in breast cancer: a microfluidic in vitro model enables examination of spatial and tempora effects. Integr. Biol. 3, 439–450 (2011).il f 17. Liu, T., Lin, B. & Qin, J. Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device. Lab Chip 10, 1671–1677 (2010).l p 18. Zhang, Q., Liu, T. & Quin, J. A microfluidic-based device for study of transendothelial invasion of tumor aggregates in realtime. Lab Chip 12, 2837–2842 (2012). p 9. Wang, L. et al. Analysis of the sensitivity and frequency characteristics of coplanar electrical cell-substrate impedance sensors Biosens. Bioelectron. 24, 14–21 (2008).l 20. Johnson, C. et al. Interleukin-6 and its receptor, key players in hepatobiliary inflammation and cancer. Transl. Gastrointest Cancer 1, 58–70 (2012). 21. Tang, Y. et al. Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β  and IL-6 signaling. Proc. Natl. Acad. Sci. USA 105, 2445–2450 (2008). 2. Liu, Y., Li, P. K., Li, C. & Lin, J. Inhibition of STAT3 signaling blocks the anti-apoptotic activity of IL-6 in human liver cancer cells. J Biol. Chem. 285, 27429–27439 (2010).f 3. Wang, D., Liebowitz, D., Kieff, E. & An, E. B. V. membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43, 831–840 (1985). 24. Baichwal, V. R. & Sudgen, B., Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene 2, 461–467 (1988). 5. Eliopoulos, A. G. et al. Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors. Oncogene 14, 2899–2916 (1997).h 26. Nakagomi, H. et al. The Epstein-barr virus latent membrane protein-1 (LMP2) induces interleukin-10 production in burkitt lymphoma lines. Int. J. Cancer 57, 240–244 (1994). 27. Lei, K. F. & Huang, C. H. Paper-based microreactor integrating cell culture and subsequent immunoassay for the investigation of cellular phosphorylation. ACS Appl. Mater. Interfaces 6, 22423–22429 (2014).ii 28. Giaever, I. & Keese, C. R. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc. Natl. Acad. References 1. Gupta, G. P. & Massague, J. Cancer metastasis: building a framework. Cell 127, 679–695 (2006). 2. Friedl, P. & Wolf, K. Plasticity of cell migration: a multiscale tuning model. J. Cell Biol. 188, 11–19 (2010). h l l k d k l h b l fl d 3. Johnson, C. et al. Interleukin-6 and its receptor, key players in hepatobiliary inflammation and cancer. Transl. Gastrointest. Cance 1, 58–70 (2012). 4. Snyder, M., Huang, X. Y. & Zhang, J. J. Signal transducers and activators of transcription 3 (STAT3) directly regulates cytokine- induced fascin expression and is required for breast cancer cell migration. J. Biol. Chem. 286, 38886–38893 (2011).h 5. Graness, A., Chwieralski, C. E., Reinhold, D., Thim, L. & Hofmann, W. Protein kinase C and ERK activation are required for TFF peptide-stimulated bronchial epithelial cell migration and tumor necrosis factor-α -induced interleukin-6 (IL-6) and IL-8 secretion J. Biol. Chem. 277, 18440–18446 (2002).il 6. McLoughlin, R. M. et al. IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation. Proc. Natl. Acad. Sci. USA 102, 9589–9594 (2005). 7. Ogata, A. et al. IL-6 triggers cell growth via the ras-dependent mitogen-activated protein kinase cascade. J. Immunology 159 2212–2221 (1997).hl 8. Sackmann, E. K., Fulton, A. L. & Beebe, D. J. The present and future role of microfluidics in biomedical research. Nature 181–189 (2014). 9. Gong, C. S. A. & Lei, K. F. Advances in miniaturized instruments for genomics. Biomed. Res. Int. 2014, 734675 (2014).l F. Microfluidic systems for diagnostic applications: a review. JALA l l y g pp 11. Mehling, M. & Tay, S. Microfluidic cell culture. Curr. Opin. Biotech. 25, 95–102 (2014). g yl p 12. Halldorsson, S., Lucumi, E., Gómez-Sjöberg, R. & Fleming, R. M. T. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 63, 218–231 (2015).l p y y , ( ) 13. Shields, C. W. IV, Reyes, C. D. & López, G. P. Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. Lab Chip 15, 1230–1249 (2015).lll 13. Shields, C. W. IV, Reyes, C. D. & López, G. P. Microfluidic cell sorting: a review of the advances in t debulking to rare cell isolation. Lab Chip 15, 1230–1249 (2015).ll l debulking to rare cell isolation. Lab Chip 15, 1230–1249 (2015) k g p , ( ) 4. Sajeesh, P. & Sen, A. K. References Sci. US 3761–3764 (1984). 29. Lei, K. F. Review on impedance detection of cellular responses in micro/nano environment. Micromachines 5, 1–12 (2014). 0. Hong, J., Kandasamy, K., Marimuthu, M., Choi, C. S. & Kim, S. Electrical cell-substrate impedance sensing as a non-invasive tool fo cancer cell study. Analyst 136, 237–245 (2011). y y , ( ) 31. Ehret, R. et al. Monitoring of cellular behavior by impedance measurements on interdigitated electrode structures. Biosens. Bioelectron. 12, 29–41 (1997). Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 7 www.nature.com/scientificreports/ 2. Thakur, M. et al. Real time monitoring of the cell viability during treatment with tumor-targeted toxins and saponins using impedance measurement. Biosens. Bioelectron. 35, 503–506 (2012). 3. Lei, K. F. & Leung, P. H. M. Microelectrode array biosensor for the detection of Legionella pneumophila. Microelectron. Eng. 91 174–177 (2012). ( ) 4. Radke, S. M. & Alocilja, E. C. A high density microelectrode array biosensor for detection of E. coli O157:H7. Biosens. Bioelectron 20, 1662–1667 (2005). Pethig, R. R. Dielectric and Electronic Properties of Biological Materi 36. Fuhr, G., Glasser, H., Muller, T. & Schnelle, T. Cell manipulation and cultivation under a.c. electric field influence in highly conductive culture media. Biochim. Biophys. Acta 1201, 353–360 (1994). Acknowledgementsh g This work was supported by the Ministry of Science and Technology, Taiwan (project no. MOST103-2221-E-182- 04-MY3) and Chang Gung Memorial Hospital, Linkou, Taiwan (Project no. BMRPC05). Author Contributions K.F. developed the concept of this work, supervised of the project, and wrote the manuscript. H.P. performed the experiment. H.P. and C.Y. analyzed data. N.M. performed statistical analysis. All authors have read and approved the final manuscript. Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Lei, K. F. et al. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device. Sci. Rep. 6, 25557; doi: 10.1038/srep25557 (2016). How to cite this article: Lei, K. F. et al. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device. Sci. Rep. 6, 25557; doi: 10.1038/srep25557 (2016). 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/ 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/ Scientific Reports | 6:25557 | DOI: 10.1038/srep25557 8
https://openalex.org/W4213168015
https://air.unimi.it/bitstream/2434/917002/2/marinedrugs-20-00135.pdf
English
null
Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae
Marine drugs
2,022
cc-by
5,826
    Citation: Carnovali, M.; Ciavatta, M.L.; Mollo, E.; Roussis, V.; Banfi, G.; Carbone, M.; Mariotti, M. Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae. Mar. Drugs 2022, 20, 135. https:// doi.org/10.3390/md20020135 Academic Editor: M. Leonor Cancela Received: 20 January 2022 Accepted: 8 February 2022 Published: 11 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 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/ Citation: Carnovali, M.; Ciavatta, M.L.; Mollo, E.; Roussis, V.; Banfi, G.; Carbone, M.; Mariotti, M. Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae. Mar. Drugs 2022, 20, 135. https:// doi.org/10.3390/md20020135 Keywords: marine natural products; bromotyrosine; osteogenesis; zebrafish; drug discovery Marta Carnovali 1 , Maria Letizia Ciavatta 2 , Ernesto Mollo 2 , Vassilios Roussis 3 , Giuseppe Banfi1,4, Marianna Carbone 2,* and Massimo Mariotti 1,5,* Marta Carnovali 1 , Maria Letizia Ciavatta 2 , Ernesto Mollo 2 , Vassilios Roussis 3 , Giuseppe Banfi1,4, Marianna Carbone 2,* and Massimo Mariotti 1,5,* 1 IRCCS Istituto Ortopedico Galeazzi, Via R. Galeazzi 4, 20161 Milano, Italy; marta.carnovali@grupposandonato.it (M.C.); banfi.giuseppe@unisr.it (G.B.) 2 Consiglio Nazionale delle Ricerche—Istituto di Chimica Biomolecolare (CNR-ICB), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy; lciavatta@icb.cnr.it (M.L.C.); emollo@icb.cnr.it (E.M.) 3 Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; roussis@pharm.uoa.gr 4 School of Medicine, Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milano, Italy 5 Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Università Degli Studi di Milano, Via della Commenda 10, 20122 Milano, Italy * Correspondence: mcarbone@icb.cnr.it (M.C.); massimo.mariotti@unimi.it (M.M.); Tel.: +39-08-1867-5227 (M.C.); +39-02-9647-4369 (M.M.) y * Correspondence: mcarbone@icb.cnr.it (M.C.); massimo.mariotti@unimi.it (M.M.); Tel.: +39-08-1867-5227 (M.C.); +39-02-9647-4369 (M.M.) * Correspondence: mcarbone@icb.cnr.it (M.C.); massimo.m Tel.: +39-08-1867-5227 (M.C.); +39-02-9647-4369 (M.M.) Abstract: Longer life expectancy has led to an increase in efforts directed to the discovery of new healing agents for disorders related to aging, such as bone diseases. Harboring an incredible variety of bioactive metabolites, marine organisms are standing out as fruitful sources also in this therapeutic field. On the other hand, the in vivo zebrafish model has proven to be an excellent low-cost screening platform for the fast identification of molecules able to regulate bone development. By using zebrafish larvae as a mineralization model, we have thus evaluated the effects of the crude acetonic extract from the marine sponge Aplysina aerophoba and its bromotyrosine components on bone development. Obtained results led to the selection of aerophobin-1 (1) as a promising candidate for applications in regenerative medicine, paving the way for the development of a novel therapeutic option in osteoporosis treatment. marine drugs marine drugs marine drugs marine drugs Article Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae Article Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae 1. Introduction The high structural diversity and bioactivity possessed by marine natural compounds have been proven straight off since the early studies leading to a relevant high number of marine metabolites that have been entered in the pharmaceutical industry pipeline [1–3]. Although treatment of infections and cancer represent the target of most of the studies aimed at exploring the pharmacological properties of compounds from the sea [4], the number of scientific papers dealing with marine natural products having modulatory effects on skeletal development has significantly increased in the recent years [5–10]. This tendency is also a consequence of the higher interest toward the discovery of new healing agents for disorders related to aging. In spite of this, however, the potential of marine chemical diversity to deliver pro-osteogenic agents still remains superficially explored [11]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. On the other hand, with the aim at ensuring the widest exploitation of pharmacolog- ical potential held in marine bioresources, efforts are continuously directed toward the development of novel strategies for the rapid detection of promising bioactive natural prod- ucts that can act as lead molecules for the development of new drugs [12]. In the classical phenotypic approaches for drug discovery, the zebrafish (Danio rerio) in vivo model offers a valid, fast, and low-cost screening platform for the identification of compounds mediating specific biological processes [13]. In particular, the rapid external development of the bone 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/marinedrugs Mar. Drugs 2022, 20, 135. https://doi.org/10.3390/md20020135 Mar. Drugs 2022, 20, 135 2 of 10 system along with the transparency of the body make zebrafish larvae especially suitable in the study of organogenesis and, in particular, osteogenesis [14]. In addition, the bone tissue of adult zebrafish is very similar to the human one for the presence of a mineralized matrix and functional osteoblasts and osteoclasts [15], while the process of embryonic osteogenesis is characterized by two different types of ossification: endochondral, which takes place from a cartilaginous scaffold, and intramembranous, which is driven by mesenchymal stem cell precursors [16,17]. 1. Introduction All these features, along with the high genetic similarity between humans and zebrafish, with 70% of zebrafish genes having an identifiable human ortholog [18], make zebrafish an ideal model for studies in osteogenesis. Capitalizing on this model, our ongoing marine bioprospecting programs are aimed at exploring in depth the potential of marine natural products as regulators of osteogenesis, including the screening of the chemical content of marine benthic invertebrates for their modulatory activity on the development of the zebrafish skeletal system. Herein we report the study of the osteogenic properties of both the crude acetonic extract from the Mediterranean sponge Aplysina aerophoba (Nardo, 1833) and its main bromotyrosine components with a special focus on intramembranous ossification. 2.1. Toxicity and Osteogenic Activity of the Sponge Extract To assess the toxicity of the crude acetone extract of A. aerophoba, zebrafish embryos were exposed from 1 h post fertilization (hpf) up to 5 days post fertilization (dpf) to the sponge extract at 1:10 serial dilutions, starting from 0 (control = vehicle DMSO only) to 300 mg/L. A significantly increased mortality compared to control was observed at concentrations ≥3 mg/L (Figure 1A). In order to investigate the effects of the sponge extract on bone development, the intramembranous ossification in the entire larval body was analyzed, focusing on vertebral mineralization. The ratio between the number of vertebrae (NV) and total body length (L) was used as an index of vertebral mineralization rate. A significant increment of NV/L compared to control was measured at 3 and 30 µg/L concentrations of A. aerophoba extract (Figure 1B), as visualized by alizarin red live staining (Figure 1C). Figure 1. Effect of different dilutions of A. aerophoba crude extract on (A) zebrafish embryos/larvae viability and (B) mineralization rate of the vertebral bodies. (C) Mineralized vertebrae visualized by alizarine red staining (purple) together with unmodified cartilage structures (blue) in control (0 concentration) and treated (30 µg/L sponge extract) larvae. Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01). Figure 1. Effect of different dilutions of A. aerophoba crude extract on (A) zebrafish embryos/larvae viability and (B) mineralization rate of the vertebral bodies. (C) Mineralized vertebrae visualized by alizarine red staining (purple) together with unmodified cartilage structures (blue) in control (0 concentration) and treated (30 µg/L sponge extract) larvae. Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01). Mar. Drugs 2022, 20, 135 3 of 10 3 of 10 . Modulatory Effects of Pure Compounds 1–8 on Zebrafish Embryo Osteogenesis 2.2. Modulatory Effects of Pure Compounds 1–8 on Zebrafish Embryo Osteogenesis Fractionation of the acetone extract of A. aerophoba was carried out by means of different chromatographic techniques, resulting in the isolation and purification of a series of bromotyrosine alkaloids, which were identified by spectroscopic methods according to data previously reported in the literature (Figure 2, Supplementary Materials S1–S16 and Materials and Methods section). Compounds 1–7, which were the main components of the bromotyrosine mixture, and the co-occurring yellow pigment uranidine (8), were assayed on zebrafish embryos/larvae to evaluate both their toxicity and pro-osteogenic properties. The viability of embryos/larvae treated with purified compounds 1–8 was evaluated during the initial developmental stage (from 1 hpf to 5 dpf) by exposing groups of 10 embryos to 1:10 serial dilutions from 1 pM to 100 µM of each compound. Slight toxic effects in the later development stage (5 dpf) were exerted by compounds 3–5 at almost all concentrations (Figure 3, Table 1), while aerophobin-1 (1) showed very low toxicity when assayed at concentrations over 1 µM. Null toxic effects were induced by aerophobin-2 (2) and LL-PAA216 (7) (Figure 3 and Table 1). Conversely, a significant toxicity profile was observed for uranidine (8) and fistularin-3 (6), both inducing early death. In particular, embryos died at all tested concentrations of compound 8, whereas treatment with fistularin- 3 (6) induced death at 48 hpf. The latter was found to cause evident pericardial edema (PE), particularly at the highest concentrations tested (Figure 3 and Table 1). Figure 2 Chemical structures of bromotyrosine alkaloids isolated from A aerophoba Figure 2. Chemical structures of bromotyrosine alkaloids isolated from A. aerophoba. Mar. Drugs 2022, 20, 135 4 of 10 Figure 3. Viability of embryos treated with different concentrations (from 1 pM to 100 µM) of the eight compounds (1–8) isolated from A. aerophoba. Bars indicate the number of live embryos at 5 dpf. Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01, *** p ≤0.001). Figure 3. Viability of embryos treated with different concentrations (from 1 pM to 100 µM) of the eight compounds (1–8) isolated from A. aerophoba. Bars indicate the number of live embryos at 5 dpf. Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01, *** p ≤0.001). Figure 3. 2.2. Modulatory Effects of Pure Compounds 1–8 on Zebrafish Embryo Osteogenesis Viability of embryos treated with different concentrations (from 1 pM to 100 µM) of the eight compounds (1–8) isolated from A. aerophoba. Bars indicate the number of live embryos at 5 dpf. Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01, *** p ≤0.001). Table 1. Toxic and osteogenic effects on developing embryos/larvae (PE, pericardial edema; NE, not evaluated). Sample Significant Toxic Effects Pro-Osteogenic Activity Minimum Dose Effect Concentration Timing Acetone extract ≥3 mg/L 5 dpf 3 µM 1 ≥1 µM 5 dpf 100 nM 2 none (until 100 µM) 5 dpf none Table 1. Toxic and osteogenic effects on developing embryos/larvae (PE, pericardial edema; NE, not evaluated). Mar. Drugs 2022, 20, 135 5 of 10 Table 1. Cont. Sample Significant Toxic Effects Pro-Osteogenic Activity Minimum Dose Effect Concentration Timing 3 ≥10 pM 5 dpf none 4 ≥1 pM 5 dpf none 5 ≥10 pM 5 dpf none 6 ≥10 pM 48 hpf (PE) NE 7 none (until 100 µM) 5 dpf none 8 ≥1 pM 1 hpf NE Based on preliminary toxicological evaluations, the study of the osteogenic properties was limited to compounds 1–5 and 7. The number of mineralized vertebrae (NV) was recorded in embryos treated with 1:10 dilutions from 1 pM to 100 µM of pure compounds 1–5 and 7 as well as in untreated embryos (control group = vehicle DMSO only). The recorded data were normalized with the total length of the larval body (L). A significant increase in the vertebral mineralization ratio (NV/L) was detected only in the embryos exposed to aerophobin-1 (1), with higher significance found at the minimum effective dose of 100 nM (Figure 4). On the other hand, as expected, an inhibitory effect on osteogenesis was reported for both aerophobin-1 (1) and (+)-aeroplysinin (3), in the range of their toxic concentrations (Figure 4). Figure 4. Mineralization rate in embryos exposed to compounds 1–5 and 7, evaluated as number of mineralized vertebrae at 5 dpf normalized for embryo length (NV/L). Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01, *** p ≤0.001). Figure 4. Mineralization rate in embryos exposed to compounds 1–5 and 7, evaluated as number of mineralized vertebrae at 5 dpf normalized for embryo length (NV/L). 3. Discussion Besides having contributed to elucidating the mechanisms of formation of the ver- tebrate skeleton [19], studies on marine sponges have highlighted the ability of these organisms or of their associated microbes to produce bioactive molecules able to interfere with bone metabolism [6–8]. Most of this research has been carried out in vitro or by using mice as in vivo models. Instead, in our search for pro-osteogenic agents in marine sponges, we have taken advantage of the zebrafish embryo model. In preliminary screening, the crude acetone extract of A. aerophoba induced pro-osteogenic effects on zebrafish larvae bone development. This prompted us to investigate the chemical content of the sponge, with the aim of identifying the active components responsible for the observed effects. The study resulted in the isolation of a pool of known bromotyrosine alkaloids (1–7), according to the literature for the genus Aplysina [20]. This class of metabolites constitutes a geo- graphically widespread class of compounds unique to the phylum Porifera, which has attracted the interest of both chemists and pharmacologists for their high structural diver- sity and bioactivity. Such a structural variety ranges from simple monomeric structures as the isolated compounds 3–5 to dimeric or more complex architectures, such as those displayed by suberedamine [21] and fistularin-3 (6), respectively. It has been postulated that these compounds are produced after sponge tissue injury as chemical defense and/or are released in seawater to protect Aplysina sponges from the invasion of microorganisms or animal predators [22,23]. Besides their ecological role, this class of marine alkaloids is characterized by a wide spectrum of pharmacological activities, including antitumor, anti-inflammatory, and neurological properties, as well as antimicrobial actions [24–26]. On the other hand, their possible effects on bone development have never been reported to date. In the present report, the bromotyrosine alkaloids 1–7 along with the yellow pigment uranidine (8) [27] have been firstly assayed for their toxicity on zebrafish embryos. Only aerophobins-1 (1) and -2 (2) and LL-PAA216 (7) displayed low toxicity, whereas compounds 4–6 and 8 were toxic in a wide spectrum of concentration, with uranidine (8) having the worst toxicity profile. It is worth noting, however, that in a recent study, fistularin-3 (6) and LL-PAA216 (7) were not toxic on adult zebrafish at 0.1 mg/mL (100µM), though exerting neurological activity resulting in a sedative effect [28]. 2.2. Modulatory Effects of Pure Compounds 1–8 on Zebrafish Embryo Osteogenesis Significant differences were evaluated by using the Student’s t-test (α = 0.05; * p ≤0.05, ** p ≤0.01, *** p ≤0.001). Mar. Drugs 2022, 20, 135 6 of 10 4.2. Biological Material The sponge A. aerophoba was collected by scuba diving at a depth of 12 m along the coast of Milos Island, Greece, during August 2014. Biological material was immediately frozen and then transferred to ICB in Naples, where it was kept at −2 ◦C until extraction. The sponge was identified by E. Mollo. A voucher sample of A. aerophoba (code Milos-P1) is available for inspection at ICB. 3. Discussion However, our evaluations were focused on the less toxic compounds only (1–5 and 7), among which only aerophobin-1 (1) induced a significant increase in vertebral mineralization rate in zebrafish embryos/larvae when proposed at the non-toxic and relatively low concentration of 100 nM. Interestingly, the non-monotonic dose-response profile in osteogenesis experiments of aerophobin-1(1) was similar to that observed for the crude acetone A. aerophoba extract, supporting the hy- pothesis that 1 represents the main component in the extract responsible for the osteogenic effect. On the other hand, the anti-osteogenic effects detected at higher concentrations for both pure aerophobin-1 (1) and the crude extract from the sponge could be due to the exceeding of the toxicity threshold, with consequent inhibition of several developmental mechanisms in zebrafish larvae. In conclusion, the use of zebrafish larvae as a mineralization model for the evaluation of bone development allowed for the fast identification of A. aerophoba as the source of a promising pro-osteogenic agent, aerophobin-1 (1). The study paves the way for further evaluations on the molecular mechanisms involved in the modulation of osteogenesis in embryos and on adult zebrafish specifically aimed at further supporting the use of 1 as an anti-osteoporotic drug. The discovery of new and more safe drugs that target bone depletion human diseases is a challenge for the scientific community but also an imperative issue for the health system. Indeed, the increasing number of patients with skeletal disorders, in part as a consequence of longer life expectancy, and to whom medical assistance has to be guaranteed [29,30], represents a severe economic burden for the whole society. 7 of 10 7 of 10 Mar. Drugs 2022, 20, 135 4.1. General Experimental Procedures Optical rotations were measured on a Jasco P-2000 digital polarimeter. ESIMS were performed on a Micromass Q-TOF MicroTM coupled with an HPLC Waters Alliance 2695. The instrument was calibrated by using a PEG mixture from 200 to 1000 MW (resolution specification 5000 FWHM, deviation < 5 ppm RMS in the presence of a known lock mass). High-resolution mass spectra (HRESIMS) were acquired on a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA). NMR experiments were recorded at the ICB-NMR Service Centre. Chemical shifts values are reported in ppm and referenced to internal signals of residual protons (CD3OD, 1H δ 3.34, 13C 49.0 ppm). 1D and 2D NMR spectra were acquired on a Bruker Avance-400 spectrometer using an inverse probe fitted with a gradient along the Z-axis, on a Bruker Avance III HD 400 MHz spectrometer equipped with a CryoProbe Prodigy, and on a DRX 600 spectrometer (600 MHz for 1H, 150 MHz for 13C) equipped with a three-channel inverse (TCI) CryoProbe. Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) chromatography was performed on an open glass column (150 cm length, 3.0 cm diameter). Silicagel chromatography was performed using pre-coated Merck F254 plates (TLC) and Merck Kieselgel 60 powder (70–230 mesh). The spots on TLC were visualized under UV light (254 nm) and then sprayed with 10% H2SO4 in water, followed by heating. 4.3. Extraction and Isolation Procedures A frozen sample of A. aerophoba (8 g, dry weight) was chopped into small pieces and immediately immersed in acetone (4 × 200 mL). Extractions were aided by using an ultrasound bath. The organic solvent from these extractions was combined, filtered, and evaporated under reduced pressure to provide 1.25 g of crude extract. The extract (1.2 g) was loaded into a Sephadex LH-20 column equilibrated with MeOH, collecting 20 fractions with 100% MeOH and the last 8 using a mixture of MeOH/H2O (1:1). The resulting 28 fractions were combined on the basis of their chromatographic homogeneity to afford twelve main fractions, from A to N, which were analyzed by 1H NMR. Aerophobin- 1 (1) and aerophobin-2 (2) were present in a mixture with other components in both fractions C (60 mg) and D (102 mg). The latter was further subjected to a silicagel column chromatography by using an increasing polarity gradient of MeOH in CHCl3, giving three subfractions (D5-D7) containing aerophobin-1 (1) and one (D9) containing aerophobin-2 (2). A portion (5.0 mg) of the main aerophobin-1 (1) containing fraction D6 (20.0 mg) was then purified on a semipreparative TLC plate using as mobile phase CHCl3/MeOH 8:2. The UV band at Rf 0.3 scratched from the plate contained pure aerophobin-1 (1, 4.0 mg). Similarly, fraction D9 (21 mg) was as well purified on a semipreparative TLC plate using a mobile phase a mixture of CHCl3/MeOH 7:3 and UV absorbing spot at Rf 0.3, affording pure aerophobin-2 (2, 3.5 mg). 1H NMR analysis of fraction E (78.2 mg) revealed the presence of (+)-aeroplysinin-1 (3), which was obtained as a pure compound (9.0 mg) by semipreparative TLC chromatography (Rf 0.75, CHCl3/MeOH 8:2). Fraction F (55.0 mg) was found to contain compound 4 by 1H NMR analysis. The subsequent purification of this fraction by semipreparative TLC (Rf 0.65 in CHCl3/MeOH 8:2) yielded 8.5 mg of pure 4. Fraction G (500.0 mg) resulted in being a complex mixture by NMR. A portion (100 mg) of this fraction was loaded onto a semipreparative SiO2 TLC plate in CHCl3/MeOH 9:1 to obtain pure hydroquinone derivative 5 (5.0 mg), fistularin-3 (6, 25.0 mg), and LLPAA216 (7, Mar. Drugs 2022, 20, 135 8 of 10 8 of 10 8.0 mg). Finally, fraction H (58 mg) was subjected to TLC chromatography on a plate to yield uranidine (8, 20.1 mg). 4.6. Zebrafish Treatment Embryos were maintained at 28 ◦C in a dark incubator in standard growing medium (E3 medium, 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) from 1 hpf up to 5 dpf. Crude extract as well as pure compounds were dissolved first in DMSO (99.9% Sigma) and then diluted in E3 medium to obtain serial concentrations as follows. Both the crude extract and pure compounds were diluted 10 times starting from 0 to 300 mg/L and from 0 to 100 µM, respectively. Each concentration was assayed on 10 embryos in triplicate. Control (CTR) embryos were exposed to an amount of DMSO equivalent to the maximum concentration used to test the compounds. We preliminarily tested also E3 medium alone that does not show any alteration both in general embryo development and mineralization (data not shown). During the experiments, embryos were observed daily under a light/fluorescence stereomicroscope (SZX-ZB7 Olympus) to follow their general health conditions and vitality and to set up times and concentrations of crude extract and pure compounds that provide teratogenic and/or toxic effects on them. 4.7. Histochemical Analysis Embryos were euthanized using a 300 mg/L tricaine methanesulfonate (Merk Life Science S.r.l., Milano, Italy) solution [33] and fixed in 3.5% formaldehyde/0.1 M sodium phosphate buffer. Soon after, double acid-free staining with Alcian Blue 8GX (Sigma) and Alizarin red S (ARS, Sigma) [34] was applied to stain cartilage and bone tissue, respectively. The quantification of osteogenesis level was measured as vertebral mineralization rate (N.V./L.), calculated as the number of mineralized vertebral bodies (N.V., positive for alizarin red S staining) normalized for the length of each embryo (L.). All embryos were examined under a light/fluorescence stereomicroscope (SZX-ZB7 Olympus), acquiring images with a Discovery CH30 camera (Tiesselab), which were ana- lyzed with ISC Capture software to take measurements. 4.5. Animals Danio rerio of AB strain was housed in ZEBTEC© Bench Top System (Tecniplast) and maintained at 28 ◦C under standard conditions [31]. Embryos have been obtained by single pair of adult fish and, before experimentation, have been checked for general health conditions under a light stereomicroscope as described [32]. 4.4. Ethics Statement In vivo experiments have been performed in the Zebrafish Laboratory (IRCCS R. Galeazzi, GSD Foundation, Milan, Italy) according to Italian and European guidelines on research (EU Directive 2010/63/EU). Tests on zebrafish and all protocols used in this study were authorized by the Ministry of Health (Italy) with n. 805/2021-PR. References 1. Jiménez, C. Marine Natural Products in Medicinal Chemistry. ACS Med. Chem. Lett. 2018, 9, 959–961. [CrossRef] [PubMed] 2. Ghareeb, M.A.; Tammam, M.A.; El-Demerdash, A.; Atanasov, A.G. Insights about clinically approved and Preclinically investi- gated marine natural products. Curr. Res. Biotechnol. 2020, 2, 88–102. [CrossRef] 1. Jiménez, C. Marine Natural Products in Medicinal Chemistry. ACS Med. Chem. Lett. 2018, 9, 959–96 J , y , , [ ] [ ] 2. Ghareeb, M.A.; Tammam, M.A.; El-Demerdash, A.; Atanasov, A.G. Insights about clinically approved and Preclinically investi- gated marine natural products. Curr. Res. Biotechnol. 2020, 2, 88–102. [CrossRef] g p , , [ ] 3. Sigwart, J.D.; Blasiak, R.; Jaspars, M.; Jouffray, J.-B.; Tasdemir, D. Unlocking the potential of marine biodiscovery. Nat. Prod. Rep. 2021, 38, 1235–1242. [CrossRef] [PubMed] B.R.; Davis, R.A.; Keyzers, R.A.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2019, 36, 122–173 4. Carroll, A.R.; Copp, B.R.; Davis, R.A.; Keyzers, R.A.; Prinsep, M.R. Marine natural products. Nat. Pro [CrossRef] 4. Carroll, A.R.; Copp, B.R.; Davis, R.A.; Keyzers, R.A.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2019, 36, 122–173. [CrossRef] 5 Ch l S R I d M M Chi l k S V M i l d N i f i F t M S i , ; pp, ; , ; y , ; p, p p , , [CrossRef] 5. Chaugule, S.R.; Indap, M.M.; Chiplunkar, S.V. Marine natural products: New avenue in treatment of osteoporosis. Front. Mar. Sci. [ ] 5. Chaugule, S.R.; Indap, M.M.; Chiplunkar, S.V. Marine natural products: New avenue in treatment of osteoporosis. Front. Mar. Sci. 2017, 4, 1–15. [CrossRef] 6. El-Desoky, A.H.; Kato, H.; Angkouw, E.D.; Mangindaan, R.E.P.; De Voogd, N.J.; Tsukamoto, S. Ceylonamides A-F, Nitrogenous Spongian Diterpenes That Inhibit RANKL-Induced Osteoclastogenesis, from the Marine Sponge Spongiaceylonensis. J. Nat. Prod. 2016, 79, 1922–1928. [CrossRef] restha, S.K.; Kim, H.; Gerwick, W.H.; Soh, Y. Kalkitoxin reduces osteoclast formation and resorption and ammatory bone loss. Int. J. Mol. Sci. 2021, 22, 2303. [CrossRef] 7. Li, L.; Yang, M.; Shrestha, S.K.; Kim, H.; Gerwick, W.H.; Soh, Y. Kalkitoxin reduces osteoclast fo protects against inflammatory bone loss. Int. J. Mol. Sci. 2021, 22, 2303. [CrossRef] 8. Wang, Q.; Chen, D.; Jin, H.; Ye, Z.; Wang, C.; Chen, K.; Kuek, V.; Xu, K.; Qiu, H.; Chen, P.; et al. 4.8. Statistical Analysis Data of each experiment carried out on 10 embryos in triplicate were used to calculate the mean value expressed as mean of the means of the 3 independent experiments ± stan- dard deviation versus control. Data were plotted on SigmaStat 3.5 software (San Jose, CA, USA) and subjected to Student’s t-test with all significance values set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20020135/s1, Figure S1: 1H NMR spectrum of aerophobin- 1 (1) (400 MHz, MeOD); Figure S2: ESI-MS spectrum of aerophobin- 1 (1); Figure S3: 1H NMR spectrum of aerophobin- 2 (2) (400 MHz, MeOD); Figure S4: ESI-MS spectrum of aerophobin- 2 (2); Figure S5: 1H NMR spectrum of (+)-aeroplysinin-1 (3) (400 MHz, CDCl3); Figure S6: ESI-MS spectrum of (+)-aeroplysinin-1 (3); Figure S7: 1H NMR spectrum of compound 4 (400 MHz, MeOD); Mar. Drugs 2022, 20, 135 9 of 10 9 of 10 Figure S8: ESI-MS spectrum of compound 4; Figure S9: 1H NMR spectrum of compound 5 (400 MHz, MeOD); Figure S10: ESI-MS spectrum of compound 5; Figure S11: 1H NMR spectrum of fistularin- 3 (6) (600 MHz, MeOD); Figure S12: ESI-MS spectrum of fistularin- 3 (6); Figure S13: 1H NMR spectrum of LL-PAA216 (7) (400 MHz, MeOD); Figure S14: ESI-MS spectrum of LL-PAA216 (7); Figure S15: 1H NMR spectrum of uranidine (8) (400 MHz, MeOD); Figure S16: ESI-MS spectrum of uranidine (8). Figure S8: ESI-MS spectrum of compound 4; Figure S9: 1H NMR spectrum of compound 5 (400 MHz, MeOD); Figure S10: ESI-MS spectrum of compound 5; Figure S11: 1H NMR spectrum of fistularin- 3 (6) (600 MHz, MeOD); Figure S12: ESI-MS spectrum of fistularin- 3 (6); Figure S13: 1H NMR spectrum of LL-PAA216 (7) (400 MHz, MeOD); Figure S14: ESI-MS spectrum of LL-PAA216 (7); Figure S15: 1H NMR spectrum of uranidine (8) (400 MHz, MeOD); Figure S16: ESI-MS spectrum of uranidine (8). Author Contributions: Conceptualization, M.M., M.C. (Marianna Carbone), and M.L.C.; methodol- ogy, M.C. (Marta Carnovali); investigation, M.C. (Marta Carnovali), M.C. (Marianna Carbone), and M.L.C.; writing—original draft preparation, M.M, M.C. (Marianna Carbone), and M.L.C.; writing— review and editing, E.M., V.R., and G.B.; funding acquisition, G.B. and M.M. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.M., M.C. (Marianna Carbone), and M.L.C.; methodol- ogy, M.C. (Marta Carnovali); investigation, M.C. 4.8. Statistical Analysis (Marta Carnovali), M.C. (Marianna Carbone), and M.L.C.; writing—original draft preparation, M.M, M.C. (Marianna Carbone), and M.L.C.; writing— review and editing, E.M., V.R., and G.B.; funding acquisition, G.B. and M.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a grant SoyFish (5 × 1000-2017-Grupposandonato Foundation) and a grant IRCCS—Ricerca Corrente from the Italian Ministry of Health. Funding: This work was supported by a grant SoyFish (5 × 1000-2017-Grupposandonato Foundation) and a grant IRCCS—Ricerca Corrente from the Italian Ministry of Health. Institutional Review Board Statement: This experimentation has been performed in the Zebrafish Laboratory (IRCCS R. Galeazzi, GSD Foundation, Milan, Italy) according to the Italian and European guidelines on research practice (EU Directive 2010/63/EU). Zebrafish experimentation protocols were approved by the Italian Ministry of Health (authorization no. 805/2021-PR). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article or supplementary files. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References The Understanding of the Metazoan Skeletal System, Based on the Initial Discoveries with y 18. Howe, K.; Clark, M.D.; Torroja, C.F.; Torrance, J.; Berthelot, C.; Muffato, M.; Collins, J.E.; Humphray, S.; McLaren, K.; Matthews, L.; et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013, 496, 498–503. [CrossRef] g g 19. Müller, W.; Schröder, H.; Wang, X. The Understanding of the Metazoan Skeletal System, Based on the Initial Discoveries with Siliceous and Calcareous Sponges. Mar. Drugs 2017, 15, 172. [CrossRef] p g g 20. Lira, N.S.; Montes, R.C.; Tavares, J.F.; da Silva, M.S.; da Cunha, E.V.L.; de Athayde-Filho, P.F.; Rodrigues, L.C.; da Silva Dias, C.; Barbosa-Filho, J.M. Brominated Compounds from Marine Sponges of the Genus Aplysina and a Compilation of Their 13C NMR Spectral Data. Mar. Drugs 2011, 9, 2316–2368. [CrossRef] p g , , [ ] 21. Tsuda, M.; Sakuma, Y.; Kobayashi, J. Suberedamines A and B, New Bromotyrosine Alkaloids from a Sponge Suberea Species. J. Nat. Prod. 2001, 64, 980–982. [CrossRef] [PubMed] 22. Weiss, B.; Ebel, R.; Elbrächter, M.; Kirchner, M.; Proksch, P. Defense metabolites from the marine sponge Verongia aerophoba. Biochem. Syst. Ecol. 1996, 24, 1–12. [CrossRef] 23. Puyana, M.; Pawlik, J.; Blum, J.; Fenical, W. Metabolite variability in Caribbean sponges of the genus Aplysina. Rev. Bras. Farmacogn. 2015, 25, 592–599. [CrossRef] ann, M.T. The Marine Bromotyrosine Derivatives. In Alkaloids: Chemistry and Biology; Elsevier: Amsterdam 05; pp. 59–262. ISBN 0124695612. g 24. Peng, J.; Li, J.; Hamann, M.T. The Marine Bromotyrosine Derivatives. In Alkaloids: Chemistry and B The Netherlands, 2005; pp. 59–262. ISBN 0124695612. pp 25. Sirimangkalakitti, N.; Olatunji, O.J.; Changwichit, K.; Saesong, T.; Chamni, S.; Chanvorachote, P.; Ingkaninan, K.; Plubrukarn, A.; Suwanborirux, K. Bromotyrosine Alkaloids with Acetylcholinesterase Inhibitory Activity from the Thai Sponge Acanthodendrilla sp. Nat. Prod. Commun. 2015, 10, 1934578X1501001. [CrossRef] p 26. Göthel, Q.; Sirirak, T.; Köck, M. Bromotyrosine-derived alkaloids from the Caribbean sponge Aply Chem. 2015, 11, 2334–2342. [CrossRef] T.; Köck, M. Bromotyrosine-derived alkaloids from the Caribbean sponge Aplysina lacunosa. Beilstein J. Org –2342. [CrossRef] 27. Cimino, G.; De Rosa, S.; De Stefano, S.; Spinella, A.; Sodano, G. The zoochrome of the sponge Verongia aerophoba (“Uranidine”). Tetrahedron Lett. 1984, 25, 2925–2928. [CrossRef] 28. De FCesário, H.P.S.; Silva, F.C.O.; Ferreira, M.K.A.; de Menezes, J.E.S.A.; dos Santos, H.S.; Nogueira, C.E.S.; de LSilva, K.S.B.; Hajdu, E.; Silveira, E.R.; Pessoa, O.D.L. References Hymenialdisine: A Marine Natural Product That Acts on Both Osteoblasts and Osteoclasts and Prevents Estrogen-Dependent Bone Loss in Mice. J. Bone Miner. Res. 2020, 35, 1582–1596. [CrossRef] 9. Zhang, Y.; Li, M.; Zhang, Q.; Wang, Z.; Li, X.; Bao, J.; Zhang, H. Arthpyrone L, a New Pyridone Alkaloid from a Deep-Sea Arthrinium sp., Inhibits Proliferation of MG63 Osteosarcoma Cells by Inducing G0/G1 Arrest and Apoptosis. Chem. Biodivers. 2021, 18, e2000639. [CrossRef] 10. Jiang, W.; Rixiati, Y.; Huang, H.; Shi, Y.J.; Huang, C.; Jiao, B. Asperolide A prevents bone metastatic breast cancer via the PI3K/AKT/mTOR/c-Fos/NFATc1 signaling pathway. Cancer Med. 2020, 9, 8173–8185. [CrossRef] 11. Xin, S.; Li, S.-M.; Gao, L.; Zheng, J.-J.; Wu, Y.-W.; Shao, C.-L.; Ren, W.-H.; Zhi, K. CHNQD-00603 Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by the miR-452-3p-Mediated Autophagy Pathway. Front. Cell Dev. Biol. 2021, 9. [CrossRef] [PubMed] 12. Liang, X.; Luo, D.; Luesch, H. Advances in exploring the therapeutic potential of marine natural products. Pharmacol. Res. 2019, 147, 104373. [CrossRef] 13. Choi, T.-Y.; Choi, T.-I.; Lee, Y.-R.; Choe, S.-K.; Kim, C.-H. Zebrafish as an animal model for biomedical research. Exp. Mol. Med. 2021, 53, 310–317. [CrossRef] [PubMed] 14. Carnovali, M.; Banfi, G.; Mariotti, M. Zebrafish Models of Human Skeletal Disorders: Embryo and Biomed. Res. Int. 2019, 2019, 1253710. [CrossRef] [PubMed] fi, G.; Mariotti, M. Zebrafish Models of Human Skeletal Disorders: Embryo and Adult Swimming Together 9, 2019, 1253710. [CrossRef] [PubMed] 15. Kimmel, C.B.; DeLaurier, A.; Ullmann, B.; Dowd, J.; McFadden, M. Modes of developmental outgrowth and shaping of a craniofacial bone in zebrafish. PLoS ONE 2010, 5, e9475. [CrossRef] 16. Tavares, B.; Santos Lopes, S. The importance of Zebrafish in biomedical research. Acta Med. Port. 2013, 26, 583–592. [CrossRef] 16. Tavares, B.; Santos Lopes, S. The importance of Zebrafish in biomedical research. Acta Med. Port. 2013, 26, 583–592. [CrossRef] 10 of 10 10 of 10 Mar. Drugs 2022, 20, 135 17. Bird, N.C.; Mabee, P.M. Developmental Morphology of the Axial Skeleton of the Zebrafish, Danio rerio (Ostariophysi: Cyprinidae). Dev. Dyn. 2003, 228, 337–357. [CrossRef] y 18. Howe, K.; Clark, M.D.; Torroja, C.F.; Torrance, J.; Berthelot, C.; Muffato, M.; Collins, J.E.; Humphray, S.; McLaren, K.; Matthews, L.; et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013, 496, 498–503. [CrossRef] 19. Müller, W.; Schröder, H.; Wang, X. References Anxiolytic-like effect of brominated compounds from the marine sponge Aplysina fulva on adult zebrafish (Danio rerio): Involvement of the GABAergic system. Neurochem. Int. 2021, 146, 105021. [CrossRef] 29. Föger-Samwald, U.; Dovjak, P.; Azizi-Semrad, U.; Kerschan-Schindl, K.; Pietschmann, P. Osteoporos therapeutic options. EXCLI J. 2020, 19, 1017–1037. [CrossRef] 30. Wang, X.; Yamauchi, K.; Mitsunaga, T. A review on osteoclast diseases and osteoclastogenesis inhibitors recently developed from natural resources. Fitoterapia 2020, 142, 104482. [CrossRef] p 31. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio Rerio), 5th ed.; University of Oregon Press: Eugene, OR, USA, 2007. 32. Kimmel, C.B.; Ballard, W.W.; Kimmel, S.R.; Ullmann, B.; Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dynam. 1995, 203, 253–310. [CrossRef] y 33. Matthews, M.; Varga, Z.M. Anesthesia and euthanasia in zebrafish. ILAR J. 2012, 53, 192–204. [CrossRef] [PubMed] 34. Walker, M.B.; Kimmel, C.B. A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech. Histochem. 2007, 82, 23–28. [CrossRef] [PubMed] 34. Walker, M.B.; Kimmel, C.B. A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech. Histochem. 2007, 82, 23–28. [CrossRef] [PubMed]
https://openalex.org/W3192362349
http://revistas.um.edu.uy/index.php/revistaderecho/article/download/810/989
es
Civil courts coping with Covid-19, de Bart Krans y Anna Nylund (Coords.), Eleven International Publishing, The Hague, 2021
Revista de derecho
2,021
cc-by
634
SANTIAGO PEREIRA CAMPOS - CIVIL COURTS COPING WITH COVID-19 - doi: https://doi.org/10.47274/DERUM/39.11 Civil courts coping with Covid-19, de Bart Krans y Anna Nylund (Coords.), Eleven International Publishing, The Hague, 20211 Santiago PEREIRA CAMPOS2 La editorial Eleven International Publishing acaba de publicar en La Haya, Países Bajos, el libro “Civil Courts coping with Covid-19”, obra colectiva en la que tuve el honor de participar. Los editores de la obra son Bart Krans (Profesor Titular en la Universidad de Leiden, Holanda, donde ocupa la cátedra de Derecho Privado y Derecho Procesal Civil) y Anna Nylund (Profesora Titular de Derecho en la Universidad de Tromsø). La imprevista pandemia de Covid-19 tuvo impactos de gran relevancia en los sistemas de justicia de todos los países, que demostraron no estar preparados para el inmenso desafío de garantizar el acceso a justicia y cumplir con las medidas sanitarias. Ello determinó que la prestación del servicio de justicia se viera gravemente afectado en casi todos los Estados. Frente a ello, la doctrina, la judicatura, la abogacía y la sociedad toda, debieron buscar rápidamente soluciones que permitieran restaurar el servicio, mediante transformaciones sin precedentes en los procesos judiciales. Los tribunales han demostrado ser creativos e innovadores en sus respuestas a la pandemia, y en su capacidad para implementar digitalización de trámites y audiencias remotas. El libro contiene un estudio comparativo de cómo los tribunales en 23 países han hecho frente a la pandemia, abordando innovaciones y adaptaciones a los procesos judiciales, que facilitan el salto digital y, a su vez, generan nuevas preocupaciones. Incluye informes de Australia, Belgica, Brasil, Canadá, China, Croacia, Dinamarca, Reino Unido, Finlandia, Francia, Alemania, Italia, Japón, Lituania, Países Bajos, Noruega, Perú, Polonia, Singapur, Slovenia, Taiwan, Uruguay y Estados Unidos y un excelente análisis comparativo de los editores. En la obra, los autores discutimos las implicaciones de la digitalización, cómo garantizar la igualdad de acceso a los tribunales, cuestiones novedosas relativas al derecho a un juicio justo en procesos telemáticos, el papel de la resolución de disputas durante la pandemia por vías diferentes al proceso judicial, y las raíces de la resistencia a la digitalización. Varias contribuciones también abordan cómo las innovaciones durante la pandemia pueden transformar el litigio civil en el futuro. 1 La obra es de open access a través del siguiente link: https://boeken.rechtsgebieden.boomportaal.nl/publicaties/9789462362048#2 2 Director de la Maestría en Derecho Procesal y Litigación, Director del Postgrado en Derecho Procesal Aplicado y Profesor Titular de Derecho Procesal en la Universidad de Montevideo. Revista de Derecho. Año XX (Julio 2021), Nº 39, pp. 253-254 | ISSN: 1510-5172 (papel) - 2301-1610 (en línea) 253 SANTIAGO PEREIRA CAMPOS - CIVIL COURTS COPING WITH COVID-19 En situaciones de especial dificultad, las instituciones y sus personas se dan por vencidas o sacan lo mejor de sí para salir adelante. Es probable que en un contexto de normalidad habría llevado décadas construir la infraestructura, implementar los nuevos procesos de trabajo y completar los otros numerosos desarrollos necesarios para dar el salto necesario e inevitable hacia la digitalización. Como señalan los editores, la pandemia ha desatado un flujo de creatividad e innovación sin precedentes entre los tribunales, la judicatura y la abogacía. Simultáneamente, la pandemia ha puesto de manifiesto en muchos casos la lentitud de los tribunales para digitalizar, así como algunas de las razones subyacentes para resistir a las audiencias remotas y la resolución de disputas en línea (ODR). Mientras que algunas de las razones de esta resistencia están bien justificadas y son oportunas, algunas otras parecen originarse de la fricción que inevitablemente conlleva un cambio culturar. Sin duda que el salto tecnológico y otros avances han abierto nuevos horizontes en el futuro de la justicia civil. De ello también trata este libro. 254 Revista de Derecho. Año XX (Julio 2021), Nº 39, pp. 253-254 | ISSN: 1510-5172 (papel) - 2301-1610 (en línea)
https://openalex.org/W4281658849
http://www.jtcvsopen.org/article/S266627362200242X/pdf
English
null
Reply From Authors: How a cell dies matters and how to evaluate it also matters
JTCVS open
2,022
cc-by
892
Satoshi Ueda, MDa Toyofumi F. Chen-Yoshikawa, MD, PhDb Satona Tanaka, MD, PhDa Yoshito Yamada, MD, PhDa Daisuke Nakajima, MD, PhDa Akihiro Ohsumi, MD, PhDa Hiroshi Date, MD, PhDa aDepartment of Thoracic Surgery Kyoto University Graduate School of Medicine Kyoto, Japan bDepartment of Thoracic Surgery Nagoya University Graduate School of Medicine Nagoya, Japan Satoshi Ueda, MDa Toyofumi F. Chen-Yoshikawa, MD, PhDb Satona Tanaka, MD, PhDa Yoshito Yamada, MD, PhDa Daisuke Nakajima, MD, PhDa Akihiro Ohsumi, MD, PhDa Hiroshi Date, MD, PhDa aDepartment of Thoracic Surgery Kyoto University Graduate School of Medicine Kyoto, Japan bDepartment of Thoracic Surgery Nagoya University Graduate School of Medicine Nagoya, Japan Satoshi Ueda, MDa Toyofumi F. Chen-Yoshikawa, MD, PhDb Satona Tanaka, MD, PhDa Yoshito Yamada, MD, PhDa Daisuke Nakajima, MD, PhDa Akihiro Ohsumi, MD, PhDa Hiroshi Date, MD, PhDa aDepartment of Thoracic Surgery Kyoto University Graduate School of Medicine Kyoto, Japan bDepartment of Thoracic Surgery Nagoya University Graduate School of Medicine Nagoya, Japan Reply to the Editor: This study aimed to investigate the effect of necrosulfo- namide (NSA), a mixed-lineage kinase domain-like protein (MLKL) inhibitor, on pulmonary ischemia-reperfusion injury (IRI) in a simple and validated in vivo model. NSA treatment showed a protective effect on pulmonary IRI, because of improvement of physiological parameters and histological findings and a lower level of proinflammatory cytokines were observed in the NSA-treated group.1 We adopted double-labeling with cleaved caspase-3 and terminal deoxynucleotidyl transferase- dUTP nick-end la- beling (TUNEL) to evaluate necroptotic/apoptotic cells, as previously described by others.2 TUNEL-positive and caspase–3-negative cells were defined as necroptotic cells.2 As Lin and colleagues reported in a “Letter to the Editor,”3 this method might count other cell death that is caspase- independent such as ferroptosis. Ferroptosis is a recently established mode of programmed cell death that is depen- dent on iron and characterized by the accumulation of lipid peroxides. A decrease in TUNEL-positive and cas- pase–3-negative cells might indicate a decrease in ferropto- sis as well as necroptosis, however, to the best of our knowledge, there are no specific markers that are applicable in rat lung tissue to distinguish ferroptosis from other types of cell death. In the past, in experimental reports describing IRI,4,5 cell death inhibited by necrostatin-1 and ferrostatin-1 Copyright  2022 The Author(s). Published by Elsevier Inc. on behalf of The American Association for Thoracic Surgery. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Thoracic: Lung Transplant: Basic Science: Letters to the Editor Thoracic: Lung Transplant: Basic Science: Letters to the Editor was defined as necroptosis and ferroptosis, respectively. Necrostatin-1 inhibits receptor-interacting protein kinase- 1 (RIPK1) and NSA inhibits MLKL, which is the down- stream of RIPK1 and regulates necroptosis. We concluded that the protective effect of NSA on pulmonary IRI was mainly caused by the inhibition of MLKL-mediated cell death, namely, necroptosis. A decrease in other types of cell death such as ferroptosis and apoptosis might be observed as a result of attenuated lung damage in the NSA-treated group. The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and re- viewers of this article have no conflicts of interest. The authors reported no conflicts of interest. The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and re- viewers of this article have no conflicts of interest. Death Receptors Death Ligands RIPK1 RIPK3 MLKL Necroptosis ↓ NSA IRI damage ↓ Apoptosis ↓? Ferroptosis ↓? Lipid peroxidation ↓ ROS ↓ REPLY FROM AUTHORS: HOW A CELL DIES MATTERS AND HOW TO EVALUATE IT ALSO MATTERS REPLY FROM AUTHORS: HOW A CELL DIES MATTERS AND HOW TO EVALUATE IT ALSO MATTERS How a cell dies matters and how to evaluate it also mat- ters. In the future, it is necessary to find a way to more rigor- ously distinguish necroptosis from ferroptosis and other cell death to advance research on programmed cell death that is applicable in various settings of experimental models. https://doi.org/10.1016/j.xjon.2022.05.016 3. Lin Y, Li H, Li J, Gu H, Li P. Caspase-independent programmed cell death is not necessarily necroptosis. J Thorac Cardiovasc Surg Open. 2022;11:349. References 1. Ueda S, Chen-Yoshikawa TF, Tanaka S, Yamada Y, Nakajima D, Ohsumi A, et al. Protective effect of necrosulfonamide on rat pulmonary ischemia-reperfusion injury via inhibition of necroptosis. J Thorac Cardiovasc Surg. 2022;163:e113-22. 2. Dong W, Zhang M, Zhu Y, Chen Y, Zhao X, Li R, et al. Protective effect of NSA on intestinal epithelial cells in a necroptosis model. Oncotarget. 2017;8:86726-35. 3. Lin Y, Li H, Li J, Gu H, Li P. Caspase-independent programmed cell death is not necessarily necroptosis. J Thorac Cardiovasc Surg Open. 2022;11:349. 4. Li W, Feng G, Gauthier JM, Lokshina I, Higashikubo R, Evans S, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart trans- plantation. J Clin Invest. 2019;129:2293-304. 5. Wang X, O’Brien ME, Yu J, Xu C, Zhang Q, Lu S, et al. Prolonged cold ischemia induces necroptotic cell death in ischemia-reperfusion injury and contributes to primary graft dysfunction after lung transplantation. Am J Respir Cell Mol Biol. 2019;61:244-56. https://doi.org/10.1016/j.xjon.2022.05.016 JTCVS Open c September 2022 350
https://openalex.org/W2196551631
https://www.qeios.com/read/IHK5GH/pdf
English
null
Uterine Corpus Cancer
Definitions
2,020
cc-by
69
Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Uterine Corpus Cancer National Cancer Institute Qeios ID: IHK5GH · https://doi.org/10.32388/IHK5GH Source National Cancer Institute. Uterine Corpus Cancer. NCI Thesaurus. Code C61574. National Cancer Institute. Uterine Corpus Cancer. NCI Thesaurus. Code C61574. A malignant neoplasm arising from the uterine corpus. This category includes endometrial carcinoma and carcinosarcoma. Qeios ID: IHK5GH · https://doi.org/10.32388/IHK5GH 1/1
https://openalex.org/W4234101010
https://peerj.com/articles/10672v0.2/submission
English
null
Peer Review #3 of "Widely assumed phenotypic associations in Cannabis sativa lack a shared genetic basis (v0.1)"
null
2,021
cc-by
16,777
Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Widely assumed phenotypic associations in Cannabis sativa 2 lack a shared genetic basis 3 4 5 Daniela Vergara1*, Cellene M. Feathers1, Ezra L. Huscher1, Ben Holmes2, Jacob A. Haas1,3, and 6 Nolan C. Kane1* 7 8 1. University of Colorado, Boulder, Department of Ecology and Evolutionary Biology 9 2. Centennial Seeds, 1216 Commerce Ct. No.7, Lafayette, CO 80026. 10 3. DabLogic, 5000 east 41st ave Denver CO 80216 11 12 13 14 *Correspondence: daniela.vergara@colorado.edu or nolan.kane@colorado.edu 15 University of Colorado Boulder 16 1900 Pleasant Street 17 Boulder, CO 80309 18 19 Keywords: cannabinoid, chemotype, geometric morphometrics, hemp, marijuana, shape 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Widely assumed phenotypic associations in Cannabis sativa lack a shared genetic basis Daniela Vergara Corresp., 1 , Cellene M Feathers 1 , Ezra L Huscher 1 , Ben Holmes 2 , Jacob A Haas 3 , Nolan C Kane 1 1 Ebio, University of Colorado at Boulder, Boulder, Colorado, United States 2 Centennial Seeds, Lafayette, Colorado, United States 3 DabLogic, 5000 east 41st ave, Denver, Colorado, USA Corresponding Author: Daniela Vergara Email address: daniela.vergara@colorado.edu The flowering plant Cannabis sativa, cultivated for centuries for multiple purposes, displays extensive variation in phenotypic traits in addition to its wide array of secondary metabolite production. Notably, Cannabis produces two well-known secondary-metabolite cannabinoids: cannabidiolic acid (CBDA) and delta-9-tetrahydrocannabinolic acid (THCA), which are the main products sought by consumers in the medical and recreational market. Cannabis has several suggested subspecies which have been shown to differ in chemistry, branching patterns, leaf morphology and other traits. In this study we obtained measurements related to phytochemistry, reproductive traits, growth architecture, and leaf morphology from 297 hybrid individuals from a cross between two diverse lineages. We explored correlations among these characteristics to inform our understanding of which traits may be causally associated. Many of the traits widely assumed to be strongly correlated did not show any relationship in this hybrid population. The current taxonomy and legal regulation within Cannabis is based on phenotypic and chemical characteristics. However, we find these traits are not associated when lineages are inter-crossed, which is a common breeding practice and forms the basis of most modern marijuana and hemp germplasms. Our results suggest naming conventions based on leaf morphology do not correspond to the chemical properties in plants with hybrid ancestry. Therefore, a new system for identifying variation within Cannabis is warranted that will provide reliable identifiers of the properties important for recreational and, especially, medical use. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed 36 Abstract 36 Abstract 37 The flowering plant Cannabis sativa, cultivated for centuries for multiple purposes, 38 displays extensive variation in phenotypic traits in addition to its wide array of secondary 39 metabolite production. Notably, Cannabis produces two well-known secondary-metabolite 40 cannabinoids: cannabidiolic acid (CBDA) and delta-9-tetrahydrocannabinolic acid (THCA), 41 which are the main products sought by consumers in the medical and recreational market. 42 Cannabis has several suggested subspecies which have been shown to differ in chemistry, 43 branching patterns, leaf morphology and other traits. In this study we obtained 44 measurements related to phytochemistry, reproductive traits, growth architecture, and leaf 45 morphology from 297 hybrid individuals from a cross between two diverse lineages. We 46 explored correlations among these characteristics to inform our understanding of which 47 traits may be causally associated. Many of the traits widely assumed to be strongly 48 correlated did not show any relationship in this hybrid population. The current taxonomy 49 and legal regulation within Cannabis is based on phenotypic and chemical characteristics. 50 However, we find these traits are not associated when lineages are inter-crossed, which is a 51 common breeding practice and forms the basis of most modern marijuana and hemp 52 germplasms. Our results suggest naming conventions based on leaf morphology do not 53 correspond to the chemical properties in plants with hybrid ancestry. Therefore, a new 54 system for identifying variation within Cannabis is warranted that will provide reliable 55 identifiers of the properties important for recreational and, especially, medical use. 56 Words: 229 57 58 Introduction 59 Phenotypic variation within and between populations is an important 60 characteristic to consider for classification purposes. In particular, if phenotypic variation 61 exists between different lineages within a species, then characterizing trait correlations can 62 shed light on how they are inherited, whether they are controlled by the same genes, and if 63 they can be used for taxonomic purposes. Wild populations may carry shared ancestral 64 traits despite being independently inherited, and therefore it may appear as if these traits 65 were associated. Additionally, selection may favor certain trait combinations. 1 Widely assumed phenotypic associations in Cannabis sativa 2 lack a shared genetic basis PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 68 of the world’s most valuable crops (Hutchison et al. 2019). Unfortunately, widespread legal 69 issues have hindered Cannabis research. 68 of the world’s most valuable crops (Hutchison et al. 2019). Unfortunately, widespread legal 69 issues have hindered Cannabis research. 70 One of the most notable characteristics of the Cannabis plant is its chemistry: the 71 production of a family of molecules known as cannabinoids which are mainly produced and 72 stored in the trichomes of female flowers (Gagne et al. 2012; Sirikantaramas et al. 2005). 73 The most studied of these cannabinoids are cannabidiolic acid (CBDA), and delta-9- 74 tetrahydrocannabinolic acid (THCA), which are produced by the enzymes CBDA and THCA 75 synthases, respectively. These two synthases are found at the final stage of the biochemical 76 pathway, along with cannabichromenic acid synthase, a third less well-studied synthase, 77 that produces Cannabichomenic acid (CBCA; Page & Stout 2017). These three synthases use 78 the same precursor molecule, cannabigerolic acid (CBGA; Laverty et al. 2019; Page & 79 Boubakir 2014; Vergara et al. 2019). The genetic sequences for the three synthases are 80 very similar and at least the genes encoding CBDA and THCA synthase are close in 81 proximity (Weilblen et al. 2015), suggesting they may have originated from the same 82 ancestor gene (Onofri et al. 2015; Padgitt-Cobb et al. 2019). Additionally, in vitro, each of 83 these synthases can produce at least eight different compounds including THCA and CBDA 84 in different ratios (Kovalchuk et al. 2020; Zirpel et al. 2018). These enzymes may be 85 classified as “promiscuous enzymes” due to their considerable similarities, the fact they act 86 on the same precursor molecule, and they can produce each other’s compounds (Auldridge 87 et al. 2006; Chakraborty et al. 2013; Franco 2011). 88 Wh h d THCA d CBDA d i h l f THC d CBD 70 One of the most notable characteristics of the Cannabis plant is its chemistry: the 71 production of a family of molecules known as cannabinoids which are mainly produced and 72 stored in the trichomes of female flowers (Gagne et al. 2012; Sirikantaramas et al. 2005). 73 The most studied of these cannabinoids are cannabidiolic acid (CBDA), and delta-9- 74 tetrahydrocannabinolic acid (THCA), which are produced by the enzymes CBDA and THCA 75 synthases, respectively. 36 Abstract 66 The angiosperm species Cannabis sativa has been cultivated for millennia for a 67 range of purposes (Li 1973; Li 1974; Russo 2007) and is currently, by some estimates, one 37 The flowering plant Cannabis sativa, cultivated for centuries for multiple purposes, 38 displays extensive variation in phenotypic traits in addition to its wide array of secondary 39 metabolite production. Notably, Cannabis produces two well-known secondary-metabolite 40 cannabinoids: cannabidiolic acid (CBDA) and delta-9-tetrahydrocannabinolic acid (THCA), 41 which are the main products sought by consumers in the medical and recreational market. 42 Cannabis has several suggested subspecies which have been shown to differ in chemistry, 43 branching patterns, leaf morphology and other traits. In this study we obtained 44 measurements related to phytochemistry, reproductive traits, growth architecture, and leaf 45 morphology from 297 hybrid individuals from a cross between two diverse lineages. We 46 explored correlations among these characteristics to inform our understanding of which 47 traits may be causally associated. Many of the traits widely assumed to be strongly 48 correlated did not show any relationship in this hybrid population. The current taxonomy 49 and legal regulation within Cannabis is based on phenotypic and chemical characteristics. 50 However, we find these traits are not associated when lineages are inter-crossed, which is a 51 common breeding practice and forms the basis of most modern marijuana and hemp 52 germplasms. Our results suggest naming conventions based on leaf morphology do not 53 correspond to the chemical properties in plants with hybrid ancestry. Therefore, a new 54 system for identifying variation within Cannabis is warranted that will provide reliable 55 identifiers of the properties important for recreational and, especially, medical use. 56 Words: 229 66 The angiosperm species Cannabis sativa has been cultivated for millennia for a 67 range of purposes (Li 1973; Li 1974; Russo 2007) and is currently, by some estimates, one PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be review Manuscript to be reviewed 91 2013; Volkow et al. 2014) and economic value (Evans 2013; Kirsch 2018), but THC has 92 been intensely selected by breeders and growers (Volkow et al. 2014) due to its 93 psychoactive effects (ElSohly & Slade 2005). Studies have also found CBDA may have 94 medicinal benefits in its acidic form (Takeda et al. 2008; Takeda et al. 2012). Differences in 95 leaf size, leaf shape, plant size, and inflorescence size are used in the Cannabis industry to 96 categorize plants and these morphological differences are thought to be useful predictors 97 of cannabinoid content. 91 2013; Volkow et al. 2014) and economic value (Evans 2013; Kirsch 2018), but THC has 92 been intensely selected by breeders and growers (Volkow et al. 2014) due to its 93 psychoactive effects (ElSohly & Slade 2005). Studies have also found CBDA may have 94 medicinal benefits in its acidic form (Takeda et al. 2008; Takeda et al. 2012). Differences in 95 leaf size, leaf shape, plant size, and inflorescence size are used in the Cannabis industry to 96 categorize plants and these morphological differences are thought to be useful predictors 97 of cannabinoid content. 98 Currently recognized lineages within the genus Cannabis include the narrowleaf 99 drug types, C. sativa ssp. sativa, the broadleaf drug type C. sativa ssp. indica, the northern 100 Eurasian wild C. sativa ssp. ruderalis, and at least one lineage of hemp (Clarke & Merlin 2013). 101 Among these subspecies, there is substantial phenotypic variation in the production of 102 multiple cannabinoids (McPartland & Russo 2001; Russo et al. 2008; Russo & McPartland 103 2003) and terpenoids (de la Fuente et al. 2020; Orser et al. 2017; Reimann-Philipp et al. 104 2019), substantial genotypic variation (Kovalchuk et al. 2020; Lynch et al. 2016; Sawler et 105 al. 2015; Vergara et al. 2016), and observed morphological variation in traits such as 106 branching, internode length, and flowering time (Clarke & Merlin 2013). 98 Currently recognized lineages within the genus Cannabis include the narrowleaf 99 drug types, C. sativa ssp. sativa, the broadleaf drug type C. sativa ssp. indica, the northern 100 Eurasian wild C. sativa ssp. ruderalis, and at least one lineage of hemp (Clarke & Merlin 2013). 101 Among these subspecies, there is substantial phenotypic variation in the production of 102 multiple cannabinoids (McPartland & Russo 2001; Russo et al. Manuscript to be reviewed These two synthases are found at the final stage of the biochemical 76 pathway, along with cannabichromenic acid synthase, a third less well-studied synthase, 77 that produces Cannabichomenic acid (CBCA; Page & Stout 2017). These three synthases use 78 the same precursor molecule, cannabigerolic acid (CBGA; Laverty et al. 2019; Page & 79 Boubakir 2014; Vergara et al. 2019). The genetic sequences for the three synthases are 80 very similar and at least the genes encoding CBDA and THCA synthase are close in 81 proximity (Weilblen et al. 2015), suggesting they may have originated from the same 82 ancestor gene (Onofri et al. 2015; Padgitt-Cobb et al. 2019). Additionally, in vitro, each of 83 these synthases can produce at least eight different compounds including THCA and CBDA 84 in different ratios (Kovalchuk et al. 2020; Zirpel et al. 2018). These enzymes may be 85 classified as “promiscuous enzymes” due to their considerable similarities, the fact they act 86 on the same precursor molecule, and they can produce each other’s compounds (Auldridge 87 et al. 2006; Chakraborty et al. 2013; Franco 2011). 88 When heated, THCA and CBDA are converted into the neutral forms THC and CBD 89 (R 2011) hi h i t t ith th h d bi id t (P t 1988 88 When heated, THCA and CBDA are converted into the neutral forms THC and CBD 89 (Russo 2011), which interact with the human endocannabinoid system (Pertwee 1988; 90 Pertwee 1997; Pertwee 2004). Both THC and CBD have medicinal (Russo 2011; Swift et al. 88 When heated, THCA and CBDA are converted into the neutral forms THC and CBD 89 (Russo 2011), which interact with the human endocannabinoid system (Pertwee 1988; 90 Pertwee 1997; Pertwee 2004). Both THC and CBD have medicinal (Russo 2011; Swift et al. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 2008; Russo & McPartland 103 2003) and terpenoids (de la Fuente et al. 2020; Orser et al. 2017; Reimann-Philipp et al. 104 2019), substantial genotypic variation (Kovalchuk et al. 2020; Lynch et al. 2016; Sawler et 105 al. 2015; Vergara et al. 2016), and observed morphological variation in traits such as 106 branching, internode length, and flowering time (Clarke & Merlin 2013). 107 The main classifications used in the modern Cannabis industry parlance are “indica”, 108 “sativa”, and “hybrids”. Sativa plants are described as tall with narrow leaves and lighter 109 density buds, allegedly producing high levels of THCA, and therefore have uplifting and 110 stimulating psychedelic effects after consumption. Indica plants are described as short with 111 broad leaves and dense buds, and produce high levels of both THCA and CBDA believed to 112 produce a relaxing effect (Clarke & Merlin 2013; McPartland 2017; Vergara et al. 2016). 113 Yet, the associations between these multiple traits have not previously been researched. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 114 Other popular ideas suggest the important distinctions between Cannabis lineages related 115 to the effects after consumption are due to differences in terpene profiles rather than to 116 cannabinoid profiles. It may be that terpene profiles are more relevant to differences in 117 Cannabis lineages than cannabinoids (de la Fuente et al. 2020; Orser et al. 2017; Reimann- 118 Philipp et al. 2019). Crosses between “sativa” and “indica” plants are referred to as 119 “hybrids” and these have variable phenotypes usually intermediate to the parents (Vergara 120 et al. 2016). Finally, the hemp group has been traditionally used for industrial purposes 121 such as fiber or oil production, however the legal definition of hemp includes any Cannabis 122 plant with less than 0.3% THC by weight. p a t t ess t a 0 3% C by e g t 123 The colloquial naming convention of “indica” and “sativa” do not correspond to the 124 scientific subspecies with similar names. Furthermore, these common distinctions do not 125 reflect evolutionary relationships (Sawler et al. 2015; Schwabe & McGlaughlin 2019; 126 Vergara et al. 2016). This misidentification can be particularly problematic for medical 127 patients who are depending on reliable and consistent products. 128 Cannabis is dioecious (Divashuk et al. 2014; van Bakel et al. 2011), although 129 monoecious plants exist, particularly in the hemp lineage (Hillig 2005; Peil et al. 2003). 130 Dioecious varieties are common for medicinal and recreational purposes, and selection in 131 domestication has been focused on females due to the production of cannabinoids (Gagne 132 et al. 2012; Sirikantaramas et al. 2005), with strong selection against males and 133 hermaphrodites. Many commercially important traits are expressed at maturity, and if 134 breeders could predict their late-stage expression through correlations among these traits 135 earlier during development, selection could be made sooner, accelerating breeding cycles. 136 Also, if traits early in the development allowed for distinguishing between sexes, males 123 The colloquial naming convention of “indica” and “sativa” do not correspond to the 124 scientific subspecies with similar names. Furthermore, these common distinctions do not 125 reflect evolutionary relationships (Sawler et al. 2015; Schwabe & McGlaughlin 2019; 126 Vergara et al. 2016). This misidentification can be particularly problematic for medical 127 patients who are depending on reliable and consistent products. 128 Cannabis is dioecious (Divashuk et al. 2014; van Bakel et al. Manuscript to be reviewed 137 could be culled before pollen production and potential female pollination. This is important 138 because females would undesirably divert energy to seeds instead of cannabinoids after 139 being pollinated (Clarke & Merlin 2013). 137 could be culled before pollen production and potential female pollination. This is important 138 because females would undesirably divert energy to seeds instead of cannabinoids after 139 being pollinated (Clarke & Merlin 2013). 137 could be culled before pollen production and potential female pollination. This is important 138 because females would undesirably divert energy to seeds instead of cannabinoids after 139 being pollinated (Clarke & Merlin 2013). 137 could be culled before pollen production and potential female pollination. This is important 138 because females would undesirably divert energy to seeds instead of cannabinoids after 139 being pollinated (Clarke & Merlin 2013). 140 In this study, we quantified 18 phenotypic traits of 297 individuals from a first- 141 generation backcross (BC1) between a female ‘Carmagnola’ hemp and a male marijuana- 142 type plant ‘Afghan Kush’. Many of the morphological traits we measured are considered 143 important by the Cannabis industry to characterize different plants. We predicted these 144 traits would vary in the BC1, and therefore allow us to investigate the pattern of possible 145 genetic correlations. Furthermore, we determined whether the association between 146 morphological traits and cannabinoid chemistry could be used to characterize Cannabis 147 lineages. Given that the current nomenclature is not supported scientific research (Lynch et 148 al. 2016; Sawler et al. 2015; Vergara et al. 2016), it is possible that erroneous associations 149 between morphological traits has contributed to the misnaming issues in the Cannabis 150 industry. 151 152 Methods 153 BC cross and measurements 154 A cross was performed between a female, narrowleaf ‘Carmagnola’ hemp plant and 155 a male broadleaf ‘Afghan Kush’ plant at Centennial Seeds in Lafayette Colorado (Figure 1A). 156 One of the female first filial (F1) offspring of this cross was backcrossed to a male sibling of 157 the parental male (Figure 1A). Two hundred ninety-seven individuals from this backcross 158 population (BC1; Figure 1A) were started indoors on April 23, 2015 and on June 6, 2015, g p ( ) 140 In this study, we quantified 18 phenotypic traits of 297 individuals from a first- 141 generation backcross (BC1) between a female ‘Carmagnola’ hemp and a male marijuana- 142 type plant ‘Afghan Kush’. Manuscript to be reviewed 2011), although 129 monoecious plants exist, particularly in the hemp lineage (Hillig 2005; Peil et al. 2003). 130 Dioecious varieties are common for medicinal and recreational purposes, and selection in 131 domestication has been focused on females due to the production of cannabinoids (Gagne 132 et al. 2012; Sirikantaramas et al. 2005), with strong selection against males and 133 hermaphrodites. Many commercially important traits are expressed at maturity, and if 134 breeders could predict their late-stage expression through correlations among these traits 135 earlier during development, selection could be made sooner, accelerating breeding cycles. 136 Also, if traits early in the development allowed for distinguishing between sexes, males 128 Cannabis is dioecious (Divashuk et al. 2014; van Bakel et al. 2011), although 129 monoecious plants exist, particularly in the hemp lineage (Hillig 2005; Peil et al. 2003). 130 Dioecious varieties are common for medicinal and recreational purposes, and selection in 131 domestication has been focused on females due to the production of cannabinoids (Gagne 132 et al. 2012; Sirikantaramas et al. 2005), with strong selection against males and 133 hermaphrodites. Many commercially important traits are expressed at maturity, and if 134 breeders could predict their late-stage expression through correlations among these traits 135 earlier during development, selection could be made sooner, accelerating breeding cycles. 136 Also, if traits early in the development allowed for distinguishing between sexes, males PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed 159 when the plants were six weeks old, they were transplanted outdoors in a field in Boulder 160 County, Colorado. 159 when the plants were six weeks old, they were transplanted outdoors in a field in Boulder 160 County, Colorado. 161 Morphological measurements including height, stalk diameter, inner-node length, 162 petiole length, leaf length and width, among other measurements, were obtained at two 163 different time points during the growing cycle (Table S1). We chose these two time points, 164 one at the beginning and one at the end of the growing season, to provide information on 165 possible trait associations during the plant’s development. The initial timepoint (IT) was 166 taken at six weeks old (June 6, 2015), and the final timepoint (FT) at 19 weeks old 167 (September 2, 2015) which corresponds to the beginning of the fall season. Additional 168 traits were measured at the FT including bud count, size of biggest bud, length of longest 169 branch, and number of buds on the longest branch (Table S1). At the FT, we also scanned a 170 representative leaf from each plant. Therefore, we had fully extended longest leaf (FELL) 171 measurements from both the IT and FT. At the FT, we determined the sex of all 297 172 individuals, and also measured the concentrations of three cannabinoids -THC, CBD, and 173 CBG- from 100 plants. Here we removed plants identified as male to avoid pollination, after 174 the representative leaf was scanned. Therefore, some of the measurements were taken on 175 fewer individuals than at the IT. 159 when the plants were six weeks old, they were transplanted outdoors in a field in Boulder 160 County, Colorado. 161 Morphological measurements including height, stalk diameter, inner-node length, 162 petiole length, leaf length and width, among other measurements, were obtained at two 163 different time points during the growing cycle (Table S1). We chose these two time points, 164 one at the beginning and one at the end of the growing season, to provide information on 165 possible trait associations during the plant’s development. The initial timepoint (IT) was 166 taken at six weeks old (June 6, 2015), and the final timepoint (FT) at 19 weeks old 167 (September 2, 2015) which corresponds to the beginning of the fall season. Manuscript to be reviewed Many of the morphological traits we measured are considered 143 important by the Cannabis industry to characterize different plants. We predicted these 144 traits would vary in the BC1, and therefore allow us to investigate the pattern of possible 145 genetic correlations. Furthermore, we determined whether the association between 146 morphological traits and cannabinoid chemistry could be used to characterize Cannabis 147 lineages. Given that the current nomenclature is not supported scientific research (Lynch et 148 al. 2016; Sawler et al. 2015; Vergara et al. 2016), it is possible that erroneous associations 149 between morphological traits has contributed to the misnaming issues in the Cannabis 150 industry. 154 A cross was performed between a female, narrowleaf ‘Carmagnola’ hemp plant and 155 a male broadleaf ‘Afghan Kush’ plant at Centennial Seeds in Lafayette Colorado (Figure 1A). 156 One of the female first filial (F1) offspring of this cross was backcrossed to a male sibling of 157 the parental male (Figure 1A). Two hundred ninety-seven individuals from this backcross 158 population (BC1; Figure 1A) were started indoors on April 23, 2015 and on June 6, 2015, PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Additional 168 traits were measured at the FT including bud count, size of biggest bud, length of longest 169 branch, and number of buds on the longest branch (Table S1). At the FT, we also scanned a 170 representative leaf from each plant. Therefore, we had fully extended longest leaf (FELL) 171 measurements from both the IT and FT. At the FT, we determined the sex of all 297 172 individuals, and also measured the concentrations of three cannabinoids -THC, CBD, and 173 CBG- from 100 plants. Here we removed plants identified as male to avoid pollination, after 174 the representative leaf was scanned. Therefore, some of the measurements were taken on 175 fewer individuals than at the IT. p g g g g 162 petiole length, leaf length and width, among other measurements, were obtained at two 163 different time points during the growing cycle (Table S1). We chose these two time points, 164 one at the beginning and one at the end of the growing season, to provide information on 165 possible trait associations during the plant’s development. The initial timepoint (IT) was 166 taken at six weeks old (June 6, 2015), and the final timepoint (FT) at 19 weeks old 167 (September 2, 2015) which corresponds to the beginning of the fall season. Additional 168 traits were measured at the FT including bud count, size of biggest bud, length of longest 169 branch, and number of buds on the longest branch (Table S1). At the FT, we also scanned a 170 representative leaf from each plant. Therefore, we had fully extended longest leaf (FELL) 171 measurements from both the IT and FT. At the FT, we determined the sex of all 297 172 individuals, and also measured the concentrations of three cannabinoids -THC, CBD, and 173 CBG- from 100 plants. Here we removed plants identified as male to avoid pollination, after 174 the representative leaf was scanned. Therefore, some of the measurements were taken on 175 fewer individuals than at the IT. 176 177 Phenotypic trait statistical analyses 178 To understand the change in individual phenotypes through the growing season, we 179 calculated the difference between the initial measurements and the final measurements 180 (delta ) for some traits. Specifically, we calculated  for the four traits that were measured 181 at both timepoints (Table S1). Manuscript to be reviewed 182 correlations for both the IT and the FT, and between-time correlations. All correlations 183 were corrected with Bonferroni for multiple comparisons (Weisstein 2004). Finally, we 184 established whether any of the measured traits differed between males and females using 185 t-tests with sex as the explanatory variable. 186 187 Leaf shape analysis 188 We carried out a geometric morphometric analysis to develop a quantifiable 189 measure of leaf shape. Specifically, we placed landmark coordinates on each leaf picture 190 with the program TPS Dig2 (Rohlf 2006). We used ten landmarks from the first, central, 191 and last leaflet (Figure 1B) which covers the whole leaf structure. Additionally, we 192 measured the length and width of each of the leaves, counted the serration number in the 193 center leaflet, and counted the number of leaflets. 194 We used the R package Geomorph (Adams & Otárola-Castillo 2013) for all geometric 195 morphometric analysis, following the methodology of Vergara et al. (2017b). A Procrustes 196 analyses was used to remove variability caused by position, orientation, and size and to 197 quantify shape variation by superimposing the objects in a joint coordinate system. Then, a 198 Principal Component Analysis (PCA) was used to identify the orthogonal structure in the 199 data and to visually explore morphological variation among individuals. 200 We performed multiple statistical tests to understand whether leaf shape was 201 related to any of the other measured traits at both timepoints and between timepoints. 202 First, we implemented several multivariate analyses of variance (MANOVAs) with shape as 203 the response variable for each of the measured traits in both timepoints and . We then 204 performed MANOVA models within each timepoint and  to understand whether the main Manuscript to be review 182 correlations for both the IT and the FT, and between-time correlations. All correlations 183 were corrected with Bonferroni for multiple comparisons (Weisstein 2004). Finally, we 184 established whether any of the measured traits differed between males and females using 185 t-tests with sex as the explanatory variable. 186 187 Leaf shape analysis 188 We carried out a geometric morphometric analysis to develop a quantifiable 189 measure of leaf shape. Specifically, we placed landmark coordinates on each leaf picture 190 with the program TPS Dig2 (Rohlf 2006). We used ten landmarks from the first, central, 191 and last leaflet (Figure 1B) which covers the whole leaf structure. Manuscript to be reviewed We then use these data to estimate the within-time point PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Additionally, we 192 measured the length and width of each of the leaves, counted the serration number in the 193 center leaflet, and counted the number of leaflets. 182 correlations for both the IT and the FT, and between-time correlations. All correlations 183 were corrected with Bonferroni for multiple comparisons (Weisstein 2004). Finally, we 184 established whether any of the measured traits differed between males and females using 185 t-tests with sex as the explanatory variable. 182 correlations for both the IT and the FT, and between-time correlations. All correlations 183 were corrected with Bonferroni for multiple comparisons (Weisstein 2004). Finally, we 184 established whether any of the measured traits differed between males and females using 185 t-tests with sex as the explanatory variable. 194 We used the R package Geomorph (Adams & Otárola-Castillo 2013) for all geometric 195 morphometric analysis, following the methodology of Vergara et al. (2017b). A Procrustes 196 analyses was used to remove variability caused by position, orientation, and size and to 197 quantify shape variation by superimposing the objects in a joint coordinate system. Then, a 198 Principal Component Analysis (PCA) was used to identify the orthogonal structure in the 199 data and to visually explore morphological variation among individuals. 200 We performed multiple statistical tests to understand whether leaf shape was 201 related to any of the other measured traits at both timepoints and between timepoints. 202 First, we implemented several multivariate analyses of variance (MANOVAs) with shape as 203 the response variable for each of the measured traits in both timepoints and . We then 204 performed MANOVA models within each timepoint and  to understand whether the main PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed 205 effects of each trait affected leaf shape. We corroborated the results using multivariate 206 multiple regressions. 207 208 Cannabinoid concentration measurements 209 The concentrations for the three cannabinoids -- CBG, THC, and CBD -- were 210 measured using gas chromatography on an SRI 86106 equipment with an MXT-35 column 211 using 197-209 mg of dried flower as described in (Brenneisen & ElSohly 1988). When 212 heated, the acidic compounds CBGA, THCA, and CBDA are turned into the neutral forms 213 CBG, THC, and CBD, which is the reason why gas chromatography only quantifies the 214 neutral forms of the compounds. 215 Given that the production of these three cannabinoids may be correlated because 216 they are part of the same biochemical pathway (Page & Boubakir 2014; Page & Stout 2017; 217 Vergara et al. 2019) and both CBDA and THCA synthases compete for the same precursor 218 molecule -CBGA-, we analyzed them using a Principal Component Analysis (PCA) to account 219 for multicollinearity and to avoid redundancies. We used a K-means cluster analysis on PC1 220 vs PC2 to visualize the different cannabinoid groups. We also added the total cannabinoid 221 concentration and measured the ratio of each cannabinoid over this total concentration 222 (Table S1). 223 224 Statistical analyses 225 We examined the associations between the production of each cannabinoid and 226 each of the measured traits at both timepoints and the . We also used cannabinoids as the 227 explanatory variables for several MANOVA models to determine whether cannabinoid 205 effects of each trait affected leaf shape. We corroborated the results using multivariate 206 multiple regressions. 207 208 Cannabinoid concentration measurements 209 The concentrations for the three cannabinoids -- CBG, THC, and CBD -- were 210 measured using gas chromatography on an SRI 86106 equipment with an MXT-35 column 211 using 197-209 mg of dried flower as described in (Brenneisen & ElSohly 1988). When 212 heated, the acidic compounds CBGA, THCA, and CBDA are turned into the neutral forms 213 CBG, THC, and CBD, which is the reason why gas chromatography only quantifies the 214 neutral forms of the compounds. 205 effects of each trait affected leaf shape. We corroborated the results using multivariate 206 multiple regressions. 214 neutral forms of the compounds. Manuscript to be reviewed 215 Given that the production of these three cannabinoids may be correlated because 216 they are part of the same biochemical pathway (Page & Boubakir 2014; Page & Stout 2017; 217 Vergara et al. 2019) and both CBDA and THCA synthases compete for the same precursor 218 molecule -CBGA-, we analyzed them using a Principal Component Analysis (PCA) to account 219 for multicollinearity and to avoid redundancies. We used a K-means cluster analysis on PC1 220 vs PC2 to visualize the different cannabinoid groups. We also added the total cannabinoid 221 concentration and measured the ratio of each cannabinoid over this total concentration 222 (Table S1). 223 224 Statistical analyses 225 We examined the associations between the production of each cannabinoid and 226 each of the measured traits at both timepoints and the . We also used cannabinoids as the 227 explanatory variables for several MANOVA models to determine whether cannabinoid PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) 215 Given that the production of these three cannabinoids may be correlated because 216 they are part of the same biochemical pathway (Page & Boubakir 2014; Page & Stout 2017; 217 Vergara et al. 2019) and both CBDA and THCA synthases compete for the same precursor 218 molecule -CBGA-, we analyzed them using a Principal Component Analysis (PCA) to account 219 for multicollinearity and to avoid redundancies. We used a K-means cluster analysis on PC1 220 vs PC2 to visualize the different cannabinoid groups. We also added the total cannabinoid 221 concentration and measured the ratio of each cannabinoid over this total concentration 222 (Table S1). Manuscript to be reviewed 228 production explained differences among the measured traits. We also corroborated the 229 MANOVA results with multivariate multiple regressions, and correlated leaf shape to 230 cannabinoid content to understand whether any association exists between those traits. 231 Finally, we generated a variance-covariance matrix to establish the association within and 232 between all phenotypic traits. 230 cannabinoid content to understand whether any association exists between those traits. 231 Finally, we generated a variance-covariance matrix to establish the association within and 232 between all phenotypic traits. 233 These data were added to the dryad repository 234 (https://doi.org/10.5061/dryad.6t1g1jwxh). Statistical analyses, including leaf geometric 235 morphometrics, were done using R (Team 2013) and the associated code is available on 236 github (https://bit.ly/38DpE8D). All figures were generated in the R Studio platform 237 version 1.1.383 (Team 2013) and enhanced with Adobe Illustrator 2019 (v23.0.6). 238 239 Results 240 241 Phenotypic trait statistical analyses (including males and females) 242 Our results show that some phenotypic traits from the IT (Table S2) are correlated 243 with each other after correcting with Bonferroni for multiple comparisons. For example, 244 height is significantly correlated to the number of branches and the number of nodes even 245 though these two traits are not significantly correlated to each other (Table S2). The 246 positive correlation between traits related to height such as number of nodes and number 247 of branches is expected. In other words, it is expected that tall plants will have multiple 248 branches and nodes. It is also expected that traits that are not related to height, such as 249 leaf-related characteristics, lack a significant correlation. 250 Similarly, the FT also shows that some traits are correlated at this stage (Table S3). 231 Finally, we generated a variance-covariance matrix to establish the association within and 232 between all phenotypic traits. 250 Similarly, the FT also shows that some traits are correlated at this stage (Table S3). 251 Some of the height-related traits show a significant correlation. For example, tall plants 250 Similarly, the FT also shows that some traits are correlated at this stage (Table S3). 251 Some of the height-related traits show a significant correlation. For example, tall plants 250 Similarly, the FT also shows that some traits are correlated at this stage (Table S3). 251 Some of the height-related traits show a significant correlation. Manuscript to be reviewed For example, tall plants PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be revie Manuscript to be reviewed 252 have long side branches as well as thicker stalks. However, as expected, some traits lack 253 association, such as stalk diameter and inflorescence number or size. 254 However, many of the significant associations within either the IT or FT are lost 255 when both timepoints are correlated between them (Table S4). These various phenotypic 256 traits are not predictive between time periods (Table S4); whether a young plant is tall or 257 short is not indicative of the adult plant’s height, and thus plants that are tall at the IT are 258 not always the same ones that are tall at the FT. Therefore, we cannot establish whether, 259 for example, tall plants also have thick stalks and numerous nodes since traits which are 260 correlated while young, are not significantly correlated while adults. In other words, the 261 plants exhibited different patterns of growth, irrespective of their initial size at the beginning of 262 the growing season. The lack of correlations between the timepoints suggests that some of 263 the statistically significant correlations may not be due to true biological variation but 264 instead due to chance and to the multiple comparisons, despite correcting with Bonferroni. 265 The lack of significance between the  correlations when compared to either the IT 266 or FT (Tables S2 and S3) suggest that some of these correlations may be spurious. The non- 267 significant correlations between the traits and their  indicates that the measured 268 characteristics do not follow a trend as they change over time. Therefore, the changes 269 during the plant’s lifetime impede future phenotypic predictions and the initial plant 270 measurements cannot be used as an indication of future success as an adult, or how the 271 phenotype will change during the plant’s lifetime. 272 Similarly, these phenotypic traits are not different between males and females 273 (Table S5). In other words, males cannot be distinguished from females with any of the 274 physical characteristics that we measured in this study. However, some trait correlations 252 have long side branches as well as thicker stalks. However, as expected, some traits lack 253 association, such as stalk diameter and inflorescence number or size. 252 have long side branches as well as thicker stalks. However, as expected, some traits lack 253 association, such as stalk diameter and inflorescence number or size. Manuscript to be reviewed 254 However, many of the significant associations within either the IT or FT are lost 255 when both timepoints are correlated between them (Table S4). These various phenotypic 256 traits are not predictive between time periods (Table S4); whether a young plant is tall or 257 short is not indicative of the adult plant’s height, and thus plants that are tall at the IT are 258 not always the same ones that are tall at the FT. Therefore, we cannot establish whether, 259 for example, tall plants also have thick stalks and numerous nodes since traits which are 260 correlated while young, are not significantly correlated while adults. In other words, the 261 plants exhibited different patterns of growth, irrespective of their initial size at the beginning of 262 the growing season. The lack of correlations between the timepoints suggests that some of 263 the statistically significant correlations may not be due to true biological variation but 264 instead due to chance and to the multiple comparisons, despite correcting with Bonferroni. 265 The lack of significance between the  correlations when compared to either the IT 272 Similarly, these phenotypic traits are not different between males and females 273 (Table S5). In other words, males cannot be distinguished from females with any of the 274 physical characteristics that we measured in this study. However, some trait correlations PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 275 do differ between the sexes (Table S6), but again are not consistent between the 276 timepoints. The only significantly different trait between both groups is the number of 277 buds in the main branch, which was taken at the FT, where males have a larger average 278 number of buds (35.75) compared to females (24.80). However, this comparison is 279 between only four males and 19 females, as most most males were removed from the field 280 before these measurements were taken, and therefore this result may again not hold any 281 true biological meaning. 282 283 Leaf shape analysis 284 Our geometric morphometric analysis on leaf shape revealed that 82.3% of all 285 variation in leaf shape is explained by the two first principal components (Figure 2). The 286 deformation grids in the top left and bottom right corners show the extreme trends in leaf 287 morphologies. Even though these morphologies are not seen in any individual plant, these 288 are the tendencies of the leaves in these furthest points of the morphospace. The 289 individuals on the top-left side of the morphospace tend to have shorter and broader 290 leaves, and as seen in the deformation grid, the first and last leaflets are pointing outwards. 291 On the other hand, individuals in the bottom right side of the morphospace tend to have a 292 longer middle leaflet, and the first and last leaflets are clumped together pointing 293 downward. Our morphometric analysis shows no significant relationship between leaf 294 shape and the plant’s sex. Therefore, both male and female plants can have similar leaf 295 shapes. 296 Our results suggest there are some trait correlations that describe leaf shape, but 297 these are not correlated to growth rates, plant size, branching architecture, 281 true biological meaning. 282 283 Leaf shape analysis 284 Our geometric morphometric analysis on leaf shape revealed that 82.3% of all 285 variation in leaf shape is explained by the two first principal components (Figure 2). The 286 deformation grids in the top left and bottom right corners show the extreme trends in leaf 287 morphologies. Even though these morphologies are not seen in any individual plant, these 288 are the tendencies of the leaves in these furthest points of the morphospace. Manuscript to be reviewed 298 phytochemistry, or plant sex (Figure S1). It appears that there could be a within-leaf effect 299 because the FELL measurements correlate within them in the IT, and serration, leaf length, 300 and number of leaflets correlate with leaf shape in the FT. However, the leaf measurements 301 show no association between timepoints (Table S7). 302 The overall trend shows leaf shape is not explained by any of the plant traits 303 measured on either timepoint (Table S7). The lack of association between a particular leaf 304 shape and plant height, or any of the other plant traits, suggests that a tall plant can have 305 broad or narrow leaves or high or low cannabinoid levels. 306 Furthermore, the MANOVA models with leaf shape as the response variable (PC1 307 and PC2, Table S7) show that the only traits that may explain leaf shape correspond to 308 other leaf measurements which are leaf length, serration, and the number of leaflets from 309 that same leaf at the FT (Figure S1). These MANOVA results confirm the within-leaf effect 310 where these measured leaf traits are associated within the same leaf. The additional 311 models that include the main effects of multiple traits support the individual MANOVA 312 results given that no significant trend in any other phenotype explains leaf shape in the IT. 313 The FT results of the within-leaf effect are confirmed with a further model including all of 314 the leaf traits (leaf length: Wilk’s λ= 0.975, F= 3.560, P=0.029740; serration: Wilk’s λ= 315 0.967, F= 4.625, P<0.012; no. leaflets Wilk’s λ = 0.8105, F= 32.483, P= 2.1e-13). However, 316 the only significant interaction effect was between leaf length and leaf width (Wilk’s λ= 317 0.967, F= 4.739, P< 0.00095). The multivariate multiple regressions confirmed the 318 MANOVA results. 319 298 phytochemistry, or plant sex (Figure S1). It appears that there could be a within-leaf effect 299 because the FELL measurements correlate within them in the IT, and serration, leaf length, 300 and number of leaflets correlate with leaf shape in the FT. However, the leaf measurements 301 show no association between timepoints (Table S7). 298 phytochemistry, or plant sex (Figure S1). It appears that there could be a within-leaf effect 299 because the FELL measurements correlate within them in the IT, and serration, leaf length, 300 and number of leaflets correlate with leaf shape in the FT. Manuscript to be reviewed The 289 individuals on the top-left side of the morphospace tend to have shorter and broader 290 leaves, and as seen in the deformation grid, the first and last leaflets are pointing outwards. 291 On the other hand, individuals in the bottom right side of the morphospace tend to have a 292 longer middle leaflet, and the first and last leaflets are clumped together pointing 293 downward. Our morphometric analysis shows no significant relationship between leaf 294 shape and the plant’s sex. Therefore, both male and female plants can have similar leaf 295 shapes. 296 Our results suggest there are some trait correlations that describe leaf shape, but 297 these are not correlated to growth rates, plant size, branching architecture, PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 321 Two clear chemotype clusters were identified in the PCA of cannabinoid chemistry 322 determined by the antagonistic relationships between THC versus CBD and CBG (Figure 3). 323 PC1 and PC2 explain 94.2% of the variation and the two distinct groups identified in figure 324 3 (represented in triangles and diamonds) covary in a pronounced way, which show 325 covariation of the original variables with two clear chemotype clusters (Figure S2). 326 The positive loadings of both CBD and CBG on PC1 (0.71 and 0.70, respectively) 327 compared to the negative loading of THC (-0.02) indicates that when CBD and CBG tend to 328 increase, THC decreases and covaries in a different direction. Therefore, the value of PC1 329 increases when CBG or CBD increase. However, on PC2, both CBG and THC have a negative 330 loading (-0.31 and -0.91 respectively), indicating a high association, while CBD has a 331 positive loading (0.28). PC2 is primarily determined by THC given its high loading value. In 332 both PC1 and PC2, CBD and THC go in different directions. 333 Cannabinoid content showed no correlation with any of the other measured 334 phenotypic traits at either time point (IT or FT) nor with the s (Table S8). These results 335 were confirmed with the MANOVAs and multivariate multiple regressions. 336 337 Leaf shape vs cannabinoid content 338 We found no relationship between leaf shape and cannabinoid content using PC1 for 339 leaf shape and PC1 for cannabinoid variation (Figure 4). Therefore, leaf shape is not 340 predictive of cannabinoid content, and individuals that are high in a particular cannabinoid 341 can have elongated or short leaves. Manuscript to be review 321 Two clear chemotype clusters were identified in the PCA of cannabinoid chemistry 322 determined by the antagonistic relationships between THC versus CBD and CBG (Figure 3). 323 PC1 and PC2 explain 94.2% of the variation and the two distinct groups identified in figure 324 3 (represented in triangles and diamonds) covary in a pronounced way, which show 325 covariation of the original variables with two clear chemotype clusters (Figure S2). 326 The positive loadings of both CBD and CBG on PC1 (0.71 and 0.70, respectively) 327 compared to the negative loading of THC (-0.02) indicates that when CBD and CBG tend to 328 increase, THC decreases and covaries in a different direction. Manuscript to be reviewed However, the leaf measurements 301 show no association between timepoints (Table S7). 302 The overall trend shows leaf shape is not explained by any of the plant traits 303 measured on either timepoint (Table S7). The lack of association between a particular leaf 304 shape and plant height, or any of the other plant traits, suggests that a tall plant can have 305 broad or narrow leaves or high or low cannabinoid levels. 304 shape and plant height, or any of the other plant traits, suggests that a tall plant can have 305 broad or narrow leaves or high or low cannabinoid levels. 306 Furthermore, the MANOVA models with leaf shape as the response variable (PC1 307 and PC2, Table S7) show that the only traits that may explain leaf shape correspond to 308 other leaf measurements which are leaf length, serration, and the number of leaflets from 309 that same leaf at the FT (Figure S1). These MANOVA results confirm the within-leaf effect 310 where these measured leaf traits are associated within the same leaf. The additional 311 models that include the main effects of multiple traits support the individual MANOVA 312 results given that no significant trend in any other phenotype explains leaf shape in the IT. 313 The FT results of the within-leaf effect are confirmed with a further model including all of 314 the leaf traits (leaf length: Wilk’s λ= 0.975, F= 3.560, P=0.029740; serration: Wilk’s λ= 315 0.967, F= 4.625, P<0.012; no. leaflets Wilk’s λ = 0.8105, F= 32.483, P= 2.1e-13). However, 316 the only significant interaction effect was between leaf length and leaf width (Wilk’s λ= 317 0.967, F= 4.739, P< 0.00095). The multivariate multiple regressions confirmed the 318 MANOVA results. Manuscript to be reviewed Manuscript to be reviewed Therefore, the value of PC1 329 increases when CBG or CBD increase. However, on PC2, both CBG and THC have a negative 330 loading (-0.31 and -0.91 respectively), indicating a high association, while CBD has a 331 positive loading (0.28). PC2 is primarily determined by THC given its high loading value. In 332 both PC1 and PC2, CBD and THC go in different directions. 321 Two clear chemotype clusters were identified in the PCA of cannabinoid chemistry 322 determined by the antagonistic relationships between THC versus CBD and CBG (Figure 3). 323 PC1 and PC2 explain 94.2% of the variation and the two distinct groups identified in figure 324 3 (represented in triangles and diamonds) covary in a pronounced way, which show 325 covariation of the original variables with two clear chemotype clusters (Figure S2). 326 The positive loadings of both CBD and CBG on PC1 (0.71 and 0.70, respectively) 327 compared to the negative loading of THC (-0.02) indicates that when CBD and CBG tend to 328 increase, THC decreases and covaries in a different direction. Therefore, the value of PC1 329 increases when CBG or CBD increase. However, on PC2, both CBG and THC have a negative 330 loading (-0.31 and -0.91 respectively), indicating a high association, while CBD has a 331 positive loading (0.28). PC2 is primarily determined by THC given its high loading value. In 332 both PC1 and PC2, CBD and THC go in different directions. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed 342 Discussion 343 In this study we examined correlations among various phenotypic traits from a 344 morphologically diverse first-generation backcross (BC1) population to understand 345 whether these multiple traits covaried with each other. We interpret these patterns of 346 correlations as being genetically-based due to our development of a diverse array of 347 progeny of known parentage all grown in a common environment. Our results suggest 348 these traits are not constrained by strong genetic correlations and the initial associations 349 between the various morphological traits in the parent generation can be broken by 350 recombination. The lack of apparent genetic correlation between these traits suggest they 351 can be selected for independently. Therefore, these traits are inherited independently, and 352 would evolve separately unless selection acts to increase or maintain correlations among 353 them. The lack of genetic correlations between the morphological traits, was also reflected 354 in a variable pattern of growth across the growing season. The dramatic trait changes and 355 the distribution variation over the course of plant growth and development (Coleman et al. 356 1994) may explain the lack of correlations between the two timepoints. This lack of 357 correlation could also signify phenotypic plasticity which is common in plants and may be a 358 form of adaptation (Schlichting 1986; Sultan 1995). Finally, in other species such as 359 sugarcane, yield has not been associated to characteristics in the stalk (ie. length, weight, 360 diameter, number) nor to other traits such as plant height (Aitken et al. 2008; Kang et al. 361 1983), so these patterns are likely not unique to Cannabis. 362 The lack of sexual dimorphism in the measured traits for this study may be specific 363 to this population, and particularly the measurements in the FT may be problematic due to 342 Discussion 342 Discussion 343 In this study we examined correlations among various phenotypic traits from a 344 morphologically diverse first-generation backcross (BC1) population to understand 345 whether these multiple traits covaried with each other. We interpret these patterns of 346 correlations as being genetically-based due to our development of a diverse array of 347 progeny of known parentage all grown in a common environment. Our results suggest 348 these traits are not constrained by strong genetic correlations and the initial associations 349 between the various morphological traits in the parent generation can be broken by 342 Discussion 343 In this study we examined correlations among various phenotypic traits from a 344 morphologically diverse first-generation backcross (BC1) population to understand 345 whether these multiple traits covaried with each other. We interpret these patterns of 346 correlations as being genetically-based due to our development of a diverse array of 347 progeny of known parentage all grown in a common environment. Our results suggest 348 these traits are not constrained by strong genetic correlations and the initial associations 349 between the various morphological traits in the parent generation can be broken by 362 The lack of sexual dimorphism in the measured traits for this study may be specific 363 to this population, and particularly the measurements in the FT may be problematic due to 364 the lack of males. Theoretical models suggest differences between males and females PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 365 particularly in wind-pollinated plants (Friedman & Barrett 2009). Additionally, sex 366 differences in Cannabis have been found in traits not measured here, such as 367 photosynthetic rates and senescence (Dzhaparidze 1969; Geber et al. 2012). Future studies 368 may include these traits to examine differences between the sexes. 365 particularly in wind-pollinated plants (Friedman & Barrett 2009). Additionally, sex 366 differences in Cannabis have been found in traits not measured here, such as 367 photosynthetic rates and senescence (Dzhaparidze 1969; Geber et al. 2012). Future studies 368 may include these traits to examine differences between the sexes. 367 photosynthetic rates and senescence (Dzhaparidze 1969; Geber et al. 2012). Future studies 368 may include these traits to examine differences between the sexes. 369 The PCA analysis facilitates the examination of shape variation for each structure 370 independently (Adams et al. 2004), allowing us to distinguish differences in leaf shape 371 (Figures 1B and 2). As size is removed during the Procrustes superimposition, it does not 372 determine the variation of the first principal component (PC1) as it does in traditional 373 morphometrics, assuring that the main source of variation explored is shape. With this 374 geometric morphometric analysis, we found that leaf shape is not related to sex (Figure 2), 375 cannabinoid production (Figure 4), or to multiple other phenotypic traits (Table S8), 376 suggesting all of these traits segregate independently. However, we did find within-leaf 377 associations between shape, leaf length, serration, and number of leaflets (Figure S1) 378 suggesting that within a single leaf some characteristics may be related to each other. 379 Because THC and CBD have attracted the most research and popular attention of all 380 the cannabinoids and their synthases both compete for the same precursor CBGA, the 381 relationship between these compounds revealed significant patterns. PC1 and PC2 for 382 cannabinoid variation (Figure 3) explain 94.2% of the variation due to the fact that there 383 are only three variables that compose the original matrix. We used a principle component 384 analysis because of the high association between these enzymes which compete for the 385 same precursor molecule (Page & Boubakir 2014; Page & Stout 2017), have similar 386 chemical structures (Brenneisen 2007; Flores-Sanchez & Verpoorte 2008) and genetic 387 sequences (Onofri et al. 2015; Vergara et al. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 2019), and may exemplify “sloppy enzymes” PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 388 (Auldridge et al. 2006; Chakraborty et al. 2013; Franco 2011). Our results show that 389 despite the evident competition for the same precursor, as seen with the negative 390 correlation between THC and CBD (Figure S2C), all of these compounds can be present 391 together. Additionally, CBG is always seen in lower levels when compared to THC and CBD 392 (Figure S2), implying that THCA and CBDA synthases are efficiently converting CBGA into 393 THCA and CBDA respectively in this population. 394 Studies suggest that THC has been selected for by breeders and growers and that 395 varieties have been bread for higher THC potency (ElSohly et al. 2016; Volkow et al. 2014). 396 Our results confirm these studies given that THC is always produced in higher quantities 397 than CBD (Figure S2), implying that THCA synthase may be a better competitor than CBDA 398 synthase in this population. 399 Variation in THC production is probably a result of gene sequence variation (Onofri 400 et al. 2015), expression levels, and gene copy number variation (Vergara et al. 2019), and 401 there are multiple genes throughout the genome associated with its production (Grassa et 402 al. 2018; Laverty et al. 2019). However, expression of these genes could be due to 403 environmental effects such as cultivation conditions (Elzinga et al. 2015), which have not 404 yet been quantified. Even though the parent plants were grown under different conditions 405 than the BC1 offspring, all of the offspring were grown under the same conditions 406 minimizing the environmental effects on the expression of these genes. 407 Although some correlations among traits are significant and make biological sense, 408 the traits that are associated with purported groups (i.e. indica and sativa) within Cannabis 409 are not correlated because of shared genetic basis. Therefore, trait correlations observed 410 are due to either shared ancestry, in the case of comparisons among subspecies or other 388 (Auldridge et al. 2006; Chakraborty et al. 2013; Franco 2011). Our results show that 389 despite the evident competition for the same precursor, as seen with the negative 390 correlation between THC and CBD (Figure S2C), all of these compounds can be present 391 together. Manuscript to be reviewed Additionally, CBG is always seen in lower levels when compared to THC and CBD 392 (Figure S2), implying that THCA and CBDA synthases are efficiently converting CBGA into 393 THCA and CBDA respectively in this population. 388 (Auldridge et al. 2006; Chakraborty et al. 2013; Franco 2011). Our results show that 389 despite the evident competition for the same precursor, as seen with the negative 390 correlation between THC and CBD (Figure S2C), all of these compounds can be present 391 together. Additionally, CBG is always seen in lower levels when compared to THC and CBD 392 (Figure S2), implying that THCA and CBDA synthases are efficiently converting CBGA into 393 THCA and CBDA respectively in this population. 394 Studies suggest that THC has been selected for by breeders and growers and that 395 varieties have been bread for higher THC potency (ElSohly et al. 2016; Volkow et al. 2014). 396 Our results confirm these studies given that THC is always produced in higher quantities 397 than CBD (Figure S2), implying that THCA synthase may be a better competitor than CBDA 398 synthase in this population. 394 Studies suggest that THC has been selected for by breeders and growers and that 395 varieties have been bread for higher THC potency (ElSohly et al. 2016; Volkow et al. 2014). 396 Our results confirm these studies given that THC is always produced in higher quantities 397 than CBD (Figure S2), implying that THCA synthase may be a better competitor than CBDA 398 synthase in this population. 399 Variation in THC production is probably a result of gene sequence variation (Onofri 400 et al. 2015), expression levels, and gene copy number variation (Vergara et al. 2019), and 401 there are multiple genes throughout the genome associated with its production (Grassa et 402 al. 2018; Laverty et al. 2019). However, expression of these genes could be due to 403 environmental effects such as cultivation conditions (Elzinga et al. 2015), which have not 404 yet been quantified. Even though the parent plants were grown under different conditions 405 than the BC1 offspring, all of the offspring were grown under the same conditions 406 minimizing the environmental effects on the expression of these genes. 399 Variation in THC production is probably a result of gene sequence variation (Onofri 400 et al. 2015), expression levels, and gene copy number variation (Vergara et al. Manuscript to be reviewed 411 major lineages, or correlated selection, in the case of modern hybrids. In other words, 412 correlations between leaf shape and phytochemistry may not be due to causal 413 relationships, but rather because breeders have intentionally (or unintentionally) selected 414 for these trait combinations. If these traits were associated due to shared ancestry or 415 correlated selection, their association can be broken by recombination. 413 relationships, but rather because breeders have intentionally (or unintentionally) selected 414 for these trait combinations. If these traits were associated due to shared ancestry or 415 correlated selection, their association can be broken by recombination. 416 This is particularly noticeable in most of the modern cultivars which are hybrids 417 from the supposed two main groups. Therefore, our study also suggests that common 418 assumptions about associations between leaf shape and chemistry may exacerbate the 419 misnaming problems of Cannabis varieties by the industry (Sawler et al. 2015; Vergara et 420 al. 2016). Given the lack of association between cannabinoids and other morphological 421 traits, the accepted standards for categorizing Cannabis types by the industry are deeply 422 flawed because their naming convention is based on sets of traits that could be 423 disassociated to each other. Additionally, other studies have shown that name is not 424 indicative of cannabinoid potency or overall chemical composition (Elzinga et al. 2015), 425 and that varieties are grouped based on reported flavors and aromas, regardless of genetic 426 relationships misclassifying closely related individuals (de la Fuente et al. 2020). This 427 misnaming problem in the Cannabis industry for both varieties and groupings (‘sativa’ and 428 ‘indica’) is greatly magnified by the fact that scientists can only study the Cannabis 429 produced by the federal government despite its inferiority in potency and diversity, and the 430 fact that it does not reflect the products distributed in consumer markets (Schwabe et al. 431 2019; Vergara et al. 2017a). It is crucial for Cannabis researchers to disseminate accurate 432 information to the public. This is not being done adequately because the scientific literature 433 is not effectively informing public policy medical decisions or providing correct 415 correlated selection, their association can be broken by recombination. 416 This is particularly noticeable in most of the modern cultivars which are hybrids 417 from the supposed two main groups. Manuscript to be reviewed 2019), and 401 there are multiple genes throughout the genome associated with its production (Grassa et 402 al. 2018; Laverty et al. 2019). However, expression of these genes could be due to 403 environmental effects such as cultivation conditions (Elzinga et al. 2015), which have not 404 yet been quantified. Even though the parent plants were grown under different conditions 405 than the BC1 offspring, all of the offspring were grown under the same conditions 406 minimizing the environmental effects on the expression of these genes. 407 Although some correlations among traits are significant and make biological sense, 408 the traits that are associated with purported groups (i.e. indica and sativa) within Cannabis 409 are not correlated because of shared genetic basis. Therefore, trait correlations observed 410 are due to either shared ancestry, in the case of comparisons among subspecies or other PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 434 information on harm reduction (Hutchison et al. 2019). This lack of information has major 435 ramifications for growers, breeders, regulators, and consumers, particularly for medical 436 patients who must understand what they are consuming to achieve the greatest benefit for 437 their individual needs. 438 In order to improve the quality and efficacy of the Cannabis consumed by medical 439 patients, it is important that unbiased, accurate, and precise chemotype testing should be 440 made mandatory. However, testing facilities do not have universally established standards, 441 as cannabinoid measurements vary widely across laboratories (Jikomes & Zoorob 2018), 442 and there are no supervising institutions that oversee testing entities or their 443 methodologies, making differences in cannabinoid reporting inevitable . 444 434 information on harm reduction (Hutchison et al. 2019). This lack of information has major 435 ramifications for growers, breeders, regulators, and consumers, particularly for medical 436 patients who must understand what they are consuming to achieve the greatest benefit for 437 their individual needs. Manuscript to be reviewed Therefore, our study also suggests that common 418 assumptions about associations between leaf shape and chemistry may exacerbate the 419 misnaming problems of Cannabis varieties by the industry (Sawler et al. 2015; Vergara et 420 al. 2016). Given the lack of association between cannabinoids and other morphological 421 traits, the accepted standards for categorizing Cannabis types by the industry are deeply 422 flawed because their naming convention is based on sets of traits that could be 423 disassociated to each other. Additionally, other studies have shown that name is not 424 indicative of cannabinoid potency or overall chemical composition (Elzinga et al. 2015), 425 and that varieties are grouped based on reported flavors and aromas, regardless of genetic 426 relationships misclassifying closely related individuals (de la Fuente et al. 2020). This 427 misnaming problem in the Cannabis industry for both varieties and groupings (‘sativa’ and 428 ‘indica’) is greatly magnified by the fact that scientists can only study the Cannabis 429 d d b th f d l t d it it i f i it i t d di it d th PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 460 Author Contributions 461 D.V. and C.M.F. analyzed the data and wrote the first draft of the manuscript; E.L.H. 462 calculated the Gmatrix, and measured BC1 plants; B.H. donated, grew, and cared for all 463 plants-parentals, F1, and BC1; J.H. grew, cared, and measured the BC1 plants; D.V and N.C.K. 464 conceived and lead the project and measured BC1 plants. All authors contributed to 465 manuscript preparation Manuscript to be reviewed Holloway and two anonymous reviewers for 458 their informative comments which greatly improved this paper. 445 Conclusions 446 The fact that most of the phenotypic traits are not genetically correlated has 447 significant implications for both Cannabis breeders and commercial growers. If these traits 448 are not linked, as previously thought, then it is possible to select for new combination of 449 traits when breeding for novel varieties. This expands the possibility of generating varieties 450 with a unique combination of traits providing unforeseen medicinal and industrial value. 451 Future breeding can be done to maximize combinations of these traits. 452 453 454 Acknowledgments 455 We would like to thank J. A. Fuentes for help with the morphometric analysis, figure 456 construction and for comments on the manuscript, to C. Pogoda, A. Schwabe, and R. Miller PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) 446 The fact that most of the phenotypic traits are not genetically correlated has 447 significant implications for both Cannabis breeders and commercial growers. If these traits 448 are not linked, as previously thought, then it is possible to select for new combination of 449 traits when breeding for novel varieties. This expands the possibility of generating varieties 450 with a unique combination of traits providing unforeseen medicinal and industrial value. 451 Future breeding can be done to maximize combinations of these traits. Manuscript to be reviewed 457 for comments on the manuscript, and to A. Holloway and two anonymous reviewers for 458 their informative comments which greatly improved this paper. 459 460 Author Contributions 461 D.V. and C.M.F. analyzed the data and wrote the first draft of the manuscript; E.L.H. 462 calculated the Gmatrix, and measured BC1 plants; B.H. donated, grew, and cared for all 463 plants-parentals, F1, and BC1; J.H. grew, cared, and measured the BC1 plants; D.V and N.C 464 conceived and lead the project and measured BC1 plants. All authors contributed to 465 manuscript preparation. 466 467 Legends Supplementary Tables and Figures 468 469 Table S1. All data. Data used for all analyses with each plant (column 1) its sex (column 470 unique id (column 3), the IT date (column 4), measurements for the IT (columns 5-12), t 471 FT (column 13), measurements for the FT (columns 14-21), deltas for four measurement 472 (columns 22-25), flower weight for cannabinoid measurement (column 26), the addition 473 all cannabinoids (column 27), the percentage of each cannabinoid over the total (column 474 28-30), the ratio of each cannabinoid to each other (columns 31-33), the PC values for th 475 cannabinoids (columns 34-36), leaf measurements for the FT (columns 37-39), and the l 476 shape PC values (columns 41-56). 477 478 Table S2 Within IT and delta () correlations. Correlations within the initial time poin 479 and  for the eight traits measured in time point one and the four  changes 480 481 Table S3 Within FT and delta () correlations. Correlations within the final time poin 482 and  for the eight traits measured in the final timepoint, the four  changes and the four 483 leaf-morphologic traits from the leaves used for geometric morphometrics (leaflet numb 484 serration in the middle leaflet, leaf width and leaf length). 485 486 Table S4 Between time-point correlations. Correlations between the eight traits from 487 time-point one against the eight traits from time point two and against the four leaf- 488 morphologic traits from the leaves used for geometric morphometrics (leaflet number, 489 serration in the middle leaflet, leaf width and leaf length). 490 457 for comments on the manuscript, and to A. Holloway and two anonymous reviewers for 458 their informative comments which greatly improved this paper. 457 for comments on the manuscript, and to A. 467 Legends Supplementary Tables and Figures 468 468 469 Table S1. All data. Data used for all analyses with each plant (column 1) its sex (column 2), 470 unique id (column 3), the IT date (column 4), measurements for the IT (columns 5-12), the 471 FT (column 13), measurements for the FT (columns 14-21), deltas for four measurements 472 (columns 22-25), flower weight for cannabinoid measurement (column 26), the addition of 473 all cannabinoids (column 27), the percentage of each cannabinoid over the total (columns 474 28-30), the ratio of each cannabinoid to each other (columns 31-33), the PC values for the 475 cannabinoids (columns 34-36), leaf measurements for the FT (columns 37-39), and the leaf 476 shape PC values (columns 41-56). 468 469 Table S1. All data. Data used for all analyses with each plant (column 1) its sex (column 2), 470 unique id (column 3), the IT date (column 4), measurements for the IT (columns 5-12), the 471 FT (column 13), measurements for the FT (columns 14-21), deltas for four measurements 472 (columns 22-25), flower weight for cannabinoid measurement (column 26), the addition of 473 all cannabinoids (column 27), the percentage of each cannabinoid over the total (columns 474 28-30), the ratio of each cannabinoid to each other (columns 31-33), the PC values for the 475 cannabinoids (columns 34-36), leaf measurements for the FT (columns 37-39), and the leaf 476 shape PC values (columns 41-56). 68 469 Table S1. All data. Data used for all analyses with each plant (column 1) its sex (column 2), 470 unique id (column 3), the IT date (column 4), measurements for the IT (columns 5-12), the 471 FT (column 13), measurements for the FT (columns 14-21), deltas for four measurements 472 (columns 22-25), flower weight for cannabinoid measurement (column 26), the addition of 473 all cannabinoids (column 27), the percentage of each cannabinoid over the total (columns 474 28-30), the ratio of each cannabinoid to each other (columns 31-33), the PC values for the 475 cannabinoids (columns 34-36), leaf measurements for the FT (columns 37-39), and the leaf 476 shape PC values (columns 41-56). 477 478 Table S2 Within IT and delta () correlations. Correlations within the initial time point 479 and  for the eight traits measured in time point one and the four  changes 480 478 Table S2 Within IT and delta () correlations. Manuscript to be reviewed 491 Table S5. t-tests between sex and phenotypic traits. t-tests between sex and the 492 phenotypic traits measured on both timepoints including leaf shape measurements in the 493 IT, FT, and . 494 493 IT, FT, and . 494 495 Table S6. Difference in trait correlations between sex. Difference between the 496 correlations of the various phenotypic traits between males and females in the IT, FT, and 497 . 498 499 Table S7. MANOVA for leaf shape against all phenotypic traits. MANOVA associations 500 between PC1 and PC2 for leaf shape against all phenotypic traits from the IT, FT, and . 501 502 Table S8. Correlations between three cannabinoids CBG, CBD, and THC, and all 503 phenotypic traits. Correlations between the three cannabinoids all phenotypic traits from 504 the IT, FT, and . 505 506 Table S8. GMatrix for variance and covariance between all phenotypic traits 507 measured. Variances and covariances between all phenotypic traits measured at both 508 timepoints. 509 510 Figure S1. PC1 for shape versus three traits. Leaf length is not correlated to PC1 (A), 511 while serration in the middle leaflet (B) and number of leaflets (C) are marginally and 512 significantly correlated, respectively. Each panel at the top has the deformation grids at the 513 minimum (left) and maximum (right) values for length (A), serration in the middle leaflet 514 (B), and number of leaflets (C). Males are shown in squares, females in open circles. The 515 insets show the p and r values and the variance and covariance between both variables, 516 also given in the variance-covariance matrix (Table S8). 517 518 Figure S2. Relationships between percent THC, CBD, and CBG. A and B show a positive 519 correlation between CBG vs CBD, and THC vs CBG, respectively. C displays the negative 520 linear relationship between THC and CBD. Notice the differences in the axis representing 521 the disparity in production of the different cannabinoids. Males are shown in squares, 522 females in open circles. The insets show the p and r values and the variance and covariance 523 between both variables, also given in the variance-covariance matrix (Table S8). 524 525 526 References 527 Adams DC, and Otárola-Castillo E. 2013. Geomorph: an R package for the collection and analysis of 528 geometric morphometric shape data. Methods in Ecology and Evolution 4:393-399. 529 Adams DC, Rohlf FJ, and Slice DE. 2004. 467 Legends Supplementary Tables and Figures 468 Correlations within the initial time point 479 and  for the eight traits measured in time point one and the four  changes 481 Table S3 Within FT and delta () correlations. Correlations within the final time point 482 and  for the eight traits measured in the final timepoint, the four  changes and the four 483 leaf-morphologic traits from the leaves used for geometric morphometrics (leaflet number, 484 serration in the middle leaflet, leaf width and leaf length). 486 Table S4 Between time-point correlations. Correlations between the eight traits from 487 time-point one against the eight traits from time point two and against the four leaf- 488 morphologic traits from the leaves used for geometric morphometrics (leaflet number, 489 serration in the middle leaflet, leaf width and leaf length). 490 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Geometric morphometrics: ten years of progress following the 530 'revolution'. Italian Journal of Zoology 71:5-16. 531 Aitken K, Hermann S, Karno K, Bonnett G, McIntyre L, and Jackson P. 2008. Genetic control of yield 532 related stalk traits in sugarcane. Theoretical and Applied Genetics 117:1191-1203. 494 495 Table S6. Difference in trait correlations between sex. Difference between the 496 correlations of the various phenotypic traits between males and females in the IT, FT, and 497 . 498 Manuscript to be reviewed Sex in plants, part II. Biochemical and physiological sex differences in dioecious 552 plants: problem of influencing sex formation. Jerusalem: IPST Press. 551 Dzhaparidze L. 1969. Sex in plants, part II. Biochemical and physiological sex differences in dioecious 552 plants: problem of influencing sex formation. Jerusalem: IPST Press. 553 ElSohly MA, Mehmedic Z, Foster S, Gon C, Chandra S, and Church JC. 2016. Changes in cannabis potency 554 over the last 2 decades (1995–2014): analysis of current data in the United States. Biological 555 psychiatry 79:613-619. 553 ElSohly MA, Mehmedic Z, Foster S, Gon C, Chandra S, and Church JC. 2016. Changes in cannabis potency 554 over the last 2 decades (1995–2014): analysis of current data in the United States. Biological 555 psychiatry 79:613-619. 556 ElSohly MA, and Slade D. 2005. Chemical constituents of marijuana: the complex mixture of natural 557 cannabinoids. Life sciences 78:539-548. 556 ElSohly MA, and Slade D. 2005. Chemical constituents of marijuana: the complex mixture of natural 557 cannabinoids. Life sciences 78:539-548. 558 Elzinga S, Fischedick J, Podkolinski R, and Raber J. 2015. Cannabinoids and terpenes as chemotaxonomic 559 markers in cannabis. Nat Prod Chem Res 3:2. 560 558 Elzinga S, Fischedick J, Podkolinski R, and Raber J. 2015. Cannabinoids and terpenes as chemotaxonomic 559 markers in cannabis Nat Prod Chem Res 3:2 560 Evans D. 2013. The economic impacts of marijuana legalization. 561 Flores-Sanchez IJ, and Verpoorte R. 2008. Secondary metabolism in cannabis. Phytochemistry reviews 562 7:615-639. 561 Flores-Sanchez IJ, and Verpoorte R. 2008. Secondary metabolism in cannabis. Phytochemistry reviews 562 7:615-639. 563 Franco OL. 2011. Peptide promiscuity: an evolutionary concept for plant defense. FEBS letters 585:995- 564 1000. 565 Friedman J, and Barrett SC. 2009. Wind of change: new insights on the ecology and evolution of 566 pollination and mating in wind-pollinated plants. Annals of Botany 103:1515-1527. 567 Gagne SJ, Stout JM, Liu E, Boubakir Z, Clark SM, and Page JE. 2012. Identification of olivetolic acid cyclase 568 from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proceedings of the 569 National Academy of Sciences 109:12811-12816. 570 Geber MA, Dawson TE, and Delph LF. 2012. Gender and sexual dimorphism in flowering plants: Springer 571 Science & Business Media. 572 Grassa CJ, Wenger JP, Dabney C, Poplawski SG, Motley ST, Michael TP, Schwartz CJ, and Weiblen GD. 573 2018. A complete Cannabis chromosome assembly and adaptive admixture for elevated 574 cannabidiol (CBD) content. 498 499 Table S7. MANOVA for leaf shape against all phenotypic traits. MANOVA associations 500 between PC1 and PC2 for leaf shape against all phenotypic traits from the IT, FT, and . 501 502 Table S8. Correlations between three cannabinoids CBG, CBD, and THC, and all 503 phenotypic traits. Correlations between the three cannabinoids all phenotypic traits from 504 the IT, FT, and . 505 531 Aitken K, Hermann S, Karno K, Bonnett G, McIntyre L, and Jackson P. 2008. Genetic control of yield 532 related stalk traits in sugarcane. Theoretical and Applied Genetics 117:1191-1203. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 533 Auldridge ME, McCarty DR, and Klee HJ. 2006. Plant carotenoid cleavage oxygenases and their 534 apocarotenoid products. Current opinion in plant biology 9:315-321. 533 Auldridge ME, McCarty DR, and Klee HJ. 2006. Plant carotenoid cleavage oxygena 533 Auldridge ME, McCarty DR, and Klee HJ. 2006. Plant carotenoid cleavage oxygenases and their 534 apocarotenoid products. Current opinion in plant biology 9:315-321. 535 Brenneisen R. 2007. Chemistry and analysis of phytocannabinoids and other Cannabis constituents. 536 Marijuana and the Cannabinoids: Springer, 17-49. 535 Brenneisen R. 2007. Chemistry and analysis of phytocannabinoids and other Cannabis constituents. 536 Marijuana and the Cannabinoids: Springer, 17-49. 537 Brenneisen R, and ElSohly MA. 1988. Chromatographic and spectroscopic profiles of Cannabis of 538 different origins: Part I. Journal of Forensic Science 33:1385-1404. 537 Brenneisen R, and ElSohly MA. 1988. Chromatographic and spectroscopic profiles of Cannabis of 538 different origins: Part I. Journal of Forensic Science 33:1385-1404. 539 Chakraborty S, Minda R, Salaye L, Dandekar AM, Bhattacharjee SK, and Rao BJ. 2013. Promiscuity-based 540 enzyme selection for rational directed evolution experiments. Enzyme Engineering: Methods 541 and Protocols:205-216. 539 Chakraborty S, Minda R, Salaye L, Dandekar AM, Bhattacharjee SK, and Rao BJ. 2013. Promiscuity-based 540 enzyme selection for rational directed evolution experiments. Enzyme Engineering: Methods 541 and Protocols:205-216. 542 Clarke R, and Merlin M. 2013. Cannabis: evolution and ethnobotany: Univ of California Press. 542 Clarke R, and Merlin M. 2013. Cannabis: evolution and ethnobotany: Univ of California Press. 543 Coleman JS, McConnaughay KD, and Ackerly DD. 1994. Interpreting phenotypic variation in plants. 544 Trends in Ecology & Evolution 9:187-191. 543 Coleman JS, McConnaughay KD, and Ackerly DD. 1994. Interpreting phenotypic variation in plants. 544 Trends in Ecology & Evolution 9:187-191. 545 de la Fuente A, Zamberlan F, Ferrán AS, Carrillo F, Tagliazucchi E, and Pallavicini C. 2020. Relationship 546 among subjective responses, flavor, and chemical composition across more than 800 547 commercial cannabis varieties. Journal of Cannabis Research 2:1-18. 545 de la Fuente A, Zamberlan F, Ferrán AS, Carrillo F, Tagliazucchi E, and Pallavicini C. 2020. Relationship 546 among subjective responses, flavor, and chemical composition across more than 800 547 commercial cannabis varieties. Journal of Cannabis Research 2:1-18. 548 Divashuk MG, Alexandrov OS, Razumova OV, Kirov IV, and Karlov GI. 2014. Molecular cytogenetic 549 characterization of the dioecious Cannabis sativa with an XY chromosome sex determination 550 system. Plos One 9:e85118. 551 Dzhaparidze L. 1969. Manuscript to be reviewed bioRxiv. 10.1101/458083 575 Hillig KW. 2005. Genetic evidence for speciation in Cannabis (Cannabaceae). Genetic Resources and Crop 576 Evolution 52:161-180. 10.1007/s10722-003-4452-y 577 Hutchison KE, Bidwell LC, Ellingson JM, and Bryan AD. 2019. Cannabis and Health Research: Rapid 578 P R i I i R h D i V l i H l h 577 Hutchison KE, Bidwell LC, Ellingson JM, and Bryan AD. 2019. Cannabis and Health Research: Rapid 578 Progress Requires Innovative Research Designs. Value in Health. 579 Jikomes N, and Zoorob M. 2018. The cannabinoid content of legal cannabis in Washington state varies 580 systematically across testing facilities and popular consumer products. Scientific Reports 8:4519. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 581 Kang MS, Miller JD, and Tai P. 1983. Genetic and Phenotypic Path Analyses and Heritability in Sugarcane 582 1. Crop Science 23:643-647. 581 Kang MS, Miller JD, and Tai P. 1983. Genetic and Phenotypic Path Analyses and Heritability in Sugarcane 582 1. Crop Science 23:643-647. 583 Kirsch J. 2018. Taxation and Welfare in the Cannabis Industry: Evidence from Colorado Edibles 2014- 584 2016. 585 Kovalchuk I, Pellino M, Rigault P, van Velzen R, Ebersbach J, R. Ashnest J, Mau M, Schranz M, Alcorn J, 586 and Laprairie R. 2020. The Genomics of Cannabis and Its Close Relatives. Annual review of plant 587 biology 71. 588 Laverty KU, Stout JM, Sullivan MJ, Shah H, Gill N, Holbrook L, Deikus G, Sebra R, Hughes TR, and Page JE. 589 2019. A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the 590 THC/CBD acid synthase loci. Genome Research 29:146-156. 591 Li HL. 1973. An archaeological and historical account of cannabis in China. Economic Botany 28:437-448. 592 Li HL. 1974. Origin and use of Cannabis in Eastern Asia; Linguistic-cultural implications. Economic Botany 593 28:293-301. 10.1007/bf02861426 592 Li HL. 1974. Origin and use of Cannabis in Eastern Asia; Linguistic-cultural implications. Economic Botany 593 28:293-301. 10.1007/bf02861426 594 Lynch RC, Vergara D, Tittes S, White K, Schwartz CJ, Gibbs MJ, Ruthenburg TC, deCesare K, Land DP, and 595 Kane NC. 2016. Genomic and Chemical Diversity in Cannabis. Critical Reviews in Plant Sciences 596 35:349-363. 10.1080/07352689.2016.1265363 Manuscript to be reviewed 628 Russo EB. 2011. Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage 629 effects. British Journal of Pharmacology 163:1344-1364. 10.1111/j.1476-5381.2011.01238.x 630 Russo EB, Jiang H-E, Li X, Sutton A, Carboni A, del Bianco F, Mandolino G, Potter DJ, Zhao Y-X, Bera S, 631 Zhang Y-B, Lue E-G, Ferguson DK, Hueber F, Zhao L-C, Liu C-J, Wang Y-F, and Li C-S. 2008. 632 Phytochemical and genetic analyses of ancient cannabis from Central Asia. Journal of 633 Experimental Botany 59:4171-4182. 10.1093/jxb/ern260 634 Russo EB, and McPartland JM. 2003. Cannabis is more than simply Δ9-tetrahydrocannabinol. 635 Psychopharmacology 165:431-432. 636 Sawler J, Stout JM, Gardner KM, Hudson D, Vidmar J, Butler L, Page JE, and Myles S. 2015. The Genetic 637 Structure of Marijuana and Hemp. Plos One 10:e0133292. 638 Schlichting CD. 1986. The evolution of phenotypic plasticity in plants. Annual Review of Ecology and 639 Systematics 17:667-693. 640 Schwabe AL, Hansen CJ, Hyslop RM, and McGlaughlin ME. 2019. Research grade marijuana supplied by 641 the National Institute on Drug Abuse is genetically divergent from commercially available 642 <em>Cannabis</em>. bioRxiv:592725. 10.1101/592725 643 Schwabe AL, and McGlaughlin ME. 2019. Genetic tools weed out misconceptions of strain reliability in 644 Cannabis sativa: implications for a budding industry. Journal of Cannabis Research 1:3. 645 10.1186/s42238-019-0001-1 646 Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, and Shoyama Y. 2005. 647 Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is 648 secreted into the storage cavity of the glandular trichomes. Plant and Cell Physiology 46:1578- 649 1582. Sultan SE. 1995. Phenotypic plasticity and plant adaptation. Acta botanica neerlandica 44: 651 Swift W, Wong A, Li KM, Arnold JC, and McGregor IS. 2013. Analysis of cannabis seizures in NSW, 652 Australia: cannabis potency and cannabinoid profile. Plos One 8:e70052. Wong A, Li KM, Arnold JC, and McGregor IS. 2013. Analysis of cannabis seizures in NSW, ustralia: cannabis potency and cannabinoid profile Plos One 8:e70052 Swift W, Wong A, Li KM, Arnold JC, and McGregor IS. 2013. Analysis of cannabis seizures i Australia: cannabis potency and cannabinoid profile. Plos One 8:e70052. 653 Takeda S, Misawa K, Yamamoto I, and Watanabe K. 2008. Cannabidiolic acid as a selective 654 cyclooxygenase-2 inhibitory component in cannabis. Drug metabolism and disposition 36:1917- 655 1921. 656 Takeda S, Okajima S, Miyoshi H, Yoshida K, Okamoto Y, Okada T, Amamoto T, Watanabe K, Omiecinski 657 CJ, and Aramaki H. 2012. 596 35:349-363. 10.1080/07352689.2016.1265363 597 McPartland JM. 2017. Cannabis sativa and Cannabis indica versus “Sativa” and “Indica”. Cannabis sativa 598 L-Botany and Biotechnology: Springer, 101-121. 599 McPartland JM, and Russo EB. 2001. Cannabis and cannabis extracts: greater than the sum of their 600 parts? Journal of Cannabis Therapeutics 1:103-132. 601 Onofri C, de Meijer EPM, and Mandolino G. 2015. Sequence heterogeneity of cannabidiolic-and 602 tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical 603 phenotype. Phytochemistry. 604 Orser C, Johnson S, Speck M, Hilyard A, and Afia I. 2017. Terpenoid Chemoprofiles Distinguish Drug-type 605 Cannabis sativa L. Cultivars in Nevada. Natural Products Chemistry and Research 6. 606 10.4172/2475-7675.1000304s 607 Padgitt-Cobb LK, Kingan SB, Wells J, Elser J, Kronmiller B, Moore D, Concepcion G, Peluso P, Rank D, 608 Jaiswal P, Henning J, and Hendrix DA. 2019. A phased, diploid assembly of the Cascade hop 609 <em>(Humulus lupulus)</em> genome reveals patterns of selection and haplotype variation. 610 bioRxiv:786145. 10.1101/786145 Page JE, and Boubakir Z. 2014. Aromatic prenyltransferase from Cannabis. Google Patents Page JE, and Stout JM. 2017. Cannabichromenic acid synthase from Cannabis sativa. Goog 613 Peil A, Flachowsky H, Schumann E, and Weber WE. 2003. Sex-linked AFLP markers indicate a 614 pseudoautosomal region in hemp (Cannabis sativa L.). Theoretical and Applied Genetics 615 107:102-109. 616 Pertwee RG. 1988. The central neuropharmcology of psychotropic cannabinoids. Pharmacology & 617 therapeutics 36:189-261. 618 Pertwee RG. 1997. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacology & therapeutics 619 74:129-180. 620 Pertwee RG. 2004. Pharmacological and therapeutic targets for Δ 9 tetrahydrocannabinol and 621 cannabidiol. Euphytica 140:73-82. 622 Reimann-Philipp U, Speck M, Orser C, Johnson S, Hilyard A, Turner H, Stokes AJ, and Small-Howard AL. 623 2019. Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey 624 of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples. 625 Cannabis and Cannabinoid Research. 626 Russo EB. 2007. History of cannabis and its preparations in saga, science, and sobriquet. Chemistry & 627 Biodiversity 4:1614-1648. 10.1002/cbdv.200790144 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed 676 Zirpel B, Kayser O, and Stehle F. 2018. Elucidation of structure-function relationship of THCA and CBDA 677 synthase from Cannabis sativa L. Journal of biotechnology 284:17-26. 678 679 680 681 682 683 684 Parental Generation F1 Generation BC1 Generation 1 2 3 4 5 7 6 8 9 10 A B 685 Figure 1. Pedigree and landmarks. Panel A: Pedigree of a first-generation backcross (BC1) between a male 686 marijuana-type Afghan Kush and a female Carmagnola hemp. The F1 generation was backcrossed with a 687 brother from the original male Afghan Kush to produce the BC1 Generation. Panel B: Exemplar leaf depicting 688 the ten points used for leaf shape analysis. The ten points measured the first, central, and last leaflets. 689 690 684 Parental Generation F1 Generation BC1 Generation 1 2 3 4 5 7 6 8 9 10 A B 685 Figure 1. Pedigree and landmarks. Panel A: Pedigree of a first-generation backcross (BC1) between a male 686 marijuana-type Afghan Kush and a female Carmagnola hemp. The F1 generation was backcrossed with a 687 brother from the original male Afghan Kush to produce the BC1 Generation. Panel B: Exemplar leaf depicting 688 the ten points used for leaf shape analysis. The ten points measured the first, central, and last leaflets. 689 684 Parental Generation F1 Generation BC1 Generation 1 2 3 4 5 7 6 8 9 10 A B 685 Figure 1. Pedigree and landmarks. Panel A: Pedigree of a first-generation backcross (BC1) between a male 686 marijuana-type Afghan Kush and a female Carmagnola hemp. The F1 generation was backcrossed with a 687 brother from the original male Afghan Kush to produce the BC1 Generation. Panel B: Exemplar leaf depicting 688 the ten points used for leaf shape analysis. The ten points measured the first, central, and last leaflets. 689 Parental Generation F1 Generation BC1 Generation 1 2 3 4 5 7 6 8 9 10 A B A 685 Figure 1. Pedigree and landmarks. Panel A: Pedigree of a first-generation backcross (BC1) between a male 686 marijuana-type Afghan Kush and a female Carmagnola hemp. The F1 generation was backcrossed with a 687 brother from the original male Afghan Kush to produce the BC1 Generation. Panel B: Exemplar leaf depicting 688 the ten points used for leaf shape analysis. Manuscript to be reviewed Cannabidiolic acid, a major cannabinoid in fiber-type cannabis, is an 658 inhibitor of MDA-MB-231 breast cancer cell migration. Toxicology letters 214:314-319. 659 Team RC. 2013. R: A language and environment for statistical computing. 660 van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, and Page JE. 2011. The draft genome 661 and transcriptome of Cannabis sativa. Genome Biology 12. 10.1186/gb-2011-12-10-r102 662 Vergara D, Baker H, Clancy K, Keepers KG, Mendieta JP, Pauli CS, Tittes SB, White KH, and Kane NC. 663 2016. Genetic and Genomic Tools for Cannabis sativa. Critical Reviews in Plant Sciences 35:364- 664 377. 10.1080/07352689.2016.1267496 665 Vergara D, Bidwell LC, Gaudino R, Torres A, Du G, Ruthenburg TC, deCesare K, Land DP, Hutchison KE, 666 and Kane NC. 2017a. Compromised External Validity: Federally Produced Cannabis Does Not 667 Reflect Legal Markets. Scientific Reports 7:46528. g f p 668 Vergara D, Fuentes JA, Stoy KS, and Lively CM. 2017b. Evaluating shell variation across different 669 populations of a freshwater snail Molluscan Research 37:120 132 668 Vergara D, Fuentes JA, Stoy KS, and Lively CM. 2017b. Evaluating shell variation across different 669 populations of a freshwater snail. Molluscan Research 37:120-132. 670 Vergara D, Huscher EL, Keepers KG, Givens RM, Cizek CG, Torres A, Gaudino R, and Kane NC. 2019. Gene 671 copy number is associated with phytochemistry in <em>Cannabis sativa</em>. bioRxiv:736181. 672 10.1101/736181 673 Volkow ND, Baler RD, Compton WM, and Weiss SRB. 2014. Adverse Health Effects of Marijuana Use. 674 New England Journal of Medicine 370:2219-2227. 10.1056/NEJMra1402309 675 Weisstein EW. 2004. Bonferroni correction. https://mathworld wolfram com/. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) 676 Zirpel B, Kayser O, and Stehle F. 2018. Elucidation of structure-function relationship of THCA and CBDA 677 synthase from Cannabis sativa L. Journal of biotechnology 284:17-26. The ten points measured the first, central, and last leaflets. 689 690 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 691 692 Figure 2: Geometric morphometric analysis of leaf shape. The two first PC explain 82.3% of the leaf shape 693 variation, which is not related to sex (males are squares, females are open circles). The deformation grids 694 mostly show the leaf deformations on PC1 which contains most of the variation. The grids show that 695 individuals in the top left have squatter, broader leaves with the first and last leaflets pointing outwards, 696 while those on the bottom right side of the morphospace have thinner leaves with a long mid-leaflet and the 697 two outer leaflets lumped together pointing downwards. 698 691 691 692 Figure 2: Geometric morphometric analysis of leaf shape. The two first PC explain 82.3% of the leaf shape 693 variation, which is not related to sex (males are squares, females are open circles). The deformation grids 694 mostly show the leaf deformations on PC1 which contains most of the variation. The grids show that 695 individuals in the top left have squatter, broader leaves with the first and last leaflets pointing outwards, 696 while those on the bottom right side of the morphospace have thinner leaves with a long mid-leaflet and the 697 two outer leaflets lumped together pointing downwards. 698 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) 701 702 Figure 3. PC1 and PC2 for cannabinoid variation. PC1 and PC2 explain 94.2% of the overall cannabinoid 703 variation for the three cannabinoids measured. There are two clear groups in the graph, squares and 704 triangles, which correspond to the two clear cannabinoid clusters. The overall trend shows that the squares 705 have low CBG and CBD and high THC. The triangles show high CBD and low THC. 706 Manuscript to be review Manuscript to be reviewed 701 702 Figure 3. PC1 and PC2 for cannabinoid variation. PC1 and PC2 explain 94.2% of the overall cannabinoid 703 variation for the three cannabinoids measured. There are two clear groups in the graph, squares and 704 triangles, which correspond to the two clear cannabinoid clusters. The overall trend shows that the squares 705 have low CBG and CBD and high THC. The triangles show high CBD and low THC. 706 707 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed 708 709 Figure 4: Correlation between leaf shape (PC1) and cannabinoid variation (PC1). Leaf shape is not 710 significantly to cannabinoid variation even at the most extreme points in the morphospace. Males are shown 711 in squares and females in open circles. 712 708 709 Figure 4: Correlation between leaf shape (PC1) and cannabinoid variation (PC1). Leaf shape is not 710 significantly to cannabinoid variation even at the most extreme points in the morphospace. Males are shown 711 in squares and females in open circles. 712 PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Figure 1 Figure 1. Pedigree and landmarks. Figure 2 Figure 2: Geometric morphometric analysis of leaf shape. Manuscript to be reviewed Figure 1. Pedigree and landmarks. Panel A: Pedigree of a first-generation backcross (BC1) between a male marijuana-type Afghan Kush and a female Carmagnola hemp. The F1 generation was backcrossed with a brother from the original male Afghan Kush to produce the BC1 Generation. Panel B: Exemplar leaf depicting the ten points used for leaf shape analysis. The ten points measured the first, central, and last leaflets. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Manuscript to be reviewed Figure 2: Geometric morphometric analysis of leaf shape. The two first PC explain 82.3% of the leaf shape variation, which is not related to sex (males are squares, females are open circles). The deformation grids mostly show the leaf deformations on PC1 which contains most of the variation. The grids show that individuals in the top left have squatter, broader leaves with the first and last leaflets pointing outwards, while those on the bottom right side of the morphospace have thinner leaves with a long mid-leaflet and the two outer leaflets lumped together pointing downwards. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Manuscript to be reviewed Figure 3 Figure 3. PC1 and PC2 for cannabinoid variation. PC1 and PC2 explain 94.2% of the overall cannabinoid variation for the three cannabinoids measured. There are two clear groups in the graph, squares and triangles, which correspond to the two clear cannabinoid clusters. The overall trend shows that the squares have low CBG and CBD and high THC. The triangles show high CBD and low THC. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020) Figure 4 Figure 4: Correlation between leaf shape (PC1) and cannabinoid variation (PC1). Figure 4: Correlation between leaf shape (PC1) and cannabinoid variation (PC1). Leaf shape is not significantly to cannabinoid variation even at the most extreme points in the morphospace. Males are shown in squares and females in open circles. PeerJ reviewing PDF | (2020:08:51989:1:0:NEW 21 Nov 2020)
https://openalex.org/W2363282561
https://www.scielo.br/j/rsbmt/a/Rx5mNrvLh3PzTCRpkXrMDdQ/?lang=en&format=pdf
English
null
Secondary transmission of cryptosporidiosis associated with well water consumption: two case studies
Revista da Sociedade Brasileira de Medicina Tropical
2,016
cc-by
1,918
ABSTRACT Cryptosporidiosis is a very prominent disease in the fi eld of public health, and usually causes diarrhea. We describe two immunocompetent patients who presented with chronic diarrhea that was ultimately found to be caused by continuous exposure to well water contaminated with the microbial cysts (oocysts) of the Cryptosporidium spp parasite. We describe the patients’ histories and possible explanations for their prolonged symptoms. Keywords: Cryptosporidium spp. Well water. Diarrhea. Keywords: Cryptosporidium spp. Well water. Diarrhea. feces and separate, hard lumps. From March 2014 onwards, the patient reported progressive worsening, with an increase in the volume of feces and number of evacuations (up to six per day), in addition to fl atulence and abdominal cramps in the lower abdomen that were alleviated after evacuation. The symptoms were even more pronounced after the ingestion of fatty or dairy foods. The patient reported weight loss of four kilograms in a month, as well as abdominal distension and rigidness. Although the patient reported that she had ceased psychiatric treatment, she had been taking trazodone hydrochloride for six months. Revista da Sociedade Brasileira de Medicina Tropical 49(2):260-262, Mar-Apr, 2016 http://dx.doi.org/10.1590/0037-8682-0232-2015 Revista da Sociedade Brasileira de Medicina Tropical 49(2):260-262, Mar-Apr, 2016 http://dx.doi.org/10.1590/0037-8682-0232-2015 Case Report Case Report INTRODUCTION Cryptosporidium spp is responsible for approximately 6% of diarrhea cases that affl ict immunocompetent individuals in developing countries(1). Generally, these individuals experience mild and self-limited diarrhea, while chronic cases are more frequent in immunocompromised individuals(2). Herein, we describe cryptosporidiosis cases caused by contaminated condominium well water in immunocompetent individuals who presented with chronic diarrhea due to recurrent infections. In these cases, the continuous exposure to contaminated water – in association with a depressive disorder and/or immunosenescence – led to the development and establishment of symptoms. A hemogram revealed the following: hemoglobin 13.6g/dL, leukocytes 5,100/mm³, segments 63%, eosinophils 4%, and platelets 250,000/mm³. Kidney function and urine samples were normal, and cluster of differentiation 4 (CD4+), cluster of differentiation 8 (CD8+), and immunoglobulin dosage were within normal levels. The patient tested negative for human immunodefi ciency virus (HIV). Secondary transmission of cryptosporidiosis associated with well water consumption: two case studies Natania Carol Cavalcante Rezende[1], Camila Loredana Pereira Alves Madeira Bezerra[1], Jéssica Jacinto Salviano de Almeida[1], Tatiane Uetti Gomes Fernandes[1] and Kleber Giovanni Luz[2] [1]. Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brasil. [2]. Instituto de Medicina Tropical do Rio Grande do Norte, Natal, Rio Grande do Norte, Brasil. CASE REPORT In one study, untreated and treated water samples were analyzed for Cryptosporidium in 66 water treatment plants in 14 sites in the United States and one Canadian province; 97% of the samples from untreated water were positive for the parasite. In the treated water, parasites were detected in 22% of the samples(4). Hence, water is an important vehicle for the transmission of this parasite. FIGURE 1 - Analysis of the condominium well water, using modifi ed cellulose membrane fi ltration and Ziehl-Neelsen methods, revealed oocysts of Cryptosporidium spp (Products and Environment Microscopic Center – LACEN/RN). Natal, State of Rio Grande do Norte, 2014. Furthermore, a decrease in the immune function of patients may have hindered the development of effi cient immune cell and humoral responses. This may have caused the prolongation of infection for approximately six months. In patients who can develop adequate immune responses, the disease is often self- limiting and resolves in approximately two weeks. In Case 1, a depressive disorder was probably the main factor causing the immune system defi ciency. Psychological stressors cause alterations that reverberate in the hormonal system and neurotransmission pathways, which directly infl uence the immune response. This change is mainly caused by the activation of the hypothalamic-pituitary-adrenal (HPA) axis and liberation of glucocorticoids during emotional stress, which can reduce the production of interleukin (IL)-1, IL-13, IL-5, IL-6, IL-8, tumor necrosis factor (TNF), and colony- stimulating factor. Additionally, migration and chemotaxis of neutrophils and eosinophils are also inhibited(5). radiotherapy and chemotherapy 17 years prior. Her symptoms began in January 2014 and involved intermittent diarrhea of yellow, aqueous feces with undigested food, and two-to-three evacuations per day. She presented with diffuse abdominal pain that improved after evacuations, fl atulence, and cold sweats. She reported a weight loss of six kilograms over six months. A diagnostic investigation was performed, and a hemogram revealed the following: hemoglobin 12g/dL, leukocytes 5,610mm³, segments 57.5%, and platelets 201 mil/mm³. CD4+, CD8+, and immunoglobulin were within normal levels, and the patient tested negative for HIV. A stool analysis test was ordered using modifi ed acid-fast staining; two out of fi ve stool samples revealed Cryptosporidium spp oocysts. The well water of the condominium was also analyzed and tested positive for the protozoan parasite. Azithromycin was prescribed to the patient and mineral water was used for cooking and bathing thereafter. The symptoms resolved after adopting these measures. CASE REPORT Case 1: A 60-year old otherwise healthy woman who is a single real estate administrator, and is a native and resident of Natal (Northeast Brazil), presented with a complaint of diarrhea for the past six months. In November 2013, the clinical case was intermittent diarrhea with aqueous feces but no mucus, blood or fat; symptoms were sometimes accompanied by abdominal cramps in the lower abdomen. In the following month, she presented with a single episode of fecal incontinence. From then until March 2014, she reported alternations between liquid Given the persistence of the symptoms, a diagnostic- epidemiological investigation was performed. When searching for the presence of Cryptosporidium spp in the feces using the Ziehl-Neelsen staining method, oocysts of this parasite were identifi ed. Analysis of the condominium well water (Figure 1) revealed the presence of the same oocysts. Therefore, the usage of contaminated water was immediately ceased, and psychiatric treatment was resumed. Currently, the patient follows medical recommendations and is asymptomatic. Case 2: An 87-year-old woman, a homemaker who is a native of and residing in the municipality of Natal, presented with diarrhea for the past six months. The patient had a background of endometrial malignant neoplasm diagnosed through biopsy after hysterectomy, and was treated with Corresponding author: Natania Carol Cavalcante Rezende. e-mail: carolcavalcante.r@gmail.com Received 10 July 2015 Accepted 28 August 2015 Corresponding author: Natania Carol Cavalcante Rezende. e-mail: carolcavalcante.r@gmail.com Received 10 July 2015 Accepted 28 August 2015 260 Rezende NCC et al. - Secondary transmission of cryptosporidiosis FIGURE 1 - Analysis of the condominium well water, using modifi ed cellulose membrane fi ltration and Ziehl-Neelsen methods, revealed oocysts of Cryptosporidium spp (Products and Environment Microscopic Center – LACEN/RN). Natal, State of Rio Grande do Norte, 2014. The water wells in the residences of both patients contained Cryptosporidium spp oocysts, which is the infectious agent in cryptosporidiosis (Figure 1). Daily consumption of this water resulted in continuous exposure to the microorganism, which is very resistant to chlorination, disinfection, and several forms of fi ltration at the oocyte stage of its biological cycle. This allows the microorganism to survive in the environment for a long period of time. Cryptosporidiosis transmission is increasingly found to be associated with the consumption of contaminated water, whether treated or not. CASE REPORT All these alterations can contribute in turn to the development of leukocytosis, a decrease in the number and activity of natural Killer (NK) cells, an increase in the CD4+/CD8+ ratio, and a decrease in the activity of T cells. Furthermore, there are alterations in the T-helper 1/T-helper 2 (Th1/Th2) balance that favor Th2, following the inhibition of IL-12, TNF-alpha, and interferon-gamma production(6). 1. Chen XM, Keithly JS, Paya CV, LaRusso NF. Cryptosporidiosis. N Engl J Med 2002; 346:1723-1731. DISCUSSION In cases of cryptosporidiosis, which is caused by an intracellular parasite, it is important to develop an effective Th1 response to combat the parasite. However, as mentioned above, immune function changes can increase Th2 at the expense of Th1; the former is more effective against extracellular pathogens(1). In sum, all these changes can contribute to an ineffi cient defense against the parasite. Cryptosporidium spp is one of the most common intestinal parasites responsible for human gastroenteritis(3). Symptoms of cryptosporidiosis depend on several factors, especially the immune competence of the individual, age, and existence of co-morbidities(1) The intestinal form of this disease has been chiefl y associated with immunosuppression. However, the cases presented herein are notable because both women were HIV-negative and immunocompetent, according to their CD4+ counts. In such cases, we postulate that the disease developed due to intense and regular exposure to Cryptosporidium spp in their condominium water supplies, and was associated with a drop in immunity because of a depressive disorder, an senescence, or chemotherapy, which made the patient more susceptible to infection. Case 2 was similar to Case 1 in terms of emotional stress, as physical or neurogenic stress activates the HPA axis causing an increase in adrenocorticotropic hormone secretion followed by the adrenocortical liberation of cortisol(6). This may explain the development of an ineffective immune cell response to the protozoan parasite, allowing for the manifestation of the disease. There are additional factors that may facilitate prolonged infection, including advanced age, previous history of cancer, and chemotherapy treatment and basal disease(1). 261 Rev Soc Bras Med Trop 49(2):260-262, Mar-Apr, 2016 It is known that the normal process of aging is accompanied by a reduction in mucus membranes that act as barriers against pathogens. Furthermore, there is a significant decrease in macrophage precursors in the bone marrow(7), as well as a decrease in the production of nitric oxide and reactive species of oxygen that are essential for the activity of these cells(8). Additionally, age-related thymic involution occurs with substitution of fatty tissue(9), causing a decrease in CD4+ T-cells, which is refl ected in the decrease in B cell activity, since these are stimulated by the former(10). These immune system alterations in the elderly may predispose such individuals to a higher number of infections, including cryptosporidiosis. 2. Wolfson J, Richter JM, Waldron MA, Webber DJ, McCarthy DM, Hopkins CC. Cryptosporidiosis in Immunocompetent Patients. N Engl J Med 1985; 312:1278-1282. DISCUSSION 3. 3. Garlipp CR, Bottini PV, Teixeira ATLS. The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia. Rev Inst Med Trop Sao Paulo 1995; 37:467-469. 4. Lima EC, Stamford TLM. Cryptosporidium spp. in aquatic environment: the principal aspects of dissemination and diagnostic methods. Cien Saude Colet 2003; 8:791-800. 5. Vismari L, Alves CJ, Palermo-Neto J. Depressão, antidepressivos e sistema imune: um novo olhar sobre um velho problema. Rev Psiquiatr Clin 2008; 35:196-204. 6. Alves CJ, Palermo-Neto J. Neuroimunomodulação: sobre o diálogo entre o sistema nervoso e imune. Rev Bras Psiquiatr 2007; 29:363-369. In conclusion, neurogenic aspects such as depression and emotional stress, along with comorbidities and a history of malignant diseases, can possibly create a state of depressed immunity. Therefore, otherwise immunocompetent individuals are susceptible to the development of diarrhea, as was the case in the patients reported here who were persistently exposed to contaminated water. 7. Ogawa T, Kitagawa M, Hirokawa K. Age-related changes of human bone marrow: a histometric estimation of proliferative cells, apoptotic cells, T cells, B cells and macrophages. Mech Ageing Dev 2000; 117:57-68. 8. Plowden J, Renshaw-Hoelscher M, Engleman C, Katz J, Sambhara S. Innate immunity in aging: impact on macrophage function. Aging Cell 2004; 3:161-167. 9. Naylor K, Li G, Vallejo AN, Lee WW, Koetz K, Bryl E, et al. The infl uence of age on T cell generation and TCR diversity. J Immunol 2005; 174:7446-7452. REFERENCES 10. Haynes L, Maue AC. Effects of aging on T cell function. Curr Opin Immunol 2009; 21:414-417. 262
https://openalex.org/W4251220385
https://www.qeios.com/read/CNCQVY/pdf
English
null
Vital Signs Consciousness State
Definitions
2,020
cc-by
57
Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Vital Signs Consciousness State National Cancer Institute National Cancer Institute Qeios ID: CNCQVY · https://doi.org/10.32388/CNCQVY Source National Cancer Institute. Vital Signs Consciousness State. NCI Thesaurus. Code C119946. C119946. The level of awareness of an organism during a vital signs assessment. 1/1
https://openalex.org/W2101810308
https://researchonline.lshtm.ac.uk/id/eprint/4651702/1/An%20analysis%20of%20timing%20and%20frequency%20of%20malaria%20infection%20during%20pregnancy%20in%20relation%20to%20the%20risk%20of%20low%20birth%20weight%2C%20anaem.pdf
English
null
An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso
Malaria journal
2,012
cc-by
6,188
* Correspondence: valinno@yahoo.fr 1Laboratory of Parasitology and Entomology, Centre Muraz, Bobo-Dioulasso, Burkina Faso Full list of author information is available at the end of the article Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Valea et al. Malaria Journal 2012, 11:71 Abstract Background: A prospective study aiming at assessing the effect of adding a third dose sulphadoxine- pyrimethamine (SP) to the standard two-dose intermittent preventive treatment for pregnant women was carried out in Hounde, Burkina Faso, between March 2006 and July 2008. Pregnant women were identified as earlier as possible during pregnancy through a network of home visitors, referred to the health facilities for inclusion and followed up until delivery. Methods: Study participants were enrolled at antenatal care (ANC) visits and randomized to receive either two or three doses of SP at the appropriate time. Women were visited daily and a blood slide was collected when there was fever (body temperature > 37.5°C) or history of fever. Women were encouraged to attend ANC and deliver in the health centre, where the new-born was examined and weighed. The timing and frequency of malaria infection was analysed in relation to the risk of low birth weight, maternal anaemia and perinatal mortality. Results: Data on birth weight and haemoglobin were available for 1,034 women. The incidence of malaria infections was significantly lower in women having received three instead of two doses of SP. Occurrence of first malaria infection during the first or second trimester was associated with a higher risk of low birth weight: incidence rate ratios of 3.56 (p < 0.001) and 1.72 (p = 0.034), respectively. After adjusting for possible confounding factors, the risk remained significantly higher for the infection in the first trimester of pregnancy (adjusted incidence rate ratio = 2.07, p = 0.002). The risk of maternal anaemia and perinatal mortality was not associated with the timing of first malaria infection. Conclusion: Malaria infection during first trimester of pregnancy is associated to a higher risk of low birth weight. Women should be encouraged to use long-lasting insecticidal nets before and throughout their pregnancy. Keywords: Malaria infection, Pregnancy, First trimester, Sulphadoxine-pyrimethamine, IPT (LBW) [2,4] and is therefore an important determinant of perinatal mortality [5-7]. To prevent and manage malaria in pregnancy, the Word Health Organization (WHO) recommends effective case management, use of insecticide-treated bed nets and intermittent preventive treatment (IPTp) with sulphadoxine-pyrimethamine (SP) [8]. Following the latest WHO guidelines, IPTp should be administered at least twice, during the second and third trimester. In Burkina Faso in 2005, the Ministry of © 2012 Valea 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. An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso Innocent Valea1*, Halidou Tinto1,2, Maxime K Drabo2, Lieven Huybregts3, Hermann Sorgho2, Jean-Bosco Ouedraogo1,2, Robert T Guiguemde1, Jean Pierre van Geertruyden4, Patrick Kolsteren3,5 and Umberto D’Alessandro6, for the FSP/MISAME study Group RESEARCH Open Access Open Access * Correspondence: valinno@yahoo.fr 1 Study settings h Two peripheral health centres (Koho and Karaba) in the Houndé health district, south-west Burkina-Faso, were selected for the study. In this area, malaria is markedly seasonal with high transmission during the rainy season (June to December) [21]. The district hospital and the 28 peripheral health facilities cover a population of approximately 247,500 people. In 2007, the number of pregnant women at risk of malaria was estimated at 12,500, while malaria was the main disease in the health district, accounting for about 38% of all consultations and 52% of hospitalizations. Study design This was part of a larger study investigating both the effect of multiple micronutrients supplementation (MMS) versus fortified food supplementation (FFS) and that of IPTp/SP, two versus three doses on the health of pregnant women and that of their offspring. Participants were randomized in permuted blocks of four to receive either two doses of SP as recommended by the National Malaria Control Pro- gramme (NMCP) or three doses. Randomization numbers were generated by a computer program, sealed in opaque envelopes and opened only when an eligible subject was identified. These numbers were then transmitted to the Study participants P y p p Pregnant women were identified through a community- based network of home visitors, as described elsewhere [22]. All women of child-bearing age in the study area were identified and paid monthly visits during which they were screened for pregnancy. Women suspected to be pregnant were referred to the health facilities for a pregnancy test. The study protocol and procedures were then explained to the potential participants in the local language and those agreeing to participate were asked to provide a written inform consent. Women with known hypersensibility to SP or not planning to stay in the study area for the next two years were excluded. Methods Study participants were recruited at ANC clinics, where the health staff recorded demographic data, medical and pregnancy history. In addition, a clinical examination was performed and vital signs, weight, height and arm circum- ference were measured. All measures were repeated at each ANC visit. Gestational age was assessed as early as possible by the study obstetrician using trans-abdominal ultrasound fetal biometry. Women included in the study were visited at home daily by the home visitors who recorded the body temperature and registered any com- plaint. In case of fever (body temperature ≥37.5°C) or his- tory of fever since the last visit, a blood sample for thick and thin film was collected and sent to the health district laboratory. All women with a confirmed malaria infection were treated with a full course of quinine (24 mg/kg/day for seven days), regardless of their gestational age. Preg- nant women were encouraged to attend their scheduled ANC visits and to deliver at the health facilities where the new-borns was examined, weighed and measured twice by two different members of the health staff. Background Each year, about 30 million pregnant women are at risk for malaria [1], with consequences of public health con- cern [2]. Indeed, malaria infection may result in mater- nal anaemia [3], pre-term delivery and low birth weight Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Page 2 of 7 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Health (MoH) replaced weekly chloroquine chemopro- phylaxis, whose efficacy had declined because of wide- spread resistance [9], with IPTp/SP [10]. Several studies have demonstrated the excellent safety and efficacy of IPTp [11-15]. Most studies used maternal anaemia and LBW as impact indicators. IPTp prevents the adverse effects of malaria during pregnancy by clearing any active infection and providing post-treatment prophy- laxis. The length of protection may become shorter and infection may occur progressively earlier as SP resistance increases [16]. As SP is given at least twice during preg- nancy, its prophylactic efficacy may vary according to both the timing of malaria infection and SP administra- tion. Unfortunately, because of fears of possible toxicity, SP cannot be given during the first trimester of preg- nancy. However, there is also little information on the consequence of malaria infection during the first trime- ster as most information available refers to infections occurring in the second and third trimester [17-20]. A randomized trial comparing two doses versus three doses of IPTp/SP was carried out in rural Burkina Faso. Participants were recruited as early as possible during pregnancy and followed-up prospectively until delivery, allowing an estimation of the effect of malaria infection at different gestational ages on the risk of maternal anaemia, LBW and perinatal mortality. Results are reported below. study pharmacist who packaged the drugs in individual plastic zip bags. Each bag was labelled with the partici- pant’s name, identification, residence, and randomization group. The field pharmacist prepared for each woman an individual schedule for SP administration according to the gestational age at randomization and transmitted it to the trained home visitors who administered both SP and either MMS or FSS. Results of the nutrition intervention have been reported elsewhere [22]. The effect of IPTp with three doses of SP versus two doses of SP on LBW and pregnancy outcomes have also been reported in a pre- vious publication [23]. Ethics The study protocol was approved by the ethical commit- tees of the Centre Muraz, Bobo-Dioulasso, Burkina Faso, and the University of Antwerp, Belgium. The trial was registered in the ClinicalTrial.gov registry (identifier: NCT00909974). The study purpose and procedures where explained to the potential participants by the study clini- cian in the local languages. Women who fulfilled the inclusion criteria and agreed to participate in the study were asked to provide a written inform consent. Study population y p p From March 2006 to July 2008, 1,296 women were ran- domized into the study. Table 1 shows the baseline characteristics of study population according to the tim- ing of first infection. Characteristics of study participants according to randomization groups were published else- where [23]. A total of 1,053 women attended at least three ANC visits while 121 and 100 attended two and one ANC visit, respectively. Analysis was performed on 1,034 women with complete records on birth weight and haemoglobin. The mean maternal age was 24.4 years and the mean gestational age at enrolment was 15.9 weeks. There was no difference in terms of base- lines characteristics between women included in the analysis and those excluded for missing data. Laboratory methods Thick and thin blood smears were collected in duplicate and stained with Giemsa 10% (pH 7.2) for 10 minutes. Parasite densities were determined on the thick smears by counting asexual parasites per 200 white blood cells Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Page 3 of 7 Page 3 of 7 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 (WBC) and assuming a WBC count of 8,000/μl. A thick blood smear was considered negative after reading 100 high-power fields. Ten percent of the slides were ran- domly selected and sent to the Laboratory of Parasitol- ogy/Centre Muraz for quality control. Haemoglobin (Hb) was measured by using a portable spectrophot- ometer (HemoCue, Angelholm, Sweden). (WBC) and assuming a WBC count of 8,000/μl. A thick blood smear was considered negative after reading 100 high-power fields. Ten percent of the slides were ran- domly selected and sent to the Laboratory of Parasitol- ogy/Centre Muraz for quality control. Haemoglobin (Hb) was measured by using a portable spectrophot- ometer (HemoCue, Angelholm, Sweden). infection was categorized according to the number of positive slides detected during the whole pregnancy, i.e. 0, 1, or ≥2. Explanatory variables and possible confoun- ders included the number of SP doses received (two or three), the nutritional supplementation (FFS or MMS), gravidity, body mass index at enrolment (underweight: body mass index < 18.5 kg/m2; or normal weigh, BMI ≥ 18.5 kg/m2), and malaria transmission season at delivery (low transmission season between December and May and high transmission season between June and November). Definitions and statistical analysis A new-born was classified as LBW if the birth weight was < 2,500 g. Women with haemoglobin levels < 11 g/dl were classified as anaemic, those with haemoglobin levels < 8 g/dl were classified as having moderate-to-severe anaemia. Neonatal death was defined as one occurring between delivery and 28 days of life. Malaria infection was defined as a slide positive for Plasmodium falci- parum, any density. Incidence rates were computed con- sidering the follow-up duration time (in months) of each woman. Women with a malaria infection after previous treatment with quinine were considered as re-infected. Time to re-infection was defined as the period between the first infection parasite clearance and the second infec- tion. As the parasite clearance was not monitored, we calculated an adjusted time to re-infection considering that all parasites were cleared after three days as indi- cated in a previous publication [24]. Incidence rates of malaria detected infections The overall incidence rate (IR) of malaria infection (expressed per 1,000 women-months) was 39.2, and was significantly higher in primi- (88.6, 95%CI: 72.6-108.1) than in secundi- (50.6, 95%CI: 39.1-65.5) and multi- gravidae (18.8, 95%CI: 14.7-24.1) (Table 2). The IR of malaria infection was significantly lower (p = 0.014) in women having received three (23.8%, 95%CI: 15.5-36.4) than two SP doses (46.6%, 95%CI: 38.3-55.4). No differ- ence between the MMS group (40.4, 95%CI: 33.5-48.6) and the FFS group (38.0%, 95%CI: 31.5-45.9) was found (p = 0.42). Data were double entered in a Microsoft Access® data- base by two data clerks. Validation and analysis were per- formed using Stata 10 IC® software. Only singleton pregnancies were included in the analysis. The Chi2 or Fisher exact test were used to compare proportions for categorical variables while a t-test for normally distribu- ted or Mann-Whitney test for non-normally distributed was used for continuous variables at baseline. A multi- variate analysis using a Poisson regression model with robust standard error estimates to evaluate the relation- ship between explanatory variables and outcomes was also performed. Crude and adjusted incidence rate ratios (IRR) with 95% CI were computed. A p-value ≤0.05 was considered as statistically significant. Rates of re-infection and length of time-to-reinfection The overall incidence of re-infections after quinine treatment was 11.4% (95%CI: 8.9-14.6) and was signifi- cantly higher (p < 0.001) in primi- (34.7, 95%CI: 25.3- 47.7) than in secundi- (11.3, 95%CI: 6.6-19.5) and multi- gravidae (3.6%, 95%CI: 2.1-6.4). Re-infection tended to be lower in women having received three (6.8%, 95%CI: 3.1-15.1) than two (14.3%, 95%CI: 10.2-19.9) SP doses (p = 0.06). No difference between MMS and FFS group was found (data not shown). The timing of malaria infection was defined according to the first infection detected at the first, second or third trimester of pregnancy. The frequency of malaria Overall, the adjusted mean time to re-infection was 59.3 days (95%CI: 45.8-72.7) with no difference by gravidity, i.e. Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Page 4 of 7 Table 1 Characteristic of study participants at inclusion by timing of first infection Characteristics No infection (n = 816) Infection in first trimester (n = 31) Infection in second trimester (n = 69) Infection in third trimester (n = 118) Study location Karaba village (n, %) 219 (26.84) 14 (45.16) 20(33.90) 40(33.94) Koho village (n, %) 597 (73.16) 17 (54,84) 49(66.10) 78(66.06) Education none (n, %) 728 (89.21) 22 (70.97) 62(89.86) 105 (88.99) primary (n, %) 69 (8.46) 8 (25.81) 7(10.14) 10 (8.47) secondary and higher (n, %) 19(2.33) 1 (3.22) 0(0) 3(2.54) Parity primigravidae (n, %) 113 (13.85) 18 (58.06) 35 (50.72) 44 (37.29) secundigravidae (n, %) 158 (19.36) 5 (16.13) 15 (21.74) 38 (32.20) multigravidae (n, %) 545 (66.79) 8 (25.81) 19 (27.54) 36 (30.51) Age mean (years) (95% CI) 25.27(24.84-25.69) 20.88 (18.96-22.80) 21.27 (20.06-22.48) 21.50 (20.63-22.37) Weight mean (kg) (95% CI] 55.67 (55.17-56.15) 55.45 (53.20-57.71) 55.24 (53.70-56.79) 55.29 (54.08-56.50) Height 162.815 163.39 162.58 161.78 mean (cm) (95% CI] (162.42-163.21) (161.72-165.08) (161.15-164.00) (160.71-162.85) BMI at enrolment 20.98 20.77 20.88 21.1 mean (95% CI] (20.83-21.12) (19.97-21.56) (20.41-21.34) (20.74-21.45) Table 1 Characteristic of study participants at inclusion by timing of first infection articipants at inclusion by timing of first infection primi- 63.7 days (95%CI: 47.2-80.2), secundi- 40.7 days (95%CI: 07.9-73.4), and multi-gravidae 51.2 days (95%CI: 3.2-99.1). Mean time to reinfection was not analysed by number of SP doses as only four women in the three-dose group were re-infected. Rates of re-infection and length of time-to-reinfection in the first (IRR = 3.56, p < 0.001) and the second tri- mester (IRR = 1.72, p = 0.034) had a significantly higher risk of delivering a LBW baby as compared to women without any infection (Table 3). After adjusting for age, parity, body mass index, number of SP doses received and malaria season at delivery, such risk remained sig- nificantly higher for women infected in the first trime- ster of pregnancy (adjusted IRR = 2.07, p = 0.002) (Table 3). Also, women infected in first trimester had a significantly higher risk of delivering a LBW when com- pared to women infected in the third trimester (Crude IRR = 3.09, 95%CI: 1.66-5.73, p < 0.01; and adjusted Effect of timing and frequency of malaria infection on low birth weight, maternal anaemia and perinatal mortality When considering the timing of the first infection, no significant difference could be found for maternal ane- mia and perinatal mortality, while women infected both Table 2 Number of cases and incidence rates of malaria infections in the study population (per 1000 women-month) Person-time* Malaria infections new cases IR (per 1000) (95% CI) IRR (95% CI) By Gravidity Overall 5541 217 39.2 (34.3-44.7) - Primigravidae 1095 97 88.6 (72.6-108.1) - Secondigravidae 1146 58 50.6 (39.1-65.56) 0.6 (0.42-0.79) Multigravidae 3300 62 18.8 (14.65-24.1) 0.2 (0.17-0.31) By SP doses received 2 doses SP 2453 113 46.1 (38.3-55.4) - 3 doses SP 884 21 23.8 (15.5-36.4) 0.5 (0.28-0.66) By Supplementation group MMS 2726 110 40.3 (33.5-48.6) - FSS 2815 107 38.0 (31.5-46.0) 0.9 (0.67-1.18) * Women-month and incidence rates of malaria infections in the study population (per 1000 women-month) Person-time* Malaria infections Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Page 5 of 7 Table 4 Crude and adjusted incident rate ratios (IRR) of low birth weight, maternal anaemia and perinatal mortality by number of detected malaria infection using Poisson regression Crude IRR (95% CI) Adjusted IRR(95% CI) Maternal anaemia No infection (391/816) 1.00 1.00 1 infection detected (79/169) 1.07 (0.91-1.26) 1.019 (0.85-1.21) ≥2 infections detected (28/49) 1.33 (1.04-1.69)a 1.236 (0.94-1.62) Low birth weight No infection (96/816) 1.00 1.00 1 infection detected (31/169) 1.56 (1.08-2.26)b 1.07 (0.73-1.58) ≥2 infections detected (12/49) 2.08 (1.29-3.52)c 1.08 (0.62-1.87) Perinatal mortality No infection (15/816) 1.00 1.00 1 infection detected (4/169) 1.28 (0.43-3.83) 0.87 (0.27-2.83) ≥2 infections detected (0/49) - - Adjusted IRR accounted for women age, parity, BMI, SP doses received and delivery season. Effect of timing and frequency of malaria infection on low birth weight, maternal anaemia and perinatal mortality a p = 0.023, b p = 0.19, c p = 0.006 Table 4 Crude and adjusted incident rate ratios (IRR) of low birth weight, maternal anaemia and perinatal mortality by number of detected malaria infection using Poisson regression Table 3 Crude and adjusted incidence rate ratios (IRR) of low birth weight, maternal anaemia and perinatal mortality by trimester of malaria infection using Poisson regression Crude IRR (95%CI) Adjusted IRR (95%CI) Maternal anaemia No infection (391/816) 1.00 1.00 Infection in 1st trimester (15/31) 1.00 (0.69-1.46) 0.98 (0.67-1.42) Infection in 2nd trimester (34/69) 1.03 (0.80-1.32) 0.99 (0.76-1.27) Infection in 3rd trimester (58/118) 1.02 (0.84-1.24) 0.97 (0.79-1.20) Low birth weight No infection(96/816) 1.00 1.00 Infection in 1st trimester (13/31) 3.56 (2.26-5.61) a 2.07 (1.30-3.27) b Infection in 2nd trimester (14/69) 1.72 (1.04-2.85) c 1.09 (0.65-1.82) Infection in 3rd trimester (16/118) 1.15 (0.70-1.88) 0.78 (0.46-1.32) Perinatal mortality No infection(15/816) 1.00 1.00 Infection in 1st trimester (0/31) - - Infection in 2nd trimester (3/69) 2.36 (0.70-7.97) 1.18 (0.31-4.47) Infection in 3rd trimester (1/118) 0.46 (0.06-3.46) 0.37 (0.04-2.81) Adjusted IRR accounted for women age, parity, BMI, SP doses received and delivery season. ap < 0.001, b p = 0.002, c p = 0.034 Table 3 Crude and adjusted incidence rate ratios (IRR) of low birth weight, maternal anaemia and perinatal mortality by trimester of malaria infection using Poisson regression Adjusted IRR accounted for women age, parity, BMI, SP doses received and delivery season. a p = 0.023, b p = 0.19, c p = 0.006 pregnancy, even after adjusting for several potential con- founding variables such as parity and number of IPTp/SP doses received. Thought the pathogenesis and immunity of malaria in pregnancy has been explored, the contribu- tion of placental immuno-pathology to anaemia and LBW is not fully understood [25]. Plasmodium falci- parum-infected erythrocytes sequester in the placenta through adhesion mechanisms, inducing placental inflammatory responses, particularly monocytes infil- trates. Inflammatory cytokines produced by T cell and macrophages, in particular Th1 responses are associated with maternal anaemia, spontaneous abortions and pre- mature deliveries. These cytokines are known to help eliminate the parasites from the placenta but their over- production can threaten the pregnancy [25,26]. Acute infection, particularly with high parasites densities have been associated with preterm delivery while chronic infection have been associated with LBW due to intrau- terine growth retardation (IUGR) and severe anaemia [27]. Effect of timing and frequency of malaria infection on low birth weight, maternal anaemia and perinatal mortality Decreased placental growth and/or decreased nutri- ent transport have been suggested as the possible final common pathways by which malaria leads to IUGR [25,28]. In this study, malaria infection during the first trimester was strongly associated with LBW. It is unclear whether this is due to a specific consequence of the infec- tion at this particular time, or whether it is related to the higher risk these women have throughout their preg- nancy. In any case, the currently recommended IPT/SP Adjusted IRR accounted for women age, parity, BMI, SP doses received and delivery season. ap < 0.001, b p = 0.002, c p = 0.034 IRR = 3.27, 95%CI: 1.78-6.01, p < 0.01) while no differ- ence was observed between women infected in the sec- ond trimester and women infected in the third trimester (Crude IRR = 1.49, 95%CI: 0.78-2.87, p = 0.23 and Adjusted IRR = 1.58, 95%CI: 0.83-2.98; p = 0.16). Most women had just one infection though some of them experienced up to four infections until delivery. Though the LBW risk seemed to be significantly higher in women with two or more infections as shown by the crude IRR, the difference disappeared after adjusting for women age, parity, body mass index, number of SP doses received and malaria season at delivery (Table 4). Simi- larly, the risk for maternal anaemia, though apparently increased by two or more infection, did not vary signifi- cantly with the number of re-infections after adjustment by the variables mentioned above. The risk of perinatal mortality was similar for women with ≥1 infection and those with no infection (adjusted IRR = 0.87, 95% CI 0.27-2.83) (Table 4). Discussion The risk of delivering a LBW baby was significantly higher in women infected during the first trimester of Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Page 6 of 7 does not cover the first trimester of pregnancy as the first SP dose should not be given before quickening, at about 20 weeks of gestation, for fear of possible toxicity for the foetus [29]. There is little information on the use of SP during the first trimester of pregnancy; available reports are limited to small case series from developed countries [15]. Therefore, the only option available for protecting pregnant women during this vulnerable period is the use of long-lasting insecticidal nets (LLIN) whose coverage and use in sub-Saharan Africa, despite major efforts such as campaigns of free mass distribution, is less than opti- mal [30,31]. A potentially interesting additional strategy might be the systematic screening and treatment of all pregnant women and this option was recently investi- gated [32,33]. However, when considering the difficulty of identifying women at the earliest stage of their preg- nancy [34], such an approach would hardly solve the pro- blem of protecting during the first trimester. Therefore, promoting LLIN through community-based promotional campaigns targeted not only at pregnant women but also at adolescents seems to be the only option available to tackle this specific problem. In conclusion, malaria infections occurring in the first trimester of pregnancy seem to have an important effect on LBW, though the mechanisms are not yet under- stood. Women should be encouraged to use LLIN throughout their pregnancy so that the deleterious effect of malaria infection during the first trimester could be prevented. Author details 1 1Laboratory of Parasitology and Entomology, Centre Muraz, Bobo-Dioulasso, Burkina Faso. 2Institut de Recherche en Sciences de la Santé, Direction Régionale de l’Ouest, Bobo-Dioulasso, Burkina Faso. 3Department of Food Safety and Food Quality, Ghent University, Ghent, Belgium. 4Department of International Health, University of Antwerp, Antwerp, Belgium. 5Unit of Nutrition and Child Health, Department of Public Health, Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium. 6Unit of Epidemiology and Control of Parasitic Diseases, Department of Parasitology, Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium. The rate of re-infection and the time length to re- infection were suggested as possible indicators to evalu- ate the preventive efficacy of SP [35]. In this study, the incidence of re-infections was much higher in women having received two as compared to three doses of SP, indicating that the latter is probably more efficacious. Nevertheless, the rate of re-infection was established after treatment with quinine and does not necessarily represent the true preventive efficacy of SP. Acknowledgements Th FSP/MISAME S d The FSP/MISAME Study Group thanks the community and especially the women from Karaba and Koho and the families who participated in the study, the health staff of the Houndé district, and the staff of Center Muraz for their great support. The FSP/MISAME Study Group thanks the community and especially the women from Karaba and Koho and the families who participated in the study, the health staff of the Houndé district, and the staff of Center Muraz for their great support. The FSP/MISAME Study Group also acknowledges the study funding organizations (Nutrition Third Word, The Belgium ministry of development, Flemish Interuniversity Council, and French Ministry of Development) for their financial support to the study. We acknowledge the FSP/MISAME field investigator team (J-P Ki and V Koudougbo/Valea, sociologists; L Toe and H Lanou, physicians; E Da, obstetrician-gynecologist; G Lougue, pharmacist; B Nagalo and O Guebe, nurses; and B Hien, laboratory technician), and the logistic team (S Ouattara, study coordinator; J Bicaba, administrative assistant; C Kouakou Yameogo; N Diallo, data clerk; M Sanou, driver; and A Hien, pharmacist). Authors’ contributions IV participated in study coordination and data cleaning, performed the data analysis and drafted the manuscript. HT participated in the design, study coordination and corrected the manuscript. MKD, HS and JPG corrected the manuscript. LH participated in data cleaning and corrected the manuscript. RTG and KP participated in the design of the study. UDA participated in the design, helped in data analysis and corrected the manuscript. All authors read and approved the final manuscript. The study has some limitations. Information on LLIN ownership and use was not collected and, knowing that this has an impact on the malaria risk during pregnancy [36], it should probably have been considered as an important confounding factor. However, it is unlikely that this has resulted in biased estimates as LLIN use in the study population was probably very low at the time of the study. In Boromo, a neighbouring district, LLIN use was estimated at about 27% in the high transmission season [21]. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 13 November 2011 Accepted: 16 March 2012 Received: 13 November 2011 Accepted: 16 March 2012 Published: 16 March 2012 Received: 13 November 2011 Accepted: 16 March 2012 Published: 16 March 2012 References Uneke CJ: Impact of placental Plasmodium falciparum malaria on pregnancy and perinatal outcome in sub-Saharan Africa: I: introduction to placental malaria. Yale J Biol Med 2007, 80:39-50. 26. Wegmann TG, Lin H, Guilbert L, Mosmann TR: Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 1993, 14:353-356. 8. WHO/AFRO: A strategic framework for malaria prevention and control during pregnancy in the African Region Word Health Organisation Regional Office for Africa. Brazzaville; 2004. 27. Brabin BJ, Romagosa C, Abdelgalil S, Menendez C, Verhoeff FH, McGready R, Fletcher KA, Owens S, d’Alessandro U, Nosten F, Fisher PR, Ordi J: The sick placenta-the role of malaria. Placenta 2004, 25:359-378. 9. Sirima SB, Sawadogo R, Moran AC, Konate A, Diarra A, Yameogo M, Parise ME, Newman RD: Failure of a chloroquine chemoprophylaxis program to adequately prevent malaria during pregnancy in Koupela District, Burkina Faso. Clin Infect Dis 2003, 36:1374-1382. 28. Rogerson SJ, Mwapasa V, Meshnick SR: Malaria in pregnancy: linking immunity and pathogenesis to prevention. Am J Trop Med Hyg 2007, 77:14-22. 10. Ministere de la Santé/Programme National de Lutte contre le Paludisme: Directives Nationales pour la prise en charge du paludisme au Burkina Faso 2006. 29. Briand V, Cottrell G, Massougbodji A, Cot M: Intermittent preventive treatment for the prevention of malaria during pregnancy in high transmission areas. Malar J 2007, 6:160. 11. Gies S, Coulibaly SO, Ouattara FT, d’Alessandro U: Individual efficacy of intermittent preventive treatment with sulfadoxine-pyrimethamine in primi- and secundigravidae in rural Burkina Faso: impact on parasitaemia, anaemia and birth weight. Trop Med Int Health 2009, 14:174-182. 30. Bourgoing R: Bednets for malaria. Reports from the field – Africa. Glob Impacts 1997, 14-15. 31. Noor AM, Mutheu JJ, Tatem AJ, Hay SI, Snow RW: Insecticide-treated net coverage in Africa: mapping progress in 2000-07. Lancet 2009, 373:58-67 12. Shulman CE, Dorman EK, Cutts F, Kawuondo K, Bulmer JN, Peshu N, Marsh K: Intermittent sulphadoxine-pyrimethamine to prevent severe anaemia secondary to malaria in pregnancy: a randomised placebo- controlled trial. Lancet 1999, 353:632-636. 32. Tagbor H, Bruce J, Agbo M, Greenwood B, Chandramohan D: Intermittent screening and treatment versus intermittent preventive treatment of malaria in pregnancy: a randomised controlled non-inferiority trial. Plos One 2010, 5:e14425. 13. References Sirima SB, Cotte AH, Konate A, Moran AC, Asamoa K, Bougouma EC, Diarra A, Ouedraogo A, Parise ME, Newman RD: Malaria prevention during pregnancy: assessing the disease burden one year after implementing a program of intermittent preventive treatment in Koupela District, Burkina Faso. Am J Trop Med Hyg 2006, 75:205-211. 33. Smith LA, Jones C, Adjei RO, Antwi GD, Afrah NA, Greenwood B, Chandramohan D, Tagbor H, Webster J: Intermittent screening and treatment versus intermittent preventive treatment of malaria in pregnancy: user acceptability. Malar J 2010, 9:18. 34. Grietens KP, Gies S, Coulibaly SO, Ky C, Somda J, Toomer E, Muela RJ, d’Alessandro U: Bottlenecks for high coverage of intermittent preventive treatment in pregnancy: the case of adolescent pregnancies in rural Burkina Faso. Plos One 2010, 5:e12013. 14. Tiono AB, Ouedraogo A, Bougouma EC, Diarra A, Konate AT, Nebie I, Sirima SB: Placental malaria and low birth weight in pregnant women living in a rural area of Burkina Faso following the use of three preventive treatment regimens. Malar J 2009, 8:224. 35. Brabin BJ, Warsame M, Uddenfeldt-Wort U, Dellicour S, Hill J, Gies S: Monitoring and evaluation of malaria in pregnancy-developing a rational basis for control. Malar J 2008, 7(Suppl 1):S6. 15. Peters PJ, Thigpen MC, Parise ME, Newman RD: Safety and toxicity of sulfadoxine/pyrimethamine: implications for malaria prevention in pregnancy using intermittent preventive treatment. Drug Saf 2007, 30:481-501. 36. d’Alessandro U: Insecticide treated bed nets to prevent malaria. BMJ 2001, 322:249-250. 16. White NJ: How antimalarial drug resistance affects post-treatment prophylaxis. Malar J 2008, 7:9. doi:10.1186/1475-2875-11-71 Cite this article as: Valea et al.: An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso. Malaria Journal 2012 11:71. doi:10.1186/1475-2875-11-71 Cite this article as: Valea et al.: An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso. Malaria Journal 2012 11:71. 17. Kalilani L, Mofolo I, Chaponda M, Rogerson SJ, Meshnick SR: The effect of timing and frequency of Plasmodium falciparum infection during pregnancy on the risk of low birth weight and maternal anemia. Trans R Soc Trop Med Hyg 2010, 104:416-422. 18. Cottrell G, Mary JY, Barro D, Cot M: The importance of the period of malarial infection during pregnancy on birth weight in tropical Africa. References Malaria infection during pregnancy was probably under- estimated as it was detected by peripheral blood smears. Placental biopsies were collected during the study but could not be analyzed due to budget constraints. 1. Dellicour S, Tatem AJ, Guerra CA, Snow RW, ter Kuile FO: Quantifying the number of pregnancies at risk of malaria in 2007: a demographic study. PLoS Med 2010, 7:e1000221. p g g p y PLoS Med 2010, 7:e1000221. 2. Desai M, ter Kuile FO, Nosten F, McGready R, Asamoa K, Brabin B, Newman RD: Epidemiology and burden of malaria in pregnancy. Lancet Infect Dis 2007, 7:93-104. 3. Menendez C, Fleming AF, Alonso PL: Malaria-related anaemia. Parasitol Today 2000, 16:469-476. 4. Guyatt HL, Snow RW: Impact of malaria during pregnancy on low birth weight in sub-Saharan Africa. Clin Microbiol Rev 2004, 17:760-9, table. 5. van Geertruyden JP, Thomas F, Erhart A, d’Alessandro U: The contribution of malaria in pregnancy to perinatal mortality. Am J Trop Med Hyg 2004, 71:35-40. 2. Desai M, ter Kuile FO, Nosten F, McGready R, Asamoa K, Brabin B, Newman RD: Epidemiology and burden of malaria in pregnancy. Lance Infect Dis 2007, 7:93-104. Despite the network of home visitors implemented and the daily visits carried out for an earlier identification of pregnant women, most of those detected during the first trimester were at ≥10 weeks of gestation, a limiting fac- tor for the assessment of infections during the first trime- ster. For some reasons, mainly socio-cultural factors [34], women were reluctant to declare their pregnancy earlier and this should be further investigated. 3. Menendez C, Fleming AF, Alonso PL: Malaria-related anaemia. Parasitol Today 2000, 16:469-476. 4. Guyatt HL, Snow RW: Impact of malaria during pregnancy on low birth weight in sub-Saharan Africa. Clin Microbiol Rev 2004, 17:760-9, table. 5. van Geertruyden JP, Thomas F, Erhart A, d’Alessandro U: The contribution of malaria in pregnancy to perinatal mortality. Am J Trop Med Hyg 2004, 71:35-40. 6. McCormick MC: The contribution of low birth weight to infant mortality and childhood morbidity. N Engl J Med 1985, 312:82-90. Page 7 of 7 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 25. Rogerson SJ, Hviid L, Duffy PE, Leke RF, Taylor DW: Malaria in pregnancy: pathogenesis and immunity. Lancet Infect Dis 2007, 7:105-117. 7. References Am J Trop Med Hyg 2007, 76:849-854. 19. Cot M, Abel L, Roisin A, Barro D, Yada A, Carnevale P, Feingold J: Risk factors of malaria infection during pregnancy in Burkina Faso: suggestion of a genetic influence. Am J Trop Med Hyg 1993, 48:358-364. 20. Huynh BT, Fievet N, Gbaguidi G, Dechavanne S, Borgella S, Guezo-Mevo B, Massougbodji A, Ndam NT, Deloron P, Cot M: Influence of the timing of malaria infection during pregnancy on birth weight and on maternal anemia in Benin. Am J Trop Med Hyg 2011, 85:214-220. 21. Coulibaly SO, Gies S, d’Alessandro U: Malaria burden among pregnant women living in the rural district of Boromo, Burkina Faso. Am J Trop Med Hyg 2007, 77:56-60. 22. Huybregts L, Roberfroid D, Lanou H, Menten J, Meda N, Van CJ, Kolsteren P: Prenatal food supplementation fortified with multiple micronutrients increases birth length: a randomized controlled trial in rural Burkina Faso. Am J Clin Nutr 2009, 90:1593-1600. 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: • 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: • 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: 23. Valea I, Tinto H, Drabo MK, Huybregts L, Henry MC, Roberfroid D, Guiguemde RT, Kolsteren P, d’Alessandro U: Intermittent preventive treatment of malaria with sulphadoxine-pyrimethamine during pregnancy in Burkina Faso: effect of adding a third dose to the standard two-dose regimen on low birth weight, anaemia and pregnancy outcomes. Malaria Journal 2010, 9:324. 24. doi:10.1186/1475-2875-11-71 Cite this article as: Valea et al.: An analysis of timing and frequency of malaria infection during pregnancy in relation to the risk of low birth weight, anaemia and perinatal mortality in Burkina Faso. Malaria Journal 2012 11:71. Valea et al. Malaria Journal 2012, 11:71 http://www.malariajournal.com/content/11/1/71 References Piola P, Nabasumba C, Turyakira E, Dhorda M, Lindegardh N, Nyehangane D, Snounou G, Ashley EA, McGready R, Nosten F, Guerin PJ: Efficacy and safety of artemether-lumefantrine compared with quinine in pregnant women with uncomplicated Plasmodium falciparum malaria: an open-label, randomised, non-inferiority trial. Lancet Infect Dis 2010, 10:762-769.
https://openalex.org/W3177604265
https://zenodo.org/records/8107097/files/2121.pdf
English
null
MODEL AND TOURISM DEVELOPMENT STRATEGY BASED ON LOCAL POTENCY IN MERANGIN REGENCY
Zenodo (CERN European Organization for Nuclear Research)
2,019
cc-by
7,325
Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 MODEL AND TOURISM DEVELOPMENT STRATEGY BASED ON LOCAL POTENCY IN MERANGIN REGENCY Rosmeli RosmeliI*, Nurhayani NurhayaniI, Sari Novita I ILecturers Faculty of Economics and Business, University of Jambi *Corresponding author: zeadevina@gmail.com Accepted Date: 29/10/2019 Published Date: 29/11/2019 Received Date: 19/08/2019 Received Date: 19/08/2019 Published Date: 29/11/2019 ABSTRACT Tourism potency and attractiveness in Merangin Regency are very diverse and spread in several districts which become tourist attractions both domestic and foreign tourists. The tourism sector in Merangin district is managed by local government, making tourism development less than optimal, it is seen with additional facilities such as home stays (hotels / inns), the mainstay products of tourist areas produced by public and access to public vehicles that can go to tourist sites. In the development of tourism, not only the government does the other parties themselves, it also contributes to infrastructure development. In developing tourism potency based on regional potential in Merangin district there are 11 alternative strategies, namely: Focusing road infrastructure to destination area, cooperation with the private sector in developing regional tourism, increasing tourism promotion in collaboration with tourism bureau in the form of Merangin tour packages, tourism promotion which is more intense, provides faster and cheaper licensing and legality for tourism bureau in Merangin Regency, repairs existing facilities and infrastructure, development of other natural tourism objects, fosters tourism-aware community development, guidance and development of Small Medium Enterprises (SMEs) by region-specific, increasing Tourism Human Resources both from the government and from the private sector and the government in cooperation with SMEs increases the promotion of local specialties. The tourism sector development model in Merangin Regency is the cooperation amongst of all existing components ranging from local government, the private sector, and the community in Merangin district itself. Keywords: Tourism, Strategy, Potency, Models and Strategies Keywords: Tourism, Strategy, Potency, Models and Strategies INTRODUCTION The tourism sector is a sector that can support regional economic growth if managed properly and will reduce the dependence of region on the exploitation of natural resources. The enhancement of tourism industry output in turn leads to an increase in the living standards of those involved (Mason, 2003). Tourism development is one of the developments that need to be developed because this sector can increase the country's foreign exchange reserves, produce rapid economic growth in providing employment, increasing income, living standards and stimulating other production factors (Lickorish, 1994). Jambi Province is a province that has a large tourism potential, some districts in Jambi Province are tourist visiting areas that are visited by numerous tourists, such as Kerinci regency, Bungo regency, Sarolangun regency and Merangin regency are areas that have great natural tourism potential. 36 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 The potential of objects and attractions in Merangin Regency which is very diverse and spread in several districts there is a tourist attraction both domestic and foreign tourists. In 2017 the number of tourists were visiting Merangin Regency consisted of 900 domestic tourists and 100 foreign tourists, by 2018 the number of foreign tourists visiting the Regency was 165 people, and more than 1,000 domestic tourists. This increase in tourist numbers is due to the promotion of both print and electronic media and social media (Dinas Kebudayaan, Pariwisata dan Olah Raga Kabupaten merangin, 2018). The tourism sector in Merangin regency is managed by local government, so that tourism development is less than optimal, it can be seen by the lack of tourist facilities such as home stays (hotel / inn), the mainstay of tourism products produced by the community and access to public vehicles that can go to tourist sites which exists. In the development of tourism, it is not only the government that conducts it-self, but other parties also contribute to development of companion infrastructure to increase revenue from the economic sector. For this development, approaches need to be made with existing tourism organizations (government and private) and related parties which are expected to support the continued development of the area's tourism. INTRODUCTION Based on the research of (Perdue, Long, & Allen, 1990), states that the population characteristics will have an impact on the development of tourism in an area, the same thing addressed by research conducted by (McGehee & Andereck, 2004) suggests that residents will receive a positive impact from tourism development and resist the impact negative of the development of tourism. Research conducted by (Binns & Nelt, 2002) concluded that the development of the tourism sector could reduce rates poverty, improve the economy of local people and reduce the dominance of apartheid in Africa South. Therefore, the development of the tourism sector will have a multiplier effect on the region for Merangin Regency development of tourism sector in Merangin Regency should involve all parties, both the district government, the private sector, the community and academics to achieve good acceleration is created from various parties and the potential of the existing tourism sector can be developed which will directly have an impact on increasing the income of local communities. In Merangin Regency, there are many natural tourism objects that have great potential, including wang bay tourism, pauh lake, masurai mountain, hesti flower garden and Merangin geopark. To all these attractions should provide great benefits, both for the community. For this reason, it is necessary to have a strategy and model in developing tourism based on the existing tourism potential. The development of the tourism sector is a good alternative to be carried out in order to increase the role of this sector in the surrounding community and the economy of Merangin Regency. The purpose of this research is first to understand and analyse the tourism sector development strategy in Merangin District and the second is analyse the model of tourism management based on regional potency in Merangin Regency TOURISM PLANNING Tourism policy provides a basic philosophy for development and determines the direction of tourism development in destination for future. A destination can be said to be developing tourism if there were previously tourist activities. In the implementation of development, planning is a factor that needs to be done and considered. According to (Inskeep & Organization, 1998) there are several approaches to be considered in planning, including: ourism policy provides a basic philosophy for development and determines the directio ourism development in destination for future. A destination can be said to be develo tourism development in destination for future. A destination can be said to be developing tourism if there were previously tourist activities. In the implementation of development, planning is a factor that needs to be done and considered. According to (Inskeep & Organization, 1998) there are several approaches to be considered in planning, including: g ) pp p g g a) Continuous Incremental, and Flexible Approach, where planning is seen as an ongoing process based on needs by monitoring existing feed backs. a) Continuous Incremental, and Flexible Approach, where planning is seen as an ongoing process based on needs by monitoring existing feed backs. em Approach, whereby tourism seen as a system relationship and needs to be planned with system analysis techniques b) System Approach, whereby tourism as with system analysis techniques b) System Approach, whereby tourism seen as a system relationship and needs to be planned as with system analysis techniques c) Comprehensive Approach, related to the system approach above, where all aspects of tourism development including institutional elements and the environment and socio- economic implications as a holistic approach c) Comprehensive Approach, related to the system approach above, where all aspects of tourism development including institutional elements and the environment and socio- economic implications as a holistic approach d) Integrated Approach related to the overall system approach where tourism is planned and developed as a system and overall where tourism is planned and developed as an integrated system in all plans and total forms of development in the area. e) Environmental and sustainable development approach, tourism is planned, developed, and managed in a way where natural and cultural resources do not experience a decline in quality and are expected to remain sustainable so an analysis of environmental carrying capacity needs to be applied to this approach. Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 TOURISM PLANNING f) Community Approach, the approach emphasizes the importance of maximizing the involvement of local communities in the planning and decision making process of tourism, to be able to increase the desires and possibilities, it is necessary to maximize community participation in the development and management carried out in tourism and its social economic benefits. g) Implementable Approach, tourism development policies, plans, and recommendations are formulated to be realistic and applicable, with the techniques used are implementation techniques including development, action programs or strategies, especially in identifying and adopting. h) Application of systematic planning approach, this approach is applied in tourism planning based on the logic of the activity. The aims to increase visitor satisfaction, diversify the tourism market, increase the contribution of tourism to the local economy, and develop the tourism potential of an area. While objectives aim to direct actions that will help achieve development goals. The Third World tourism industry will be threatened by many of the problems that have plagued other outward-oriented development strategies in the South during the postwar era, alternative tourism development strategies must be designed either alone or together with the main tourism, so that tourism development can develop simultaneously and can reduce the negative impacts and increase the positive impact of the development of tourism (Brohman, 1996). According Jovanovic (2008), Geographic Information Systems (GIS) , in the tourism industry used to provide: (a) A digital map base for printed maps; (b) Digital files for internet mapping; (c) Digital files for mobile mapping; (d) Attractions map and (e) Website with interactive mapping. GIS technology offers great opportunities for the development of modern tourism application using maps. TOURISM According to law number 10/2009 concerning tourism are all activities related to tourism and are multidimensional and multidisciplinary in nature which emerge as a manifestation of the needs of each person and the State as well as interactions between tourists and the local community, fellow tourists, the government, regional governments and entrepreneurs. There are 4 components forming tourism, through the tourism industry, tourist destinations, marketing and tourism institutions. All efforts undertaken in the development of tourism are aimed at maximizing tourist visits or also called tourism offers. 37 COLLECTING DATA METHOD Primary data collected directly to the object of research by the method: Primary data collected directly to the object of research by the method: 1. Observation Method Observe the existing tourist objects regarding infrastructure and infrastructure that is in the vicinity of the tourist area. Observe the existing tourist objects regarding infrastructure and infrastructure that is i the vicinity of the tourist area. 2. Questionnaire Method There are 5 research sampel, Tourism institution, travel agent, academia, public works office and regional office for planning and development. 3. Interview Method Interviews were conducted concurrently with respondents to better understand the conditions of tourism in the Merangin Regency TOURISM SUSTAINABLE DEVELOPMENT MODEL According to World Commission on Environment and Development (WCED) cited in Dewi (2011), sustainable development is development that meets the necessary of the present without compromising the ability of future generations to meet their needs. To carry out sustainable development, development must increase efficiency economically, protect and restore the ecological system and improve the welfare of humanity. 38 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Whereas the tourism sustainable development is sustainable development achieved through tourism. Sustainable development is a long-term economic development, which balances economic benefits with environmental and social costs. Sustainable tourism development aims to realize sustainable tourism growth. Sustainable tourism development is achieved by balancing three main elements in sustainable tourism development which are environmental, economic and social which show general principles in sustainable tourism, through: g p p 1. Balancing the use of environment with the economic benefits of tourism. 2. Balancing the use of environmental resources with changes in social and community values caused by the reduction of environmental resources, 2. Balancing the use of environmental resources with changes in social and community values caused by the reduction of environmental resources, y 3. Balancing the economic growth and the impact of economic growth on social and economic values. 3. Balancing the economic growth and the impact of economic growth on social and economic values. The term sustainable tourism is an industry that strives to make the least impact on the environment and local culture, while helping to bring in income, create jobs, and conserve ecosystems. This responsibility encompasses a sensitive environment and culture. The WTP on its website explains that sustainable tourism as tourism which directs the management of all sources is carried out in such a way that needs, economic, social and beauty can be met while maintaining cultural integrity, essential ecological processes, biodiversity and life support systems in the environment concerned (Ernawati, 2010). According to (Lee & Kim, 2016), the results of the analysis state that community attachment and community involment are the main factors that have an impact on sustainable tourism development. TYPE AND DATA SOURCE This research utilising primary and secondary data. Primary data regarding information about the potentials of attractions. The data was obtained directly from observations and interviews with related agencies. Secondary data regarding the development of these attractions. The data was obtained from published by statistics bureau, the compilation of data from Merangin Regency Tourism and Sports Youth Office and other relevant publications and sources. Secondary data regarding the development of these attractions. The data was obtained from published by statistics bureau, the compilation of data from Merangin Regency Tourism and Sports Youth Office and other relevant publications and sources. 4. Development Strategy Decision Making Stage After matching the internal and external factors, a decision is made to determine the main strategy through the Quantitative Strtaegic Planning Matrix (QSPM) analysis tool. QSPM uses input from stage 1 (IFE dan EFE matrix) and matching results from stage 2 (SWOT). After the scores fro IFE and EFE is revealed, than those scores will be calculated with the questionnaire score from SWOT which shows the strategic alternative. The result of the calculation will be captured in Total Attractiveness Score (TAS). The highest score of TAS presents the strategic priority that should be done. To develop an integrated model of tourism development. As for the stages as follows: (1). Qualitative Descriptive Analysis (stage 1), (2) Focus Group Discussion (FGD), stages 2 and (3) Conclusions Model of tourism development in Merangin Regency in Jambi Province (Phase 3). DATA ANALYSIS METHOD Data analysis method used in this research are descriptive-qualitative. Qualitative descriptive method is used by describing writing based on interpretation based on the current situation. To formulate a strategy for developing tourism sector in Merangin regency, data analysis was carried out using a SWOT analysis. According to David (2006), there were three stages in the formulation of the strategy, through: The First Stage is the input stage, such as External Factor Evaluation Matrix (EFE) matrix is a tool used to examine the company’s external environment and to identify opportunities and threats that exist, Compotititive Profile Matriks (CPM) is tool that compares companies and their competitors and reveals their relative strengths and 39 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 weaknesses, and the lnternal Factor Evaluation Matrix (IFE) is a tool used to evaluate a company’s internal environment and reveal its strengths and weaknesses ; 2) matching stages, such as Strenght-Weaknesses-Opportunities-Threat Matrix (SWOT), and the Strategies Pasition and Action Evaluation Matrix (SPACE) is one of the matriks used by a company to determine strategy is most appropriate to be applied, Boston Consulting Group Matrix (BCG) is an analytical tool used to assist companies in considering growth opportunities with long-term strategic planning and assist in the allocation of appropriate resource locations, Internal- External Matrix (IE) include the company’s internal strengths and external influences faced by the company to obtain business strategies, and grand strategy matrix; and 3) the decision stage, such as the quantitative strategy planning matrix (QSPM) is a matrix that constructs alternative strategies, based on the key internal and external success factors identified earlier. According to (Rangkuti, 2014), the steps taken in the formulation of the strategy are as follows: ccording to (Rangkuti, 2014), the steps taken in the formulation of the strategy are as foll According to (Rangkuti, 2014), the steps taken in the formulation of the strategy are as follows: 1. Data collection stage This stage is basically not just data collection, but also a classification and pre-analysis activity. At this stage the data can be divided into two namely internal and external data. The model used at this stage is the analysis of internal and external factors by compiling in the Internal Factor Analysis Summary (IFAS)table. 2. 4. Threats a. Lack of community support for tourism facilities b. Poorest Road Infrastructure c. The existence of Travel Destinations in Other Regencies Alternative Formulation of Tourism Development Strategy in Merangin Regency After identifying and analyzing the internal and external environmental conditions of tourism development based on regional potency in Merangin regencyt, the next step is the formulation of strategies to be carried out. The strategy formulation is carried out through three stages, namely the input stage which includes the IFE matrix and EFE matrix, the matching stage which includes the IE matrix and the SWOT matrix, and the decision-making stage using the QSPM matrix. 3. Opportunities c. There are SMEs that provide local specialties DATA ANALYSIS METHOD Analysis Phase After all data affecting the development of coffee commodities in Jambi Province are collected, proceed to the analysis stage through a strategy formulation model with a SWOT matrix. 3. Phase Matching Strategy for tourism development in Merangin Regency From the SWOT matrix we will get alternative strategies as shown in the following figure : Table 1. Matriks SWOT IFA/EFA STRENGHTS (S) WEAKNES (W) OPPORTUNITIES (O) SO Strategy WO Strategy Creating a strategy use force to utilize opportunity if it's on quadrant I Creating a strategy minimize weaknesses to utilize opportunity. Use if is in quadrant III TREATHS (T) ST Strategy WT Strategy Creating a strategy use force to overcome threats. Used if located at quadrant II Creating a strategy minimize weaknesses and avoid threats. Used if located at quadrant IV 40 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 This research discusses only on tourism strategy and development in Merangin regency based on its tourism potential. Further, this research will discuss about tourism promotion based on digital promotion. Identification of Strengths, Weaknesses, Opportunities and Threats Identification of strengths, weaknesses, opportunities, and threats are important factors in conducting a SWOT analysis. For the analysis of the internal environment, the identification made is a strength factor and a weakness factor, while for an external environmental analysis the factors identified are the opportunity factor and the threat factor. After these factors are identified, it will produce various alternative strategies, then the best strategy is chosen. 1. Strength 1. Strength The strengths possessed by Merangin tourism are: a. A beautiful tourist attraction, original and has special characteristics b. Freash Air c. Profound Government Support 1. Strength The strengths possessed by Merangin tourism are: The strengths possessed by Merangin tourism are: a. A beautiful tourist attraction, original and has special characteristics b. Freash Air c. Profound Government Support d. Good Security Conditions 2. Weakness 2. Weakness Weaknesses in tourism development in Merangin district are: a. Tourism promotion is still low b. Inadequate facilities and supporting infrastructure for tourism c. The absence of public transportation to reach tourist attractions d. Lack of HR in Tourism e. The government budget is still limited to the tourism sector 2. Weakness Weaknesses in tourism development in Merangin district are: b. Inadequate facilities and supporting infrastructure for tourism c. The absence of public transportation to reach tourist attraction e. The government budget is still limited to the tourism sector 3. Opportunities a. There are still numerous natural tourism objects that have not yet been developed b. The existence of tour and travel agents c. There are SMEs that provide local specialties 1. Matriks IFE The IFE matrix is used to find out how big the role of internal factors is in the development of regional potential based tourism in Merangin District. 41 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Table 2. IFE Matrix Model and Tourism-Based Development Strategy Regional Potential in Merangin Regency Factor Strategis Internal Point Rate Score Strength 1. Attractions beautiful, original and has a characteristic 0,108 3,750 0,404 2. Cool air 0,134 3,000 0,401 3. Large Government Support 0,083 3,750 0,365 4. Good security conditions 0,097 3,500 0,345 Weakness 1. Tourism promotion is still low 0,128 2,000 0,257 2. Inadequate facilities and supporting infrastructure for tourism 0,097 1,750 0,170 3. The absence of public transportation to reach tourist attractions 0,095 2,000 0,191 4. Lack of human resources in the Field of Tourism 0,161 1,750 0,283 5. The government budget is still limited to the tourism sector 0,095 1,500 0,143 Total 1 2,522 Source: Processed from Questioners, 2019 odel and Tourism-Based Development Strategy Regional Potential in Based on IFE table above, it is known that the strength factor that has the highest score is a beautiful, original tourist attraction and has a unique characteristic with a score of 0,404. The second highest score is owned by the cool air with a score of 0,401, this is not surprising because natural tourism in the Merangin Regency is still original and beautiful as well as tourist destinations close to Mount Masurai in the Merangin District. Government support obtained a score of 0,365 which is in the third position of strength owned by the Merangin district. The Merangin district government provides strong support for the development of the tourism sector in this area which can be seen from the regional vision and mission. The main weakness in development of tourism in the Merangin regency is the limited government budget with a score of 0,143. With a limited budget command the tourism sector will require a long time in the provision of tourism facilities, facilities and infrastructure, the provision of tourism facilities, facilities and infrastructure will have an impact on tourism development more quickly, therefore the need for investment from the private sector in tourism management areas in Merangin Regency. 1. Matriks IFE The second weakness in tourism development in Merangin Regency is the inadequate tourism supporting facilities and infrastructure with a score of 0,170, the existing tourism facilities and infrastructure will be very closely related to the number of tourist visits to the region. Overall, the total weighted score of strengths and weaknesses in IFE matrix is 2,522. Thus, the internal condition of regional potential based tourism development in Merangin district is above the average value of 2,50. Based on the total weighted values, it can be concluded that the development of tourism in the Merangin district is in a strong position in utilizing the strengths it has and is capable enough to overcome weaknesses. 2. Matriks EFE The EFE matrix is used to determine the effect of external factors faced in tourism development in Merangin District. seen in Table 4.2. 42 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Table. 3. EFE Matrix Model and Tourism Development Strategy Based On Regional Potency in Merangin Regency Factor Strategy External Weight Rating weight score Opportunity 1. There are still many natural tourism objects that have not yet been developed 0,171 4 0,683 2. The existence of tour and travel agents 0,171 2,25 0,384 3. The existence of micro and small businesses that provide local specialties 0,188 2,75 0,516 Threat 1. Lack of community support for tourism facilities 0,158 2,5 0,396 2. Road infrastructure that is not good yet 0,092 2,5 0,229 3. The existence of tourist destinations in other districts 0,221 2,25 0,497 Source: Processed from Questioners, 2019 Based on EFE matrix table, the main opportunity is there are still numerous non-developed tourism objects with the highest score of 0,683. This score shows that tourism development in Merangin district is still wide open with many natural tourism desires that have not yet been developed, even have not been touched by the local government at all. A score of 0,516 is owned by the presence of SMEs that provide local specialties. The main threat factor that must be faced in the development of tourism in the Merangin district is the unfavorable road infrastructure with a score of 0,229, the main road infrastructure leading to the tourist destination is still not good, this is seen by the still many rocky and perforated roads coupled with terrain quite a terrible road. Another threat faced in the development of tourism in Merangin district is the lack of community support for tourism facilities. Overall, the total weighted score from three opportunities and three threats in the EFE matrix is 2,705 or above the average value of 2,50. Based on the total weighted values, it can be concluded that the development of regional-based tourism potential in Merangi Regency is able to respond to the external environment by utilizing the opportunities they have to face threats. 43 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 3. IE Matrix IE matrix is arranged based on the analysis of internal and external factors combined from the IFE matrix and the EFE matrix. IE matrix can be seen in Figure 4.1 below: Total Score IFE Strong Average Weak 4,00 3,00 2,00 1,00 I II III High 3,00 IV V VI Middle 2,00 VII VIII IX Low 1,00 Figure 1. IE Matrix Tourism Development in Merangin Regency Jambi Province T O T A L S C O R E E F E 2,522 2,.7052 Figure 1. IE Matrix Tourism Development in Merangin Regency Jambi Province The results of internal factor analysis by using the IFE matrix obtained a weighted total score of 2,545. While the results of the analysis of external factors using the EFE matrix obtained a weighted total score of 2,705. Based on the total weighted score from the IFE and EFE matrix, the development of regional potential based tourism in Merangin Regency is in cell V in the IE matrix. The strategy that can be taken at the position of the cell is the Hold and Maintain Strategy. 4. SWOT Matrix The SWOT matrix is compiled based on the results of the identification and analysis of internal and external factors, including strengths and weaknesses that are owned as well as opportunities and threats that must be faced. The integration of internal and external factors in the SWOT matrix will result in several alternative strategies that can be used in the development of regional-based tourism potential in the Merangin regency. The SWOT matrix can produce four sets of possible alternative strategies namely S-O strategy, S-T strategy, W-O strategy and W-T strategy. The strategies generated from the SWOT matrix can be seen below. 44 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Table 4. SWOT Matrix Tourisn Development Based On Local Potency in Merangin Regency Source: Processed from Questioners, 2019 Internal Factors External Factors Strength (S) 1.A beautiful, original and distinctive attraction 2. Cool air 3. Large Government Support 4. Good security condition Weakness (W) 1. Tourism promotion is still low 2. Inadequate tourism supporting facilities and infrastructure 3. The absence of public transportation to reach tourist attractions 4. Lack of human resources in the field of tourism 5. The government budget is still limited to the tourism sector Opportunity 1. There are still natural tourism objects that have not yet been developed 2. The existence of tour and travel agents 3. The existence of SMEs that provide local specialties S – O Strategy 1. Development of other natural attractions (S1 and O1) 2. The government works in cooperation with SMEs to increase the promotion of regional specialties (S3 and O3) 3. Provide faster and cheaper licensing and legality for tourism bureaus in Merangin District (S3, S4 and W2) W – O Strategy 1. Increased tourism promotion in collaboration with travel agencies in the form of Merangin tour packages (S1 & O2) 2. Improvement of existing facilities and infrastructure (S2, O1 and O2) 3. Increasing Tourism Human Resources both from the government apparatus and from the private sector (S4 and S2) 4. Coaching and developing SMEs by - by special regions (S2 and O3) Threat 1. The lack of community support in tourism area 2. Low quality of road infrastructure 3. There are another tourism areas in other regions S – T Strategy 1. Developing tourism-aware communities (S3 and T1) 2. 4. SWOT Matrix Focusing road infrastructure to tourist destination areas (S3, T1 and T2) W – T Srategy 1. Cooperation with the private sector in the development of regional tourism (W 2 and W5, T2) 2. More intensive tourism promotion (W1 and T3) e 4. SWOT Matrix Tourisn Development Based On Local Potency in Merangin Regency Table 4. SWOT Matrix Tourisn Development Based On Local Potency in Merangin Strength (S) 1.A beautiful, original and distinctive attraction 2. Cool air 3. Large Government Support 4. Good security condition 3. The absence of public transportation to reach tourist attractions S – O Strategy W – O Strategy 1. Increased tourism promotion in collaboration with travel agencies in the form of Merangin tour packages (S1 & O2) ) 2. The government works in cooperation with SMEs to increase the promotion of regional specialties (S3 and O3) 2. Improvement of existing facilities and infrastructure (S2, O1 and O2) 3. The existence of SMEs that provide local specialties 3. Increasing Tourism Human Resources both from the government apparatus and from the private sector (S4 and S2) Source: Processed from Questioners, 2019 Source: Processed from Questioners, 2019 Source: Processed from Questioners, 2019 5. QSPM Matrix The final stage of the strategy formulation analysis is the decision making stage, through the selection of best strategy according to priority using the QSPM matrix. After obtaining several alternative strategies for developing regional-based tourism potential in Merangin District, the selection of alternative strategies is prioritized to be implemented using the QSPM matrix. The strategy chosen to be implemented is based on the results of the QSPM analysis calculations shown in the table below. 45 Asian Journal of Social Science Research (e-ISSN: 2600-9706) V l 2 I 2 2019 Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Table 5. QSPM Matrix Alternative for Tourism Sector Development Strategy Based on Regional Potential in Merangin Regency Strategy Relationship TAS Total Attractiveness Score Rank 1. Development of other natural attractions (S1 and O1) 4,315 7 2. The government cooperates with SMEs to increase the promotion of regional specialties (S3 and O3) 3,829 11 3. Provide faster and cheaper licensing and legality for tourism bureaus in Merangin Regency (S3, S4 and W2) 4,386 5 4. Increasing tourism promotion in collaboration with travel agencies in the form of Merangin tour packages (S1and O2) 4,663 3 5. Repair of existing facilities and infrastructure (S2, O1 and O2) 4,384 6 6. Increasing Tourism Human Resources both from the government apparatus and from the private sector (S4 and S2) 3,886 10 7. Coaching and developing SMEs by - by special regions (S2 and O3) 3,912 9 8. Grow and develop tourism-aware society (S3 and T1) 4,160 8 9. Focusing road infrastructure on tourist destination areas (S3, T1 and T2) 4,956 1 10. Cooperation with the private sector in developing regional tourism (W 2 and W5, T2) 4,890 2 11. More intensive tourism promotion (W1 and T3) 4,659 4 Source: Processed from Questioners, 2019 QSPM Matrix Alternative for Tourism Sector Development Strategy Based on Regional al in Merangin Regency Based on the QSPM matrix above, alternative priority strategies that can be carried out by the Merangin district government in developing the tourism sector based on regional potential are: 1. On focusing of road infrastructure to tourist destination areas with a TAS value of 4.956. This strategy is a priority strategy in the development of the tourism sector because with a good road infrastructure, tourists will become more interested in visiting these attractions. 2. Cooperation with the private sector in development of regional tourism with a TAS value of 4,890. 5. QSPM Matrix It means that there is a need for financial assistance from the private sector both from the domestic private sector or through foreign investment. p g g 3. Increased tourism promotion in collaboration with travel agencies in the form of Merangin tour packages with a TAS value of 4.663. Offering Merangin tour packages through travel agents will be more attractive for tourists 4. More vigorous tourism promotion is a strategy to develop the fourth tourism sector with a TAS value of 4.659. With the promotion of tourism that is more intense the number of tourists who come to be more and the development of the tourism sector in the Merangin district can run faster. 46 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 5. Providing faster and cheaper licensing and legality for tourism agency in Merangin regency is the 5th strategy with a TAS value of 4.386, this will have an impact on the emergence of new tourism bureaus and improve performance at bureaus tourism in attracting tourist visits and will have an impact on the development of the tourism sector in Merangin regency. 5. Providing faster and cheaper licensing and legality for tourism agency in Merangin regency is the 5th strategy with a TAS value of 4.386, this will have an impact on the emergence of new tourism bureaus and improve performance at bureaus tourism in attracting tourist visits and will have an impact on the development of the tourism sector in Merangin regency. p p g g y 6. The 6th strategy in developing the tourism sector is the improvement of existing facilities and infrastructure, with the improvement of tourist facilities and infrastructure, the tourists feel comfortable, happy and want to return to visit the tourist destination 6. The 6th strategy in developing the tourism sector is the improvement of existing facilities and infrastructure, with the improvement of tourist facilities and infrastructure, the tourists feel comfortable, happy and want to return to visit the tourist destination 7. Development of other natural attractions with a TAS value of 4,315. Merangin Regency still has tourism objects that have not yet been developed that have the same potential as other tourism objects that already exist today. 5. QSPM Matrix The final strategy in the development of the tourism sector in Merangin Regency is that the Government in cooperation with SMEs promotes the promotion of local specialties, making regional souvenirs one of the icons sought by tourists. 5. QSPM Matrix If the local government develops tourism objects with the necessary facilities and infrastructure, then the opportunity to attract greater tourist arrivals to come to the Merangin district is more open. 7. Development of other natural attractions with a TAS value of 4,315. Merangin Regency still has tourism objects that have not yet been developed that have the same potential as other tourism objects that already exist today. If the local government develops tourism objects with the necessary facilities and infrastructure, then the opportunity to attract greater tourist arrivals to come to the Merangin district is more open. 8. Developing tourism-aware society is the 8th strategy with a TAS value of 4.160. Currently the community tourism awareness movement in Merangin Regency is one of the efforts made by the local government through the rural tourism community 8. Developing tourism-aware society is the 8th strategy with a TAS value of 4.160. Currently the community tourism awareness movement in Merangin Regency is one of the efforts made by the local government through the rural tourism community 9. The 9th tourism sector development strategy is the fostering and development of SMEs by regional specialists with a TAS value of 3,912. Development of SMEs by local specialties can be carried out with training and product marketing development, this needs to be done because local specialties in Merangin district are still very narrow in their marketing reach and are still difficult to find. 10. Improving of Tourism HR both from the government apparatus and from the private sector with a TAS value of 3,886. This increase in HR will have an impact on the development of the tourism sector by making tourism management safer, more comfortable and sustainable. 10. Improving of Tourism HR both from the government apparatus and from the private sector with a TAS value of 3,886. This increase in HR will have an impact on the development of the tourism sector by making tourism management safer, more comfortable and sustainable. 11. The final strategy in the development of the tourism sector in Merangin Regency is that the Government in cooperation with SMEs promotes the promotion of local specialties, making regional souvenirs one of the icons sought by tourists. 11. ONAL POTENCY BASED TOURISM SECTOR DEVELOPMENT MODEL IN MERANGIN REGENCY With this tourism sector development model, it can contribute to involve the sectors in the area in tourism development and novelty in this research is a new tourism development model. ACKNOWLEDGEMENT The authors thank the university for the support of the publication. The authors thank the university for the support of the publication. ONAL POTENCY BASED TOURISM SECTOR DEVELOPMENT MODEL IN MERANGIN REGENCY The development of the tourism sector must involve all parties, ranging from the central government, local governments, the private sector and the community around the tourist attraction. The development of the tourism sector will have an impact on increasing local revenue, empowering the surrounding community and increasing people's income from the existence of these attractions. Figure 2. Model Tourism Development Based on Local Potency in Merangin Regency Local Goverment Private Sector Community Road Tourism Aware Community Facilities Tour Agent SMEs Enterprises) Tourism Objectives Tourism Development Road Local Goverment Facilities Tourism Development Tourism Objectives Private Sector Development Tour Agent Tourism Aware Community Community Figure 2. Model Tourism Development Based on Local Potency in Merangin Regency Enterprises) 47 47 Asian Journal of Social Science Research (e-ISSN: 2600-9706) Volume 2, Issue 2, 2019 DOI: https://doi.org/10.5281/zenodo.8107097 There are 3 components involved in development of tourism sector in Merangin regency, through local government, private sector and community. The local government provides the main road infrastructure to attractions with good conditions, as well as the roads are around the tourist attraction, in addition to providing road infrastructure, the local government can also provide facilities and infrastructure for tourist facilities around the tourist attraction. In addition to the local government, the private sector can also participate in the development of tourism in the Merangin regency through travel bureaus by providing tour packages, and investing in tourism in the provision of tourism facilities and infrastructure. In addition to the local government and the private sector, the community is also an important part in the development of tourism in the Merangin district. Tourism-aware community movement is one form of community movement to participate in the tourism sector by maintaining facilities and infrastructure, helping tourists visiting tourist objects, making community homes as home stays and making communities to form SMEs based on results. regional agriculture. In many cases, alternative tourism strategirs ought to be designed, either by themselves or in concert with mainstream tourism, to provide more appropriate forms of development that reduce the negative impacts and increase the positive effects tof tourism. These include a stress on small-scale, localy owned developments that increase local multiplier and spread effects, greather community participation in tourism planning, and more attention for the culture and environmental sustainability of tourism projects (Brohman, 1996). CONCLUSION ased on the results of research and discussion, the following conclusions are obtained: 1. In developing tourism potency based on regional potential in Merangin regency there are 11 alternative strategies, namely: Focusing road infrastructure to the destination area, cooperation with the private sector in developing regional tourism, increasing tourism promotion in collaboration with tourism bureaus in the form of Merangin tour packages, promotion of tourism that is more intense, provides licensing and legality faster and cheaper for tourism bureaus in Merangin District, improvement of existing facilities and infrastructure, development of other natural tourism objects, fostering tourism-aware communities, fostering and developing SMEs by - by local specialties, increasing Tourism Human Resources both from the government apparatus and from the private sector and the government in collaboration with SMEs increases the promotion of regional specialties. 2. The tourism sector development model in Merangin regency is the cooperation amongst of all existing components starting from the local government, the private sector and the community in Merangin district itself. 2. The tourism sector development model in Merangin regency is the cooperation amongst of all existing components starting from the local government, the private sector and the community in Merangin district itself. 2. The tourism sector development model in Merangin regency is the cooperation amongst of all existing components starting from the local government, the private sector and the community in Merangin district itself. CONTRIBUTION OF AUTHOR All authors are participants in the data collection, analysis, writing and revising the manuscript. All authors are participants in the data collection, analysis, writing and revising the manuscript. CONFLICT OF INTEREST The authors declare that they have no conflicts of interest regarding this manuscript. The authors declare that they have no conflicts of interest regarding this manuscript. 48 REFERENCEES Binns, T., & Nelt, E. (2002). Tourism as an development strategy in south Africa. The Geographical Journal, 168(3 September), 235–247. Brohman, J. (1996). New directions in tourism for Third world development. Annals of Tourism Research, 23(1), 48–70. David, F. (2006). Manajemen Strategi: Konsep (Terjemahan) Jilid 1 (10th ed.). Jakarta: Salemba Empat. Dewi., I. janita. (2011). Implementasi dan Implikasi Pemasaran Pariwisata Yang Bertanggungjawab (Responsible Tourism Marketing). Ike janita dewi, (2011). Implementasi dan Implikasi Pemasaran Pariwisata Yang Bertanggungjawab (Responsible Tourism Marketing). Kementrian Kebudayaan dan Pariwisata Republik Indonesia, Jakarta. Dinas Pariwisata Kabupaten Merangin, 2018. Data Jumlah Wisatawan Kabupaten Merangin Ernawati, N. M. (2010). Tingkat Kesiapan Desa Tihingan-Klungkung, Bali Sebagai Tempat Wisata Berbasis Masyarakat. Jurnal Analisis Pariwisata, 10(1), 1–8. Inskeep, E., & Organization, W. T. (1998). Guide for Local Authorities on Developing Sustainable Tourism. Madrid, Spain: World Tourism Organization. Jovanovic, Verka . (2008). The Applocation of GIS and Its Components In Tourism. Yugoslav Journal of Operations Research, 18(2), 261 - 272 Lee, B. K., & Kim, H. J. (2016). Influence Analysis of community resident support for sustainable development. Influence Analysis of Community Resident Support for Sustainable Development., 1–10. Lickorish, leonard j et al, (1994). Developing tourism destination : policies and perspective, Harlow, longman. Mason, P. (2003). Tourism impact, planning and management. United Kingdom (UK): Butterworth-HeinemannAn imprint of Elsevier. McGehee, N. G., & Andereck, K. L. (2004). Factors Predicting Rural Residents Support of Tourism. Journal of Travel Research, 43(2), 188–200. Perdue, R. R., Long, P. T., & Allen, L. R. (1990). Resident support for Tourism Development. Annals of Tourism Research, 17(4), 586–599. Rangkuti, F. (2014). Analisis Swot Teknik Pembeda Kasus Bisnis. Jakarta: PT. Gramedia Pustaka Utama. Undang-Undang Republik Indonesia No. 10 Tahun 2009 Tentang Kepariwisataan. 49
https://openalex.org/W4384401211
https://pure.ulster.ac.uk/files/123141545/bjep.12625.pdf
English
null
International comparisons of the home mathematics environment and relations with children's mathematical achievement
British journal of educational psychology
2,023
cc-by
9,442
General rights Th i ht General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the docum Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of the output without obtaining the permission of the author(s). If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. Document Licence: CC BY Document Licence: CC BY General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. International comparisons of the home mathematics environment and relations with children's mathematical achievement Ellis, A., Cosso, J., Duncan, R. J., Susperreguy, M. I., Simms, V., & Purpura, D. J. (2023). International comparisons of the home mathematics environment and relations with children's mathematical achievement. British Journal of Educational Psychology, 93(4), 1-17. Article e12625. Advance online publication. https://doi.org/10.1111/bjep.12625 Link to publication record in Ulster University Research Portal Published in: British Journal of Educational Psychology Publication Status: Published online: 15/07/2023 DOI: 10.1111/bjep.12625 Document Version Publisher's PDF, also known as Version of record 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. © 2023 The Authors. British Journal of Educational Psychology published by John Wiley & Sons Ltd on behalf of British Psychological Society. Take down policy Take down policy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 Received: 18 March 2022  |  Accepted: 20 May 2023 DOI: 10.1111/bjep.12625 Received: 18 March 2022  |  Accepted: 20 May 2023 DOI: 10.1111/bjep.12625 Alexa Ellis1   | Jimena Cosso2   | Robert J. Duncan3   | María Inés Susperreguy4,5   | Victoria Simms6   | David J. Purpura3 Alexa Ellis1   | Jimena Cosso2   | Robert J. Duncan3   | María Inés Susperreguy4,5   | Victoria Simms6   | David J. Purpura3 1Human Development and Family Studies, The University of Alabama, Tuscaloosa, Alabama, USA 2Department of Educational Psychology, Counseling, and Special Education, The Pennsylvania State University, University Park, Pennsylvania, USA 3Human Development and Family Science, Purdue University, West Lafayette, Indiana, USA 4Facultad de Educación, Pontificia Universidad Católica de Chile, Santiago, Chile 5Millennium Nucleus for the Study of the Development of Early Math Skills (MEMAT), Santiago, Chile 6School of Psychology, Ulster University, Coleraine, UK Correspondence Alexa Ellis, The University of Alabama, 651 Peter Bryce Blvd, Tuscaloosa, AL 35401, USA. Email: alexa.ellis@ua.edu 1Human Development and Family Studies, The University of Alabama, Tuscaloosa, Alabama, USA 2Department of Educational Psychology, Counseling, and Special Education, The Pennsylvania State University, University Park, Pennsylvania, USA 3Human Development and Family Science, Purdue University, West Lafayette, Indiana, USA 4Facultad de Educación, Pontificia Universidad Católica de Chile, Santiago, Chile 5Millennium Nucleus for the Study of the Development of Early Math Skills (MEMAT), Santiago, Chile 6School of Psychology, Ulster University, Coleraine, UK Correspondence Alexa Ellis, The University of Alabama, 651 Peter Bryce Blvd, Tuscaloosa, AL 35401, USA. Email: alexa.ellis@ua.edu wileyonlinelibrary.com/journal/bjep  |  1 of 17 Br J Educ Psychol. 2023;00:e12625. https://doi.org/10.1111/bjep.12625 p g p p y Authors. British Journal of Educational Psychology published by John Wiley & Sons Ltd on behalf of British Psychological Societ K E Y W O R D S home mathematics environment, international perspective, TIMSS BACKGROUND Research emphasizing the importance of children's early mathematical skills prior to school-­based in- struction (Davis-­Kean et al., 2021; Watts et al., 2014) has fuelled an interest in the diverse mathematical activities that children engage with in the home environment (hereinafter the home mathematics envi- ronment (HME); LeFevre et al., 2010; Niklas et al., 2018). Most commonly, HME research has focused on parent–­child interactions surrounding numerical activities as measured by the frequency of engaging in such activities (LeFevre et al., 2009). Research has identified a small, yet significant relation between the frequency of HME activities and children's mathematical understanding (Daucourt et al., 2021; Mutaf-­Yıldız et al., 2020), suggesting that the home may provide the primary avenue for parents to con- tribute to children's mathematical skills. However, much of the published research examining the HME is comprised of samples from a limited range of countries (15 represented in Daucourt et al., 2021). In this study, we broaden international representation and address a current gap in the literature by testing to what extent, if at all, there are differences in the HME across countries—­in terms of retrospective average activity reports, the reliability of measurement, and the relation between the HME and chil- dren's mathematics performance. Abstract Abstract Background: Home mathematics environment (HME) re- search has focused on parent–­child interactions surround- ing numerical activities as measured by the frequency of engaging in such activities. However, HME survey ques- tions have been developed from limited perspectives (e.g., Early Childhood Research Quarterly, 27, 2012, 231; Journal of Social Issues, 64, 2008, 95; Early childhood mathematics education research: Learning trajectories for young children, Routledge, New York, 2009), by researchers from a small subset of countries (15; Psychological Bulletin, 147, 2020, 565), which may skew our interpretations. Aims and Sample: This study broadened international representation by leveraging secondary data from the 2019 TIMSS to examine the variation of the frequency and reli- ability of the HME scale and its relation to children's math- ematical achievement. Across 54 countries, 231,138 parents and children (Mage = 10.22 years; 51% male) participated in the larger study. Correspondence Alexa Ellis, The University of Alabama, 651 Peter Bryce Blvd, Tuscaloosa, AL 35401, USA. Email: alexa.ellis@ua.edu Methods: Parents completed a retrospective home envi- ronment survey and children were assessed on mathemat- ics skills. Basic frequency descriptive statistics, Cronbach's alpha reliability coefficients, and Pearson's r correlation co- efficients were used to assess variability across countries. Results: Findings suggested that families in certain coun- tries engaged in home mathematics activities more frequently than families in other countries; however, the HME scale demonstrated acceptable internal consistency across fami- lies in all countries (M α = .79; range = [.73, .89]). Further, the average relation between HME and mathematical wileyonlinelibrary.com/journal/bjep  |  1 of 17 Br J Educ Psychol. 2023;00:e12625. https://doi.org/10.1111/bjep.12625 Br J Educ Psychol. 2023;00:e12625. https://doi.org/10.1111/bjep.12625 1 of 17 2 of 17 ELLIS et al. achievement was r = .15 with a range between r = .02 to r = .41. Conclusion: Our results indicate substantial variation across countries in the HME-­mathematical achievement association. These findings underscore the importance of international representation in advancing research on the diversity of a child's home environment. The home mathematics environment Many studies have recently focused on how the HME relates to children's mathematical skills (Daucourt et al., 2021; Elliott & Bachman, 2018; Mutaf-­Yıldız et al., 2020). However, findings from the existing liter- ature are mixed (Blevins-­Knabe, 2016; Mutaf-­Yıldız et al., 2020), with some studies suggesting both early home mathematics and literacy activities were positively related to children's fourth-­grade mathematical achievement (Gustafsson, 2013; Punter et al., 2016). Other studies indicate the HME has no relation to children's mathematical performance at age 3 and 5 (Blevins-­Knabe et al., 2000; Missall et al., 2015). Most recently, a meta-­analysis was conducted to synthesize the extant literature and estimate the average overall effect size of the relation between the HME and children's mathematical development (Daucourt et al., 2021). Across 15 countries, 631 effect sizes, and 68 independent samples, Daucourt et al. (2021) reported an overall small (r = .13, 95% CI: [.09, .17]), yet statistically significant, correlation that translates to 2% of the common variance between the HME and children's mathematical achievement. However, most of the samples included in the meta-­analysis came from countries often overrepresented in the lit- erature, most specifically the United States of America and Canada. Notably, countries overrepresented in the literature do not capture the vast differences in educational conditions, cultural values, or beliefs present across the globe, and prior research suggests interactions in the home environment vary interna- tionally (Cahoon et al., 2021; LeFevre et al., 2010; Susperreguy et al., 2020). Thus, there is a clear need to understand child development and parenting more globally (Lansford et al., 2016, 2019). INTERNATIONAL HME TIMSS 3 of 17 An international perspective Given that prior research has been restricted by the number of countries present in the literature, the current study responds to a recent call for expanding the measurement of the HME to include more diverse and internationally representative populations (Hornburg et al., 2021). Many questions about HME activities used in prior studies were developed by researchers in the United States of America (Sarama & Clements, 2009) and Canada (LeFevre et al., 2009). This inherently sets those populations as the norm. Therefore, it is possible that certain survey questions may or may not be appropriate for capturing all international home mathematics activities. For example, a HME survey item such as, ‘play board or card games’ first requires board games to be a relevant activity in each country and second requires parents to understand which board or card games the question is referencing. Hence, it is es- sential to increase the number of countries represented in the literature to better understand the role of diverse backgrounds and experiences in the HME. Although Daucourt et al. (2021) suggest a small, average HME-­achievement relation in the ex- isting literature, due to the small representation of countries outside of North America, the authors were unable to include country as a potential moderator of this relation. In fact, country of origin was originally a preregistered moderator that had to be excluded from analyses as to not lead to ungeneralizable findings based on skewed samples (Daucourt et al., 2021). Therefore, examining the relation between the HME and children's mathematical achievement by country would address an important gap in the literature. Importantly, analysing and descriptively reporting these relations from more countries (not included in prior research) provides opportunities for new populations to be represented and contribute to our broader understanding of the HME and children's mathemat- ical achievement. Data source and sample Data analysed in the present study were obtained from the TIMSS 2019. TIMSS includes data from children at fourth and eighth grades every 4 years since 1995 in 64 participating countries. All countries in the TIMSS data set are OECD (Organization for Economic Co-­operation and Development) countries. The TIMSS data set is designed to examine trends in student mathematics and science achievement in countries around the world. Each country participating in the study de- velops and implements a national sampling plan with the country's National Research Coordinator and TIMSS sampling experts. The sampling procedure was upheld by Statistics Canada and advised by National Research Coordinators to ensure that the national sampling plan conformed to TIMSS standards (LaRoche et al., 2020). In addition to monitoring children's trends in mathematics and sci- ence, TIMSS also assesses children's contexts for learning through parents', teachers' and principals' questionnaires. The TIMSS data set is optimal for this study as it is the only data set that includes questions about the HME from different countries, and at the same time, provides measures of children's mathematical achievement. The sample for the present study consisted of fourth-­grade children (Mage[SD] = 10.22 [.66]; 51% male) that completed the mathematical achievement test and their parents that responded to the early learning survey. The final sample consisted of 231,138 chil- dren from 54 countries across six continents. The present investigation The main contribution of this study is to test to what extent there are international differences in the frequency of engagement in a specified set of HME activities. The current study was preregistered (https://osf.io/q7w3n) and utilized data from the Trends in International Mathematics and Science Study (TIMSS) 2019 wave, focusing on the mathematical questions provided in a retrospective home questionnaire. We were able to utilize data from 54 countries present in the TIMSS data set, to address our three main research questions: (1) Do families across countries differ in how frequently they partici- pate in mathematical activities in the home? (2) Do the HME survey questions demonstrate acceptable reliability across all countries? (3) Finally, how does the overall HME activity score relate to fourth-­ grade mathematical achievement across different countries? Prior studies have suggested the HME may vary across broader international contexts (Cahoon et al., 2021; Susperreguy et al., 2020), therefore, we first expect that the frequency in which parents report participating in each of these activities will differ by country. Second, based on prior work in the TIMSS data set, results have shown that the reliability of the overall home environment survey has been reported as acceptable across all countries (Mullis & Fishbein, 2020), which means the items are consistent with one another and measure the same construct. However, given the scale was initially developed using literacy and mathematics together, the current study examines a subset of questions from the larger survey. Thus, we found it important to examine the reliability of the subset of items as this approach was novel. We hypothesize that the HME scale will vary across countries but continue to demonstrate acceptable reliability across the subset of items. Finally, we expect that the relation be- tween the HME and children's mathematical achievement will vary across countries but demonstrate an overall positive, small relation based on the prior HME meta-­analysis (r = .13; based on Daucourt et al., 2021). 4 of 17 ELLIS et al. 4 of 17 Measures Home mathematics environment Home mathematics environment The HME questions were asked retrospectively and taken from the TIMSS 2019 Early Learning Survey (ELS), which was developed based on previous research documenting the importance of early child- hood learning activities. To read more about the development of the TIMSS home context question- naire, citations and outlines can be found online under the Assessment Framework chapters of the TIMSS 2019 technical report (Mullis & Martin, 2017). The ELS consists of 18 items asking parents or guardians about the home resources to support their children learning, their demographic information, their opinion about the school and pre-­primary education, retrospective reports of the home activities related to literacy and numeracy before the child enters formal school and their opinion about children's literacy and numeracy abilities at first grade. The TIMSS & the Progress in International Reading Literacy Study (PIRLS) International Study centre conducted extensive analyses and reported accept- able reliability and validity across all countries (Yin & Fishbein, 2020). For the purpose of the present study, a subset of nine of the 18 items in the ELS measuring the HME were used. When children were in fourth grade, parents were asked to retrospectively report on aspects of their home learning environment. The ELS prompts, ‘Before your child began primary or elemen- tary school, how often did you or someone else in your home do the following activities with him or her?’ Items included ‘say counting rhymes or sing counting songs’ (ASBH01J), ‘play with number toys’ (ASBH01K), ‘count different things’ (ASBH01L), ‘play games with involving shapes’ (ASBH01M), ‘play with building blocks or construction toys’ (ASBH01N), ‘play board or card games’ (ASBH01O), ‘write numbers’ (ASBH01P), ‘draw shapes’ (ASBH01Q), ‘measure or weigh things’ (ASBH01R). Parents could respond by selecting often (1), sometimes (2), or never or almost never (3). Responses were reverse-­ coded such that higher scores reflected more of a given activity. Countries adapted the ELS based on their native language. 5 of 17 INTERNATIONAL HME TIMSS Mathematical achievement The TIMSS & PIRLS International Study Center at Boston College developed and managed the meas- urement and assessments of mathematical achievement for each year of the TIMSS (Cotter et al., 2020). Children's mathematical skills were measured at fourth grade using an item response theory (IRT) scaling approach in which five plausible values were determined. The test assessed four mathematical domains: numeracy, geometry, shape and measurement, and data display (Mullis & Martin, 2017) and consisted of 175 total items. However, items were separated into 14 achievement scales with the aim that children would respond to only a subset of items (between 20 and 28 total items) and IRT would be used to provide five plausible mathematical achievement values. Items were multiple-­choice (worth one point) and other constructed responses (e.g., writing words or numbers, drawing, or sorting; worth two points; Mullis & Martin, 2017). The final achievement measure accounted for sampling design, sampling weights and plausible values specific to TIMSS (International Association for the Evaluation of Educational Achievement, 2021). Country The TIMSS 2019 label of country identification was used as a grouping variable (IDCNTRY). The TIMSS allows all participating countries to complete the ELS. Of the 64 participating TIMSS coun- tries, 54 countries completed the survey (see Table 2). Analytic plan The first research question was examined using basic frequency descriptive statistics (mean and stand- ard deviation across each of the home mathematics items and average by country) to assess how fre- quently families across countries reported engaging in mathematical activities in the home. The second research question was examined using Cronbach's alpha reliability coefficient to assess to what extent the internal consistency of the HME items vary by country. Finally, the third research question was examined using Pearson's r correlation coefficients to assess to what extent the overall HME score was related to children's mathematical achievement by country. The International Association for the Evaluation of Educational Achievement (IEA) International Database Analyzer (International Association for the Evaluation of Educational Achievement, 2021) software was used to create SPSS syntax. This syntax considers the sampling design, sampling weights, and plausible values reported in TIMSS. Analyses conducted using a program that does not account for the special structure of the TIMSS design would produce biased results (International Association for the Evaluation of Educational Achievement, 2021). Thus, all related syntax and analyses used SPSS ver- sion 26.0.0.0 (IBM Corporation, 2019) and are available on the OSF project page (https://osf.io/c5h7). We include Bonferroni correction for multiple comparisons given the number of estimates. Although p-­values are reported in the manuscript, we emphasize focusing on the magnitude of effect sizes across countries. Global HME Separating the nine items that focused on home mathematics activities from the larger ELS, we cal- culated the means and standard deviations for each of the nine items across all countries (Table 1) and each country across all nine items (Table 2). Across the nine items, we found that the most common ELLIS et al. 6 of 17 6 of 17 TA BL E 1  Average global descriptive statistics of the HME items. M (SD) Min Max Count things 2.55 (.57) 1.95 2.81 Building blocks 2.53 (.60) 1.84 2.85 Games with shapes 2.50 (.60) 1.78 2.79 Write numbers 2.43 (.61) 2.07 2.79 Draw shapes 2.41 (.63) 1.97 2.68 Board or card games 2.30 (.64) 1.68 2.73 Number toys 2.29 (.67) 1.79 2.65 Counting songs 2.25 (.68) 1.79 2.73 Weigh or measure things 1.79 (.69) 1.40 2.07 HME total 2.34 (.39) 1.86 2.54 activity parents reported engaging their children in when pooled was counting things in the home (M [SD] = 2.55 [.57]), and the least common activity parents engaged their children in was weighing or measuring things (M [SD] = 1.79 [.69]). Overall, most parents participated in home mathematics activi- ties with their children between sometimes and often (M [SD] = 2.34 [.39]). However, there was substan- tial variation in the frequency of activities across countries. For example, families in Morocco engaged in home mathematics activities the least often (M [SD] = 1.86 [.55]), whereas families in Northern Ireland engaged in home mathematics activities the most often (M [SD] = 2.54 [.37]), and these were significantly different (t[8330] = 49.71, p < .001). Reliability of the HME Table 2 displays the reliability of the HME across each country for the nine home mathematics ac- tivities. The results suggest that items for all 54 countries demonstrated acceptable (α > .70; DeVellis & Thorpe, 2021) internal consistency (Mα = .79) suggesting that the nine HME items are closely related as a group regardless of country. Notably, HME survey items for families in Georgia, Italy, Kosovo and Oman demonstrated the lowest, yet still acceptable Cronbach's alpha coefficient (α = .74), and survey items for families in Turkey demonstrated the highest coefficient (α = .90). Relation between the HME and mathematical achievement Examining the correlation between preschool home mathematics activities and children's mathematical achievement in fourth grade, we found a small, positive average relation across the included countries (r = .15) that was consistent with the meta-­analytic effects reported by Daucourt et al. (2021), albeit representing a much wider geographical spread. Table 2 also displays Pearson's r coefficient for each country. Due to the large sample size and sampling weights applied in IDB Analyzer, all but one cor- relation was statistically significant in Table 2 (p < .05). Children in Turkey and Bulgaria demonstrated the strongest relation between home mathematics activities and mathematical achievement (r = .41, p < .001), and children in Georgia demonstrated the smallest positive correlation between home math- ematics activities and achievement (r = .02, p = .25). The substantial variation in the HME-­mathematical achievement relation led us to conduct sensi- tivity analyses to examine item-­level HME-­mathematical achievement correlations across countries. A heatmap of these individual correlations can be seen in Table 3. Results showed that all items tended to be moderately related to mathematics for children in Bulgaria and Turkey, suggesting the larger correla- tions in these countries (r = .41) are a product of generally stronger relations across items rather than a |  7 of INTERNATIONAL HME TIMSS TA BL E 2  Country HME and mathematical achievement statistics. Relation between the HME and mathematical achievement Country Country ID HME M (SD) N observations HME rank Cronbach's alpha Alpha rank HME-­math achievement correlation Correlation rank Albania ALB 2.45 (.40) 3957 14 .79 24 .22* 4 Armenia ARM 2.38 (.40) 4835 25 .77 35 .11* 41 Austria AUT 2.35 (.36) 3854 29 .75 46 .13* 29 Azerbaijan AZE 2.23 (.42) 4468 44 .76 41 .17* 17 Bahrain BHR 2.39 (.38) 4985 21 .78 28 .11* 41 Belgium BFL 2.23 (.39) 4311 45 .77 35 .13* 29 Bosnia and Herzegovina BIH 2.43 (.35) 5218 16 .76 41 .12* 35 Bulgaria BGR 2.25 (.49) 4059 40 .84 5 .41* 1 Canada CAN 2.47 (.38) 9240 10 .82 11 .11* 41 Chile CHL 2.28 (.42) 3698 37 .80 18 .20* 7 Chinese Taipei CHN 2.19 (.43) 3584 47 .86 4 .20* 7 Croatia CRO 2.48 (.35) 3649 7 .78 28 .16* 22 Cyprus CYP 2.40 (.41) 3744 20 .82 11 .18* 14 Czech Republic CZE 2.43 (.33) 3901 16 .75 46 .07* 49 Denmark DNK 2.30 (.35) 1886 34 .75 46 .11* 41 Finland FIN 2.25 (.35) 4105 40 .76 41 .11* 41 France FRA 2.39 (.38) 3656 21 .76 41 .19* 10 Georgia GEO 2.30 (.38) 3295 35 .74 51 .02 54 Germany GRM 2.36 (.36) 2187 28 .75 46 .10* 47 Hong Kong HKG 2.18 (.41) 2663 49 .84 5 .17* 17 Hungary HUN 2.51 (.34) 4147 3 .75 46 .13* 29 Iran IRN 2.16 (.43) 5475 50 .80 18 .17* 17 Ireland IRL 2.48 (.39) 4228 7 .82 11 .19* 10 Italy ITA 2.37 (.37) 3501 27 .74 51 .12* 35 Japan JPN 2.12 (.42) 3840 51 .81 14 .16* 22 INTERNATIONAL HME TIMSS 7 of 17 (Continues) TA BL E 2  Country HME and mathematical achievement statistics. 8 of 17 ELLIS et al. Relation between the HME and mathematical achievement 17  |      ELLIS Country Country ID HME M (SD) N observations HME rank Cronbach's alpha Alpha rank HME-­math achievement correlation Correlation rank Kazakhstan KAZ 2.47 (.35) 4346 10 .78 28 .12* 35 Korea KOR 2.39 (.42) 3798 21 .86 3 .21* 6 Kosovo KSV 2.41 (.35) 3932 19 .74 51 .13* 29 Kuwait KWT 2.33 (.41) 3263 31 .79 24 .11* 41 Latvia LVA 2.39 (.36) 4212 21 .78 28 .10* 47 Lithuania LTU 2.38 (.35) 3008 25 .78 28 .07* 49 Malta MLT 2.50 (.37) 2573 6 .80 18 .18* 14 Montenegro MNE 2.44 (.37) 4629 15 .78 28 .15* 25 Morocco MRC 1.86 (.55) 6511 54 .84 5 .22* 4 New Zealand NZL 2.51 (.39) 2018 3 .84 5 .12* 35 North Macedonia NMC 2.46 (.38) 2733 13 .81 14 .20* 7 Northern Ireland NIR 2.54 (.37) 1821 1 .83 10 .14* 27 Norway NOR 2.30 (.36) 2291 35 .77 35 .12* 35 Oman OMN 2.25 (.38) 6094 40 .74 51 .19* 10 Pakistan PAK 2.00 (.45) 2963 53 .77 35 .17* 17 Philippines PHL 2.25 (.39) 4788 40 .81 14 .18* 14 Poland POL 2.51 (.32) 4543 3 .77 35 .05* 53 Portugal PRT 2.35 (.37) 3986 29 .78 28 .16* 22 Qatar QAT 2.31 (.41) 3785 33 .80 18 .17* 17 Russian Federation RUS 2.52 (.35) 3912 2 .81 14 .07* 49 Saudi Arabia SAU 2.27 (.42) 3333 39 .79 24 .13* 29 Serbia SRB 2.48 (.35) 4127 7 .77 35 .24* 3 Singapore SGP 2.28 (.45) 5737 37 .87 2 .14* 27 Slovak Republic SVK 2.47 (.37) 3970 10 .80 18 .13* 29 INTERNATIONAL HME TIMSS 9 of 17 INTERNATIONAL HME TIMSS ntry Country ID HME M (SD) N observations HME rank Cronbach's alpha Alpha rank HME-­math achievement correlation Correlation rank h Africa SAF 2.21 (.44) 9126 46 .80 18 .19* 10 SPA 2.33 (.37) 8326 31 .76 41 .15* 25 en SWE 2.19 (.40) 3176 47 .79 24 .07* 49 y TUR 2.10 (.56) 3663 52 .90 1 .41* 1 d Arab Emirates UAE 2.43 (.41) 11,988 16 .84 5 .12* 35 after Bonferroni adjustment. Relation between the HME and mathematical achievement L E 2  (Continued) ntry Country ID HME M (SD) N observations HME rank Cronbach's alpha Alpha rank HME-­math achievement correlation Correlation rank h Africa SAF 2.21 (.44) 9126 46 .80 18 .19* 10 SPA 2.33 (.37) 8326 31 .76 41 .15* 25 en SWE 2.19 (.40) 3176 47 .79 24 .07* 49 y TUR 2.10 (.56) 3663 52 .90 1 .41* 1 d Arab Emirates UAE 2.43 (.41) 11,988 16 .84 5 .12* 35 after Bonferroni adjustment. L E 2  (Continued) Country Country ID HME M (SD) N observations HME rank Cronbach's alpha Alpha rank HME-­math achievement correlation Correlation rank South Africa SAF 2.21 (.44) 9126 46 .80 18 .19* 10 Spain SPA 2.33 (.37) 8326 31 .76 41 .15* 25 Sweden SWE 2.19 (.40) 3176 47 .79 24 .07* 49 Turkey TUR 2.10 (.56) 3663 52 .90 1 .41* 1 United Arab Emirates UAE 2.43 (.41) 11,988 16 .84 5 .12* 35 p< 05 after Bonferroni adjustment 10 of 17 ELLIS et al. | TA BL E 3  Heatmap of HME item and mathematical achievement correlation coefficients by country. Relation between the HME and mathematical achievement Country ID Count songs Number toys Count things Games with shapes Building blocks Board and card games Write numbers Draw shapes Weigh or measure things ALB .09* .13* .19* .20* .19* .11* .15* .19* .01 ARM .04 .09* .09* .12* .05 .06* .04 .06* .02 AUT .08* .02 .17* .15* .19* .13* −.02 .02 .04 AZE .06* .07* .09* .17* .14* .09* .10* .10* .05 BHR .07* .06* .10* .11* .10* .05 .06* .04 .00 BFL .07* .04 .12* .19* .18* .14* −.03 −.01 .04 BIH .06* .09* .13* .12* .18* .06* −.03 .03 −.01 BGR .22* .31* .35* .39* .34* .28* .29* .22* .13* CAN .02 .08* .10* .14* .10* .08* .07* .07* .02 CHL .11* .17* .15* .21* .19* .12* .08* .10* .02 CHN .13* .15* .17* .20* .23* .14* .10* .11* .03 CRO .07* .12* .13* .15* .13* .11* .09* .05 .03 CYP .10* .15* .14* .16* .17* .13* .08* .05 .07* CZE .00 .00 .11* .06 .07* .06 .00 −.01 .07* DNK .05 .02 .14* .07 .08 .10* .05 .01 .07 FIN .01 .00 .15* .15* .16* .11* .06 −.01 .05 FRA .07* .15* .17* .18* .17* .14* .03 .06 .08* Legend GEO −.08* .02 .11* .09* .05 .02 −.06 −.04 −.02 .45 GRM .04 .00 .12* .11* .16* .11* −.04 .00 .09* .40 HKG .11* .13* .15* .17* .16* .10* .08* .03 .03 .35 HUN .01 .09* .13* .20* .16* .13* −.04 .04 .07* .30 IRN .11* .09* .14* .18* .18* .12* .04 .13* −.05 .25 IRL .09* .09* .13* .16* .13* .10* .12* .13* .16* .20 ITA .04 .07* .11* .15* .16* .08* .01 .02 −.01 .15 JPN .03 .08* .10* .14* .12* .09* .14* .09* .10* .10 INTERNATIONAL HME TIMSS 11 of 17 Country ID Count songs Number toys Count things Games with shapes Building blocks Board and card games Write numbers Draw shapes Weigh or measure things KAZ .05 .11* .09* .13* .12* .11* .04 .07* −.05 .05 KOR .15* .15* .16* .20* .18* .15* .12* .12* .10* .00 KSV .07* .10* .16* .14* .12* .02 .05 .04 .00 −.05 KWT .09* .06 .12* .08* .07* .02 .10* .06 −.01 −.10 LVA .01 .04 .08* .14* .12* .15* −.01 −.01 .04 −.15 LTU −.01 .07 .06 .07 .14* .12* −.04 .01 .01 −.20 MLT .16* .13* .17* .14* .15* .09* .05 .08* .08* −.25 MNE .08* .10* .12* .11* .18* .10* .02 .08* .04 −.30 MRC .15* .18* .16* .17* .16* .15* .17* .16* .03 −.35 NZL .05 .05 .14* .13* .14* .07 .06 .05 .06 −.40 NMC .16* .13* .15* .22* .21* .07 .10* .08* .03 −.45 NIR .06 .08 .12* .13* .12* .09 .06 .07 .10* NOR .06 .07 .11* .11* .09* .08 .05 .03 .09* OMN .09* .12* .14* .16* .13* .11* .13* .09* .02 PAK .13* −.05 .12* .13* .16* .04 .10* .07 .11* PHL .14* .16* .14* .19* .15* .06* .07* .13* −.04 POL .02 .05 .06* .06* .04 .07* .00 −.02 .03 PRT .10* .16* .17* .18* .17* .09* .02 .03 .02 QAT .13* .14* .17* .21* .19* .04 .09* .03 −.02 RUS −.02 .07* .08* .09* .07* .10* .01 .00 .01 SAU .11* .07* .11* .11* .11* .07* .11* .10* −.07* SRB .10* .14* .17* .30* .25* .10* .07* .10* .04 SGP .07* .11* .11* .15* .19* .10* .09* .04 .01 SVK .08* .08* .13* .20* .19* .04 −.03 .01 .04 (Continues) TA BL E 3  (Continued) 12 of 17 ELLIS et al. Exploratory grouping analyses As part of our preregistration, we discussed grouping countries to explain differences in the HME. Each of the considered grouping variables can be seen on the project's OSF page (https://osf.io/c5h7x/). These included groups such as Gross Enrollment Ratio in Tertiary Education, Western, Human Development Index (HDI), TIMSS Math Achievement Groupings, and the Gini Index. There were multiple reserva- tions about the grouping variables for reasons ranging from some countries missing data or informa- tion, which resulted in them being excluded from group analyses (i.e., Gini Index), to unequal groupings (i.e., HDI), to random cut-­offs (i.e., 50% for Gross Enrollment Ratio in Tertiary Ed). For these reasons, the authors decided not to go forward with any exploratory grouping analyses in the final manuscript. Relation between the HME and mathematical achievement Country ID Count songs Number toys Count things Games with shapes Building blocks Board and card games Write numbers Draw shapes Weigh or measure things SAF .15* .15* .15* .19* .17* .12* .03 .07* −.02 SPA .02 .16* .12* .18* .18* .06* .06* .03 .01 SWE −.03 −.02 .15* .10* .12* .08* .03 −.02 .03 TUR .27* .30* .34* .38* .31* .30* .27* .31* .21* UAE .10* .12* .11* .14* .16* .05* .03 .04* −.03 p < .05 after Bonferroni adjustment. Country ID Count songs Number toys Count things Games with shapes Building blocks Board and card games Write numbers Draw shapes Weigh or measure things SAF .15* .15* .15* .19* .17* .12* .03 .07* −.02 SPA .02 .16* .12* .18* .18* .06* .06* .03 .01 SWE −.03 −.02 .15* .10* .12* .08* .03 −.02 .03 TUR .27* .30* .34* .38* .31* .30* .27* .31* .21* UAE .10* .12* .11* .14* .16* .05* .03 .04* −.03 *p < .05 after Bonferroni adjustment. TA BL E 3  (Continued) INTERNATIONAL HME TIMSS 13 of 17 specific item. Further, parent engagement in items such as ‘count things’, ‘games with shapes’, ‘building blocks’, and ‘board and card games’ were positively related to children's mathematical achievement across all countries. Whereas parent engagement in items such as ‘counting songs’, ‘number toys’, ‘write numbers’, ‘draw shapes’, and ‘weigh or measure things’ showed very small positive to non-­existent as- sociations with children's mathematical achievement across countries. However, it is important to note that although some effects remained statistically significant, many of these relations are small, near zero, effect sizes. specific item. Further, parent engagement in items such as ‘count things’, ‘games with shapes’, ‘building blocks’, and ‘board and card games’ were positively related to children's mathematical achievement across all countries. Whereas parent engagement in items such as ‘counting songs’, ‘number toys’, ‘write numbers’, ‘draw shapes’, and ‘weigh or measure things’ showed very small positive to non-­existent as- sociations with children's mathematical achievement across countries. However, it is important to note that although some effects remained statistically significant, many of these relations are small, near zero, effect sizes. DISCUSSION Utilizing data from a large, international data set, the present study examined the HME across a broader representation of countries. By including more countries in our analyses than any prior research, our study presents an essential first step in studying the HME from a broader international perspective and analysing the same measure across multiple countries in the same age group. Findings from our study showed that families in certain countries engaged in home mathematics activities (as measured by this scale) more frequently than families in other countries, and all countries demonstrated acceptable reli- ability across the nine home mathematical survey items. Further, our findings highlight that although the global average relation between the HME and children's mathematical achievement was like that of previous research (Daucourt et al., 2021), we observed substantial variation around this average relation across countries. These results demonstrate that international research is both beneficial and imperative for theoretical and practical implications to further our understanding of how the HME contributes to children's development of early mathematics skills in different contexts. Importantly, our findings demonstrated a large amount of variability surrounding family reports of engaging in HME activities. Survey questions in the HME area have inherently been developed from a limited perspective (Anders et al., 2012; Melhuish et al., 2008; Sarama & Clements, 2009), which re- stricts our knowledge of the types of mathematics activities that may be present across homes globally. Therefore, reported differences in activity frequency may not solely reflect the wide range of the HME in different country contexts, but also emphasize that these measures do not adequately represent the full range of HME activities across different settings. It could be some items might be more prone to variations in interpretation between countries than other items. One future direction to better capture the international approach in the home environment could be to use participatory research methods to develop country-­specific research tools. For example, Cahoon et al. (2021) took a similar approach to capture this diversity in Northern Ireland and found that other activities and topics emerged when par- ents were prompted to talk about their engagement with their children in mathematics. Thus, consid- erations for examining country and cultural factors in the measurement and contribution of the home environment on children's mathematical performance are important areas for future research. DISCUSSION Consistent with our hypotheses, the reliability across the subset of nine HME items was acceptable in all countries. Notably, the reliability of a measure tells us the degree to which the interrelation among 14 of 17 ELLIS et al. the items on a scale is consistent with one another implying that the items are measuring the same construct. In the case of this study, acceptable reliability was observed across all countries suggesting that all items were related. However, what is unknown is whether a specific item from the survey con- sistently performs worse across all countries or differently between countries. Future research should examine whether there are specific items that might be better suited to measure the HME for everyone or whether these items are potentially invariant in other countries. the items on a scale is consistent with one another implying that the items are measuring the same construct. In the case of this study, acceptable reliability was observed across all countries suggesting that all items were related. However, what is unknown is whether a specific item from the survey con- sistently performs worse across all countries or differently between countries. Future research should examine whether there are specific items that might be better suited to measure the HME for everyone or whether these items are potentially invariant in other countries. Finally, this study demonstrated positive although very weak (i.e., most lie between .10 and .20) significant associations between the frequency of home mathematics activities families reported and children's mathematical achievement. This finding aligns with earlier studies that have examined the HME (e.g., Daucourt et al., 2021; Mutaf-­Yıldız et al., 2020). However, one important note from the cur- rent study was again the large level of variability in this association across the 54 countries. The small relation between the HME and children's mathematical achievement in countries such as Georgia com- pared to the moderate relation in Turkey and Bulgaria emphasizes that the role of the HME in children's mathematical development differs between these countries. The range of this relation across countries led us to examine the relation between individual HME items and children's mathematical achievement. DISCUSSION These sensitivity analyses demonstrated parent engagement in some items, such as ‘playing with blocks’ and ‘games with shapes’, showed relatively stronger correlations with achievement, which would suggest these activities can be related back to the well-­documented relation between spatial abilities and math- ematics in the literature (Hawes et al., 2022; Wolfgang et al., 2001). However, the analyses also demon- strated that parent engagement in some items, such as ‘counting songs’, ‘number toys’, ‘write numbers’, ‘draw shapes' and ‘weigh or measure things’ showed differential (very small positive to non-­existent) relations to children's mathematical achievement across countries. Therefore, fostering parent engage- ment in home activities such as these might be more important for children's mathematical achievement in some countries compared to others. Furthermore, the variation in the association suggests that there are important cross-­cultural differences in the HME and mathematical achievement. To create better recommendations tailored to a population of interest, future work should conduct deeper investigations of the HME within countries. The substantial variability across countries in both the frequency of engaging in HME activities and the HME-­achievement relation presents the field with many questions for future work. These dif- ferences may be due to a plethora of potential factors. For example, differences in parent beliefs about mathematics may differ across cultural groups (del Río et al., 2017; LeFevre et al., 2010). Alternatively, resources in the home (Melhuish et al., 2008) or differences in caregiver-­educator communications (Lin et al., 2019) may also substantially vary across countries. Finally, differences across the frequency of engagement could also be due to parents engaging in certain activities because they see the need for further development in that specific area for their child. Future research is needed to understand the ways in which specific countries rely on specific aspects of the home environment as an educational tool and how and why these variations take place. INTERNATIONAL HME TIMSS 15 of 17 Although the current study includes 54 countries across the globe, it is limited by those who par- ticipated in the larger TIMSS data collection. Many of the countries that participate in the TIMSS demonstrate lower economic inequality, and all are part of the OECD. In addition, out of the 64 countries that participated in the TIMSS, 10 countries did not participate in the home survey. Therefore, although this study is broader internationally than previous research, it does not capture the large variation in economic inequality present in our world. Future research should continue to diversify research to include the other 75% of countries in the world to better understand global perspectives on mathematics. CONFLICT OF INTEREST STATEMENT All authors declare no conflict of interest. AUTHOR CONTRIBUTIONS Alexa Ellis: Conceptualization; data curation; formal analysis; investigation; methodology; soft- ware; validation; visualization; writing –­ original draft; writing –­ review and editing. Jimena Cosso: Conceptualization; investigation; validation; writing –­ review and editing. Robert J. Duncan: Conceptualization; investigation; supervision; validation; writing –­ review and editing. María Inés Susperreguy: Conceptualization; investigation; supervision; writing –­ review and editing. Victoria Simms: Conceptualization; investigation; supervision; writing –­ review and editing. David J. Purpura: Conceptualization; funding acquisition; investigation; supervision; writing –­ review and editing. Conclusion Prior research has highlighted the importance of emphasizing diversity in participants in our research samples (Henrich et al., 2010), particularly in the HME literature (e.g., Hornburg et al., 2021). Given the existing survey questions that assess the HME have been developed from limited perspectives (e.g., LeFevre et al., 2009; Melhuish et al., 2008; Sarama & Clements, 2009), our knowledge of specific activi- ties and responses for different countries has been restricted. Thus, using a large, international data set that sampled families across 54 countries, this study increases diversity and representation in the litera- ture that has not previously existed. Our findings showed substantial variability across countries in the frequency of families engaging in HME activities and the HME-­mathematical achievement relation. Overall, this study highlights the importance of taking a broader, international approach to not just participant recruitment, but also the measurement of various constructs (e.g., children's home learning environments). ACK NO​WLE​DGE​MENTS Data was used from IEA's Trends in International Mathematics and Science Study—­TIMSS 2019 Copyright © 2021 International Association for the Evaluation of Educational Achievement (IEA). Publisher: TIMSS & PIRLS International Study Center, Lynch School of Education and Human Development, Boston College. The research reported here was supported by the National Science Foundation Award #1749294 to Purdue University, as well as ANID – MILENIO – NCS2021_014. The opinions expressed are those of the authors and do not represent the views of the funding sources. This article has earned Open Data and Preregistered Research Designs badges. Data and the preregis- tered design and analysis plan are available at https://osf.io/c5h7x/. Limitations The current study makes a clear contribution to the literature in demonstrating international variability of the HME. However, by using retrospective reports from parents, the accuracy of reporting may have been affected by the time since the activities occurred. Interestingly, it should be noted that the average relation seen in the current study is similar to that reported by Daucourt et al. (2021), which included the use of different methods (both concurrent and retrospective survey responses). Thus, a more important limitation here is the use of surveys to assess the home environment. Parent reports can be biased towards higher amounts of activities than normal to show favourable engagement (Johnson & Christensen, 2019). Future research should, therefore, perhaps combine parent reports with natu- ralistic observations of the home environment across different countries to increase the accuracy of measurement. INTERNATIONAL HME TIMSS INTERNATIONAL HME TIMSS ORCID Alexa Ellis  https://orcid.org/0000-0001-7481-5788 Jimena Cosso  https://orcid.org/0000-0001-8313-8547 Robert J. Duncan  https://orcid.org/0000-0001-6900-0322 María Inés Susperreguy  https://orcid.org/0000-0001-5584-2692 Victoria Simms  https://orcid.org/0000-0001-5664-6810 David J. Purpura  https://orcid.org/0000-0002-9427-914X R EFER ENCES Anders, Y., Rossbach, H.-­G., Weinert, S., Ebert, S., Kuger, S., Lehrl, S., & von Maurice, J. (2012). Home and preschool learning environments and their relations to the development of early numeracy skills. Early Childhood Research Quarterly, 27, 231–­ 244. https://doi.org/10.1016/j.ecresq.2011.08.003 Anders, Y., Rossbach, H.-­G., Weinert, S., Ebert, S., Kuger, S., Lehrl, S., & von Maurice, J. (2012). Home and preschool learning environments and their relations to the development of early numeracy skills. Early Childhood Research Quarterly, 27, 231–­ 244. https://doi.org/10.1016/j.ecresq.2011.08.003 Blevins-­Knabe, B. (2016). Early mathematical development: How the home environment matters. In B. Blevins-­Knabe & A. M. B. Austin (Eds.), Early childhood mathematics skill development in the home environment (pp. 7–­28). Springer International Publishing. https://doi.org/10.1007/978-­3-­319-­43974​-­7_2 Blevins-­Knabe, B. (2016). Early mathematical development: How the home environment matters. In B. Blevins-­Knabe & A. M. B. Austin (Eds.), Early childhood mathematics skill development in the home environment (pp. 7–­28). Springer International Publishing. https://doi.org/10.1007/978-­3-­319-­43974​-­7_2 Blevins-­Knabe, B., Austin, A. B., Musun, L., Eddy, A., & Jones, R. M. (2000). Family home care providers' and parents' beliefs and practices concerning mathematics with young children. Early Child Development and Care, 165(1), 41–­58. https://doi. org/10.1080/03004​43001​650104 Blevins-­Knabe, B., Austin, A. B., Musun, L., Eddy, A., & Jones, R. M. (2000). Family home care providers' and parents' beliefs and practices concerning mathematics with young children. Early Child Development and Care, 165(1), 41–­58. https://doi. org/10.1080/03004​43001​650104 Cahoon, A., Cassidy, T., Purpura, D., & Simms, V. (2021). Developing a rigorous measure of the pre-­school h environment. Journal of Numerical Cognition, 7(2), 172–­194. https://doi.org/10.23668/​psych​archi​ves.4778 Cotter, K. E., Centurino, V. A. S., & Mullis, I. V. S. (2020). Developing the TIMSS 2019 mathematics and science achieve- ment instruments. In M. O. Martin, M. von Davier, & I. V. S. Mullis (Eds.), Methods and procedures: TIMSS 2019 technical report (pp. 1.1–­1.36). TIMSS & PIRLS International Study Center. https://timss​andpi​rls.bc.edu/timss​2019/metho​ds/ chapt​er-­1.html ment instruments. In M. O. Martin, M. von Davier, & I. V. S. Mullis (Eds.), Methods and procedures: TIMSS 2019 technical report (pp. 1.1–­1.36). TIMSS & PIRLS International Study Center. https://timss​andpi​rls.bc.edu/timss​2019/metho​ds/ chapt​er-­1.html p Daucourt, M. C., Napoli, A. R., Quinn, J. M., Wood, S. G., & Hart, S. A. (2021). The home math environment and math achieve- ment: A meta analysis Psychological Bulletin 147(6) 565 https://doi org/10 1037/bul0000330 Daucourt, M. C., Napoli, A. R., Quinn, J. M., Wood, S. G., & Hart, S. A. (2021). The home math environment and math achieve- ment: A meta-­analysis. R EFER ENCES Psychological Bulletin, 147(6), 565. https://doi.org/10.1037/bul00​00330 Davis-­Kean, P. E., Domina, T., Kuhfeld, M., Ellis, A., & Gershoff, E. T. (2021). It matters how you start: Early numeracy mas- tery predicts high school math course-­taking and college attendance. Infant and Child Development, 31(2), e2281. del Río, M. F., Susperreguy, M. I., Strasser, K., & Salinas, V. (2017). Distinct influences of mothers and fathers on kinder- gartners' numeracy performance: The role of math anxiety, home numeracy practices, and numeracy expectations. Early Education and Development, 28, 939–­955. https://doi.org/10.1080/10409​289.2017.1331662 p p g DeVellis, R. F., & Thorpe, C. T. (2021). Scale development: Theory and applications. Sage Publications. DeVellis, R. F., & Thorpe, C. T. (2021). Scale development: Theory and applications. Sage Publications. hman, H. J. (2018). How do parents foster young children's math skills? Child Development Perspectives, 12, 16–­21. org/10.1111/cdep.12249 Elliott, L., & Bachman, H. J. (2018). How do parents foster young children's math skills? Child Development Pers https://doi.org/10.1111/cdep.12249 Gustafsson, J. E. (2013). Causal inference in educational effectiveness research: A comparison of three methods to investi- gate effects of homework on student achievement. School Effectiveness and School Improvement, 24(3), 275–­295. https://doi. org/10.1080/09243​453.2013.806334 g Hawes, Z. C. K., Gilligan-­Lee, K. A., & Mix, K. S. (2022). Effects of spatial training on mathematics performance: A meta-­ analysis. Developmental Psychology, 58(1), 112–­137. https://doi.org/10.1037/dev00​01281 Henrich, J., Heine, S. J., & Norenzayan, A. (2010). The weirdest people in the world? Behavioral and Brain Science https://doi.org/10.1017/S0140​525X0​999152X e, S. J., & Norenzayan, A. (2010). The weirdest people in the world? Behavioral and Brain Sciences, 33(2–­3), 61–­83. org/10.1017/S0140​525X0​999152X Borriello, G. A., Kung, M., Lin, J., Litkowski, E., Cosso, J., Ellis, A., King, Y. A., Zippert, E., Cabrera, N. J., Hornburg, C. B., Borriello, G. A., Kung, M., Lin, J., Litkowski, E., Cosso, J., Ellis, A., King, Y. A., Zippert, E., Cabrera, N. J., Davis-­Kean, P., Eason, S. H., Hart, S. A., Iruka, I. U., LeFevre, J.-­A., Simms, V., Susperreguy, M. I., Cahoon, A., Chan, W. W. L., … Purpura, D. J. (2021). Next directions in measurement of the home mathematics environment: An international and interdisciplinary perspective. Journal of Numerical Cognition, 7(2), 195–­220. https://doi.org/10.5964/jnc.6143 g g J J g pp J Davis-­Kean, P., Eason, S. H., Hart, S. A., Iruka, I. U., LeFevre, J.-­A., Simms, V., Susperreguy, M. I., Cahoon, A., Chan, W. W. L., … Purpura, D. J. (2021). DATA AVAIL ABILITY STATEMENT The data that support the findings of this study are openly available from IEA's Trends in International Mathematics and Science Study—­TIMSS 2019 at https://timss​2019.org/inter​natio​nal-­datab​ase/. This article has earned Open Data and Preregistered Research Designs badges. Data and the preregis- tered design and analysis plan are available at https://osf.io/c5h7x/. 16 of 17 ELLIS et al. DATA AVAIL ABILITY STATEMENT R EFER ENCES Probing the relationship between home numeracy and chil- dren's mathematical skills: A systematic review. Frontiers in Psychology, 11, 1–­21. https://doi.org/10.3389/fpsyg.2020.02074 Niklas F Cohrssen C Vidmar M Segerer R Schmiedeler S Galpin R Klemm V V Kandler S Tayler C (2018) Niklas, F., Cohrssen, C., Vidmar, M., Segerer, R., Schmiedeler, S., Galpin, R., Klemm, V. V., Kandler, S., … Tayler, C. (2018). Early childhood professionals' perceptions of children's school readiness characteristics in six countries. International Journal of Educational Research, 90, 144–­159. https://doi.org/10.1016/j.ijer.2018.06.001 J f p g j j Punter, R. A., Glas, C. A., & Meelissen, M. R. (2016). Psychometric framework for modeling parental involvement and reading literacy. Springer. https://doi.org/10.1007/978-3-319-28064-6 p g p g Sarama, J., & Clements, D. H. (2009). Early childhood mathematics education research: Learning trajectories for young chil Sarama, J., & Clements, D. H. (2009). Early childhood mathematics education research: Learning trajectories for young children. Routledge. Susperreguy, M. I., Douglas, H., Xu, C., Molina-­Rojas, N., & LeFevre, J. A. (2020). Expanding the home numeracy model to Chilean children: Relations among parental expectations, attitudes, activities, and children's mathematical outcomes. Early Childhood Research Quarterly, 50, 16–­28. https://doi.org/10.1016/j.ecresq.2018.06.010 Susperreguy, M. I., Douglas, H., Xu, C., Molina-­Rojas, N., & LeFevre, J. A. (2020). Expanding the home numeracy model to Chilean children: Relations among parental expectations, attitudes, activities, and children's mathematical outcomes. Early Childhood Research Quarterly, 50, 16–­28. https://doi.org/10.1016/j.ecresq.2018.06.010 Q y, , p g j q Watts, T. W., Duncan, G. J., Siegler, R. S., & Davis-­Kean, P. E. (2014). What's past is prologue: Relations between early math- ematics knowledge and high school achievement. Educational Researcher, 43(7), 352–­360. https://doi.org/10.3102/00131​ 89X14​553660 Q y p g j q Watts, T. W., Duncan, G. J., Siegler, R. S., & Davis-­Kean, P. E. (2014). What's past is prologue: Relations between early math- ematics knowledge and high school achievement. Educational Researcher, 43(7), 352–­360. https://doi.org/10.3102/00131​ 89X14​553660 Watts, T. W., Duncan, G. J., Siegler, R. S., & Davis-­Kean, P. E. (2014). What's past is prologue: Relations between early math- ematics knowledge and high school achievement. Educational Researcher, 43(7), 352–­360. https://doi.org/10.3102/00131​ 89X14​553660 Wolfgang, C. H., Stannard, L. L., & Jones, I. (2001). Block play performance among preschoolers as a predictor of later school achievement in mathematics. Journal of Research in Childhood Education, 15(2), 173–­180. https://doi.org/10.1080/02568​54010​ 9594958 Wolfgang, C. H., Stannard, L. L., & Jones, I. (2001). Block play performance among preschoolers as a predictor of later school achievement in mathematics. R EFER ENCES https://doi.org/10.1037/ipp00​00102 g g y p nal Perspectives in Psychology: Research, Practice, Consultation, 8(1), 1–­13. https://doi.org/10.1037/ipp00​00102 LaRoche, S., Joncas, M., & Foy, P. (2020). Sample design in TIMSS 2019. In M. O. Martin, M. von Davier, & I. V. S. Mullis (Eds.), Methods and procedures: TIMSS 2019 technical report (pp. 3.1–­3.33). TIMSS & PIRLS International Study Center. https://timss​andpi​rls.bc.edu/timss​2019/metho​ds/chapt​er-­3.html p p lyzoi, E., Skwarchuk, S.-­L., Fast, L., & Sowinski, C. (2010). Do home numeracy and literacy practices of Greek LeFevre, J.-­A., Polyzoi, E., Skwarchuk, S.-­L., Fast, L., & Sowinski, C. (2010). Do home numeracy and literacy practices of Greek and Canadian parents predict the numeracy skills of kindergarten children? International Journal of Early Years Education, 18, 55–­70. https://doi.org/10.1080/09669​76100​3693926 p g LeFevre, J.-­A., Skwarchuk, S.-­L., Smith-­Chant, B. L., Fast, L., Kamawar, D., & Bisanz, J. (2009). Home numeracy experiences and children's math performance in the early school years. Canadian Journal of Behavioural Science, 41, 55–­66. https://doi. org/10.1037/a0014532 g Lin, J., Litkowski, E., Schmerold, K., Elicker, J., Schmitt, S. A., & Purpura, D. J. (2019). Parent–­educator communication linked to more frequent home learning activities for preschoolers. Child & Youth Care Forum, 48(5), 757–­772. https://doi. org/10.1007/s1056​6-­019-­09505​-­9 g Melhuish, E. C., Phan, M. B., Sylva, K., Sammons, P., Siraj-­Blatchford, I., & Taggart, B. (2008). Effects of the home learning environment and preschool center experience upon literacy and numeracy development in early primary school. Journal of Social Issues, 64, 95–­114. https://doi.org/10.1111/j.1540-­4560.2008.00550.x g j Missall, K., Hojnoski, R. L., Caskie, G. I. L., & Repasky, P. (2015). Home numeracy environments of preschoolers: Examining relations among mathematical activities, parent mathematical beliefs, and early mathematical skills. Early Education and Development, 26, 356–­376. https://doi.org/10.1080/10409​289.2015.968243 p p g Mullis, I. V. S., & Fishbein, B. (2020). Updating the TIMSS 2019 instruments for describing the contexts for student learning. In M. O. Martin, M. von Davier, & I. V. S. Mullis (Eds.), Methods and procedures: TIMSS 2019 technical report (pp. 2.1–­2.9). TIMSS & PIRLS International Study Center. https://timss​andpi​rls.bc.edu/timss​2019/metho​ds/chapt​er-­2.html y p p p Mullis, I. V. S., & Martin, M. O. (Eds.). (2017). TIMSS 2019 assessment frameworks. TIMSS & PIRLS International Study Center. http://timss​andpi​rls.bc.edu/timss​2019/frame​works/ p p Mutaf-­Yıldız, B., Sasanguie, D., De Smedt, B., & Reynvoet, B. (2020). Probing the relationship between home numeracy and chil- dren's mathematical skills: A systematic review. Frontiers in Psychology, 11, 1–­21. https://doi.org/10.3389/fpsyg.2020.02074 Mutaf-­Yıldız, B., Sasanguie, D., De Smedt, B., & Reynvoet, B. (2020). R EFER ENCES Next directions in measurement of the home mathematics environment: An international d i t di ipli p p ti J l f N i l C iti 7(2) 195 220 http //d i /10 5964/j 6143 , P., Eason, S. H., Hart, S. A., Iruka, I. U., LeFevre, J.-­A., Simms, V., Susperreguy, M. I., Cahoon, A., Chan, W. urpura, D. J. (2021). Next directions in measurement of the home mathematics environment: An international IBM Corporation. (2019). IBM SPSS statistics for windows (version 26.0) [computer software]. IBM Corporation. IBM Corporation. (2019). IBM SPSS statistics for windows (version 26.0) [computer software]. IBM Corporation. I i l A i i f h E l i f Ed i l A hi (2021) IEA IDB l ( i 4 0) [ p t ft International Association for the Evaluation of Educational Achievement. (2021). IEA IDB analyzer (version ware]. IEA Hamburg. Retrieved from https://www.iea.nl/data International Association for the Evaluation of Educational Achieve ware]. IEA Hamburg. Retrieved from https://www.iea.nl/data International Association for the Evaluation of Educational Achievement. (2021). IEA IDB analyzer (version 4.0) [computer soft- ware]. IEA Hamburg. Retrieved from https://www.iea.nl/data J h R B & Ch i t L (2019) Ed ti l h Q tit ti lit ti d i d pp h S P bli ti Johnson, R. B., & Christensen, L. (2019). Educational research: Quantitative, qualitative, and mixed approaches. Sage P Johnson, R. B., & Christensen, L. (2019). Educational research: Quantitati Lansford, J. E., Bornstein, M. H., Deater-­Deckard, K., Dodge, K. A., Al-­Hassan, S. M., Bacchini, D., Bombi, A. S., Chang, L., Chen, B., Di Giunta, L., Malone, P. S., Oburu, P., Patorelli, C., Skinner, A. T., Sorbring, E., Steinberg, L., Tapanya, S., Alampay, L. P., Uribe Tirado, L. M., & Zelli, A. (2016). How international research on parenting advances understanding of child development. Child Development Perspectives, 10(3), 202–­207. https://doi.org/10.1111/cdep.12186 17 of 17 INTERNATIONAL HME TIMSS Lansford, J. E., Gauvain, M., Koller, S. H., Daiute, C., Hyson, M., Motti-­Stefanidi, F., Smith, O., Verma, S., & Zhou, N. (2019). The importance of international collaborative research for advancing understanding of child and youth development. Lansford, J. E., Gauvain, M., Koller, S. H., Daiute, C., Hyson, M., Motti-­Stefanidi, F., Smith, O., Verma, S., & Zhou, N. (2019). The importance of international collaborative research for advancing understanding of child and youth development. International Perspectives in Psychology: Research, Practice, Consultation, 8(1), 1–­13. R EFER ENCES Journal of Research in Childhood Education, 15(2), 173–­180. https://doi.org/10.1080/02568​54010​ 9594958 Yin, L., & Fishbein, B. (2020). Creating and interpreting the TIMSS 2019 context questionnaire scales. In M. O. Martin, M. von Davier, & I. V. S. Mullis (Eds.), Methods and procedures: TIMSS 2019 technical report (pp. 16.1–­16.331). TIMSS & PIRLS International Study Center. https://timss​andpi​rls.bc.edu/timss​2019/metho​ds/chapt​er-­16.html How to cite this article: Ellis, A., Cosso, J., Duncan, R. J., Susperreguy, M. I., Simms, V., & Purpura, D. J. (2023). International comparisons of the home mathematics environment and relations with children's mathematical achievement. British Journal of Educational Psychology, 00, e12625. https://doi.org/10.1111/bjep.12625
https://openalex.org/W4391050553
https://www.biorxiv.org/content/biorxiv/early/2024/01/20/2024.01.17.575815.full.pdf
English
null
The neocortical infrastructure for language involves region-specific patterns of laminar gene expression
bioRxiv (Cold Spring Harbor Laboratory)
2,024
cc-by
18,099
Keywords Cerebral cortex, language network, spatial transcriptomics, laminar expression, cortical layers, structural connectivity, dyslexia. Abstract The language network of the human brain has core components in the inferior frontal cortex and superior/middle temporal cortex, with left-hemisphere dominance in most people. Functional specialization and interconnectivity of these neocortical regions is likely to be reflected in their molecular and cellular profiles. Excitatory connections between cortical regions arise and innervate according to layer-specific patterns. Here we generated a new gene expression dataset from human postmortem cortical tissue samples from core language network regions, using spatial transcriptomics to discriminate gene expression across cortical layers. Integration of these data with existing single-cell expression data identified 56 genes that showed differences in laminar expression profiles between frontal and temporal language cortex together with upregulation in layer II/III and/or layer V/VI excitatory neurons. Based on data from large-scale genome-wide screening in the population, DNA variants within these 56 genes showed set-level associations with inter-individual variation in structural connectivity between left-hemisphere frontal and temporal language cortex, and with predisposition to dyslexia. The axon guidance genes SLIT1 and SLIT2 were consistently implicated. These findings identify region-specific patterns of laminar gene expression as a feature of the brain’s language network. #Correspondence to: Prof. Clyde Francks, Max Planck Institute for Psycholinguistics, Wundtlaan 1, Nijmegen 6525XD, The Netherlands. Email: clyde.francks@mpi.nl #Correspondence to: Prof. Clyde Francks, Max Planck Institute for Psycholinguistics, Wundtlaan 1, Nijmegen 6525XD, The Netherlands. Email: clyde.francks@mpi.nl . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint The neocortical infrastructure for language involves region-specific patterns of laminar gene expression Maggie M.K. Wong1*, Zhiqiang Sha1*, Lukas Lütje1, Xiang-Zhen Kong1,2,3, Niels Velthuijs4,5, Sabrina van Heukelum1,6, Wilma D.J. van de Berg7,8, Laura E. Jonkman7,8,9, Simon E. Fisher1,6, Clyde Francks1,6,10# 1Language & Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands. 2Department of Psychology and Behavioral Sciences, Zhejiang University, Hangzhou, China. 3State Key Lab of Brain-Machine Intelligence, Zhejiang University, Hangzhou, China. 4Epigenomics & Single Cell Biophysics Group, Department of Cell Biology FNWI, Radboud University, Nijmegen, The Netherlands. 5Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands. 6Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands. 7Section Clinical Neuroanatomy and Biobanking, Department of Anatomy and Neurosciences, Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. 8Amsterdam Neuroscience, Neurodegeneration, Amsterdam, The Netherlands. 9Amsterdam Neuroscience, Brain Imaging, Amsterdam, The Netherlands. 10Department of Cognitive Neuroscience, Radboud University Medical Center, Nijmegen, The Netherlands. *Equal contributions. Introduction The human capacity for language relies on a distributed network of brain regions, core to which are the inferior frontal gyrus and superior/middle temporal cortex (1-3). In most people, there is functional dominance of the left-hemisphere regions for language, especially for sentence production (4). A major challenge for biological studies of language is to understand how this regional specialization is supported by distinct molecular and cytoarchitectonic profiles. For example, it is likely that molecules such as transcription factors, neurotransmitter receptors, ion channels, and synaptic adhesion proteins affect neural signaling and inter-areal connectivity, to influence regional functional specialization (5-7). 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Postmortem transcriptomic analysis generates a quantitative profile of gene expression values across thousands of active genes within a tissue sample. This approach relies on sampling tissue from defined anatomical regions within a few hours of death, before levels of messenger RNA (mRNA) become too degraded (8-10). Through combining this type of postmortem data with brain maps derived from non-invasive imaging of living individuals, numerous studies have found that patterns of gene expression across the cerebral cortex co-vary with anatomical and functional organization (6, 7, 11-19). Some general principles are that i) cortical transcription profiles exhibit macroscale gradients that correlate with hierarchical specialization, from primary sensorimotor to multimodal association regions, and ii) cortical regions with relatively higher inter-connectivity tend to have somewhat similar gene expression profiles, even when spatially separated. However, previous analysis that linked postmortem gene expression to connectivity within the human brain’s left-hemisphere language network relied on measurements derived from entire tissue blocks, i.e. spanning all cortical layers and often including some underlying white matter (7). Laminar structure is a fundamental organizing principle of the cerebral cortex that relates to local micro-circuitry and inter-regional connectivity (20-23). In particular, inter-regional feedforward and feedback excitatory neural projections originate preferentially from supragranular (upper) layers II & III, and infragranular (lower) layers V & VI, respectively (23-27). Spatial transcriptomics is a technique that can discriminate the expression levels of thousands of genes while maintaining positional information across tissue sections on a microscope slide (28, 29). Introduction With this technique it is possible to discern differences of transcriptomic profiles across cortical layers (30). It is known that laminar profiles of gene expression can vary between cortical regions (31), but although spatial transcriptomics has been applied to samples from the middle temporal cortex (30, 32), no such studies have yet included samples from the inferior frontal gyrus. Contrasting laminar profiles of gene expression between frontal and temporal regions of the core language network has therefore not been possible. In the present study we applied 10x Genomics Visium spatial transcriptomics (28, 29) to discriminate laminar gene expression profiles in human cerebral cortical samples, derived from the left-hemisphere inferior frontal gyrus and superior temporal sulcus of three neurotypical adult donors (Figure 1). For human cerebral cortical tissue sections, each spot on a Visium spatial transcriptomic slide indexes the gene expression from an average of 3-5 cells (30). We used our data to identify genes that showed region-specific laminar expression profiles. Specifically, we sought differences between the frontal and temporal cortical regions in terms of supragranular layer II/III versus infragranular layer V/VI expression patterns. As connections between distant regions of the cerebral cortex are primarily made through excitatory projection neurons from these layers, we then integrated our spatial transcriptomic data with existing single-cell transcriptomic data from the human cerebral cortex, to identify genes that are expressed more highly in layer II/III and/or layer V/VI excitatory neurons than other cortical cell types, in addition to showing fronto-temporal differences in their infragranular versus supragranular expression patterns. We reasoned that the laminar expression patterns of such genes may be adapted to support frontal-temporal connectivity within the left-hemisphere language network. To test this hypothesis for the genes that we identified, we then analyzed genetic association data from a neuroimaging genetics study of 30,814 adults (33), with respect to structural connectivity of the main nerve fiber tract that links frontal and temporal regions of the core language network – the arcuate fasciculus. In addition, to test the possible relevance for language-related cognition of the genes we identified, we analyzed data from the largest genome-wide association studies of language-related traits performed to date: word reading ability in 33,959 individuals (34), and dyslexia in 51,800 adults who reported having a diagnosis versus 1,087,070 controls (35) (the latter based on data from 23andMe, Inc.). Data-driven cluster analysis based on gene expression defines cortical laminar structure Separately for each of the three donors (Supplementary Table 1), tissue sectioning was performed using two blocks from the inferior frontal gyrus and two blocks from the posterior part of the superior temporal sulcus (Figure 1; Supplementary Figure 1). Separately for each block, we obtained two pairs of directly adjacent 2 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint sections, resulting in a total of 48 sections (3 donors × 4 tissue blocks × 4 sections). After quality control (Methods), we measured expression levels for 22,170 genes across 140,192 spots on the Visium slides, corresponding to all 48 sections together (Supplementary Table 2, Supplementary Figures 2-3). Spots failing quality control in our data were especially concentrated in white matter, perhaps due to relatively low mRNA levels or diffusivity in the myelinated axons of neurons, while grey matter was well measured (Figure 2, Supplementary Figures 2-3). sections, resulting in a total of 48 sections (3 donors × 4 tissue blocks × 4 sections). After quality control (Methods), we measured expression levels for 22,170 genes across 140,192 spots on the Visium slides, corresponding to all 48 sections together (Supplementary Table 2, Supplementary Figures 2-3). Spots failing quality control in our data were especially concentrated in white matter, perhaps due to relatively low mRNA levels or diffusivity in the myelinated axons of neurons, while grey matter was well measured (Figure 2, Supplementary Figures 2-3). Figure 1. Schematic overview of the main steps in this study. Step 1: We acquired postmortem tissue sections from core regions of the left-hemisphere language network: the inferior frontal gyrus and posterior superior temporal sulcus, from three neurotypical donors. We performed spatial transcriptomics for each of 48 cortical tissue sections (3 donors x 2 regions x 2 blocks x 2 pairs of adjacent sections). Data-driven cluster analysis based on gene expression defines cortical laminar structure Step 2: The resulting spot-level transcriptomic data were normalized to perform data-driven clustering analysis across all 48 sections, to identify layer-like clusters of spots in grey matter that matched across sections. Step 3: We annotated the data-driven clusters to cortical layers based on the spatial expression profiles of known layer marker genes, as well as classical cytoarchitecture. For each section, we pseudo-bulked the transcriptomic data by summing the unique molecular identifier counts across spots within layers II/III to define one supragranular bulked cluster, and within layers V/VI to define one infragranular bulked cluster. Step 4: We tested for genes showing layer*lobe interaction effects, i.e. genes whose supra- versus infragranular contrasts were different between the frontal and temporal cortex of the left-hemisphere. By reprocessing a previously published single-cell transcriptomic dataset generated from postmortem human cortical samples, we also identified genes that showed upregulation in layer II/III excitatory neurons and/or layer V/VI cortico-cortical projection neurons compared to other cortical cell types, as well as showing layer*lobe interactions. The laminar expression patterns of such genes may be adapted to support connectivity within the core language network. Step 5: The genes identified in step 4 were defined as a set, to test for a set-level association with white matter connectivity between core language-related brain regions, based on imaging genetics analysis in 30,814 individuals. We also tested for a set-level association with respect to word reading ability and dyslexia, based on previous large-scale genetic studies of these traits. Figure 1. Schematic overview of the main steps in this study. Step 1: We acquired postmortem tissue sections from core regions of the left-hemisphere language network: the inferior frontal gyrus and posterior superior temporal sulcus, from three neurotypical donors. We performed spatial transcriptomics for each of 48 cortical tissue sections (3 donors x 2 regions x 2 blocks x 2 pairs of adjacent sections). Step 2: The resulting spot-level transcriptomic data were normalized to perform data-driven clustering analysis across all 48 sections, to identify layer-like clusters of spots in grey matter that matched across sections. Step 3: We annotated the data-driven clusters to cortical layers based on the spatial expression profiles of known layer marker genes, as well as classical cytoarchitecture. For each section, we pseudo-bulked the transcriptomic data by summing the unique molecular identifier counts across spots within layers II/III to define one supragranular bulked cluster, and within layers V/VI to define one infragranular bulked cluster. Data-driven cluster analysis based on gene expression defines cortical laminar structure CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint highly in cluster 3 (Supplementary Figure 5), and layer VI marker CCK was expressed most highly in cluster 1 (Supplementary Figure 5). All of these marker-based annotations matched the expected order of spatial layering from upper to lower layers. Additional clusters were located in white matter, with higher expression of marker MBP than other clusters (Supplementary Figure 5), or were based on a smaller number of sporadically distributed spots (Figure 2B, Supplementary Figure 4). highly in cluster 3 (Supplementary Figure 5), and layer VI marker CCK was expressed most highly in cluster 1 (Supplementary Figure 5). All of these marker-based annotations matched the expected order of spatial layering from upper to lower layers. Additional clusters were located in white matter, with higher expression of marker MBP than other clusters (Supplementary Figure 5), or were based on a smaller number of sporadically distributed spots (Figure 2B, Supplementary Figure 4). Figure 2. Data-driven analysis based on gene expression profiles identifies layer-like clustering in cerebral cortical tissue samples. (A) Uniform manifold approximation and projection mapping (UMAP) helps to visualize the similarities and differences between 12 data-driven clusters, based on combined analysis of data from 48 tissue sections from the inferior frontal gyrus and superior temporal sulcus. Each dot in the UMAP plot represents a single spatial transcriptomic spot from one of the 48 sections. Each spot captures transcriptomic expression from an average of 3-5 cells that correspond to that spot’s position on the slide. Data-driven cluster analysis based on gene expression defines cortical laminar structure (B) Layer-like spatial patterns of data-driven clusters based on gene expression profiles, in six example sections of human cerebral cortex. For all 48 sections see Supplementary Figure 4. There were seven layer-like clusters in grey matter, and five clusters that were located in white matter or sporadically located without laminar appearances. Cluster-layer correspondence was determined through known layer marker genes and cytoarchitecture, whereby layer I = cluster 9; layer II = cluster 10; layer III = clusters 2 &11; layer IV = cluster 6; layer V = cluster 3; layer VI = cluster 1 (see main text). (C) Principal component analysis (PCA) of pseudo-bulked data from five clusters corresponding to cortical layers II, III, V and VI. The first i i l ( ) l i d f i i h i i d ( i d d f h f Figure 2. Data-driven analysis based on gene expression profiles identifies layer-like clustering in cerebral cortical tissue samples. (A) Uniform manifold approximation and projection mapping (UMAP) helps to visualize the similarities and differences between 12 data-driven clusters, based on combined analysis of data from 48 tissue sections from the inferior frontal gyrus and superior temporal sulcus. Each dot in the UMAP plot represents a single spatial transcriptomic spot from one of the 48 sections. Each spot captures transcriptomic expression from an average of 3-5 cells that correspond to that spot’s position on the slide. (B) Layer-like spatial patterns of data-driven clusters based on gene expression profiles, in six example sections of human cerebral cortex. For all 48 sections see Supplementary Figure 4. There were seven layer-like clusters in grey matter, and five clusters that were located in white matter or sporadically located without laminar appearances. Cluster-layer correspondence was determined through known layer marker genes and cytoarchitecture, whereby layer I = cluster 9; layer II = cluster 10; layer III = clusters 2 &11; layer IV = cluster 6; layer V = cluster 3; layer VI = cluster 1 (see main text). (C) Principal component analysis (PCA) of pseudo-bulked data from five clusters corresponding to cortical layers II, III, V and VI. The first principal component (PCA 1) explained 23% of variance in the transcriptomic data (as indexed from the top 10% of variable genes) and supported a primary distinction of supragranular from infragranular clusters in terms of transcriptomic profiles. Data-driven cluster analysis based on gene expression defines cortical laminar structure Step 4: We tested for genes showing layer*lobe interaction effects, i.e. genes whose supra- versus infragranular contrasts were different between the frontal and temporal cortex of the left-hemisphere. By reprocessing a previously published single-cell transcriptomic dataset generated from postmortem human cortical samples, we also identified genes that showed upregulation in layer II/III excitatory neurons and/or layer V/VI cortico-cortical projection neurons compared to other cortical cell types, as well as showing layer*lobe interactions. The laminar expression patterns of such genes may be adapted to support connectivity within the core language network. Step 5: The genes identified in step 4 were defined as a set, to test for a set-level association with white matter connectivity between core language-related brain regions, based on imaging genetics analysis in 30,814 individuals. We also tested for a set-level association with respect to word reading ability and dyslexia, based on previous large-scale genetic studies of these traits. Data were normalized and harmonized for sample effects (Methods). We then performed clustering analysis using the data from all sections together, using BayesSpace (36). This approach identified corresponding data- driven clusters of spots across sections that were matched based on their gene expression profiles. The main data-driven clusters had clear layer-like appearances in all sections (Figure 2B, Supplementary Figure 4). UMAP analysis confirmed that the top components of variation in the transcriptomic data were related to layer- like differences and the grey-white matter distinction (Fig. 2). We then annotated the data-driven clusters with reference to known layer marker genes (30, 37, 38) and structural cytoarchitecture visible by Nissl staining (Methods, Figure 3A, Supplementary Figures 5-6). The cerebral cortex is classically divided into six layers (39, 40). AQP4, FABP7 and RELN, as markers of layer I, showed highest expression in cluster 9 that was defined at the outermost surface (Figure 2B, Supplementary Figure 5). Directly underlying this, cluster 10 was enriched for the expression of layer II markers, such as ENC1 (Supplementary Figure 5). Our data-driven analysis based on transcriptomic profiles distinguished two clusters corresponding to layer III (clusters 2 & 11), and both of these clusters showed higher expression of layer III marker ADCYAP1 than other clusters (Supplementary Figure 5). RORB, as a canonical marker of layer IV, was expressed most highly in cluster 6 (Supplementary Figure 5). Layer V marker TRABD2A was expressed most 3 . Data-driven cluster analysis based on gene expression defines cortical laminar structure CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supragranular versus infragranular layers As excitatory neural projections from supragranular layers II & III, and infragranular layers V & VI, are primarily responsible for inter-areal projections (23), we focused on these four layers for our subsequent analyses. We therefore left aside data from layer I (i.e. cluster 9), and the granular layer IV (named for its granular appearance under light microscopy) that separates the supragranular from infragranular layers. This meant focusing on data from five clusters: supragranular cluster 10 (layer II), supragranular clusters 2 & 11 (layer III), infragranular cluster 3 (layer V) and infragranular cluster 1 (layer VI) (Figures 2B, 3A). Separately for each of the 48 tissue sections and each of these five clusters, we performed “pseudo-bulking” by summing and normalizing the unique molecular identifier counts across all spots for each gene (Methods), to generate a single section- and cluster-specific expression value per gene (therefore 240 values per gene, from 48 sections x 5 clusters). Principal component analysis based on the 10% of most variable genes (Methods) showed how the sections and clusters related in terms of transcriptional profile similarity (Figure 2C). The first component explained 23% of the variance and distinguished supragranular clusters 1 & 3 from infragranular clusters 2, 10 & 11 (Figure 2B, 2C). The second component explained 9% of the variance and captured laminar differentiation in a manner that reflected proximity to the granular layer (Figure 2B, 2C). These findings further confirmed that cortical layers can be meaningfully defined based on transcriptional profiles, and supported a primary distinction of supragranular layers II/III from infragranular layers V/VI at the mRNA level. The findings also indicated that the stereotypical layer arrangement, as reflected by overall transcriptional profiles, is conserved between the inferior frontal gyrus and superior temporal sulcus of the left-hemisphere. We therefore expected that any inter-areal differences of laminar gene expression between these regions would be relatively subtle, and/or restricted to a minority of genes. Data-driven cluster analysis based on gene expression defines cortical laminar structure The second principal component (PCA 2) explained 9% of the variance and reflected proximity to the granular layer Figure 2. Data-driven analysis based on gene expression profiles identifies layer-like clustering in cerebral cortical tissue samples. (A) Uniform manifold approximation and projection mapping (UMAP) helps to visualize the similarities and differences between 12 data-driven clusters, based on combined analysis of data from 48 tissue sections from the inferior frontal gyrus and superior temporal sulcus. Each dot in the UMAP plot represents a single spatial transcriptomic spot from one of the 48 sections. Each spot captures transcriptomic expression from an average of 3-5 cells that correspond to that spot’s position on the slide. (B) Layer-like spatial patterns of data-driven clusters based on gene expression profiles, in six example sections of human cerebral cortex. For all 48 sections see Supplementary Figure 4. There were seven layer-like clusters in grey matter, and five clusters that were located in white matter or sporadically located without laminar appearances. Cluster-layer correspondence was determined through known layer marker genes and cytoarchitecture, whereby layer I = cluster 9; layer II = cluster 10; layer III = clusters 2 &11; layer IV = cluster 6; layer V = cluster 3; layer VI = cluster 1 (see main text). (C) Principal component analysis (PCA) of pseudo-bulked data from five clusters corresponding to cortical layers II, III, V and VI. The first principal component (PCA 1) explained 23% of variance in the transcriptomic data (as indexed from the top 10% of variable genes) and supported a primary distinction of supragranular from infragranular clusters in terms of transcriptomic profiles. The second principal component (PCA 2) explained 9% of the variance and reflected proximity to the granular layer. 4 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . Identifying genes that show different laminar expression patterns between frontal and temporal language cortex (C) SOSTDC1 showed similar supragranular and infragranular levels in the inferior frontal gyrus, but markedly higher infragranular than supragranular expression in the superior temporal sulcus. For comparable plots for all 56 genes that showed significant layer*lobe interaction effects as well as upregulation in layer II/III and/or layer V/VI excitatory neurons compared to other cortical cell types see Supplementary Figure 7 3. Identiying genes with supra- versus infragranular contrasts that differ between frontal and tempor Figure 3. Identiying genes with supra- versus infragranular contrasts that differ between front i f th l ft h i h l t k (A) S h ti f th d b lki d Figure 3. Identiying genes with supra- versus infragranular contrasts that differ between frontal and temporal regions of the left-hemisphere language network. (A) Schematic of the pseudo-bulking procedure that integrated the spatial transcriptomic data from spot-level to layer-level within each cortical tissue section, separately for supragranular layers II/III versus infragranular layers V/VI. The pseudo-bulked data were then used in linear mixed effects modelling to test each gene for a layer*lobe interaction effect (supra- vs infragranular)*(frontal vs temporal), while controlling for donor, tissue block and adjacent sections. (B & C) Expression data for two genes with the most significant layer*lobe interaction effects, where each dot represents one of 48 sections for each lobe and layer combination: (B) PTPRK showed markedly higher supragranular than infragranular expression in the inferior frontal gyrus, but supragranular and infragranular levels were similar in the superior temporal sulcus. (C) SOSTDC1 showed similar supragranular and infragranular levels in the inferior frontal gyrus, but markedly higher infragranular than supragranular expression in the superior temporal sulcus. For comparable plots for all 56 genes that showed significant layer*lobe interaction effects as well as upregulation in layer II/III and/or layer V/VI excitatory neurons compared to other cortical cell types, see Supplementary Figure 7. Identifying genes that show different laminar expression patterns between frontal and temporal language cortex CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Figure 3. Identiying genes with supra- versus infragranular contrasts that differ between frontal and temporal regions of the left-hemisphere language network. (A) Schematic of the pseudo-bulking procedure that integrated the spatial transcriptomic data from spot-level to layer-level within each cortical tissue section, separately for supragranular layers II/III versus infragranular layers V/VI. The pseudo-bulked data were then used in linear mixed effects modelling to test each gene for a layer*lobe interaction effect (supra- vs infragranular)*(frontal vs temporal), while controlling for donor, tissue block and adjacent sections. (B & C) Expression data for two genes with the most significant layer*lobe interaction effects, where each dot represents one of 48 sections for each lobe and layer combination: (B) PTPRK showed markedly higher supragranular than infragranular expression in the inferior frontal gyrus, but supragranular and infragranular levels were similar in the superior temporal sulcus. (C) SOSTDC1 showed similar supragranular and infragranular levels in the inferior frontal gyrus, but markedly higher infragranular than supragranular expression in the superior temporal sulcus. For comparable plots for all 56 genes that showed significant layer*lobe interaction effects as well as upregulation in layer II/III and/or layer V/VI excitatory neurons compared to other cortical cell types, see Supplementary Figure 7. Figure 3. Identiying genes with supra- versus infragranular contrasts that differ between frontal and temporal regions of the left-hemisphere language network. (A) Schematic of the pseudo-bulking procedure that integrated the spatial transcriptomic data from spot-level to layer-level within each cortical tissue section, separately for supragranular layers II/III versus infragranular layers V/VI. The pseudo-bulked data were then used in linear mixed effects modelling to test each gene for a layer*lobe interaction effect (supra- vs infragranular)*(frontal vs temporal), while controlling for donor, tissue block and adjacent sections. (B & C) Expression data for two genes with the most significant layer*lobe interaction effects, where each dot represents one of 48 sections for each lobe and layer combination: (B) PTPRK showed markedly higher supragranular than infragranular expression in the inferior frontal gyrus, but supragranular and infragranular levels were similar in the superior temporal sulcus. Identifying genes that show different laminar expression patterns between frontal and temporal language cortex For the following analyses we retained 12,656 genes for which canonical transcripts were defined according to the human reference genome GRCh38. For each of the 48 tissue sections separately, we pseudo-bulked data from spots in clusters 2, 10 & 11 to create a single supragranular layer II/III bulked cluster, and from clusters 1 & 3 to create a single infragranular layer V/VI bulked cluster (Methods). This resulted in 96 expression values per gene (48 sections x 2 bulked clusters). We then used linear mixed effect modeling to test for genes that showed layer*lobe interaction effects, i.e. genes whose supra- versus infragranular contrasts were different between the inferior frontal gyrus and superior temporal sulcus (Figure 3). We controlled for donor and tissue block as fixed effects, while also accounting for adjacent sections in the model (Methods). Seventy-two genes showed significant layer*lobe interaction effects at FDR p<0.01 (Supplementary Table 3). Two genes particularly stood out in terms of statistical significance: PTPRK (t=9.20, p=1.05x10-14) and SOSTDC1 (t=8.58, p=2.07x10-13) (Supplementary Table 3) (Figure 3B, 3C). In the inferior frontal gyrus, PTPRK showed markedly higher supragranular than infragranular expression, whereas in the superior temporal sulcus this gene showed similar expression in supragranular and infragranular layers, thus giving rise to a layer*lobe statistical interaction (Figure 3B). PTPRK encodes a signaling molecule of the protein tyrosine phosphatase family. For SOSTDC1, there was markedly higher infragranular than supragranular expression in the superior temporal sulcus, while in the inferior frontal gyrus the expression level was similar in infragranular and supragranular layers (Figure 3C). SOSTDC1 encodes a secreted protein of the sclerostin family that functions as an antagonist to growth factors of the bone morphogenetic protein (BMP) family, and also affects Wnt signalling (41). The 72-gene set showed enrichments at FDR p<0.01 for thirteen biological processes defined in the Gene Ontology (42), most significantly for functions related to neuron projection/axon guidance, driven by the genes encoding signalling molecules NELL1, PTPRM, SLIT1 and SLIT2 (Supplementary Table 4) (43). Other implicated processes included ion transport and regulation of catecholamine secretion (Supplementary Table 4). 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . Genetic association analysis with respect to brain and behavioural variability A previous genome-wide association study in 30,810 adults from the UK Biobank dataset mapped associations of common genetic variants in the population with individual differences in brain-wide structural connectivity, as determined by tractography based on diffusion imaging (33). We made use of the genome-wide summary statistics from that study, which index the extent to which each common variant in the genome is associated with a given structural connection in the brain. For the present study, we extracted data for four frontal-temporal connections that link the approximate left-hemisphere regions from which we sampled postmortem brain tissue for spatial transcriptomics: i.e. between the pars opercularis and superior temporal cortex, pars triangularis and superior temporal cortex, pars opercularis and middle temporal cortex, and pars triangularis and middle temporal cortex (Figure 1, Supplementary Figure 1, Supplementary Figure 8). These white matter connections are primarily via the arcuate fasciculus (33) (Supplementary Figure 8). We focussed on the 56 genes that showed significant evidence in the present study for having different laminar patterns between frontal and temporal cortex, and upregulation in excitatory neurons of layer II/III and/or layer V/VI cortico-cortical projection neurons compared to other cortical cell types, i.e. the neurons that primarily form inter-areal connections. We applied the Gene-set Association analysis Using Sparse Signals (GAUSS) software (47), which tests for gene-set level association based on genome-wide summary statistics, and also identifies the specific genes that drive a set-level association. The 56 genes showed a significant set-level associaton with white matter connectivity between the pars triangularis and middle temporal cortex (unadjusted p=0.004, Bonferroni-adjusted p=0.016 for testing four connectivities) (Supplementary Table 9). GAUSS identified 26 genes driving this significant association, with 6 genes individually at p<0.05: BHLHE22, COL5A2, NELL2, RYR3, SLIT1, SLIT2 (Supplementary Table 9). Connectivity between the pars triangularis and superior temporal cortex also showed nominally significant set-level association with the 56 genes (unadjusted p=0.02), but this did not survive Bonferroni correction. In terms of language-related behavioural traits, by far the largest genome-wide association studies performed to date have been of word reading ability in 33,959 individuals (34) and dyslexia in 51,800 adults who reported having a diagnosis versus 1,087,070 controls (35). Genes upregulated in layer II/III or layer V/VI excitatory neurons To focus on the laminar profiles of genes expressed in the cells that primarily form inter-areal projections, we then used existing single-cell RNA sequencing data to identify genes with higher expression in excitatory neurons of layers II/II and/or layers V/VI compared to other cortical cell types. We reprocessed single cell data derived from the dorsolateral prefrontal cortex and anterior cingulate cortex from 31 donors (44) (Methods). This dataset included post-quality control gene expression data from 104,559 nuclei that had been annotated to 17 defined cell types. For each of 12,155 genes that overlapped with those in our spatial transcriptomics dataset, we extracted and processed single cell gene expression data to aggregate unique molecular identifier counts for each of the 17 defined cell types (44) (Methods). These profiles were then used for differential expression analysis to identify genes that were expressed more highly in the cell type ‘layer II/III excitatory neurons’ and/or the cell type ‘layer V/VI cortico-cortical projection neurons’ compared to all other cell types combined (see Methods). At FDR p<0.01 there were 2219 genes upregulated in layer II/III excitatory neurons, and 1732 genes 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint upregulated in layer V/VI cortico-cortical projection neurons (Supplementary Tables 5 and 6), which yielded a union list of 2622 genes showing upregulation in one or both of these cell types. Among the 2622 genes, there were 56 genes that showed significant layer*lobe interactions at FDR p<0.01, i.e. Genes upregulated in layer II/III or layer V/VI excitatory neurons 56 genes with supra- versus infragranular contrasts that differed between the inferior frontal gyrus and superior temporal sulcus, in addition to showing upregulation in excitatory neurons of layer II/III and/or layer V/VI cortico-cortical projection neurons (Supplementary Figure 7; Supplementary Table 7). These again included PTPRK and SOSTDC1 as the two most significant genes with layer*lobe interaction effects (Supplementary Table 7), and gene ontology enrichment analysis again pointed most significantly to neuron projection/axon guidance, driven by NELL1, NELL2, SLIT1 and SLIT2 (Supplementary Table 8). Magnesium ion responsiveness was also implicated, driven by RYR3 and SNCA (Supplementary Table 8). Other individual genes among the set of 56 included the transcription factors LMO3 and LMO4; the latter might be involved in the patterning of fetal brain left-right asymmetry (45), and CDH10 encoding a cell adhesion molecule of the cadherin family that is involved in layer and circuit formation (46) (Supplementary Table 8). All 56 genes that showed upregulation in excitatory neurons of layer II/III and/or layer V/VI cortico-cortical projection neurons, together with significant layer*lobe interactions at FDR p<0.01, can be found in Supplementary Table 7. Discussion In this study we created a new spatial transcriptomics dataset from postmortem human cerebral cortical tissue, focused on core regions of the left-hemisphere language network. We identified genes with region-specific laminar expression profiles, i.e. that showed supragranular versus infragranular contrasts that differed between the inferior frontal gyrus and superior temporal sulcus. In combination with single cell transcriptomic data, there were 56 genes that additionally showed upregulation in layer II/III excitatory neurons and/or layer V/VI excitatory cortico-cortical projection neurons - the cell types that are primarily responsible for inter-areal connections - compared to other cortical cell types. Consistent with this, the 56 genes showed a set-level association with frontal-temporal white matter connectivity, and also with the language- and reading-related disorder dyslexia, in large-scale genome-wide association data. Genes involved in axon guidance were especially implicated by these analyses, including SLIT1 and SLIT2. Spatial transcriptomic data-driven clustering revealed robustly matching layers across cortical tissue sections from frontal and temporal regions, which indicates that laminar patterns are in fact largely similar between the inferior frontal gyrus and superior temporal sulcus, in terms of overall gene expression profiles. However, we aimed to identify genes that do not conform to this overall homogeneity in the present study. This was because language-related cognition is likely to be supported by specialized areal functions, and uniquely adapted inter- areal connectivity, between frontal and temporal regions of the left hemisphere (in addition to pan-cortical aspects of laminar organization). Our findings confirm the existence of regional variation in laminar expression profiles for a minority of genes, following pioneering work based on a smaller number of genes and other cortical regions (31). These findings therefore establish regional variation of laminar gene expression as an organizing principal in the human cerebral cortex. Invasive, in vivo axon tracing, especially in the visual system of macaque monkeys, has shown that inter- regional feedforward and feedback connections tend to originate from upper and lower cortical layers respectively (23, 24). Clearly such invasive tracing methods cannot be applied in humans, which means that layer-specific frontal-temporal connections within the human language network have not been directly observed. Nonetheless, the frontal and temporal regions of the core language network are connected by prominent white matter tracts, notably the arcuate fasciculus that has undergone specific left-lateralized anatomical changes in humans compared to macaques (48). Genetic association analysis with respect to brain and behavioural variability We tested the 56 genes for a set-level association with word reading ability and separately also with dyslexia, based on the genome-wide association summary statistics from those studies, for SNPs spanning the genomic locus of each gene plus 50 kilobases upstream and downstream (34, 35). The 56 genes showed no significant set-level association with word reading ability, but a highly significant association with dyslexia (p=6.22x10-9) (Supplementary Table 10). GAUSS identified 24 genes driving the association with dyslexia, with 14 genes individually at p<0.05: BHLHE22, CDH10, DAB1, DIAPH1, FBXO32, GABRD, GPR26, KCNH5, KIRREL3, NEFH, OXR1, SLIT1, SLIT2, SNCA (Supplementary Table 10). Three of these 14 genes were also among the 6 associated with structural connectivity between the pars triangularis and middle temporal cortex, suggesting that region-specific laminar expression patterns of these genes might be especially important for supporting language-related cognition: BHLHE22 which encodes a neural transcription factor, and SLIT1 and SLIT2 which encode axon guidance molecules. 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Discussion CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint cortex may therefore affect synaptic plasticity and stability. Our data suggest that such roles contribute to cortical regional and laminar specialization of function. cortex may therefore affect synaptic plasticity and stability. Our data suggest that such roles contribute to cortical regional and laminar specialization of function. As regards dyslexia, this disorder of reading is closely linked to linguistic cognition in many affected individuals, including reduced phonological awareness and sometimes language impairment (54-56). Dyslexia has also been linked to altered frontal-temporal structural connectivity via the arcuate fasciculus, although results on this have been inconsistent across studies (57, 58). While reading is a cultural innovation, it recruits much of the neural circuitry that underpins the human capacity for oral language (59). The present study found that genes with laminar expression patterns that differ between the frontal and temporal language cortex, and are also upregulated in excitatory neurons of layers II/III and/or layers V/VI, show a highly significant set-level association with dyslexia. For this analysis we used data from a genetic study of 51,800 adults who reported having had a diagnosis, versus 1,087,070 controls (35). It is likely that many of the 51,800 individuals received their diagnoses during childhood, although information on this was not recorded, and some may have received diagnoses in adulthood. For SLIT genes and other genes involved in axon guidance, their contributions to dyslexia may occur to a large extent during neurodevelopment, but perhaps also via functions in the adult brain as discussed above, consistent with the neural expression of such genes in the adult cerebral cortex. The lack of a gene set-level association with word reading ability may have been due to the smaller sample size of the genome-wide association study from which we used summary statistics for that trait (33,959 individuals with quantitative data on word reading skills) (34), compared to the larger study of dyslexia (~1.14 million individuals) (35). Future studies would benefit from larger-scale GWAS of additional language-related traits such as expressive and receptive language abilities. Discussion Transcription from genomic DNA to mRNA occurs in the cell nucleus, and translation from mRNA to proteins occurs prominently in the cell body, from where proteins can be transported along axons and dendrites to other locations where they are needed - for example presynaptic boutons. However, movement of mRNA molecules along axons and dendrites also occurs, to support local translation distantly from the cell body (60). In the present study we interpreted laminar gene expression profiles as reflecting primarily cell body gene expression, which for inter-areal projection neurons implies the origins of connections, rather than their terminals. Also we used single-nucleus gene expression data to inform our analyses, which necessarily misses mRNA in axons and dendrites. In adult brain tissue, the extent to which neural translation occurs locally in the neuropil versus in the cell body is uncertain (60), but translation in the neuropil may be a substantial contributor for some proteins (61). With the spatial transcriptomic technology used in the present study, each spot has previously been shown to measure mRNA molecules from an average of 3-5 cell bodies in cerebral cortical tissue sections, but a contribution from neuropil is also expected, and a minority of spots that are not overlain by cell bodies may measure neuropil mRNAs only (30). In this case, laminar differences of gene expression may relate partly to the terminals of inter-areal connections, as well as the abundances of different cell types or the levels of expression within those cell types. To summarize, this study made several contributions to understanding the cerebral cortical infrastructure underlying language. We identified genes with region-dependent laminar expression profiles that may support regional functional specialization. Ours is among a small number of studies to have shown cortical regional variation in laminar gene expression, and helps to establish this as an organizing principal of the human brain more generally. Through additionally focusing on upregulation of transcription in excitatory projection neurons, our study identified genes that may affect inter-areal connectivity within the left-hemisphere language network. This possibility was supported by genetic association data that implicated several of the relevant genes in fronto- temporal white matter connectivity, and the language-related trait dyslexia. Discussion This is consistent with regional and network-level specialization of the frontal and temporal regions in the left-hemisphere, to support language-related cognition. The present study provides indirect evidence that inter-areal connections within the left-hemisphere language network are supported by region-dependent laminar profiles, because genomic variants within the implicated genes showed association with inter-individual variation in frontal-temporal white matter connectivity. However, we cannot exclude that genes with regional variation of laminar expression may affect inter-areal connectivity through other mechanisms, not only related to their region-dependent laminar expression profiles. It also remains unclear to what extent feedforward and feedback connectivity may operate between the frontal and temporal regions of the left-hemisphere language network. These mechanisms are most easily understood with respect to bottom-up and top-down transmission of information between regions on different hierarchical levels, from sensory cortex to association cortex. The inferior frontal gyrus and superior temporal sulcus are both implicated in high-level linguistic functions, including sentence-level processing (1-3). Therefore these regions may be broadly comparable in terms of hierarchy within the language network. Layer-specific functional MRI has been applied to dissociate top-down and bottom-up signal contributions to the left occipitotemporal sulcus during word reading (49), but this region does not overlap with the temporal region analyzed in the present study. The genes that we implicated in this study showed their most significant Gene Ontology set-level enrichment for functioning in axon guidance. For example, SLIT1 and SLIT2 are well known as extracellular axon guidance molecules that are involved in the patterning of brain networks during development (43), as well as other aspects of neocortical formation (50). Less is known of the roles of these genes in mature adult brains, long after inter- areal nerve fiber connections are established. They may have roles in the regulation of tissue integrity and homeostasis (51, 52), through mechanisms similar to their developmental roles, for example in regulating cell- cell adhesion. Consistent with this, SLIT proteins have been implicated in synaptogenesis through forming complexes with presynaptic Neurexin and postsynaptic Robo proteins (53), and their expression in the adult 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . Sectioning and spatial transcriptomic data generation From each tissue block we took two pairs of adjacent 10μm-thick tissue sections that would be used for spatial transcriptomics, resulting in a total of 48 tissue sections: 3 donors x 2 lobes (frontal and temporal) x 2 blocks x 2 adjacent pairs of sections. The section pairs were separated by 300μm (Figure 1). The sectioning was performed within a cryostat (Thermo Fisher NX70) following the manufacturer’s protocol (Visium Tissue Preparation Guide, 10x Genomics CG000240 RevA), and aimed to span across the cortical layers. Frozen 10μm- thick tissue sections were mounted on Tissue Optimization Slides (Visium Spatial Tissue Optimization Slide & Reagent Kit, 10x Genomics 1000193) and Gene Expression Slides (Visium Spatial Gene Expression Slide & Reagent Kit, 10x Genomics, 1000184) and stored at -80oC until further processing. Additional sections from within the 300μm interval were taken for other parts of the study (see below) and stored at -80oC until use. Permeabilization time was optimized following the manufacturer’s protocol (Visium Spatial Tissue Optimization, 10x Genomics CG000238 RevA). Fluorescence images of sections for tissue optimization were taken using a AxioScan Z1 SlideScanner (Zeiss) with Hamamatsu Orca Flash camera, a Cy5 filter, a Plan- Apochromat 20x/0.8 M27 objective and Zeiss Zen v.2.6 (Blue Edition) software. A permeabilization time of 18-minutes was used for the Visium Spatial Gene Expression workflow. Frozen tissue sections on gene expression slides were processed for spatial transcriptomics using the Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics, 1000184) following the manufacturer’s protocols (Visium Spatial Gene Expression User Guide,10x Genomics CG000239 RevA). Briefly, brightfield images of H&E-stained sections were acquired using an AxioScan Z1 slide scanner (Zeiss) with a Hitachi HV-F292SCL camera, a Plan-Apochromat 20x/0.8 M27 objective and Zeiss Zen v.2.6 (Blue Edition) software. H&E-stained sections were then permeabilized for 18 minutes, followed by reverse transcription, second-strand synthesis, denaturation, cDNA amplification and quality control, and library construction. qPCR was performed using the KAPA SYBR FAST kit (KAPA Biosystems) and BioRad CFX96 Real-Time System. The cDNA amplification cycle number was determined at ~25% of the peak fluorescence value. The final libraries were processed and a BioRad T100 Thermal Cycler was used for PCR reactions during library construction. The final libraries were sequenced on a NovaSeq 6000 System (Illumina) for 150 bp pair-end reads. Sectioning and spatial transcriptomic data generation The sequencing reads were then trimmed to read 1: 28 cycles; i7 index read: 10 cycles; i5 index read: 10 cycles; and read 2: 91 cycles, followed by demultiplexing using Space Ranger software v.1.2.2 (10x Genomics). The 48 libraries were sequenced to a median depth of 469×10-6 reads. Donors and sampling This study was approved by the Ethics Committee Faculty of Social Sciences, Radboud University Nijmegen, and the ethics and Biobank Review Committee, Free University Medical Center Amsterdam. All donors gave written informed consent to the human body bequest program at the department of Anatomy and Neurosciences, Amsterdam UMC – location VUmc (governed by the human tissue act (‘ter beschikking stelling’, Artikel 18, lid 1 en 19 van de Wet op de Lijkbezorging, 1991)). Collection of brain tissue was facilitated though the Normal 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Aging Brain Collection Amsterdam (NABCA) biobank. The standardized protocol at NABCA includes craniotomy and dissection of left hemisphere tissue blocks followed by freezing (liquid nitric oxygen), with a postmortem delay to autopsy of around 8 hours (8). For the present study we obtained postmortem brain tissue from three neurotypical donors: a 59-year-old male, a 59-year-old female, and a 63-year-old female (Supplementary Table 1). All three postmortem brains were confirmed to have minimal pathology by macro- and microscopic investigation, and RNA integrity numbers of at least 7 (Supplementary Table 1) (8, 62). From each donor we sampled from four left-hemisphere tissue blocks of roughly 1.5cm3 each (containing both grey and white matter): two blocks from the inferior frontal gyrus and two blocks from the posterior superior temporal sulcus (see Supplementary Figure 1 for the sampled regions). Mapping data-driven clusters to cortical layers We extracted the expression data for 12 genes that have been reported as robust layer markers in human cerebral cortical tissue (30), and plotted them separately across each of the twelve clusters (Supplementary Figure 5). This revealed the expected pattern of upper-to-lower layer identities as described in the Results section (above), whereby the following correspondences were identified: layer I = cluster 9; layer II = cluster 10; layer III = clusters 2 &11; layer IV = cluster 6; layer V = cluster 3; layer VI = cluster 1 (Figure 2B, Figure 3A, Supplementary Figure 5). Spatial transcriptomic data processing and quality control We processed the raw FASTQ files and H&E histology images of sections with the Space Ranger software v.1.2.2, using STAR v.2.5.1b (63) for alignment against the Cell Ranger reference genome refdata-cellranger- GRCh38-3.0.0, available at: http://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-3.0.0.tar.gz. H&E histology images were rotated and resized to 2000 x 2000 pixels using FIJI/ImageJ (v.1.53t). Default parameters for spaceranger count v.1.2.2 was used to generate count matrix files and QC metrics (https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/using/count). QC metrics returned by this software are available in Supplementary Table 2. This processing resulted in unique molecular identifier (UMI)/feature-barcode matrices for each of the 48 sections. We read the raw feature-barcode matrix from each section, coupled with its corresponding histology image, to construct a customized object using the SummarizedExperiment R package (64). The data across all 48 sections were then aggregated to form one SingleCellExperiment object (65). There were 170,157 detected spots corresponding to all 48 sections together, with a mean of 6783 UMIs per spot, and a mean of 2782 genes per spot. We excluded spots according to spot-wise quality control metrics using the default settings of the 10 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint perCellQCMetrics and quickPerCellQC functions from the Scran v1.18.7 R Bioconductor package, which considers the log-total UMI count, log-number of detected features, and percentage of counts in specified ‘control’ gene sets (mitochondrial genes, spike-in transcripts) (66). We also excluded spots in parts where the sections had folded over on the spatial transcriptomic slides (Supplementary Figure 3). We then excluded genes that were expressed in less than 0.01% of spots across all of the 48 samples combined, as well as 13 mitochondrial genes. These steps left 140,192 spots from which a total of 22,170 genes were measured. Data-driven clustering to match across sections We used the functions quickCluster and computeSumFactors within Scran (66), and the logNormCounts function within the Scater package v1.18.6 (67), to compute log normalized gene expression counts at the spot level. The normalized data were then used to fit a model with respect to gene mean expression and variance using the modelGeneVar function of Scran (66), followed by identifying the top 10% of variable genes. This set of highly variable genes was used to compute principal components using the runPCA function within Scater (67), generating the top 50 components. We used Harmony (68), an algorithm that can perform integration of gene expression data from multiple spatial transcriptomics datasets, to adjust for batch effects on these 50 components. Harmonized components were then used to perform clustering analysis with BayesSpace (36), a deep-learning and Bayesian algorithm that identifies clusters of spots based on transcriptomic profile similarity, but also gives higher weight for clustering to physically close spots. We performed clustering across all 48 sections in a single analysis, which had the advantage of identifying matching layer-like clusters across sections, regardless of variability in terms of the exact orientation of sectioning across layers (Figure 2B, Supplementary Figure 4). We set the clustering implementation to return twelve clusters, which identified seven layer-like clusters within grey matter that were consistently located across sections, and five clusters that were located either in white matter or sporadically distributed without layer-like appearances, the latter based on small numbers of spots (Figure 2, Supplementary Figure 4). In this way we were confident to have identified the main layer-like clusters within grey matter, which were the focus of our study. The outermost layer-like cluster, i.e. cluster 9 (corresponding to cortical layer I on the basis of marker gene expression and histological analysis – see below), was not identified in all 48 sections (Supplementary Figure 4), probably due to surface tissue loss during freezing/handling/sectioning. We also generated a two-dimensional map based on uniform manifold approximation and projection (UMAP) with the Scater package (69) across all spots, to visualize how the clusters compared to each other in terms of transcriptional similarity (Figure 2A). Supragranular versus infragranular expression We focused our subsequent analyses on supragranular layers II/III (clusters 2, 10, 11) and infragranular layers V/VI (clusters 1, 3). For each gene, we pseudo-bulked the spot-level data into cluster-level data (70-72), by summing the raw gene-expression counts across all spots in a given section and cluster, resulting in a new SingleCellExperiment object containing 22,170 genes across 240 clusters (i.e. 48 sections × 5 clusters). This pseudo-bulking procedure reduced sparsity and increased the coverage of genes compared to spot-wise data. Using the same software and functions as described above with respect to the processing for data-driven clustering analysis, we normalized the pseudo-bulked, cluster-enriched gene expression matrix, identified the top 10% of variable genes, and computed the top 2 principal components to visualize how the 48 sections x 5 clusters relate in terms of transcriptome profile similarity. This supported a primary distinction between the supragranular and infragranular clusters (Figure 2C), and we therefore repeated the pseudo-bulking, but now for spots assigned to supragranular clusters 2, 10 and 11 all together, and spots assigned to infragranular clusters 1 and 3 all together, resulting in a new SingleCellExperiment object that comprised gene expression data from 48 sections x 2 pseudo-bulked clusters. We only retained genes with canonical transcripts defined in reference human genome GRCh38, https://ftp.ncbi.nlm.nih.gov/refseq/MANE/MANE_human/release_0.93/, for a total of 12,656 genes. With the normalized pseudo-bulk profiles, we computed the correlation structure between spatially adjacent sections using the duplicateCorrelation function of the Limma software v3.46.0 (73). We then applied linear mixed effect modelling using the lmFit and eBayes functions of Limma (73), to test a model for each gene whereby its expression level varied depending on the main effect of layer (supra- versus infragranular expression, i.e. a binary variable), lobe (frontal versus temporal, another binary variable), the layer*lobe interaction (our primary interest was to find genes with different laminar patterns between the frontal and temporal regions), with donor and tissue block as fixed effects, and blocking the 24 pairs of spatially adjacent sections according to their correlation coefficient matrix. In fitting such a model, Limma uses data from all genes to improve the variance estimate for any given gene, which helps to overcome relatively small sample sizes in transcriptomic studies (73). FDR correction was applied at adjusted P<0.01 to identify genes with significant layer*lobe interaction effects. Supragranular versus infragranular expression The list of genes with FDR-adjusted P<0.01 was input to the software Enrichr (74) (as implemented at https://maayanlab.cloud/Enrichr/ ) for a descriptive Gene Ontology analysis. Cresyl-violet/Nissl staining In addition, we used extra sections taken from the 300μm intervals between spatially adjacent section pairs from three tissue blocks, to perform Cresyl-violet/Nissl staining. Fresh-frozen sections (10 µm) adjacent to the section used for Visium spatial transcriptomics was used for Cresyl-violet/Nissl staining. Sections were first fixated in 4% paraformaldehyde (in PBS, pH=7.4) for 10 minutes at room temperature. Fixated sections were washed twice for 5 minutes each with 1x PBS, followed by serial hydration in 70% ethanol, 50% ethanol and demi water, 2 minutes each. Sections then went through rapid dehydration in 70%, 96% and 100% ethanol, and were air-dried. Air-dried sections were stained in 0.1% Cresylviolet (Sigma) in MilliQ water at 56oC for 20 minutes, then incubated in 96% ethanol for 1 minute to remove excess staining. Stained sections were dehydrated in 96% ethanol for 2 minutes twice, in 100% ethanol for 2 minutes three times, in xylol (Sigma) for 2 minutes three times, and mounted in Entellen New (Sigma). Brightfield images were acquired using a AxioScan Z1 SlideScanner (Zeiss) with a Hitachi HV-F292SCL camera, a Plan-Apochromat 20x/0.8 M27 objective and Zeiss 11 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Zen v.2.6 (Blue Edition) software. Images were analysed with FIJI/ImageJ (v.1.53t) and QuPath0.2.3. Trained experimenters from different institutes (authors SvH, LEJ) then independently confirmed layer identification according to the classical six-layer schema, based on structural cytoarchitecture and manual boundary definitions (Supplementary Figure 6). Zen v.2.6 (Blue Edition) software. Images were analysed with FIJI/ImageJ (v.1.53t) and QuPath0.2.3. Trained experimenters from different institutes (authors SvH, LEJ) then independently confirmed layer identification according to the classical six-layer schema, based on structural cytoarchitecture and manual boundary definitions (Supplementary Figure 6). Gene-set association analysis with brain and behavioural variability We used previously published SNP-wise summary statistics from a genome-wide association study in 30,810 adults from the UK Biobank population dataset, to test whether the 56 genes defined in the section above contain genetic variants that are associated with white matter connectivity between left-hemisphere core language network regions (33) (four connectivity metrics: pars opercularis – superior temporal cortex; pars opercularis – middle temporal cortex; pars triangularis – superior temporal cortex; pars triangularis – middle temporal cortex) (Supplementary Figure 8). Genetic variants were mapped to each of the 56 genes based on National Center for Biotechnology Information build 37.3 gene definitions as implemented in the MAGMA software (75), including 50 kb upstream and 50 kb downstream of each gene. Separately for each white matter connectivity metric we then used MAGMA to derive a single gene-based association P value per gene, using the default single- nucleotide polymorphism (SNP)-wise mean model. This analysis combined the association signals of all SNPs within a given gene, while considering the linkage disequilibrium between SNPs. Separately for each of the four white matter connectivity metrics, the resulting gene-based association P values were used as input to test for a set-level association of the 56 genes defined in the previous section (above), using the GAUSS software (47). GAUSS has the advantage that it additionally identifies the subset of ‘driver genes’ with the maximal evidence of association, that best account for a significant set-level association. GAUSS uses a self-constrained null model for well-controlled type I error. We then adjusted the set-level association P values by Bonferroni correction for the four white matter connectivity metrics. Finally, we ran the same procedure for the 56-gene set in relation to genetic association summary statistics from an analysis of word reading ability in 33,959 individuals carried out by the GenLang Consortium (34), and a study of dyslexia in 51,800 adults who self-reported having a diagnosis versus 1,087,070 controls (35), carried out using data from 23andMe, Inc. We then adjusted the set-level association P values by Bonferroni correction for these two language- and reading-related traits. (Adjusting the set-level association P values for six traits, i.e. four white matter connectivity metrics and two reading-related behavioural traits, made no difference to which associations were significant at adjusted P<0.05). Disclosures The authors declare no competing interests. Acknowledgements This research was funded by the Max Planck Society (Germany) and the Netherlands Organization for Scientific Research (Language in Interaction consortium: Gravitation grant number 024.001.006). The funders had no role in study design, data collection and analysis, the decision to publish or preparation of the manuscript. We would like to thank the research participants and employees of 23andMe, Inc. for making this work possible. Cell-type analysis It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint For each of these 2622 genes, we returned to our spatial transcriptomic data and extracted the layer*lobe interaction P value, and identified 56 genes whose layer*lobe interaction P values were significant at FDR<0.01, in addition to being upregulated in layer II/III excitatory neurons and/or layer V/VI cortico-cortical projection neurons (Supplementary Table 7). Cell-type analysis We next made use of an existing single-cell transcriptomic dataset based on 104,559 nuclei from 41 postmortem tissue samples from the dorsolateral prefrontal cortex or anterior cingulate cortex of 15 individuals with autism and 16 neurotypical controls (44). That study had used the single-cell gene expression data to define 17 major cell types: fibrous astrocytes, protoplasmic astrocytes, endothelial, parvalbumin interneurons, somatostatin interneurons, SV2C interneurons, VIP interneurons, layer II/III excitatory neurons, layer IV excitatory neurons, layer V/VI corticofugal projection neurons, layer V/VI cortico-cortical projection neurons, microglia, maturing neurons, NRGN-expressing neurons I, NRGN-expressing neurons II, oligodendrocytes and oligodendrocyte progenitor cells. We aggregated UMI counts from all 104,559 nuclei to create cell type-specific log-transformed normalized counts for each of the 17 cell types, resulting in a set of pseudo-bulked profiles for all unique donor- region-cell type combinations. For each gene, we then used its pseudo-bulked profiles to test for upregulation in a given cell type compared to all other cell types, using the lmFit and eBayes functions in Limma (73), while adjusting for the fixed effects of brain region, age, sex and diagnosis, and a random intercept of donor. We then extracted the results of interest for the present study, i.e. testing for upregulation in layer II/III excitatory neurons or layer V/VI cortico-cortical projection neurons. Separately for each of these two cell types, we defined upregulated genes as those with FDR-adjusted P<0.01 and a positive t-score (expressed higher in this cell type against the others) (Supplementary Tables 5, 6). We then created a union gene set that included 2622 genes upregulated in layer II/III excitatory neurons and/or layer V/VI cortico-cortical projection neurons. 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Data and Materials Availability The spatial transcriptomic dataset created for this study can be found within the Gene Expression Omnibus with accession number <<TO BE ADDED>>. Genome-wide association summary statistics from the 23andMe study of dyslexia should be requested from 23andMe (https://research.23andme.com/dataset-access/). Other data sources are cited in the Methods section and can be accessed via the corresponding publications. This study used openly available software as cited in the Methods section. Additional code that supported the analyses in this study is available at <<LINK TO BE ADDED>>. 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Author Contributions Conceptualization: SEF, WDJvdB, LEJ, CF. Data curation: MMKW, ZS, NV, SvH, LEJ. Formal analysis: MMKW, ZS. Funding acquisition: SEF, CF. Investigation: MMKW, ZS, LL, XZK, NV, SvH, LEJ. Methodology: MMKW, ZS, LL, XZK, NV, SvH, WDJvdB, LEJ. Project administration: CF. Resources: MM, WDJvdB, LEJ, SEF, CF. Supervision: SEF, CF. Visualization: MMKW, XZK, ZS, NV. Writing - original draft: MMKW, ZS, CF. Writing - review & editing: All authors. Conceptualization: SEF, WDJvdB, LEJ, CF. Data curation: MMKW, ZS, NV, SvH, LEJ. Formal analysis: MMKW, ZS. Funding acquisition: SEF, CF. Investigation: MMKW, ZS, LL, XZK, NV, SvH, LEJ. Methodology: MMKW, ZS, LL, XZK, NV, SvH, WDJvdB, LEJ. Project administration: CF. Resources: MM, WDJvdB, LEJ, SEF, CF. Supervision: SEF, CF. Visualization: MMKW, XZK, ZS, NV. Writing - original draft: MMKW, ZS, CF. Writing - review & editing: All authors. References Correlated gene expression supports synchronous ctivity in brain networks. Science 348(6240):1241-1244. Kong X-Z, et al. (2020) Gene Expression Correlates of the Cortical Network Underlying Sentence Processing. Neurobiology of Language 1(1):77-103. 8. Jonkman LE, et al. (2019) Normal Aging Brain Collection Amsterdam (NABCA): A comprehensive collection of postmortem high-field imaging, neuropathological and morphometric datasets of non- neurological controls. NeuroImage: Clinical 22:101698. 9. Keo A, et al. (2020) Transcriptomic signatures of brain regional vulnerability to Parkinson’s disease. Communications biology 3(1):101. 9. Keo A, et al. (2020) Transcriptomic signatures of brain regional vulnerability to Parkinson’s disease. Communications biology 3(1):101. 10. Cappelletti C, et al. (2023) Transcriptomic profiling of Parkinson's disease brains reveals disease stage specific gene expression changes. Acta Neuropathologica 146(2):227-244. 10. Cappelletti C, et al. (2023) Transcriptomic profiling of Parkinson's disease brains reveals disease stage specific gene expression changes. Acta Neuropathologica 146(2):227-244. 11. Vertes PE, et al. (2016) Gene transcription profiles associated with inter-modular hubs and connection distance in human functional magnetic resonance imaging networks. Philos Trans R Soc Lond B Biol Sci 371(1705). 12. Romme IA, de Reus MA, Ophoff RA, Kahn RS, & van den Heuvel MP (2017) Connectome Disconnectivity and Cortical Gene Expression in Patients With Schizophrenia. Biol Psychiatry 81(6):495-502. 13. Romero-Garcia R, Warrier V, Bullmore ET, Baron-Cohen S, & Bethlehem RAI (2018) Synaptic and transcriptionally downregulated genes are associated with cortical thickness differences in autism. Mol Psychiatry. 14. McColgan P, et al. (2018) Brain Regions Showing White Matter Loss in Huntington's Disease Are Enriched for Synaptic and Metabolic Genes. Biol Psychiatry 83(5):456-465. 15. Bartres-Faz D, et al. (2019) Characterizing the molecular architecture of cortical regions associated with high educational attainment in older individuals. J Neurosci. 16. Burt JB, et al. (2018) Hierarchy of transcriptomic specialization across human cortex captured by structural neuroimaging topography Nature Neuroscience 21(9):1251 1259 with high educational attainment in older individuals. J Neurosci. 16. Burt JB, et al. (2018) Hierarchy of transcriptomic specialization across human cortex captured by structural neuroimaging topography. Nature Neuroscience 21(9):1251-1259. 17. Fulcher BD & Fornito A (2016) A transcriptional signature of hub connectivity in the mouse connectome. Proc Natl Acad Sci U S A 113(5):1435-1440. 18. Fornito A, Arnatkeviciute A, & Fulcher B (2019) Bridging the Gap between Connectome and Transcriptome. Trends in Cognitive Sciences 23(1):34-50. 18. Fornito A, Arnatkeviciute A, & Fulcher B (2019) Bridging the Gap between Connectome and Transcriptome. Trends in Cognitive Sciences 23(1):34-50. 19. References 1. Labache L, et al. (2019) A SENtence Supramodal Areas AtlaS (SENSAAS) based on multiple task- induced activation mapping and graph analysis of intrinsic connectivity in 144 healthy right-handers. Brain Struct Funct 224(2):859-882. 2. Friederici AD & Gierhan SM (2013) The language network. Curr Opin Neurobiol 23(2):250-254. 3. Malik-Moraleda S, et al. (2022) An investigation across 45 languages and 12 language families reveals a universal language network. Nat Neurosci 25(8):1014-1019. 4. Mazoyer B, et al. (2014) Gaussian Mixture Modeling of Hemispheric Lateralization for Language in a Large Sample of Healthy Individuals Balanced for Handedness. PLoS ONE 9(6):e101165. 5. Moodie JE, et al. (2023) General and specific patterns of cortical gene expression as substrates of complex cognitive functioning. bioRxiv:2023.2003.2016.532915. 6. Richiardi J, et al. (2015) BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks. Science 348(6240):1241-1244. 7. Kong X-Z, et al. (2020) Gene Expression Correlates of the Cortical Network Underlying Sentence Processing. Neurobiology of Language 1(1):77-103. 8. Jonkman LE, et al. (2019) Normal Aging Brain Collection Amsterdam (NABCA): A comprehensive collection of postmortem high-field imaging, neuropathological and morphometric datasets of non- neurological controls. NeuroImage: Clinical 22:101698. 9 K A l (2020) i i i f b i i l l bili ki ’ di 1. Labache L, et al. (2019) A SENtence Supramodal Areas AtlaS (SENSAAS) based on multiple task- induced activation mapping and graph analysis of intrinsic connectivity in 144 healthy right-handers. Brain Struct Funct 224(2):859-882. 1. Labache L, et al. (2019) A SENtence Supramodal Areas AtlaS (SENSAAS) based on multiple task- induced activation mapping and graph analysis of intrinsic connectivity in 144 healthy right-handers. Brain Struct Funct 224(2):859-882. 2. Friederici AD & Gierhan SM (2013) The language network. Curr Opin Neurobiol 23(2):250-254. 3. Malik-Moraleda S, et al. (2022) An investigation across 45 languages and 12 language families reveals a universal language network. Nat Neurosci 25(8):1014-1019. 4. Mazoyer B, et al. (2014) Gaussian Mixture Modeling of Hemispheric Lateralization for Language in a Large Sample of Healthy Individuals Balanced for Handedness. PLoS ONE 9(6):e101165. 5. Moodie JE, et al. (2023) General and specific patterns of cortical gene expression as substrates of complex cognitive functioning. bioRxiv:2023.2003.2016.532915. 6. Richiardi J, et al. (2015) BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks. Science 348(6240):1241-1244. 7. Kong X-Z, et al. (2020) Gene Expression Correlates of the Cortical Network Underlying Sentence Richiardi J, et al. (2015) BRAIN NETWORKS. References Sha Z, Schijven D, Fisher SE, & Francks C (2023) Genetic architecture of the white matter connectome of the human brain. Science advances 9(7):eadd2870. 34. Eising E, et al. (2022) Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people. Proc Natl Acad Sci U S A 119(35):e2202764119. 35. Doust C, et al. (2022) Discovery of 42 genome-wide significant loci associated with dyslexia. Nat Genet 54(11):1621-1629. 36. Zhao E, et al. (2021) Spatial transcriptomics at subspot resolution with BayesSpace. Nature Biotechnology 39(11):1375-1384. 37. He Z, et al. (2017) Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques. Nature Neuroscience 20(6):886-895. 38. Hevner RF (2007) Layer-Specific Markers as Probes for Neuron Type Identity in Human Neocortex and Malformations of Cortical Development. Journal of Neuropathology & Experimental Neurology 66(2):101-109. 39. Molnár Z & Rockland KS (2020) Chapter 5 - Cortical columns. Neural Circuit and Cognitive 32. Jorstad NL, et al. (2023) Transcriptomic cytoarchitecture reveals principles of human neocortex organization. Science 382(6667):eadf6812. 33. Sha Z, Schijven D, Fisher SE, & Francks C (2023) Genetic architecture of the white matter connectome of the human brain. Science advances 9(7):eadd2870. 33. Sha Z, Schijven D, Fisher SE, & Francks C (2023) Genetic architecture of the white matter connectome of the human brain. Science advances 9(7):eadd2870. 34. Eising E, et al. (2022) Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people. Proc Natl Acad Sci U S A 119(35):e2202764119. 34. Eising E, et al. (2022) Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people. Proc Natl Acad Sci U S A 119(35):e2202764119. 35. Doust C, et al. (2022) Discovery of 42 genome-wide significant loci associated with dyslexia. Nat Genet 54(11):1621-1629. 35. Doust C, et al. (2022) Discovery of 42 genome-wide significant loci associated with dyslexia. Nat Genet 54(11):1621-1629. 36. Zhao E, et al. (2021) Spatial transcriptomics at subspot resolution with BayesSpace. Nature Biotechnology 39(11):1375-1384. 36. Zhao E, et al. (2021) Spatial transcriptomics at subspot resolution with BayesSpace. Nature Biotechnology 39(11):1375-1384. 37. He Z, et al. (2017) Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques. Nature Neuroscience 20(6):886-895. 37. He Z, et al. (2017) Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques. Nature Neuroscience 20(6):886-895. 38. References Wagstyl K, et al. (2022) Transcriptional Cartography Integrates Multiscale Biology of the Human Cortex. bioRxiv:2022.2006.2013.495984. 19. Wagstyl K, et al. (2022) Transcriptional Cartography Integrates Multiscale Biology of the Human Cortex. bioRxiv:2022.2006.2013.495984. 14 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint 20. Rockland KS & Pandya DN (1979) Laminar origins and terminations of cortical connections of the occipital lobe in the rhesus monkey. Brain Res 179(1):3-20. 21. Felleman DJ & Van Essen DC (1991) Distributed hierarchical processing in the primate cerebral cortex. Cereb Cortex 1(1):1-47. 22. Hilgetag CC, O'Neill MA, & Young MP (1996) Indeterminate organization of the visual system. Science 271(5250):776-777. 23. Goulas A, Zilles K, & Hilgetag CC (2018) Cortical Gradients and Laminar Projections in Mammals. Trends Neurosci 41(11):775-788. 24. Burt JB, et al. (2018) Hierarchy of transcriptomic specialization across human cortex captured by structural neuroimaging topography. Nat Neurosci 21(9):1251-1259. 25. Kral A & Eggermont JJ (2007) What's to lose and what's to learn: development under auditory deprivation, cochlear implants and limits of cortical plasticity. Brain Res Rev 56(1):259-269. 26. Bastos AM, et al. (2015) Visual areas exert feedforward and feedback influences through distinct frequency channels. Neuron 85(2):390-401. 27. Markov NT & Kennedy H (2013) The importance of being hierarchical. Current opinion in neurobiology 23(2):187-194. 28. Salmen F, et al. (2018) Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections. Nature protocols 13(11):2501-2534. 29. Stahl PL, et al. (2016) Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353(6294):78-82. 30. Maynard KR, et al. (2021) Transcriptome-scale spatial gene expression in the human dorsolateral prefrontal cortex. Nat Neurosci 24(3):425-436. 31. Zeng H, et al. (2012) Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures. Cell 149(2):483-496. 32. Jorstad NL, et al. (2023) Transcriptomic cytoarchitecture reveals principles of human neocortex organization. Science 382(6667):eadf6812. 33. References Bishop DVM, Snowling MJ, Thompson PA, Greenhalgh T, & consortium atC- (2017) Phase 2 of CATALISE: a multinational and multidisciplinary Delphi consensus study of problems with language 51. Laws KM & Bashaw GJ (2022) Diverse roles for axon guidance pathways in adult tissue architecture and function. Natural Sciences 2(4):e20220021. 52. Li J-c, et al. (2015) Increased Permeability of the Blood-Brain Barrier and Alzheimer's Disease-Like Alterations in Slit-2 Transgenic Mice. Journal of Alzheimer's Disease 43:535-548. 52. Li J-c, et al. (2015) Increased Permeability of the Blood-Brain Barrier and Alzheimer's Disease-Like Alterations in Slit-2 Transgenic Mice. Journal of Alzheimer's Disease 43:535-548. 53. Blockus H, et al. (2021) Synaptogenic activity of the axon guidance molecule Robo2 underlies hippocampal circuit function. Cell Reports 37(3):109828. 53. Blockus H, et al. (2021) Synaptogenic activity of the axon guidance molecule Robo2 underlies hippocampal circuit function. Cell Reports 37(3):109828. 54. Bishop DVM, Snowling MJ, Thompson PA, Greenhalgh T, & consortium atC- (2017) Phase 2 of CATALISE: a multinational and multidisciplinary Delphi consensus study of problems with language development: Terminology. Journal of Child Psychology and Psychiatry 58(10):1068-1080. 54. Bishop DVM, Snowling MJ, Thompson PA, Greenhalgh T, & consortium atC- (2017) Phase 2 of CATALISE: a multinational and multidisciplinary Delphi consensus study of problems with language development: Terminology. Journal of Child Psychology and Psychiatry 58(10):1068-1080. 55. Bishop DVM & Snowling MJ (2004) Developmental Dyslexia and Specific Language Imp or Different? Psychological bulletin 130:858-886. 56. Knoop-van Campen CAN, Segers E, & Verhoeven L (2018) How phonological awareness mediates the relation between working memory and word reading efficiency in children with dyslexia. Dyslexia (Chichester, England) 24(2):156-169. 56. Knoop-van Campen CAN, Segers E, & Verhoeven L (2018) How phonological awareness mediates the relation between working memory and word reading efficiency in children with dyslexia. Dyslexia (Chichester, England) 24(2):156-169. 57. Van Der Auwera S, Vandermosten M, Wouters J, Ghesquière P, & Vanderauwera J (2021) A three- time point longitudinal investigation of the arcuate fasciculus throughout reading acquisition in children developing dyslexia. NeuroImage 237:118087. 57. Van Der Auwera S, Vandermosten M, Wouters J, Ghesquière P, & Vanderauwera J (2021) A three- time point longitudinal investigation of the arcuate fasciculus throughout reading acquisition in children developing dyslexia. NeuroImage 237:118087. 58. Meisler SL & Gabrieli JDE (2021) A Large-Scale Investigation of White Matter Microstructural Associations with Reading Ability. bioRxiv:2021.2008.2026.456137. 58. Meisler SL & Gabrieli JDE (2021) A Large-Scale Investigation of White Matter Microstructural Associations with Reading Ability. References bioRxiv:2021.2008.2026.456137. 59. Woolnough O, et al. (2023) Spatiotemporally distributed frontotemporal networks for sentence reading. Proceedings of the National Academy of Sciences 120(17):e2300252120. 60. Spaulding EL & Burgess RW (2017) Accumulating Evidence for Axonal Translation in Neuronal Homeostasis Frontiers in Neuroscience 11 59. Woolnough O, et al. (2023) Spatiotemporally distributed frontotemporal networks for sentence reading. Proceedings of the National Academy of Sciences 120(17):e2300252120. 60. Spaulding EL & Burgess RW (2017) Accumulating Evidence for Axonal Translation in Neuronal 59. Woolnough O, et al. (2023) Spatiotemporally distributed frontotemporal networks for sentence reading. Proceedings of the National Academy of Sciences 120(17):e2300252120. 60. Spaulding EL & Burgess RW (2017) Accumulating Evidence for Axonal Translation in Neuronal Homeostasis. Frontiers in Neuroscience 11. ead g oceed gs of t e at o a cade y of Sc e ces 0( ) e 300 5 0 60. Spaulding EL & Burgess RW (2017) Accumulating Evidence for Axonal Translation in Neuronal Homeostasis. Frontiers in Neuroscience 11. 61. Glock C, et al. (2021) The translatome of neuronal cell bodies, dendrites, and axons. Proc Natl Acad Sci U S A 118(43). l ( ) i l i i l h i ' i i id li f h 61. Glock C, et al. (2021) The translatome of neuronal cell bodies, dendrites, and axons. Proc Natl Acad Sci U S A 118(43). ( ) 62. Hyman BT, et al. (2012) National Institute on Aging–Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease. Alzheimer's & Dementia 8(1):1-13. 63. Dobin A, et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15-21. 62. Hyman BT, et al. (2012) National Institute on Aging–Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease. Alzheimer's & Dementia 8(1):1-13. 63. Dobin A, et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15-21. 64. Morgan M, Obenchain V, Hester J, & Pagès H (2017) SummarizedExperiment: SummarizedExperiment container. R package version 1(0). 64. Morgan M, Obenchain V, Hester J, & Pagès H (2017) SummarizedExperiment: SummarizedExperiment container. R package version 1(0). 65. Amezquita RA, et al. (2020) Orchestrating single-cell analysis with Bioconductor. Nat Methods 65. Amezquita RA, et al. (2020) Orchestrating single-cell analysis with Bioconductor. Nat Methods 17(2):137-145. 66. Lun AT, McCarthy DJ, & Marioni JC (2016) A step-by-step workflow for low-level analysis of single- cell RNA-seq data with Bioconductor. F1000Res 5:2122. 66. References Hevner RF (2007) Layer-Specific Markers as Probes for Neuron Type Identity in Human Neocortex and Malformations of Cortical Development. Journal of Neuropathology & Experimental Neurology 66(2):101-109. 38. Hevner RF (2007) Layer-Specific Markers as Probes for Neuron Type Identity in Human Neocortex and Malformations of Cortical Development. Journal of Neuropathology & Experimental Neurology 66(2):101-109. 39. Molnár Z & Rockland KS (2020) Chapter 5 - Cortical columns. Neural Circuit and Cognitive Development (Second Edition), eds Rubenstein J, Rakic P, Chen B, & Kwan KY (Academic Press), pp 103-126. 39. Molnár Z & Rockland KS (2020) Chapter 5 - Cortical columns. Neural Circuit and Cognitive Development (Second Edition), eds Rubenstein J, Rakic P, Chen B, & Kwan KY (Academic Press), pp 103-126. 40. Rajkowska G & Goldman-Rakic PS (1995) Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria. Cereb Cortex 5(4):307-322. 40. Rajkowska G & Goldman-Rakic PS (1995) Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria. Cereb Cortex 5(4):307-322. 41. Tong X, et al. (2022) Role of Sostdc1 in skeletal biology and cancer. Frontiers in Physiology 13. 42. Ashburner M, et al. (2000) Gene Ontology: tool for the unification of biology. Nature Genetics 25(1):25-29. 43. Muzio G, et al. (2023) networkGWAS: A network-based approach to discover genetic associations. Bioinformatics. 43. Muzio G, et al. (2023) networkGWAS: A network-based approach to discover genetic associations. Bioinformatics. 15 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint 44. Velmeshev D, et al. (2019) Single-cell genomics identifies cell type-specific molecular changes in autism. Science 364(6441):685-689. 45 S T t l (2005) E l A t f G T i ti i E b i H L ft d Ri ht 45. Sun T, et al. References (2005) Early Asymmetry of Gene Transcription in Embryonic Human Left a Cerebral Cortex. Science 308(5729):1794-1798. 46. Polanco J, Reyes-Vigil F, Weisberg SD, Dhimitruka I, & Brusés JL (2021) Differential Spatiotemporal Expression of Type I and Type II Cadherins Associated With the Segmentation of the Central Nervous System and Formation of Brain Nuclei in the Developing Mouse. Frontiers in molecular neuroscience 14. 46. Polanco J, Reyes-Vigil F, Weisberg SD, Dhimitruka I, & Brusés JL (2021) Differential Spatiotemporal Expression of Type I and Type II Cadherins Associated With the Segmentation of the Central Nervous System and Formation of Brain Nuclei in the Developing Mouse. Frontiers in molecular neuroscience 14. 47. Dutta D, et al. (2021) A powerful subset-based method identifies gene set associations and improves interpretation in UK Biobank. Am J Hum Genet 108(4):669-681. 47. Dutta D, et al. (2021) A powerful subset-based method identifies gene set associations and improves interpretation in UK Biobank. Am J Hum Genet 108(4):669-681. 48. Eichert N, et al. (2019) What is special about the human arcuate fasciculus? Lateralization, projections, and expansion. Cortex 118:107-115. 48. Eichert N, et al. (2019) What is special about the human arcuate fasciculus? Lateralization, projections, and expansion. Cortex 118:107-115. 49. Sharoh D, et al. (2019) Laminar specific fMRI reveals directed interactions in distributed networks during language processing. Proceedings of the National Academy of Sciences:201907858. 50. Gonda Y, Namba T, & Hanashima C (2020) Beyond Axon Guidance: Roles of Slit-Robo Signaling in , ( ) p during language processing. Proceedings of the National Academy of Sciences:201907858. 50. Gonda Y, Namba T, & Hanashima C (2020) Beyond Axon Guidance: Roles of Slit-Robo Signaling in Neocortical Formation. Frontiers in cell and developmental biology 8. , ( ) p during language processing. Proceedings of the National Academy of Sciences:201907858. 50. Gonda Y, Namba T, & Hanashima C (2020) Beyond Axon Guidance: Roles of Slit-Robo Signaling in Neocortical Formation. Frontiers in cell and developmental biology 8. Neocortical Formation. Frontiers in cell and developmental biology 8. 51. Laws KM & Bashaw GJ (2022) Diverse roles for axon guidance pathways in adult tissue architecture and function. Natural Sciences 2(4):e20220021. 52. Li J-c, et al. (2015) Increased Permeability of the Blood-Brain Barrier and Alzheimer's Disease-Like Alterations in Slit-2 Transgenic Mice. Journal of Alzheimer's Disease 43:535-548. 53. Blockus H, et al. (2021) Synaptogenic activity of the axon guidance molecule Robo2 underlies hippocampal circuit function. Cell Reports 37(3):109828. 54. References It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Wong & Sha et al., The cerebral cortical infrastructure for language involves region-specific patterns of laminar gene expression Wong & Sha et al., The cerebral cortical infrastructure for language involves region-specific patterns of laminar gene expression References Lun AT, McCarthy DJ, & Marioni JC (2016) A step-by-step workflow for low-level analysis of single- cell RNA-seq data with Bioconductor. F1000Res 5:2122. 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint 67. McCarthy DJ, Campbell KR, Lun AT, & Wills QF (2017) Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33(8):1179-1186. 68. Korsunsky I, et al. (2019) Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16(12):1289-1296. 69. McInnes L, Healy J, & Melville J (2018) Umap: Uniform manifold approximation and projection for dimension reduction. arXiv preprint arXiv:1802.03426. 70. Lun ATL & Marioni JC (2017) Overcoming confounding plate effects in differential expression analyses of single-cell RNA-seq data. Biostatistics 18(3):451-464. 71. Kang HM, et al. (2018) Multiplexed droplet single-cell RNA-sequencing using natural genetic variation. Nat Biotechnol 36(1):89-94. 72. Crowell HL, et al. (2020) muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data. Nat Commun 11(1):6077. 73. Ritchie ME, et al. (2015) limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43(7):e47. 74. Kuleshov MV, et al. (2016) Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Research 44(W1):W90-W97. 75. de Leeuw CA, Mooij JM, Heskes T, & Posthuma D (2015) MAGMA: generalized gene-set analysis of GWAS data. PLoS Comput Biol 11(4):e1004219. McCarthy DJ, Campbell KR, Lun AT, & Wills QF (2017) Scater: pre-processing, quality control, ormalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33(8):1179-1186 75. de Leeuw CA, Mooij JM, Heskes T, & Posthuma D (2015) MAGMA: generalized gene-set analysis of GWAS data. PLoS Comput Biol 11(4):e1004219. 17 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Contents: - Supplementary Figures 1-8 on pages 2-9. - Supplementary Tables: 10 tables as different worksheets in one separate Excel file. Page 1 of 9 Page 1 of 9 Page 1 of 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 1. Schematic of the left-hemisphere regions sampled in this study. Two tissue blocks were taken from the inferior frontal gyrus. Blocks were approximately centred on the yellow spots indicated (block ‘gfi1’ more anterior/inferior, block ‘gfi3’ more posterior/superior). Two tissue blocks were also taken from the superior temporal sulcus. Again, blocks were approximately centred on the yellow spots indicated (block ‘gts4’ more anterior/inferior, block ‘gts5’ more posterior/superior). The broader coloured regions around the yellow spots represent four areas defined in the SENtence Supramodal Areas AtlaS (SENSAAS) (Labache et al. (2019) – see reference in the main manuscript). Supplementary Figure 1. Schematic of the left-hemisphere regions sampled in this study. Two tissue blocks were taken from the inferior frontal gyrus. Blocks were approximately centred on the yellow spots indicated (block ‘gfi1’ more anterior/inferior, block ‘gfi3’ more posterior/superior). Two tissue blocks were also taken from the superior temporal sulcus. Again, blocks were approximately centred on the yellow spots indicated (block ‘gts4’ more anterior/inferior, block ‘gts5’ more posterior/superior). The broader coloured regions around the yellow spots represent four areas defined in the SENtence Supramodal Areas AtlaS (SENSAAS) (Labache et al. (2019) – see reference in the main manuscript). Supplementary Figure 1. Schematic of the left-hemisphere regions sampled in this study. Two tissue blocks were taken from the inferior frontal gyrus. Blocks were approximately centred on the yellow spots indicated (block ‘gfi1’ more anterior/inferior, block ‘gfi3’ more posterior/superior). Two tissue blocks were also taken from the superior temporal sulcus. Again, blocks were approximately centred on the yellow spots indicated (block ‘gts4’ more anterior/inferior, block ‘gts5’ more posterior/superior). The broader coloured regions around the yellow spots represent four areas defined in the SENtence Supramodal Areas AtlaS (SENSAAS) (Labache et al. (2019) – see reference in the main manuscript). Page 2 of 9 Page 2 of 9 Page 2 of 9 . Contents: CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 2. Unique molecular identifier counts across all spots for 48 cortical tissue sections. The pink/purple areas were outside of the capture area for spatial transcriptomics. The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Supplementary Figure 2. Unique molecular identifier counts across all spots for 48 cortical tissue sections. The pink/purple areas were outside of the capture area for spatial transcriptomics. The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Page 3 of 9 Page 3 of 9 Page 3 of 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. Contents: ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 3. Spots excluded during spot-level quality control. Spots coloured red were excluded according to the process described in the main text (Methods). The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Supplementary Figure 3. Spots excluded during spot-level quality control. Spots coloured red were excluded according to the process described in the main text (Methods). The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Page 4 of 9 Page 4 of 9 Page 4 of 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 4. Spatial distributions of gene expression data-driven clusters of spots across 48 tissue sections. The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Supplementary Figure 4. Contents: Spatial distributions of gene expression data-driven clusters of spots across 48 tissue sections. The labels of the 48 cortical tissue sections are given as Donor_Block_Section, where the three donors were anw082, anw797 and anw945, the blocks were gfi1 & gf13 from the inferior frontal gyrus and gts4 & gts5 from the superior temporal sulcus, and the tissue sections from each block are called a1 & a2 (the first adjacent pair of sections) and a3 & a4 (second pair of adjacent sections). Page 5 of 9 Page 5 of 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 5. The expression levels of layer marker genes in data-driven clusters shows the expression of a single marker gene, with the x-axis showing the data-driven clusters an showing the normalized gene expression across 48 tissue sections. Clusters are ordered from according to their laminar spatial locations from upper to lower. These data support th correspondence: layerI=cluster9, layerII=cluster10, layerIII=clusters11&2, layerIV=cluster 6, layer layerVI=cluster1, with other clusters corresponding to white matter or with sporadic spatial distrib Supplementary Figure 5. The expression levels of layer marker genes in data-driven clusters. Each panel shows the expression of a single marker gene, with the x-axis showing the data-driven clusters and the y-axis showing the normalized gene expression across 48 tissue sections. Clusters are ordered from left-to-right according to their laminar spatial locations from upper to lower. These data support the following correspondence: layerI=cluster9, layerII=cluster10, layerIII=clusters11&2, layerIV=cluster 6, layerV=cluster 3, layerVI=cluster1, with other clusters corresponding to white matter or with sporadic spatial distributions. y Figure 5. The expression levels of layer marker genes in data-driven clusters. Each panel Supplementary Figure 5. The expression levels of layer marker genes in data-driven cluste Supplementary Figure 5. The expression levels of layer marker genes in data-driven clusters. Contents: Each panel shows the expression of a single marker gene, with the x-axis showing the data-driven clusters and the y-axis showing the normalized gene expression across 48 tissue sections. Clusters are ordered from left-to-right according to their laminar spatial locations from upper to lower. These data support the following correspondence: layerI=cluster9, layerII=cluster10, layerIII=clusters11&2, layerIV=cluster 6, layerV=cluster 3, layerVI=cluster1, with other clusters corresponding to white matter or with sporadic spatial distributions. Page 6 of 9 Page 6 of 9 Page 6 of 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 6. Cluster-layer correspondence assessed through cytoarchitecture. Comparison between data-driven gene expression clusters from the spatial transcriptomic data, and manually-defined cortical layers based on cytoarchitecture, across three pairs of tissue sections. Supplementary Figure 6. Cluster-layer correspondence assessed through cytoarchitecture. Comparison between data-driven gene expression clusters from the spatial transcriptomic data, and manually-defined cortical layers based on cytoarchitecture, across three pairs of tissue sections. Page 7 of 9 Page 7 of 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 7. Contents: Significant layer*lobe interaction effects for 56 genes that also showe upregulation in Layer II/III excitatory neurons and/or Layer V/VI cortico-cortical projection neuron Each panel shows the expression of a single gene, with the x-axis showing four pseudo-bulked clusters (se main text) and the y-axis showing the normalized gene expression across 48 tissue sections. Supplementary Figure 7. Significant layer*lobe interaction effects for 56 genes that also showed upregulation in Layer II/III excitatory neurons and/or Layer V/VI cortico-cortical projection neurons. Each panel shows the expression of a single gene, with the x-axis showing four pseudo-bulked clusters (see main text) and the y-axis showing the normalized gene expression across 48 tissue sections. Page 8 of 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint Supplementary Figure 8. White matter connectivity between four regions of the core left-hemisphere language network. (A) The four cortical regions are shown in different shades of blue, as defined in the Automated Anatomical Labelling brain atlas (Tzourio-Mazoyer et al. 2002), and applied in the genome-wide association study of Sha et al. (2023) in 30,810 adults (see reference list in the main manuscript). Also shown is an abstract network representation where the four cortical regions are nodes and the structural connections between them are edges. In the present study we were interested in the four frontal-temporal network edges, i.e. pars opercularis - superior temporal cortex; pars triangularis - superior temporal cortex; pars opercularis - middle temporal cortex; and pars triangularis - middle temporal cortex. (B) Visualization of the white matter connections between the four cortical regions in an example individual, with gold representing connections between the pars opercularis and middle temporal cortex, blue representing connections between the pars opercularis and superior temporal cortex, purple representing connections between the pars triangularis and middle temporal cortex, and yellow representing connections between the pars triangularis and superior temporal cortex. Also shown are connections between the pars opercularis and pars triangularis (red), and connections between the middle temporal cortex and superior temporal cortex (green), but these within-lobe connections were not considered in the present study. Contents: Figure reproduced from Sha et al, (2023) under an open access Creative Commons Attribution License 4.0 (CC BY) (see reference list in the main manuscript). Supplementary Figure 8. White matter connectivity between four regions of the core left-hemisphere language network. (A) The four cortical regions are shown in different shades of blue, as defined in the Automated Anatomical Labelling brain atlas (Tzourio-Mazoyer et al. 2002), and applied in the genome-wide association study of Sha et al. (2023) in 30,810 adults (see reference list in the main manuscript). Also shown is an abstract network representation where the four cortical regions are nodes and the structural connections between them are edges. In the present study we were interested in the four frontal-temporal network edges, i.e. pars opercularis - superior temporal cortex; pars triangularis - superior temporal cortex; pars opercularis - middle temporal cortex; and pars triangularis - middle temporal cortex. (B) Visualization of the white matter connections between the four cortical regions in an example individual, with gold representing connections between the pars opercularis and middle temporal cortex, blue representing connections between the pars opercularis and superior temporal cortex, purple representing connections between the pars triangularis and middle temporal cortex, and yellow representing connections between the pars triangularis and superior temporal cortex. Also shown are connections between the pars opercularis and pars triangularis (red), and connections between the middle temporal cortex and superior temporal cortex (green), but these within-lobe connections were not considered in the present study. Figure reproduced from Sha et al, (2023) under an open access Creative Commons Attribution License 4.0 (CC BY) (see reference list in the main manuscript). Page 9 of 9 Page 9 of 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 20, 2024. ; https://doi.org/10.1101/2024.01.17.575815 doi: bioRxiv preprint
https://openalex.org/W4303415151
https://www.nogr.org/jour/article/download/2048/1831
Russian
null
Risk factors of malignancy
Èksperimentalʹnaâ i kliničeskaâ gastroènterologiâ
2,022
cc-by
11,215
Факторы риска развития онкопатологии Друк И. В., Семенова Е. В., Логинова Е. Н., Кореннова О. Ю., Семенкин А. А., Лялюкова Е. А., Надей Е. В. Друк И. В., Семенова Е. В., Логинова Е. Н., Кореннова О. Ю., Семенкин А. А., Лялюкова Е. А., Надей Е. В. Федеральное государственное бюджетное образовательное учреждение высшего образования «Омский государственный медицинский университет» Министерства здравоохранения Российской Федерации, 644099, Омск, ул. Ленина, д. 12, Россия Федеральное государственное бюджетное образовательное учреждение высшего образования «Омский государственный медицинский университет» Министерства здравоохранения Российской Федерации, 644099, Омск, ул. Ленина, д. 12, Россия Для цитирования: Друк И. В., Семенова Е. В., Логинова Е. Н., Кореннова О. Ю., Семенкин А. А., Лялюкова Е. А., Надей Е. В. Факторы риска развития онкопатологии. Экспериментальная и клиническая гастроэнтерология. 2022;205(9): 116–128. DOI: 10.31146/1682-8658-ecg-205-9-116-128  Для переписки: Семенова Елена Владимировна sel.92@mail.ru Друк Инна Викторовна, д. м. н., доцент, заведующая кафедрой внутренних болезней и семейной медицины ДПО Семенова Елена Владимировна, к. м. н., врач-кардиолог клиники Логинова Екатерина Николаевна, к. м. н., доцент, доцент кафедры внутренних болезней и семейной медицины ДПО Кореннова Ольга Юрьевна, д. м. н., профессор, профессор кафедры внутренних болезней и семейной медицины ДПО Семенкин Александр Анатольевич, д. м. н., профессор, профессор кафедры внутренних болезней и семейной медицины ДПО Лялюкова Елена Александровна, д. м. н., доцент, профессор кафедры внутренних болезней и семейной медицины ДПО Надей Елена Витальевна, к. м. н., доцент кафедры внутренних болезней и семейной медицины ДПО обзор review обзор review https://doi.org/10.31146/1682-8658-ecg-205-9-116-128 https://doi.org/10.31146/1682-8658-ecg-205-9-116-128 Risk factors of malignancy Korennova, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0001–8047–5521 p Elena V. Semenova, Cardiologist of Univercity Clinic; ORCID: 0000–0003–1375–017X Alexandre A. Semyonkin, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0002–3786–9995, Researcher ID: C-7195–2014 Alexandre A. Semyonkin, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0002–3786–9995, Researcher ID: C-7195–2014 Elena A. Lyalyukova, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Associate Professor; ORCID: 0000–0003–4878–0838 Elena V. Nadey, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD; ORCID: 0000–0003–0440–7118 Elena V. Nadey, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD; ORCID: 0000–0003–0440–7118 Summary Prevention strategies aim to change environmental and lifestyle risk factors that contribute to the development of cancer. Screening detects abnormalities before they become clinically apparent, allowing intervention either before cancer develops or at an early stage when treatment is most likely to be eff ective. Despite reliable data on factors that reduce the risk of neo- plasms, in routine clinical practice, the eff ectiveness of cancer prevention is still not high enough. This determines the high importance of the topic of this review, which summarizes the current scientifi c data on risk factors for the development of oncopathology and ways to infl uence them. Such risk factors as lifestyle, alcohol and tobacco use, physical activity, nutrition and other environmental factors are considered. A large role is given to infections, which is also considered in this review. The article highlights the intake of vitamins and microelements for the purpose of cancer prevention, as well as the infl uence of certain drugs taken to treat non-oncological diseases on the development of oncopathology. The purpose of the review is to present an analysis of current literature data on the methods of cancer prevention. Keywords: cancer prevention, risk factors, oncology Keywords: cancer prevention, risk factors, oncology Keywords: cancer prevention, risk factors, oncology Confl ict of interest. Authors declare no confl ict of interest. Confl ict of interest. Authors declare no confl ict of interest. рака [1, 2]. Несмотря на достоверные данные о фак- торах, снижающих риск возникновения новоо- бразований, в рутинной клинической практике эффективность канцерпревенции по-прежнему недостаточно высока [3, 4]. Это определяет высокую значимость темы настоящего обзора, в котором суммированы актуальные научные данные о фак- торах риска развития онкопатологии и способах влияния на них. Повлиять на смертность от многих видов рака можно как с помощью скрининга и профилакти- ки. Скрининг выявляет аномалии до того, как они станут клинически очевидными, что позволяет проводить вмешательство либо до развития рака, либо на ранней стадии, когда лечение чаще всего эффективно. Стратегии профилактики направ- лены на изменение условий окружающей среды и образа жизни, которые способствуют развитию рака [1, 2]. Несмотря на достоверные данные о фак- торах, снижающих риск возникновения новоо- бразований, в рутинной клинической практике эффективность канцерпревенции по-прежнему недостаточно высока [3, 4]. Это определяет высокую значимость темы настоящего обзора, в котором суммированы актуальные научные данные о фак- торах риска развития онкопатологии и способах влияния на них. Резюме Стратегии профилактики направлены на изменение факторов риска окружающей среды и образа жизни, которые способствуют развитию рака. Скрининг выявляет аномалии до того, как они станут клинически очевидными, что позволяет проводить вмешательство либо до развития рака, либо на ранней стадии, когда лечение чаще всего эффек- тивно. Несмотря на достоверные данные о факторах, снижающих риск возникновения новообразований, в рутинной клинической практике эффективность канцерпревенции по-прежнему недостаточно высока. Это определяет высокую значимость темы настоящего обзора, в котором суммированы актуальные научные данные о факторах риска развития онкопатологии и способах влияния на них. Рассмотрены такие факторы риска как образ жизни, употребление алкоголя и табакокурение, физическая активность, питание и другие факторы внешней среды. Большая роль отдается инфекциям, что также рассмотрено в настоящем обзоре. Освещен прием витаминов и микроэлементов с целью профилактики рака, а также влияние некоторых препаратов, принимаемым для лечения неонкологических заболеваний, на развитие онкопатологии. EDN: ZEAKCC Цель обзора — представить анализ современных литературных данных о способах канцеропревенции. Ключевые слова: профилактика рака, факторы риска, онкология Ключевые слова: профилактика рака, факторы риска, онкология Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. 116 обзор | review https://doi.org/10.31146/1682-8658-ecg-205-9-116-128 Risk factors of malignancy I. V. Druk, E. V. Semenova, E. N. Loginova, O. Yu. Korennova, A. A. Semyonkin, E. A. Lyalyukova, Е. V. Nadey Omsk State Medical University, 12, Lenina str., Omsk, 644099 Russia Risk factors of malignancy Risk factors of malignancy I. V. Druk, E. V. Semenova, E. N. Loginova, O. Yu. Korennova, A. A. Semyonkin, E. A. Lyalyukova, Е. V. Nadey Omsk State Medical University, 12, Lenina str., Omsk, 644099 Russia For citation: Druk I. V., Semenova E. V., Loginova E. N., Korennova O. Yu., Semyonkin A. A., Lyalyukova E. A., Nadey Е. V. Risk factors of malignancy. Experimental and Clinical Gastroenterology. 2022;205(9): 116–128. (In Russ.) DOI: 10.31146/1682-8658-ecg-205-9-116-128  Corresponding author: Elena V. Semenova sel.92@mail.ru Inna V. Druk, Head of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Associate Professor; ORCID: 0000–0001–8317–7765, Scopus Author ID: 56165927600 Elena V. Semenova, Cardiologist of Univercity Clinic; ORCID: 0000–0003–1375–017X Ekaterina N. Loginova, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD, Associate Professor; ORCID: 0000–0002–0601–7044, Scopus Author ID: 57190962149, Researcher ID: ABH-4244–2020 Olga Yu. Korennova, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0001–8047–5521 Alexandre A. Semyonkin, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0002–3786–9995, Researcher ID: C-7195–2014 Elena A. Lyalyukova, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Associate Professor; ORCID: 0000–0003–4878–0838 Elena V. Nadey, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD; ORCID: 0000–0003–0440–7118 Inna V. Druk, Head of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Associate Professor; ORCID: 0000–0001–8317–7765, Scopus Author ID: 56165927600 Elena V. Semenova, Cardiologist of Univercity Clinic; ORCID: 0000–0003–1375–017X Ekaterina N. Loginova, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD, Associate Professor; ORCID: 0000–0002–0601–7044, Scopus Author ID: 57190962149, Researcher ID: ABH-4244–2020 Olga Yu. Korennova, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0001–8047–5521 Alexandre A. Semyonkin, Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Professor; ORCID: 0000–0002–3786–9995, Researcher ID: C-7195–2014 Inna V. Druk, Head of the Department of Internal Diseases and Family Medicine (Postgraduate education), MD, Associate Professor; ORCID: 0000–0001–8317–7765, Scopus Author ID: 56165927600 Professor; ORCID: 0000–0001–8317–7765, Scopus Author ID: 56165927600 Elena V. Semenova, Cardiologist of Univercity Clinic; ORCID: 0000–0003–1375–017X Ekaterina N. Loginova, Associate Professor of the Department of Internal Diseases and Family Medicine (Postgraduate education), PhD, Associate Professor; ORCID: 0000–0002–0601–7044, Scopus Author ID: 57190962149, Researcher ID: ABH-4244–2020 Olga Yu. Образ жизни неправильное питание и отсутствие физической активности являются причиной большинства смертей от рака [4, 5]. Мировые данные свиде- тельствуют о том, что аналогичный повышенный риск смертности от рака связан с  курением, Образ жизни связан с различными злокачествен- ными новообразованиями, в том числе такими как рак легких, колоректальный рак, рак предстатель- ной железы и молочной железы [3]. Такие факторы риска, как употребление табака, избыточный вес, 117 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 соблюдение диеты, богатой фруктами, овощами и цельнозерновыми продуктами, с низким со- держанием насыщенных/трансжиров, красного мяса и переработанного мяса; соблюдение диеты, богатой фруктами, овощами и цельнозерновыми продуктами, с низким со- держанием насыщенных/трансжиров, красного мяса и переработанного мяса; употреблением алкоголя, диетой с низким содер- жанием фруктов и овощей, избыточным весом, малоподвижным образом жизни, загрязнением воздуха в городах, использованием твердого то- плива и инфекциями [3]. Таким образом, влияя на перечисленные факторы, риск рака может быть значительно снижен [6–8]. соблюдение диеты, богатой фруктами, овощами и цельнозерновыми продуктами, с низким со- держанием насыщенных/трансжиров, красного мяса и переработанного мяса; употреблением алкоголя, диетой с низким содер- жанием фруктов и овощей, избыточным весом, малоподвижным образом жизни, загрязнением воздуха в городах, использованием твердого то- плива и инфекциями [3]. Таким образом, влияя на перечисленные факторы, риск рака может быть значительно снижен [6–8]. ограничение употребления алкоголя; ограничение употребления алкоголя; защита от инфекций, передающихся половым путем, включая вакцинацию против вируса па- пилломы человека (ВПЧ); путем, включая вакцинацию против вируса па- пилломы человека (ВПЧ); К общим рекомендациям изменения образа жиз- ни относят: защита от солнца и ограничение соляриев; регулярные обследования на рак молочной же- регулярные обследования на рак молочной же- лезы, шейки матки, колоректальный рак и рак легких (если применимо, на основании курения в анамнезе) [1, 7, 9]. • полное ограничение табакокурения; • физическая активность; • поддержание здорового веса; Физическая активность как для проксимального, так и для дистального рака толстой кишки [44]. В другом метаанализе повышенная физическая активность была связана с 16-процентным снижением риска развития аде- номатозных полипов толстой кишки [45]. Снижение физической активности связано с по- вышенным риском развития рака и, по мнению ученых, малоподвижный образ жизни связан с 5% случаев смерти от рака [29–32]. Однако оп- тимальная продолжительность, интенсивность и  частота физической активности, которые могут обеспечить защиту от рака, неизвестны. Физическая активность в определенные перио- ды жизни, например, в подростковом возрасте, может обеспечить дополнительную защиту от болезней, особенно от рака молочной железы [8, 33, 34]. Наибольший и достоверный защит- ный эффект физической активности выявлен в отношении риска развития колоректального рака. К категории «вероятно», отнесено влияние физической активности на риск развития рака молочной железы после менопаузы и рака эндо- метрия [33, 34]. Было предложено несколько механизмов для объяснения возможного защитного эффекта фи- зической активности, включая снижение цирку- лирующих уровней инсулина, гормонов и других факторов роста; влияние на уровень простаглан- динов; улучшение иммунной функции и измене- ние метаболизма желчных кислот [46–48]. Избыточный вес связан с повышенным риском развития 13 видов рака [49]. Использование ме- тодов снижения веса, включая бариатрическую хирургию, также приводит к снижению смертно- сти от рака. В ретроспективном когортном иссле- довании бариатрическая хирургия была связана с 60-процентным снижением смертности от рака (5,5 против 13,3 на 10 000 человеко-лет) в течение семи лет наблюдения [50]. В другом ретроспектив- ном когортном исследовании бариатрическая хи- рургия была связана с 33-процентным снижением общего риска рака, с более значительным снижени- ем на 40–55% конкретных видов рака, связанных с массой тела: рак толстой кишки, постменопау- зальный рак молочной железы, рак эндометрия и рак поджелудочной железы [51]. Большая физическая активность связана со снижением риска многих различных видов рака, но наиболее убедительные данные связаны со снижением риска рака толстой кишки и молочной железы [31, 35, 38–43]. Например, в метаанали- зе 52 исследований риск развития рака толстой кишки снизился на 24% при сравнении наиболее и наименее активных людей [35]. В последующем метаанализе, включавшем 21 исследование, были получены аналогичные данные о снижении риска Алкоголь действовать как раздражитель, вызывая увели- чение деления клеток; как переносчик канцеро- генов; как ингибитор метилирования ДНК; или в качестве прометаболита для идентифициро- ванных канцерогенов, таких как ацетальдегид [26–28]. Определено несколько механизмов, объясняю- щих канцерогенность алкоголя [26]. Его раство- ряющие свой ства могут позволить канцерогенам проникать через клеточные мембраны. Алкоголь повышает уровень эстрогена и влияет на метабо- лизм фолиевой кислоты. Алкоголь также может Табакокурение канцерогенеза; он доставляет канцерогены не- посредственно в ткани, вызывает раздражение и воспаление, нарушает естественные защитные барьеры организма [8]. Опасность табака чаще всего связана с курением сигарет, но также воз- никает с сигарами, трубками, бездымным таба- ком и воздействием окружающего (пассивного) табачного дыма [13–25]. Табаококурение является одной из наиболее важных предотвратимых причин развития рака, в мире на его долю приходится до 30% всех смертей от рака [10, 11]. Примерно половина всех курильщиков умирает от болезней, связан- ных с табаком, а взрослые курильщики теряют в среднем 13 лет жизни из-за употребления та- бака [12]. Табак действует на несколько стадий Рацион питания Объединенный анализ 14 когортных исследований (n>750 000), свидетельствует о том, что употребление более 800 г фруктов и овощей в день по сравнению с потреблением менее 200 г в день снижает риск развития рака дистального отдела толстой кишки. (относительный риск [RR] 0,74), но не для проксимального рака [8, 9, 72, 73]. Последующий метаанализ 19 когортных исследова- ний пришел к выводу, что более слабый защитный эффект, при этом большая часть снижения риска связана с гораздо более низким пороговым значени- ем (100 г/день) потребления фруктов и овощей [74]. Согласно рекомендациям Американского он- кологического общества от 2020 года по диете и физической активности для профилактики рака, в которых рекомендуется здоровое питание, опре- деляемое как употребление разнообразных овощей (темно- зеленые, красные и оранжевые, бобовые, богатые клетчаткой, и другие), фрукты и цельные зерна, а также ограничение или исключение крас- ного и обработанного мяса, подслащенных саха- ром напитков или продуктов с высокой степенью переработки и продуктов из очищенного зерна [54]. Метаанализ проспективных исследований «здо- рового» и «нездорового» режима питания выявил благоприятную связь между здоровым питанием и более низким риском некоторых видов рака [55]. В большом когортном исследовании средиземно- морской диеты (т. е. высокое потребление фруктов, овощей, орехов, бобовых, цельнозернового хлеба, рыбы и оливкового масла) увеличение соблюдения диеты на два балла (по шкале 10 баллов) было свя- зано со снижением риска развития рака на 4–12% [56, 57]. В исследовании риска рака и смертности смертность от всех причин была на 17 процентов ниже при вегетарианской диете, чем у невегета- рианцев [58]. у Доказательства более убедительны, хотя и слабы в отношении возможной защитной связи между раком предстательной железы и потреблением то- матных продуктов. Первоначальные исследования дали противоречивые результаты, а систематиче- ский обзор, проведенный Управлением по сани- тарному надзору за качеством пищевых продуктов и медикаментов США (FDA), обнаружил «очень ограниченные доказательства» в поддержку свя- зи между потреблением помидоров и снижением риска рака предстательной железы или других видов рака (яичников, желудка и поджелудочной железы) [75]. Анализ проспективной когорты из 51 529 мужчин из исследования Health Professionals Follow-up Study показал, что потребление ликопина с пищей связано с более низкой заболеваемостью раком предстательной железы и сниженным ри- ском летального рака предстательной железы [76]. Анализ опухолевых биомаркеров согласовывался с возможной ролью ингибирования опухолевого неоангиогенеза как механизма, лежащего в основе этих наблюдений [76, 77]. В другом когортном исследовании диеты с высо- ким содержанием ультрапереработанных продук- тов были связаны с более чем 10-процентным уве- личением риска всех видов рака и рака молочной железы [59]. Рацион питания развития рака. В целом не всегда было доказано, что диетические жиры, фрукты и овощи влияют на Конкретные компоненты диеты, а также общие режимы питания были изучены в связи с риском 118 обзор | review риск развития рака [52, 53]. Однако определенные режимы питания, а также потребление других питательных веществ, особенно некоторых ми- кроэлементов, могут обеспечить определенную степень защиты от ряда злокачественных новоо- бразований [54, 55]. проспективного исследования рака и питания (EPIC), когортного исследования почти 500 000 взрослых европейцев, за которым наблюдали в те- чение девяти лет, выявили лишь слабую связь меж- ду повышенным потреблением фруктов и овощей и общим риском развития рака [53]. р р р При этом, многие эпидемиологические иссле- дования, хотя и не все, указывают на слабую связь между потреблением пищи с высоким содержанием фруктов и овощей и защитой от колоректального рака [69–71]. Объединенный анализ 14 когортных исследований (n>750 000), свидетельствует о том, что употребление более 800 г фруктов и овощей в день по сравнению с потреблением менее 200 г в день снижает риск развития рака дистального отдела толстой кишки. (относительный риск [RR] 0,74), но не для проксимального рака [8, 9, 72, 73]. Последующий метаанализ 19 когортных исследова- ний пришел к выводу, что более слабый защитный эффект, при этом большая часть снижения риска связана с гораздо более низким пороговым значени- ем (100 г/день) потребления фруктов и овощей [74]. Доказательства более убедительны, хотя и слабы в отношении возможной защитной связи между раком предстательной железы и потреблением то- матных продуктов. Первоначальные исследования дали противоречивые результаты, а систематиче- ский обзор, проведенный Управлением по сани- тарному надзору за качеством пищевых продуктов и медикаментов США (FDA), обнаружил «очень ограниченные доказательства» в поддержку свя- зи между потреблением помидоров и снижением риска рака предстательной железы или других видов рака (яичников, желудка и поджелудочной железы) [75]. Анализ проспективной когорты из 51 529 мужчин из исследования Health Professionals Follow-up Study показал, что потребление ликопина с пищей связано с более низкой заболеваемостью раком предстательной железы и сниженным ри- ском летального рака предстательной железы [76]. Анализ опухолевых биомаркеров согласовывался с возможной ролью ингибирования опухолевого неоангиогенеза как механизма, лежащего в основе этих наблюдений [76, 77]. В й б При этом, многие эпидемиологические иссле- дования, хотя и не все, указывают на слабую связь между потреблением пищи с высоким содержанием фруктов и овощей и защитой от колоректального рака [69–71]. Рацион питания К ультраобработанным продуктам от- носились упакованный хлеб массового производ- ства, упакованные закуски, газированные напитки, восстановленные мясные продукты с нитритными консервантами, супы быстрого приготовления, замороженные блюда и другие [60]. Несмотря на то, что была обнаружена связь меж- ду режимом питания и заболеваемостью раком, ни одно из исследований не следует рассматривать как поддерживающее использование пищевых добавок для профилактики рака. Некоторые из компонентов пищевых добавок могут быть токсич- ными (флавоноиды могут вызывать печеночную недостаточность), а прием некоторых соединений (например, витамина Е) связан с повышением за- болеваемости раком [55]. В ряде исследований было показано, что упо- требление соевых продуктов может снизить риск развития рака легких и груди у некурящих жен- щин, особенно при агрессивных опухолях [78, 79]. Продемонстирован также защитный эффект, связанный с потреблением молока, флавоноидов, кальция и витамина D, снижающими риск разви- тия рака молочной железы и других локализаций [80–116]. Пищевые жиры широко изучались как возмож- ный фактор, объясняющий различия в показателях заболеваемости раком в разных странах; однако данные в поддержку этой теории ограничены [59]. Пищевые жиры широко изучались как возмож- ный фактор, объясняющий различия в показателях заболеваемости раком в разных странах; однако данные в поддержку этой теории ограничены [59]. Потребление красного и переработанного мяса связано с повышенным риском развития колорек- тального рака и прогрессирующего рака предста- тельной железы [61–63]. Международное агент- ство по изучению рака (IARC) классифицирует переработанное мясо как канцероген группы 1 для человека [64]. Потребление пищевых волокон связано со сни- жением заболеваемости колоректальным раком [90]. Несмотря на то, что в результатах отдельных обсервационных исследований наблюдались не- которые расхождения, метаанализ «доза-эффект» в рамках Cancer Research Fund Continuous Update Project выявлено снижение риска колоректального рака с увеличением потребления клетчатки. Этот анализ, включающий 15 исследований с общим чис- лом случаев 14 876, показал снижение риска забо- левания на 9% при употреблении каждых 10 г/день продуктов, содержащих клетчатку. Аналогичные Потребление красного и переработанного мяса связано с повышенным риском развития колорек- тального рака и прогрессирующего рака предста- тельной железы [61–63]. Международное агент- ство по изучению рака (IARC) классифицирует переработанное мясо как канцероген группы 1 для человека [64]. Доказательства того, что большее потребление фруктов и овощей снижает риск развития рака, противоречивы [65–69]. Данные европейского 119 experimental & clinical gastroenterology | № 205 (9) 2022 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 обратные ассоциации были обнаружены в популя- циях Северной Америки и Европы [91]. рандомизированное исследование показало увели- чение риска развития рака у женщин, получавших омега-3 жирные кислоты, но не у мужчин [93]. Снижение веса в период менопаузы В немногочисленных исследованиях был показано положительное влияние достижения оптимальной массы тела у женщин в постменопаузе на снижение риска развития рака молочной железы и рака эндо- метрия [128–130]. Инсоляция Данные исследований свидетельствуют о риске развития новообразований при чрезмерной инсоляции. Более того, загар в солярии защищает от повреждения кожи на фоне воздействия солнца. По мнению большинства авторов, следует отговаривать молодых людей от использования оборудования для загара, и следует серьезно рассмотреть вопрос об ограничении доступа несовершеннолетних к соляриям [131–133]. Инфекции Рацион питания Жирные кислоты омега-3 и диетическая рыба. Систематический обзор проспективных исследова- ний, оценивающих влияние потребления жирных кислот омега-3 на заболеваемость раком, пришел к выводу, что нет никакой связи между жирными кислотами омега-3 и риском рака для 11 различных типов рака [92]. Десять исследований, рассмотрен- ных в этом обзоре, сообщили о значительных ре- зультатах, но отдельные исследования указывали как на повышенный, так и на сниженный риск без последовательной закономерности. Последующее Кофе и кофеин. Потребление кофе связано с бо- лее низким риском развития некоторых видов рака, включая рак эндометрия и рак печени [94]. Мета- анализ World Cancer Research Fund Continuous Update Project выявил значительное снижение ри- ска рака эндометрия на 7% и снижение риска рака печени на 1,4% на каждую чашку кофе в день. Для рака эндометрия результаты были аналогичными для кофе без кофеина, риск значительно ниже на 8% на один чашка/день [95]. Витамины и микроэлементы Многочисленные обсервационные исследования показали, что более высокое потребление кальция (как с пищей, так и в виде пищевых добавок) свя- зано со снижением риска развития колоректаль- ного рака [105, 106]. В объединенной когорте из исследований Health Professionals Follow-up Study и Nurse’s Health Study риск развития дистального, но не проксимального рака толстой кишки был снижен у пациентов, которые потребляли более 1250 мг/день элементарного кальция по сравнению с 500 мг/день [107]. Добавки кальция также, по-ви- димому, предотвращают рецидив колоректальных аденом. Мета-анализы показали снижение риска рецидива аденомы на 12–13 процентов пациентов, рандомизированных для приема добавок каль- ция [108–109]. Однако защитный эффект добавок кальция не был продемонстрирован Инициативой женского здоровья, которая не обнаружила зна- чительного снижения заболеваемости или стадии колоректального рака в группе, получавшей 500 мг кальция и 200 международных единиц витамина D дважды в день по сравнению с плацебо [110]. В целом группа экспертов Всемирного фонда иссле- дований рака пришла к выводу, что «прием добавок кальция, вероятно, защищает от колоректального рака» [1]. Многочисленные наблюдательные и проспектив- ные исследования, а также систематические обзоры приема дополнительных витаминов и минералов для профилактики рака преимущественно показа- ли отрицательные результаты [96–100], за редким исключением [101,102]. Тем не менее, витамины и минералы могут быть назначены для пациентов, рацион которых может быть оценен как недоста- точный по тем или иным причинам. Всемирный фонд исследования рака и Американский инсти- тут исследования рака не рекомендуют исполь- зовать добавки для профилактики рака [1, 2]. Рекомендуется придерживаться здоровой диеты для удовлетворения потребностей в питании [54]. Витамин D. Взаимосвязи между потреблением витамина D или уровнями 25(OH)D в сыворотке крови и риском развития рака в исследованиях также противоречивы, несмотря на обсуждаемость данной темы [103–106]. Исследования различаются в зависимости от участников (пол, исходные уров- ни в сыворотке крови), оцениваемые типы рака и дозы витамина D. Кальций. Повышенное потребление кальция связано со снижением риска колоректального рака, но может быть связано с повышенным риском рака предстательной железы. Может существовать ми- нимальный уровень потребления кальция, около 700 мг/день, который обеспечивает защиту от ко- лоректального рака без значительного увеличения риска рака предстательной железы [107–116]. Данные в отношении профилактической пользы витаминов Е, А, селена, фолиевой кислоты, железа противоречивы и недостаточны для формирова- ния выводов и рекомендаций [111–127]. Инфекции Выделяют несколько вирусов, прямо ассо- циированных с определенным видом рака: ви- рус папилломы человека (ВПЧ) с раком шейки матки и другими видами аногенитального рака, а также плоскоклеточным раком головы и шеи, вирус гепатита В (ВГВ) и вирус гепатита С (ВГС) с гепатоцеллюлярной карциномой, Т-клеточный Подсчитано, что 13 процентов всех новых случа- ев рака во всем мире связаны с инфекциями [9]. Вирусы имеют несколько механизмов повышения риска развития рака, влияя на клеточную транс- формацию, нарушая контроль клеточного цикла, увеличивая скорость оборота клеток и подавления иммунитета [10]. 120 обзор | review лимфотропный вирус человека типа 1 (HTLV-1) при Т-клеточном лейкозе взрослых, вирус иммун- нодефицита человека (ВИЧ) с саркомой Капоши, а также с неходжкинской лимфомой и с множе- ственными злокачественными новообразования- ми, определяющими синдром неприобретенного иммунодефицита (СПИД), вирус герпеса человека 8 (HHV-8) с саркомой Капоши и первичной выпот- ной лимфомой, вирус Эпштейна- Барр с лимфомой Беркитта [134–144]. регулирование нанесения татуировок, активное информирование о барьерных методах контрацеп- ции и пропаганда безопасного секса, постоянный скрининг доноров крови, органов и спермы, а так- же разработка продуктов искусственной крови. Кроме того, существуют рекомендации по вак- цинации. Вакцинация против ВПЧ рекомендуется всем детям, а также молодым женщинам и юно- шам, которые не были вакцинированы в детстве. Вакцинация против ВГВ внесена в национальный календарь прививок для детей и рекомендована для взрослых с высоким риском инфицирования ВГВ для уменьшения вирусной нагрузки и после- дующей гепатоцеллюлярной карциномы. Скрининг на ВГС рекомендуется всем взрослым в возрасте от 18 до 79 лет [143]. Кроме вирусов, изучены связи с некоторыми бактериями, так, коллонизация Helicobacter pylori связана со злокачественными новообразованиями желудочно- кишечного тракта, включая рак желуд- ка [140] и лимфомы лимфоидной ткани, ассоци- ированной со слизистой оболочкой. Печеночные двуустки связаны с холангиокарциномой и гепа- тоцеллюлярной карциномой [141]. Чрезмерное употребление алкоголя может играть роль в развитии рака у пациентов с хроническими инфекциями ВГВ и ВГС, и его следует избегать. Предварительные данные свидетельствуют о том, что противовирусная терапия может снизить риск развития рака у пациентов с хроническими инфек- циями ВГС за счет снижения уровня РНК ВГС, но долгосрочный эффект противовирусной терапии на риск развития рака неизвестен [145–147]. Большинство инфекций, связанных с раком, вызваны вирусами, которые распространяются при контакте с зараженной кровью или биологиче- скими жидкостями [142, 143]. Для предотвращения такой передачи введено использование стериль- ных одноразовых игл для одного пациента в ме- дицинских учреждениях, программы обмена игл, Роль лекарственных средств в профилактике рака Аспирин и другие нестероидные противовоспал В исследованиях показано, что регулярное приме- нение аспирина и других нестероидных противо- воспалительных препаратов (НПВП) снижает риск аденоматозных полипов и колоректального рака на 20–40% [165–168]. Рандомизированное контро- лируемое исследование показало, что у пациентов с высоким риском синдрома Линча прием 600 мг аспирина в день снижает риск колоректального рака на 60 процентов [169]. Однако оптимальная доза аспирина для пациентов с более низким ри- ском не установлена [170]. Аспирин и другие нестероидные противовоспалительные препараты может влиять на внутриклеточную передачу сиг- налов посредством ингибирования активности фосфолипазы. Касательно эффективности аспи- рина в профилактике других видов рака данные ограничены [171]. может влиять на внутриклеточную передачу сиг- налов посредством ингибирования активности фосфолипазы. Касательно эффективности аспи- рина в профилактике других видов рака данные ограничены [171]. При подборе рекомендаций и анализе получае- мых пациентом препаратов необходимо сопостав- лять потенциальные преимущества долгосрочной терапии с потенциальными побочными эффектами (гастродуоденальная токсичность и повышенный сердечно- сосудистый риск при использовании неа- спириновых НПВП), учитывая наличие скрининга колоректального рака [165–171]. Было предложено несколько теорий, почему аспирин и другие НПВП эффективны в снижении риска колоректального рака и, возможно, эффек- тивны при других видах рака. Согласно одной из них НПВП могут вызывать остановку клеточного цикла или апоптоз аномальных клеток. Согласно другой – снижение риска может быть связано с не- обратимым ингибированием циклооксигеназы 2 (ЦОГ-2). Ингибирование этого фермента снижает синтез простагландинов, что может замедлять рост опухоли. Третья теория говорит о том, что аспирин Необходимы дальнейшие исследования, чтобы понять, нужны ли высокие дозы аспирина, а также определение видимых преимуществ и противо- показаний для групп пациентов высокого риска возникновения рака ЖКТ, таких как люди с коло- ректальным раком или аденомой в анамнезе. При составлении рекомендаций относительно аспирина также необходимо учитывать его пользу для про- филактики ишемической болезни сердца. Нарушения углеводного обмена мероприятий (повышение физической актив- ности и диета с высоким содержанием фруктов и овощей и низким содержанием красного мяса и продуктов, подвергшихся интенсивной обра- ботке) [148–164]. Большинство исследователей свидетельствуют о высоком риске развития новообразований у па- циентов с нарушением углеводного обмена, гипе- ринсулинемией и сахарным диабетом, что диктует необходимость проведения профилактических Литература  References the EPIC study. Am J Clin Nutr. 2012;96(1):150–163. doi: 10.3945/ajcn.111.031674 1. Colditz G. A., Wolin K. Y., Gehlert S. Applying what we know to accelerate cancer prevention. Sci Transl Med. 2012;4(127):127rv4. doi: 10.1126/scitranslmed.3003218 1. Colditz G. A., Wolin K. Y., Gehlert S. Applying what we know to accelerate cancer prevention. Sci Transl Med. 2012;4(127):127rv4. doi: 10.1126/scitranslmed.3003218 1. Colditz G. A., Wolin K. Y., Gehlert S. Applying what we know to accelerate cancer prevention. Sci Transl Med. 2012;4(127):127rv4. doi: 10.1126/scitranslmed.3003218 the EPIC study. Am J Clin Nutr. 2012;96(1):150–163. doi: 10.3945/ajcn.111.031674 7. Ford E. S., Bergmann M. M., Kröger J., et al. Healthy living is the best revenge: fi ndings from the European Prospective Investigation Into Cancer and Nutrition- Potsdam study. Arch Intern Med. 2009;169(15):1355– 1362. doi: 10.1001/archinternmed.2009.237 2. Emmons K. M., Colditz G. A. Realizing the Potential of Cancer Prevention – Th e Role of Implementation Science. N Engl J Med. 2017;376(10):986–990. doi: 10.1056/NEJMsb1609101 3. Bray F., Ferlay J., Soerjomataram I., et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424. doi: 10.3322/ caac.21492 8. Li Y., Schoufour J., Wang D. D., et al. Healthy lifestyle and life expectancy free of cancer, cardiovascular dis- ease, and type 2 diabetes: prospective cohort study. BMJ. 2020;368: l6669. doi: 10.1136/bmj.l6669 9. de Martel C., Georges D., Bray F., et al. Global burden of cancer attributable to infections in 2018: a worldwide in- cidence analysis. Lancet Glob Health. 2020;8(2):180–190. doi: 10.1016/S2214–109X(19)30488–7 4. Colditz G. A., Atwood K. A., Emmons K., et al. Harvard report on cancer prevention volume 4: Harvard Cancer Risk Index. Risk Index Working Group, Harvard Center for Cancer Prevention. Cancer Causes Control. 2000;11(6):477–488. doi: 10.1023/a:1008984432272 10. Brawley O. W. Avoidable cancer deaths globally. CA Cancer J Clin. 2011;61(2):67–68. doi: 10.3322/caac.20108 5. Danaei G., Vander Hoorn S., Lopez A. D., et al. Comparative Risk Assessment collaborating group (Cancers). Causes of cancer in the world: comparative risk assessment of nine behavioural and environmen- tal risk factors. Lancet. 2005;366(9499):1784–1793. doi: 10.1016/S0140–6736(05)67725–2 11. Henley S. J., Th omas C. C., Sharapova S. R., et al. Vital Signs: Disparities in Tobacco- Related Cancer Incidence and Mortality – United States, 2004–2013. MMWR Morb Mortal Wkly Rep. 2016;65(44):1212–1218. doi: 10.15585/ mmwr.mm6544a3 12. Centers for Disease Control and Prevention (CDC). State-specifi c smoking- attributable mortality and years of potential life lost – United States, 2000–2004. Варфарин заболеваемость всеми видами рака, диагности- рованными по крайней мере через два года после начала приема варфарина [182]. Исследования показывают смешанные результаты в отношении связи между использованием вар- фарина и риском развития рака [182, 183]. В попу- ляционном когортном исследовании, в котором приняли участие более одного миллиона человек в Норвегии, у принимавших варфарин (в тече- ние не менее шести месяцев) была более низкая Известно, что варфарин блокирует онкогенез у мышей, и постулируется, что его биохимические действия усиливают противоопухолевый иммун- ный надзор [183]. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 раком поджелудочной железы и печени, а так- же незначительное снижение заболеваемости раком молочной железы, толстой кишки и про- статы. В двух последующих метаанализах у па- циентов с диабетом была выявлена связь между применением метформина и снижением риска колоректального рака и снижением риска коло- ректального рака, рака печени и легких [175, 176]. Следует заметить, что эффект имел тенденцию к дозозависимости. раком поджелудочной железы и печени, а так- же незначительное снижение заболеваемости раком молочной железы, толстой кишки и про- статы. В двух последующих метаанализах у па- циентов с диабетом была выявлена связь между применением метформина и снижением риска колоректального рака и снижением риска коло- ректального рака, рака печени и легких [175, 176]. Следует заметить, что эффект имел тенденцию к дозозависимости. Эта гипотеза подтверждается данными проспек- тивного голландского исследовании 1353 пациен- тов с диабетом 2 типа, наблюдавшихся в течение 9,6 лет, среди которых смертность от рака была снижена у тех, кто принимал метформин [173]. В регистре случай- контроль в Великобритании применение метформина ассоциировалось со зна- чительным снижением риска рака поджелудоч- ной железы у женщин, но не влияло на риск рака у мужчин [177]. В обсервационном исследовании 4085 паци- ентов в Великобритании, которые принимали метформин для лечения диабета 2 типа с 1994 по 2003 год, заболеваемость раком была на 40 про- центов ниже, чем среди пациентов с диабетом, которые не принимали метформин (7,3 процента против 11,6 процента, отношение рисков) [172]. Среди предполагаемых механизмов эффектив- ности метформина против рака – ингибирование роста раковых клеток, подавление сверхэкспрессии HER2 и ингибирование mTOR [178–180]. Однако перечисленные исследования имею ограничение в виде возможно повышенного риска смерти из-за использования других схем лечения диабета. Статины исследованиях; однако такие данные могут быть искажены факторами риска и сопутствующи- ми заболеваниями [171]. Напротив, метаанализ 27 рандомизированных исследований статинов (в которых профилактика рака не была основным результатом) не показал, что статины снижают заболеваемость каким-либо раком [181]. Исследования статинов показывают смешанные результаты; нет убедительных доказательств того, что статины влияют на риск развития рака. Связь между приемом статинов и снижением риска раз- личных видов рака (особенно рака желудочно- кишечного тракта и молочной железы) была предложена в многочисленных обсервационных Метформин вано 4042 случая рака и 529 смертей у пациен- тов с диабетом, как заболеваемость раком, так и смертность от рака среди пациентов, получа- ющих метформин снизилась на 30 процентов [172–174]. Наблюдалось снижение заболеваемости Метформин связан со снижением заболеваемости несколькими типами рака у пациентов с диабе- том 2 типа. Согласно систематическому обзору и метаанализу, включавшему 11 независимых исследований, в которых было зарегистриро- 121 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Литература  References Recreational physical activity and risk of prostate cancer in a large cohort of U.S. men. Cancer Epidemiol Biomarkers Prev. 2005; 14:275–279. 19. Macacu A., Autier P., Boniol M., Boyle P. Active and pas- sive smoking and risk of breast cancer: a meta-analysis. Breast Cancer Res Treat. 2015; 154:213–224. doi: 10.1007/ s10549–015–3628–4 38. Colditz G. A., Cannuscio C. C., Frazier A. L. Physical activity and reduced risk of colon cancer: implications for prevention. Cancer Causes Control. 1997; 8:649–667. 20. National Center for Chronic Disease Prevention and Health Promotion (US) Offi ce on Smoking and Health. The Health Consequences of Smoking-50 Years of Progress: A Report of the Surgeon General. Atlanta (GA): Centers for Disease Control and Prevention (US); 2014. 39. Dallal C. M., Sullivan- Halley J., Ross R. K., et al. Long- term recreational physical activity and risk of inva- sive and in situ breast cancer: the California teachers study. Arch Intern Med. 2007; 167:408–415. doi: 10.1001/ archinte.167.4.408 21. Machii R., Saika K. Mortality attributable to tobacco by region based on the WHO Global Report. Jpn J Clin Oncol. 2012 May;42(5):464–465. doi: 10.1093/jjco/hys063 40. Chao A., Connell C. J., Jacobs E. J., et al. Amount, type, and timing of recreational physical activity in relation to colon and rectal cancer in older adults: the Cancer Prevention Study II Nutrition Cohort. Cancer Epidemiol Biomarkers Prev. 2004; 13:2187–2195. 22. Samet J. M. Th e 1990 Report of the Surgeon General: Th e Health Benefi ts of Smoking Cessation. Am Rev Respir Dis. 1990; 142:993–994. doi: 10.1164/ajrccm/142.5.993 23. Vineis P., Alavanja M., Buffl er P., et al. Tobacco and cancer: recent epidemiological evidence. J Natl Cancer Inst. 2004; 96:99–106. doi: 10.1093/jnci/djh014 41. Martínez M. E., Giovannucci E., Spiegelman D., et al. Leisure-time physical activity, body size, and co- lon cancer in women. Nurses’ Health Study Research Group. J Natl Cancer Inst. 1997; 89:948–955. doi: 10.1093/ jnci/89.13.948 24. Haughton B., Stang J. Population risk factors and trends in health care and public policy. J Acad Nutr Diet. 2012;112(3 Suppl): S35–46. doi: 10.1016/j.jand.2011.12.011 25. A clinical practice guideline for treating tobacco use and dependence: A US Public Health Service report. Th e Tobacco Use and Dependence Clinical Practice Guideline Panel, Staff , and Consortium Representatives. JAMA. 2000; 283:3244–3254. doi:10.1001/jama.283.24.3244 42. Kehm R. D., Genkinger J. M., MacInnis R.J. et al. Литература  References MMWR Morb Mortal Wkly Rep. 2009;58(2):29–33. 6. Romaguera D., Vergnaud A. C., Peeters P. H., et al. Is con- cordance with World Cancer Research Fund/American Institute for Cancer Research guidelines for cancer pre- vention related to subsequent risk of cancer? Results from 122 обзор | review 13. Th un M. J., Heath C. W. Jr. Changes in mortality from smoking in two American Cancer Society prospective studies since 1959. Prev Med. 1997;26(4):422–426. doi: 10.1006/pmed.1997.0182 31. Inoue M., Yamamoto S., Kurahashi N., et al. Daily total physical activity level and total cancer risk in men and women: results from a large- scale population- based co- hort study in Japan. Am J Epidemiol. 2008; 168:391–403. doi: 10.1093/aje/kwn146 14. Sasco A. J., Secretan M. B., Straif K. Tobacco smoking and cancer: a brief review of recent epidemiological ev- idence. Lung Cancer. 2004; 45 Suppl 2: S3. doi: 10.1016/j. lungcan.2004.07.998 32. Harvard Report on Cancer Prevention. Volume 1: Causes of human cancer. Cancer Causes Control. 1996; 7 Suppl 1: S3. doi: 10.1007/BF02352719 33. Maruti S. S., Willett W. C., Feskanich D., et al. A pro- spective study of age-specific physical activity and premenopausal breast cancer. J Natl Cancer Inst. 2008; 100:728–737. doi: 10.1093/jnci/djn135 15. Brownson R. C., Novotny T. E., Perry M. C. Cigarette smoking and adult leukemia. A meta-analysis. Arch Intern Med. 1993; 153:469–475. 16. Murphy A. B., Akereyeni F., Nyame Y. A., et al. Smoking and prostate cancer in a multi- ethnic cohort. Prostate. 2013; 73:1518–1528. doi: 10.1002/pros.22699 34. Bernstein L. Exercise and breast cancer prevention. Curr Oncol Rep. 2009; 11:490–496. doi: 10.1007/s11912–009– 0066–7 17. Kenfi eld S. A., Stampfer M. J., Chan J. M., Giovannucci E. Smoking and prostate cancer survival and recurrence. JAMA. 2011; 305:2548–2555. doi: 10.1001/jama.2011.879 35. Wolin K. Y., Yan Y., Colditz G. A., Lee I. M. Physical ac- tivity and colon cancer prevention: a meta-analysis. Br J Cancer. 2009; 100:611–616. doi: 10.1038/sj.bjc.6604917 18. Islami F., Moreira D. M., Boff etta P., Freedland S. J. A sys- tematic review and meta-analysis of tobacco use and prostate cancer mortality and incidence in prospec- tive cohort studies. Eur Urol. 2014; 66:1054–1064. doi: 10.1001/jama.2011.879 36. Antonelli J. A., Jones L. W., Bañez L. L., et al. Exercise and prostate cancer risk in a cohort of veterans undergoing prostate needle biopsy. J Urol. 2009; 182:2226–2231. doi: 10.1016/j.juro.2009.07.028 37. Patel A. V., Rodriguez C., Jacobs E. J., et al. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 47. Giovannucci E., Ascherio A., Rimm E. B., et al. Physical activity, obesity, and risk for colon cancer and adeno- ma in men. Ann Intern Med. 1995; 122:327–334. doi: 10.7326/0003–4819–122–5–199503010–00002 64. Bouvard V., Loomis D., Guyton K. Z., et al. Carcino- genicity of consumption of red and processed meat. Lancet Oncol. 2015; 16:1599–1600. doi: 10.1016/S1470– 2045(15)00444–1 48. Martínez M. E., Heddens D., Earnest D. L., et al. Physical activity, body mass index, and prostaglandin E2 levels in rectal mucosa. J Natl Cancer Inst. 1999; 91:950–953. doi: 10.1093/jnci/91.11.950 65. Benetou V., Orfanos P., Lagiou P., et al. Vegetables and fruits in relation to cancer risk: evidence from the Greek EPIC cohort study. Cancer Epidemiol Biomarkers Prev. 2008; 17:387–392. doi: 10.1016/S1470–2045(15)00444–1 49. Lauby- Secretan B., Scoccianti C., Loomis D., et al. Body Fatness and Cancer – Viewpoint of the IARC Working Group. N Engl J Med. 2016; 375:794. doi: 10.1093/ jnci/91.11.950 66. Takachi R., Inoue M., Ishihara J., et al. Fruit and vege- table intake and risk of total cancer and cardiovascular disease: Japan Public Health Center- Based Prospective Study. Am J Epidemiol. 2008; 167:59–70. doi: 10.1093/ aje/kwm263 50. Genser L., Barrat C. Long term outcomes aft er bariatric and metabolic surgery. Presse Med. 2018;47(5):471–479. French. doi: 10.1016/j.lpm.2018.01.003 67. Hung H. C., Joshipura K. J., Jiang R., et al. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst. 2004; 96:1577–1584. doi: 10.1093/jnci/djh296 51. Schauer D. P., Feigelson H. S., Koebnick C., et al. Bariatric Surgery and the Risk of Cancer in a Large Multisite Cohort. Ann Surg. 2019; 269:95–101. doi: 10.1097/ SLA.0000000000002525 68. George S. M., Park Y., Leitzmann M. F., et al. Fruit and vegetable intake and risk of cancer: a prospective cohort study. Am J Clin Nutr. 2009; 89:347–353. doi: 10.3945/ ajcn.2008.26722 52. Aune D., Giovannucci E., Boff etta P., et al. Fruit and veg- etable intake and the risk of cardiovascular disease, total cancer and all-cause mortality-a systematic review and dose-response meta-analysis of prospective studies. Int J Epidemiol. 2017;46(3):1029–1056. doi: 10.1093/ije/dyw319 69. Feskanich D., Ziegler R. G., Michaud D. S., et al. Prospective study of fruit and vegetable consump- tion and risk of lung cancer among men and women. J Natl Cancer Inst. 2000; 92:1812–1823. doi: 10.1093/ jnci/92.22.1812 53. Boff etta P., Couto E., Wichmann J., et al. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Fruit and veg- etable intake and overall cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). J Natl Cancer Inst. 2010; 102:529–537. doi: 10.1093/jnci/djq072 70. Smith- Warner S.A., Spiegelman D., Yaun S. S., et al. Intake of fruits and vegetables and risk of breast can- cer: a pooled analysis of cohort studies. JAMA. 2001; 285:769–776. doi: 10.1001/jama.285.6.769 71. Michels K. B., Edward Giovannucci, Joshipura K. J., et al. Prospective study of fruit and vegetable consumption and incidence of colon and rectal cancers. J Natl Cancer Inst. 2000; 92:1740–1752. doi: 10.1093/jnci/92.21.1740 54. Rock C. L., Th omson C., Gansler T., et al. American Cancer Society guideline for diet and physical activity for cancer prevention. CA Cancer J Clin. 2020; 70:245–271. doi: 10.3322/caac.21591 55. Grosso G., Bella F., Godos J., et al. Possible role of diet in cancer: systematic review and multiple meta-analyses of dietary patterns, lifestyle factors, and cancer risk. Nutr Rev. 2017; 75:405–419. doi: 10.1093/nutrit/nux012 72. Koushik A., Hunter D. J., Spiegelman D., et al. Fruits, vegetables, and colon cancer risk in a pooled analysis of 14 cohort studies. J Natl Cancer Inst. 2007; 99:1471–1483. doi: 10.1093/jnci/djm155 56. Benetou  V., Trichopoulou A., Orfanos P., et al. Conformity to traditional Mediterranean diet and can- cer incidence: the Greek EPIC cohort. Br J Cancer. 2008; 99:191–195. doi: 10.1038/sj.bjc.6604418 73. Lee J. E., Chan A. T. Fruit, vegetables, and folate: cultivat- ing the evidence for cancer prevention. Gastroenterology. 2011; 141:16–20. doi: 10.1053/j.gastro.2011.05.020 74. Aune D., Chan D. S., Lau R., et al. Dietary fi bre, whole grains, and risk of colorectal cancer: systematic review and dose-response meta-analysis of prospective studies. BMJ. 2011; 343: d6617. doi:10.1136/bmj.d6617 57. Couto E., Boff etta P., Lagiou P., et al. Mediterranean dietary pattern and cancer risk in the EPIC cohort. Br J Cancer. 2011; 104:1493–1499. doi: 10.1038/bjc.2011.106 75. Kavanaugh C. J., Trumbo P. R., Ellwood K. C. Th e U. S. Food and Drug Administration’s evidence- based re- view for qualifi ed health claims: tomatoes, lycopene, and cancer. J Natl Cancer Inst. 2007; 99:1074–1085. doi: 10.1093/jnci/djm037 58. Le L. T., Sabaté J. Beyond meatless, the health eff ects of vegan diets: fi ndings from the Adventist cohorts. Nutrients. 2014; 6:2131–2147. doi: 10.3390/nu6062131 59. Brennan S. F., Cantwell M. M., Cardwell C. R., et al. Dietary patterns and breast cancer risk: a systematic review and meta-analysis. Am J Clin Nutr. 2010; 91:1294– 1302. doi: 10.3945/ajcn.2009.28796 76. Литература  References Recreational Physical Activity Is Associated with Reduced Breast Cancer Risk in Adult Women at High Risk for Breast Cancer: A Cohort Study of Women Selected for Familial and Genetic Risk. Cancer Res. 2020;80(1):116–125. doi: 10.1158/0008–5472.CAN-19– 1847 26. Seitz H. K., Stickel F. Molecular mechanisms of alcohol- mediated carcinogenesis. Nat Rev Cancer. 2007; 7:599– 612. doi: 10.1038/nrc2191 43. Simon T. G., Kim M. N., Luo X., et al. Physical activity compared to adiposity and risk of liver- related mor- tality: Results from two prospective, nationwide co- horts. J Hepatol. 2020;72(6):1062–1069. doi: 10.1016/j. jhep.2019.12.022 27. Theruvathu J. A., Jaruga P., Nath R. G., et al. Poly- ami nes stimulate the formation of mutagenic 1, N2-propanodeoxyguanosine adducts from acetaldehyde. Nucleic Acids Res. 2005; 33:3513–3520. doi: 10.1093/nar/ gki661 44. Boyle T., Keegel T., Bull F., et al. Physical activity and risks of proximal and distal colon cancers: a systemat- ic review and meta-analysis. J Natl Cancer Inst. 2012; 104:1548–1561. doi: 10.1093/jnci/djs354 28. Boff etta P., Hashibe M. Alcohol and cancer. Lancet Oncol. 2006; 7:149–156. doi: 10.1016/S1470–2045(06)70577–0 29. Matthews C. E., Moore S. C., Arem H., et al. Amount and Intensity of Leisure- Time Physical Activity and Lower Cancer Risk. J Clin Oncol. 2020; 38:686–697. doi: 10.1200/JCO.19.02407 45. Wolin K. Y., Yan Y., Colditz G. A. Physical activity and risk of colon adenoma: a meta-analysis. Br J Cancer. 2011; 104:882–885. doi: 10.1038/sj.bjc.6606045 46. McKeown- Eyssen G. Epidemiology of colorectal cancer revisited: are serum triglycerides and/or plasma glucose associated with risk? Cancer Epidemiol Biomarkers Prev. 1994; 3:687–695. 30. Moore S. C., Lee I. M., Weiderpass E., et al. Association of Leisure- Time Physical Activity With Risk of 26 Types of Cancer in 1.44 Million Adults. JAMA Intern Med. 2016; 176:816–825. doi: 10.1001/jamainternmed.2016.1548 123 experimental & clinical gastroenterology | № 205 (9) 2022 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Milk, milk products and lactose intake and ovarian cancer risk: a meta-analy- sis of epidemiological studies. Int J Cancer. 2006; 118:431– 441. doi: 10.1002/ijc.21305 89. Mommers M., Schouten L. J., Goldbohm R. A., van den Brandt P. A. Dairy consumption and ovarian cancer risk in the Netherlands Cohort Study on Diet and Cancer. Br J Cancer. 2006; 94:165–170. doi: 10.1038/sj.bjc.6602890 104. Freedman D. M., Looker A. C., Abnet C. C., et al. Serum 25-hydroxyvitamin D and cancer mortality in the NHANES III study (1988–2006). Cancer Res. 2010; 70: 8587–97. doi: 10.1158/0008–5472.CAN-10–1420 90. World Cancer Research Fund. Colorectal cancer: How diet, nutrition and physical activity aff ect colorectal (bowel) cancer risk. Available at: https://www.wcrf.org/ dietandcancer/colorectal- cancer Accessed: 31.03.2022. 105. Pietinen P., Malila N., Virtanen M., et al. Diet and risk of colorectal cancer in a cohort of Finnish men. Cancer Causes Control. 1999; 10: 387–96. doi: 10.1023/a:1008962219408 91. Norat T., Vieira A. R., Abar L., et al. World Cancer Research Fund international systematic literature re- view: Th e associations between food, nutrition and phys- ical activity and the risk of colorectal cancer. Available at: https://www.wcrf.org/sites/default/fi les/colorectal- cancer-slr.pdf Accessed: 31.03.2022. 106. Garland C., Shekelle R. B., Barrett- Connor E., et al. Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men. Lancet. 1985; 1: 307–9. doi: 10.1016/s0140–6736(85)91082–7 107. Wu K., Willett W. C., Fuchs C. S., et al. Calcium intake and risk of colon cancer in women and men. J Natl Cancer Inst. 2002; 94: 437–46. doi: 10.1093/jnci/94.6.437 92. MacLean C.H., Newberry S. J., Mojica W. A., et al. Eff ects of omega-3 fatty acids on cancer risk: a systematic review. JAMA. 2006; 295:403–415. doi: 10.1001/jama.295.4.403 108. Bonovas S., Fiorino G., Lytras T., et al. Calcium supple- mentation for the prevention of colorectal adenomas: A systematic review and meta-analysis of randomized controlled trials. World J Gastroenterol. 2016; 22: 4594– 603. doi: 10.3748/wjg.v22.i18.4594 93. Andreeva V. A., Touvier M., Kesse- Guyot E., et al. B vita- min and/or ω-3 fatty acid supplementation and cancer: ancillary fi ndings from the supplementation with folate, vitamins B6 and B12, and/or omega-3 fatty acids (SU. FOL.OM3) randomized trial. Arch Intern Med. 2012; 172:540–547. doi: 10.1001/archinternmed.2011.1450 109. Veettil S. K., Ching S. M., Lim K. G., et al. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Zu K., Mucci L., Rosner B. A., et al. Dietary lycopene, angiogenesis, and prostate cancer: a prospective study in the prostate- specifi c antigen era. J Natl Cancer Inst. 2014; 106: djt430. doi: 10.1093/jnci/djt430 60. Fiolet T., Srour B., Sellem L., et al. Consumption of ultra- processed foods and cancer risk: results from NutriNet- Santé prospective cohort. BMJ. 2018; 360: k322. doi: 10.1136/bmj.k322 77. Jung S., Spiegelman D., Baglietto L., et al. Fruit and vegetable intake and risk of breast cancer by hormone receptor status. J Natl Cancer Inst. 2013; 105:219–236. doi: 10.1093/jnci/djs635 61. Chan D. S., Lau R., Aune D., et al. Red and processed meat and colorectal cancer incidence: meta-analysis of prospective studies. PLoS One. 2011; 6: e20456. doi: 10.1371/journal.pone.0020456 78. Wu A. H., Yu M. C., Tseng C. C., Pike M. C. Epidemiology of soy exposures and breast cancer risk. Br J Cancer. 2008; 98:9–14. doi: 10.1038/sj.bjc.6604145 62. Wu K., Spiegelman D., Hou T., et al. Associations be- tween unprocessed red and processed meat, poultry, seafood and egg intake and the risk of prostate cancer: A pooled analysis of 15 prospective cohort studies. Int J Cancer. 2016; 138:2368–2382. doi: 10.1002/ijc.29973 79. Yang G., Shu X. O., Chow W. H., et al. Soy food intake and risk of lung cancer: evidence from the Shanghai Women’s Health Study and a meta-analysis. Am J Epidemiol. 2012; 176:846–855. doi: 10.1093/aje/kws168 80. Fink B. N., Steck S. E., Wolff M. S., et al. Dietary fl avonoid intake and breast cancer risk among women on Long 63. Wolk A. Potential health hazards of eating red meat. J Intern Med. 2017; 281:106–122. doi: 10.1111/joim.12543 124 обзор | review Island. Am J Epidemiol. 2007; 165:514–523. doi: 10.1093/ aje/kwk033 96. Vastag B. Nutrients for prevention: negative trials send researchers back to drawing board. J Natl Cancer Inst. 2009; 101:446–448, 451. doi: 10.1093/jnci/djp073 81. Shin M. H., Holmes M. D., Hankinson S. E., et al. Intake of dairy products, calcium, and vitamin d and risk of breast cancer. J Natl Cancer Inst. 2002; 94:1301–1311. doi: 10.1093/jnci/94.17.1301 97. Coulter I. D., Hardy M. L., Morton S. C., et al. Antioxidants vitamin C and vitamin e for the preven- tion and treatment of cancer. J Gen Intern Med. 2006; 21:735–744. doi: 10.1111/j.1525–1497.2006.00483.x 82. Knekt P., Järvinen R., Seppänen R., et al. Intake of dairy products and the risk of breast cancer. Br J Cancer. 1996; 73:687–691. doi: 10.1038/bjc.1996.119 98. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Lin J., Cook N. R., Albert C., et al. Vitamins C and E and beta carotene supplementation and cancer risk: a randomized controlled trial. J Natl Cancer Inst. 2009; 101:14–23. doi: 10.1093/jnci/djn438 83. Boyd N. F., Martin L. J., Noff el M., et al. A meta-analysis of studies of dietary fat and breast cancer risk. Br J Cancer. 1993; 68:627–636. doi: 10.1038/bjc.1993.398 99. Neuhouser M. L., Wassertheil- Smoller S., Th omson C., et al. Multivitamin use and risk of cancer and cardiovas- cular disease in the Women’s Health Initiative cohorts. Arch Intern Med. 2009; 169:294–304. doi: 10.1001/archin- ternmed.2008.540 84. Lin J., Manson J. E., Lee I. M., et al. Intakes of calci- um and vitamin D and breast cancer risk in women. Arch Intern Med. 2007; 167:1050–1059. doi: 10.1001/ archinte.167.10.1050 100. Park S. Y., Murphy S. P., Wilkens L. R., et al. Multivitamin use and the risk of mortality and cancer incidence: the multiethnic cohort study. Am J Epidemiol. 2011; 173:906–914. doi: 10.1093/aje/kwq447 85. Missmer S. A., Smith- Warner S.A., Spiegelman D., et al. Meat and dairy food consumption and breast cancer: a pooled analysis of cohort studies. Int J Epidemiol. 2002; 31:78–85. doi: 10.1093/ije/31.1.78 101. Gaziano J. M., Sesso H. D., Christen W. G., et al. Multivitamins in the prevention of cancer in men: the Physicians’ Health Study II randomized controlled trial. JAMA. 2012; 308: 1871–80. doi: 10.1001/jama.2012.14641 86. Qin L. Q., Xu J. Y., Wang P. Y., et al. Milk/dairy prod- ucts consumption, galactose metabolism and ovarian cancer: meta-analysis of epidemiological studies. Eur J Cancer Prev. 2005; 14:13–19. doi: 10.1097/00008469– 200502000–00003 102. Qiao Y. L., Dawsey S. M., Kamangar F., et al. Total and cancer mortality aft er supplementation with vita- mins and minerals: follow-up of the Linxian General Population Nutrition Intervention Trial. J Natl Cancer Inst. 2009; 101: 507–18. doi: 10.1093/jnci/djp037 87. Genkinger J. M., Hunter D. J., Spiegelman D., et al. Dairy products and ovarian cancer: a pooled analysis of 12 cohort studies. Cancer Epidemiol Biomarkers Prev. 2006; 15:364–372. doi: 10.1158/1055–9965.EPI-05–0484 103. Chung M., Lee J., Terasawa T., et al. Vitamin D with or without calcium supplementation for prevention of cancer and fractures: an updated meta-analysis for the U. S. Preventive Services Task Force. Ann Intern Med. 2011; 155: 827–38. doi: 10.7326/0003–4819–155–12– 201112200–00005 88. Larsson S. C., Orsini N., Wolk A. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 127. Vinceti M., Filippini T., Del Giovane C., et al. Selenium for preventing cancer. Cochrane Database Syst Rev. 2018; 1: CD005195. doi: 10.1002/14651858.CD005195.pub4 observational studies. Nutr Cancer. 2008;60(4):421–41. doi: 10.1080/01635580801911779 observational studies. Nutr Cancer. 2008;60(4):421–41. doi: 10.1080/01635580801911779 112. Giovannucci E., Rimm E. B., Wolk A., et al. Calcium and fructose intake in relation to risk of prostate cancer. Cancer Res. 1998; 58:442–447. 128. Eliassen A. H., Colditz G. A., Rosner B., et al. Adult weight change and risk of postmenopausal breast cancer. JAMA. 2006; 296: 193–201. doi: 10.1001/jama.296.2.193 113. Chan J. M., Stampfer M. J., Ma J., et al. Dairy products, calcium, and prostate cancer risk in the Physicians’ Health Study. Am J Clin Nutr. 2001; 74: 549–54. doi: 10.1093/ajcn/74.4.549 129. Luo J., Chlebowski R. T., Hendryx M., et al. Intentional Weight Loss and Endometrial Cancer Risk. J Clin Oncol. 2017; 35: 1189–1193. doi: 10.1200/JCO.2016.70.5822 114. Rodriguez C., McCullough M.L., Mondul A. M., et al. Calcium, dairy products, and risk of prostate cancer in a prospective cohort of United States men. Cancer Epidemiol Biomarkers Prev. 2003; 12:597–603. 130. Chlebowski R. T., Luo J., Anderson G. L., et al. Weight loss and breast cancer incidence in postmenopaus- al women. Cancer. 2019; 125: 205–212. doi: 10.1002/ cncr.31687 115. Schuurman A. G., van den Brandt P. A., Dorant E., Goldbohm R. A. Animal products, calcium and pro- tein and prostate cancer risk in Th e Netherlands Cohort Study. Br J Cancer. 1999; 80: 1107–13. doi: 10.1038/ sj.bjc.6690472 131. Armstrong B. K., Cust A. E. Sun exposure and skin cancer, and the puzzle of cutaneous melanoma: A per- spective on Fears et al. Mathematical models of age and ultraviolet eff ects on the incidence of skin cancer among whites in the United States. American Journal of Epidemiology. 1977; 105: 420–427. Cancer Epidemiol. 2017; 48: 147–156. doi: 10.1016/j.canep.2017.04.004 116. Chan J. M., Pietinen P., Virtanen M., et al. Diet and pros- tate cancer risk in a cohort of smokers, with a specifi c fo- cus on calcium and phosphorus (Finland). Cancer Causes Control. 2000; 11: 859–67. doi: 10.1023/a:1008947201132 132. Whiteman D. C., Whiteman C. A., Green A. C. Childhood sun exposure as a risk factor for mela- noma: a systematic review of epidemiologic stud- ies. Cancer Causes Control. 2001; 12: 69–82. doi: 10.1023/a:1008980919928 117. Lippman S. M., Klein E. A., Goodman P. J., et al. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Eff ect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2009; 301: 39–51. doi: 10.1001/jama.2008.864 133. International Agency for Research on Cancer Working Group on artifi cial ultraviolet (UV) light and skin cancer. Th e association of use of sunbeds with cutaneous ma- lignant melanoma and other skin cancers: A systematic review. Int J Cancer. 2007; 120: 1116–22. doi: 10.1002/ ijc.22453 118. Klein E. A., Thompson I. M. Jr., Tangen C. M., et al. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2011; 306: 1549–56. doi: 10.1001/jama.2011.1437 119. Moyer V. A. U. S. Preventive Services Task Force. Vitamin, mineral, and multivitamin supplements for the primary prevention of cardiovascular disease and cancer: U. S. Preventive services Task Force recommen- dation statement. Ann Intern Med. 2014; 160: 558–64. doi: 10.7326/M14–0198 134. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum. 2007;90:1–636. 135. Hepatitis viruses. IARC Monogr Eval Carcinog Risks Hum. 1994;59:1–255. 120. van Asperen I. A., Feskens E. J., Bowles C. H., Kromhout D. Body iron stores and mortality due to cancer and isch- aemic heart disease: a 17-year follow-up study of elderly men and women. Int J Epidemiol. 1995; 24: 665–70. doi: 10.1093/ije/24.4.665 136. Human T-cell lymphotropic viruses. IARC Monogr Eval Carcinog Risks Hum. 1996;67:261–390. 137. Deeken J. F., Tjen- A-Looi A., Rudek M. A., et al. Th e rising challenge of non- AIDS-defi ning cancers in HIV- infected patients. Clin Infect Dis. 2012 Nov;55(9):1228– 35. doi: 10.1093/cid/cis613 121. Knekt P., Reunanen A., Takkunen H., et al. Body iron stores and risk of cancer. Int J Cancer. 1994; 56: 379–82. doi: 10.1002/ijc.2910560315 138. Parkin D. M. The global health burden of infection- associated cancers in the year 2002. Int J Cancer. 2006;118(12):3030–44. doi: 10.1002/ijc.21731 122. Zacharski L. R., Chow B. K., Howes P. S., et al. Decreased cancer risk aft er iron reduction in patients with periph- eral arterial disease: results from a randomized trial. J Natl Cancer Inst. 2008; 100: 996–1002. doi: 10.1093/ jnci/djn209 139. Malnati M. S., Dagna L., Ponzoni M., Lusso P. Human herpesvirus 8 (HHV-8/KSHV) and hematologic malig- nancies. Rev Clin Exp Hematol. 2003; 7:375–405. 140. Schistosomes, liver fl ukes and Helicobacter pylori. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Eff ects of calcium on the incidence of recurrent colorectal ad- enomas: A systematic review with meta-analysis and trial sequential analysis of randomized controlled tri- als. Medicine (Baltimore). 2017; 96: e7661. doi: 10.1097/ MD.0000000000007661 94. World Cancer Research Fund. Endometrial cancer: How diet, nutrition and physical activity aff ect endometrial (womb) cancer risk. Available at: https://www.wcrf.org/ dietandcancer/endometrial- cancer Accessed: 31.03.2022. 110. Wactawski- Wende J., Kotchen J. M., Anderson G. L., et al. Calcium plus vitamin D supplementation and the risk of colorectal cancer. N Engl J Med. 2006; 354(10):1102. 95. Vieira A. R., Abar L., Chan D. S.M., et al. Foods and beverages and colorectal cancer risk: a systematic re- view and meta-analysis of cohort studies, an update of the evidence of the WCRF-AICR Continuous Update Project. Ann Oncol. 2017;28(8):1788–1802. doi: 10.1093/ annonc/mdx171 111. Huncharek M., Muscat J., Kupelnick B. Dairy products, dietary calcium and vitamin D intake as risk factors for prostate cancer: a meta-analysis of 26,769 cases from 45 125 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7–14 June 1994. IARC Monogr Eval Carcinog Risks Hum. 1994; 61:1–241. 123. Duffi eld- Lillico A.J., Shureiqi I., Lippman S. M. Can selenium prevent colorectal cancer? A signpost from epidemiology. J Natl Cancer Inst. 2004;96(22):1645–7. doi: 10.1093/jnci/djh332 141. Xia J., Jiang S. C., Peng H. J. Association between Liver Fluke Infection and Hepatobiliary Pathological Changes: A Systematic Review and Meta- Analysis. PLoS One. 2015; 10: e0132673. doi: 10.1371/journal.pone.0132673 124. Combs G. F. Jr. Current evidence and research needs to support a health claim for selenium and cancer preven- tion. J Nutr. 2005; 135: 343–7. doi: 10.1093/jn/135.2.343 125. Steevens J., van den Brandt P. A., Goldbohm R. A., Schouten L. J. Selenium status and the risk of esopha- geal and gastric cancer subtypes: the Netherlands co- hort study. Gastroenterology. 2010; 138: 1704–13. doi: 10.1053/j.gastro.2009.12.004 142. American College of Obstetricians and Gynecologists. ACOG practice bulletin. Cervical Cytology screening. Number 45, August 2003. Int J Gynaecol Obstet. 2003; 83: 1409–1420. doi: 10.1097/AOG.0b013e3181c6f8a4 143. US Preventive Services Task Force, Owens D. K., Davidson K. W., et al. Screening for Hepatitis C Virus Infection in Adolescents and Adults: US Preventive Services Task Force Recommendation Statement. JAMA. 2020; 323: 970–975. doi: 10.1001/jama.2020.1123 126. Sayehmiri K., Azami M., Mohammadi Y., et al. Th e association between Selenium and Prostate Cancer: a Systematic Review and Meta- Analysis. Asian Pac J Cancer Prev. 2018;19(6):1431–1437. doi: 10.22034/ APJCP.2018.19.6.1431 126 обзор | review 144. Vangipuram R., Tyring S. K. AIDS-Associated Malignancies. Cancer Treat Res. 2019;177:1–21. doi: 10.1007/978–3–030–03502–0_1 Women’s Health Study. J Natl Cancer Inst. 2004; 96:229– 233. doi: 10.1093/jnci/djh020 161. Slattery M. L., Curtin K., Ma K., et al. Diet activity, and lifestyle associations with p53 mutations in co- lon tumors. Cancer Epidemiol Biomarkers Prev. 2002; 11:541–548. 145. Sung J. J., Tsoi K. K., Wong V. W., et al. Meta-analysis: Treatment of hepatitis B infection reduces risk of hepa- tocellular carcinoma. Aliment Pharmacol Th er. 2008; 28: 1067–77. doi: 10.1111/j.1365–2036.2008.03816.x 162. Terry P. D., Jain M., Miller A. B., et al. Glycemic load, carbohydrate intake, and risk of colorectal cancer in women: a prospective cohort study. J Natl Cancer Inst. 2003; 95:914–916. doi: 10.1093/jnci/95.12.914 146. Soriano V., Puoti M., Peters M., et al. Care of HIV pa- tients with chronic hepatitis B: updated recommenda- tions from the HIV-Hepatitis B Virus International Panel. AIDS. 2008;22(12):1399–410. doi: 10.1097/ QAD.0b013e3282f8b46f 163. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Dietary car- bohydrates, fi ber, and breast cancer risk. Am J Epidemiol. 2004; 159: 732–9. doi: 10.1093/aje/kwh112 169. Chan A. T., Lippman S. M. Aspirin and colorectal can- cer prevention in Lynch syndrome. Lancet. 2011; 378: 2051–2. doi: 10.1016/S0140–6736(11)61216–6 155. Nielsen T. G., Olsen A., Christensen J., et al. Dietary car- bohydrate intake is not associated with the breast cancer incidence rate ratio in postmenopausal Danish women. J Nutr. 2005; 135: 124–8. doi: 10.1093/jn/135.1.124 170. Bosetti C., Santucci C., Gallus S., et al. Aspirin and the risk of colorectal and other digestive tract cancers: an updated meta-analysis through 2019. Ann Oncol. 2020;31(5):558–568. doi: 10.1016/j.annonc.2020.02.012 156. Higginbotham S., Zhang Z. F., Lee I. M., et al. Dietary glycemic load and breast cancer risk in the Women’s Health Study. Cancer Epidemiol Biomarkers Prev. 2004; 13: 65–70. doi: 10.1158/1055–9965.epi-03–0066 171. Gronich N., Rennert G. Beyond aspirin- cancer preven- tion with statins, metformin and bisphosphonates. Nat Rev Clin Oncol. 2013; 10: 1620–5. doi: 10.2337/dc08–2175 157. Romieu I., Ferrari P., Rinaldi S., et al. Dietary glycemic index and glycemic load and breast cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). Am J Clin Nutr. 2012; 96: 345–355. doi: 10.3945/ajcn.111.026724 172. Libby G., Donnelly L. A., Donnan P. T., et al. New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes. Diabetes Care. 2009; 32: 1620–5. doi: 10.2337/dc08–2175 173. Landman G. W., Kleefstra N., van Hateren K. J., et al. Metformin associated with lower cancer mortality in type 2 diabetes: ZODIAC-16. Diabetes Care. 2010; 322–6. doi: 10.2337/dc09–1380 158. Dong J. Y., Qin L. Q. Dietary glycemic index, glycemic load, and risk of breast cancer: meta-analysis of prospec- tive cohort studies. Breast Cancer Res Treat. 2011; 126: 287–94. doi: 10.1007/s10549–011–1343–3 174. Decensi A., Puntoni M., Goodwin P., et al. Metformin and cancer risk in diabetic patients: a systematic re- view and meta-analysis. Cancer Prev Res (Phila). 2010; 3:1451–61. doi: 10.1158/1940–6207.CAPR-10–0157 159. Shikany J. M., Flood A. P., Kitahara C. M., et al. Dietary carbohydrate, glycemic index, glycemic load, and risk of prostate cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort. Cancer Causes Control. 2011; 22: 995–1002. doi: 10.1007/ s10552–011–9772–1 175. Zhang Z. J., Zheng Z. J., Kan H., et al. Reduced risk of colorectal cancer with metformin therapy in patients with type 2 diabetes: a meta-analysis. Diabetes Care. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 Michaud D. S., Fuchs C. S., Liu S., et al. Dietary glycemic load, carbohydrate, sugar, and colorectal cancer risk in men and women. Cancer Epidemiol Biomarkers Prev. 2005; 14:138–147. 147. Applegate T. L., Fajardo E., Sacks J. A. Hepatitis C Virus Diagnosis and the Holy Grail. Infect Dis Clin North Am. 2018;32(2):425–445. doi: 10.1016/j.idc.2018.02.010 164. Aune D., Chan D. S., Lau R., et al. Carbohydrates, gly- cemic index, glycemic load, and colorectal cancer risk: a systematic review and meta-analysis of cohort studies. Cancer Causes Control. 2012; 23: 521–35. doi: 10.1007/ s10552–012–9918–9 148. Giovannucci E. Insulin and colon cancer. Cancer Causes Control. 1995; 6: 164–79. doi: 10.1007/BF00052777 149. Giovannucci E., Harlan D. M., Archer M. C., et al. Diabetes and cancer: a consensus report. CA Cancer J Clin. 2010; 60: 1674–85. doi: 10.2337/dc10–0666 165. Li P., Wu H., Zhang H., et al. Aspirin use aft er diagnosis but not prediagnosis improves established colorectal cancer survival: a meta-analysis. Gut. 2015;64(9):1419– 25. doi: 10.1136/gutjnl-2014–308260 150. Handelsman Y., Leroith D., Bloomgarden Z. T., et al. Diabetes and cancer – an AACE/ACE consensus state- ment. Endocr Pract. 2013 Jul- Aug;19(4):675–93. doi: 10.4158/EP13248.CS 166. Figueiredo J. C., Jacobs E. J., Newton C. C., et al. Associations of Aspirin and Non- Aspirin Non- Steroidal Anti- Infl ammatory Drugs With Colorectal Cancer Mortality Aft er Diagnosis. J Natl Cancer Inst. 2021;113(7):833–840. doi: 10.1093/jnci/djab008 151. George S. M., Mayne S. T., Leitzmann M. F., et al. Dietary glycemic index, glycemic load, and risk of can- cer: a prospective cohort study. Am J Epidemiol. 2009; 169: 462–72. doi: 10.1093/aje/kwn347 152. Augustin L. S., Dal Maso L., La Vecchia C., et al. Dietary glycemic index and glycemic load, and breast cancer risk: a case-control study. Ann Oncol. 2001; 12: 1533–8. doi: 10.1023/a:1013176129380 167. Jacobs E. J., Th un M. J., Bain E. B., et al. A large cohort study of long-term daily use of adult- strength aspirin and cancer incidence. J Natl Cancer Inst. 2007; 99:608– 15. doi: 10.1093/jnci/djk132 153. Jonas C. R., McCullough M.L., Teras L. R., et al. Dietary glycemic index, glycemic load, and risk of incident breast cancer in postmenopausal women. Cancer Epidemiol Biomarkers Prev. 2003; 12:573–577. 168. Serrano D., Patrignani P., Stigliano V., et al. Aspirin Colorectal Cancer Prevention in Lynch Syndrome: Recommendations in the Era of Precision Medicine. Genes (Basel). 2022;13(3):460. doi: 10.3390/ genes13030460 154. Holmes M. D., Liu S., Hankinson S. E., et al. экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 2011; 34:2323–8. doi: 10.2337/dc11–0512 160. Higginbotham S., Zhang Z. F., Lee I. M., et al. Dietary glycemic load and risk of colorectal cancer in the 127 experimental & clinical gastroenterology | № 205 (9) 2022 экспериментальная и клиническая гастроэнтерология | № 205 (9) 2022 experimental & clinical gastroenterology | № 205 (9) 2022 176. Noto H., Goto A., Tsujimoto T., Noda M. Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis. PLoS One. 2012; 7: e33411. doi: 10.1371/journal.pone.0033411 metformin: new insights into the molecular manage- ment of breast cancer resistance to the HER2 tyrosine kinase inhibitor lapatinib (Tykerb). Clin Transl Oncol. 2009; 11:455–459. doi: 10.1007/s12094–009–0384–0 177. Bodmer M., Becker C., Meier C., et al. Use of antidiabet- ic agents and the risk of pancreatic cancer: a case-con- trol analysis. Am J Gastroenterol. 2012; 107:620–626. doi: 10.1038/ajg.2011.483 181. Cholesterol Treatment Trialists’ (CTT) Collaboration, Emberson J. R., Kearney P. M., et al. Lack of eff ect of lowering LDL cholesterol on cancer: meta-analysis of individual data from 175,000 people in 27 randomised trials of statin therapy. PLoS One. 2012; 7: e29849. doi: 10.1371/journal.pone.0029849 178. Alimova I. N., Liu B., Fan Z., et al. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle. 2009; 8:909–915. doi: 10.4161/cc.8.6.7933 182. Haaland G. S., Falk R. S., Straume O., Lorens J. B. Association of Warfarin Use With Lower Overall Cancer Incidence Among Patients Older Th an 50 Years. JAMA Intern Med. 2017; 177:1774–1780. doi: 10.1001/jamain- ternmed.2017.5512 179. Vazquez- Martin A., Oliveras- Ferraros C., Menendez J. A. Th e antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR eff ector p70S6K1 in human breast carcinoma cells. Cell Cycle. 2009; 8:88–89. doi: 10.4161/cc.8.1.7499 183. Tagalakis V., Blostein M., Robinson- Cohen C., Kahn S. R. Th e eff ect of anticoagulants on cancer risk and survival: systematic review. Cancer Treat Rev. 2007; 33:358–368. doi: 10.1016/j.ctrv.2007.02.004 180. Vázquez- Martín A., Oliveras- Ferraros C., del Barco S., et al. mTOR inhibitors and the anti-diabetic biguanide 128
https://openalex.org/W4288622881
https://www.scielo.br/j/rsp/a/74nG4bbB8ddwrVfG7pnYv7g/?lang=en&format=pdf
Latin
null
Cognitive function among older adults
Revista de saúde pública/Revista de Saúde Pública
2,019
cc-by
5,271
http://www.rsp.fsp.usp.br/ Rev Saude Publica. 2018;52 Suppl 2:4s Rev Saude Publica. 2018;52 Suppl 2:4s Supplement ELSI-Brazil Original Article Supplement ELSI-Brazil Original Article Correspondence: Erico Castro-Costa Av. Augusto de Lima, 1715 30190-002 Belo Horizonte, MG, Brasil E-mail: ericocastrocosta@gmail.com How to cite: Castro-Costa E, Lima- Costa MF, Bof de Andrade F, Souza- Junior PRB, Ferri CP. Cognitive function among older adults: ELSI- Brazil results. Rev Saude Publica. 2018;52 Suppl 2:4s. Cognitive function among older adults: ELSI-Brazil results Erico Castro-CostaI, Maria Fernanda Lima-CostaI,II, Fabíola Bof de AndradeI,II, Paulo Roberto Borges de Souza JuniorIII, Cleusa Pinheiro FerriIV I Fundação Oswaldo Cruz. Instituto René Rachou. Núcleo de Estudos em Saúde Pública e Envelhecimento. Belo Horizonte, MG, Brasil , , II Fundação Oswaldo Cruz. Instituto René Rachou. Programa de Pós-Graduação em Saúde Coletiva. Belo Horizonte, MG, Brasil , III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de Janeiro, RJ, Brasil , III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de Janeiro, RJ, Brasil J , J, IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil J , J, IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil Data Source The ELSI-Brazil is a population-based cohort study, designed to represent the Brazilian population aged 50 years or older, and with the objective of investigating the dynamics of aging in the Brazilian population and its determinants. The baseline was established between the years 2015/2016. More details can be found on the research’s homepagea and in another publication21. ELSI-Brazil baseline data collection included: 1) a household interview; 2) an individual interview with the participant; 3) physical measurements; 4) laboratory tests; 3) storage of blood aliquots for future analysis. a Fundação Oswaldo Cruz. Brazilian Longitudinal Study of Aging. Rio de Janeiro; c2015 [cited 2017 Nov 28]. Available from: http://elsi.cpqrr.fiocruz.br INTRODUCTION Cognitive function is an important determinant of independence and better quality of life among older adults1. According to recent projections, the elderly population will triple in Brazil and will increase from 19.6 million in 2010 to 66.5 million in 20502, making it the sixth largest elderly population in the world3. Aging is a complex phenomenon and its impact on health conditions and functionality occurs heterogeneously among older adults. These differences are associated with genetic4, environmental and social determinants5-8, and individual characteristics of the elderly9,10. Previous population-based studies with national samples from several countries have shown that socio-demographic factors11,12 and differences between rural and urban residence areas13,14 are related to alterations in cognitive function among older adults, showing important cultural and geographic variations11,12, 15,16. Brazil is a country of continental proportions, has one of the highest levels of inequality17, occupies the 75th position in the Human Development Index (HDI)18 and presents differences for this index among its macroregions19. There has been an increasing number of studies on the cognitive function of Brazilian older adults20. However, it was observed that the majority (72%) was done in the Southeast and none of these studies had a design that allowed the comparison of cognitive function among Brazilian macroregions20. The Brazilian Longitudinal Study of Aging (ELSI-Brazil) used a national sample representative of individuals aged 50 years or older and allows us to investigate the cognitive function of older Brazilian adults. Thus, the objective of the present study was: 1) to investigate the cognitive function of older adults stratified by the Brazilian macroregions; 2) to compare the association between sociodemographic factors and the place of residence with cognitive function and the variations between the macroregions; 3) to investigate whether the variations in the sociodemographic structure and the place of residence observed are totally or partially responsible for the potential differences in cognitive function among macroregions. ABSTRACT ABSTRACT OBJECTIVE: To investigate macroregional variations in cognitive function in a national sample representative of the Brazilian population aged 50 years and older. METHODS: Data from the baseline of the Longitudinal Study of Brazilian Elderly (ELSI-Brazil), collected between 2015 and 2016, were used. Memory was measured by means of a 10-word list and executive function, by semantic verbal fluency, based on the naming of animals. Gender, age, education, and rural or urban residence were potentially confounding RESULTS: Among the 9,412 ELSI-Brazil participants, 9,085 were included in the analysis; 53.9% were women and the average age was 63.0 (0.42) years. After adjusting for potential confounding variables, average scores for memory and verbal fluency were lower in the Northeast region and higher in the Midwest and Southeast, respectively. In the South region, higher scores were found for immediate and combined memory. In all regions, older participants and those with lower schooling had worse scores for memory and verbal fluency. CONCLUSIONS: There are differences in cognitive function among older adults in the different macroregions, independent of age, gender, schooling, and rural or urban residence. Correspondence: Erico Castro-Costa Av. Augusto de Lima, 1715 30190-002 Belo Horizonte, MG, Brasil E-mail: ericocastrocosta@gmail.com DESCRIPTORS: Aged. Cognition. Memory. Socioeconomic factors. Received: Dec 18, 2017 Approved: Mar 18, 2018 How to cite: Castro-Costa E, Lima- Costa MF, Bof de Andrade F, Souza- Junior PRB, Ferri CP. Cognitive function among older adults: ELSI- Brazil results. Rev Saude Publica. 2018;52 Suppl 2:4s. Copyright: 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 that the original author and source are credited. 1s Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Ethical Aspects The ELSI-Brazil Project was approved by the Ethics Commission of Fiocruz, Minas Gerais (CAAE 34649814.3.0000.5091). All participants signed separate informed consent forms for all research procedures. Cognitive Function The study adopts a conceptual framework common to other large-scale longitudinal studies of aging in the world, such as the Health and Retirement Study21. This specific concept of the cognitive function module in ELSI-Brazil allows direct comparison of Brazilian results with results found in other countries, such as: China [the China Health and Retirement Longitudinal Study (https://g2aging.org/?section=study&studyid=4)], the United States [the US Health and Retirement Study (http://hrsonline. isr.umich.edu)], England [the English Longitudinal Study of Ageing (http://www.natcen.ac.uk/elsa)], Mexico [Mexican Health and Aging Study(mhasweb.org/Resources.aspx)] etc. Cognitive function among older Brazilian adults Castro-Costa E et al. In the present analysis, we selected two variables that represent three well-established domains of cognitive function: memory, language and executive function. Memory was evaluated through the word list learning test, in which 10 words were read to the participants. Immediate memory was defined as the repetition of the words immediately after their reading. Late memory (delayed recall) was their repetition five minutes after presentation. The participant was kept busy filling out the questionnaire during the interval between the two tasks. Combined memory was obtained by the sum of the scores for immediate and late memory. Language and executive function were measured by the semantic verbal fluency test (animal category), in which participants were asked to say the name of as many animals as they could in a one-minute period. Statistical Analyses Statistical analyzes were conducted in the Stata 14.1 program. Due to the complex design of the sample with stratification, all analyzes were done using weighting factors for each stratum, thus obtaining in the final results the contribution of each stratum according to its actual weight, and not through its participation in the sample. The use of Stata’s svy command allowed the output of robust standardized error. To compare the characteristics of the participants among the macroregions, the analysis of variance (ANOVA) was weighted for continuous variables and the Pearson’s chi-squared test was weighted for the categorical variables. Multivariate linear regressions with a 95% confidence interval were performed to estimate the relationship between memory (immediate, late and combined) and verbal fluency with gender, age, schooling and place of residence for each macroregion. Finally, the average of the variables memory (immediate, late and combined) and verbal fluency was standardized separately and then simultaneously, by gender, age, schooling and place of residence, using the direct method22 for each macroregion. Other Variables Also considered were the variables gender, age (50-59, 60-69, 70-79, 80 years or older), schooling (< 4, 4-7, 8-11, > 11) and place of residence (rural or urban). Ethical Aspects RESULTS Of the 9,412 ELSI-Brazil baseline participants, 9,085 (96.5%) presented complete information for all variables and were included in this analysis. The main characteristics of the participants by region are presented in Table 1. In the total sample, the average age was 63.0 years (SE = 0.42) and the majority were female (53.9%) and had less than 11 years of schooling (76.2%). Table 2 shows the average scores and multivariate analyzes of the association between memory and verbal fluency scores stratified by macroregions. Residents in the Southeast, South and Midwest regions had the best performances, both in the memory and in the verbal fluency evaluation. Older adults in the Southern macroregion had the best performance in the variables immediate memory and combined memory, while the best performance in late memory and verbal fluency occurred among residents of the Midwest and Southeast, respectively. Older residents with low schooling had worse memory performance (immediate, late and combined) and worse verbal fluency in all macroregions. On the other hand, rural participants showed worse performance in immediate and Cognitive function among older Brazilian adults Castro-Costa E et al. combined memory in the Northeast and Southeast, and in late memory in the Southeast. In the Midwest, rural residents performed better in late and combined memory. Regarding verbal fluency, participants from the rural area of the South and Midwest macroregion, and women from the North and Midwest, presented worse performance of this function. Table 1. Characteristics of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. RESULTS Characteristic Total North Northeast Southeast South Midwest p n = 9,085 n = 713 n = 2,416 n = 3,825 n = 1,234 n = 897 Gender: female (%) 53.9 49.9 54.6 54.5 53.6 52.1 0.934 Age – average (SE) 63.0 (0.42) 62.1 (0.67) 63.3 (0.75) 63.0 (0.72) 63.0 (0.92) 62.3 (1.1) 0.945 Education (years) (%) < 0.0001 > 11 23.8 26.3 18.3 26.3 24.2 23.7 8–10 11.9 15.6 8.9 12.9 12.2 11.3 4–7 31.4 25.4 23.2 34.4 36.9 31.8 < 4 32.8 32.7 49.5 26.4 26.7 33.2 Place of residence: Rural (%) 15.3 17.7 30.3 6.5 21.6 5.6 0.006 Memory - mean (SE) Immediate 4.3 (0.05) 4.2 (0.11) 3.8 (0.08) 4.5 (0.06) 4.6 (0.07) 4.3 (0.07) < 0.0001 Late 2.9 (0.06) 2.7 (0.15) 2.4 (0.09) 3.0 (0.07) 3.0 (0.09) 3.1 (0.13) < 0.0001 Combined 7.3 (0.10) 7.0 (0.24) 6.2 (0.17) 7.5 (0.13) 7.7 (0.16) 7.6 (0.19) < 0.0001 Verbal fluency - mean (SE) 12.6 (0.29) 11.4 (0.37) 11.4 (0.85) 13.2 (0.41) 12.6 (0.40) 12.9 (0.38) 0.012 All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a complex sample. All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a complex sample. Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores an chooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores and the variables gender, age, schooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. F: female; M: male All multivariate regressions were adjusted for gender, age, schooling and place of residence. RESULTS 4s Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Standardized average memory (immediate, late and combined) and verbal fluency scores are arranged and compared between the macroregions in Table 3. The direct standardization of memory and verbal fluency averages by the individual effect of gender, age, schooling and place of residence and by the simultaneous effect of these four variables did not alter the pattern of difference between the regions. The values of the averages observed and simultaneously standardized by the four variables are shown in the Figure. s of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. RESULTS Cognitive function Standardized by gender Standardized by age Standardized by schooling Standardized by place of residence Standardized by the 4 variables Average Score 95%CI Average Score 95%CI Average Score 95%CI Average Score 95%CI Average Score 95%CI Immediate memory North 4.24 4.00–4.47 4.03 3.85–4.20 4.11 3.91–4.31 4.23 4.03–4.43 4.08 3.92–4.24 Northeast 3.83 3.65–4.00 3.74 3.60–3.88 3.97 3.87–4.07 3.89 3.71–4.08 3.82 3.72–3.93 Southeast 4.51 4.40–4.63 4.44 4.34–4.55 4.37 4.30–4.44 4.45 4.36–4.54 4.38 4.31–4.45 South 4.60 4.45–4.74 4.50 4.32–4.68 4.46 4.35–4.57 4.62 4.50–4.74 4.50 4.38–4.61 Midwest 4.43 4.29–4.57 4.29 4.18–4.40 4.37 4.33–4.42 4.41 4.29–4.54 4.33 4.25–4.40 Late Memory North 2.76 2.45–3.07 2.56 2.24–2.87 2.65 2.26–3.05 2.76 2.44–3.09 2.62 2.25–2.99 Northeast 2.42 2.24–2.61 2.34 2.20–2.48 2.54 2.41–2.68 2.48 2.31–2.66 2.42 2.32–2.52 Southeast 3.05 2.91–3.20 2.97 2.86–3.08 2.89 2.80–2.99 2.98 2.86–3.11 2.90 2.81–2.99 South 3.10 2.92–3.28 2.99 2.81–3.17 2.96 2.81–3.11 3.12 2.98–3.27 3.01 2.86–3.16 Midwest 3.15 2.90–3.42 2.99 2.81–3.18 3.10 2.98–3.22 3.18 2.94–3.41 3.09 2.96–3.22 Combined Memory North 7.01 6.55–7.48 6.60 6.17–7.03 6.78 6.18–7.37 7.01 6.52–7.49 6.72 6.21–7.22 Northeast 6.26 5.92–6.61 6.09 5.83–6.35 6.52 6.30–6.74 6.38 6.04–6.73 6.25 6.06–6.43 Southeast 7.58 7.33–7.83 7.42 7.22–7.62 7.28 7.13–7.43 7.44 7.24–7.65 7.29 7.14–7.44 South 7.70 7.39–8.01 7.50 7.16–7.84 7.43 7.18–7.67 7.75 7.50–7.99 7.52 7.27–7.76 Midwest 7.60 7.22–7.99 7.29 7.02–7.57 7.49 7.35–7.62 7.62 7.28–7.96 7.46 7.28–7.64 Verbal fluency North 11.44 10.66–12.23 11.23 10.73–11.73 11.30 10.98–11.71 11.53 10.83–12.24 11.30 10.70–11.89 Northeast 11.41 9.69–13.13 11.23 9.79–12.66 11.79 9.88–13.71 11.60 9.66–13.54 11.54 9.53–13.55 Southeast 13.21 12.41–14.01 13.33 12.34–14.32 13.26 12.10–14.41 13.26 12.31–14.21 13.41 11.94–14.89 South 12.62 11.83–13.40 12.48 11.69–13.26 12.39 11.85–12.92 12.74 12.23–13.26 12.41 11.91–12.91 Midwest 12.97 12.22–13.71 12.75 12.07–13.44 12.86 12.32–13.40 12.87 12.14–13.60 12.77 12.11–13.44 Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitu 2015-2016. 16 14 12 10 18 16 14 12 0 North observed immediate memory Northeast Southeast South Midwest standardized late memory observed verbal fluency standardized immediate memory observed combined memory observed late memory standardized combined memory standardized verbal fluency Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. 16 14 12 10 18 16 14 12 0 North observed immediate memory Northeast Southeast South Midwest standardized late memory observed verbal fluency standardized immediate memory observed combined memory observed late memory standardized combined memory standardized verbal fluency Figure. RESULTS Cognitive function North Northeast Southeast South Midwest Average 95%CI Average 95%CI Average 95%CI Average 95%CI Average 95%CI Memory Immediate 4.22 3.99–4.45 3.83 3.65–4.00 4.51 4.40–4.63 4.60 4.45–4.75 4.43 4.30–4.56 Late 2.76 2.45–3.07 2.42 2.23–2.60 3.05 2.90–3.20 3.09 2.92–3.27 3.15 2.89–3.41 Combined 6.99 6.53–7.46 6.26 5.92–6.60 7.58 7.32–7.83 7.70 7.39–8.01 7.60 7.22–7.99 Verbal fluency 11.52 10.78–12.27 11.41 9.73–13.09 13.24 12.42–14.06 12.64 11.84–13.44 13.01 12.25–13.77 β 95%CI β 95%CI β 95%CI β 95%CI β 95%CI Immediate Memory Gender (F versus M) 0.22 -0.15–0.59 0.01 -0.10–0.13 0.07 -0.07–0.21 0.04 -0.17–0.27 0.03 -0.13–0.19 Age (≥ 75 versus < 75) -1.95 -2.33– -1.57 -1.31 -1.45– -1.18 -1.20 -1.36– -1.03 -1.55 -1.90– -1.19 -1.49 -1.16– -1.36 Education (< 11 versus ≥ 11) -0.96 -1.28– -0.64 -1.17 -1.36– -0.97 -1.02 -1.13– -0.91 -1.01 -1.19– -0.82 -1.07 -1.49– -0.66 Place of residence (rural versus urban) -0.06 -0.30–0.16 -0.26 -0.39– -0.13 -0.51 -0.66– -0.36 -0.04 -0.32–0.24 0.01 -0.07–0.09 Late Memory Gender (F versus M) 0.09 -0.17–0.35 0.07 -0.07–0.21 0.07 -0.08–0.23 0.26 0.06–0.46 0.00 -0.44–0.44 Age (≥ 75 versus < 75) -1.76 -2.23– -1.30 -1.13 -1.30– -0.96 -1.52 -1.70– -1.33 -1.54 -1.76– -1.32 -1.45 -1.70– -1.20 Education (< 11 versus ≥ 11) -1.02 -1.27– -0.76 -1.22 -1.41– -1.03 -1.21 -1.35– -1.06 -1.11 -1.32– -0.89 -1.12 -1.57– -0.67 Place of residence (rural versus urban) 0.13 -0.25–0.53 -0.24 -0.53–0.05 -0.54 -0.77– -0.30 -0.22 -0.45–0.02 0.39 0.25–0.52 Combined Memory Gender (F versus M) 0.31 -0.28–0.90 0.08 -0.14–0.31 0.14 -0.12–0.42 0.31 -0.11–0.73 0.03 -0.55–0.61 Age (≥ 75 versus < 7 5) -3.72 -4.52– -2.92 -2.44 -2.70– -2.19 -2.72 -3.04– -2.40 -3.09 -3.66– -2.53 -2.94 -3.25– -2.62 Education (< 11 versus ≥ 11) -1.99 -2.51– -1.46 -2.39 -2.73– -2.05 -2.23 -2.47– -1.99 -2.12 -2.46– -1.78 -2.20 -3.06– -1.34 Place of residence (rural versus urban) 0.06 -0.50–0.63 -0.50 -0.86– -0.15 -1.05 -1.34– -0.76 -0.26 -0.74–0.22 0.40 0.22–0.58 Verbal Fluency Gender (F versus M) -1.24 -2.04– -0.44 0.02 -2.07–2.13 -1.44 -3.34–0.45 -0.46 -0.96–0.03 -0.99 -1.72– -0.27 Age (≥ 75 versus < 75) -1.84 -2.66– -1.03 -2.33 -4.35– -0.31 3.74 -2.23–9.72 -2.04 -2.71– -1.38 -2.09 -2.76– -1.42 Education (< 11 versus ≥ 11) -1.57 -2.44– -0.70 -1.75 -4.04–0.52 -2.71 -3.53– -1.88 -3.60 -4.24– -2.97 -2.45 -4.15– -0.75 Place of residence (rural versus urban) -0.20 -1.18–0.77 -1.07 -3.53–1.39 0.90 -3.97–5.78 -0.73 -1.46– -0.01 -1.10 -1.84– -0.36 F: female; M: male All multivariate regressions were adjusted for gender, age, schooling and place of residence. DISCUSSION DISCUSSION This is the first national study with a representative sample of the population aged 50 years or older that compared cognitive function in the five Brazilian macroregions, where the same tests were applied in the same period. Residents of the Southeast, South and Midwest regions presented better cognitive function in all the applied tests. However, there were variations on the ranking, depending on the ability evaluated. In all regions, there was a worsening of the three types of memory and verbal fluency correlated with the oldest age and low level of schooling, except for the Southeast macroregion, where the worsening of verbal fluency was not correlated with older age. Regarding the place of residence, we observed, inconsistently, that residents of the rural area exhibited lower scores for tests that evaluate memory and verbal fluency. However, residents of the rural area of the Midwest performed better in late memory than those in the urban area. The female gender presented the worst performance in verbal fluency tests in the North and Midwest macroregions. Finally, the standardized averages of the three types of memory and of verbal fluency by the effect of gender, age, schooling and place of residence showed that these variables did not explain all the differences found among the Brazilian macroregions. This study has several advantages such as: 1) the use of a representative sample of Brazilian adults aged 50 and older; 2) the direct evaluation of cognitive function through validated and applied tests in the same period, which allows comparisons between the Brazilian macroregions and with other countries; 3) training and certification of the interviewers according to the protocols developed for the study; 4) quality assurance and control of data collection by conducting previous pilot studies in order to identify and correct potential problems in procedures and interviews. However, the study also has limitations that must be considered when interpreting the results. The use of a weighted sample may underestimate the averages for cognitive function due to its design effect. However, the use of specific analyzes for weighting is likely to overcome this limitation. Although all dimensions of cognitive function were not evaluated in this study, scores of our measures (10-word list and verbal fluency test) were normally distributed with no evidence of floor or ceiling effect23, commonly found in low education populations. RESULTS Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. DISCUSSION The results of the present study are directly comparable with studies conducted in high- income countries (Health Retirement Study [HRS], English Longitudinal Study of Ageing [ELSA], the Irish Longitudinal Study on Ageing [TILDA])11,12 and in middle income countries, such as (Mexican Health and Aging Study [MHAS] and the Chinese Health and Retirement Longitudinal Study [CHARLS])15,16, in which cognitive function was assessed using the same measure for memory (a list of 10 words, with the exception of the MHAS study, which used a list of eight words) and verbal fluency (animal category semantic fluency test). ELSI-Brazil, even with a younger sample due to the inclusion of middle-aged adults, presented lower averages for all abilities evaluated (immediate, late and combined memory) when compared to the three studies in high-income countries (HRS, ELSA, TILDA)11,12 which used a sample of elderly individuals aged 60 years or older. Probably, this occurred due to the great difference in schooling between these samples, since this factor plays an important role in cognitive function24. In ELSI-Brazil, 76.2% of the sample of Brazilians aged 50 and older have less than 12 years of schooling; in HRS, ELSA and TILDA, rates are 56%, 48% and 44% for those aged 65 years and older, and 39%, 28%, 28% for those aged 57-64 years, respectively11,12. Compared with studies in middle- and low-income countries, where low schooling was similar between ELSI-Brazil, MHAS and CHARLS15,16. It was observed that in the Mexican study, in spite of presenting an older population (adults 60 years old or older), performance in all three skills was better compared to our findings (immediate memory: 4.8 versus 4.3; late memory: 4.4 versus 2.9, and verbal fluency: 15.3 versus 12.6). This was because the participants in the MHAS sample were healthier than those in the ELSI-Brazil since the tests for cognitive evaluation were applied only to those who had not had a stroke or did Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. not present depressive symptoms16. In addition, the memory was tested by a list of eight words, which probably influenced the results obtained with this version, overestimating the performance of the participants16. Regarding the Chinese study15, combined memory performance was worse than in ELSI-Brazil (3.3 versus 7.3), probably because the majority of participants were from the rural area13,14. DISCUSSION Comparisons with other Brazilian studies are limited due to the differences between the age groups and the outcomes studied. In a study with employees aged 35-74 at six universities in the Northeast, Southeast and South macroregions, memory was evaluated by a list of 10 words applied at three different occasions (immediate, late, recognition), while semantic verbal fluency was evaluated by the animal category test25. The averages for late memory ranged from four to eight in men and five to eight in women, while the average values for verbal fluency ranged from 12 to 21 among men and 13 to 21 among women according to participants’ schooling25. Like the Longitudinal Study on Adult Health (ELSA-Brazil), the present study also demonstrated that older participants with low educational level had worse performance in memory and verbal fluency. However, the scores were lower in all tests when compared to those found in ELSA-Brazil. This discrepancy probably occurred because ELSA-Brazil was conducted exclusively in the urban area (six capitals), while ELSI-Brazil was conducted both with participants living in the urban area and with those living in rural areas. Memory and Verbal Fluency assessments, in the present study, are part of the cognitive assessment of the Consortium to Establish the Registry for Alzheimer’s Disease (CERAD)26, translated and validated to Brazilian Portuguese for carrying out in Brazilian elderly population27. Previous studies have shown that older people with lower levels of schooling and those coming from rural areas have worse cognitive performance11-16,27; data for the gender variable are inconsistent11,12,15,16,28. Our findings replicated the associations previously demonstrated for memory and verbal fluency in other studies with population-based samples11- 16,26,27, with the exception of results for late and combined memory, which showed that participants from rural areas in the Midwest had the best performance. This is likely to have happened because the main activity in this macroregion is farming, which possibly favors better socioeconomic conditions, which in turn may have contributed to this performance. Finally, the results of the present study showed that the performance of adults aged 50 years or older on cognitive function tests is worse than that found in high-income countries (United States, England and Ireland) and inconsistent with that of middle-income countries (worse than that found in Mexico and better than that found in China). We observed that the association between sociodemographic, settlement factors and cognitive function had consistent variation patterns in the Brazilian macroregions. DISCUSSION However, as these factors do not fully explain the differences in cognitive function in adults aged 50 years or older observed between regions, other contextual and cultural factors not investigated here may play a relevant role in the differences found. REFERENCES 1. Cigolle CT, Langa KM, Kabeto MU, Tian Z, Blaum CS. Geriatric conditions and disability: the Health and Retirement Study. Ann Intern Med. 2007;147(3):156-64. https://doi.org/10.7326/0003-4819-147-3-200708070-00004 2. Instituto Brasileiro de Geografia e Estatística, Coordenação de Geografia. Brasil: uma visão geográfica e ambiental no início do século XXI. Rio de Janeiro: IBGE; 2016. 2. Instituto Brasileiro de Geografia e Estatística, Coordenação de Geografia. Brasil: uma visão geográfica e ambiental no início do século XXI. Rio de Janeiro: IBGE; 2016. 3. World health Organization. World report on aging and health. Geneva: WHO; 2015 [cited 2017 Nov 25]. Availabe from: http://www.who.int/entity/ageing/publications/world-report-2015/en/ 3. World health Organization. World report on aging and health. Geneva: WHO; 2015 [cited 2017 Nov 25]. Availabe from: http://www.who.int/entity/ageing/publications/world-report-2015/en/ 4. Steves CJ, Spector TD, Jackson SH. Aging, genes, environment and epigenetic: what twin studies tell us now, and in the future. Age Aging. 2012;41(5):581-6. https://doi.org/10.1093/ageing/afs097 5. Naughton C, Drennan J, Treacy MP, Laerty A, Lyons I, Phelan A, et al. Abuse and neglect of o older people in Ireland: report on the national study of elder abuse and neglect. Dublin: National Centre for the Protection of Older People; 2010 [cited 2017 Nov 23]. Available from: http://www.ncpop.ie/userfiles/file/ncpop%20reports/Study%203%20Prevalence.pdf Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. 6. Wu L, Chen H, Hu Y, Xiang H, Yu X, Zhang T, et al. Prevalence and associated factors of elder mistreatment in a rural community in People’s Republic of China: a cross-sectional study. PLoS One. 2012;7(3):e33857. https://doi.org/101371/journal.pone.0033857 7. Shankar A, McMunn A, Banks J, Steptoe A. Loneliness, social isolation, and behavioral and biological health indicators in older adults. Health Psychol. 2011;30(4):377-85. https://doi.org/101037/a0022826 8. Iliffe S, Kharicha K, Harari D, Swift C, Gillmann G, Stuck AE. Health risk appraisal in older people 2: the implications for clinicians and commissioners of social isolation risk in older people. Br J Gen Pract. 2007;57(537):277-82. 9. World Health Organization. Closing the gap in a generation: health equity through action on social determinants of health: final report of the Commission on Social Determinants of Health. Geneva: WHO; 2008 [cited 2017 Nov 23]. Available from: http://whqlibdoc.who.int/ publications/2008/9789241563703_eng.pdf 10. Dannefer D. Cumulative advantage/disadvantage and the life course: cross-fertilizing age and social science theory. J Gerontol B Psychol Sci Soc Sci. 2003;58(6):S327-37. https://doi.org/10.1093/geronb/58.6.s327 11. Langa KM, Llewellyn DJ, Lang IA, Weir DR, Wallace RB, Kabeto MU, et al. REFERENCES Cognitive health among older adults in the United States and in England. BMC Geriatr. 2009;9:23. https://doi.org/10.1186/1471-2318-9-23 12. Savva GM, Maty SC, Setti A, Feeney J. Cognitive and physical health of the older populations of England, the United States, and Ireland: international comparability of the Irish Longitudinal Study on Ageing. J Am Geriatr Soc. 2013;61 Suppl 2:s291-8. https://doi.org/10.111/jgs12196 13. Saenz JL, Downer B, Garcia MAG, Wong R. Cognition and context: rural-urban differences in cognitive aging among older Mexican adults. J Aging Health. 2017 Apr 1::898264317703560. https://doi.org/10.1177/0898264317703560 14. Cassarino M, O’Sullivan V, Kenny RA, Setti A. Environment and cognitive aging: a cross-sectional study of place residence and cognitive performance in the Irish Longitudinal Study on Aging. Neuropsychology. 2016;30(5):543-57. https://doi.org/10.1037/neu0000253 15. Lei X, Smith JP, Sun X, Zhao Y. Gender differences in cognition in China and reasons for change over time: evidence from CHARLS. J Econ Aging. 2014:4:46-55. https://doi.org/10.1016/j.jeoa.2013.11.001 16. Mejía-Arango S, Wong R, Michaels-Obregón A. Normative and standardized data for cognitive measures in the Mexican Health and Aging Study. Salud Publica Mex. 2015;57 Suppl 1:S90-6. 17. World Bank. Gini Index (World Bank estimate) 2011-2015. Washington (DC); 2015. [cited 2017 Nov 23]. Available from: https://data.worldbank.org/indicator/SI.POV.GINI 18. United Nations Development Programme. Human Development Reports. New York: UNDP [cited 2017 Nov 23]. Available from: http://hdr.undp.org/en 19. Programa das Nações Unidas para o Desenvolvimento. Desenvolvimento humano nas macrorregiões brasileiras: 2016. Brasília (DF): PNUD, IPEA, FJP; 2016. 20. Martins NIM, Caldas PR, Cabral ED, Lins CCSA, Coriolano MGWS. Instrumentos de avaliação cognitiva utilizados nos últimos 5 anos em idosos brasileiros. Cienc Saude Coletiva. 2017 out [cited 2017 Nov 23]:0402. Available from: http://www.cienciaesaudecoletiva.com.br/artigos/instrumentos- de-avaliacao-cognitiva-utilizados-nos-ultimos-5-anos-em-idosos-brasileiros/16454?id=16454 21. Lima-Costa MF, Andrade FB, Souza Jr PRB, Neri AL, Duarte YAO, Castro-Costa E, de Oliveira C. The Brazilian Longitudinal Study of Aging (ELSI-Brazil): objectives and design. Am J Epidemiol. 2018 Jan 31. https://doi.org/10.1093/aje/kwx387 22. National Institute on Aging; World Health Organization. Global Health and Aging. Bethesda (MD): National Institute of Health; 2011 [cited 2017 Nov 23]. (NIH publication, 11-773). Available from: http://www.who.int/ageing/publications/global_health.pdf 23. Batty GD, Deary IJ, Zaninotto P. Association of cognitive function with cause-specific mortality in middle and older age: follow-up of participants in the English Longitudinal Study of Ageing. Am J Epidemiol. 2016;183(3):183-90. https://doi.org/10.1093/aje/kwv139 24. Park DC. The basic mechanism accounting for age-related decline in cognitive function. In: Park DC, Schwarz N, editors. Cognitive aging: a primer. New York: Psychology Press; 2000. p.3-21. 25. Cognitive function among older Brazilian adults Castro-Costa E et al. 28. Ferreira L, Ferreira Santos-Galduróz R, Ferri CP, Fernandes Galduróz JC. Rate of cognitive decline in relation to sex after 60 years-of-age: a systematic review. Geriatr Gerontol Int. 2014;14(1):23- 31. https://doi.org/10.1111/ggi.12093 26. Morris JC, Heyman A, Mohs RC, Hughs JP, Van Belle G, Fillenbaum G, et al. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheirmer’s disease. Neurology. 1989;39(9):1159-65. https://doi.org/10.1212/WNL.39.9.1159 27. Bertolucci PHF, Okamoto IH, Toniolo Neto J, Ramos LR, Brucki SMD. Desempenho da população brasileira na bateria neuropsicológica do Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Rev Psiq Clin (São Paulo). 1998;25(2):80-3. Cognitive function among older Brazilian adults Castro-Costa E et al. REFERENCES Passos VMA, Giatti L, Bensenor I, Tiemeier H, Ikram MA, Figueiredo RC, et al. Education plays a greater role than age in cognitive test performance among participants of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). BMC Neurol. 2015;15:191. https://doi.org/10.1186/s12883-015-0454-6 8 https://doi.org/10.11606/S1518-8787.2018052000629 8s Funding: The ELSI-Brazil baseline study was supported by the Brazilian Ministry of Health (DECIT/SCTIE – Department of Science and Technology from the Secretariat of Science, Technology and Strategic Inputs (Grant 404965/2012-1); COSAPI/DAPES/SAS – Healthcare Coordination of Older Adults, Department of Strategic and Programmatic Actions from the Secretariat of Health Care) (Grants 20836, 22566, and 23700); and the Brazilian Ministry of Science, Technology, Innovation and Communication. Authors’ Contribution: Data analysis and interpretation: ECC, MFLC, FBA, PRBSJ, CPF. Manuscript writing: ECC, MFLC, FBA, PRBSJ, CPF. Manuscript critical review: ECC, MFLC, FBA, PRBSJ, CPF. All authors have approved the final version of the manuscript and assume public responsibility for its content. Conflict of Interest: The authors declare no conflict of interest. 9s
W2315992156.txt
https://zenodo.org/records/1642604/files/article.pdf
de
Vergleichende Studien über den Stoffwechsel verschiedener Tierarten. II. Mitteilung.
Hoppe-Seyler's Zeitschrift für Physiologische Chemie
1,909
public-domain
716
Vergleichende Studien Ober den Stoffwechsel verschiedener Tierarten. II. Mitteilung. Von Emil Abderhalden und Carl Brahm. (Aus dem physiologischen Institut der tierärztlichen Hochschule, Berlin). (Der Redaktion zugegangen am 16. August 1909.) Wir hatten vor kurzem1) in Übereinstimmung mit His u. Hofmeister nachgewiesen, daß der Organismus des Hundes nach Zufuhr von Pyridin Methylpyridin ausscheidet, Kaninchen dagegen waren nicht imstande, die Methylierung auszuführen. Wir stellten damals Versuche in Aussicht, die feststellen sollten, ob eine Erscheinung vorliegt, die auf der Art der Ernährung beruht, oder aber, ob der Organismus des Hundes an und für sich ein anderes Verhalten gegenüber Pyridin zeigt. Wir haben zunächst Pyridin an zwei junge Hunde verfüttert, welche bis zum Beginn des Versuches nur Muttermilch aufgenommen hatten. Sie erhielten ausschließlich Kuhmilch und mittels Schlundsonde Pyridin. Es ließ sich keine Bildung von Methylpyridin nachweisen. Das Pyridin ging unverändert in den Urin über. Es wurde in Form eines Pikrates isoliert. Die Tiere erhielten nunmehr gekochtes Pferdefleisch. Nach Eingabe von Pyridin erschien neben Pyridin Methylpyridin im Harn. Damit steht ein zweiter Versuch im Einklang. Ein junger Hund, der erst seit wenigen Tagen Fleischnahrung aufgenommen hatte, schied ebenfalls Methylpyridin aus. Dieses wurde durch die Darstellung des Platinsalzes des Methylpyridylammoniumhydroxydes identifiziert. Orangerote Tafeln, F. 212 «, 32,88 °/o Pt, statt der berechneten 32,6?o/0 Pt. l ) Emil Abderhalden t Carl Brahm und Alfred Schittenhelm, I. Mitteil., Diese Zeitschrift, Bd. LIX, S. 32, 1909. Brought to you by | Purdue University Libra Authenticated Download Date | 5/26/15 5:58 AM Emil Abderhalden und Carl Brahm, Nach diesem Ergebnisse versuchten wir nunmehr festzustellen, ob es gelingt, durch Ernährung mit bestimmten Nahrungsmitteln, das Vermögen, Methylpyridin aus Pyridin zu bilden, zu beeinflussen. Wir fütterten einen Hund 8 Wochen lang ausschließlich mit Milch. Nach Eingabe von Pyridin wurde ausschließlich Methylpyridin ausgeschieden. Kaninchen antworten, wie oben erwähnt, auf Eingabe von Pyridin nicht mit der Bildung von Methylpyridin. Es gelingt nicht, durch lange dauernde Ernährung mit Milch eine Bildung von Methylpyridin zu veranlassen. Ein Kaninchen erhielt 3 Wochen und ein anderes 8 Wochen ausschließlich Milch. Nach Pyridineingabe erschien Pyridin im Harn und kein Methylpyridin. Endlich fütterten wir ein Kaninchen 8 Tage mit Pferdefleisch. Auch hier blieb die Methylierung aus. Aus diesen Versuchen geht hervor, daß junge Hunde, die nur Milchnahrung aufgenommen haben, bei Eingabe von Pyridin kein Methylpyridin bilden. Die Methylierung tritt ein, sobald die Tiere einige Zeit Fleisch aufgenommen haben. Später kann durch länger dauernde, ausschließliche Ernährung mit Milch die Methylierung des Pyridins nicht mehr verhindert werden. Kaninchen bilden kein Methylpyridin, auch dann nicht, wenn sie längere Zeit mit Fleisch resp. Milch ernährt werden. Nicht ausgeschlossen ist durch diese Versuche, daß vielleicht eine noch länger dauernde ausschließliche Ernährung mit Milch schließlich doch einen Einfluß gewinnt. Starke Diarrhöen setzen derartigen Versuchen meist eine Grenze. Versuche. 1. Kaninchen, 1712,0 g: 20. L —28.1. mit Pferdefleisch gefüttert. 1,0 g Pyridin als salzsaures Salz eingeführt. Im Harn nur Pyridin vorhanden. 2. K a n i n c h e n , 2345,0g: 30. L—19. H. mit Milch gefüttert. 1,0 g Pyridin als salzsaures Salz eingeführt. Kein Methylpyridin im Harn. 3. Kaninchen, 2330,0g: 21. V. —21. VII. mit Milch ernährt. Zweimal je l g Pyridin gegeben. Kein Methylpyridin im Harn. Brought to you by | Purdue University Libra Authenticated Download Date | 5/26/15 5:58 AM Über den Stoffwechsel verschiedener Tierarten. II. 135 1. Hund: 28. IV. —1. VII. ausschließlich mit Milch ernährt. Am 2. VII. 1,0 g Pyridin als salzsaures Salz und am 3. VII. wiederum die gleiche Dosis mit der Schlundsonde eingeführt. Methylpyridin im Harn. 5. 14. VII. 09. Zwei junge, ausschließlich mit Milch ernährte Hunde erhalten je 0,25 g salzsaures Pyridin. 15. VII. 09. Wieder je 0,25 g salzsaures Pyridin. 16. VII. 09. > > 0,25 » 17. VII. 09. Die Versuchstiere erbrechen. 18. VII. 09. Je 0,25 g salzsaures Pyridin. 19. VII. 09. » 0,25 » 20. VII. 09. » 0,25 * 21. VII. 09. » 0,25 » Während der ganzen Periode konnte kein Methylpyridin, wohl aher Pyridin im Harn nachgewiesen werden. 22. VII. 09. Die Hunde erhalten gekochtes Pferdefleisch bis zum 9. VIII. 09. 2. VIII. 09—9. VIII. 09. Je 0,25 g salzsaures Pyridin. Der Harn am 2. und 3. VIII. enthielt kein Methylpyridin. Vom 7. VIII. an trat neben freiem Pyridin regelmäßig Methylpyridin auf. Brought to you by | Purdue University Libra Authenticated Download Date | 5/26/15 5:58 AM
https://openalex.org/W2360860258
https://www.shs-conferences.org/articles/shsconf/pdf/2016/02/shsconf_sshe2016_02007.pdf
English
null
Survey on multicultural education situation of schools in Miao nationality areas in south Sichuan province
SHS web of conferences
2,016
cc-by
5,093
DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 201 /201 shsconf 0 00 (201 ) SHS Web of Conferences 0 0 , 2 2 6 6 6 4 4 2 2 07 7 DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 201 /201 shsconf 0 00 (201 ) SHS Web of Conferences 0 0 , 2 2 6 6 6 4 4 2 2 07 7 1 INTRODUCTION For a long time, the research on China’s multicultural education is generally fixed on some “bright spots”, the core areas in minority regions, while the study of multicultural education in scattered and mixed areas seems to have become a “blind spot” to the academic circle. The teacher-and-student structures, school-and- community relations as well as local policies in scat- tered and mixed areas are significantly different with those in core areas. The paths and means in core areas to implement multicultural education are not com- pletely suitable for the school education in scattered and mixed areas. Therefore, for schools in scattered and mixed areas, it is neither a good choice to entirely follow the paths and means in core areas, nor to com- pletely ignore their own responsibility for multicultur- al education. To this end, the author chooses primary and secondary schools in Hele Township of Miao nationality in Xuyong County in Sichuan as a case to do the survey on multicultural education situation. According to the survey, the author found that the education purposes, curriculum plans and teaching methods are not typical features of multicultural edu- Survey on multicultural education situation of schools in Miao ity areas in south Sichuan province Tao Yu1,2 & Yuan Zhang3* 1Center for Studies of Education and Psychology of Minorities in Southwest China, Southwest University, Chongqing, China 2School of Humanities and Management Sciences, Sichuan Medical University, Luzhou, Sichuan, China 3Ideological and Political Department, Sichuan Medical University, Luzhou, Sichuan, China Tao Yu1,2 & Yuan Zhang3* 1Center for Studies of Education and Psychology of Minorities in Southwest China, Southwest University, Chongqing, China 2School of Humanities and Management Sciences, Sichuan Medical University, Luzhou, Sichuan, China 3Ideological and Political Department, Sichuan Medical University, Luzhou, Sichuan, China ABSTRACT: In the long run, the school education in scattered and mixed areas not features with multicultural education, but implements a unified school system as well as knowledge contents. And this unified school system does have some negative impacts on the inheritance of the ethnic culture, the academic achievements and the self-identification of the minority children. Multicultural education is essential to motivate the comprehensive development and freedom of people. And the need to implement multicultural education for schools in scattered Miao Region is more urgent than the centered ethnic minority areas. In order to change this situation, we should change the idea of rationalism monism in education. Keywords: multicultural education; scattered ethnic region; Miao ethnic group; value orientation of education Keywords: multicultural education; scattered ethnic region; Miao ethnic group; value orientation of education cation but tend to be the unified value orientation. *Corresponding author: 184627669@qq.com Article available at http://www.shs-conferences.org or http://dx.doi.org/10.1051/shsconf/20162402007 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited.    SHS Web of Conferences ernment-sponsored festivals of Miao, such as Stepping Mountain Festival and Miao’s customs festival. Every year, Hele Township of Miao nationality holds various cultural festivals with their own features, and has cul- tural and art performances at opening and closing ceremonies. The schools are asked to organize stu- dents to prepare dancing and singing programs em- bodying Miao’s culture. But the schools face difficul- ties in organizing such activities due to the policy issues and lack of funds. And both teachers and stu- dents are perfunctory in the preparation, not treating these festival celebrations seriously as parts of multi- cultural education programs. with Han nationality in some respects such as the use of teaching materials, examination systems and teach- ing methods, using standard teaching materials, par- ticipating in unified examinations, and nearly using the same teaching methods. The survey found that the people there have become the real “minority” in the residential places since the scattered and mixed areas are not the core areas, and their cultural traditions, languages, customs and his- torical knowledge also have been marginalized from local social and cultural structures, constantly sur- rounded by the strong culture (host culture in the resi- dence places). Taking Hele Township of Miao nation- ality as an example, the spread breadth and depth of Miao’ culture in the main group’s (Han nationality) students are far worse than the impact of Han’s culture on Miao students, obviously staying disadvantaged. In language, Miao students are required to master and neatly use Han’s language, characters and related courses, and also use Han’s characters in exams, while Han’s students actually do not have such problems. They generally do not understand Miao’s language and cultural traditions (such as Lusheng, Miao em- broidery, etc.), and are incapable of taking the initia- tive to communicate with Miao People. The one-way cultural output in school education design makes Miao students get used to accepting Han’s culture but lack capacities to output their own culture, resulting in the very obvious cultural convergence and educational homogeneity. Overall, the ethnic education activities in the center school nearly have no connection with local Miao community. The construction of campus culture, the interest groups for Miao’s culture as well as the cele- bration activities related to local Miao’s festivals, all of these are the school’s independent behaviors. SHS Web of Conferences Alt- hough there are teachers and students from both Miao and Han nationality participating in them, the families of Miao students and local Miao People are not di- rectly involved in the school’s ethnic educational ac- tivities. These activities deviate away from the daily life and cultural activities of Miao community, be- longing to a kind of “official” Miao’s cultural educa- tional activity different from local culture. 3 THE NEGATIVE IMPACT OF UNIFIED EDUCATIONAL VALUE ORIENTATION ON MIAO COMMUNITY’S CULTURE 2.2 Multicultural education becomes formalistic and cut from the community culture, not being really internalized into students’ cultural traditions 3.1 School education isolates from community education, even clashes with the community education, accelerating the ablation of Miao’s culture The survey shows that there are three main forms for multicultural education carried out in primary and secondary schools in Hele Township of Miao nation- ality: (1) showing ethnic cultural features in the con- struction of campus culture. For example, organizing students to learn “Bamboo Dance” instead of radio exercise during long breaks, and posting and hanging ornaments with Miao’s cultural features (Lusheng, Ox head, Miao embroidery patterns, etc.); (2) creating extracurricular interest groups related to Miao’s cul- ture. Currently, in the four schools of Hele Township of Miao nationality, only the center school has created three groups of extracurricular activity associated with Miao’s culture, involving Miao’s language, musical instruments of Miao nationality and Lusheng dance. However, according to the survey, although the three interest groups have been set up, there are no full-time instructors, no clear membership structures and no activities till now. That is, these are literally interest groups, not playing any roles in cultural activities on campus. While the other three schools in Hele Town- ship of Miao nationality even have no such formal interest groups; (3) as required by local governments, the schools organize students to participate in gov- There are no written words of Miao’s culture, and most of them are inherited by words of mouth, em- bedded in daily lives, festivals, weddings and funerals, customs and traditions, and passed down by imparting and learning through generations. Therefore, Miao’s culture, under the impact of current dominant culture, is very easy to lose and disappear, although Miao People in scattered and mixed areas are still keeping a clear and strong sense of identity to their local culture to some extent. For instance, in New Year parties, the elders in Miao’s families always like to talk about the resplendence and precious value of their ethnic culture, and constantly tell their children to “be polite” and “do things well”1. However, compared with core areas, the impact of Han’s culture are much stronger and quick- er. First of all, the people in scattered and mixed areas live dispersedly and have worked together with Han People for a long time, suffering a deeper effect of Han’s culture. 2.1 The education homogenization is obvious and the local knowledge becomes aphasiac 2.1 The education homogenization is obvious and the local knowledge becomes aphasiac According to the survey, the primary and secondary schools in Hele Township of Miao nationality have not effectively followed the basic concepts of multi- cultural education to implement education policies and teaching programs in line with their local conditions for the purpose of suiting cultural needs of different nationalities, they instead choose to implement a uni- fied school system as well as knowledge contents. For instance, the curricula, all the school courses in Hele Township of Miao nationality are arranged in accord- ance with currently unified requirements of the state, having no specialized courses manifesting local Mi- ao’s culture, even in music, sports and art, which can reflect cultural features. In a word, there are nearly no contents related to Miao’s music, traditional sport events as well as arts. Besides, they follows the pace This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits distribution, and reproduction in any medium, provided the original work is properly cited.    SSHE 2015 ceremonies, but now they appear in the occasions such as Miao’s cultural festivals, various celebrations and entertainments held by local governments. In the meantime, schools organizing students to play music using Lusheng is just for the purpose of festival cere- monies. It only involves melodies and dance moves, making Lusheng lost its function to transfer Miao’s knowledge and moral culture through funeral and sacrifice rites. Therefore, it’s not surprised that Lusheng lost its status as a representative in traditional Miao’s culture. The second we should worry about is to mechanically introduce Miao’s culture from other areas. For instance, the “Bamboo Dance” which has been adopted as the physical exercise during long break time by the center school in Hele Township. There is a big difference, both physically and spiritu- ally, from the earlier “Bamboo Dance”2 existed in local community. Han’s culture. In the 1930s, Mr. Yifu Rui wrote down his preliminary impression on the Miao nationality in Southern Sichuan after doing the investigation, “The group, Yaque Miao, in terms of the material culture, has been completely influenced to some extent by Han nationality in addition to women’s clothing, and as for the social organization, they have little significant difference from local farmers. However, they still keep a variety of original manners as well as customs” only in the ceremonies in marriage, funeral and wor- ship [1]. In language, all of the adults in Hele Town- ship of Miao nationality are generally able to speak Chinese, while rare Han’s adults know about Miao’s language. Second, the schools in scattered and mixed areas attach great importance to the dominant culture, giv- ing lessons in Chinese, and mainly teaching students Han’s culture and knowledge (Chinese), modern sci- ence (mathematics, biology, physics, chemistry, etc.) as well as social and civic knowledge (life safety, legality, morality, etc.). All of these invisibly force the young generation in Miao nationality to get away from their ethnic culture, making it difficult to be passed down. 3 THE NEGATIVE IMPACT OF UNIFIED EDUCATIONAL VALUE ORIENTATION ON MIAO COMMUNITY’S CULTURE Historically, the entire Miao nationality in Southern Sichuan has been deeply influenced by 02007-p.2 4 THE NEGATIVE IMPACT OF UNIFIED EDUCATIONAL VALUE ORIENTATION ON MIAO STUDENTS The school education under the guidance of monistic theory not only affects the heritage of Miao commu- nity’s culture, but also has a bad influence on Miao students. In the survey, we found that Miao students in scattered and mixed areas of Miao nationality in Southern Sichuan generally have two problems. One is the worse educational background of Miao students. When they enter into the dominant society, they get fewer opportunities to go in upper classes and cannot obtain recognition by the dominant society. After graduating from junior high school or high school, they have to work outside or go back to farm, getting no more chances for social promotion through school education. The other is, since the students have not mastered their ethnic traditional culture in growing years and lacked ethnic self-identity, it is hard for them to get recognized by other ethnic members after returning home, eventually becoming the “cultural marginalized people”. Third, school education occupies most of students’ time, and all of their learning and life have to go around it. To complete the teaching task of the school, teachers and students have to focus on the study, making students have no spare time to receive family and community education, even no consistent time to learn ethnic culture, such as Lusheng, ancient songs, Miao embroidery, etc. The survey found that children in Miao nationality usually go to school at a very early age. Some children accept pre-school education since the age of three, so they have no opportunities and time to learn Lusheng, Miao embroidery, etc. In Hele Township of Miao nationality, the primary and sec- ondary students are rarely capable of playing Lusheng compared with their forefathers. For the adults in local Miao community, parents fully expect their children to inherit the ethnic culture, and become a qualified Miao People to “be polite” and “do things well”. But the current situation of inheriting the traditional culture by teenagers really makes them feel worried but. 4.2 Miao students are prone to suffer cultural identity problems In school education, the unified value orientation makes Miao students can’t get access to the infor- mation related to their own culture, such as Miao’s language, ethnic dances, customs, taboos, ethnic fes- tivals and lack opportunities to acquire their own cul- tural traditions and histories, resulting in the gap be- tween Miao students and their group. Also, it makes them become “outsiders” after growing up and unable to internalize group’s culture, history and traditions as parts of their identity. In recent decades, although Miao groups have established good relations with Han community through long-term exchanges, and the people in Hele Township of Miao nationality have gotten accustomed to communicating with local Han People in a kind of Sichuan dialect (an entering tone spoken in a small independent area in southern Si- chuan) free of obstacles, Miao’s adults still feel a sense of isolation from Han People. In the meantime, the adults in Hele Township of Miao nationality are easier to accept those Han People who can speak sim- ple Miao’s language, considering them as “insiders” and more willing to carry out in-depth conversations and exchanges with them. Obviously, their senses of ethnic identity are very distinct. However, the Miao students accepting school education are more inter- ested in modern civilization and dominant culture (such as computer games, mobile games, popular mu- sic, etc.), not expecting to learn Lusheng and Miao’s language as well as wearing Miao’s clothes. SHS Web of Conferences for their children 3. As for the language, in southern Sichuan, the word “Miao” means negative and usually stands for a strange, brutal and violent personality (for example, if someone is considered as “Miao”, he or she is deemed to be strange and hard to get along with.). To this end, local Miao People are used to calling “Miao nationality” as “the ethnic”, and calling “speaking Miao’s language” as “speaking a kind of ethnic dialect”. Besides, they are quite disgusted to be called “Miao Zi” (negative words) by other nationali- ties. Due to the lack of multicultural education, Miao students can’t correctly understand their own culture and its relationships with other ethnic cultures. It is one of the reasons why they lack the sense of identity and belonging to their own group and ethnic culture, and also why they can’t form a correct sense of iden- tity as an ethnic member. A growing number of Mi- ao’s teenagers can’t establish an effective sense of ethnic recognition and identity. On one hand, they know there is a deep ethnic cultural imprint on them, making them different from local Han People. On the other hand, they can’t accept, respect and love Miao’s culture from their hearts. Since they have not known well about their own culture, they are considered as “outsiders” by other members of the group, living in an awkward “cultural marginalized” situation. Therefore, the school-age children have to face a problem since entering school, namely, mastering a new language in a short time. It directly affects stu- dents’ interest and confidence in learning. Compared with their classmates who come from Han group, they are easier to be self-abased and more prone to be weary of learning, which are obviously not helpful for them to achieve good results in examinations, entrance exams and other competitions. In the long run, after entering the society, Miao students with worse educa- tional background are generally not easy to improve their status through excellent academic performance and get recognition from the dominant society. 5 ENLIGHTENMENT OF MULTICULTURAL EDUCATION ON SCHOOLS IN SCATTERED AND MIXED AREAS 5.1 Multicultural education is the essential demand for individuals to achieve free and comprehensive development 3.2 Heritage of ethnic culture has been misplaced The survey found that although local schools intend to allow students to have more opportunities to experi- ence and understand Miao’s culture, they do not effec- tively investigate the culture in local Miao community in the process of trying to carry out ethnic cultural educational activities, but mechanically introduce cultural traditions from other Miao groups which ac- tually do not suit them, leading to the dislocated her- itage of ethnic culture. The first we should care about is the dislocated heritage in existing culture. In Hele Township of Miao nationality, Lusheng and cow leather drums are only used in funeral and sacrifice To the minority students in scattered and mixed areas, the language, culture and knowledge they receive after entering school are completely different from what they have learned in childhood. The preschoolers in Hele Township learn Miao’s language since they were born, and they usually speak Miao’s language in daily life. Thus they know little about Chinese. However, to boost the merging of Miao students into the dominant culture, schools give lessons in Chinese and require teachers and students to speak Mandarin or Sichuan dialect for communication during break periods. 02007-p.3 SHS Web of Conferences SHS Web of Conferences SSHE 2015 5.2 Scattered and mixed areas are more needed to implement multicultural education concepts than core areas 5.2 Scattered and mixed areas are more needed to implement multicultural education concepts than core areas First, unlike the schools in core areas where most of the students are the minorities, the number of Miao students in the schools in Hele Township of Miao nationality is nearly the same with the number of Han’s students. Thus it is necessary to consider both Miao students and Han’s in terms of teaching materi- als and ethnic cultural courses. Second, according to the survey, due to the lack of ethnic identity and the experience of long-term sufferings of discrimination mentioned above, Miao students are not willing to independently learn and show Miao’s culture in front of Han’s students, such as playing Lusheng, learning Miao’s dance and wearing Miao’s clothes, making students lack motivation in schools’ multicultural education activities. Third, being affected by the dominant culture, Miao students and their families are more inclined to study for entrance exams and future employments. It can be said that they are not very concerned about the ethnic culture taught in school. But, on the other side, Miao People in scattered and mixed areas still have a strong and clear sense of their ethnic culture identity, and very much hope that their children can inherit the ethnic culture and Ābe polite” and “do things well” in the name of a qualified Miao People to. Therefore, the situation in scattered and mixed areas is more in line with the essential require- ments of multiculturalism and multicultural education, and they are more needed to implement the basic con- cepts of multicultural education. 5.1 Multicultural education is the essential demand for individuals to achieve free and comprehensive development Currently, multicultural education in Western coun- tries has already evolved into an effectively national education movement and a necessary mean to promote the healthy development of the multicultural society. For China, it is important to learn ideas and means from western countries combining with the education situations in minority areas to achieve the multicultur- al education. It has been generally agreed that multi- culturalism is human’s essential demand, and also is the prerequisite for human’s independent life, self-determination and self-selection. Further, the purpose of multicultural education is consistent with the essential demand of human’s free and comprehen- sive development, which is the basic idea supposed to be implemented in education. The real multicultural education is the premise for people to live a good, self-determining and prosperous life, and helps people realize their equal status and rights for free develop- ment in the country and society. While for scattered and mixed areas of Miao nationality in southern Si- chuan, first of all, cultivating the capacity of students’ self-selection and self-determination is the primary task of school education, for which the multicultural According to the survey, due to historical reasons, the impact of it still remains even though the discrim- ination of local people against Miao groups has dis- appeared, making Miao People in scattered and mixed areas have less sense of ethnic pride and self-acceptance. For example, the mixed marriage between Han and Miao nationalities in the 556 fami- lies in Fuxing Village in Hele Township of Miao na- tionality, there are only six families choosing to marry with Han People, accounting for 1% in the total num- ber of the families in this village. Despite that the state has special care policies to the minority, two thirds of the six families preferred to choose “Han nationality” 02007-p.4 ACKNOWLEDGEMENT This project is supported by the Center for Rural Childhood Education Research. SSHE 2015 education being implemented by schools must be open, recognizing and clarifying the objectivity of co-existence of local Han’s culture and Miao’s culture, recognizing the equal status of different cultures and the significance to the development of modern society brought by mutual influence and promotion of differ- ent cultures, and offering students necessary and real options for self-selection and self-determination. Sec- ond, the content of multicultural education should be multicultural in some ways. That is, the multicultural education in schools must provide chances to the stu- dents to experience and understand different cultures that exist in local areas, and respect the unique values of different ethnic groups and cultural groups in the ways of life, value orientation and behavior patterns represented by the multi ethnic cultures. And the ob- jects of multicultural education not only include Miao students, but also Han’s and other ethnic students. the school education in scattered and mixed areas of Miao nationality in southern Sichuan can’t bear the primary responsibility of multicultural education, and the ethnic education activities are mostly formalistic or perfunctory. Besides, those activities have been separated for a long time from the ethnic community life, even clashed with it. For all of these, we can find out many reasons such as the lack of policies, schools’ weak consciousness for multicultural education, or the neglect of multicultural education by Miao students and their family. However, from a more deeply aspect, all of the phenomena neglecting the multicultural ed- ucation are caused by a fundamentally thinking mode, namely the rationalistic monism which underlies the educational value orientation of schools in scattered and mixed areas of Miao nationality in southern Si- chuan, and it has become the ultimate guiding idea that determines the educational methods, tools and purposes. In other words, for schools, multicultural education is just the surface, but the educational thoughts based on monistic theory actually play a fundamental role in local education system. For stu- dents, it is still a long way to cultivate the inclusive, open-minded and independent outlook on value. Cur- rently, the educational idea based on monism is con- tinued to be implemented, and its adverse conse- quences are increasingly prominent, even aggravated, producing a seriously adverse effect both on the cul- tural heritage of Miao community and the personal growth of Miao students in scattered and mixed areas in Southern Sichuan. SHS Web of Conferences Information Table of Fuxing Village in Hele Town- ship of Miao Nationality”. Deadline: May 21, 2014. presents. In the process of “giving presents”, the guest who gave presents and the internal director sang and responded to each other, occasionally lasting for an hour. Sometimes, there were lots of guests, so they had to wait for a long time outside the house to give the present. Meanwhile, their presents would always block the way so that people had to jump across the bamboo poles (being used to place the presents). Gradually, it evolved into “Bamboo Dance” along with the melodies of Lusheng. “Bamboo Dance” was to lay the bamboo or bamboo poles parallel on the ground, and people walked and danced, jumping across the bamboo or bamboo poles. It is obviously different from the modern “Bamboo Dance” which needs someone to lift, down, separate and close the bamboo, and in the meantime dancers jump and move along with the opening and closing rhythm. Li once has seen the old “Bamboo Dance” in the participation of Stepping Mountain Festival in childhood (about 40 years ago), but now nobody can dance. Dictation time: September 17, 2014. Information Table of Fuxing Village in Hele Town- ship of Miao Nationality”. Deadline: May 21, 2014. presents. In the process of “giving presents”, the guest who gave presents and the internal director sang and responded to each other, occasionally lasting for an hour. Sometimes, there were lots of guests, so they had to wait for a long time outside the house to give the present. Meanwhile, their presents would always block the way so that people had to jump across the bamboo poles (being used to place the presents). Gradually, it evolved into “Bamboo Dance” along with the melodies of Lusheng. “Bamboo Dance” was to lay the bamboo or bamboo poles parallel on the ground, and people walked and danced, jumping across the bamboo or bamboo poles. It is obviously different from the modern “Bamboo Dance” which needs someone to lift, down, separate and close the bamboo, and in the meantime dancers jump and move along with the opening and closing rhythm. Li once has seen the old “Bamboo Dance” in the participation of Stepping Mountain Festival in childhood (about 40 years ago), but now nobody can dance. Dictation time: September 17, 2014. NOTES 1. The people in Hele Township of Miao nationality generally call the sacrifice rites like “uat bles (A kind of superstitious activity in funeral ceremonies asking monks or Taoists to recite scriptures to save the dead from misery)” and “uat beuk” as “do things”. “Do things well” means to understand various rites in funeral ceremonies, and it also means being able to participate in the specific matters such as playing Lusheng and saying sacrifice words in sacrifice rites. Such kind of people are respected by family. 1. The people in Hele Township of Miao nationality generally call the sacrifice rites like “uat bles (A kind of superstitious activity in funeral ceremonies asking monks or Taoists to recite scriptures to save the dead from misery)” and “uat beuk” as “do things”. “Do things well” means to understand various rites in funeral ceremonies, and it also means being able to participate in the specific matters such as playing Lusheng and saying sacrifice words in sacrifice rites. Such kind of people are respected by family. 2. According to X Li’s words of mouth, a villager from Miao nationality, the “Bamboo Dance” in Hele Township of Miao nationality originated in the process of “giving presents” in marriage and funeral ceremonies. In the marriage and funeral ceremonies, the guests brought some presents, such as corns, rice, wine, etc., and the host asked a specific person (called “internal director”) to be responsible for handling the 5.3 The lack of features of multicultural education in school education at scattered and mixed areas is fundamental because of the impact of rationalistic monism 5.3 The lack of features of multicultural education in school education at scattered and mixed areas is fundamental because of the impact of rationalistic monism According to the survey of fieldwork, we can see that According to the survey of fieldwork, we can see that 02007-p.5 REFERENCES [1] Yifu Rui & Donggui Guan. 1941. The Information about Marriage and Funeral Rites in Yaque Miao Nationality in Southern Sichuan. Taipei, Taiwan: the Twenty-three of A-Type of Single Publication by History and Lan- guage Institution of Chinese Academia. pp.3. [1] Yifu Rui & Donggui Guan. 1941. The Information about Marriage and Funeral Rites in Yaque Miao Nationality in Southern Sichuan. Taipei, Taiwan: the Twenty-three of A-Type of Single Publication by History and Lan- guage Institution of Chinese Academia. pp.3. [2] Berlin Isaiah. 2009. Crooked Timber of Humanity. Translated by Xiukun Yue. Nanjing: Yilin Press. [3] James A Banks. 1994. An Introduction to Multicultural Education. Boston: Allyn and Bacon. [4] Fang Liu. 2009. The Context of cultural factors in changes in scattered and mixed areas of minorities -survey and analysis of Jiancao Gaoshan Miao National- ity in Xuyong County in Sichuan by Ye Tian. Journal of Southwest University for Nationalities (Humanities and Social Sciences Edition), (10). [4] Fang Liu. 2009. The Context of cultural factors in changes in scattered and mixed areas of minorities -survey and analysis of Jiancao Gaoshan Miao National- ity in Xuyong County in Sichuan by Ye Tian. Journal of Southwest University for Nationalities (Humanities and Social Sciences Edition), (10). [5] Yuan Zhang & Tao Yu. 2013. Analysis of the minority on safeguarding their rights through the movie Warriors of the Rainbow: Seediq Bale. Guizhou Ethnic Studies. [5] Yuan Zhang & Tao Yu. 2013. Analysis of the minority on safeguarding their rights through the movie Warriors of the Rainbow: Seediq Bale. Guizhou Ethnic Studies. 3. The statistic data come from “Regular Residence 02007-p.6
https://openalex.org/W2904249487
http://scholar.colorado.edu/downloads/r207tq060
English
null
Intratumoral heterogeneity of endogenous tumor cell invasive behavior in human glioblastoma
Scientific reports
2,018
cc-by
8,682
Authors Jonathon J Parker, Peter Canoll, Lee Niswander, B K Kleinschmidt-DeMasters, Kara Foshay, and Allen Waziri Intratumoral heterogeneity of endogenous tumor cell invasive behavior in human glioblastoma Received: 1 August 2018 Accepted: 9 November 2018 Published: xx xx xxxx Received: 1 August 2018 Accepted: 9 November 2018 Published: xx xx xxxx Jonathon J. Parker1, Peter Canoll2, Lee Niswander3, B. K. Kleinschmidt-DeMasters4,5, Kara Foshay6 &AllenWaziri6 Intratumoral genetic heterogeneity is a widely accepted characteristic of human cancer, including the most common primary malignant brain tumor, glioblastoma. However, the variability in biological behaviors amongst cells within individual tumors is not well described. Invasion into unaffected brain parenchyma is one such behavior, and a leading mechanism of tumor recurrence unaddressed by the current therapeutic armamentarium. Further, providing insight into variability of tumor cell migration within individual tumors may inform discovery of novel anti-invasive therapeutics. In this study, ex vivo organotypic slice cultures from EGFR-wild type and EGFR-amplified patient tumors were treated with the EGFR inhibitor gefitinib to evaluate potential sub-population restricted intratumoral drug-specific responses. High-resolution time-lapse microscopy and quantitative path tracking demonstrated migration of individual cells are punctuated by intermittent bursts of movement. Elevation of population aggregate mean speeds were driven by subpopulations of cells exhibiting frequent high- amplitude bursts, enriched within EGFR-amplified tumors. Treatment with gefitinib specifically targeted high-burst cell subpopulations only in EGFR-amplified tumors, decreasing bursting frequency and amplitude. We provide evidence of intratumoral subpopulations of cells with enhanced migratory behavior in human glioblastoma, selectively targeted via EGFR inhibition. These data justify use of direct human tumor slice cultures to investigate patient-specific therapies designed to limit tumor invasion. The innate ability of glioblastoma to infiltrate normal brain is a clinical challenge, which limits efficacy of surgi- cal resection, radiotherapy, and chemobiotherapies. Consortium based efforts utilizing large-scale data analyses reveal extensive GBM heterogeneity at the inter-tumoral level, and several molecular subtypes have been defined based on commonly observed genetic and epigenetic changes1,2. While detection of IDH1 mutation and/or meth- ylation of the MGMT promoter are now correlated to increased overall survival3–6, the prognostic value of other common genetic mutations, including amplification of the EGFR locus, remains unclear7. The ability to under- stand the network of connections between genetic heterogeneity, tumor cell phenotype, and disease progression, has potential to improve therapeutic targeting via increasing accuracy of predictions of drug response. p p p g g g y p g p Our lab recently demonstrated phenotypic heterogeneity in GBM migratory potential, which correlates to patient-specific EGFR amplification status. Authors www.nature.com/scientificreports www.nature.com/scientificreports Received: 1 August 2018 Accepted: 9 November 2018 Published: xx xx xxxx Intratumoral heterogeneity of endogenous tumor cell invasive behavior in human glioblastoma Amplification at this locus, which is detected in 40–50% of GBM tissues8, is typically mosaic and believed to enhance pro-invasive signaling through EGFR. Interestingly, clinical imaging suggests this subset of receptor-amplified cells is enriched at the infiltrative tumor edge9,10. Supporting these data, our ex vivo slice cultures demonstrated increased tumor cell migration in EGFR-amplified tumors, and blockade of EGFR signaling with the small molecule inhibitor, gefitinib, induced a statistically significant reduc- tion in migratory behavior within the same sample set11. Identification of this link between genetic heterogeneity and tumor cell behavior provides a paradigm for the mechanistic link between molecular variation and cellular behavioral changes which dictate therapeutic response. 1Stanford University School of Medicine, Department of Neurosurgery, Stanford, CA, USA. 2Columbia University College of Physicians and Surgeons, Department of Pathology and Cell Biology, New York, NY, USA. 3University of Colorado, Department of Molecular, Cellular & Developmental Biology, Boulder, CO, USA. 4University of Colorado School of Medicine, Department of Pathology, Anschutz Medical Campus, Aurora, CO, USA. 5University of Colorado School of Medicine, Department of Neurosurgery, Anschutz Medical Campus, Aurora, CO, USA. 6Inova Neuroscience and Spine Institute, Inova Health Systems, Falls Church, VA, USA. Correspondence and requests for materials should be addressed to K.F. (email: kara.foshay@inova.org) SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 1 www.nature.com/scientificreports/ Despite evidence supporting the pro-migratory role of EGFR in GBM progression, trials of gefitinib and other targeted receptor tyrosine kinase (RTK) therapies, alone or in combination, failed to extend patient survival12. This disconnect between in vitro drug studies and in vivo efficacy led the field to consider the prevalence of molecular heterogeneity within individual tumors as a mechanism of treatment resistance. Integrated analysis of primary GBM revealed significant gene expression changes within samples isolated from different regions of the same tumor13. These findings were confirmed at the cellular level through single-cell RNA-seq, which identified cell-to-cell variation in regulation of growth, metabolism, and immune response trasncripts14. More recently, single-cell sequencing highlighted differential expression in cells of the tumor core as compared to those of the infiltrated penumbra15. i p To date, the extent to which genetically or epigenetically distinct subsets of cells, present within individual human GBM tumors, contribute to overall variation in cell behavior and drug response in vivo remains unclear. Results H Time-lapse microscopy revealed cell-to-cell variability in direction- ality, and in 5 out of 7 tumors analyzed we found a significant positive correlation with mean migration speed (Spearman r ranged 0.45 to 0.65, mean 0.56) (Fig. 1D, GBM-8 shown). Thus, the fastest migrating tumor cells are also the most efficient in their tissue-traversing path. These data confirm the existence of cell-to-cell heterogeneity in patterns of migration with dynamic evidence shown for the first time within patient-derived living GBM tissue. While the observation of intratumoral migratory heterogeneity in human GBM is novel, tissue level spatial variation in proliferative behavior has been reported around histological structures24. While PDGF-driven mouse models suggest tumor cells simultaneously maintain high rates of proliferation and migration23, the “go or grow” hypothesis suggests mitotically active tumor cells are less migratory25. Thus, we used time-lapse images of active cell division to evaluate mitotic variation as one plausible explanation for observed migrational heterogeneity in our slice model (Fig. 1E). Dividing tumor cells paused only transiently from their migratory program, retracted filopodia, completed mitosis, and re-initiated migration in less than 3 hours (Fig. 1E). Cell division is less preva- lent in human tumor slice cultures as compared to mouse models, but observed mitotic events were not restricted spatially, and recurrent divisions in individual cells were not observed during the imaging period. These data suggest cell division does not explain the “pause and burst” pattern of movement, or the widespread intratumoral heterogeneity observed with respect to migration speed. i While the observation of intratumoral migratory heterogeneity in human GBM is novel, tissue level spatial variation in proliferative behavior has been reported around histological structures24. While PDGF-driven mouse models suggest tumor cells simultaneously maintain high rates of proliferation and migration23, the “go or grow” hypothesis suggests mitotically active tumor cells are less migratory25. Thus, we used time-lapse images of active cell division to evaluate mitotic variation as one plausible explanation for observed migrational heterogeneity in our slice model (Fig. 1E). Dividing tumor cells paused only transiently from their migratory program, retracted filopodia, completed mitosis, and re-initiated migration in less than 3 hours (Fig. 1E). Cell division is less preva- lent in human tumor slice cultures as compared to mouse models, but observed mitotic events were not restricted spatially, and recurrent divisions in individual cells were not observed during the imaging period. Intratumoral heterogeneity of endogenous tumor cell invasive behavior in human glioblastoma However, in the PDGF-driven rat glioma model, two distinct tumor-associated cell populations exhibit disparate migratory potentials in response to PDGF secretion, suggesting that a particular sub-population can dominantly contribute to the invasiveness of the tumor, as a whole16. Indeed, differential amplification of RTKs, including EGFR, PDGFR, and MET was observed within tumor cells isolated from distinct regions of multifocal GBM in individual patients9,17,18. Our previous studies utilized low-resolution path-tracking that was sufficient to detect inter-patient but not intratumoral migrational heterogeneity. We hypothesize that intratumoral molecular heter- ogeneity may manifest as measurable differences in migratory potential within human GBM cell subpopulations. g y yf g y p p p In the current study, we perform high temporal resolution path-tracking analysis to gain insight into the divergence of migratory behavior within individual tumors. We demonstrate the presence of small, fast moving subpopulations of cells that dictate overall tumor invasiveness. Interestingly these fast cells are more prevalent within EGFR-amplified tissues and, preferentially respond to gefitinib, emphasizing the clinical implications of intratumoral heterogeneity. Whether molecularly distinct subpopulations arise early in disease progression, or from treatment-resistant cells, their presence necessitates consideration in predicting response to, and failure of, targeted therapeutics. Results H Human glioblastoma slice cultures reveal intratumoral variation in cell migration. While cell- to-cell variation in gene expression and receptor tyrosine kinase amplification within GBM are well recognized, it remains unclear how genetically and epigenetically distinct subpopulations contribute to variation in cell behav- ior. We hypothesize that intratumoral molecular heterogeneity manifests as quantifiable variation in migratory behavior within the tumor microenvironment. Indeed, our previous analysis of 20 human GBM tumors revealed inter-patient differences in cell migration and highly variable migration patterns within individual tumors, sug- gesting intrinsic heterogeneity in migratory potential from cell to cell (Fig. 1A,B)11. To assess patterns of migration in individual cells, we tracked cell movement over 11 hours using time-lapse confocal microscopy of human GBM slice cultures. Proliferative cells within the tumor were preferentially labeled by transduction with an MMLV-retroviral vector constitutively expressing ZsGreen. Migrating tumor cells moved intermittently, pausing to dynamically probe the environment with filopodia, followed by bursts of rapid move- ment mediated by filopodia retraction and nuclear deformation. This saltatory pattern of movement was previ- ously described in neural progenitors cells, GBM xenografts, and PDGF-driven GBM mouse models, suggesting it may be intrinsic to GBM invasion19–23. Notably, migratory cells moved at high speeds passing stationary cells, which did not move over the imaging time frame, resulting in single-cell migration speeds that varied widely. When represented graphically, mean speeds approximate a log normal distribution with a long tail suggestive of a small subpopulation of fast migrating cells (Fig. 1C). In addition to speed, a cell’s directionality, described by the ratio of cell displacement over total distance traveled, contributes to migration efficiency. Time-lapse microscopy revealed cell-to-cell variability in direction- ality, and in 5 out of 7 tumors analyzed we found a significant positive correlation with mean migration speed (Spearman r ranged 0.45 to 0.65, mean 0.56) (Fig. 1D, GBM-8 shown). Thus, the fastest migrating tumor cells are also the most efficient in their tissue-traversing path. These data confirm the existence of cell-to-cell heterogeneity in patterns of migration with dynamic evidence shown for the first time within patient-derived living GBM tissue. In addition to speed, a cell’s directionality, described by the ratio of cell displacement over total distance traveled, contributes to migration efficiency. Results H These data suggest cell division does not explain the “pause and burst” pattern of movement, or the widespread intratumoral heterogeneity observed with respect to migration speed. Bursts of tumor cell movement contribute to increased overall migration speed. Our studies confirmed significant heterogeneity in the mean speed and directionality of cells migrating within GBM tissue. Given the “pause and burst” nature of tumor cell migration, we postulated that the frequency and amplitude of bursts, may contribute significantly to overall cell speed. Indeed, others have suggested that high speed move- ments are predictive of migratory behavior over long cell tracks26 and of overall tumor invasiveness22. p g y g To explore variation in bursting behavior we performed high temporal resolution tracking of tumor in our cohort of GBM slice cultures (n = 7, Supplemental Fig. 1C). The instantaneous speed of each cell SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. High temporal resolution path tracking highlights intratumoral heterogeneity of speed and directionality of tumor cell migration in focal micro-regions. (A) 10x field view of tumor cells in GBM-8 by transduction of an MMLV-retroviral vector constitutively expressing ZsGreen, and (B) accompanying migration paths after 11 hours of imaging. Scale bars represent 50 μm. Tumor cell location was tracked e 11 minutes. Cells with long, direct paths are observed migrating past stationary cells. (C) Cell speeds are distributed log-normally (R2 = 0.95) in this representative population (GBM-8). The red line represents t fit regression to a log-normal base model. (D) Correlation between directionality and speed in a represen tumor (GBM-8, r = 0.65, p < 0.0001). Similar results were obtained in 5 out of 7 tumors analyzed (Spearm r correlation coefficient ranged from 0.45 to 0.65). (E) A representative tumor cell tracked over 10 hours, in 55-minute intervals, demonstrating rapid cell division during migration (GBM-13), coupled with the instantaneous speed over migration time for this cell (right). Figure 1. High temporal resolution path tracking highlights intratumoral heterogeneity of speed and directionality of tumor cell migration in focal micro-regions. (A) 10x field view of tumor cells in GBM-8 labeled by transduction of an MMLV-retroviral vector constitutively expressing ZsGreen, and (B) accompanying migration paths after 11 hours of imaging. Scale bars represent 50 μm. Tumor cell location was tracked every 11 minutes. Cells with long, direct paths are observed migrating past stationary cells. Results H (C) Cell speeds are distributed log-normally (R2 = 0.95) in this representative population (GBM-8). The red line represents the best fit regression to a log-normal base model. (D) Correlation between directionality and speed in a representative tumor (GBM-8, r = 0.65, p < 0.0001). Similar results were obtained in 5 out of 7 tumors analyzed (Spearman r correlation coefficient ranged from 0.45 to 0.65). (E) A representative tumor cell tracked over 10 hours, in 55-minute intervals, demonstrating rapid cell division during migration (GBM-13), coupled with the instantaneous speed over migration time for this cell (right). Fi 1 Hi h t l l ti th t ki hi hli ht i t t l h t it f d d Figure 1. High temporal resolution path tracking highlights intratumoral heterogeneity of speed and directionality of tumor cell migration in focal micro-regions. (A) 10x field view of tumor cells in GBM-8 labeled by transduction of an MMLV-retroviral vector constitutively expressing ZsGreen, and (B) accompanying migration paths after 11 hours of imaging. Scale bars represent 50 μm. Tumor cell location was tracked every 11 minutes. Cells with long, direct paths are observed migrating past stationary cells. (C) Cell speeds are distributed log-normally (R2 = 0.95) in this representative population (GBM-8). The red line represents the best fit regression to a log-normal base model. (D) Correlation between directionality and speed in a representative tumor (GBM-8, r = 0.65, p < 0.0001). Similar results were obtained in 5 out of 7 tumors analyzed (Spearman r correlation coefficient ranged from 0.45 to 0.65). (E) A representative tumor cell tracked over 10 hours, in 55-minute intervals, demonstrating rapid cell division during migration (GBM-13), coupled with the instantaneous speed over migration time for this cell (right). calculated utilizing high granularity tracking, every 11 minutes, and plotted in three dimensions to render a sur- face topography map (Fig. 2A,B). Within each tumor analyzed, we noted high-amplitude peaks that represent bursts of migration much faster than “baseline” migration speed. Some cells had numerous high amplitude peaks, calculated utilizing high granularity tracking, every 11 minutes, and plotted in three dimensions to render a sur- face topography map (Fig. 2A,B). Within each tumor analyzed, we noted high-amplitude peaks that represent bursts of migration much faster than “baseline” migration speed. Some cells had numerous high amplitude peaks, SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 3 www.nature.com/scientificreports/ Figure 2. Results H Surface topography analysis highlights a subpopulation of glioblastoma cells with increased bursting activity. (A,C). Line based schematics of differences in Euclidean distance traversed per unit time during a burst of cell migration (red line). (B) Surface topography map demonstrates bursting behavior of cells from a representative tumor cell population (GBM-8 shown, analysis performed in all tumors n = 7). Peak height represents speed (μm/hr) with a color-coded heat map (right). (D). Cells with frequent bursting behavior (increased standard deviation of instantaneous speed) represent a small subpopulation within the tumor. (E) There is a positive correlation between standard deviation of instantaneous speed and mean effective speed for individual cells (Spearman correlation coefficient, r = 0.93, p < 0.0001). Figure 2. Surface topography analysis highlights a subpopulation of glioblastoma cells with increased bursting activity. (A,C). Line based schematics of differences in Euclidean distance traversed per unit time during a burst of cell migration (red line). (B) Surface topography map demonstrates bursting behavior of cells from a representative tumor cell population (GBM-8 shown, analysis performed in all tumors n = 7). Peak height represents speed (μm/hr) with a color-coded heat map (right). (D). Cells with frequent bursting behavior (increased standard deviation of instantaneous speed) represent a small subpopulation within the tumor. (E) There is a positive correlation between standard deviation of instantaneous speed and mean effective speed for individual cells (Spearman correlation coefficient, r = 0.93, p < 0.0001). while other cells had few or none (Fig. 2C and Supplemental Fig. 1A,B). This variability, or oscillation from “base- line”, can be represented by the standard deviation of the instantaneous speed. Cells with the greatest standard deviation in instantaneous speed, or the largest bursts of movement, represented less than 5 percent of the total population (Fig. 2D). Among these cells, there was a positive correlation between standard deviation of instan- taneous speed and effective migration speed of the cell (r = 0.93, p < 0.0001) (Fig. 2E). These data confirm that the bursting behavior of a small population of fast cells within the tumor contributes to effective migration and overall tumor speed. EGFR amplification contributes to increased intratumoral heterogeneity with respect to cell migration. Genomic amplification of the WT EGFR receptor is common in GBM and displays intratumoral cell-to-cell heterogeneity. Our previous work demonstrated that despite heterogeneity, EGFR-amplified tumors migrate faster than EGFR non-amplified tumors at the population level11. Results H Therefore, we sought to determine whether EGFR amplification correlated with heterogeneity in individual tumor cell migration patterns. Again using instantaneous speed surface topography plots, we identified significantly more high amplitude peaks for cells tracked in EGFR-amplified tumors (Fig. 3A, GBM-8 shown), representing an increased percentage of fast migrating cells when compared to non-amplified cells (Fig. 3B, GBM-5 shown). Across all tumors, these data support an association between EGFR amplification and fast migratory behavior of cells, which contributes to overall tumor invasiveness.if Gefitinib, a known inhibitor of EGFR signaling, selectively decreases the overall effective migration speed of EGFR-amplified tumors11. This prompted us to investigate whether targeting EGFR with gefitinib alters migra- tion parameters within slice cultures on a per cell basis (n = 6 individual tumor slice cultures). Utilizing surface topography analysis, we observed that gefitinib treatment qualitatively decreased high amplitude peaks only in EGFR-amplified tumors (Fig. 3C,D). Quantitatively, there was no change in the overall number of observed cell movements. However, we observed a nearly 50% decrease in the frequency of high-amplitude peaks per cell (over 20 μm/hr) in gefitinib treated EGFR-amplified tumors, suggesting this molecule can effectively slow migration of fast moving cells (Fig. 3E,F). In amplified tumors, we identified a significant decrease in the standard deviation of instantaneous speed and in the distance traveled by each tumor cell, per peak, with no significant effects observed SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 4 www.nature.com/scientificreports/ Figure 3. Gefitinib treatment disrupts high-speed burst behavior selectively among tumor cell populations w EGFR-amplified glioblastoma. (A–D). Topography graphs of instantaneous tumor cell speed over time, wher rows represent individual cells. For all treated samples (n = 6) cells were tracked every 11 minutes and ordered increasing standard deviation of instantaneous speed. Peak height and color-coded heat-map shading both re cell speed, representative data from GBM-5 and GBM-8 are shown. Gefitinib treatment (10 μM) for 11 hours, in a qualitative decrease in high-amplitude peaks in an EGFR-amplified slice (GBM-8, A,C). Non-significant were observed after treatment of a non-amplified tumor slice (GBM-5, B,D). (E) Movements per hour (i.e. pe did not change between control (DMSO, black bars) and gefitinib (grey bars) treatment in pooled cell populat Figure 3. Gefitinib treatment disrupts high-speed burst behavior selectively among tumor cell populations within EGFR-amplified glioblastoma. (A–D). Topography graphs of instantaneous tumor cell speed over time, where rows represent individual cells. Discussion An increasing wealth of in vitro and in vivo data from human tissue describes glioblastoma as a paradigm of continually evolving heterogeneity at the cellular, genomic, transcriptomic, and proteomic levels14,28–30. Thus, highlighting a widely supported mechanism underlying treatment resistance and disease recurrence that plagues GBM patients. Efforts to trace the evolution of GBM reveal that many mutations present in the initial tumor are not present in secondary tumors and satellite lesions31,32. Not surprisingly, population level molecular char- acterization of primary GBM tumors, which is now widespread, has little impact on clinical treatment plan or improving patient prognosis.f Even as our understanding of molecular heterogeneity grows, tumor cell invasion remains a barrier to effective treatment in all patients, and no approved therapeutics limit this behavior. Our GBM slice culture model focuses on observation of these clinically important invasive behaviors within the native tumor microenvironment. For the first time, we present dynamic data demonstrating intrinsic heterogeneity in tumor cell migration within liv- ing human GBM tissue, which is characterized by juxtaposition of migratory and non-migratory cells, along with variations in speed, directionality, and temporal bursts of increased migration. p y p g We suspect heterogeneity in migratory behavior is a manifestation of the evolving genomic landscape as GBM progresses, and thus could influence clinical prognosis and therapeutic efficacy. Cells within a given GBM population often harbor amplification of up to 3 oncogenic receptor tyrosine kinases, but the majority of cells demonstrate mutually exclusive receptor amplification17,18,33. While amplification and mutation of the EGFR locus is the most common genetic alteration in GBM8,34, recent phylogenetic analysis of primary tissue suggests this may be a late-occurring change32, enriched within cells near the infiltrative tumor edge or within satellite lesions9,10,33. These data suggest EGFR may drive invasion but not gliomagenesis. Indeed, exogenous overexpres- sion of WT-EGFR in oligodendrocyte precursor cells in vivo is sufficient to drive cell motility, but insufficient for reliable tumorigenesis35.i Human tumors harboring EGFR-amplification are more invasive and demonstrate increased overall migration speed in slice cultures11. Our current study reveals a small subpopulation of cells within EGFR-amplified tum- ors, characterized by high mean speeds driven by bursting behavior. These short periods of movement correlate with more effective migration, as measured by directionality and displacement, and contribute to widespread heterogeneity in migratory parameters. www.nature.com/scientificreports/ in non-amplified tumors (Fig. 3G,H). Combined, these data indicate that gefitinib blocks an EGFR dependent bursting behavior present within a subset of migratory tumor cells.ii g p g y Within EGFR-amplified tumors, cells possess varying levels of receptor amplification and EGFR receptor activation27,28, which may explain in part the observed heterogeneity in migratory behaviors. To assess heter- ogeneity across samples, we plotted individual cells, from either EGFR-amplified or non-amplified tumors, as points defined by two behavioral characteristics of migration. Displacement was used to capture the effective migration potential of the cell, and standard deviation of instantaneous speed served as a surrogate of the cell’s bursting behavior. Inhibition of EGFR reduced fast bursting behavior in a subset of cells, thus, we hypothesized that overall population heterogeneity based on these migration parameters would decrease accordingly with gefitinib treatment.ii gi In both EGFR-amplified and non-amplified tumors there was a linear relationship between displacement and standard deviation of instantaneous speed, again suggesting that cell bursting behavior contributes to effective migration. However, the distribution of cells in EGFR-amplified tumors was elongated, with more cells falling in the right upper quadrant (both high effective speed and high amplitude movement bursts), representing an aug- mented subset of cells with enhanced migration potential. In non-amplified tumors treated with gefitinib there was no obvious change in the shape of this distribution, or the centroid position of the population (defined by the mean values for each parameter) (Fig. 4A,B). In striking contrast, treatment of EGFR-amplified tumors with gefitinib induced a visible downward shift in the shape of the distribution and in the centroid position (Fig. 4A,B). The change in heterogeneity was assessed using Levene’s test for variance equality. In EGFR-amplified tumors, the variance of displacement was significantly decreased (Levene’s statistic, 10.5, p = 0.0013) along with the standard deviation of instantaneous speed for each cell (15.2, p = 0.0001). However, among non-amplified tumors there was no significant change in the variance of displacement (Levene’s statistic 1.2, p = 0.27) or standard deviation of instantaneous speed (1.6, p = 0.20).i p p As predicted, cells from EGFR-amplified tumors appear more heterogeneous in their baseline migratory behavior, with an expansion in the sub-population of migrating tumor cells that travel the farthest and fastest. www.nature.com/scientificreports/ The presence of this key subpopulation is selectively decreased by gefitinib treatment, supporting the notion that drugs targeting a single pro-migratory pathway in GBM, may have limited effects on the overall population of tumor cells. Although, experiments to confirm that the subpopulation of cells harboring amplification of the EGFR locus corresponds specifically to the affected subpopulation of highly invasive cells are underway, our data highlight the potential relevance of personalized therapeutics based upon patient-specific genetic alterations. Results H For all treated samples (n = 6) cells were tracked every 11 minutes and ordered by increasing standard deviation of instantaneous speed. Peak height and color-coded heat-map shading both represent cell speed, representative data from GBM-5 and GBM-8 are shown. Gefitinib treatment (10 μM) for 11 hours, resulted in a qualitative decrease in high-amplitude peaks in an EGFR-amplified slice (GBM-8, A,C). Non-significant changes were observed after treatment of a non-amplified tumor slice (GBM-5, B,D). (E) Movements per hour (i.e. peaks/hr) did not change between control (DMSO, black bars) and gefitinib (grey bars) treatment in pooled cell populations from all EGFR-amplified or non-amplified tumors (p > 0.05, n = 3 amplified tumors, n = 3 non-amplified tumors). (F–H) High-speed movements, standard deviation of instantaneous speed, and distance traveled by the cell per movement were significantly decreased in EGFR-amplified tumors (n = 3, p < 0.05), with no effect in non-amplified tumors (n = 3, p > 0.05). Error bars represent standard error of the mean (SEM). Figure 3. Gefitinib treatment disrupts high-speed burst behavior selectively among tumor cell populations within EGFR-amplified glioblastoma. (A–D). Topography graphs of instantaneous tumor cell speed over time, where rows represent individual cells. For all treated samples (n = 6) cells were tracked every 11 minutes and ordered by increasing standard deviation of instantaneous speed. Peak height and color-coded heat-map shading both represent cell speed, representative data from GBM-5 and GBM-8 are shown. Gefitinib treatment (10 μM) for 11 hours, resulted in a qualitative decrease in high-amplitude peaks in an EGFR-amplified slice (GBM-8, A,C). Non-significant changes were observed after treatment of a non-amplified tumor slice (GBM-5, B,D). (E) Movements per hour (i.e. peaks/hr) did not change between control (DMSO, black bars) and gefitinib (grey bars) treatment in pooled cell populations from all EGFR-amplified or non-amplified tumors (p > 0.05, n = 3 amplified tumors, n = 3 non-amplified tumors). (F–H) High-speed movements, standard deviation of instantaneous speed, and distance traveled by the cell per movement were significantly decreased in EGFR-amplified tumors (n = 3, p < 0.05), with no effect in non-amplified tumors (n = 3, p > 0.05). Error bars represent standard error of the mean (SEM). SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 5 www.nature.com/scientificreports/ SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 Discussion (B) Cell population centroids (bars represent standard deviation) are coincident in receptor non-amplified tumors (left) whereas the population centroid moves to the lower left in amplified tumors (right) after gefitinib treatment. Figure 4. Gefitinib treatment selectively disrupts EGFR-Amplification migration behaviors at the tumor cell population level. High temporal resolution cell path tracking occurred every 11 minutes in EGFR-amplified (n = 3) and non-amplified tumors (n = 3). Cells from were pooled across tumors for analysis under control (152 EGFR-amp cells, 162 non-amp) and gefitinib (10 μM) treatment conditions (129 EGFR-amp, 163 non-amp), and were grouped based on tissue level receptor amplification status of the originating tumor. Error bars represent standard deviation (SD). (A) Individual cells are displayed according to displacement in micrometers (i.e. total distance traveled during imaging) and standard deviation of instantaneous speed (a metric of an individual cell’s speed variability at anytime during migration) from receptor non-amplified slices (left) demonstrate minimal change in migratory properties despite gefitinib treatment. A dropout of cells with efficient and increased bursting behavior is observed in EGFR-amplified slices (right). (B) Cell population centroids (bars represent standard deviation) are coincident in receptor non-amplified tumors (left) whereas the population centroid moves to the lower left in amplified tumors (right) after gefitinib treatment. Figure 4. Gefitinib treatment selectively disrupts EGFR-Amplification migration behaviors at the tumor cell population level. High temporal resolution cell path tracking occurred every 11 minutes in EGFR-amplified (n = 3) and non-amplified tumors (n = 3). Cells from were pooled across tumors for analysis under control (152 EGFR-amp cells, 162 non-amp) and gefitinib (10 μM) treatment conditions (129 EGFR-amp, 163 non-amp), and were grouped based on tissue level receptor amplification status of the originating tumor. Error bars represent standard deviation (SD). (A) Individual cells are displayed according to displacement in micrometers (i.e. total distance traveled during imaging) and standard deviation of instantaneous speed (a metric of an individual cell’s speed variability at anytime during migration) from receptor non-amplified slices (left) demonstrate minimal change in migratory properties despite gefitinib treatment. A dropout of cells with efficient and increased bursting behavior is observed in EGFR-amplified slices (right). (B) Cell population centroids (bars represent standard deviation) are coincident in receptor non-amplified tumors (left) whereas the population centroid moves to the lower left in amplified tumors (right) after gefitinib treatment. behavior of the population. Discussion Within EGFR-amplified tumors, not all cells harbor receptor amplifica- tion, therefore we postulated increased EGFR gene dose supports augmented cell migration capability. We were unable to directly confirm EGFR copy number in a per cell fashion with live imaging. However, we observed that upon slice culture treatment with gefitinib, displacement and peaking behavior are homogenized. After treat- ment, the centroid of the EGFR-amplified population moved, roughly approximating the location of the EGFR non-amplified population centroid, suggesting a return to a “baseline” migratory behavior. We believe this rep- resents a selective effect on EGFR-amplified cells, as EGFR non-amplified cells likely exhibit minimal response to gefitinib treatment.f gi To effectively design therapeutics limiting GBM invasion, we must understand the molecular mediators underlying heterogeneity in migratory behavior. This study suggests that targeting EGFR in human GBM slices selectively inhibits migration of receptor-amplified GBM cells, resulting in homogenization of the migratory SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 6 www.nature.com/scientificreports/ Figure 4. Gefitinib treatment selectively disrupts EGFR-Amplification migration behaviors at the tumor cell population level. High temporal resolution cell path tracking occurred every 11 minutes in EGFR-amplified (n = 3) and non-amplified tumors (n = 3). Cells from were pooled across tumors for analysis under control (15 EGFR-amp cells, 162 non-amp) and gefitinib (10 μM) treatment conditions (129 EGFR-amp, 163 non-amp), an were grouped based on tissue level receptor amplification status of the originating tumor. Error bars represent Figure 4. Gefitinib treatment selectively disrupts EGFR-Amplification migration behaviors at the tumor cell population level. High temporal resolution cell path tracking occurred every 11 minutes in EGFR-amplified (n = 3) and non-amplified tumors (n = 3). Cells from were pooled across tumors for analysis under control (152 EGFR-amp cells, 162 non-amp) and gefitinib (10 μM) treatment conditions (129 EGFR-amp, 163 non-amp), and were grouped based on tissue level receptor amplification status of the originating tumor. Error bars represent standard deviation (SD). (A) Individual cells are displayed according to displacement in micrometers (i.e. total distance traveled during imaging) and standard deviation of instantaneous speed (a metric of an individual cell’s speed variability at anytime during migration) from receptor non-amplified slices (left) demonstrate minimal change in migratory properties despite gefitinib treatment. A dropout of cells with efficient and increased bursting behavior is observed in EGFR-amplified slices (right). Materials and Methods Organotypic tumor slice culture preparation. Human glioblastoma tumor tissue was acquired with informed consent by all patients or their legally authorized representative, under an IRB approved protocol at the University of Colorado Hospital. All subsequent experiments were performed in accordance with IRB reg- ulations. Human glioblastoma tissue from contrast enhancing regions was obtained intraoperatively utilizing surgical navigation technology43–45. Slice culture generation is described elsewhere11,46. Briefly, tumor tissue was embedded in low melting temperature agarose (Invitrogen) and sliced 300–350 μm thick using a VT100S Vibratome (Leica). Tumor slices were cultured on PTFE inserts (Millipore) maintained in a humidified incubator (37 °C and 5% CO2) using slice culture medium as described elsewhere11. Pathology reports for patient samples (n = 7) provided population-level EGFR-amplification binary scoring, but did not assess levels in individual cells. EGFR amplification status was assessed via fluorescence in situ hybridization performed and scored by an accred- ited cytogenetics laboratory at the University of Colorado Hospital. All samples were IDH1 wild-type (assessed by IHC) and EGFRviii status was not assessed. Individual patient demographic and tumor genetic information is summarized in Supplemental Table 1. Retroviral infection of slice cultures. Actively proliferating cells were labeled with ZsGreen1 (Clonetech) via transduction of slice cultures with MMLV-based retroviral particles (104 viral particles/uL) in unsupple- mented Neurobasal medium added dropwise to the surface of the tissue slice, 11 days after generation. Viral production is described elsewhere11,46. Slices were imaged 72 hours after infection. Time-lapse laser scanning confocal imaging of organotypic human tumor slices. Imaging was performed as previously described11,46, using a stage-top incubator (Pecon) maintained at 37 °C and 5% CO2. Tissue was imaged with a 488 nm laser and 10x air objective (c-Apochromat NA0.45) on a Zeiss LSM510. Imaging fields spanned a 900 μm by 900 μm region between the center and slice edge, and depth varied from 150–250 μm, with a 10 μm Z-step and 11 minute interval between field-scans. Tumor cell migration tracking and processing. Two-dimensional maximum intensity projections were generated from three-dimensional Z-stacks using Zeiss Zen software (Zeiss Inc.) Manual cell-tracking was per- formed by one observer (J.J.P.) marking the centroid of the tumor cell body. Cell position was recorded every 11 minutes using ImageJ (NIH) and MTrackJ47. Discussion This is supported by the observations of early effects on the tumor cell speed of tumors within minutes to 1 hour (Supplemental Fig. 2). Although we did not quantitatively assess changes in proliferation rate or cell death, other studies targeting EGFR within in vivo glioblastoma models resulted in min- imal effects on proliferation, and a predominant effect on tumor invasion36. In this in vivo study, tumors quickly resumed their infiltrative behavior upon treatment withdrawal, indicating that EGFR inhibition was not sufficient to kill invasive cells, but instead transiently slowed invasion36. SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 7 www.nature.com/scientificreports/ While the EGFR pathway appears a promising target for reduction of migration, many parameters remain unexplored. Among studies showing similar bursting migratory behavior in GBM cells19–23, some suggest this movement pattern is dependent on the molecular motor myosin II21. This implicates myosin II and its regula- tors as physiologically relevant targets to limit invasion of GBM cells with augmented migratory capabilities37. Enhanced signaling through other RTKs may also contribute to heterogeneity23,34,38, and these pathways may change over the course of tumor progression. Independent of the chosen target, factors such as optimal dose, penetration of the brain in vivo, and the potential need for long-term treatment must be considered.l p p g Although controversial, aggressive strategies including supratotal39,40 and fluorescence–guided tumor resec- tion41 as well as wide-field FLAIR-based radiation boosting42 have recently demonstrated a small but limited potential to control the most infiltrative component of GBM. However, a long-term, patient-specific therapeutic agent that effectively controls GBM infiltration is desperately needed. By focusing on the reduction of observable, clinically important behaviors in actual GBM tissue, our model provides an ideal platform for pinpointing the molecular pathways that lead to disease spread and recurrence. Our hope is that similar studies using the slice cul- ture model will refine our approach to developing and choosing treatments, allowing us to predict and overcome possible mechanisms of resistance within subsets of tumor cells. Though in vivo experiments are necessary, this proof of principle study, demonstrates inhibition of a key node in the molecular migration pathway, homogenizes migratory behavior, representing an important step toward improved treatment for GBM patients. Further, ex vivo imaging of organotypic slice cultures to quantitate cell behavior responses to candidate therapies is a prom- ising strategy to develop personalized therapy across the spectrum of human cancers. Materials and Methods Data analysis was performed on cell tracks with high- (11 minute intervals) or low-temporal resolution (55 minute intervals) using the Chemotaxis and Migration Tool (Ibidi) to calculate mean migration speed (μm/hr), instantaneous migration speed (μm/hr), total path length (μm), and net path length (μm). Directionality, was calculated as the ratio of net path length (displacement) to total path length (μm). Mean effective speed, was calculated as the product of mean cell speed and directionality. Inherent in the transformation of 3-D to 2-D tracking data, all calculated distances and subsequent speeds underestimate actual values. Cell track analysis and visualization. Individual cell tracks were analyzed utilizing Prism 6 (GraphPad) to calculate the standard deviation of the instantaneous speed (ratio of the distance traveled to length of the imaging interval). Movement peaks were defined as an instantaneous speed(s) above 0 μm/hr, with at least two adjoining data points, and number of peaks was calculated for each cell using the area under curve function. A secondary metric, “high-speed” peaks, was defined as instantaneous speed greater than 20 μm/hr. We visualized the cell track, time, and instantaneous speed in the x, y, and z axis, respectively, by rendering three-dimensional surface maps (MATLAB) to evaluate cell movement topography. The functional contribution of each peak to overall migration of the cell, was determined by calculating distance traveled per movement, as defined by the area under the curve for each peak. Human tumor slice culture gefitinib treatment. Slices were imaged for 11 hours under DMSO (1:1000) control conditions, followed by 11 hours of 10 μm gefitinib (Iressa, Tocris). Between imaging periods slice cul- ture inserts were briefly removed from the stage incubator, media was exchanged with temperature and CO2 SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 8 www.nature.com/scientificreports/ equilibrated gefitinib containing media, and the exact imaging field re-located. Identical imaging parameters were set for both imaging periods. equilibrated gefitinib containing media, and the exact imaging field re-located. Identical imaging parameters were set for both imaging periods. Statistical analysis. Statistical analyses were performed using Prism 6 (GraphPad) and data is repre- sented as mean ± SEM unless otherwise noted. Two-tailed t-tests were used for comparison of two groups of means, while Spearman’s correlation was used to assess the correlation between two individual cell parameters (non-Gaussian distributions; r- and p-values reported). MATLAB R2010a (MathWorks) was used to perform Levene’s test for the equality of variances where appropriate. Data Availability d fil d y Raw video files and coordinate based cellular path tracking data for all experiments are available upon request. References References 1. Verhaak, R. G. W. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA IDH1 EGFR and NF1 Cancer Cell 17 98 110 (2010) 1. Verhaak, R. G. W. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).h 1. Verhaak, R. G. W. et al. Integrated genomic analysis identifies clinically relevant subtypes of gli abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).h 2. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013). 2. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013). h 3. Williams Parsons, D. et al. An Integrated Genomic Analysis of Human Glioblastoma Multiforme. Science 321, 1807–1812 (2008). 4. Sanson, M. et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J. Clin. Oncol. 27, 4150–4154 (2009).h h . Williams Parsons, D. et al. An Integrated Genomic Analysis of Hu 5. Zhang, K., Wang, X.-Q., Zhou, B. & Zhang, L. The prognostic value of MGMT promoter methylation in Glioblastoma multiforme a meta-analysis. Fam. Cancer 12, 449–458 (2013).h y 6. Binabaj, M. M. et al. The prognostic value of MGMT promoter methylation in glioblastoma: A meta-analysis of clinical trials. J. Cell. Physiol. https://doi.org/10.1002/jcp.25896 (2017). y p g j p ( ) 7. Haynes, H. R., Camelo-Piragua, S. & Kurian, K. M. Prognostic and predictive biomarkers in adult and pediatric gliomas: toward personalized treatment. Front. Oncol. 4, 47 (2014).i p 8. Smith, J. S. et al. PTEN mutation, EGFR amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J. Natl. Cancer Inst. 93, 1246–1256 (2001).l p 8. Smith, J. S. et al. PTEN mutation, EGFR amplification, and o multiforme. J. Natl. Cancer Inst. 93, 1246–1256 (2001). i multiforme. J. Natl. Cancer Inst. 93, 1246–1256 (2001). 9. Okada, Y. et al. Selection pressures of TP53 mutation and microenvironmental location in gene amplification in human glioblastomas. Cancer Res. 63, 413–416 (2003). 9. Okada, Y. et al. Selection pressures of TP53 mutation and microenvironmental location influence epidermal growth factor receptor gene amplification in human glioblastomas. Cancer Res. 63, 413–416 (2003). 9. Okada, Y. et al. Selection pressures of TP53 mutation and g pi g 10. Aghi, M. et al. References Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma define subpopulations with distinct growth factor response. Proc. Natl. Acad. Sci. USA 109, 3041–3046 (2012). 9 B D J l P d li d i li bl h i ll l i d f d b i l 9. Brat, D. J. et al. Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population. Cancer Res. 64, 920–927 (2004). g g 0. Giese, A., Bjerkvig, R., Berens, M. E. & Westphal, M. Cost of migration: invasion of malignant gliomas and implications for treatment. J. Clin. Oncol. 21, 1624–1636 (2003). 20. Giese, A., Bjerkvig, R., Berens, M. E. & Westphal, M. Cost of migration: invasion of malignant gliomas and implications for treatment. J. Clin. Oncol. 21, 1624–1636 (2003). 21 L T Ch K R T A S H & T Y Di l t l ti b t i l h l lik ll d it l 1. Lan, T., Cheng, K., Ren, T., Arce, S. H. & Tseng, Y. Displacement correlations between a single mesenchymal-like cell and its nucleus effectively link subcellular activities and motility in cell migration analysis. Sci. Rep. 6, 34047 (2016). f 2. Nathanson, D. A. et al. Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA Science 343, 72–76 (2014). 3. Müller, S. et al. Single-cell sequencing maps gene expression to mutational phylogenies in PDGF- and EGF-driven gliomas. Mol Syst. Biol. 12, 889 (2016). y ( ) 24. de Aquino, P. F. et al. A Time-Based and Intratumoral Proteomic Assessment of a Recurrent Glioblastoma Multiforme. Front. Oncol. 6, 183 (2016).h 5. Wei, W. et al. Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma. Cancer Cell 29, 563–573 (2016). 6. Johnson, B. E. et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 343, 189–193 (2014). 27. Eskilsson, E. et al. EGFRvIII mutations can emerge as late and heterogenous events in glioblastoma development and promote angiogenesis through Src activation. Neuro. Oncol. 18, 1644–1655 (2016).i g g g 8. Snuderl, M. et al. Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma. Cancer Cell 20, 810–817 (2011). SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 References Magnetic resonance imaging characteristics predict epidermal growth factor receptor amplification status in glioblastoma. Clin. Cancer Res. 11, 8600–8605 (2005).ii g 11. Parker, J. J. et al. Gefitinib selectively inhibits tumor cell migration in EGFR-amplified human glioblastoma. Neuro. Onco 1048–1057 (2013). 12. Day, S. E. & Waziri, A. Clinical trials of small molecule inhibitors in high-grade glioma. Neurosurg. Clin. N. Am. 23, 407–416 (2012). 13 Sottoriva A et al Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics Proc Natl Acad Sci Day, S. E. & Waziri, A. Clinical trials of small molecule inhibitors in 2. Day, S. E. & Waziri, A. Clinical trials of small molecule inhibitors in high grade glioma. Neurosurg. Clin. N. Am. 23, 407 416 (2012) 3. Sottoriva, A. et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc. Natl. Acad. Sci USA 110, 4009–4014 (2013). 14. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014). 15 D i S t l Si l C ll RNA S A l i f I fil i N l i C ll h Mi i F f H Gli bl C ll . Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heter 14. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014). 15. Darmanis, S. et al. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma. Cell Rep. 21, 1399–1410 (2017). 14. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014). 15. Darmanis, S. et al. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma. Cell p ( ) 6. Massey, S. C., Assanah, M. C., Lopez, K. A., Canoll, P. & Swanson, K. R. Glial progenitor cell recruitment drives aggressive glioma growth: mathematical and experimental modelling. J. R. Soc. Interface 9, 1757–1766 (2012).i 17. Little, S. E. et al. Receptor tyrosine kinase genes amplified in glioblastoma exhibit a mutual exclusivity in variable proportions reflective of individual tumor heterogeneity. Cancer Res. 72, 1614–1620 (2012).ii l g y 18. Szerlip, N. J. et al. Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma d subpopulations with distinct growth factor response. Proc. Natl. Acad. Sci. USA 109, 3041–3046 (2012). 8. Szerlip, N. J. et al. Acknowledgementsh g The work in this manuscript was supported by start-up funds provided to A. Waziri from the Department of Neurosurgery at the University of Colorado School of Medicine. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 37. Duma, C. M. et al. Upfront boost Gamma Knife ‘leading-edge’ radiosurgery to FLAIR MRI-defined tumor migration pathways in 174 patients with glioblastoma multiforme: a 15-year assessment of a novel therapy. J. Neurosurg. 125, 40–49 (2016). l l h d d ll l d l f b l l 38. Aryal, M. P. et al. Dynamic contrast enhanced MRI parameters and tumor cellularity in a rat model of cerebral glioma at 7 T. M Reson. Med. 71, 2206–2214 (2014).f 9. Ellingson, B. M. et al. Validation of functional diffusion maps (fDMs) as a biomarker for human glioma cellularity. J. Magn. Reson Imaging 31, 538–548 (2010).f g g ( ) 0. Sugahara, T. et al. Usefulness of diffusion-weighted MRI with echo-planar technique in the evaluation of cellularity in gliomas. J Magn. Reson. Imaging 9, 53–60 (1999). g g g 41. Parker, J. J., Lizarraga, M., Waziri, A. & Foshay, K. M. A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-InvasiveDrugs. J. Vis. Exp. https://doi.org/10.3791/53557 (2017). g pif g J p p g ( ) 42. Meijering, E., Dzyubachyk, O. & Smal, I. Methods for cell and particle tracking. Methods Enzymol. 504, 183–200 (2012). j g y y p g y 43. Farin, A. et al. Transplanted glioma cells migrate and proliferate on host brain vasculature: a dynamic analysis. Glia 53, 799–808 (2006). 44. Suzuki, S. O. & Goldman, J. E. Multiple Cell Populations in the Early Postnatal Subventricular Zone Take Distinct Migr Pathways: A Dynamic Study of Glial and Neuronal Progenitor Migration. J. Neurosci. 23, 4240–4250 (2003).h y y y g g 5. Beadle, C. et al. The role of myosin II in glioma invasion of the brain. Mol. Biol. Cell 19, 3357–3368 (2008).i h y g ( ) 46. Fayzullin, A. et al. Time-lapse phenotyping of invasive glioma cells ex vivo reveals subtype-specific movement patterns guided by tumor core signaling. Exp. Cell Res. 349, 199–213 (2016). g g p 7. Assanah, M. C. et al. PDGF stimulates the massive expansion of glial progenitors in the neonatal forebrain. Glia 57, 1835–1847 (2009). Author Contributions Experimental design was conceived by J. Parker, P. Canoll, and A. Waziri. Experiments and data analysis were led by J. Parker with contributions from L. Niswander and B.K. DeMasters. Data interpretation and writing of the manuscript were led by J. Parker, K. Foshay, and A. Waziri. References 9 I d M M t l T h t it i ti i t i d b t t EGFR i d d t ki i it i li bl t 28. Snuderl, M. et al. Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma. Cance 28. Snuderl, M. et al. Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma. Cancer Cell 20, 810–817 (2011). 29. Inda, M.-M. et al. Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev. 24, 1731–1745 (2010).f i 29. Inda, M.-M. et al. Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev. 24, 1731–1745 (2010).f 30. Ivkovic, S., Canoll, P. & Goldman, J. E. Constitutive EGFR signaling in oligodendrocyte progenitors leads to diffuse hyperplasia in postnatal white matter. J. Neurosci. 28, 914–922 (2008).i p 1. Talasila, K. M. et al. EGFR wild-type amplification and activation promote invasion and development of glioblastoma independen of angiogenesis. Acta Neuropathol. 125, 683–698 (2013).f g g p 32. Ivkovic, S. et al. Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens. Mol. Biol. Cell 23, 533–542 (2012). G l h h l l d ll l b f l bl G ( ) 33. Dunn, G. P. et al. Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev. 26, 756–784 (2012).hl 34. Li, Y. M., Suki, D., Hess, K. & Sawaya, R. The influence of maximum safe resection of gliobl we do better than gross-total resection? J. Neurosurg. 124, 977–988 (2016).h 34. Li, Y. M., Suki, D., Hess, K. & Sawaya, R. The influence of maximum safe resection of glioblastoma on survival in 1229 patients: Can we do better than gross-total resection? J. Neurosurg. 124, 977–988 (2016).h 35. Esquenazi, Y. et al. The Survival Advantage of ‘Supratotal’ Resection of Glioblastoma Using Selective Cortical Mapping and the Subpial Technique. Neurosurgery 81, 275–288 (2017).il p q g y ( ) 6. Neira, J. A. et al. Aggressive resection at the infiltrative margins of glioblastoma facilitated by intraoperative fluorescein guidance. J Neurosurg. 127, 111–122 (2017). SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 9 © The Author(s) 2018 SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36280-9 Competing Interests: The authors declare no competing interests. ublisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. 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 Cre- ative 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 per- mitted 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/. © The Author(s) 2018 SCIeNtIFIC REPOrtS | (2018) 8:18002 | DOI:10.1038/s41598-018-36280-9 10
https://openalex.org/W4389238739
https://www.nature.com/articles/s41398-023-02666-1.pdf
English
null
Drug memory reconsolidation: from molecular mechanisms to the clinical context
Translational psychiatry
2,023
cc-by
14,104
INTRODUCTION pavlovian or instrumental, might provide targets for new treatment development [30–36]. A similar conceptualisation of other ‘maladaptive memory disorders’ such as specific phobia [37, 38] led to the development of treatments such as ‘cue exposure’ or ‘prolonged exposure’ therapy. Prolonged exposure therapy involves the repeated re-exposure of individuals to pavlovian CSs, previously associated with motivationally relevant outcomes, in the absence of that outcome [37]. For example, a phobic stimulus (e.g. a spider, normally eliciting fear in an individual with arachnophobia) is repeatedly presented while the individual works through guided exercises to control their fear (e.g. relaxation exercises). These repeated exposures can become progressively more proximal in a process referred to as ‘systematic desensitisation’ [39]. On successful completion of the therapy, the individual should no longer show the pavlovian conditioned response (i.e. fear) to the stimulus. However, despite its success for disorders such as phobia [40], the efficacy of prolonged exposure therapy for addiction is limited and mixed, with meta-analyses suggesting little to no effect on treatment outcomes [41–44]. Drug addiction, or substance use disorder (SUD) is a chronic relapsing disorder that is estimated to annually cost the UK National Health Service in excess of £30 billion [1, 2], and to have an economic impact in the United States in excess of $440 billion per year [3]. Individuals addicted to drugs show high motivation for the drug(s) of abuse, impaired control over drug use, and persistent use of drug despite adverse physical, psychological and social consequences of drug use [4]. Addiction is a complex disorder, involving the hijacking of neural circuitry that has evolved to support motivated behaviour relevant to survival of the individual and propagation of the species. A prominent view is that learning mechanisms, originally evolved to support foraging behaviour, become maladaptively recruited to drug-seeking and drug-taking, allowing addiction to be conceptualised as a disorder of maladaptive learning and memory [5, 6]. This involves the aberrant engagement of both pavlovian and instrumental learning mechanisms. The acts of drug-seeking and drug-taking are instrumental, initially being goal-directed but, in a subset of individuals [7, 8], ultimately becoming habitual and compulsive [9, 10]. However, drug-seeking and drug-taking occur in specific environments (contexts) and in the presence of people and paraphernalia (i.e. discrete cues) that become associated with the drug high in a pavlovian manner [5]. Translational Psychiatry Translational Psychiatry www.nature.com/tp EXPERT REVIEW OPEN Drug memory reconsolidation: from molecular mechanisms to the clinical context Amy L. Milton 1✉ © The Author(s) 2023 Amy L. Milton Since its rediscovery at the beginning of the 21st Century, memory reconsolidation has been proposed to be a therapeutic target for reducing the impact of emotional memories that can go awry in mental health disorders such as drug addiction (substance use disorder, SUD). Addiction can be conceptualised as a disorder of learning and memory, in which both pavlovian and instrumental learning systems become hijacked into supporting drug-seeking and drug-taking behaviours. The past two decades of research have characterised the details of the molecular pathways supporting the reconsolidation of pavlovian cue-drug memories, with more recent work indicating that the reconsolidation of instrumental drug-seeking memories also relies upon similar mechanisms. This narrative review considers what is known about the mechanisms underlying the reconsolidation of pavlovian and instrumental memories associated with drug use, how these approaches have translated to experimental medicine studies, and the challenges and opportunities for the clinical use of reconsolidation-based therapies. Translational Psychiatry (2023) 13:370 ; https://doi.org/10.1038/s41398-023-02666-1 Received: 13 April 2023 Revised: 7 November 2023 Accepted: 13 November 2023 THE RECONSOLIDATION OF PAVLOVIAN DRUG-ASSOCIATED MEMORIES Targeting the consolidation of memories underlying addiction – their initial storage within the brain, hypothesised to rely upon synaptic plasticity changes [52] and the formation of memory traces or ‘engrams’ [53, 54] – is not a feasible therapeutic strategy. Many who use drugs of abuse will ultimately not become addicted [55, 56] and thus, any globally-applied treatment strategy would target a large proportion of the drug-using population who will never develop SUD. Furthermore, even for those who would benefit from disrupting the consolidation of drug-associated memories, the window for treatment opportunity is very small, encompassing only a few hours (typically thought to be around 6 h, and certainly within 24 h - see ref. [57] for review). Although memory consolidation presents a challenging target for disrupting drug-associated memories, the rediscovery of memory reconsoli- dation at the beginning of the 21st Century reignited interest in memory-based treatments for addiction. Drug-associated CSs are powerful precipitators of relapse in individuals trying to maintain abstinence [13–15, 17] with imaging studies showing that activation of the limbic corticostriatal circuitry in response to drug CSs is predictive of subsequent relapse for individuals addicted to alcohol [11, 12], nicotine [19–21], opiates [22] and psychostimulants [23, 24]. Disrupting the reconsolidation of drug-associated CSs therefore presents a potential treatment target for reducing the impact of these CSs on relapse behaviour in the long term. p g Numerous studies have investigated the impact of disrupting the reconsolidation of pavlovian drug-associated cues, in both self-administration and conditioned place preference (CPP; see below) procedures. Consistent with the activation of the limbic corticostriatal circuitry by drug-associated cues [13–15, 17], the reconsolidation of pavlovian CS-drug memories relies upon plasticity mechanisms within regions such as the basolateral amygdala (BLA), hippocampus and nucleus accumbens [69] (Fig. 2). The specific regions involved depend upon whether the memory being targeted for disruption is required for the association between contextual or discrete CSs and the drug outcome (unconditioned stimulus, US), and whether specific psychological processes (e.g. conditioned reinforcement) or the contribution of multiple psychological processes are being targeted. Indeed, even within regions, different behavioural procedures recruit specific ensembles that can be distinguished within the same animals using engram-labelling techniques [70]. INTRODUCTION Therefore targeting multiple associations, whether simultaneously through reactivating by re-exposure to the drug US [63, 64], or sequentially by reactivating different CSs individually, may be the optimal approach for reconsolidation- based therapies. Fig. 1 Simplified representation of associations acquired during learning about a drug of abuse. Associations are made between initially neutral stimuli (both discrete and contextual) that ultimately become associated with the drug of abuse, acting as an uncondi- tioned stimulus, in a pavlovian manner. These associations can be affective or predictive, leading to actions (reflexive motor responses or more flexible instrumental behaviour). Additionally, instrumental associations are acquired between the action and the outcome (goal-directed responses) where individuals will work for the unconditioned stimulus if they are motivated to do so, and there is an instrumental contingency between the response and the unconditioned stimulus. Instrumental responding can also be supported by habitual, stimulus-response associations between conditioned stimuli and the action. Magenta lines represent learned associations. p The past two decades have seen a concerted research effort to determine whether reconsolidation-based approaches could be used to treat SUD, primarily in animal models, but also in a growing number of studies in humans. This review will focus upon treatments aiming to disrupt the reconsolidation of pavlovian memories (which should reduce the capacity of drug-associated CSs and contexts to precipitate relapse) and the disruption of instrumental memories (which should reduce drug-seeking behaviour directly) in both conditioned place preference (CPP) and self-administration models, and also consider how these proof-of-principle approaches have translated to clinical populations. capacity of a pavlovian CS to acquire affective properties related to the primary reinforcer, and subsequently to support responding for the CS in its own right, allowing the cue to bridge long delays to primary reinforcement [51]. The fact that the CS becomes reinforcing in its own right is a major challenge to therapies based upon extinction learning. INTRODUCTION 1 Simplified representation of associ learning about a drug of abuse. Associati initially neutral stimuli (both discrete and co become associated with the drug of abuse tioned stimulus, in a pavlovian manner. Th affective or predictive, leading to actions (re or more flexible instrumental behaviour). Ad associations are acquired between the ac (goal-directed responses) where individu unconditioned stimulus if they are motivat is an instrumental contingency between unconditioned stimulus. Instrumental res supported by habitual, stimulus-response conditioned stimuli and the action. Magenta associations. 2 2 2 Fig. 1 Simplified representation of associations acquired during learning about a drug of abuse. Associations are made between initially neutral stimuli (both discrete and contextual) that ultimately become associated with the drug of abuse, acting as an uncondi- tioned stimulus, in a pavlovian manner. These associations can be affective or predictive, leading to actions (reflexive motor responses or more flexible instrumental behaviour). Additionally, instrumental associations are acquired between the action and the outcome (goal-directed responses) where individuals will work for the unconditioned stimulus if they are motivated to do so, and there is an instrumental contingency between the response and the unconditioned stimulus. Instrumental responding can also be supported by habitual, stimulus-response associations between conditioned stimuli and the action. Magenta lines represent learned associations. 2 change in the reinforcement contingency for instrumental memories [65, 66]. Importantly, reactivation of the memory occurs under conditions in which there is a ‘mismatch’ between what is expected and what actually occurs [61], although the relationship between memory retrieval, memory destabilisation/reconsolida- tion and extinction learning is not straightforward [67] and likely interacts with prior learning history [68]. Thus, although reconsolidation-based approaches are simple in principle – induce lability of a well-established memory and administer an amnestic agent or behavioural interference procedure to disrupt its reconsolidation, thereby weakening it in the long-term - the practicalities of disrupting pavlovian and instrumental drug- associated memories has presented multiple challenges, including identification of the optimal associations to target for disruption (Fig. 1). In principle, these associations can be dissociated experimentally and could be independently targeted for disrup- tion under the appropriate conditions of memory reactivation. In practice, these associations act simultaneously in the real world to support ongoing drug-seeking behaviour and to promote relapse during abstinence [31]. INTRODUCTION These pavlovian drug-associated conditioned stimuli (‘CS-drug memories’) become powerful precipitators of relapse in those trying to maintain abstinence [11–24], and in experimental animal models of addiction, can be used to promote drug-seeking behaviour [25–29]. Prolonged exposure therapy may be less effective in the treatment of addiction because rather than ‘erasing’ or ‘over- writing’ the original memory, the mechanism underlying pro- longed exposure creates a new, inhibitory ‘cue-no outcome’ extinction memory that competes with the original for beha- vioural expression [45, 46]. While extinction learning is effective for disorders such as phobia [47], there are issues around its contextual specificity [46] (though see ref. [48] for methods to minimise the impact of contextual influences). However, the CSs associated with drugs of abuse often develop conditioned reinforcing properties that make them remarkably resistant to extinction [49, 50]. Conditioned reinforcement refers to the Conceptualising addiction as a disorder of learning and memory raises the prospect that drug-associated memories, whether A.L. Milton capacity of a pavlovian CS to acquire affec to the primary reinforcer, and subsequently for the CS in its own right, allowing the cu to primary reinforcement [51]. The fact reinforcing in its own right is a major challe upon extinction learning. Targeting the consolidation of memories their initial storage within the brain, hypo synaptic plasticity changes [52] and the traces or ‘engrams’ [53, 54] – is not a feasib Many who use drugs of abuse will ultimate [55, 56] and thus, any globally-applied tre target a large proportion of the drug-usin never develop SUD. Furthermore, even benefit from disrupting the consolidatio memories, the window for treatment opp encompassing only a few hours (typically 6 h, and certainly within 24 h - see ref. [57 memory consolidation presents a challengi drug-associated memories, the rediscovery dation at the beginning of the 21st Centu memory-based treatments for addiction. Reconsolidation is hypothesised to be memories can be updated under certain and almost since its rediscovery in 2000 [58 as a means by which old, well-establi maladaptive) memories could be tar [31, 33, 59, 60]. By reactivating the maladap to drug-seeking, it should be possible to m susceptible to disruption through admi agents, or behavioural interference. R mechanistically, destabilisation) of the me in different ways, but typically involves brie the CS [61, 62] or the US [63, 64] for pavlov to as ‘CS-based’ and ‘US-based’ reactivati Fig. THE RECONSOLIDATION OF PAVLOVIAN DRUG-ASSOCIATED MEMORIES However, for all these associations, the necessity of plasticity processes including activation of the NMDA subtype of glutamate y Reconsolidation is hypothesised to be the process by which memories can be updated under certain conditions of retrieval, and almost since its rediscovery in 2000 [58] it has been proposed as a means by which old, well-established (and potentially maladaptive) memories could be targeted for disruption [31, 33, 59, 60]. By reactivating the maladaptive memories relevant to drug-seeking, it should be possible to make them once again susceptible to disruption through administration of amnestic agents, or behavioural interference. Reactivation (or, more mechanistically, destabilisation) of the memory can be achieved in different ways, but typically involves brief re-exposure to either the CS [61, 62] or the US [63, 64] for pavlovian memories (referred to as ‘CS-based’ and ‘US-based’ reactivation respectively), and a Translational Psychiatry (2023) 13:370 A.L. Milton 3 Fig. 2 Neural regions implicated in drug memory reconsolidation. a Sagittal schematic of the rat brain, showing key regions of the corticolimbic striatal circuitry implicated in drug memory reconsolidation. b Connection of key brain regions implicated in drug memory reconsolidation, denoting associations supported by each structure and the type of memory reactivation session required to recruit it. Abbreviation: Amy amygdala, DH dorsal hippocampus, DS dorsal striatum, NAcb, nucleus accumbens. Reproduced, with permission, from ref. [69]. Fig. 2 Neural regions implicated in drug memory reconsolidation. a Sagittal schematic of the rat brain, showing key regions of the corticolimbic striatal circuitry implicated in drug memory reconsolidation. b Connection of key brain regions implicated in drug memory reconsolidation, denoting associations supported by each structure and the type of memory reactivation session required to recruit it. Abbreviation: Amy amygdala, DH dorsal hippocampus, DS dorsal striatum, NAcb, nucleus accumbens. Reproduced, with permission, from ref. [69]. receptor (NMDAR), protein kinase activation and protein synthesis has been demonstrated. associate a specific context with the effects of an experimenter- administered drug, and subsequently manifests as a preference for that context over an alternative context paired with a control injection [75]. By contrast, context-induced reinstatement involves training animals to self-administer drug (typically intravenously, but orally in the case of alcohol) in a specific context, and testing the capacity of that context to support recovery of drug-seeking compared to control contexts [76]. THE RECONSOLIDATION OF PAVLOVIAN DRUG-ASSOCIATED MEMORIES The memory representing the association of the context and the drug outcome is likely supported by the BLA [77–80], as it is for discrete cues [81–83]. Disrupting context-drug memories However, the context representation itself depends upon the dorsal hippocampus, similar to the circuitry supporting contextual fear conditioning [84–86]. preventing its interaction with the scaffolding protein GRIP1 [99]. Epigenetic regulation is also critical for drug memory reconsolida- tion, as activation of the histone demethylase KDM6B in the medial prefrontal cortex [100] and Tet3 in the dorsal hippocampus [101] are both required for the reconsolidation of the memories underlying cocaine CPP. Disrupting the contextual memories underlying drug-conditioned place preference. The memories underlying drug CPP can be disrupted by targeting biochemical pathways that ultimately lead to immediate early gene expression and protein synthesis (Fig. 3). These manipulations produce amnesia more reliably when the reactivation session is reinforced with an injection of the drug of abuse (i.e. ‘US-based’ reactivation is used). Protein synthesis inhibitors disrupt both cocaine [87] and morphine [87, 88] CPP when administered systemically in conjunction with reinforced re- exposure to the drug-paired context, as do local infusions of protein synthesis inhibitors intracerebroventricularly [89] or within the BLA [88] (though see ref. [90]), central amygdala [91], dorsal hippocampus [88] and nucleus accumbens core [88]. Upstream of protein synthesis, the reconsolidation of drug CPP memories depends upon the activation of transcriptional and translational regulators including eIFα [92], circTmeff-1 [93] and mTORC1 [94], and protein kinases including ERK [87], p70S6 kinase [94] and GSK3β [95, 96]. Protein phosphatases, such as protein phospha- tase 1, are also necessary for the reconsolidation of cocaine CPP memories [97]. Furthermore, the knockdown of the immediate early gene zif268 in the BLA or the nucleus accumbens disrupts the reconsolidation of the memories underlying cocaine CPP [98]. However, some proteases are also necessary for the reconsolida- tion of CPP memories, as the inhibition of the calcium-dependent cysteine protease calpain in the nucleus accumbens core prevents the reconsolidation of the memories underlying cocaine CPP by y g At the level of cell surface receptors, catecholaminergic signalling appears necessary for the reconsolidation of the memories underlying drug CPP. Mice with a constitutive genetic knockout of the dopamine D3 receptor show impaired reconso- lidation of cocaine CPP memories [102], as do wild type mice administered with the D3 receptor antagonist PG01037 [102] or the D1 receptor antagonist SCH23390 [103]. Furthermore, the enhancement of dopaminergic signalling with amphetamine facilitates the reconsolidation of the memories underlying morphine CPP [104]. Another catecholamine, noradrenaline, is also required for drug memory reconsolidation. Disrupting context-drug memories Studies of the impact of contextual cues on drug-seeking, and whether reconsolidation of these memories could be a target for reducing that impact, have relied primarily upon the conditioned place preference (CPP) procedure, or have explicitly assessed the impact of context on the self-administration of drugs of abuse (e.g. refs. [71–74]). The CPP procedure involves training animals to Translational Psychiatry (2023) 13:370 Fig. 3 Crosstalk between molecular pathways modulating drug memory reconsolidation. Drug memory reconsolidation is modulated at the cell-surface, intracellular and nuclear levels, with crosstalk between the individual signalling molecules that have been investigated to date. Solid lines represent activation/enhancement of signalling; dashed lines represent inhibition. Abbreviations: AC adenylyl cyclase, AMP adenosine monophosphate, AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptor, βAR β-adrenergic receptor, cAMP cyclic adenosine monophosphate, CaM calmodulin, CaMKs calcium-calmodulin dependent kinases, CaN calcineurin, D1R D1 dopamine receptor, D3R D3 dopamine receptor, eIFα eukaryotic initiation factor α, Epac exchange protein activated by cAMP, ERK extracellular signal-regulated kinase, GRIP1 glutamate receptor interacting protein 1, GSK3β glycogen synthase kinase β, IEG immediate early gene, KDM6B lysine demethylase 6B, mTORC1 mammalian target of rapamycin complex 1, NMDAR N-methyl-D-aspartate subtype of glutamate receptor, nNOS neuronal nitric oxide synthase, NO nitric oxide, p70S6K p70-S6 kinase, PKA protein kinase A, SO2 sulphur dioxide, SFKs src-family kinases, Tet3 tet methylcytosine dioxygenase 3. A.L. Milton A.L. Milton A.L. Milton 4 Fig. 3 Crosstalk between molecular pathways modulating drug memory reconsolidation. Drug memory reconsolidation is modulated at the cell-surface, intracellular and nuclear levels, with crosstalk between the individual signalling molecules that have been investigated to date. Solid lines represent activation/enhancement of signalling; dashed lines represent inhibition. Abbreviations: AC adenylyl cyclase, AMP adenosine monophosphate, AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptor, βAR β-adrenergic receptor, cAMP cyclic adenosine monophosphate, CaM calmodulin, CaMKs calcium-calmodulin dependent kinases, CaN calcineurin, D1R D1 dopamine receptor, D3R D3 dopamine receptor, eIFα eukaryotic initiation factor α, Epac exchange protein activated by cAMP, ERK extracellular signal-regulated kinase, GRIP1 glutamate receptor interacting protein 1, GSK3β glycogen synthase kinase β, IEG immediate early gene, KDM6B lysine demethylase 6B, mTORC1 mammalian target of rapamycin complex 1, NMDAR N-methyl-D-aspartate subtype of glutamate receptor, nNOS neuronal nitric oxide synthase, NO nitric oxide, p70S6K p70-S6 kinase, PKA protein kinase A, SO2 sulphur dioxide, SFKs src-family kinases, Tet3 tet methylcytosine dioxygenase 3. However, the context representation itself depends upon the dorsal hippocampus, similar to the circuitry supporting contextual fear conditioning [84–86]. Disrupting context-drug memories The glucocorticoid receptor antagonist, mifepristone, enhanced the reconsolidation of a context-cocaine memory [142] when admi- nistered directly to the BLA, similar to the effect of administering corticotropin-releasing factor (CRF) in female, but not male, rats [73]. CRF signalling in the BLA appears necessary for reconsolida- tion of the drug-context memory in both sexes, as administration of the CRF-1 receptor antagonist antalarmin at reactivation reduced subsequent context-induced cocaine-seeking in both males and females [73]. This activation of the HPA axis appears to interact with signalling at endocannabinoid receptors within the BLA. Systemic antagonism of CB1 receptors prior to, but not after, memory reactivation impairs the reconsolidation of the drug- context memory, and reduces the expression of immediate early genes including zif268 in the BLA [143]. Furthermore, while direct antagonism of CB1 receptors within the BLA leaves drug-context memory reconsolidation unimpaired (suggesting that systemic administration of CB1 receptor antagonists exerts amnestic effects via structures other than the BLA), administration of the CB1 receptor agonist AM251 during memory reactivation facilitates subsequent context-induced cocaine-seeking [74], and increases plasma corticosterone levels in a manner comparable to return to the drug-associated context [74]. This may indicate that CB1 receptors modulate the degree of HPA axis activation during drug- associated memory reconsolidation. There has been great interest in whether behavioural interven- tions, aimed at exploiting the updating function of reconsolidation [126, 127], might reduce the strength of context-drug memories. Adapting the ‘retrieval-extinction’ procedure first used for updating fear memories by Monfils and colleagues [128] – in which a brief memory reactivation session, followed after a short delay by extinction training led to long-term reductions in fear memory – several studies have investigated whether reactivation of the drug- conditioned place preference memory can return it to a state in which it is susceptible to updating with the information that the context is no longer associated with drug. Xue and colleagues [129] reported reductions in both cocaine CPP and morphine CPP when CS-based reactivation of the CPP memory was followed by extinction training, though it has been reported that the initial reductions in place preference produced by the retrieval-extinction procedure can subsequently recover [130]. Differences in training history (e.g. the strength of conditioning) appear to alter the boundary conditions relevant to retrieval-extinction [131], allowing behaviour to be reduced either by a reconsolidation-dependent mechanism, or by facilitation of extinction. (Note that these two mechanisms are difficult to distinguish behaviourally [132]). Disrupting context-drug memories Agonising α2 receptors with clonidine disrupts cocaine CPP [105], and antagonising β-adrenergic receptors with systemic propranolol during a single CPP memory reactivation session impairs subsequent CPP for cocaine [106], morphine [107] and nicotine [108], although not alcohol [109]. However, propranolol appears less effective when the drug CPP memory is strong, particularly if the memory has been recently acquired [110]. This boundary condition relating to memory strength may account for why some studies required multiple reactivation sessions under propranolol to observe subsequent amnesia [111]. Where propranolol is effective, it appears to exert its effects on memory via central noradrenergic signalling mechanisms. Administration of nadolol, a β-adrenergic receptor antagonist that does not cross Translational Psychiatry (2023) 13:370 A.L. Milton 5 the blood-brain barrier, does not disrupt the reconsolidation of the memories underlying morphine CPP under reactivation conditions for which propranolol is effective [107]. Furthermore, direct targeting of antagonists against α1 and β2 adrenergic receptors in the BLA disrupts the reconsolidation of the memories underlying cocaine CPP [112], as do both propranolol and nadolol when administered directly to the BLA [113]. β1 receptors in the central nucleus of the amygdala are also necessary for the reconsolidation of the memories underlying cocaine CPP [91]. Furthermore, administration of propranolol directly to the prelimbic cortex produces effects on both the retrieval and reconsolidation of cocaine CPP memories [113], perhaps indicat- ing that some of the apparent discrepancies produced by studies using systemic propranolol can be attributed to effects in different neural structures. contexts paired with non-contingently administered drug, con- textual cues also influence the propensity to seek drugs and to relapse following abstinence in individuals that have acquired drug self-administration [76]. Consistent with CPP studies, the reconsolidation of the memories underlying the influence of the drug-paired context on instrumental drug-seeking behaviour also relies upon protein synthesis [136], supported by intracellular signalling pathways activated by signalling at cell surface receptors (Fig. 3). p g Consistent with its well-established role in representing contexts, much research into contextual influences on drug self- administration has focused upon the dorsal hippocampus. The projections between the dorsal hippocampus and basolateral amygdala are critical for the reconsolidation of memories under- lying contextual influences on behaviour [72, 137], and disconnec- tion of these structures during memory reactivation impairs subsequent context-induced reinstatement of drug-seeking [72]. Disrupting context-drug memories Dorsal hippocampal activity is necessary for the reconsolidation of context-drug memories, as pharmacological inactivation with tetradotoxin [71] or optogenetic inhibition [138] of the dorsal hippocampus at reactivation impairs subsequent context-induced recovery of drug-seeking. However, inhibition of dorsal hippo- campal protein synthesis with anisomycin at reactivation does not affect the capacity of the context to support the reinstatement of drug-seeking [71], although preventing signalling at GluN2A- containing NMDARs and activation of Src-family kinases does [139], suggesting engagement of some synaptic plasticity processes. As context-induced reinstatement of drug-seeking is subsequent impaired when anisomycin is delivered to the BLA [136] at reactivation, this has led to the hypothesis that the dorsal hippocampus modulates the reconsolidation of the context-drug memory, which may be stored within the BLA [138]. The reconsolidation of the memories underlying drug CPP also depends upon activation at the NMDA subtype of glutamate receptor. Administration of the non-competitive NMDA receptor (NMDAR) antagonist MK-801 (dizocilpine) disrupts the reconsoli- dation of memories underlying cocaine CPP, usually with a single session of memory reactivation [114–116], but sometimes requiring multiple reactivation sessions [117]. Ketamine, another non-competitive NMDAR antagonist, disrupts the reconsolidation of memories underlying morphine CPP [118]. Amongst their many intracellular actions (Fig. 3), signalling at NMDARs leads to activation of neuronal nitric oxide synthase (nNOS) [119] and inhibition of nNOS also disrupts the reconsolidation of memories underlying cocaine CPP [115, 120]. Sulphur dioxide, which acts on nNOS pathways and affects neuronal function [121], impairs the reconsolidation of the memories underlying both cocaine and morphine CPP [122]. Finally, astrocytic lactate signalling, which likely exerts effects on synaptic plasticity via NMDARs [123], is also necessary for the reconsolidation of the memories underlying cocaine CPP [124, 125]. memory, which may be stored within the BLA [138]. The plasticity mechanisms underlying the reconsolidation of the context memory within the BLA are like those described for the reconsolidation of the memories underlying CPP (Fig. 3). In addition to the requirement for protein synthesis [136], protein kinases including ERK [140] and PKA [141] (though not CaMKII [141]) are necessary. In contrast to studies of CPP, which have focused primarily upon monoaminergic and glutamatergic signalling at the cell surface level, studies of context-induced reinstatement have considered more extensively the requirement for glucocorticoid-mediated signalling and endocannabinoids. Disrupting context-drug memories Although the apparent discrepancies in the persistence of reduced preference for the drug-paired context following retrieval- extinction are unlikely due to the strength of conditioning (due to similar training protocols across studies), there may have been more subtle individual differences underlying the difference in treatment outcome [133]. An alternative updating approach, using counterconditioning (pairing a previously reward-associated CS to an aversive, rather than appetitive outcome) rather than extinction during the reconsolidation window, led to persistent reductions in both cocaine CPP [134] and alcohol CPP [135]. Disrupting the contextual memories influencing drug self- administration. In addition to supporting a preference for A small number of studies have investigated the impact of behavioural interventions on the reconsolidation of drug-context Translational Psychiatry (2023) 13:370 A.L. Milton 6 memories. As for CPP [134, 135], aversive counterconditioning following a memory reactivation session can subsequently reduce context-induced alcohol-seeking [135]. Post-retrieval extinction produces a similar reduction in alcohol-seeking [144], although as a similar outcome is observed when extinction precedes memory reactivation, it is not clear that this effect depends upon memory reconsolidation rather than a facilitation of extinction learning [132, 133, 144]. approach memory, only sign-tracking, and not goal-tracking, was impaired [161]. This has led to speculation that propranolol may impair the emotional component of the CS-US memory, without affecting the predictive value of CS [161]. This view would be consistent with studies of propranolol in fear-conditioned humans, in which automatic reflexive behaviours produced by an emotional CS were reduced by propranolol administration at reactivation, but the expectation that the CS would be associated with shock was unaffected [162]. Disrupting discrete CS-drug memories influencing drug self- administration. Discrete pavlovian CS-drug memories interact with the instrumental acts of drug-seeking and drug-taking in multiple psychologically and neurobiologically dissociable ways [31]. Three such processes that have been studied in the context of reconsolidation are conditioned reinforcement, conditioned approach, and conditioned motivation. Pavlovian conditioned approach can be observed in different individuals as ‘sign- tracking’ or ‘goal-tracking’, reflecting a tendency to approach the pavlovian CS or the location of US delivery when the CS is presented, respectively (see ref. [145] for review). Conditioned motivation is usually assessed through ‘pavlovian-instrumental transfer’ (PIT) procedures, and describes the capacity of reward- associated cues to influence instrumental behaviour associated with the same or different rewards (see refs. [146, 147] for review). Disrupting context-drug memories These processes can be studied in isolation using specific behavioural procedures [148], though in reality drug-seeking will be influenced by all of these processes. This can be addressed experimentally with the use of reinstatement procedures [149], which have also been used to interrogate the mechanisms underlying the reconsolidation of pavlovian CS-drug memories. Most studies assessing the mechanisms underlying the reconsolidation of CS-drug memories have used more transla- tionally relevant procedures, in which the instrumental self- administration response for the drug produces both the drug reinforcer and the drug-associated CS, and the same response is tested following the manipulation aiming to disrupt reconsolida- tion [149, 163]. Reinstatement can subsequently be assessed through spontaneous recovery of previously acquired drug- seeking, or CS-induced or drug-induced reinstatement [163]. Like the memories underlying CPP, CS-drug memories acquired during self-administration depend upon protein synthesis [70, 164–166] and expression of the immediate early gene zif268 in the basolateral amygdala [167]. Prevention of transcription by inhibiting the dephosphorylation of eIFα also disrupts CS- cocaine memory reconsolidation [92]. Epigenetic changes occur during the reconsolidation of CS-drug memories, as inhibition of DNA methyltransferase (DNMT) during reactivation impairs sub- sequently both CS-induced and drug-induced reinstatement for heroin [168] and cocaine [169]. Administration of garcinol, which reduces histone acetylation, impairs subsequent CS-induced reinstatement and the expression of the immediate early genes arc and zif268 when administered into the lateral amygdala in conjunction with CS-cocaine memory reactivation [170]. Further- more, garcinol can disrupt simultaneously the reconsolidation of multiple CS-cocaine memories when it is administered in conjunction with US-based reactivation [64]. The memories underlying the conditioned reinforcing proper- ties of cocaine-associated cues, like those underlying CPP (Fig. 3), depend upon protein synthesis [62] and the expression of immediate early genes including zif268 [62]. However, unlike CPP, the reconsolidation of the memory underlying conditioned reinforcement requires Zif268 expression in the BLA, but not the nucleus accumbens core [98]. Upstream of gene expression, the reconsolidation of the conditioned reinforcement memory depends upon PKA [150] and the activation of NMDA receptors [151] and β-adrenergic receptors (for both cocaine cues [152] and alcohol cues [153]), with the administration of the adrenergic prodrug dipivefrine enhancing reconsolidation of a CS-alcohol memory when given in conjunction with memory reactivation [153]. Disrupting context-drug memories Numerous protein kinases are required for the reconsolidation of CS-drug memories, including glycogen synthase kinase 3β (GSK3β), inhibition of which within the BLA, but not the CeN, impairs both subsequent CS-induced and drug-induced reinstate- ment [171]. Systemic administration of rapamycin, which inhibits mTOR signalling, similarly impairs the reconsolidation of CS- cocaine memories [172], and CaMKII inhibition not only impairs CS-cocaine memory reconsolidation, but also appears to facilitate its extinction [173]. Inhibition of PKA with Rp-cAMPS impairs CS- cocaine memory reconsolidation [150], as does activation of the exchange protein directed activated by cAMP (Epac) [174]. As for CPP, protein phosphatases are required for CS-drug memory reconsolidation, with inhibition of calpain in the nucleus accumbens core at reactivation reducing subsequent CS-induced cocaine-seeking [99]. Activation of the protein phosphatase calcineurin not only appears to disrupt the reconsolidation of CS-drug memory, but also appears to facilitate its extinction [175], similar to the effects of inhibiting CaMKII [173]. Less is known about the mechanisms underlying the reconso- lidation of conditioned approach and conditioned motivation memories for drug-associated cues, which have been more extensively studied with natural (food) reinforcers [154–156]. However, it has been shown that the reconsolidation of pavlovian conditioned approach memories for CSs associated with alcohol [157] and sucrose [155, 156, 158–160] depend upon NMDAR activation, for both sign-tracking and goal-tracking responses. Although early studies suggested the memories underlying the goal-tracking responses were resistant to reconsolidation block- ade [154], it has subsequently been shown that goal-tracking memories will reconsolidate under specific conditions [155, 158], and that the destabilisation of these memories relies upon dopaminergic signalling from the ventral tegmental area [159]. β-adrenergic receptor antagonism at reactivation has produced mixed results on the reconsolidation of conditioned approach memories, in contrast to its effects on the memories underlying conditioned reinforcement. Propranolol did not disrupt the reconsolidation of the memories underlying conditioned approach for either alcohol [157] or sucrose [156], and also did not disrupt the reconsolidation of memories underlying pavlovian- instrumental transfer under reactivation conditions in which NMDAR antagonism produced amnesia [156, 157]. Even when propranolol did disrupt the reconsolidation of a conditioned g Monoaminergic signalling is also required for drug memory reconsolidation. Compared to CPP, dopaminergic signalling has been much less studied using self-administration procedures, but both D1 and D3-mediated signalling are necessary for the reconsolidation of CS-cocaine memories in mice [102, 176]. THE RECONSOLIDATION OF INSTRUMENTAL DRUG- ASSOCIATED MEMORIES The acts of drug-seeking and drug-taking are supported by instrumental associations, and can be considered to be a form of aberrantly engaged, maladaptive foraging behaviour [5]. These associations are initially goal-directed (i.e. an association forms between the drug-seeking action and the drug outcome), but ultimately become habitual and elicited by environmental, pavlovian CSs (i.e. become supported by ‘stimulus-response’ associations) [10]. In a subpopulation of individuals, these drug- seeking habits ultimately become uncontrolled and compulsive in nature [9, 10]. Pavlovian and instrumental memories interact to support ongoing drug-seeking behaviour and to promote relapse during attempted abstinence (Fig. 1; see refs. [31, 32], for further detail) and many studies, including those discussed in the previous section, have measured reductions in the capacity of pavlovian CSs to influence instrumental responding following a disruption of pavlovian memory reconsolidation. Far fewer studies have attempted to disrupt the memories underlying the instru- mental associations. However, disrupting the reconsolidation of the instrumental memories supporting drug-seeking and drug- taking behaviour would be an exciting therapeutic prospect. taking behaviour would be an exciting therapeutic prospect. It is likely that the relative paucity of studies of instrumental memory reconsolidation is at least partly due to an early study [192] in which an instrumental memory appeared not to undergo protein synthesis-dependent reconsolidation following retrieval. There may also be differences in the boundary conditions determining whether instrumental memories reconsolidate, as compared to pavlovian memories. Whereas pavlovian memories are susceptible to disruption following CS-based reactivation, instrumental memories may require a ‘mismatch’ in the type of reinforcement contingency experienced at reactivation [65, 66]. A predictable reinforcement schedule (i.e. additional sessions of training) did not render an instrumental memory underlying saccharin-seeking susceptible to disruption with cycloheximide [193] and studies investigating well-established cocaine-seeking memories in self-administering rats have indicated that a change in reinforcement schedule at reactivation, from a predictable ‘fixed-ratio’ schedule to a less predictable ‘variable ratio’ schedule of reinforcement, is required to induce susceptibility to disruption with the NMDAR antagonist MK-801, with non-reinforced memory reactivation sessions being insufficient to destabilise the instru- mental memory [65, 66]. However, other studies of instrumental sucrose-seeking and nicotine-seeking have shown that non- reinforced memory reactivation sessions can lead to memory lability [194–196]. A detailed analysis of procedural differences that may have contributed to these apparent differences in boundary conditions is considered by Piva et al. Disrupting context-drug memories Whether the source of noradrenaline input determines whether noradrenergic signalling influences memory destabilisation or restabilisation remains a question for future research. whether the retrieval-extinction procedure is supported by reconsolidation or extinction mechanisms [133]. Disrupting context-drug memories By contrast, noradrenergic signalling has been more extensively investigated, but has produced some apparently conflicting results in the literature that remain to be reconciled. Despite only being necessary for one of the three ‘routes to relapse’ [31] when CS-based reactivation procedures are used [152, 156, 157], signal- ling at β-adrenergic receptors is more effective in impairing CS- drug memory reconsolidation in reinstatement procedures when administered in conjunction with US-based reactivation. Propra- nolol administration in conjunction with a CS-based memory reactivation session gave mixed effects on the subsequent Translational Psychiatry (2023) 13:370 A.L. Milton 7 reinstatement of drug-seeking, producing no effect on the reinstatement of cocaine-seeking after enforced abstinence [177] or extinction of cocaine-seeking responses [164], but reducing subsequent alcohol-seeking after enforced abstinence [178] (with repeated rounds of propranolol administration and reactivation), and heroin-seeking in an extinction-reinstatement procedure [179]. Whether this reflects a difference in the noradrenergic mechanisms underlying the reconsolidation of memories asso- ciated with psychostimulants and depressants, or is related to the specific behavioural procedures used, warrants further investiga- tion. However, when exposure to the drug US was used to reactivate the memory, β1 adrenergic receptor antagonism within the CeN impaired the reinstatement of cocaine-seeking after instrumental extinction [91], and administration of propranolol directly to the BLA reduced subsequent alcohol self- administration when alcohol reinforcers were available during the reactivation session [180]. The key adrenergic innervation supporting reconsolidation within the BLA appears to come from the nucleus of the solitary tract (NST) rather than the locus coeruleus (LC), as reactivation of CS-morphine memories led to increases in activation (measured through immediate early gene expression) in the NST and BLA, but not the LC [181]. Moreover, chemogenetic activation of NST-BLA projections (but not LC-BLA projections) led to greater sensitivity of the CS-drug memory to protein synthesis inhibition, while inhibition of these projections prevented the amnestic effect usually produced by protein synthesis inhibition [181]. However, these latter data are more consistent with noradrenaline from the NST supporting the destabilisation of the CS-drug memory, in contrast to the effects on restabilisation of the CS-drug memory reported above (see also refs. [113, 182]). They also stand in contrast to findings from the fear memory reconsolidation literature, where inhibiting noradre- nergic signalling via LC-BLA projections rescued sensitivity to protein synthesis inhibition in a strong fear memory that was otherwise resistant [183]. CONCLUSIONS Th t li The conceptualisation of addiction as a disorder of learning and memory, combined with the re-emergence of research into the mechanisms underlying memory reconsolidation, provides the potential for the development of novel therapeutic approaches for treating addiction. There are a multitude of maladaptive emotional memories that contribute to the persistence of drug- seeking and that promote relapse following a period of abstinence, including pavlovian (contextual and discrete CSs) and instrumental memories. While there are many reports that reactivating drug memories through presentation of the US (i.e. allowing the administration of a small dose of the drug of abuse) allows for widespread disruption within the drug-associated memory network, this approach would present the ethical challenge of administering an (often illegal) drug of abuse to a patient who is trying to maintain abstinence. Whether drugs with a similar pharmacological action could substitute for the drug of abuse in a ‘US-based’ reactivation session is worthy of further study. Antagonism at β-adrenergic receptors has been used to target reconsolidation of addictive drug memories in both small-scale human trials and larger clinical trials. Despite initial reports that propranolol was ineffective at disrupting the reconsolidation of drug-associated memories in smokers when smoking-related CSs were used to reactivate the memory [207], subsequent studies using US-based reactivation of the nicotine memory showed a subsequent reduction in cigarette use in the group that had received propranolol [108]. The requirement for US-based reactivation to destabilise the memory may be specific to the strong interoceptive cues associated with smoking [208, 209], as reactivation of cocaine-associated memories through exposure to cocaine CSs was sufficient to induce sensitivity to disruption with propranolol in cocaine users [210, 211]. p p , Numerous studies have investigated whether behavioural interventions might be used to interfere or alter reconsolidation of drug memories in humans. The types of interventions used include extinction training following memory reactivation (the ‘retrieval-extinction’ procedures discussed previously), counter- conditioning, and cognitive reappraisal. The first demonstration of retrieval-extinction in drug users was that of Xue et al. [129], who built upon their rodent work to show in outpatient heroin users that a brief re-exposure to heroin-associated CSs, followed by prolonged exposure therapy, was sufficient to reduce CS-induced craving for at least 6 months [129]. A subsequent study showed that retrieval-extinction was also effective at reducing self- reported cigarette consumption in a population of smokers [212], although there were no effects on physiological reactivity to smoking-related CSs. THE RECONSOLIDATION OF INSTRUMENTAL DRUG- ASSOCIATED MEMORIES [197], and may be related to the dose of amnestic agent administered, the potential engagement of metaplasticity mechanisms [194], the specific reinforcer, the strength of training, changes in reactivation context [198] or the passage of time between learning and reactivation [199]. It may be that a better understanding of the relationship between prior learning – which generates the expectations that determine whether a ‘mismatch’ or prediction error is detected [200] – and the reactivation session itself will be necessary to resolve these apparent differences [68]. Glutamatergic signalling is critical for cue-drug memory reconsolidation, and silent synapses within the nucleus accum- bens appear to be unsilenced during drug memory reconsolida- tion, via a mechanism dependent upon the intracellular signalling molecule Rac1 [184]. Systemic antagonism of NMDARs at reactivation impairs cue-alcohol [166, 185, 186] and cue-cocaine [151] memory reconsolidation, as does antagonism of NMDARs within the BLA (which also reduces the expression of Zif268 in a reactivation-dependent manner) [151] and antagonism of GluN2A-containing NMDARs in the infralimbic cortex [187]. Systemic enhancement of NMDAR-mediated signalling with the partial agonist D-cycloserine enhances the reconsolidation of CS- cocaine memories, and increases Zif268 expression within the BLA [188]. Inhibition of lactate signalling at reactivation also reduces subsequent cocaine-seeking [125], potentially via effects on NMDARs. As for CPP procedures, there has been interest in non- pharmacological interventions targeting cue-drug memory recon- solidation, including retrieval-extinction. As for CPP, the data have been mixed and may have been influenced by subtle differences in procedure (previous reviews have considered extensively procedural differences and their impact on retrieval-extinction outcomes for seeking behaviour for natural [189] and drug- associated [190] reinforcers) or individual differences in boundary conditions [133]. While the first demonstration of reduced recovery of drug-seeking following retrieval-extinction was effective for both opiate and cocaine memories [129], there have also been reports that a retrieval-extinction procedure that successfully impairs CS-nicotine memories is ineffective for CS- cocaine memories [191]. More research is needed to determine If US-based reactivation session sessions are necessary to induce the destabilisation of instrumental memories, then this could present a challenge to clinical translation. Although prolonged exposure therapy often involves the handling of drug-associated paraphernalia [129] and, sometimes, limited self- administration of legal drugs such as nicotine (e.g. ref. [108]), a requirement to self-administer the drug of abuse would be Translational Psychiatry (2023) 13:370 A.L. RECONSOLIDATION IN THE CLINICAL CONTEXT To what extent can the findings discussed above, primarily from animal models relevant to drug addiction, be extrapolated to humans and to the clinical context? In light of the more extensive animal literature on disrupting the reconsolidation of pavlovian drug-associated memories, the majority of studies in humans have focused upon reducing reactivity to drug-associated CSs following reconsolidation-based manipulations. Many of these have recruited participants using legal drugs (alcohol and nicotine) but a small number of studies and clinical trials have focused upon psychostimulant and opiate users. p y p As for animals, pavlovian CS-drug memory reconsolidation depends upon NMDA receptor-mediated signalling in humans. The engagement of plasticity mechanisms appears to be important, as simply reducing neuronal activation with intrave- nous lidocaine infusions during reactivation had no effect on subsequent CS-induced cocaine craving [202].The administration of the NMDA receptor antagonist ketamine, in conjunction with a memory reactivation session that engaged prediction error, reduced subsequent alcohol-seeking in a population of hazardous drinkers [203], as did administration of the non-competitive NMDA receptor inhibitor nitrous oxide [204]. By contrast, another non- competitive NMDA receptor antagonist, memantine, given at memory reactivation was ineffective at reducing subsequent cigarette intake in a population of smokers [205]. However, despite both being NMDA receptor antagonists, memantine and ketamine have distinct effects on NMDA receptors [206] that may explain this apparent discrepancy. THE RECONSOLIDATION OF INSTRUMENTAL DRUG- ASSOCIATED MEMORIES Milton 8 challenging for ethical and safeguarding reasons in the case of illicit drugs. Whether non-contingent administration of the drug US (see Fig. 1), or use of pharmacological agents with similar neurochemical effects but reduced abuse liability (e.g. methyl- phenidate for psychostimulants [63] or methadone for opioids [201]), would be sufficient to destabilise the instrumental memory underlying drug-seeking remains an important outstanding question. the reduction in cigarettes consumed in the retrieval-extinction group. However, they found that administering a stressful treatment (the Montreal Imaging Stress Test) prior to extinction itself led to reduced cigarette consumption. This is consistent with stress facilitating extinction learning, as has been observed previously for fear memories [214]. p y Several studies have investigated whether post-reactivation counterconditioning might be used to reduce subsequent drug use. In a population of hazardous drinkers, re-exposure to alcohol- associated CSs combined with visual counterconditioning (expo- sure to unpleasant images from the International Affective Picture Scale [215]) and gustatory counterconditioning (exposure to solutions laced with Bitrex) was sufficient to reduce alcohol consumption [216] for at least 9 months follow-up [217]. The effects of counterconditioning appeared stronger than those of cognitive reappraisal following memory reactivation, which reduced scores on an alcohol fluency task, but not subsequent alcohol consumption or attentional bias towards alcohol- associated CSs [218]. However, this intervention was brief compared to the procedures usually used in cognitive therapy, and likely did not reproduce the therapeutic relationship necessary for the effectiveness of cognitive therapy (see ref. [219] for further discussion). More intensive and extensive cognitive therapy with cocaine use disorder patients showed enhanced efficacy of cognitive therapy when combined with memory reactivation procedures [220, 221]. These findings suggest that the addition of a brief CS reminder session to already established therapies, such as prolonged exposure and cognitive therapy, could markedly enhance patient outcomes, though more research is needed to test this in larger scale clinical populations. Translational Psychiatry (2023) 13:370 REFERENCES Grüsser SM, Wrase J, Klein S, Hermann D, Smolka MN, Ruf M, et al. Cue-induced activation of the striatum and medial prefrontal cortex is associated with sub- sequent relapse in abstinent alcoholics. Psychopharmacology. 2004;175:296–302. 38. Wolitzky-Taylor KB, Horowitz JD, Powers MB, Telch MJ. Psychological approa- ches in the treatment of specific phobias: a meta-analysis. Clin Psychol Rev. 2008;28:1021–37. e J. Psychotherapy by reciprocal inhibition. Cond Reflex. 1968;3:234 39. Wolpe J. Psychotherapy by reciprocal inhibition. Cond Reflex. 12. Seo D, Lacadie CM, Tuit K, Hong KI, Constable RT, Sinha R. Disrupted ven- tromedial prefrontal function, alcohol craving, and subsequent relapse risk. JAMA Psychiatry. 2013;70:727–39. 40. Holmes EA, Craske MG, Graybiel AM. A call for mental-health science. Nature. 2014;511:287–9. 41. Mellentin AI, Skøt L, Nielsen B, Schippers GM, Nielsen AS, Stenager E, et al. Cue exposure therapy for the treatment of alcohol use disorders: a meta-analytic review. Clin Psychol Rev. 2017;57:195–207. 13. Childress AR, Mozley PD, McElgin W, Fitzgerald J, Reivich M, O’Brien CP. Limbic activation during cue-induced cocaine craving. Am J Psychiatry. 1999;156:11–18. 14. Gawin, FH & Kleber, HD in Clinician’s Guide to Cocaine Addiction: Theory, Research and Treatment, pp. 33–52 (The Guildford Press, 1992). 42. Kiyak C, Simonetti ME, Norton S, Deluca P. The efficacy of cue exposure therapy on alcohol use disorders: a quantitative meta-analysis and systematic review. Addict Behav. 2022;139:107578. 15. Grant S, London ED, Newlin DB, Villemagne VL, Liu X, Contoreggi C, et al. Activation of memory circuits during cue-elicited cocaine craving. Proc Natl Acad Sci. 1996;93:12040–5. 43. Conklin CA, Tiffany ST. Applying extinction research and theory to cue-exposure addiction treatments. Addiction. 2002;97:155–67. 16. O’Brien CP, Testa T, O’Brien TJ, Greenstein R. Conditioning in human opiate addicts. Pavlovian J Biol Sci. 1976;11:195–202. 44. Mayet, S, Farrell, M, Ferri, M, Amato, L & Davoli, M Psychosocial treatment for opiate abuse and dependence. Cochrane Database Syst Rev 4 (2004). 17. O’Brien CP, Childress AR, McClellan TA, Ehrman R. Classical conditioning in drug dependent humans. Ann N. Y Acad Sci. 1992;654:400–15. 45. Bouton ME, Swartzentruber D. Sources of relapse after extinction in pavlovian and instrumental learning. Clin Psychol Rev. 1991;11:123–40. 18. Ehrman RN, Robbins SJ, Childress AR, O’Brien CP. Conditioned responses to cocaine-related stimuli in cocaine abuse patients. Psychopharmacology. 1992;107:523–9. 46. Bouton ME. Context, ambiguity, and unlearning: sources of relapse after behavioral extinction. Biol Psychiatry. 2002;52:976–86. 47. Wechsler TF, Kümpers F, Mühlberger A. REFERENCES 29. Feltenstein MW, See RE. Potentiation of cue-induced reinstatement of cocaine- seeking in rats by the anxiogenic drug yohimbine. Behav Brain Res. 2006;174:1–8. 1. Public Health England. An evidence review of the outcomes that can be expected of drug misuse treatment in England. (2017). 1. Public Health England. An evidence review of the outcomes that can be expected of drug misuse treatment in England. (2017). 2. Public Health England. The public health burden of alcohol and the effective- ness and cost-effectiveness of alcohol control policies: an evidence review. (2018). 30. Lee JLC, Everitt BJ. In Memories: Molecules and Circuits Research and Perspectives in Neurosciences (eds B Bontempi, AJ Silva, & Y Christen) 83–98 (Springer-Verlag, 2007). 3. The Secretary General. Facing addiction in America: The Surgeon General’s spotlight on opioids. (2018). 31. Milton AL, Everitt BJ. The psychological and neurochemical mechanisms of drug memory reconsolidation: implications for the treatment of addiction. Eur J Neurosci. 2010;31:2308–19. 4. American Psychiatric Association. The Diagnostic and Statistical Manual of Mental Disorders (5th ed.). (American Psychiatric Publishing, 2013). Disorders (5th ed.). (American Psychiatric Publishing, 2013). 32. Milton AL, Everitt BJ. The persistence of maladaptive memory: addiction, drug memories and anti-relapse treatments. Neurosci Biobehav Rev. 2012;36:1119–39. 5. Everitt BJ, Dickinson A, Robbins TW. The neuropsychological basis of addictive behaviour. Brain Res Rev. 2001;36:129–38. 6. Hyman SE. Addiction: a disease of learning and memory. Am J Psychiatry. 2005;162:1414–22. 33. Torregrossa MM, Corlett PR, Taylor JR. Aberrant learning and memory in addiction. Neurobiol Learn Mem. 2011;96:609–23. 7. Deroche-Gamonet V, Belin D, Piazza PV. Evidence for addiction-like behavior in the rat. Science. 2004;305:1014–7. 34. Tronson NC, Taylor JR. Addiction: a drug-induced disorder of memory recon- solidation. Curr Opin Neurobiol. 2013;23:573–80. 8. Pelloux Y, Everitt BJ, Dickinson A. Compulsive drug seeking by rats under punishment: effects of drug taking history. Psychopharmacology. 2007;194:127–37. 35. Torregrossa MM, Taylor JR. Learning to forget: manipulating extinction and reconsolidation processes to treat addiction. Psychopharmacology. 2013;226:659–72. 9. Everitt BJ, Robbins TW. Neural systems of reinforcement for drug addiction: from actions to habits to compulsion. Nat Neurosci. 2005;8:1481–9. 36. Dunbar AB, Taylor JR. Reconsolidation and psychopathology: moving towards reconsolidation-based treatments. Neurobiol Learn Mem. 2016;142:162–71. 10. Everitt BJ, Robbins TW. Drug addiction: updating actions to habits to compul- sions ten years on. Annu Rev Psychol. 2016;67:8.1–8.28. 37. Watson JP, Gaind R, Marks IM. Prolonged exposure: a rapid treatment for phobias. Br Med J. 1971;1:13–15. 11. CONCLUSIONS Th t li This lack of physiological modulation was also found in a recent study [213], which also failed to replicate y The clinical trials of reconsolidation-based intervention for the treatment of addiction have been relatively small-scale, but overall there is cause for optimism. Future clinical studies will need to address a number of research questions, including the optimal reactivation procedure(s) to induce drug memory destabilisation (and ideally, this would include the development of methods to measure online and in real-time the induction of memory lability [68]), whether there are predictable individual differences in patients that could be used to tailor the reactivation procedure [222], and whether the effects seen to date generalise to larger patient populations. However, the encouraging data to date suggest that there is potential for reconsolidation-based interven- tions in providing a greater range of treatment options for patients. Questions also remain within the preclinical literature, including whether behavioural interference approaches such as retrieval- extinction are acting via reconsolidation or extinction mechanisms A.L. Milton 9 [133] and whether reconsolidation-based interventions are effective in animal models of compulsive drug-seeking [7, 8]. Demonstrations that instrumental drug-seeking memories can be disrupted [65, 66, 223] raises the exciting prospect that it may be possible to return drug-seeking from being habitual to goal- directed, although whether individuals treated in this fashion would reacquire habitual and compulsive drug-seeking remains to be established. At the very least, it seems feasible that reconsolidation-based interventions would serve to reduce the pernicious effects of drug memories in eliciting automatic drug- seeking behaviours sufficiently to allow other, complementary therapies such as contingency management or cognitive beha- vioural therapy to have a greater chance of success. 23. Kosten TR, Scanley BE, Tucker KA, Oliveto A, Prince C, Sinha R, et al. Cue-induced brain activity changes and relapse in cocaine-dependent patients. Neu- ropsychopharmacology. 2006;31:644–50. 24. Regier PS, Jagannathan K, Franklin TR, Wetherill RR, Langleben DD, Gawyrsiak M, et al. Sustained brain response to repeated drug cues is associated with poor drug-use outcomes. Addict Biol. 2021;26:e13028. 25. de Wit H, Stewart J. Reinstatement of cocaine-reinforced responding in the rat. Psychopharmacology. 1981;75:134–43. 26. Arroyo M, Markou A, Robbins TW, Everitt BJ. Acquisition, maintenance and reinstatement of intravenous cocaine self-administration under a second-order schedule of reinforcement in rats: effects of conditioned cues and continuous access to cocaine. Psychopharmacology. 1998;140:331–44. access to cocaine. Psychopharmacology. 1998;140:331–44. 27. Crombag HS, Shaham Y. CONCLUSIONS Th t li Renewal of drug seeking by contextual cues after prolonged extinction in rats. Behav Neurosci. 2002;116:169–73. 28. Ito R, Dalley JW, Robbins TW, Everitt BJ. Dopamine release in the dorsal striatum during cocaine-seeking behavior under the control of a drug-associated cue. J Neurosci. 2002;22:6247–53. REFERENCES Garcinol blocks the reconsolidation of multiple cocaine- paired cues after a single cocaine-reactivation session. Neuropsychopharma- cology. 2017;42:1884–92. 65. Exton-McGuinness MTJ, Patton RC, Sacco LB, Lee JLC. Reconsolidation of a well- learned instrumental memory. Learn Mem. 2014;21:468–77. 89. Robinson MJF, Franklin KBJ. Effects of anisomycin on consolidation and reconsolidation of a morphine-conditioned place preference. Behav Brain Res. 2007;178:146–53. 66. Exton-McGuinness MTJ, Lee JLC. Reduction in responding for sucrose and cocaine reinforcement by disruption of memory reconsolidation. eNeuro. 2015;2:e0009–0015.2015. 90. Yim AJ, Moraes CRG, Ferreira TL, Oliveira MGM. Protein synthesis inhibition in the basolateral amygdala following retrieval does not impair expression of morphine-associated conditioned place preference. Behav Brain Res. 2006;171:162–9. 67. Vaverková Z, Milton AL, Merlo E. Retrieval-dependent mechanisms affecting emotional memory persistence: reconsolidation, extinction, and the space in between. Front Behav Neurosci. 2020;14:574358. 91. Zhu H, Zhou Y, Liu Z, Chen X, Li Y, Liu X, et al. β1-adrenoceptor in the central amygdala is required for unconditioned stimulus-induced drug memory reconsolidation. Int J Neuropsychopharmacol. 2018;21:267–80. 68. Milton AL, Das RK, Merlo E. The challenge of memory destabilisation: from prediction error to prior expectations and biomarkers. Brain Res Bull. 2023;194:100–4. 92. Jian M, Luo YX, Xue YX, Han Y, Shi HS, Liu JF, et al. eIF2α dephosphorylation in basolateral amygdala mediates reconsolidation of drug memory. J Neurosci. 2014;34:10010–21. 69. Taujanskaitė U, Cahill EN, Milton AL. Targeting drug memory reconsolidation: a neural analysis. Curr Opin Pharmacol. 2021;56:7–12. 93. Shen Q, Xie B, Galaj E, Yu H, Li X, Lu Y, et al. CircTmeff-1 in the nucleus accumbens regulates the reconsolidation of cocaine-associated memory. Brain Res Bull. 2022;185:64–73. 70. Xue YX, Chen YY, Zhang LB, Zhang LQ, Huang GD, Sun SC, et al. Selective inhibition of amygdala neuronal ensembles encoding nicotine-associated memories prevents nicotine seeking and relapse. Biol Psychiatry. 2017;82:781–93. 94. Shi X, von Weltin E, Fitzsimmons E, Do C, Caban Rivera C, Chen C, et al. Reac- tivation of cocaine contextual memory engages mechanistic target of rapa- mycin/S6 kinase 1 signaling. Front Pharmacol. 2022;13:976932. 71. Ramirez DR, Bell GH, Lasseter HC, Xie X, Traina SA, Fuchs RA. Dorsal hippocampal regulation of memory reconsolidation processes that facilitate drug context- induced cocaine-seeking behavior in rats. Eur J Neurosci. 2009;30:901–12. 95. Shi X, Miller JS, Harper LJ, Poole RL, Gould TJ, Unterwald EM. Reactivation of cocaine reward memory engages the Akt/GSK3/mTOR signaling pathway and can be disrupted by GSK3 inhibition. Psychopharmacology. 2014;231:3109–18. 72. REFERENCES Inferiority or even superiority of virtual reality exposure therapy in phobias? - A systematic review and quantitative meta-analysis on randomized controlled trials specifically comparing the efficacy of virtual reality exposure to gold standard in vivo exposure in agoraphobia, specific phobia, and social phobia. Front Psychol. 2019;10:1758. 19. Janes AC, Pizzagalli DA, Richardt S, Frederick BD, Chuzi S, Pachas G, et al. Brain reactivity to smoking cues prior to smoking cessation predicts ability to main- tain tobacco abstinence. Biol Psychiatry. 2010;67:722–9. 20. McClernon FJ, Hiott FB, Liu J, Salley AN, Behm FM, Rose JE. Selectively reduced respones to smoking cues in amygdala following extinction-based smoking cessation: results of a preliminary functional magnetic resonance imaging study. Addiction Biol. 2007;12:503–12. 48. Craske MG, Treanor M, Conway CC, Zbozinek T, Vervliet B. Maximizing exposure therapy: an inhibitory learning approach. Behav Res Ther. 2014;58:10–23. 21. Versace F, Engelmann JM, Robinson JD, Jackson EF, Green CE, Lam CY, et al. Prequit fMRI responses to pleasant cues and cigarette-related cues predict smoking cessation outcome. Nicotine Tob Res. 2014;16:697–708. 49. Di Ciano P, Everitt BJ. Conditioned reinforcing properties of stimuli paired with self-adminstered cocaine, heroin or sucrose: implications for the persistence of addictive behavior. Neuropharmacology. 2004;47:202–13. 22. Li Q, Li W, Wang H, Wang Y, Zhang Y, Zhu J, et al. Predicting subsequent relapse by drug-related cue-induced brain activation in heroin addiction: an event- related functional magnetic resonance imaging study. Addiction Biol. 2015;20:968–78. 50. Tunstall BJ, Kearns DN. Cocaine can generate a stronger conditioned reinforcer than food despite being a weaker primary reinforcer. Addict Biol. 2016;21:282–93. 51. Mackintosh, NJ. The Psychology of Animal Learning. (Academic Press, 1974). Translational Psychiatry (2023) 13:370 A.L. Milton 10 52. Martin SJ, Morris RGM. New life in an old idea: the synaptic plasticity and memory hypothesis revisited. Hippocampus. 2002;12:609–36. 80. Fuchs RA, Weber SM, Rice HJ, Neisewander JL. Effects of excitotoxic lesions of the basolateral amygdala on cocaine-seeking behavior and cocaine conditioned place preference in rats. Brain Res. 2002;929:15–25. 53. Josselyn SA, Köhler S, Frankland PW. Finding the engram. Nat Rev Neurosci. 2015;16:521–34. 81. Everitt, BJ, Cardinal, RN, Hall, J, Parkinson, JA, & Robbins, TW. Differential involvement of amygdala subsystems in appetitive conditioning and drug addiction. In The Amygdala: A Functional Analysis (2nd Ed.) pp. 353–90 (Oxford University Press, 2000). 54. Tonegawa S, Pignatelli M, Roy DS, Ryan TJ. Memory engram storage and retrieval. Curr Opin Neurobiol. 2015;35:101–9. 55. REFERENCES Anthony JC, Warner LA, Kessler RC. Comparative epidemiology of dependence on tobacco, alcohol, controlled substances, and inhalants: findings from the National Comorbidity Survey. Exp Clin Psychopharmacol. 1994;166:244–68. 82. Burns LH, Everitt BJ, Robbins TW. Effects of excitotoxic lesions of the basolateral amygdala on conditional discrimination learning with primary and conditioned reinforcement. Behav Brain Res. 1999;100:123–33. 56. Schlag AK. Percentages of problem drug use and their implications for policy making: a review of the literature. Drug Sci, Policy Law. 2020;6:1–9. 83. Whitelaw RB, Markou A, Robbins TW, Everitt BJ. Excitotoxic lesions of the basolateral amygdala impair the acquisition of cocaine-seeking behaviour under a second-order schedule of reinforcement. Psychopharmacology. 1996;127:213–24. 57. McGaugh JL. Memory - a century of consolidation. Science. 2000;287:248–51. 58. Nader K, Schafe GE, LeDoux JE. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature. 2000;406:722–6. 84. Selden NRW, Everitt BJ, Jarrard LE, Robbins TW. Complementary roles for the amygdala and hippocampus in aversive conditioning to explicit and contextual cues. Neuroscience. 1991;42:335–50. 59. Dębiec, J & Altemus, M Toward a new treatment for traumatic memories. Cer- ebrum. 2006:2–11. 60. Hernandez PJ, Kelley AE. Cracking addiction the second time around: recon- solidation of drug-related memories. Neuron. 2005;47:772–5. 85. Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci. 1992;106:274–85. 61. Pedreira ME, Pérez-Cuesta LM, Maldonado H. Mismatch between what is expected and what actually occurs triggers memory reconsolidation or extinc- tion. Learn Mem. 2004;11:579–85. 86. Ito R, Robbins TW, McNaughton BL, Everitt BJ. Selective excitotoxic lesions of the hippocampus and basolateral amygdala have dissociable effects on appetitive cue and place conditioning based on path integration in a novel Y-maze pro- cedure. Eur J Neurosci. 2006;23:3071–80. 62. Lee JLC, Di Ciano P, Thomas KL, Everitt BJ. Disrupting reconsolidation of drug memories reduces cocaine seeking behavior. Neuron. 2005;47:795–801. 63. Luo YX, Xue YX, Liu JF, Shi HS, Jian M, Han Y, et al. A novel UCS memory retrieval-extinction procedure to inhibit relapse to drug seeking. Nat Commun. 2015;6:7675. 87. Valjent E, Corbillé AG, Bertran-Gonzelez J, Hervé D, Girault JA. Inhibition of ERK pathway or protein synthesis during reexposure to drugs of abuse erases pre- viously learned place preference. Proc Natl Acad Sci. 2006;103:2932–7. 88. Milekic MH, Brown SD, Castellini C, Alberini CM. Persistent disruption of an established morphine conditioned place preference. J Neurosci. 2006;26:3010–20. 64. Dunbar AB, Taylor JR. REFERENCES Wells AM, Lasseter HC, Xie X, Cowhey KE, Reittinger AM, Fuchs RA. Interaction between the basolateral amygdala and dorsal hippocampus is critical for cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior in rats. Learn Mem. 2011;18:693–702. 96. Wu P, Xue YX, Ding ZB, Xue LF, Xu CM, Lu L. Glycogen synthase kinase 3B in the basolateral amygdala is critical for the reconsolidation of cocaine reward memory. J Neurochem. 2011;118:113–25. 73. Ritchie JL, Walters JL, Galliou JM, Christian RJ, Qi S, Savenkova MI, et al. Baso- lateral amygdala corticotropin-releasing factor type 1 regulates context-cocaine memory strength during reconsolidation in a sex-dependent manner. Neuro- pharmacology. 2021;200:108819. 97. Shi X, von Weltin E, Barr JL, Unterwald EM. Activation of GSK3β induced by recall of cocaine reward memories is dependent on GluN2A/B NMDA receptor sig- naling. J Neurochem. 2019;151:91–102. 98. Théberge FR, Milton AL, Belin D, Lee JLC, Everitt BJ. The basolateral amygdala and nucleus accumbens core mediate dissociable aspects of drug memory reconsolidation. Learn Mem. 2010;17:444–53. 74. Higginbotham JA, Jones NM, Wang R, Christian RJ, Ritchie JL, McLaughlin RJ, et al. Basolateral amygdala CB1 receptors gate HPA axis activation and context- cocaine memory strength during reconsolidation. Neuropsychopharmacology. 2021;46:1554–64. 99. Liang J, Li JL, Han Y, Luo YX, Xue YX, Zhang Y, et al. Calpain-GRIP signaling in nucleus accumbens core mediates the reconsolidation of drug reward memory. J Neurosci. 2017;37:8938–51. 75. Schechter MD, Calcagnetti DJ. Trends in place preference conditioning with a cross-indexed bibliography; 1957-1991. Neurosci Biobehav Rev. 1993;17:21–41. 100. Zhang YX, Akumuo RC, España RA, Yan CX, Gao WJ, Li YC. The histone deme- thylase KDM6B in the medial prefrontal cortex epigenetically regulates cocaine reward memory. Neuropharmacology. 2018;141:113–25. 76. Crombag HS, Bossert JM, Koya E, Shaham Y. Context-induced relapse to drug seeking: a review. Philos Trans R Soc B. 2008;363:3233–43. 77. Everitt BJ, Morris KA, O’Brien A, Robbins TW. The basolateral amygdala-ventral striatal system and conditioned place preference: further evidence of limbic- striatal interactions underlying reward-related processes. Neuroscience. 1991;42:1–18. 101. Liu C, Sun X, Wang Z, Le Q, Liu P, Jiang C, et al. Retrieval-induced upregulation of Tet3 in pyramidal neurons of the dorsal hippocampus mediates cocaine- associated memory reconsolidation. Int J Neuropsychopharmacol. 2018;21:255–66. 78. White NM, McDonald RJ. Acquisition of a spatial conditioned place preference is impaired by amygdala lesions and improved by fornix lesions. Behav Brain Res. 1993;55:269–81. 102. Yan Y, Kong H, Wu EJ, Newman AH, Xu M. REFERENCES Dopamine D3 receptors regulate reconsolidation of cocaine memory. Neuroscience. 2013;241:32–40. 79. Brown EE, Fibiger HC. Differential effects of excitotoxic lesions of the amygdala on cocaine-induced conditioned locomotion and conditioned place preference. Psychopharmacology. 1993;113:123–30. 103. Marion-Poll L, Besnard A, Longueville S, Valjent E, Engmann O, Caboche J, et al. Cocaine conditioned place preference: unexpected suppression of preference due to testing combined with strong conditioning. Addict Biol. 2019;24:364–75. Translational Psychiatry (2023) 13:370 Translational Psychiatry (2023) 13:370 Translational Psychiatry (2023) 13:370 A.L. Milton 11 104. Blaiss CA, Janak PH. Post-training and post-reactivation administration of amphetamine enhances morphine conditioned place preference. Behav Brain Res. 2006;171:329–37. 131. Chang H-A, Dai W, Hu SS-J. Dose-dependent effect of retrieval-extinction on preventing reinstatement of cocaine-associated memory in mice. Chin J Physiol. 2022;65:159–70. 105. Denny RR, Unterwald EM. Clonidine, an α2 receptor agonist, disrupts reconso- lidation of a cocaine-paired environmental memory. Behav Pharmacol. 2019;30:529–33. 132. Cahill EN, Milton AL. Neurochemical and molecular mechanisms underlying the retrieval-extinction effect. Psychopharmacology. 2019;236:111–32. 133. Kuijer, EJ, Ferragud, A & Milton, AL. Retrieval-extinction and relapse prevention: rewriting maladaptive drug memories? Front Behav Neurosci. 2020;14:23. 106. Bernardi RE, Lattal KM, Berger SP. Postretrieval propranolol disrupts a cocaine conditioned place preference. Neuroreport. 2006;17:1443–7. 134. Goltseker K, Bolotin L, Barak S. Counterconditioning during reconsolidation prevents relapse of cocaine memories. Neuropsychopharmacology. 2017;42:716–26. 107. Robinson MJF, Franklin KBJ. Central but not peripheral beta-adrenergic antag- onism blocks reconsolidation for a morphine place preference. Behav Brain Res. 2007;182:129–34. 135. Goltseker, K, Handrus, H & Barak, S Disruption of relapse to alcohol seeking by aversive counterconditioning following memory retrieval. Addict Biol. 2020:e12935. 108. Xue YX, Deng JH, Chen YY, Zhang LB, Wu P, Huang GD, et al. Effect of selective inhibition of reactivated nicotine-associated memories with propranolol on nicotine craving. JAMA Psychiatry. 2017;74:224–32. 136. Fuchs RA, Bell GH, Ramirez DR, Eaddy JL, Su Z-I. Basolateral amygdala involve- ment in memory reconsolidation processes that facilitate drug context-induced cocaine seeking. Eur J Neurosci. 2009;30:889–900. 109. Font L, Cunningham CL. Post-retrieval propranolol treatment does not modulate reconsolidation or extinction of ethanol-induced conditioned place preference. Pharmacol Biochem Behav. 2012;101:222–30. 137. Mamiya N, Fukushima H, Suzuki A, Matsuyama Z, Homma S, Frankland PW, et al. Brain region-specific gene expression activation required for reconsolidation and extinction of contextual fear memory. J Neurosci. 2009;29:402–13. 110. Robinson MJF, Franklin KBJ. Reconsolidation of a morphine place preference: impact of the strength and age of memory on disruption by propranolol and midazolam. REFERENCES Behav Brain Res. 2010;213:201–7. 138. Qi S, Tan SM, Wang R, Higginbotham JA, Ritchie JL, Ibarra CK, et al. Optogenetic inhibition of the dorsal hippocampus CA3 region during early-stage cocaine- memory reconsolidation disrupts subsequent context-induced cocaine seeking in rats. Neuropsychopharmacology. 2022;47:1473–83. 111. Fricks-Gleason AN, Marshall JF. Post-retrieval b-adrenergic receptor blockade: effects on extinction and reconsolidation of cocaine-cue memories. Learn Mem. 2008;15:643–8. 112. Bernardi RE, Ryabinin AE, Berger SP, Lattal KM. Post-retrieval disruption of a cocaine conditioned place preference by systemic and intrabasolateral amyg- dala b2 and a1-adrenergic antagonists. Learn Mem. 2009;16:777–89. 139. Wells AM, Xie X, Higginbotham JA, Arguello AA, Healey KL, Blanton M, et al. Contribution of an SFK-mediated signaling pathway in the dorsal hippocampus to cocaine-memory reconsolidation in rats. Neuropsychopharmacology. 2016;41:675–85. 113. Otis JM, Dashew KB, Mueller D. Neurobiological dissociation of retrieval and reconsolidation of cocaine-associated memory. J Neurosci. 2013;33:1271–81. 140. Wells AM, Arguello AA, Xie X, Blanton MA, Lasseter HC, Reittinger AM, et al. Extracellular signal-regulated kinase in the basolateral amygdala, but not the nucleus accumbens core, is critical for context-response-cocaine memory reconsolidation in rats. Neuropsychopharmacology. 2013;38:753–62. 114. Kelley JB, Anderson KL, Itzhak Y. Long-term memory of cocaine-associated context: disruption and reinstatement. Neuroreport. 2007;18:777–80. 115. Itzhak Y. Role of the NMDA receptor and nitric oxide in memory reconsolidation of cocaine-induced conditioned place preference in mice. Ann N. Y Acad Sci. 2008;1139:350–7. 141. Arguello AA, Hodges MA, Wells AM, Lara H, Xie X, Fuchs RA. Involvement of amygdalar protein kinase A, but not calcium/calmodulin-dependent protein kinase II, in the reconsolidation of cocaine-related contextual memories in rats. Psychopharmacology. 2014;231:55–65. 116. Brown TE, Lee BR, Sorg BA. The NMDA antagonist MK-801 disrupts reconsoli- dation of a cocaine-associated memory for conditioned place preference but not for self-administration in rats. Learn Mem. 2008;15:857–65. 142. Stringfield SJ, Higginbotham JA, Wang R, Berger AL, McLaughlin RJ, Fuchs RA. Role of glucocorticoid receptor-mediated mechanisms in cocaine memory enhancement. Neuropharmacology. 2017;123:349–58. 117. Sadler R, Herzig V, Schmidt WJ. Repeated treatment with the NMDA antagonist MK-801 disrupts reconsolidation of memory for amphetamine-conditioned place preference. Behav Pharmacol. 2007;18:699–703. 143. Higginbotham JA, Wang R, Richardson BD, Shiina H, Tan SM, Presker MA, et al. CB1 receptor signaling modulates amygdalar plasticity during context-cocaine memory reconsolidation to promote subsequent cocaine seeking. J Neurosci. 2021;41:613–29. 118. Zhai H, Wu P, Chen S, Li F, Liu Y, Lu L. Effects of scopolamine and ketamine on reconsolidation of morphine conditioned place preference in rats. REFERENCES Behav Pharmacol. 2008;19:211–6. 119. Garthwaite J. Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 1991;14:60–67. 144. Millan EZ, Milligan-Saville J, McNally GP. Memory retrieval, extinction, and reinstatement of alcohol seeking. Neurobiol Learn Mem. 2013;101:26–32. 120. Itzhak Y, Anderson KL. Memory reconsolidation of cocaine-associated context requires nitric oxide signaling. Synapse. 2007;61:1002–5. 145. Flagel SB, Akil H, Robinson TE. Individual differences in the attribution of incentive salience to reward-related cues: implications for addiction. Neuro- pharmacology. 2009;56:139–48. 121. Yun Y, Yao G, Yue H, Guo L, Qin G, Li G, et al. SO2 inhalation causes synaptic injury in rat hippocampus via its derivatives in vivo. Chemosphere. 2013;93:2426–32. 146. Cartoni E, Balleine B, Baldassarre G. Appetitive pavlovian-instrumental transfer: a review. Neurosci Biobehav Rev. 2016;71:829–48. 122. Rulan D, Zhenbang Y, Yipu Z, Yuan G, Galaj E, Xiaorui S, et al. Exogenous SO2 donor treatment impairs reconsolidation of drug reward memory in mice. Eur J Pharmacol. 2021;896:173911. 147. Corbit LH, Balleine BW. Learning and motivational processes contributing to pavlovian-instrumental transfer and their neural bases: dopamine and beyond. Curr Top Behav Neurosci. 2015;27:259–89. 123. Yang J, Ruchti E, Petit JM, Jourdain P, Grenningloh G, Allaman I, et al. Lactate promotes plasticity gene expression by potentiating NMDA signaling in neu- rons. Proc Natl Acad Sci. 2014;111:12228–33. 148. Rutherford LG, Milton AL. Deconstructing and reconstructing behaviour relevant to mental health disorders: the benefits of a psychological approach, with a focus on addiction. Neurosci Biobehav Rev. 2022;133:104514. 124. Boury-Jamot B, Carrard A, Martin JL, Halfon O, Magistretti PJ, Boutrel B. Dis- rupting astrocyte-neuron lactate transfer persistently reduces conditioned responses to cocaine. Mol Psychiatry. 2016;21:1070–6. 149. Reichel CM, Bevins RA. Forced abstinence model of relapse to study pharma- cological treatments of substance use disorder. Curr Drug Abus Rev. 2009;2:184–94. 150. Sanchez H, Quinn JJ, Torregrossa MM, Taylor JR. Reconsolidation of a cocaine- associated stimulus requires amygdalar protein kinase A. J Neurosci. 2010;30:4401–7. 125. Zhang Y, Xue Y, Meng S, Luo Y, Liang J, Li J, et al. Inhibition of lactate transport erases drug memory and prevents drug relapse. Biol Psychiatry. 2016;79:928–39. 126. Lee JLC, Nader K, Schiller D. An update on memory reconsolidation updating. Trends Cogn Sci. 2017;21:531–45. 151. Milton AL, Lee JLC, Butler VJ, Gardner RJ, Everitt BJ. Intra-amygdala and systemic antagonism of NMDA receptors prevents the reconsolidation of drug-associated memory and impairs subsequently both novel and previously acquired drug- seeking behaviors. J Neurosci. 2008;28:8230–7. 127. REFERENCES J Neurosci. 2013;33:14205–10. 183. Haubrich J, Bernabo M, Nader K. Noradrenergic projections from the locus coeruleus to the amygdala constrain fear memory reconsolidation. eLife. 2020;9:e57010. 184. Wright WJ, Graziane NM, Neumann PA, Hamilton PJ, Cates HM, Fuerst L, et al. Silent synapses dictate cocaine memory destabilization and reconsolidation. Nat Neurosci. 2020;23:32–46. 160. Drame ML, Balaet M, Lee JLC. Memory reconsolidation impairments in sign- tracking to an audiovisual compound stimulus. Behav Brain Res. 2020;393:112774. 161. Cogan ES, Shapses MA, Robinson TE, Tronson NC. Disrupting reconsolidation: memory erasure or blunting of emotional/motivational value? Neuropsycho- pharmacology. 2019;44:399–407. 185. Puaud M, Ossowska Z, Barnard J, Milton AL. Saccharin fading is not required for the acquisition of alcohol self-administration, and can alter the dynamics of cue- alcohol memory reconsolidation. Psychopharmacology. 2018;235:1069–82. 162. Kindt M, Soeter M, Vervliet B. Beyond extinction: erasing human fear responses and preventing the return of fear. Nat Neurosci. 2009;12:256–8. 186. Vengeliene V, Olevska A, Spanagel R. Long-lasting effect of NMDA receptor antagonist memantine on ethanol-cue association and relapse. J Neurochem. 2015;135:1080–5. 163. Sanchis-Segura C, Spanagel R. Behavioural assessment of drug reinforcement and addictive features in rodents: an overview. Addict Biol. 2008;11:2–38. 187. Hafenbreidel M, Rafa Todd C, Mueller D. Infralimbic GluN2A-containing NMDA receptors modulate reconsolidation of cocaine self-administration memory. Neuropsychopharmacology. 2017;42:1113–25. 164. Dunbar AB, Taylor JR. Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory. Learn Mem. 2016;23:391–8. 188. Lee JLC, Gardner RJ, Butler VJ, Everitt BJ. D-cycloserine potentiates the recon- solidation of cocaine-associated memories. Learn Mem. 2009;16:82–85. 165. Sorg BA, Todd RP, Slaker M, Churchill L. Anisomycin in the medial prefrontal cortex reduces reconsolidation of cocaine-associated memories in the rat self- administration model. Neuropharmacology. 2015;92:25–33. 189. Kredlow MA, Unger LD, Otto MW. Harnessing reconsolidation to weaken fear and appetitive memories: a meta-analysis of post-retrieval extinction effects. Psychological Bull. 2016;142:314–36. 166. von der Goltz C, Vengeliene V, Bilbao A, Perreau-Lenz S, Pawlak CR, Kiefer F, et al. Cue-induced alcohol seeking behaviour is reduced by disrupting the reconsolidation of alcohol-related memories. Psychopharmacology. 2009;205:389–97. 190. Hutton-Bedbrook K, McNally GP. The promises and pitfalls of retrieval-extinction procedures in preventing relapse to drug-seeking. Front Psychiatry. 2013;4:14. 191. Struik RF, De Vries TJ, Peters J. Detrimental effects of a retrieval-extinction procedure on nicotine seeking, but not cocaine seeking. Front Behav Neurosci. 2019;13:243. 167. Lee JLC, Milton AL, Everitt BJ. REFERENCES Cue-induced cocaine seeking and relapse are reduced by disruption of drug memory reconsolidation. J Neurosci. 2006;26:5881–7. 192. Hernandez PJ, Kelley AE. Long-term memory for instrumental responses does not undergo protein synthesis-dependent reconsolidation upon retrieval. Learn Mem. 2004;11:748–54. 168. Qian S, Shi C, Huang S, Yang C, Luo Y. DNA methyltransferase activity in the basolateral amygdala is critical for reconsolidation of a heroin reward memory. Front Mol Neurosci. 2022;15:1002139. 193. Mierzejewski P, Korkosz A, Rogowski A, Korkosz I, Kostowski W, Scinska A. Post- session injections of a protein synthesis inhibitor, cycloheximide do not alter saccharin self-administration. Prog Neuro-Psychopharmacol Biol Psychiatry. 2009;33:286–9. 169. Shi HS, Luo YX, Yin X, Wu HH, Xue G, Geng XH, et al. Reconsolidation of a cocaine associated memory requires DNA methyltransferase activity in the basolateral amygdala. Sci Rep. 2015;5:13327. 170. Monsey MS, Ruiz SG, Taylor JR. Regulation of garcinol on histone acetylation in the amygdala and on the reconsolidation of a cocaine-associated memory. Front Behav Neurosci. 2020;13:281. 194. Piva A, Gerace E, Di Chio M, Osanni L, Padovani L, Caffino L, et al. The meta- plastic effects of NMDA receptors blockade on reactivation of instrumental memories in rats. Neurobiol Learn Mem. 2018;154:87–96. 171. Xie Y, Zhang Y, Hu T, Zhao Z, Liu Q, Li H. Inhibition of glycogen synthase kinase 3β activity in the basolateral amygdala disrupts reconsolidation and attenuates heroin relapse. Front Mol Neurosci. 2022;15:932939. 195. Tedesco V, Mutti A, Auber A, Chiamulera C. Nicotine-seeking reinstatement is reduced by inhibition of instrumental memory reconsolidation. Behav Phar- macol. 2014;25:725–31. 172. Zhang F, Huang S, Bu H, Zhou Y, Chen L, Kang Z, et al. Disrupting reconsoli- dation by systemic inhibition of mTOR kinase via rapamycin reduces cocaine- seeking behavior. Front Pharmacol. 2021;12:652865. 196. Cahill EN, Vousden GH, Exton-McGuinness MT, Beh IR, Swerner CB, Macak M, et al. Knockdown of zif268 in the posterior dorsolateral striatum does not enduringly disrupt a response memory of a rewarded T-maze task. Neu- roscience. 2018;370:112–20. 173. Rich MT, Abbott TB, Chung L, Gulcicek EE, Stone KL, Colangelo CM, et al. Phosphoproteomic analysis reveals a novel mechanism of CaMKIIα regulation inversely induced by cocaine memory extinction versus reconsolidation. J Neurosci. 2016;36:7613–27. 197. Piva A, Pintori N, Padovani L, Chiamulera C. Protocols for instrumental memory reconsolidation in rodents: a methodological review. J Neurosci Methods. 2020;342:108766. 198. Piva A, Gerace E, Di Chio M, Padovani L, Paolone G, Pellegrini-Giampietro DE, et al. REFERENCES Lee JLC. Reconsolidation: maintaining memory relevance. Trends Neurosci. 2009;32:413–20. 128. Monfils MH, Cowansage KK, Klann E, LeDoux JE. Extinction-reconsolidation boundaries: key to persistent attenuation of fear memories. Science. 2009;324:951–5. 152. Milton AL, Lee JLC, Everitt BJ. Reconsolidation of appetitive memories for both natural and drug reinforcement is dependent on β-adrenergic receptors. Learn Mem. 2008;15:88–92. 129. Xue YX, Luo YX, Wu P, Shi HS, Xue LF, Chen C, et al. A memory retrieval- extinction procedure to prevent drug craving and relapse. Science. 2012;336:241–5. 153. Schramm MJW, Everitt BJ, Milton AL. Bidirectional modulation of alcohol- associated memory reconsolidation through manipulation of adrenergic sig- naling. Neuropsychopharmacology. 2016;41:1103–11. 130. Ma X, Zhang J-J, Yu L-C. Post-retrieval extinction training enhances or hinders the extinction of morphine-induced conditioned place preference in rats dependent on the retrieval-extinction interval. Psychopharmacology. 2012;221:19–26. 154. Blaiss CA, Janak PH. Post-training, but not post-reactivation, administration of amphetamine and anisomycin modulates pavlovian conditioned approach. Neurobiol Learn Mem. 2007;87:644–58. Translational Psychiatry (2023) 13:370 A.L. Milton 12 179. Chen L, Huang S, Yang C, Wu F, Zheng Q, Yan H, et al. Blockade of β-adrenergic receptors by propranolol disrupts reconsolidation of drug memory and attenuates heroin seeking. Front Pharmacol. 2021;12:686845. 155. Reichelt AC, Lee JLC. Appetitive Pavlovian goal-tracking memories reconsolidate only under specific conditions. Learn Mem. 2012;20:51–60. 156. Lee JLC, Everitt BJ. Reactivation-dependent amnesia in pavlovian approach and instrumental transfer. Learn Mem. 2008;15:597–602. 180. Chesworth R, Corbit LH. Noradrenergic β-receptor antagonism in the basolateral amygdala impairs reconsolidation, but not extinction, of alcohol self- administration. Neurobiol Learn Mem. 2018;151:59–70. 157. Milton AL, Schramm MJ, Wawrzynski JR, Gore F, Oikonomou-Mpegeti F, Wang NQ, et al. Antagonism at NMDA receptors, but not β-adrenergic receptors, disrupts the reconsolidation of pavlovian conditioned approach and instru- mental transfer for ethanol-associated conditioned stimuli. Psychopharmacol- ogy. 2012;219:751–61. 181. Zheng W, Wu C, Du WJ, Li Y, Shen F, Haghparast A, et al. Differential involve- ment of nucleus tractus solitarius projections and locus coeruleus projections to the basolateral amygdala in morphine-associated memory destabilization. Pro- gress in Neuropsychopharmacology &. Biol Psychiatry. 2022;115:110496. 158. Reichelt AC, Lee JLC. Over-expectation generated in a complex appetitive goal- tracking task is capable of inducing memory reconsolidation. Psychopharma- cology. 2013;226:649–58. ess in Neuropsychopharmacology &. Biol Psychiatry. 2022;115:110496 182. Otis JM, Werner CT, Mueller D. Noradrenergic regulation of fear and drug-associated memory reconsolidation. Neuropsychopharmacology. 2015;40:793–803. 159. Reichelt AC, Exton-McGuinness MT, Lee JLC. Ventral tegmental dopamine dys- regulation prevents appetitive memory destabilization. REFERENCES Reconsolidation of sucrose instrumental memory in rats: the role of retrieval context. Brain Res. 2019;1714:193–201. 174. Wan X, Torregrossa MM, Sanchez H, Nairn AC, Taylor JR. Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsoli- dation of a memory associated with self-administered cocaine. PLoS One. 2014;9:e107359. 199. Cheng C, Exton-McGuinness MTJ, Lee JLC. Procedures between training and reactivation influence the destabilization of instrumental sucrose memory. Front Behav Neurosci. 2022;16:953629. 175. Rich MT, Huang YH, Torregrossa MM. Calcineurin promotes neuroplastic chan- ges in the amygdala associated with weakened cocaine-cue memories. J Neu- rosci. 2020;40:1344–54. 200. Gershman SJ, Monfils MH, Norman KA, Niv Y. The computational nature of memory modification. eLife. 2017;6:e23763. 176. Yan Y, Newman AH, Xu M. Dopamine D1 and D3 receptors mediate reconsoli- dation of cocaine memories in mouse models of drug self-administration. Neuroscience. 2014;278:154–64. 201. Yue JL, Yuan K, Bao YP, Meng SQ, Shi L, Fang Q, et al. The effect of a methadone- initiated memory reconsolidation updating procedure in opioid use disorder: a translational study. eBioMedicine. 2022;85:104283. 177. Milton AL, Everitt BJ. NMDA receptors and beta-adrenergic receptors as mole- cular targets for the prevention of relapse to drug-seeking. Eur Neuropsycho- pharmacol. 2009;19:S86–S87. 202. Becker JE, Price JL, Leonard D, Suris A, Kandil E, Shaw M, et al. The efficacy of lidocaine in disrupting cocaine cue-induced memory reconsolidation. Drug Alcohol Depend. 2020;212:108062. 178. Wouda JA, Diergaarde L, Riga D, Mourik Y V, Schoffelmeer AN, De Vries TJ. Disruption of long-term alcohol-related memory reconsolidation: role of β-adrenoceptors and NMDA receptors. Frontiers in. Behav Neurosci. 2010;4:179. 203. Das RK, Gale G, Walsh K, Hennessy VE, Iskandar G, Mordecai LA, et al. Ketamine can reduce harmful drinking by pharmacologically rewriting drinking memories. Nat Commun. 2019;10:5187. Translational Psychiatry (2023) 13:370 Translational Psychiatry (2023) 13:370 A.L. Milton 13 pilot trial of memory-focused cognitive therapy. J Exp Psychopathol. 2019;10:1–14. 204. Das RK, Walsh K, Hannaford J, Lazzarino AI, Kamboj SK. Nitrous oxide may interfere with the reconsolidation of drinking memories in hazardous drinkers in a prediction-error-dependent manner. Eur Neuropsychopharmacol. 2018;28:828–40. 222. Bui, UTD & Milton, AL. Making leaps and hitting boundaries in reconsolidation: overcoming boundary conditions to increase clinical translatability of reconsolidation-based therapies. Neuroscience. 2023;519:198–206. 205. Das RK, Hindocha C, Freeman TP, Lazzarino AI, Curran HV, Kamboj SK. Assessing the translational feasibility of pharmacological drug memory reconsolidation blockade with memantine in quitting smokers. Psychopharmacology. 2015;232:3363–74. 223. REFERENCES Exton-McGuinness MTJ, Drame ML, Flavell CR, Lee JLC. On the resistance to relapse to cocaine-seeking following impairment of instrumental cocaine memory reconsolidation. Front Behav Neurosci. 2019;13:242. 206. Glasgow NG, Povysheva N V, Azofeifa AM, Johnson JW. Memantine and keta- mine differentially alter NMDA receptor desensitization. J Neurosci. 2017;37:9686–704. COMPETING INTERESTS The author declares no competing interests. 212. Germeroth LJ, Carpenter MJ, Baker NL, Froeliger B, LaRowe SD, Saladin ME. Effect of a brief memory updating intervention on smoking behavior: a ran- domized clinical trial. JAMA Psychiatry. 2017;74:214–23. ADDITIONAL INFORMATION 213. Barnabe A, Gamache K, de Camargo JVP, Allen-Flanagan E, Rioux M, Pruessner J, et al. A novel stress-based intervention reduces cigarette use in non-treatment seeking smokers. Neuropsychopharmacology. 2023;48:308–16. Correspondence and requests for materials should be addressed to Amy L. Milton. Reprints and permission information is available at http://www.nature.com/ reprints ACKNOWLEDGEMENTS 207. Pachas GN, Gilman J, Orr SP, Hoeppner B, Carlini SV, Loebl T, et al. Single dose propranolol does not affect physiologic or emotional reactivity to smoking cues. Psychopharmacology. 2015;232:1619–28. ALM’s research is supported by UKRI (MR/N02530X/1 and BB/W001195/1). ALM is the Ferreras-Willetts Fellow at Downing College, University of Cambridge. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. 208. Murray JE, Bevins RA. Acquired appetitive responding to intravenous nicotine reflects a pavlovian conditioned association. Behav Neurosci. 2009;123:97–108. 209. Naqvi NH, Bechara A. The insula and drug addiction: an interoceptive view of pleasure, urges, and decision-making. Brain Struct Funct. 2010;214:435–50. AUTHOR CONTRIBUTIONS 210. Saladin ME, Gray KM, McRae-Clark AL, LaRowe SD, Yeatts SD, Baker NL, et al. A double blind, placebo-controlled study of the effects of post-retrieval propra- nolol on reconsolidation of memory for craving and cue reactivity in cocaine dependent humans. Psychopharmacology. 2013;226:721–37. ALM was responsible for the manuscript conceptualisation, drafting, critical editing, figure visualisation and acquisition of funding. ALM was responsible for the manuscript conceptualisation, drafting, critical editing, figure visualisation and acquisition of funding. 211. Lonergan M, Saumier D, Tremblay J, Kieffer B, Brown TG, Brunet A. Reactivating addiction-related memories under propranolol to reduce craving: a pilot ran- domized controlled trial. J Behav Ther Exp Psychiatry. 2016;50:245–9. Reprints and permission information is available at http://www.nature.com/ reprints 214. Meir Drexler S, Merz CJ, Wolf OT. Preextinction stress prevents context-related renewal of fear. Behav Ther. 2018;49:1008–19. 215. Bradley, MM & Lang, PJ The International Affective Picture System (IAPS) in the study of emotion and attention. In Handbook of emotion elicitation and assessment (eds JA Coan & JJB Allen), 29-46 (Oxford University Press, 2007). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 216. Das RK, Lawn W, Kamboj SK. Rewriting the valuation and salience of alcohol- related stimuli via memory reconsolidation. Transl Psychiatry. 2015;5:e645. 217. Gale G, Walsh K, Hennessy VE, Stemerding LE, Ni KS, Thomas E, et al. Long-term behavioural rewriting of maladaptive drinking memories via reconsolidation- update mechanisms. Psychol Med. 2021;51:2875–85. 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/. 218. Hon T, Das RK, Kamboj SK. The effects of cognitive reappraisal following retrieval-procedures designed to destabilize alcohol memories in high-risk drinkers. Psychopharmacology. 2016;233:851–61. 219. Lane RD, Ryan L, Nadel L, Greenberg L. Memory reconsolidation, emotional arousal, and the process of change in psychotherapy: new insights from brain science. Behav Brain Sci. 2015;38:e1. 220. Marsden J, Goetz C, Meynen T, Mitcheson L, Stillwell G, Eastwood B, et al. Memory-Focused Cognitive Therapy for Cocaine Use Disorder: theory, proce- dures and preliminary evidence from an external pilot randomised controlled trial. EBioMedicine. 2018;29:177–89. 221. Goetz C, Meynen T, Mitcheson L, Grey N, Eastwood B, Strang J, et al. Does craving for cocaine mediate cocaine use? Analysis of a randomized controlled © The Author(s) 2023 © The Author(s) 2023 Translational Psychiatry (2023) 13:370
https://openalex.org/W2492705943
https://www.biodiversitylibrary.org/itempdf/59942
English
null
Animal competitors; profit and loss from the wild four-footed tenants of the farm
null
1,911
public-domain
68,384
THE -YOUNG- THE -YOUNG- THE YOUNG FARMER'S PRACTICAL LIBRARY EDITED BY ERNEST INGERSOLL THE YOUNG FARMER'S PRACTICAL LIBRARY EDITED BY ERNEST INGERSOLL The Yotmg Farmer's Practical Library EDITED BY ERNEST INGERSOLL EDITED BY ERNEST INGERSOLL EDITED BY ERNEST INGERSOLL Cloth i6mo Illustrated each 75 cents net. From Kitchen to Garret. By VIRGINIA TERHUNE VAN DE WATER. Neighborhood Entertainments. By RENEE B. STERN, of the Congressional Library. Home Waterworks. By CARLETON J. LYNDE, Professor of Physics in Mac- donald College, Quebec. Animal Competitors. By ERNEST INGERSOLL. The rarm Mechanic. By L. W. CHASE, Professor of Farm Mechanics in the University of Nebraska. The Satisfactions of Country Life. By DR. JAMES W. ROBERTSON, Principal of Macdonald College, Quebec. Roads, Paths and Bridges. By L. W. PAGE, Chief of the Office of Public Roads, U. S. Department of Agriculture. Health on the Farm. By DR. L. F. HARRIS, Secretary Georgia State Board of Health. Electricity on the Farm. By FREDERICK M. CONLEE. Co-operation Among Farmers. By JOHN LEK COULTER. L UNIVERSITY of CALIFORNIA AT LOS ANGELES LIBRARY ANIMAL COMPETITORS PROFIT AND LOSS FROM THE WILD FOUR-FOOTED TENANTS OF THE FARM BY 3L2 >> *t -2. ERNEST INGERSOLL EDITOR OF THE YOUNG FARMER'S PRACTICAL LIBRARY, AND AUTHOR OF " THE LIFE OF MAMMALS," " WIT OF THE WILD," "WILD LIFE OF ORCHARD AND FIELD," ETC. ILLUSTRATED Hew Korft STURGIS & WALTON COMPANY 1911 All rights reserved Copyright 1911 By STURGIS & WALTON COMPANY Set up and electrotyped. Published April, 1911 SB BY THE GENERAL EDITOR This is the day of the small book. There is much to be done. Time is short. Information is earnestly desired, but it is wanted in compact form, confined directly to the subject in view, authenticated by real knowledge, and, withal, gracefully delivered. It is to fulfill these con- ditions that the present series has been pro- jected to lend real assistance to those who are looking about for new tools and fresh ideas. g It is addressed especially to the man and woman at a distance from the libraries, exhibi- tions, and daily notes of progress, which are the main advantage, to a studious mind, of liv- ing in or near a large city. The editor has had in view, especially, the farmer and villager who is striving to make the life of himself and his family broader and brighter, as well as to increase his bank account; and it is therefore in the humane, rather than in a commercial di- rection, that the Library has been planned. v v INTRODUCTION yi The average American little needs advice on the conduct of his farm or business ; or, if he thinks he does, a large supply of such help in farming and trading as books and periodicals can give, is available to him. But many a man who is well to do and knows how to continue to make money, is ignorant how to spend it in a way to bring to himself, and confer upon his wife and children, those conveniences, comforts and niceties which alone make money worth acquiring and life worth living. He hardly realizes that they are within his reach. y For suggestion and guidance in this direction there is a real call, to which this series is an answer. It proposes to tell its readers how they can make work easier, health more secure, and the home more enjoyable and tenacious of the whole family. No evil in American rural life is so great as the tendency of the young people to leave the farm and the village. The only way to overcome this evil is to make rural life less hard and sordid ; more comfortable and attractive. It is to the solving of that problem that these books are addressed. BY THE GENERAL EDITOR Their central idea is to show how country life may be made INTRODUCTION vii richer in interest, broader in its activities and its outlook, and sweeter to the taste. , To this end men and women who have given each a lifetime of study and thought to his or her speciality, will contribute to the Library, and it is safe to promise that each volume will join with its eminently practical information a still more valuable stimulation of thought. EBNEST INGEBSOLL. PREFATORY NOTE The writer could hardly claim much original- ity for this book, were he so disposed. His aim has not been a literary one, but rather to compose a useful handbook of the mammals the wild four-footed tenants of our American farm-lands, from the point of view of the agriculturist, orchardist and ranchman. The United States Department of Agriculture, through its various departments and publica- tions, has from time to time issued information vast in its sum in respect to economic zoology ; and most of the Agricultural Colleges and Experiment Stations in the several States have repeated and supplemented this exten- sively. The bulk of this proffered matter, how- ever, relates to the ravages of injurious insects, or to the beauty and usefulness of birds sub- jects which may receive attention in future volumes in this Library. y The economic importance of the mammals ix ix PEEFATOEY NOTE x the rats, field-mice, rabbits, gophers, ground- squirrels, muskrats, etc.; the fox, the wolves and the fur-bearers; the deer and their kin have been appreciated by very few; yet the harm done annually by one unchecked class of them entails a vast waste, while the benefit which might be obtained from another class is lost because their lives are little cared for and their capabilities for profitable exploitation al- most wholly neglected. It is hoped that this book will lead to a re- versal of this wasteful and negligent state of affairs; and that by its help the farmer's friends among the wild animals about him may be encouraged and his foes subdued. Thus the account of the agriculturist with his four- footed competitors may be changed from a need- lessly heavy balance on the loss side, to one of profit, reckoned partly in savings and partly in "new business." My sources of statistical information, espe- cially for the West, have been largely reports of investigations conducted by the Biological Survey. These reports, it is true, have been widely distributed during the past ten years, PREFATORY NOTE xi but they have gone out as chapters in forbid- ding public documents, or else separately in loose pamphlets which in most cases have been speedily lost. It is impracticable for the or- dinary man to get copies of them now if he tries, and their usefulness has therefore come to an untimely end. Among them are original and valuable essays by Dr. C. CONTENTS THE PEST OF RATS PAGE Varieties of rats Cost of their board Destructiveness Carriers of disease Breeders of bubonic plague Pre- cautions and suppression Need of cooperation . . 3 PREFATORY NOTE Hart Merriam, Chief of the Biological Survey until his resig- nation in 1910, when H. W. Henshaw succeeded to his office; Vernon Bailey, the assistant in charge of field investigations ; David E. Lantz, Wilfred H. Osgood, E. W. Palmer, Stanley E, Piper, E. W. Nelson, Edward A. Goldman, and others attached to the Department. p Knowing the accuracy and importance of this half-lost material, and also aware that nothing better could be furnished in its stead, I have not hesitated to make liberal use of it, often in its own well-chosen language. It was writ- ten for the benefit of the public ; and I am con- fident the gentlemen above mentioned will gladly see it renew its usefulness in the per- manent form a bound book affords, and rejoice in the greater force their facts and recommen- PREFATORY NOTE xii dations will obtain by being associated in an orderly array. To them belongs credit for the larger part of the facts presented in the pages that follow. I have simply arranged and enforced the material anew in the most suitable form I could devise. Attention may be called, further, to one novel feature in the book, namely, the detailed in- structions as to the cultivation of certain wild animals in captivity as an industry. Among those recommended for this purpose are the deer, for sale alive to parks, and to furnish venison to market; the muskrat for food and skins; the silver fox for its costly pelt, and such other fur-bearers as the mink and skunk. All over the country young men are so situated as to be able to add one or more of these enter- prises to their year's work, and to derive from them an attractive addition to the annual in- come, while contributing in no small degree to the general wealth and welfare of the country. New York, Jan. 1, 1911. New York, Jan. 1, 1911. CHAPTER III THE MEADOW-MOUSE AND ITS MISCHIEF American voles Prairie-mice and Pine-mice Multiplica- tion into plagues Prevention of plagues Damage to crops considered Protection of young trees ... 48 ANIMAL COMPETITOES ANIMAL COMPETITOES CHAPTER I aa 3 <f X THE PEST OF EATS WE have in the United States three foreign rats, all injurious to health and property. 1. The brown house-rat (Mus norvegicus), called also gray rat, house-rat, barn-rat, wharf-rat and Norway rat, and, in England, Hanoverian rat. Its average total length is about 16.4 inches, of which 7 inches belongs to the tail, and it usually weighs less than a pound, though specimens have been known so much larger as to weigh 24 to 28 ounces. The gen- eral color is grayish-brown above and whitish below, the long overhairs of the back having black tips. The head is shorter, the muzzle more blunt, the ears smaller and the tail rela- tively shorter than in the other species. It is 3 3 ANIMAL COMPETITORS 4 spread all over the continent, except the Utah basin. 2. The black rat (Mus rattus), smaller than the brown rat, and sooty or slaty black, paler on the under parts. Like the brown rat, it is of Oriental origin and seems to have pre- ceded the former ur its immigration into western Europe and thence to this continent. It was carried from Europe to Spanish Amer- ica about three and a half centuries ago, and thence spread northward to the English col- onies. Upon the arrival of the brown rat in North America toward the end of the 18th century it began to decrease, and is now rare, surviving only in scattered colonies, but re- main numerous in many parts of the West Indies, Middle and South America, Hawaii, etc. 3. The roof-rat (Mus alexandrinus) , simi- lar to the brown rat in form and habits, but grayer above, and yellowish white on the feet and abdomen. Its history is much like that of the black rat, but it has held its own better against the dominance of the brown rat, in- habits sea-going ships, and has established THE PEST OF BATS 5 itself in all the warmer parts of the world. It is still prevalent in our South Atlantic States, in the West Indies and in South America. The tame white rats sold as pets are mostly of this and the black species. In habits these three rats are similar, with the important exception that the black rat and the roof-rat (which some zoologists consider merely varieties of one species), do not bur- row under foundations, etc., as does the brown rat. CHAPTER I aa 3 <f X THE PEST OF EATS On the other hand they are far more agile and addicted to climbing, a decided ad- vantage in the tropics where a large part of their food is obtained from trees, in whose branches they frequently lodge their nests ; and are less able to withstand cold than the brown rat, which survives arctic winters in whaling ships, apparently without distress. They are also less prolific, having only ten mammae to the brown rat's twelve, and bearing on the average only about five young at a birth to the other's eight. This difference in prolificacy alone would account for the great dominance of the brown rat, at least in North America; and it is to that species the rat, par excellence ANIMAL COMPETITORS 6 that we devote our attention in considering the relation it bears to human welfare, espe- cially on the farm. "The rat," says Lantz, succinctly, "is the worst mammalian pest known to man. Its depredations throughout the world result in losses amounting to hundreds of millions of dollars annually. But these losses, great as they are, are of less importance than the fact that rats carry from house to house and from seaport to seaport the germs of the dreaded plague. ' ' This enormous evil can be cured only by be- ing prevented ; and it is not only to the personal interest of every man, but a part of his public duty, to do all in his power to stamp out a pest which is not only costing the country many millions of dollars in damages annually, but is constantly threatening each of us with horrid diseases. History. In order to destroy the rats we have, and to guard against their increase on our own premises, at least, we must become acquainted with the haunts and habits of the animal. THE PEST OF BATS 7 The early history of the brown rat is prac- tically unknown. Various modern writers have asserted that it came originally from Persia or India ; but W. T. Blanford, a leading zoolo- gist of British India, states that it is at pres- ent unknown in Persia, and that, as concerns India, the black rat is the generally distributed species, while the brown rat is found only along the coast and the navigable rivers. CHAPTER I aa 3 <f X THE PEST OF EATS This seems to imply that the latter is a compara- tively recent immigrant into India; and other evidence seems to show that its original home was northward of the Himalayan ranges. Its resistance to cold supports this hypothesis. It seems to have entered Europe first by cross- ing the Volga into Eussia in hunger-driven hordes in 1727, but it reached England from some eastern port a year or two later, coinci- dent with the accession of George I to the British throne. The general, but erroneous, be- lief in Great Britain that it was introduced from Norwegian timber-ships gives it the name "Norway" rat there, as I explained in my Life of Mammals. "It reached our eastern ports in The early history of the brown rat is prac- tically unknown. Various modern writers have asserted that it came originally from Persia or India ; but W. T. Blanford, a leading zoolo- gist of British India, states that it is at pres- ent unknown in Persia, and that, as concerns India, the black rat is the generally distributed species, while the brown rat is found only along the coast and the navigable rivers. This seems to imply that the latter is a compara- tively recent immigrant into India; and other evidence seems to show that its original home was northward of the Himalayan ranges. Its resistance to cold supports this hypothesis. It seems to have entered Europe first by cross- ing the Volga into Eussia in hunger-driven hordes in 1727, but it reached England from some eastern port a year or two later, coinci- dent with the accession of George I to the British throne. The general, but erroneous, be- lief in Great Britain that it was introduced from Norwegian timber-ships gives it the name "Norway" rat there, as I explained in my Life of Mammals. "It reached our eastern ports in 1775 and was popularly credited to the hated ANIMAL COMPETITORS 8 Hessian soldiers, a queer echo of the London idea that it came there with the Hanoverian train of the present reigning house. CHAPTER I aa 3 <f X THE PEST OF EATS By 1830 it had reached the Mississippi, and by 1857, at least, was numerous in California." Now no part of the country save the western deserts is free from these pests ; and competent judges estimate their numbers as at least five times that of the human population, with which they more than keep pace as widening civilization more and more favors their support and in- crease. Fecundity of the rat. A consequence is that from time to time there is an overflow of rats from one locality or region to another which gives us a glimpse of the unseen crowd in the midst of which we live. "In 1903, a multi- tude of migrating rats spread over several counties of western Illinois. They were no- ticed especially in Mercer and Eock Island counties. For several years prior to this in- vasion no abnormal numbers were seen, and their coming was remarkably sudden. An eye- witness to the phenomenon informed the writer THE PEST OF RATS 9 that as he was returning to his home by moon- light he heard a general rustling in the field near by, and soon a vast army of rats crossed the road in front of him, all going in one direc- tion. The mass stretched away as far as could be seen in the dim light. These animals re- mained on the farms and in the villages of the surrounding country, and during the winter and summer of 1904 were a veritable plague. A local newspaper stated that between March 20 and April 20, 1904, Mr. F. U. Montgomery of Preemption, Mercer county, killed 3,435 rats on his farm." This enormous multiplication is due to the animal's adaptability to climate, its omnivo- rousness, its habit of burrowing, its strength and cunning in withstanding and outwitting enemies, and, most of all, to its astonishing fecundity, especially where food is abundant. This rat breeds in the temperate parts of this country from three to five times a year, the female bringing forth each time from 6 to 20 young. Mr. Lantz concludes from such data as are available that in the vicinity of Wash- ANIMAL COMPETITORS 10 ington the average litter is 10. A pair and their progeny breeding three times a year would, thus, if all remained alive, produce a population of more than 20,000,000. "Of course, such results never occur in nature. CHAPTER I aa 3 <f X THE PEST OF EATS Apparently not nearly half the rats born are females ; at least, among mature rats the males greatly predominate. Then, too, the life of young rats, as well as that of the old, is a con- tinuous struggle for existence. Disease, the elements, natural enemies, the devices and cun- ning of man, and even cannibalism are contin- ually at work to reduce their numbers. ' ' The young are born, after a gestation period of 21 days, in a burrow dug in the ground under buildings, piles of lumber or wood, beneath strawstacks, etc., or simply bored into a stream- bank. They are naked and blind at birth, but develop with great rapidity. p g p y What it costs to board our rats. The dam- age done by rats over so great an area as the United States or Canada, is incalculable. David E. Lantz, in the document from which I am quoting freely, summarizes their destruc- tion thus : THE PEST OF RATS 11 "The brown rat is practically omnivorous. The statement applies as well to the black rat and the roof rat. Their bill of fare includes seeds and grains of all kinds, flour, meal, and food products made from them; fruits and garden vegetables; mush- rooms; bark of growing trees; bulbs, roots, stems, leaves, and flowers of herbaceous plants; eggs, chicks, ducklings, young pigeons, and young rabbits; milk, butter, and cheese; fresh meat and carrion; mice, rats, fish, frogs, and mussels. This great variety of food explains the ease with which rats adapt them- selves to almost every environment. every "Experiments show that the average quantity of grain consumed by a full-grown rat is fully 2 ounces daily. A half-grown rat eats about as much as an adult. Fed on grain, a rat eats 45 to 50 pounds a year, worth about 60 cents if wheat, or $1.80 if oat- meal. Fed on beefsteaks worth 25 cents a pound, or on young chicks or squabs with a much higher prospective value, the cost of maintaining a rat is proportionately increased. Granted that more than half the food of our rats is waste, the average cost of keeping one rat is still upward of 25 cents a year. CHAPTER I aa 3 <f X THE PEST OF EATS They devoured everything that came in the way fruits, plants, and even trees. Where corn was sown they would come by troops in the night and scratch it out of the ground ; 'nay,' writes a contemporary chronicler, 'they so de- voured the fruits of the earth that the people were des- titute of bread for a year or two.' Every expedient was tried to destroy them. Dogs were trained to hunt them, who would kill a score or more in an hour. Cats, both wild and tame, were employed in large numbers for the same purpose ; poisons and traps every man having to set twelve traps were brought into requisition; and even woods were set on fire, to help to exterminate them. Every letter written at this period by the plague-stricken colonists contains some account of the dreadful scourge. 'Our great enemies the rats threaten the subversion of the plan- tation,' writes one colonist in July, 1616. 'Rats are a great judgment of God upon us,' wrote another a year later. 'At last it pleased God, but by what Shortly after the settlement of the Bermudas by the British, the colony was infested with rats, which, in the space of two years, had increased so alarm- ingly that none of the islands were free from them, and even fish were taken with rats in their bellies. A writer in the Academy recalls some of the horrors of this plague of rats. The rats, we are told, had nests in almost every tree, and burrowed in most places in the ground like rabbits. They devoured everything that came in the way fruits, plants, and even trees. Where corn was sown they would come by troops in the night and scratch it out of the ground ; 'nay,' writes a contemporary chronicler, 'they so de- voured the fruits of the earth that the people were des- titute of bread for a year or two.' Every expedient was tried to destroy them. Dogs were trained to hunt them, who would kill a score or more in an hour. Cats, both wild and tame, were employed in large numbers for the same purpose ; poisons and traps every man having to set twelve traps were brought into requisition; and even woods were set on fire, to help to exterminate them. CHAPTER I aa 3 <f X THE PEST OF EATS y "If an accurate census of the rats of the United States were possible, a reasonably correct calculation of the minimum cost of feeding them could be made from the above data. If the number of rats sup- ported by the people throughout the United States were equal to the number of domestic animals on the farms horses, cattle, sheep, and hogs the minimum ANIMAL COMPETITORS 12 cost of feeding them on grain would be upward of $100,000,000 a year. To some such enormous total every farmer, and indeed every householder who has rats upon his premises, contributes a share. p p , "But the actual depredations of rats are by no means confined to what they eat. They destroy fully as much grain as they consume, and they pollute and render unfit for human consumption a much larger proportion of all other food materials that they at- tack. In addition, the damage they do to property of other kinds is often as great as that done to food supplies. ' ' Destructiveness of rats in the fields. The rat in America is usually thought of as vermin in the house and barn, so that little notice is taken of its destructiveness in the fields which Europeans understand very well. Cultivated grains may be regarded as the favorite food. The animals dig the seed from the ground as soon as sown, eat the tender sprouts when they appear, and later feast upon the maturing crop. After harvest they attack grain in shock, stack, and mow, and when thrashing is over, in crib, granary, elevator, mill, and warehouse. In- dian corn seems especially to suffer from their depredations. They climb the stalks and strip the cobs of the milky kernels; and if cut corn THE PEST OF EATS 13 is left in shocks, especially near drains or other rat-harbors, it is likely to be ruined. Shortly after the settlement of the Bermudas by the British, the colony was infested with rats, which, in the space of two years, had increased so alarm- ingly that none of the islands were free from them, and even fish were taken with rats in their bellies. A writer in the Academy recalls some of the horrors of this plague of rats. The rats, we are told, had nests in almost every tree, and burrowed in most places in the ground like rabbits. CHAPTER I aa 3 <f X THE PEST OF EATS Every letter written at this period by the plague-stricken colonists contains some account of the dreadful scourge. 'Our great enemies the rats threaten the subversion of the plan- tation,' writes one colonist in July, 1616. 'Rats are a great judgment of God upon us,' wrote another a year later. 'At last it pleased God, but by what ANIMAL COMPETITORS 14 means is not well known, to take them away, inso- much that the wild cats and many dogs that lived on them were famished.' There was universal joy at the sudden removal of such destructive vermin; and the all but despairing planters were enabled once more to resume their neglected occupations with spirit and energy. gy Much more recently, rats became such a plague in the sugar-plantations of the West Indies, and especially in Jamaica, that the East Indian mungoos a fierce, weasel-like civet was introduced. This ani- mal cleared out the rats, but speedily became in other directions such a nuisance that its destruction had to be effected in order to save the poultry and birds of the Island. Eats often damage corn in cribs. Too fre- quently these receptacles for grain are built close to the ground, and rats live under the floor, and soon get access to the grain. They shell the corn, eating the softer part of the kernel and wasting much more than they con- sume. They carry the grain to subterranean burrows and bring up into the crib moist soil, which induces mold. Similarly they eat the small grains in the field and take toll of the granary and feed-box, often 5 to 10 per cent. THE PEST OF EATS 15 of feedstuffs, malt and the like; while no pest of the sugar-cane is much more to be feared. The damage done by rats to fruits and vege- tables while stored in cellars and pits is well known. They attack ripe tomatoes, melons, cantaloupes, squashes, pumpkins, sweet corn, and many other vegetables in the field, and the depredations are often attributed to rabbits. Rats are fond of nearly all small fruits, even climbing grape-vines, blackberry-canes, and currant-bushes to obtain the ripe fruit; and often feed upon ripe apples, pears, cherries, and so forth. Bats are recognized pests of the greenhouse and the plant-propagating pit, where they at- tack seeds, bulbs, leaves, stems and flowers. CHAPTER I aa 3 <f X THE PEST OF EATS Of flowering bulbs the tulip suffers most and hyacinths also are eaten, while narcissus bulbs are apparently immune to attack. Carnations seem especially liable to destruction. Destructiveness to poultry and game. Very serious is the loss due to rats entering badly constructed hen and pigeon houses, probably greater, in Mr. Lantz's opinion, than that in- ANIMAL COMPETITORS 16 flicted by foxes, minks, weasels, skunks, hawks and owls combined; but mostly one or all of these are made to take the blame. "Not long since, in a published account of depre- dations on poultry, the damage was attributed to a skunk. The statement was made that both eggs and young chicks were taken from under a sitting hen without disturbing her. This is a trick peculiar to the rat, and it is evident that a mistake was made as to the identity of the thief. "Where rats are numerous in springtime, they often prey upon young chicks, capturing them in the nest and in and around the coops. I have known them to take nearly all the chicks on a large poultry ranch, and, in the same neighborhood and over a large territory, to destroy nearly 50 per cent, of the season's hatching. Young ducks, turkeys, and pi- geons are equally liable to attack, and where rats are numerous are safe only in rat-proof coops. "A writer in a western agricultural paper states that in 1904 rats robbed him of an entire summer's hatching of three or four hundred chicks. A cor- respondent of another journal says, 'Rats destroyed enough grain and poultry on this place in one season to pay our taxes for three years.' When it is re- membered that the poultry and eggs produced each year from the farms of the United States have a value of over $600,000,000, it will be seen that even a small percentage of loss aggregates a large sum." THE PEST OF RATS 17 In Europe the rat is the bane of gamekeepers who try to preserve broods of pheasants 'and other game. Our wild game-birds are less molested and perhaps better able to protect themselves; yet our grouse and quail must suffer, for rats eat the eggs of ground-nesting song-birds, but the real offender is seldom even suspected. Bats often gnaw the hoofs of horses until the feet bleed. Brushing the hoofs with dilute carbolic acid is a preventive. CHAPTER I aa 3 <f X THE PEST OF EATS They have been known to kill young lambs and pigs, and to at- tack very fat hogs and eat holes in their bodies, causing death. Farrowing sows have been killed by rats gnawing their teats until blood poisoning resulted. Rats damage buildings and stored goods. Interest in the damage done to stored goods and merchandise belongs more to the city ware- houseman than to the countryman, but the latter is well aware that old harness and gear of all sorts with leather about it, any grain- bags and similar articles must be protected from rats. Damage to houses and barns is, ANIMAL COMPETITORS 18 however, a matter of grave interest to inhabit- ant^ of the village as well as of the city. Quot- ing Lantz again, "The damage to houses and furniture by rats con- stitutes a large item. They burrow under founda- tions or through the plaster in a stone wall and admit streams of water that eventually weaken or undermine the structure itself. They seem, to be able to penetrate almost everything except stone, brick, cement, glass, and iron. They gnaw into a grain bin, or through a wainscoting, a floor, or a door in a single night In the same way they enter chests, wardrobes, bookcases, closets, barrels, and boxes for the stores within. Almost every old dwelling in the country bears abundant evidence of its former or present occupancy by rats. Eats gnaw through lead pipes or wooden tanks to obtain water, and sometimes before the leak is discovered, ceilings, wall decora- tions, and floor coverings are flooded and practically ruined. All this is waste of a tangible kind and a constant drain on the prosperity of the people." Then there is the ever-menacing devastation from fires due to rats carrying matches into their nests and there igniting them by chewing them, or simply by overheating ; or due to their gnawing the insulation from electric wires a surprisingly frequent origin of fires of late years. THE PEST OF RATS 19 Rats as carriers of disease. Finally, rats are always a menace to health, and may become the agents of the dissemination of the most dreadful and virulent of diseases the Asiatic plague, which has more than once decimated the civilized world. CHAPTER I aa 3 <f X THE PEST OF EATS It has been calculated that 25,000,000 of persons perished in an epidemic of this character which swept over the world in the 14th century; and it did not require the literary genius of a De Foe to perpetuate the memory of the awful visitation which almost depopulated London and set all Europe in mourning toward the end of the 16th century. Even then, in the cloud of mystery, supersti- tion and horror of fear which made most men blind and helpless, the truth was dimly recog- nized by a few, namely that it was not the wrath of God nor the malignancy of some evil spirit nor a miasm from earth or sea that struck men down, but the communication of disease from the sick to the well. This, it was observed, could be effected not only by contact with human victims, but that the contagion was caught and passed on by all the small animals about a house. Hence orders were issued that Rats as carriers of disease. Finally, rats are always a menace to health, and may become the agents of the dissemination of the most dreadful and virulent of diseases the Asiatic plague, which has more than once decimated the civilized world. It has been calculated that 25,000,000 of persons perished in an epidemic of this character which swept over the world in the 14th century; and it did not require the literary genius of a De Foe to perpetuate the memory of the awful visitation which almost depopulated London and set all Europe in mourning toward the end of the 16th century. Even then, in the cloud of mystery, supersti- tion and horror of fear which made most men blind and helpless, the truth was dimly recog- nized by a few, namely that it was not the wrath of God nor the malignancy of some evil spirit nor a miasm from earth or sea that struck men down, but the communication of disease from the sick to the well. This, it was observed, could be effected not only by contact with human victims, but that the contagion was caught and passed on by all the small animals about a house. Hence orders were issued that ANIMAL COMPETITORS 20 not only rats, mice, and small vermin of all sorts should be killed, but also dogs and cats. CHAPTER I aa 3 <f X THE PEST OF EATS An ordinance by the authorities at Winchester, England, in 1583, is typical of many others issued in British towns, viz. : "That if any house within this cytie shall happen to be infected with the Plague, that thene every per- sone to keepe within his or her house every his or her dogg, and not to suffer them to goo at large. And if any dogge be then founde at large, it shall be lawful for the Beadle or any other person to kill Ihe same dogg, and that any owner of such dogg going at large shall lose six shillings." Among the records of King's Lynn, under May, 1585, appears this: "For as muche as it hath pleased Allmightie God to begynn to send us his visitacion with sickness amongst us, and that dogges and cattes are thought verie unfitt to be suffered in this tyme. Therefore Mr. Maior, aldermen, and common councell have or- dered and decreed that every inhabitant within the same Towne shall forthwith take all their dogges and yappes and hange them or kill them and carrye them to some out-place and burye them for breadings of a great annoyance. And likewise for cattes, if there be any nigh unto any house or houses visited with sick- ness. ... It is ordered that the cattes shall furth- with be killed in all such places. ' ' An exception was THE PEST OF RATS 21 made in favor of any "dogge of accompte." Such a one was allowed to be kept if "kenelled or tied up or led in a lease." As often happens, a fact was clearly per- ceived and acted upon beneficially long before the philosophy of it was comprehended. p p y p Rats responsible for the plague. It was not until the very end of the last century scarcely a dozen years ago, that the suspected truth of the real nature of the plague was dis- covered through scientific studies of the disease which then appeared in a most threatening form in India. It was determined that of the several phases of plague the most common is that which produces swellings or "buboes" on the body of the victim, and hence is called bubonic plague. CHAPTER I aa 3 <f X THE PEST OF EATS This is rarely communicated direct from man to man, but through the me- dium of insects which suck the patient's blood and then, filled with the diseased blood in which are floating the deadly bacilli (Bacillus pestis) which produce the disturbance, pierce the skin of some other creature and leave more or less of these plague-germs in the puncture. p g g p Any blood-sucking bug, as, for example, the ANIMAL COMPETITORS 00 bed-bug, may do this; but the most common agent is the flea. Another fact is that the rat seems especially susceptible to the disease ; and, indeed, it is be- MOUTH-PARTS OF A BAT-FLEA, SHOWING WHERE BACILLI MAY CLING AND BE CARRIED INTO THE NEXT WOUND. From Doane's "Insects and Disease." By Permission of Henry Holt & Co. MOUTH-PARTS OF A BAT-FLEA, SHOWING WHERE BACILLI MAY CLING AND BE CARRIED INTO THE NEXT WOUND. From Doane's "Insects and Disease." By Permission of Henry Holt & Co. lieved that it was originally a disease of this rodent. Rats abound in fleas, and, as is the case with most furry or feathered animals, have a species peculiar to their race. This lieved that it was originally a disease of this rodent. Rats abound in fleas, and, as is the case with most furry or feathered animals, have a species peculiar to their race. This THE PEST OF EATS 23 rat-flea will bite and communicate the disease from rat to rat, and an outbreak of plague among men is usually preceded by an epidemic among the rats. The rat-flea does not bite man; but those which live on human beings will thrive on rats and may return from an in- fected rat to a human host if opportunity offers. The fleas of dogs and cats will temporarily live on the skin of both rodents and human beings, and may thus take a part in the transmission of plague. The fleas usually leave a rat or other animal as soon as it dies, and, with their stomachs full of plague-bacilli, with others clinging to their proboscis and sucking lips, they seek new hosts. CHAPTER I aa 3 <f X THE PEST OF EATS The new host, whether rat, or some other animal, or perchance a human being, is soon bitten with these infected mouths, and thus receives the germs of the malady y Those who wish to pursue the study of this matter in further detail will find a very full exposition of it, and of the general relations of insects to common diseases, in R. W. Doane's Insects and Disease (New York, Holt, 1910). 24 ANIMAL COMPETITORS It is now understood that the first thing to do when a case of plague is brought to some port in a ship sailing from the Orient is to exterminate the rats of the locality; and the best preventive against this and other afflictions getting a foothold anywhere, is to keep the rats down. The Japanese were quick to take ad- vantage of the new knowledge, and by the fierce crusade they waged against the wharf-rats in their ports prevented a spread of bubonic plague, always threatening them, in the armies they sent into Manchuria. In this way, too, by a vigorous crusade against the animal in Cali- fornia, in which many hundreds of thousands were trapped or poisoned, the plague was re- cently eradicated in San Francisco before it had reached alarming proportions. Rats and trichina. But rats disseminate diseases other than bubonic plague. Trich- inosis among swine is probably perpetuated entirely by rats, since trichinae in the hog can result only from its eating the flesh of animals infested with the parasite. The only two an- imals of the farm known to be subject to this THE PEST OF RATS 25 parasite are the rat and the hog itself. Pork becomes trichinous, then, only when swine eat the flesh of infected rats or hogs. Country slaughter-houses, where rats are abundant and swine are fed on offal, are the chief sources of trichinous pork. That the danger from this source has not been confined to the rural slaughtering-places alone, is shown by the in- vestigation conducted by the Biological Survey in 1909 into the "rat-nuisance," said to exist about the great packing-houses in Chicago and St. Louis. The older establishments were found to be infested with rats, causing a se- rious aggregate loss, and endangering both the health of the workmen and the wholesomeness of the product; but this state of things has been greatly improved, and new buildings are designed to be rat-proof. CHAPTER I aa 3 <f X THE PEST OF EATS g p Rats creep through drains and step about in all sorts of filth; and to their feet and fur clings slime which may be loaded with germs of typhoid, diptheria and any other of the malignant list of diseases due to bacilli that develop in darkness and filth. Consequently ANIMAL COMPETITORS 26 no household is safe into which rats may wan- der and leave the seeds of disease brought from the gutter. In view of these facts it would seem impor- tant that every man should attempt to free his property of these undesirable tenants, which, so far as we can see, make no return whatever for the damage and depredation of which they are guilty. Methods of suppression. It is perhaps too late to get rid of the rat altogether, but it is not too late to subdue him and prevent a great part of the evils that follow his presence. How shall it be done? First, try to destroy or drive away those rats you have. Seek out their holes, runways and lodging-places, clean them out and stop them up so far as you are able. The cunning of the rascals is great and they will shift their quarters or invent new means of access and ways of attack with discouraging ingenuity and persistence, which you. must endeavor to match. Untiring watchfulness and work will win. Trapping, if intelligently pursued, will cap- THE PEST OF EATS 27 ture a great many. The old-fashioned figure-4 trap, dropping a box, or better, a deadfall, is often highly effective. Several sorts of steel traps may be used to advantage; and in the last chapter of this book will be found descrip- tions of various forms and directions for bait- ing and setting them. Poisoning will clear out the creatures more rapidly and effectively but can hardly be used except about barns and out-buildings, and even there should be done intelligently and with cer- tain precautions. Therefore instructions as to the best means and methods of poisoning will also be found in the last chapter. p While endeavoring to kill off the rats by these various methods, precautions should be taken against their return. Their runways, and harboring places must be sought out and made untenable. The wisdom of stopping up all holes by which they enter houses, barns or cellars, need hardly be mentioned to common- sense readers. CHAPTER I aa 3 <f X THE PEST OF EATS Freshly slaked lime placed in their dry burrows and runs is effective; or fresh thin whitewash to be poured into them. A strong solution of copperas is good, and gas- ANIMAL COMPETITORS 28 tar daubed about their holes, as also is caustic potash. Where burrows are discovered in banks or fields the inmates may be suffocated by pushing into the holes wads of rags satu- rated with bisulphide of carbon, as is practiced against gophers ; but this is of little use in buildings, for it escapes too easily. Rat-proof construction. All new or recon- structed buildings should be made rat-proof. This is best done by the use of cement. Even then, when foundations and walls are made of tight concrete, care must be taken lest drains and other openings admit them. Outer doors, especially those that give upon alleys, should not be left open. Basement and cellar win- dows of barns, stables, chicken-houses, etc., should be screened with wire, so that they may be left ajar for ventilation without danger. Inner doors to vestibules are of great assist^ ance. Even old cellars may be made rat-proof by the use of cement at small expense. y p When wooden walls are built upon proper foundations, the building may be made proof against these and other noxious visitors by THE PEST OF EATS 29 filling the space between the sheathing and the lath for about a foot with concrete. Bats frequently enter houses from sewers by way of soil-pipes leading into water-closets, but this can be guarded against by care in construc- tion and the use of water-traps. "Almost everywhere, in country, village, and city, the wooden floors of sidewalks, areas, and porches are commonly laid upon timbers resting upon the ground. Under these floors rats are safe from most of their enemies. Only municipal action can com- pletely remedy these conditions, but all such rat-har- bors should be destroyed and replaced by cement floors. Considering durability, healthfulness, and other advantages, this material is the cheapest that can be used. The floors of wooden porches should always be well above the ground. Rats often under- mine brick walks or areas. "Granaries, corncribs, and poultry-houses may be made rat-proof by a liberal use of concrete in the foundations and floors; or the floors may be of wood resting upon concrete. CHAPTER I aa 3 <f X THE PEST OF EATS Objection has been urged against the use of concrete floors for horses, cattle, and poultry, because the material is too good a con- ductor of heat, and the health of the animals suffers from contact with floors of this kind. In poultry- houses, dry soil or sand may be used as a covering for the cement floor; and in stables, a wooden floor ANIMAL COMPETITORS 30 resting on the concrete is just as satisfactory so far as the exclusion of rats is concerned." Keeping food from rats. The general rat- proofing of buildings is the most important step in limiting the food supply of rats. The effect of an abundance of food. on the breeding of rodents has already been mentioned. Well-fed rats mature quickly, breed often, and have large litters of young. Besides limiting reproduc- tion, scarcity of food will make the measures to destroy the animals by traps, poisons, or bacterial cultures far more effective. But since much of the animals' food consists of garbage and other waste materials, offal of any kind must be so disposed of that rats can not obtain it. The best method is by burning it. The management of slaughter-houses in the country, in particular, needs reform. It is a common practice to leave offal of slaughtered animals to be eaten by both rats and swine. Such places are not only centers of rat-propaga- tion, but are the chief means of perpetuating trichinae in pork. All this should be changed in fact and by law. The offal should be promptly cremated or otherwise disposed of. There is Keeping food from rats. The general rat- proofing of buildings is the most important step in limiting the food supply of rats. The effect of an abundance of food. on the breeding of rodents has already been mentioned. Well-fed rats mature quickly, breed often, and have large litters of young. Besides limiting reproduc- tion, scarcity of food will make the measures to destroy the animals by traps, poisons, or bacterial cultures far more effective. But since much of the animals' food consists of garbage and other waste materials, offal of any kind must be so disposed of that rats can not obtain it. The best method is by burning it. The management of slaughter-houses in the country, in particular, needs reform. CHAPTER I aa 3 <f X THE PEST OF EATS It is a common practice to leave offal of slaughtered animals to be eaten by both rats and swine. Such places are not only centers of rat-propaga- tion, but are the chief means of perpetuating trichinae in pork. All this should be changed in fact and by law. The offal should be promptly cremated or otherwise disposed of. There is THE PEST OF EATS 31 no reason why country slaughter-houses should not be as cleanly as are the abattoirs of a modern city. Disposal of dead rats. Finally, the bodies of dead rats should never be handled with the bare fingers, or thrown out to be eaten by dogs or pigs or other animals; for they may con- tain, as has been shown, the germs of dreadful diseases. They should be burned, or else turned to account by being buried at the foot of grape-vines or young trees, for which they will make an excellent fertilizer. Four-footed enemies of the rat. A word as to the assistance animals may give in killing off and keeping down the rats. How greatly the increase of all rodents is due to the destruc- tion of the various wild mammals, birds and reptiles, that prey upon them, will be shown hereafter. Hawks, owls, weasels and skunks dispose of a great number of rats in rural dis- tricts, and might take many more if they were permitted. Skunks in particular are a most valuable help in this direction both the large northern skunks and the small spotted species of the South and West and will, if allowed, ANIMAL COMPETITORS 32 speedily clear a place of its rats and mice. Un- fortunately they are seldom allowed to tenant the premises without being molested by either dogs or men. When thus disturbed, the skunks emit the characteristic secretion, which is al- most their only defense against enemies. Un- disturbed, they are quite inoffensive and will stay about the farm-buildings until rats and mice are no longer to be had. Skunks usually hunt by night, and hence poultry properly housed is safe from them. It is the loose, un- cared-for hens that suffer. The same may be said of weasels, which will follow a rat into its burrow, and seem to take such delight in slaughtering it that no rats can be found shortly after a weasel or two have taken up their quarters in the place. CHAPTER I aa 3 <f X THE PEST OF EATS The drawback to their good work is, that they are fond of poultry and clever in getting it. The same may be said of minks; but a rat-proof hen-house is also weasel-proof. p Farm ferrets, like weasels (of which they are a larger cousin) are inveterate foes of rals, but their value under ordinary circumstances is overestimated. THE PEST OF BATS 33 "For effective work," says one who knows, "they require experienced handling and the ad- ditional services of a dog or two. Dogs and ferrets must be thoroughly accustomed to each other, and the former must be quiet and steady instead of noisy and excitable. The ferret is used only to bolt the rats, which are killed by the dogs. If unmuzzled ferrets are sent into rat retreats, they are apt to make a kill and then lie up after sucking the blood of their vic- tim. Sometimes they remain for hours in the burrows or escape by other exits and are lost. There is danger that these lost ferrets may adapt themselves to wild conditions and become a pest by preying upon poultry and birds." p y p y g p p y Cats, as a rule, are not of much use. Most of them are too well-fed, and will be afraid of, or not take the trouble to pursue rats, although they may be excellent mousers. y y A couple of good terriers, however, will work wonders in freeing one's premises if trained to rat-catching. The ordinary farmer's big cur is of no use for this purpose and little for any other; but a Scotch, Irish or fox terrier, properly taught, will take pride in the work, ANIMAL COMPETITORS 34 and catch a surprising number of victims until all are frightened away. g y Cooperation necessary to subdue the pest. Little that is really effective can be done, how- ever, without cooperation in each district. 1 To destroy the animals on the premises of a single farmer in a community has little perma- nent value, since they are soon replaced from near-by farms. If, however, the farmers of an entire township or county unite in efforts to get rid of rats, much more lasting results may be attained. Such organized efforts repeated with reasonable frequency are very effective. q y y Cooperative efforts to destroy rats have taken various forms in different localities. i See Cooperation among Farmers, by Prof. John Lee Coul- ter. In this Library, 1911, 75 cents. CHAPTER I aa 3 <f X THE PEST OF EATS In cities municipal employes have occasionally been set at work hunting rats from their re- treats with at least temporary benefit to the community. Thus, in 1904, at Folkestone, Eng- land, a town of about 25,000 inhabitants, the corporation employes, helped by dogs, in three days killed 1,645 rats. A better example is re- ported from India, where cooperative work THE PEST OF BATS 35 has prevailed over large districts. Thus in the Punjab more than 625 centers of popula- tion, including large towns, were systematically cleared of rats in 1908, the actual number known to have been destroyed reaching 4,116,- 334, while large numbers were poisoned and escaped to die. The result in diminution of the endemic plague and other diseases was most marked. Side-hunts in which rats are the only animals that count in the contest have sometimes been organized and successfully carried out. At New Burlington, Ohio, a rat-hunt took place November 26, 1866, in which each of the two sides killed over 8,000 rats, the beaten party serving a Thanksgiving banquet to the winners. At about the same period county agricultural societies sometimes offered prizes to the family presenting the largest number of rats' tails as evidence that the animals had been destroyed. Even as late as May 2, 1907, in one of the coun- ties of Kentucky, by general consent, the day was set apart for killing rats, and, according to newspaper report, was quite generally observed. p p p q g y There is danger that organized rat-hunts will ANIMAL COMPETITORS 36 be followed by long intervals of indifference and inaction. This may be prevented by offer- ing prizes covering a definite period of effort. Such prizes accomplish more than municipal bounties, because they secure a friendly rivalry which stimulates the contestants to do their utmost to win. In England and some of its colonies contests for prizes have been organized to promote the destruction of the European house-sparrow, but many of the so-called *' sparrow clubs" are really sparrow and rat clubs, for the destruc- tion of both pests are avowed objects of the or- ganization. A sparrow club in Kent, England, secured the destruction of 28,000 sparrows and 16,000 rats in three seasons, by the annual ex- penditure of but $29.20 in prize money. CHAPTER I aa 3 <f X THE PEST OF EATS Had ordinary bounties been paid for this destruc- tion, the tax on the community would have been about $1,200. THE PANTEY MOUSE THE house-mouse (Mus musculus) needs no description. The only native species with which it can be confused is the harvest-mouse, from which it may be readily distinguished by its larger size and by the plain or ungrooved upper incisors. Like the rat it is a native of the Old World, very fertile, adaptable and hardy, and from time immemorial has followed civilization so closely that it soon becomes es- tablished in any settled region. It is therefore a world-wide nuisance, but by no means so great or difficult a one as is the rat. g Characteristics. The little house-mouse can hardly be confused with any other, for its ash- gray coat, becoming gradually lighter and often yellowish on the under parts, has fur- nished the language with a distinctive term, "mouse-color"; and its pointed nose, large 37 ANIMAL COMPETITORS 38 ears, half an inch long, very small eyes, and long naked tail, are unlike those of any native mouse. Its total length is about 7 inches, 31/2 of which belong to the tail. Its molar teeth have each three tubercles, instead of two, as in our own wood-mice ; and its incisors are un- grooved, by which, at any rate with the aid of a magnifying glass, the marks of its biting are readily identified. Its gray protective coat has seemed satis- factory under all circumstances, for there is no perceptible difference between representatives of the species in the four quarters of the globe. In general the type seems singularly invariable, only one or two varieties having arisen, such as the queer rhinoceros mice which appeared in England some years ago, and took their name from their hairless, deeply-folded skin which gave them the appearance of miniature rhinos. This fixity of type may be due in part to the fact that every country has received a con- stant immigration of fresh blood by means of ships and other conveyances. y Japanese dancing-mice. One strange vari- ety, however, has arisen, probably in China THE PANTRY MOUSE 39 from which it spread long ago to Japan, whence we have lately derived the specimens now com- monly sold in the animal-stores of our cities under the name of dancing or waltzing mice. They are small in size, pied black and white in a great variety of patterns, and are ex- tremely agile and amusing. THE PANTEY MOUSE Their distinguish- ing peculiarity, however, is their constant whirling about, so that a lot of them together seem like a company of dancers waltzing busily to some music unheard by us. y The origin and extraordinary behavior of this astonishing race of mice has been the sub- ject of much study, which has been summed up and extensively added to by Prof. Eobert M. Yerkes of Harvard University in a book en- titled The Dancing Mouse, a Study in Animal Behavior (New York, 1907). He regards it as highly probable that the Chinese took advan- tage of some deviation in captive mice from the usual form to develop a special race by means of careful and patient natural selection. "The dancing tendency is such in nature as to unfit an individual for the usual conditions of mouse existence, hence, in all probability, human care ANIMAL COMPETITORS 40 alone could have produced and preserved the race of dancers." That it originated in a "freak" seems very likely, since mere cultivation of the familiar white mice (albinos), beloved of children, never develops into a habit of whirling. A German naturalist inbred albino mice for 28 generations without producing any hint of such a pecul- iarity. Prevalence of mice. House-mice are very prolific. They will begin to produce young when only three months old, and continue to breed at intervals of two or three months all the year round. It is not known whether any proper pairing takes place probably not. The period of gestation is 25 days, five to ten young are produced at a birth minute, pink, blind, hairless things and are weaned after about two weeks. There are few houses or barns in which mice do not make themselves at home, racing from cellar to garret in the hollow walls and par- titions, and gnawing passageways wherever they think it worth while. Too often they penetrate where food is kept, and besides what THE PANTRY MOUSE 41 they eat spoil large quantities by trampling and dragging their tails over or through it, and leaving their acrid-smelling traces. Where they are numerous, this becomes a very serious pest; and it is only the most slovenly house- keepers who will permit their presence. THE PANTEY MOUSE A good cat, kept hungry enough to make her eager to go a-mousing, is probably the best safeguard ; but traps are useful especially the cheap and handy little guillotine traps de- scribed in the last chapter. Of course a wise person will stop up all holes, clean out the nests which may be found in an extraordinary variety of snug places, and make the little beasts as unwelcome as possible. They carry fleas and other parasites; and are often sorely afflicted with warbles ; but it is not known that they transport the flea which communicates the microbe of the plague. However, their pretty feet often dabble in filth and may bring into the house dangerous germs, so that it is not well to permit them the freedom of your kitchen or p y Mice carrying pathogenic bacilli. In a re- port of an investigation of the transmission of ANIMAL COMPETITORS 42 disease by house-mice made by Dr. P. Bara- baschi, and published in an Italian medical journal in 1909 (see Experiment Station Record, Vol. XXII, No. 7), it is stated that I>r. Barabaschi has found many bacilli within their bodies and excreta. Among these were the pneumococcus to which croupous pneumonia is due, the bacillus of anthrax, that of ery- sipelas, those to be found in abcesses, boils, etc., and other pathogenic germs. The mice with the pneumococcus were caught in private houses where there had recently been pneu- monia. The excreta of the mice "mice dirt" drying and scattering in dust, may transmit infection even without more direct contact. The greatest danger from this source is in- curred by persons working in granaries, etc., where mice abound and their droppings are scattered over the substances handled. It may be added that an American physician asserts that the microbe of measles comes from mice. In houses left untenanted for a time mice In houses left untenanted for a time mice frequently do considerable damage by tearing holes in blankets, bedding and clothing, to get material for their nests. The writer has him- THE PANTRY MOUSE 43 self suffered decidedly from their work in a summer bungalow while it was unoccupied during the winter, finding beautiful nests among the bed-clothing, made from his blan- kets. Apart from their mischief and dirt, mice are pretty little creatures and make interesting pets for the little folks. THE PANTEY MOUSE Caught young they are easily kept alive and comfortable in a roomy cage and exhibit many interesting ways. Boiled oats are a favorite food, and they like to nibble at grain and at pieces of bone with shreds of meat left on them; they also catch flies and other insects. Mr. Cram is of the opinion that, in cold weather at least, most of the house-mice live almost wholly upon insects, as flies, spiders, wasps and the like, that have packed themselves away snugly for the winter in secret crannies between the boards, some- times hundreds of them closely huddled to- gether gether. Musical mice. One of the most curious and remarkable facts in the history of the house- mouse is its so-called singing. Many instances are on record, of which the following related in ANIMAL COMPETITORS 44 The Scientific American some years ago is typ- ical: The Scientific American some years ago is typ- ical: "A few winters since, while one of his family was amusing herself at the piano, a mouse made his ap- pearance on the threshold of the apartment, and, un- dismayed by the light or the presence of the family, chirped and carolled with intense satisfaction to itself, and to the great delight of its audience. Fre- quently afterward, but always in the evening, the rare songster repeated his performance. The piano keys were never struck that the mouse did not fol- low; but when the instrument was not touched, the music from the mouse would come, as if for a re- minder. Sometimes the little animal made himself visible and sometimes was hidden in the pantry which, for reasons obvious to housekeepers, he, she, or it had selected as an abode. One evening the mouse was traced to the stairway. Under the carpet sat the little creature, throwing his soul into his song. A lamp was placed beside him, and the family stood and looked and listened for half an hour or more. His head was up, and the movements of the muscles of his throat were plainly visible. Unfortunately our correspondent undertook to capture the singer. Many mice were caught and each was given twenty-four hours grace to sing for its life. But never after the treachery of the trap was the sound of the mouse's carol heard. THE PANTEY MOUSE If caught he died and made no THE PANTRY MOUSE 45 More lately (1909), a gentleman at Hamilton, Ontario, sent me the following account of a sim- ilar case: "Some months ago in one of the current magazines there appeared an article on "A Singing Mouse." The story related told of a gentleman whose atten- tion was attracted by a peculiar little singing noise, heard in one place, then in another part of his house. Curiosity led him to make a search, which ended, as he told his readers, in the finding in the wood-shed of one or more mice, which, no doubt, were the guilty parties. . . . parties. "Some few nights ago, in our own house, we heard a peculiar noise. At first we believed it due to some mischievous boys playing the old-time trick of 'Tick- a-tack' on the window, as the sound proceeded from that part of the room. The sound resembled it some- what. The following evening the same sounds pro- ceeded from another part of the house. I was not present, but those who heard it dwelt on the musical quality of the thing, and declared the noise consisted of distinct notes, and ventured to add that it was pretty. Well, we were becoming interested. The third night I was home, and, filled with a bold de- termination to do or die, went at an investigation. , g "The noise proceeded from the kitchen, and one declared it came from beneath the sink. Upon re- moving a brush in the corner, on the floor, a little mouse ran out. The story in the magazine at once ANIMAL COMPETITORS 46 came to my mind. I was satisfied as to the 'singing mouse' being a reality. came to my mind. I was satisfied as to the 'singing mouse' being a reality. "The next appearance of our little singer was a night or so after the sink episode, when in the pantry, I and another saw it under a shelf, and it did not show much fear at our presence. It is not unlike other gray mice, only in the song it sings. This even- ing it was heard in the pantry. ' ' Many other instances might be quoted, some from writings more than a century old. Dr. Elliott Coues wrote an extensive article about these singing mice several years ago in The American Naturalist. THE PANTEY MOUSE His conclusion was that the sound was due to an asthmatic affection of the throat and vocal organs. An interesting narrative of various exhibitions of this faculty is also included in Dr. Merriam's admirable book on the mammals of the Adirondacks (Trans. Linnaean Soc. of N. Y., Vol. II.) ; and in Vol. V. of The American Naturalist, the late Eev. Samuel Lockwood gave a most pleasing history of a white-footed (wild) mouse which was kept in a cage, and was a persistent and prolonged singer, having two or more regular tunes, the music of which he gives. The sim- ilar performance of a captive house-mouse is THE PANTRY MOUSE 47 related in Vol. V. of The Standard Natural History. All observers speak of the trilling, warbling, bird-like character of the notes. "It was not much of a song," writes a Detroit lady, "as songs go, but still a distinct musical effort. Sometimes it would run up an octave and end with a decided attempt at a trill. Sometimes it would try- to trill all the notes. ... Its favorite position when singing was an erect one, standing on its hind feet, and holding by its forward ones to the wall or a bracket, almost invariably turning its face towards us. It remained with us several weeks, and at length became so familiar as to appear to enjoy company, seemingly putting forth all its strength to amuse us with its little song, which improved daily in tone and volume, but not in compass. Its voice became so clear that we could frequently hear it in the parlor that opened out of the dining room." Most persons regard this singing as not due to disease, but quite natural. "There seems good reason for believing," in the language of Ernest Thompson Seton, "that house-mice, and, indeed, all mice, will at times express their sense of well-being in a series of complicated sounds that correspond in every way with the singing of birds." THE MEADOW-MOUSE AND ITS MISCHIEF WHILE the alien rats and mice are working costly mischief about the house, stable, and granary, their native cousins, the wild mice, are doing vast harm in garden, orchard and field. Naturalists count 200 or more species of these animals in North America, but we need concern ourselves with only certain types, since, from the farmer's point of view, the ac- tions of all are much alike, and the principal damage is caused by those of a single group the short-tailed meadow-mice of the genus Microtus. To this genus alone David E. Lantz has devoted a treatise of 64 pages in the pub- lications (Bulletin 31) of the U. S. Biological Survey from which, as before, I shall quote freely. He prefaces this treatise with the statement that the mice of this genus alone cause an average annual damage to American farmers of not less than $3,000,000. It is this 48 49 ANIMAL COMPETITORS 50 kind of mouse which now and then in this country, and still more frequently in eastern Europe, appears suddenly in such vast num- bers as to constitute a veritable plague, ruining the produce of the year in all directions. p y Sometimes wild animals increase in numbers so suddenly that the change has been likened to a tidal wave, and ignorant people have re- garded the invasion as of miraculous origin. The belief that crickets, locusts, frogs, and even mice sometimes fall from the clouds is still held in many countries. "The careful observer, however, sees little mystery in the phenomena mentioned. He has studied the general habits of animals their food, their powers of reproduction, their migrations, the checks on their increase due to natural enemies, disease, and varying climate and consequently he attributes sudden changes in their numbers to known causes. In such changes he recognizes, especially, the influence of man, both direct and indirect, and his responsibility for interferences that greatly modify the operations of nature." American voles or meadow-mice. The mice of the genus Microtus (formerly Arricola) rep- resent a group which embraces a large number MISCHIEF OF MEADOW-MOUSE 51 of forms of' small and very similar rodents which in some respects resemble true mice, but are readily distinguished by the robust body, thick head, very short ears, blunt muzzle and short, hairy tail. There seems to be no entirely appropriate vernacular name for .these mice. THE MEADOW-MOUSE AND ITS MISCHIEF The French call them campagnols, the Germans wuhlmduse. English-speaking people outside the United States call them voles. In the United States they are variously designated as meadow-mice or field-mice, and locally as bear-mice, bull- mice, buck-tailed mice, mole-mice, and so forth. Meadoiv-mice would do very well if it were not that several of our four-score species belong to the high dry plains of the West. As, however, the typical meadow-mouse of the east ranges over nearly the whole country, its name may well be adopted for the whole genus. The three species most frequently met with in connection with damage are : 1. Common meadow-mouse (Microtus penn- sylvanicus.) y 2. Prairie-mouse (M. ochrogaster.) 3. Pine-mouse (M. pinetorum.) 2. Prairie-mouse (M. ochrogaster.) 2. Prairie-mouse (M. ochrogaster.) 3. Pine-mouse (M. pinetorum.) 3. Pine-mouse (M. pinetorum.) 52 ANIMAL COMPETITORS The common meadow-mouse (No. 1) is 6y2 inches long, of which the tail takes 1% inches ; and has eight teats. Its fur is long, overlain with coarse black hairs, and in winter almost conceals the ears. The usual color above is a dark brown, against which the black hairs are not conspicuous. This shades off gradually into gray or tawny on the under parts. This species may be said to inhabit the whole con- tinent, though in the mountainous parts of the West other species are more numerous and conspicuous. This mouse has its natural habitat in moist meadows and grassy borders of swamps, but it habitually extends its range into neighboring cultivated fields and waste lands. Nearly all meadows are full of the animals. On parting the thick grass almost anywhere one can find the smooth trails, and where the grass is thin they are often plainly visible. After the melt- ing of deep snow, or where the dry grass has been, burned, the network of runways is espe- cially conspicuous to the eye. In swamps the paths* cross soft mud, and where a green scum of minute floating plants covers stagnant MISCHIEF OF MEADOW-MOUSE 53 water, the trails are often defined across it by streaks where the animals swim from side to side. The normal number of young averages about six, brought forth in an underground burrow. The normal number of young averages about six, brought forth in an underground burrow. Prairie and pine mice. The prairie-mouse (No. 2) differs only slightly from No. 1. THE MEADOW-MOUSE AND ITS MISCHIEF It is a little smaller, the rough tail is shorter, its two colors are more sharply contrasted, and the general pelage coarser. The color of the under parts shades into a buff or cinnamon, and in winter becomes gray. It is confined to the central Mississippi valley, where is produced more than half the corn, oats and winter wheat of the country ; and two closely related species extend the range of mischief to the dry plains west and northwest. These prairie-mice have an especially fine opportunity for damage, and in the winter are more destructive to trees than the M. pennsylvanicus. Fortunately, there- fore, they are less productive, having usually only three or four young at a time, and the long summer droughts and extreme winters of the interior West further limit reproduction. The i mouse or red backed mouse The typical pine mouse or red-backed mouse ANIMAL COMPETITORS 54 (No. 3) is found only in the Carolinas and Georgia, but several varieties widen the specific range from southern New York to Oklahoma, south of the latitude of Lake Erie. The typical Georgian pine mouse has glossy, mole-like fur of a bright russet-brown color. The variety of the blue-grass region is darker and very glossy, while that of the states west of the Mississippi, between southern Iowa and northern Texas, is deep chestnut. In all, the skull is wide and flat, and the fur short, dense and glossy. g y Owing to their peculiar habits, pine mice are not so well known as are the northern meadow- mice. Their natural habitat is the forest, al- though they are by no means restricted to pine- woods or forested areas. The life of the pine mice is largely spent in underground tunnels, which so closely resemble those of the mole that generally they are mistaken for the work of that animal; but the inner diameter of the mouse-tunnels is less. When moles and pine mice live in the same vicinity, the mice often use the runways made by the former and this habit has helped to bring moles into disrepute with farmers. MISCHIEF OF MEADOW-MOUSE 55 From their intricate tunnels under the leaf- mold frequent burrows descend into the soil, some of which are utilized as nesting places. Nests are built also at the surface of the ground, under fallen logs, brush-heaps, flat stones, fences, or other shelter. THE MEADOW-MOUSE AND ITS MISCHIEF The number of young at a birth evidently averages less than is usual in the genus Microtus, as is shown by the small number of mammae ; but this is coun- terbalanced by the safer underground exist- ence; so that within their range pine mice are about as abundant as other field-mice. Quick and Butler, writing of the food-habits of the pine mouse in Indiana, state that it lives upon the tender roots of young hickories, the young sprouts of white clover, the fruit of the red haw, and the tuberous roots of the wild violet. These writers found all but the fruit buried, some in deposits of a gallon in a burrow, and the caches sometimes extending 18 inches below the surface of the ground. Violet roots predominated in these stores. Kennicott also states that pine mice store acorns and nuts in burrows for winter use. Living in concealment neither their presence ANIMAL COMPETITORS 56 nor the injury they inflict is suspected. Bulbs planted hopefully in autumn, appear not at all in spring, or only in the shape of sickly plants. Nursery and orchard trees fail without reason until their roots are examined and the work of this hidden nibbler is disclosed. General wild habits. It appears then that in habits there is considerable variety among the mice of this group (Microtince), but least in the matter of diet. While some species have a vastly widespread range, others are confined to very limited localities. Some species prefer high and dry ground, while others live in low, moist places. Except in cold weather, nearly all species can temporarily adapt themselves to moist surroundings; but a few seem to be al- most as aquatic as the nearly-allied muskrat. Some dwell in forests, others in the open prairies; some burrow under the ground like moles, while others make smooth paths or trails upon its surface. p The nests of meadow-mice are compact bunches or globes, composed chiefly of grass blades and other dry vegetable fibers. They are placed in depressions in the ground, in MISCHIEF OF MEADOW-MOUSE 57 shallow burrows, or supported on grass stems of brush-piles above the ground. Sometimes they are placed under flat stones or logs or under shocks of grain. THE MEADOW-MOUSE AND ITS MISCHIEF The structures are so slight that a day's sunshine will dry them out after a storm, and yet they are so compact that the animals pass the coldest weather snugly housed in them under the snow. The young of most kinds are born in underground nests and are at first hairless and blind. When discov- ered in the nest the mother vole slips noise- lessly away, sometimes carrying the young at- tached to her mammae. The breeding-season includes most months of the year, except mid-winter in cold latitudes and periods of long-continued drought. The number of litters in a year thus depends on climate, and especially upon the character and length of the winter. In temperate latitudes in normal seasons from four to six litters are produced ; but the variation in the same species is remarkable, and depends partly upon climate, but probably more on the scarcity or abundance of food. The period of gestation is about twenty days. ANIMAL COMPETITORS 58 These mice, like their European relatives, the voles and lemmings, at times increase in num- bers abnormally, but the causes are little un- derstood. At such times they multiply with amazing rapidity, and begin breeding when only six months old. y "If a thousand pairs of field-mice," remarks Mr. Lantz, "survive the winter in any neigh- borhood, the potential conditions for a vole plague are present. If, now, instead of normal reproduction, circumstances bring about a con- siderable increase both in the number of young at a time and in the number of litters in a sea- son, the probability of a plague is greatly in- creased." Plagues of field-mice. Swarms of mice dev- astating the fields have been seen by very few American farmers, though occasional se- vere outbreaks of this kind are on record in Nova Scotia and in various eastern states. They have been all too frequent in all parts of the Old World from the earliest times, and used to be regarded superstitiously as punishments sent from on high. The valley of the Danube and the plains of southern Russia seem to have MISCHIEF OF MEADOW-MOUSE 59 been especially subject to these devastations, which have occurred most terribly even within the past few years. AN ALFALFA FIELD DEVOURED AND HONEYCOMBED BY FIELD-MICE. AN ALFALFA FIELD DEVOURED AND HONEYCOMBED BY FIELD-MICE. THE MEADOW-MOUSE AND ITS MISCHIEF In 1907-8 an outbreak of field-mice in Ne- vada, Utah and northeast California, threat- ANIMAL COMPETITORS 60 ened to develop into a plague as great as any recorded, and the facts concerning it have been studied and preserved in a pamphlet by Stan- ley E. Piper of the Biological Survey. The species was the black or Carson mouse (Micro- tus montanus), which is widely prevalent west of the Rocky Mountains. "The greatest loss occurred in the rich fields of alfalfa bordering Humboldt Kiver for the last ten or twelve miles of its course. Noticeable here through gradually increasing damage during 1906, the field- mice appeared early in the summer of 1907 in alarm- ing numbers. By November they had overrun a large part of the cultivated area, and on many large ranches were estimated to number from 8,000 to 12,000 to the acre. Fields were literally honey- combed by their holes, which numbered about 24,000 to the acre. During the summer they ruined one- third of the alfalfa, destroyed three-fourths of the potatoes, and severely injured root-crops, as beets and carrots. Upon the disappearance of green food in the fall they attacked the roots of alfalfa, so as to render many alfalfa fields a total loss. They girdled and killed most of the young shade-trees planted along ditches and about the borders of fields, while small orchards suffered severely." Decline of the visitation. By January, 1908, the ravages had extended over considerably MISCHIEF OF MEADOW-MOUSE 61 more of the district, and the main body of mice was gradually progressing to fresh fields. From this time, however, the abatement of the plague was rapid. By March 15, the invasion of fresh lands had ceased, though mice contin- ued considerably in excess of normal abundance until May. By August they had practically disappeared from the valley. This scourge left a dismal scene of destruction over four-fifths of the cultivated area in the district. Of 20,- 000 acres in alfalfa, 15,000ywere so completely destroyed as to require replanting. Consider- ing the actual losses in crops and the cost of restoring the alfalfa fields, and allowing for the value of the wheat which replaced alfalfa in most of the ruined fields for the season of 1908, Mr. THE MEADOW-MOUSE AND ITS MISCHIEF Piper estimates the loss in this district at $250 000 Some interesting particulars are recorded as to the diminution of the horde, which was preyed upon by a gathered crowd of predatory birds, mammals and reptiles, as well as ex- tensively poisoned, yet succumbed at last mainly to natural mortality, not to any spe- cific bacterial disease. 02 ANIMAL COMPETITORS "In the spring," Mr. Piper relates, "the mice in this locality failed to reproduce, while the same spe- cies was breeding prolifically in other localities. In March several hundred females were examined in Humboldt Valley, of which very few were pregnant. Moreover, the mice themselves presented a different appearance from those seen when the plague was at its height a fact noted by many ranchmen in the valley. During the fall of 1907 larger and much more vigorous individuals predominated, while in the spring of 1908 scarcely any of these remained. They continued in destructive numbers until . . . May. But they did not noticeably breed with the return of favorable weather and by August had practically disappeared." Prevention of plagues of mice. Unfortu- nately the liability to such " plagues" in- creases with the spread of settlement and cultivation. "Agricultural development dis- tinctly increases the danger by furthering the destruction of their natural enemies, by furnish- ing a great abundance of food, and by increas- ing the area in which they find favorable homes." On the other hand, the prevention of plagues is comparatively easy. Systematic poisoning must be relied upon to repress them when they are obviously on the increase, but ANIMAL COMPETITORS 64 there are many inexpensive methods for pre- venting this increase. The destruction of rank grasses and weeds along fences and ditches, and particularly, in the West, the pasturing off of the last growth of alfalfa in fall, thus ex- posing the mice to the sight of predaceous en- emies, are important. Winter-burning of dry vegetation on wild hay lands, on strips border- ing fields, and on swampy or otherwise waste areas in and about cultivated fields, will aid materially in controlling them. The survivors may invade cultivated fields, but there they can be more readily poisoned. Flooding the fields in cold winter weather, when the mice quickly perish from exposure, is an effective method in irrigated lands. THE MEADOW-MOUSE AND ITS MISCHIEF Plows turn out the bur- rows and nests of practically all the mice pres- ent and render them easy victims for dogs, which when trained to kill mice can not be too highly recommended as effective and inexpen- sive aids in controlling the pests. That hawks, owls, gulls, crows, ravens, and herons among birds, and skunks, weasels, foxes, and badgers among mammals, are persistent enemies of field-mice and other rodent pests has been often. MISCHIEF OF MEADOW-MOUSE 65 pointed out. The protection and encourage- ment of these valuable allies of the farmer can not be too strongly advocated. Trapping, systematically continued, is of great service; and advice upon it is given at the end of this book, as also for poisoning these small pests. Food of wild mice. Returning now to a further consideration of the mice in the normal numbers which are always with us, an under- standing of their feeding is most important as a preliminary to repressive measures. p y p In summer the principal food is green vege- tation and unripe seeds of grain and grasses. As the season advances, ripe grain and seeds take the place of the immature; and in winter bulbous and other roots are in part substituted for stems and leaves. It is mainly in winter that apple orchards and young forest trees suf- fer, for meadow-mice invade cleanly cultivated fields only under shelter of snow. Unlike the foreign voles, our American species do not, as a rule, lay up winter-stores in any considerable quantities, as do some other American mice the deer-mice, for instance. Instead, our mead- ANIMAL COMPETITORS 66 ow-mice are active all winter not hibernating, but gathering food from day to day and wan- dering widely. Yet in the far North the climate has compelled habits of winter provision in the tundra vole (Microtus operarius) which is small, inhabits the mossy tundras of western Alaska, and gathers stores of small bulbous roots, sometimes placing a peck or more in a single cavity just below the surface on a mossy knoll or slope. In autumn, shortly before the first snowfall, the Eskimo women and children discover these stores by means of pointed sticks. In this way considerable quantities of food are gathered, which are boiled and eaten as a delicacy. THE MEADOW-MOUSE AND ITS MISCHIEF "The boiled roots have a flavor like a boiled unripe sweet potato, and are very palatable during the long winter fare of meat and fish," according to E. ~W. Nelson. g Damage from murine voracity. Complaints of damage to meadows and pastures have been steadily increasing, with occasional reports of the total ruin of a red-clover field. More grass is cut down and left than is eaten. In winter, haystacks are attacked and sometimes so rid- dled as to be spoiled for market. MISCHIEF OF MEADOW-MOUSE 67 Growing grain of all kinds is' destroyed. Field-mice injure early peas and other vege- tables, and pine mice often destroy potatoes in the ground. In the fall vegetables piled on the ground or stored in pits are liable to at- tack, and especially celery. Apples, pears, and other fruits are eaten also, including melons. g Blackberries, raspberries, grapes, currants, gooseberries, and strawberries are often badly damaged by field-mice, and when the animals are abundant whole plantations are ruined. Strawberries are especially liable to injury be- cause of winter mulching and also because the plants themselves furnish excellent food and shelter for the animals. Winter mulching of small fruits increases the damage unless care- fully guarded by clean surrounding areas. y Damage to standing nursery stock is done usually under cover of snow; and in addition to girdling trees above the surface meadow- mice, sometimes dig down and attack the roots. Pine mice usually begin their evil work with the sprouting grain, and, in the case of fall- sown wheat and rye, continue it during the en- itire winter. Much greater damage is done ANIMAL COMPETITORS 68 when the grain is nearly mature, as stalks are then cut down; and after harvest the animals attack the shocked grain. In shocks and stacks the mice are perfectly at home, and multiply with such rapidity that within a few weeks a pair and their progeny may totally ruin 'an entire shock of wheat, oats or corn. In view of this situation it is a question whether the farmer who hastens to market his crop is not, on the whole, a gainer over his neighbor who waits for more favorable prices. In these and other ways the annual de- struction of grain and forage throughout the country is enormous; nor is the injury all done by the short-tailed meadow-mice. ' ' One of the most common mistakes made by sports- men in the supposed interests of game protection," THE MEADOW-MOUSE AND ITS MISCHIEF Deer- mice (Peromyscus), pocket-mice (Perogna- thus), harvest-mice (Reithrodontomys), and ordinary house-mice are also concerned in the damage. Throughout the country the brown rat and in the Southwest the cotton-rat (Sig- modon) are serious field-pests. p General preventive measures. The forego- ing testimony sufficiently shows the noxious character of these small rodents ; and suggests the query : * ' How shall it be stopped ? ' ' MISCHIEF OF MEADOW-MOUSE MISCHIEF OF MEADOW-MOUSE 69 In view of their wide distribution, the nature of their habits and the abundance of shelter and food everywhere in America, it is impos- sible to get rid of them; but it is not hopeless to reduce their ability for damage to a negligi- ble quantity. Thorough and clean cultivation, with frequent plowing, is perhaps the most effective general remedy, and where this is done throughout a group of adjoining farms, and the roadside weeds and fence-tangles are regularly cut, or burned over, little trouble will be ex- perienced within the district. p Next to this is the preservation of the birds and other animals which prey upon mice, and which have been so ruthlessly killed off in most rural districts, partly through the insane tend- ency to kill every living thing which animates many country boys and men, and partly through mistaken ideas as to the harm such an- imals do. Even persons who ought to know better engage in this miscellaneous destruction of the best friends a farmer can have, proof of which will appear later in this book. ANIMAL COMPETITORS 70 remarks D. E. Lantz, "is the offer of prizes for the destruction of alleged 'game-destroying' mammals and birds. In one instance nine competitors for a club's prizes destroyed during twelve months 184 weasels, 48 foxes, 54 minks, 343 skunks, 15 great horned owls, 6 'common owls,' and 148 hawks. The fact that only 21 owls were killed in an entire year by nine men trying for a record reveals a scarcity of these useful birds that is not complimentary to the intelligence of the community. The large num- ber of skunks killed indicates ignorance or disregard of the usefulness of that animal in destroying insects and mice. Apparently, too, there was no discrim- ination as to the species of hawks destroyed, and it is probably safe to say that field-mice in a single year have damaged the farmers of the region concerned a hundredfold more than the value of all the game and poultry saved through the offer of prizes. ' ' Protection of orchards and nurseries. In- jury to orchards and nurseries by field-mice may generally be prevented by forethought and the exercise of ordinary care. Of first impor- tance, always, is clean tillage. No grass or weeds should be left in or near the nursery. MISCHIEF OF MEADOW-MOUSE So well is this understood by the majority of experienced nurserymen that by clean tillage they secure practical immunity from the rav- ages of mice except in winters of deep and long- MISCHIEF OF MEADOW-MOUSE 71 lying snow. Unfortunately, nurserymen can not control the lands which environ their trees, and when snow falls to a considerable depth prompt measures are sometimes necessary to keep mice from destroying them. y g This can be accomplished most readily by dragging a heavy log several times around each block of trees, packing the snow so firmly that mice cannot tunnel under it. If this be done promptly after the first snowfall, subsequent falls will require little attention. Under no circumstances should matted grass or litter be allowed around the trunks of trees or along the borders of the orchard. In the absence of snow a cleared space of about 18 inches radius about each trunk is enough to prevent damage. This space should be as smooth and clean as possible. Tree-protectors and washes considered. If any part of the orchard is so located as to be subject to snowdrifts, and mice are abundant in the vicinity, tree-protectors should be used. These may be had of dealers for 60 to 75 cents per hundred, or they may be made by the farmer. Strips of wire cloth make excellent ANIMAL COMPETITORS 72 protectors, and tarred paper is a favorite with some horticulturists. The wire cloth or paper AN APPLE-TREE GNAWED BY MEADOW-MICE. cut into b t i h id AN APPLE-TREE GNAWED BY MEADOW-MICE. is cut into strips about 7 inches wide and at least 15 inches long. A strip is secured around MISCHIEF OF MEADOW-MOUSE 73 each tree with wire or cord. Tarred paper should never be used on very young trees, and when used on others should not be left in place during the summer, since it may injure the growing tree. Various paints and washes have been recom- mended to prevent attacks of mice and rabbits in orchards. The majority of these are with- out merit and some of them are liable to kill young trees. Some of the washes require re- newal after every hard rain. MISCHIEF OF MEADOW-MOUSE In experiments with a wash of whale-oil soap, crude carbolic acid, and water, for apple trees, it was found that in about forty-eight hours the carbolic acid had so far evaporated that mice renewed their work upon the bark. Blood and grease, said to give immunity from rabbit attacks, would invite the attention of field-mice. Eeports recently received by the Biological Survey seem to indicate that the ordinary lime- and-sulphur wash, recommended for the winter spraying of trees to destroy the San Jose scale, is an effective preventive of the attacks of both mice and rabbits. The wash is very cheap (from 1 to 2 cents a gallon when prepared in ANIMAL COMPETITORS 74 45 to 50 gallon quantities) and is easily applied to the trunks of trees either in the form of a spray or by the use of a brush. One thorough application in November would probably be ef- fective for the entire winter. The ingredients of the wash are 20 pounds of unslaked lime, 15 pounds flowers of sulphur, and water to make 45 to 50 gallons. The mixture should be boiled in an iron kettle at least an hour and applied to the trees while warm. pp Winter mulching of trees is dangerous, un- less the neighborhood is known to be free from mice. Mulch containing straw may be placed in the orchard in spring, but it should be re- moved before the approach of cold weather. Fine, thoroughly rotted manure may be used in the orchard with but little danger. Lime or ashes about the trunks of trees has some value in keeping off mice, but clean cultivation is equally or more effective. q y Remedies for injured trees. When trees are girdled by mice, portions of the inner bark (cambium layer) are often left, partly cover- ing the hard wood below. If sunlight and wind have free access to the injury, the remaining MISCHIEF OP MEADOW-MOUSE 75 bark dries up and the tree dies. If light and air are excluded, new bark will form and the wound quickly heal over. To facilitate the healing process, it is important that wounds be covered as soon as possible. All that is needed is to mound up the soil about the trunk of the tree high enough to cover the wound, and the covering should remain all summer. MOUSE CAUGHT IN A GUILLOTINE TRAP. MISCHIEF OF MEADOW-MOUSE This is the simplest, cheapest, most humane, and on the whole most effective trap for catching and killing rats, wild mice or house-mice. MOUSE CAUGHT IN A GUILLOTINE TRAP. This is the simplest, cheapest, most humane, and on the whole most effective trap for catching and killing rats, wild mice or house-mice. PROFIT FROM THE MUSKRAT IN Europe some of the best-known species of meadow-mouse are large and aquatic, as, for example, the common water-rat of England. Of the same sort, on a larger scale, is our American muskrat a huge, water-dwelling vole with a tail compressed into a sculling-oar, whose appearance and manner of life are fa- miliar to most of us. Mischief done by muskrats. In the Eastern States muskrats do little damage, although everywhere abundant, except occasionally by opening a dike that protects a New England salt-meadow from high tides. West of the Al- leghanies, however, they often cause great an- noyance to the owners of canals and reservoirs by their burrowing; and frequently enter gar- dens near the water-side and devour fruit and vegetables to a considerable extent, while corn- fields sometimes suffer much when the corn 76 PROFIT FROM THE MUSKRAT 77 is in the roasting-ear stage. At times, also, they have ruined ornamental ponds by eating out of them the lilies and similar plants of whose bulbs they are fond. But this sort of destruction is rarely noticed except in the neighborhood of extensive marshes. g Far more serious, however, is the trouble and loss the busy animals occasion by perforating the dams and embankments of mill-ponds, ice-ponds, irrigation ditches and reservoirs. Every canal suffers breaks due to them, as well as to brown rats, gophers, mice, crayfish and moles. In the rice plantations of the Gulf coast they are a serious nuisance by cutting the em- bankments and flooding or draining the rice- fields at the wrong time; and this has resulted in Louisiana in laws protecting the alligators in some parishes because they kill the rats. So serious was the situation in Plaquemine Parish, La., in 1908-9, that a general slaughter of muskrats took place, and fully half a million are said to have been killed. The sale of their pelts produced about $100,000. p p Trapping, shooting and poisoning may all be made effective to a certain extent against ANIMAL COMPETITORS 78 muskrats, but should only be employed in ex- ceptional circumstances. The most valuable works open to their attacks should be con- structed of or faced with rubble or concrete to a proper depth. Muskrats worth far more than the damage they do. PROFIT FROM THE MUSKRAT As a matter of fact the harm done by muskrats is, on the whole, far outbalanced by their value in fur, so that in Canada, and in many states of the Union, the animal is pro.- tected during a close season (April to Novem- ber), when its young are being nurtured and the fur is not " prime," i. e., in good condition for market. These states are Delaware, Illi- nois, Iowa, Maine, Maryland (coast counties), Michigan, Minnesota, New Jersey, New York, North Carolina (coast counties), South Dakota, Virginia (coast counties) and Wisconsin. This protection, however, ends too early in the fall. The open season should not begin in the United States before December 15, and should close by March 15; in Canada it may be slightly pro- longed g The muskrat supplies one of the most useful and dependable sources of fur for clothing, and ANIMAL COMPETITORS 80 also a good flesh-food which is not utilized to the extent it deserves. Excellence of muskrat flesh. The Indian aborigines habitually ate this flesh, especially in winter, and taught the colonists how to cook it, boiled with corn, into a toothsome dish. The early western hunters and explorers were glad to get it, liking it roasted over a slow fire. Lantz tells us that in the retail markets of Philadelphia, Baltimore, Wilmington and other cities, these animals are sold as " marsh rab- bits," but no attempt is made to conceal the fact that they are muskrats. "They are bought and eaten both by well-to-do citizens and by the poorer people who seldom indulge in high-priced game. The animals are trapped pri- marily for their pelts, but after they are skinned, the additional labor of preparing the meat for mar- ket is so slight that they can be sold very cheaply. "In the Baltimore markets, February 21, 1908, I found muskrats for sale at various stalls. The retail price was 10 cents each. At the commission houses I learned that several firms receive them regularly from the lower Chesapeake. . . . p "In February, 1907, the Philadelphia Record stated that a single dealer on Dock street in that city sold about 3,000 muskrats a week for food. The chief PROFIT FROM THE MUSKRAT 81 source of this supply was stated to be in the vicinity of Salem, N. J. PROFIT FROM THE MUSKRAT The Saginaw (Mich.) Courier-Her- ald states that in the season of 1907-8 dressed musk- rats at that place retailed at from 15 to 20 cents each, and that dealers had ready sale for all they could provide. Muskrat is said to be a favorite dish at dinners given by church societies in Delaware and Maryland, and annual muskrat banquets are a fea- ture with certain gun clubs in the West. Those of the Monroe (Mich.) Marsh Club have been celebrated for many years. Nearly a dozen years ago, when the club desired the enactment of a law to protect the muskrat on the marshes adjoining the Great Lakes, they went to Lansing, taking with them their own chefs, and invited the entire Legislature to partake of their hospitality. . . . The law was passed without opposition. . . . pp "The flesh of the muskrat is dark red in color, but fine-grained and tender. Unfavorable opinions as to its flavor arise, probably, from lack of skill in cook- ing or from carelessness in skinning the animal. In the usual method of skinning, the hair-side of the pelts does not come in contact with the flesh, the musk-glands often come off with the skin, and only in summer does the musky odor pervade the flesh. An unskilled person is more likely to leave some of the odor, but in winter it may all be removed by washing [as, also, the gamey flavor, when too strong for one's taste, by soaking in salt water.] The novice should be careful to keep the fur from touching the flesh, to avoid cutting into the musk-glands, and to ANIMAL COMPETITORS 82 trim off any subcutaneous glands that may adhere to the meat." The following published recipes for cooking muskrat are credited to George T. Bowen, a caterer of Baltimore, Md. : "Fried Muskrat. Wash the meat thoroughly and cut in quarters. Let it lie in salt water for an hour or more, then wash, dry with a cloth, and season. Dip the pieces in a prepared egg-batter and dust them with flour or meal. Place the lard in a frying-pan and let it get hot. Then put in the muskrat and fry very slowly for an hour. Prepare a gravy of milk, butter, flour, and parsley, and season it to taste. After it thickens pour it over the cooked muskrat. p "Roast Muskrat. PROFIT FROM THE MUSKRAT Wash the meat thoroughly, let it lie for an hour or more in salt water, and then wash again. Put it in a pan with water, salt, pepper, butter, and a little onion; sprinkle flour over it, and baste it until it is thoroughly done. g y "Stewed Muskrat. Wash the meat thoroughly, cut it in pieces, and let it lie in salt water for an hour. Then wash again, put it in a saucepan, and season with butter, salt, and pepper to taste. Let it simmer slowly, and when nearly done put parsley and a lit- tle chopped onion into it. When entirely dore thicken with a gravy of flour and water, as for stewed chicken." Steady demand for muskrat fur. It is, how- ever, for its pelt that the mu.skrat is chiefly PROFIT FROM THE MUSKKAT 83 valued, and should be preserved and cultivated under properly restrictive conditions. Com- pared with most other furs of small size, musk- rat furs are of excellent quality and durability ; their cheapness is chiefly the result of their abundance. Properly dyed and made up, they are difficult to distinguish from sealskin, but their wearing qualities are greatly inferior. The modern dresser and dyer have found means of imitating nearly all the more costly furs with that of this animal, and have thus created a continuous demand for the pelts. p Notwithstanding that during the past 150 years nearly 250 millions of muskrats have been trapped, vast numbers of these pelts reach market annually. The sales at the great Lon- don auctions (which determine prices for the world) for 1909 were 3,771,000, at higher prices than at any time previously. Many fur-buying establishments advertise most alluringly, in order to induce consignments from local deal- ers, or from individual trappers; but in many cases they grade the furs so low that the re- turns are far below expectation. It is prob- ably better policy, as a rule, for the amateur ANIMAL COMPETITORS 84 rural trapper to sell his pelts to a local buyer whom he knows and trusts than to ship them to a distant dealer. In any event he ought to un- derstand the points of quality by which his furs are graded, and keep himself informed as to current prices. p Trapping the muskrat. Muskrats are not suspicious and are easily trapped. PROFIT FROM THE MUSKRAT They take any suitable bait readily, especially in winter and early spring, when green food is scarce. A strong steel trap breaks the leg bone and in struggling the animal is apt to tear loose, leav- ing a foot, or part of it, in the trap. For this reason the traps should be set so that the cap- tives will quickly drown. The best baits are carrots, sweet apples, parsnips, turnips, or pieces of squash. Many trappers use scent to attract the animals, but the practice is of doubt- ful utility. y Most muskrat trappers use the ordinary steel trap (No. 1). The manner of setting it depends upon the situation, and the skill of the trapper is best displayed in selecting this. Muskrat trails may be found along the banks of all streams and ponds which they inhabit, and the PROFIT FROM THE MUSKRAT 85 practiced eye can often trace them into shallow water. Sink the trap in the trail, partly in the mud or sand where the water is two or three inches deep, and fasten the chain to a stake, or, better still, to a slender pole, reaching into deep water. Fasten the bait to a stick set in the mud, so that the bait is about a foot above the pan of the trap. The animal in reaching for the bait sets the hind foot upon the pan and is caught more securely than if taken by the fore foot. It immediately plunges into deep water, sliding the chain along the pole as far as it will go, and soon drowns. If the chain is fastened to a stake, it should be planted in water a foot or more in depth, so that the animal will drown. p Besides this water-set for the steel trap, other situations will suggest themselves to the intelligent trapper. One of the best is in the opening of the animal's burrow in the bank. Here no bait is required. Sometimes a spade is needed to cut out a piece of turf and make room for the trap, the top of which should be at least two inches under water. When ponds are frozen over, traps are often set in the muskrat houses, the trapper going ANIMAL COMPETITORS 86 to them on the ice; but this practice destroys the houses and is not to be commended. "The box trap is a favorite with some trappers. They use a long wooden box whose cross section inside is about 6 by 6 inches and which has a gate at each end. The gates are of wire and arranged to swing in- ward but not outward. The box is set just under water with one end at the entrance to a muskrat burrow. PROFIT FROM THE MUSKRAT Trap- ping near the houses in open water is far better. When the houses are not far from the bank, a long plank may be used advantageously as a support for traps. It is moored to the shore by a wire passed through a staple driven into one end of the plank, while the other end projects into the pond or rests against the side of the muskrat house. Light cleats are nailed to the upper side of the plank at intervals of a foot with space enough between them to hold a trap when set. The ring at the end of each trap chain is fastened to the plank by a staple. Baits of carrot or apple may be scattered along the plank; but they are not necessary, since the animals will use such a plank as a highway to the shore, and are almost sure to be caught. Most of the occupants of a house may some- times be taken on one plank in a single night. PROFIT FROM THE MUSKRAT 87 The animal lifts the gate on leaving the burrow and is imprisoned and drowned. Others follow until per- haps all the occupants of the burrow are caught. A similar trap may be made entirely of heavy wire net- ting of half-inch mesh, bent to shape. These traps are well adapted to very narrow streams or ditches favorite runways of the animals. y "An open barrel sunk near the bank of the stream or pond frequented by muskrats is said to be an ef- fective trap. The top of the barrel should be level with the surface of the ground. The barrel is half full of water, upon which pieces of carrot or apple are floating. A piece of board about 8 inches square, or a few floating chips, will delude the animals into jumping into the barrel to secure the food. Musk- rats taken alive should be killed by a sharp blow across the back of the head. "A floating barrel is said to be a good substitute for a sunken barrel. A hole 8 to 12 inches square is sawed in the side of a barrel having both ends in- tact. A strong cleat is nailed across each end, pro- jecting 6 or 8 inches on the sides. Upon the projecting cleats boards as long or somewhat longer than the barrel are nailed. PROFIT FROM THE MUSKRAT Enough water is placed in the barrel to make it float with the outer platform level with the surface of the pond say, with about one-third of the surface of the barrel exposed. p "Another way of taking the muskrat is to spear it inside its winter house. This is a common Indian method; but it should not be encouraged. Not only are the pelts injured by the spear, but when the ANIMAL COMPETITORS 88 ponds are ice-bound, the animals that escape the spear often perish after their houses are destroyed." Trapping was at one time a popular calling in the United States; but fur-bearing animals have so decreased in numbers that nowadays few persons earn a livelihood by trapping alone. A large part of the supply of muskrat fur is taken by boys, who adopt this method of earn- ing a little extra spending money. They often attend school, and look after the traps in the morning and evening. g g Preparation of the pelt. Muskrat skins in- tended for market should be "cased," not opened along the belly. In skinning begin at the heel and slit up the middle of one hind leg to the tail, around it, and then down the other leg to the heel. The skin may then be easily (but gently) turned back over the body, leaving the fur side inward. Next, cut closely and cautiously around ears, nose and lips, and scrape off adhering bits of flesh. The skin, inside out, is then stretched over a thin board shaped like a rifle-cartridge, and a tack or two is inserted to keep it in position until dry, the drying should be in the open air, not before PROFIT FEOM THE MUSKRAT 89 a fire or in the sun, and not exposed to rain. Formerly many of these skins were dressed at home, but the process is complicated and diffi- cult. Cultivation of muskrats. In view of the real value, continuous demand, growing difficulty of obtaining prime pelts and consequent steady enhancement of price, it has occurred to many persons that the rearing of muskrats in pro- tected marshes and under favoring conditions would be profitable. Some of the experiments already tried in this direction have succeeded well, but they are in reality little jnore than a cooperative protection of certain extensive haunts against over-trapping as well as against poaching. PROFIT FROM THE MUSKRAT Large tracts of marshes at the western end of Lake Erie, controlled by sporting clubs, are thus governed and yield a substantial revenue under wise management. g In this way a new value has been given to extensive areas of marsh, liable to tidal over- flow, along the western margin of Chesapeake Bay, where landowners now lease the trapping privilege, and trappers and owners unite to protect the marshes from poaching. The owner ANIMAL COMPETITORS 90 receives half the fur caught, while the trapper gets the other half and all he can realize from the sale of the meat. In the short season of seventy-four days, January 1 to March 15, dur- ing 1908 and 1909, trappers easily made from $400 to $900 each. The demand for the meat is growing, and all of it is utilized. The Baltimore market takes about 30,000 animals during a season, the bulk of which come from Dorchester County, Va. y, The editor of the Cambridge Record, a local newspaper, stated (1909) that the muskrat in- dustry of Porchester brings into the county about $100,000 annually. This would indicate that about a quarter-million of the animals are trapped each season. The danger of exhaust- ing the supply by continued close trapping has been discussed in Dorchester County, but trap- pers there maintain that with the long closed season, March 15 to January 1, little ground for anxiety on this score exists. However, it is worth keeping in mind. p g Possibilities of this business. There are many places in all the eastern half of the United States where a similar industry might PROFIT FROM THE MUSKRAT 91 be developed, even though on a smaller scale; and it is a very suitable investment and occupa- tion for men and boys who might organize small local companies to carry it on effectively. As it is winter work, little time useful for any- thing else would be needed. The chief require- ments are protection for the animals during a close season, and from poaching in winter ; and a suitable limitation of the number taken, based upon local circumstances. p In their natural haunts no feeding is re- quired ; and it may often be advisable in places to enlarge the area suitable to the animals by damming the outlet so as to flood a wider area. PROFIT FROM THE MUSKRAT The rats increase rapidly when encouraged, though the varying testimony on the subject seems to show that they differ in this respect in different parts of the country. Seton says that in Manitoba there are commonly said to be three litters a year, of four to nine each, and that the first litter of the year themselves have young in the autumn. The period of gestation is about 30 days. y Suitable places and proper care. There are few swamps and marshy streams in which ANIMAL COMPETITORS 92 these rodents are not naturally numerous, and they persist in the midst of civilized districts in a marvelous way. There would seem to be no difficulty then in colonizing a new artificial marsh or pond for the sake of rearing them. Such a place, however, must have a natural or planted growth of suitable food-plants lilies, arums, sedges, etc. whose roots form their winter subsistence. Among the principal of these plants are yellow and white pond-lilies, the golden clubhead or river-bulrush, a large sedge whose fleshy tubers are nutritious, and the lotus (Nelumbo). In summer they feed upon a far more extensive list of aquatic plants and shore grasses, vegetables and fruits, and also largely on mussels, snails, crayfish, caddis- worms, sluggish fish, like carp, and now and then catch ducks and other small animals. The feeding-habits of the wild muskrats of the locality should be carefully considered. y y The highest usefulness of a "muskrat-farm" would be gained, however, by improving the stock in size and color. To do this selective breeding should be attempted. The larger and blacker the pelt the higher price it will bring. PROFIT FROM THE MUSKRAT 93 It would be well, then, to trap as many of the year's crop as possible alive, and to put back any notably large or very black ones to act as breeders, while the small and light-colored examples were steadily weeded out. In this way only a few years would be required, in a restricted community, to produce a notable im- provement in your muskrats. It would be well, then, to trap as many of the year's crop as possible alive, and to put back any notably large or very black ones to act as breeders, while the small and light-colored examples were steadily weeded out. PROFIT FROM THE MUSKRAT In this way only a few years would be required, in a restricted community, to produce a notable im- provement in your muskrats. p y As little expense or trouble is required, muskrat-farming ought to be a very profitable enterprise in many places. CAN THE BEAVER BE SAVED? HEEE, too, may be considered the beaver, with reference to the possibility of preserving it from extinction, and cultivating it for fur. In most of our states and provinces this animal is more or less under legal protection, and scattered colonies flourish throughout the moun- tainous parts of the West, while several zoolog- ical parks and some private estates have colonies. These thrive, and increase so fast that from time to time it is necessary to thin out the band. A newspaper reports that about 100 were in this way culled out of the colony in Algonkin Park, a national reserve in north- ern Ontario, during 1909. g It would seem entirely feasible, then, for any- one having a favorable place on his estate to rear beavers. A swampy valley is usually of little usefulness otherwise. The cost of con- fining and protecting the colony would, how- 94 CAN THE "BEAVER BE SAVED? 95 A BEAVER-DAM NEAR LAKE TEMISCAMING, ONTARIO. From a Photograph by George S. Bryan. ASKATCHEWAN A BEAVER-DAM NEAR LAKE TEMISCAMING, ONTARIO. From a Photograph by George S. Bryan. A BEAVER-DAM NEAR LAKE TEMISCAMING, ONTARIO. From a Photograph by George S. Brya ASKATCHEWAN ASKATCHEWAN ANIMAL COMPETITORS 96 ever, be considerable in most situations. A strong iron or wire fence which would resist their jaws, and which would also keep out bad dogs, would be a large item if it enclosed an area spacious enough for extensive operations. Only a short time would elapse before the beavers had cut down and used up all the trees and bushes which were not jacketed with stout wire higher than they could reach; and after that it would be needful to feed them with fresh tree-limbs of suitable kinds. Lastly, if the colony amounted to anything it would doubtless be necessary in most places to guard it well against human marauders who would kill the animals for their valuable pelts. It is to be noted that these animals will not eat the bark of evergreen (coniferous) trees of any kind. ever, be considerable in most situations. A strong iron or wire fence which would resist their jaws, and which would also keep out bad dogs, would be a large item if it enclosed an area spacious enough for extensive operations. "They are singular," he says, "with respect to their ingenuity and great labor in the construction CAN THE BEAVER BE SAVED? Only a short time would elapse before the beavers had cut down and used up all the trees and bushes which were not jacketed with stout wire higher than they could reach; and after that it would be needful to feed them with fresh tree-limbs of suitable kinds. Lastly, if the colony amounted to anything it would doubtless be necessary in most places to guard it well against human marauders who would kill the animals for their valuable pelts. It is to be noted that these animals will not eat the bark of evergreen (coniferous) trees of any kind. g y It is proper also to add a caution quoted from Vernon Bailey's notes on Texas mammals (N. A. Fauna, No. 25.) "In talking with John Seavel, an old beaver trap- per, I asked him why it would not pay to protect the beaver in a pond like that above Pecos bridge (over the Rio Grande), and let them multiply. The idea was not new to him, for he had talked it over with other trappers and all agreed that it was not worth CAN THE BEAVER BE SAVED? 97 trying because they considered the beaver naturally ferocious, to a great extent solitary and a slow breeder. Seavel says that two old beavers rarely live together in one house or even in one small pond ; that they fight and chase away any newcomers; that if a family grows up and is undisturbed in a pond or a deep bend of the river, its members keep all others of the species away, and that they attack and kill any one of their number that is found in a trap or sick or crippled. While he thinks that systematic breeding for fur is out of the question, he admits that the beaver should be protected all over the country until the few that remain increase and restock the streams." If this is generally true, it may be found that the most profitable course for a beaver-culti- vator is to acquire control of a stream already tenanted by beaver, and guard them there in their natural life, taking only a proper propor- tion each year. CHAPTER VI WOOD-EATS, PACK-EATS, COTTON- EATS, ETC. IN the South and West are to be found a great number of species of rodents called wood- rats (Neotoma) which are pretty, interesting and amusing rather than harmful in their mag- pie-like mischief. They are rat-like in form, with long, scantily-haired tails, but squirrel-like in agility and climbing power; and more in- clined to go abroad in the dusk of evening and morning than during the brighter hours of the day. Florida and Texas species. One species is well known in wooded country from New Jersey southward, and is 13 inches long, including a 5-inch tail, brownish gray, the sides tawny, the belly and feet all white. Bartram described the animal and its home, as he met with them in Florida almost 150 years ago. "They are singular," he says, "with respect to their ingenuity and great labor in the construction 98 WOOD-EATS, PACK-RATS, ETC. 99 of their habitations, which are* conical pyramids about three feet high, constructed with dry branches, which they collect with great labor and perseverance, and pile up without any apparent order; yet they are so interwoven with one another, that it would take a bear or wildcat some time to pull one of these castles to pieces." p "The very playful character of this species," re- marked Audubon, "its cleanly habits, its mild, promi- nent and bright eyes ; together with its fine form and easy susceptibility of domestication, would render it a far more interesting pet than many others." West and southwest of the Plains live many closely related species, some of which inhabit the mountains, others only timbered valleys, while still others are restricted to desert val- leys. All erect more or less elaborate and often conspicuous houses, sometimes in the branches of trees a safer place than on the ground. g Vernon Bailey discusses upon them thus : ' ' For houses they heap up a bushel or more of sticks, stones, cow-chips, cactus, bones, or other materials which the animals can carry and pile up as a protect- ing cover for their nests and burrows in the ground be- neath. Cactus and thorny branches, if available, are always a conspicuous part of the building material. The house is usually occupied by one old rat, a pair, or ANIMAL COMPETITORS 100 a family, but never -by a colony. CHAPTER VI Wood-rats are social and visit back and forth from one house to another until well-worn trails often connect the houses and lead to the feeding grounds. The food of these ani- mals is mainly seeds, berries, and many kinds of green foliage. Where the houses are located near the edges of fields, grain, fruits, and vegetables are sometimes FIELD-NEST OF BAILEY'S WOOD-RAT. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by H. W. Nash. FIELD-NEST OF BAILEY'S WOOD-RAT. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by H. W. Nash. eaten or carried away and stored up for food, but fortunately the rats are never sufficiently numerous to do serious damage. Their houses are easily de- stroyed and the occupants captured by a few min- utes' w rork with a shovel, or the rats can readily be trapped or poisoned. They frequently enter cabins WOOD-RATS, PACK-RATS, ETC. 101 or camps not permanently occupied and eat or carry away provisions. They sometimes cause great annoy- ance by cutting leather harnesses or saddles. There is rarely more than one animal responsible for the mischief in a camp, however, and a rat-trap will usually prevent further trouble. It is unfortunate that the odious name of rat has become attached to these bright and interesting little animals, as other- wise they might become a table delicacy. They are cleanly in habits and are strictly vegetarian in diet. Their flesh is as white and delicate as that of the quail and finer in flavor than that of the squirrel or rabbit. ' ' The mountain pack-rat. The best-known of these wood-rats is that yellowish-gray one of the Rocky Mountain region, with the very bushy tail, known as pack-rat, trade-rat or bush-rat (Neotoma cinerea), and its reputa- tion is an evil one. As Warren remarks: "While the warm weather lasts (in Colorado), they do not trouble habitations very much, but when in the mountains the weather begins to get colder the rat looks out for a warm place for his winter residence, and often selects the miner's cabin or some ranch-house." It soon makes its presence known by carrying away any portable articles, and it makes no difference ANIMAL COMPETITORS 102 whether they are of any use to the animal or not. It has been known to carry off even sticks of dynamite. CHAPTER VI Its nests are frequently lined by shredding gunny-sacking or clothing. Rolled or folded blankets have been completely riddled by them in their search for stuff to make their beds, which often are placed in queer situations. A correspondent in British Columbia tells of one which inhabited a letter-box nailed against a tree in a lonely locality. "I visited him -often/' he writes, "and on opening the door, his head, with its big round eyes and great round ears, would appear out of his warm bed with an expression of inquiry, but with no sign of fear. He had brought everything in through a knot hole, apparently too small to admit even his body. Before his nest was stored a pile of Oregon grapes and green leaves, but I could not discover that he ate any of them, although occupying the box for some weeks. ' ' On the Pacific coast they are fond of estab- lishing themselves in the sod roofs of log cabins or used to be, when such structures were more common than nowadays and become a nuisance. A capable thief. The following account, WOOD-RATS, PACK-RATS, ETC. 103 WOOD-RATS, PACK-RATS, ETC. 103 103 communicated in 1877 to The American Journal of Science by A. W. Chase, shows what the an- imal is capable of in the way of mischief. The tale relates to a dwelling-house near a disused sawmill in Oregon: "This house was left uninhabited for two years, and, being at some distance from the little settlement, it was frequently broken into by tramps who sought a shelter for the night. When I entered this house I was astonished to see an immense rat's nest on the empty stove. On examining this nest, which was about five feet in height, and occupied the whole top of the stove (a large range), I found the outside to be composed entirely of spikes, all laid with sym- metry, so as to present the points of the nails out- ward. In the center of this mass was the nest, com- posed of finely divided fibers of the hemp packing. Interlaced with the spikes we found the following: About three dozen knives, forks and spoons, all the butcher knives, three in number, a large carving knife, fork and steel, several large plugs of tobacco; the outer casing of a silver watch was disposed in one part of the pile, the glass of the same watch in another, and the works in still another; an old purse containing some silver, matches and tobacco; nearly all the small tools from the tool closets, among them several large augers. Altogether it was a very curi- ous mixture of different articles, all of which must have been transported some distance, as they were originally stored in different parts of the house. ANIMAL COMPETITORS 104 "The ingenuity and skill displayed in the construc- tion of this nest; and the curious taste for articles of iron, many of them heavy, for component parts, struck me with surprise. The articles of value were, A ROCKY-MOUNTAIN PACK-RAT. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by II. W. Nash. A ROCKY-MOUNTAIN PACK-RAT. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by II. W. Nash. I think, stolen from the men who had broken into the house for temporary lodging. I have preserved a sketch of this iron-clad nest, which I think unique in natural history. "Many curious facts have since been related to me WOOD-RATS, PACK-RATS, ETC. WOOD-RATS, PACK-RATS, ETC. 103 105 concerning the habits of this little creature. A miner told me the following: He once, during the mining excitement in Siskiyou County, became, in California parlance, 'dead broke,' and applied for and obtained employment in a mining camp, where the owner's hands and all slept in the same cabin. Shortly after his arrival small articles commenced to disappear; if a whole plug of tobacco were left on the table it would be gone in the morning. Finally a bag, containing one hundred dollars or more in gold dust, was taken from a small table at the head of a bunk in which one of the proprietors of the claim slept. Suspicion fell on the newcomer, and he would perhaps have fared hardly, for with those rough miners punish- ment is short and sharp ; but just in time a large rat's nest was discovered in the garret of the cabin, and in it was found the missing money, as well as the tobacco and other articles supposed to have been stolen." The destructive cotton-rat. It would be pos- sible to write extensively, and perhaps enter- tainingly of a long list of other wild mice and rats, .such as our very pretty, white-footed wood-mouse (Peromyscus) which, in its vari- ous species and subspecies, is scattered all over the continent; but few of them have sufficient economic interest to justify it. There are times when any or all may become dangerous by ANIMAL COMPETITORS 106 overmultiplication ; but the only sort now of importance in that direction is the southern cotton-rat (genus Sigmodon.) The common species (S. hispidum) is typically a denizen of the Atlantic coast from North Carolina to Florida, but its varieties extend the specific range westward to Mexico. The total length is 10-101/4 inches, two-fifths of which belongs to the tail. The color varies a good deal, but in general is a yellowish grizzle above, and ashy to whitish below. y Their natural habits much resemble those of their neighbors, the pine mice, and like them they have not only surface runways but long galleries and nesting-places under the soil. They may be very numerous without attracting much attention because of this cryptic manner of life, and still more because they rarely come abroad until after dark. WOOD-RATS, PACK-RATS, ETC. 103 There have been times, as in 1889 in western-central Texas, when they swarmed in a regular "plague," and played havoc with the corn-crop. They are especially numerous, always, along the borders of cotton-fields, and Bailey records that in Texas their runways are often fairly lined with WOOD-RATS, PACK-EATS, ETC. 107 cotton that has been pulled from the bolls and dragged under cover where its seeds can be eaten with safety, while a small amount is car- ried away for bedding. Considering the great area of the cotton-growing country, all infested with these busy pilferers, the aggregate loss of cotton must represent a large sum. "A simple and effective remedy," as Bailey reminds the planter, "would be to clean out the borders of fields by burning the weeds, grass and rubbish accumulating along the fences year after year as a harbor for various rodent and insect pests and a perennial source of sup- ply for weed-seeds. If these borders were burned yearly, mowed and raked, treated with oil or chemicals to prevent weed-growth, closely pastured or thoroughly cultivated, the hawks and owls would quickly dispose of the rodents which would then have no protecting cover. ' ' Jumping-mice, Allied to the true or murine mice, and even more nearly to the Old World jerboas, are the jumping-mice, pocket-mice and kangaroo-rats of the family Zapodidce, all in- teresting and beautiful little animals but not requiring much attention here, because their ANIMAL COMPETITORS 108 presence affects humanity in a very trifling de- gree. The only representative in the East is the common tawny-red, large-eared jumping-mouse, often seen rushing away from under foot in amazing leaps. The hind legs and feet are tremendously developed, while the fore pair are exceedingly small and delicate. The nose is pointed, and the hairless tail very long, meas- uring 5 inches from root to tip, while the length of the body is only 3 inches. They sub- sist almost exclusively on weed-seeds, and go early to bed in warm grass-nests underground, where they remain in deep hibernation until late in the spring. In the arid West and in Mexico live numer- ous small cousins of the genus Perognathus, whose hind legs are less lengthened, and which have fur-lined pockets in their cheeks, tiny ears, gray or yellowish coats, pure white feet and under parts, and tails about the length of their bodies. WOOD-RATS, PACK-RATS, ETC. 103 They are rarely seen, because nocturnal; burrow in dry ground; and lay up stores of small seeds. They are easily caught in small traps baited with rolled oats (of which WOOD-RATS, PACK-EATS, ETC. 109 all mice are extremely fond) ; and "unless as the result of a great reduction of mouse-hunt- ing birds and mammals, they will never be a pest." They are styled kangaroo-mice, or elf- mice. Western kangaroo-rats. A third group is that of the kangaroo-rats, found in all the arid valleys west of the Plains and south of northern Idaho and the Sacramento river. While not Qven remotely related to either kan- garoos or rats, they have been thus named on account of their long hind legs and tails, small hands, and their method of progressing by hops. One species, the desert kangaroo-rat, (Dipodomys) is distinguished by its large size, about twice that of the others (Perodipus). This large four-toed kangaroo-rat of the Great Basin and southern California is the one of most interest. It is about 5 inches from tip of nose to base of tail, and the tail is about 8 inches long. Its legs and hind feet are dispro- portionately long, in striking contrast to the tiny front feet, or hands. The large head, prominent black eyes, and short ears give the animal a quaint appearance. The glossy coat ANIMAL COMPETITORS 110 is light sand-color over the upper parts and pure white below. The tail is white along the sides and for an inch at the tip. The fur-lined cheek-pouches are large enough to admit the tip of the little finger. These, and the smaller kangaroo-rats, are common over the drier parts of the valley coun- try, especially in the mellowest and sandiest soil; and Ernest Thompson Seton has made them the subject of one of his most accurate and charming descriptions of animal life. They are strictly nocturnal and are rarely seen alive, but their round burrows are conspicuous, and the paired tracks of their long hind feet may be seen every morning on the naked sands. The manner of traveling is by hops, or long leaps on the hind feet, while the tail serves as a balance and rudder, the tiny front paws being used only as hands. The burrows usually en- ter the side of a sandy hillock, dune, or embank- ment, and often extend 10 or 20 feet. WOOD-RATS, PACK-RATS, ETC. 103 They do not go deep into the ground, but if started at the base of an embankment they may penetrate through it below the water-level and tap the ditches, mischief wholly accidental. Usually WOOD-EATS, PACK-BATS, ETC. I Ill on entering the burrow before daylight in the morning they securely close the opening behind them by packing it full of fresh sand, doubtless to keep out snakes, weasels, and other unwel- come intruders. Their food consists mainly of the small seeds of native desert plants, but also includes a little grain. A part of the food is carried into their burrows for future use. They are never suffi- ciently abundant to seriously injure crops, but a year or two ago were found damaging vine- yards in the Santa Cruz Mountains, California, by biting off fruit-buds. The trouble was easily disposed of by scattering poisoned grain near their burrows. This course will always free a locality from them whenever they may become a little harmful. THE GRAY GOPHERS THIS brings us to another group of burrow- ing and pouched rodents, which, however, are far from harmless the gophers. This term is given in the Gulf States to a burrowing turtle, and in the Northwest to a striped ground- squirrel, but those here in view are the chunked, short-legged, blunt-nosed, short-tailed ground- diggers of the family Geomyidce. gg y y They inhabit nearly the whole of the open country west of the Mississippi river not lifted into mountain ranges, and one species, locally called salamander (Geomys tuza), occupies large areas in Florida, Georgia, and Alabama. The western plains are the home of two promi- nent species, the dark "common" one (Geomys bursarius), formerly spread as far east of the Mississippi as the prairies extended and now dwelling between that river and the Rocky Mountains from near the Canadian line to the 112 THE GRAY GOPHERS 113 Gulf coast; the yellower, larger, chestnut- faced gopher (Cratogeomys castanops), occur- ring from Colorado southwestward ; and the NORTHERN GRAY GOPHER (THOMOMYS TALPOIDES). Photographed hy H. K. Jb, and reproduced by permission. NORTHERN GRAY GOPHER (THOMOMYS TALPOIDES). Photographed hy H. K. Jb, and reproduced by permission. smaller northern gray gopher, or mole-go- pher (Thomomys talpoides), which is the fa- miliar of the Dakotas, Wyoming and the Cana- ANIMAL COMPETITORS 114 dian plains. West of the Eockies occur a large number of species, some very difficult to dis- tinguish from one another, of which the reddish Thomomys douglasi of the Columbia River valley, and T. bottce (chestnut above, reddish brown below) of central and southern Cali- fornia, are most notable. In many regions they have increased rather than diminished with civilization, owing to the destruction of their natural enemies, to the loosening of the soil by plowing and to the vast increase of food afforded them by orchards, gardens and crops. Though several genera and species are sepa- rated by zoologists, from the farmer's point of view there is a substantial likeness, not only in their yellowish-gray or brownish, unmarked coats ; their big, thick heads ; their short strong legs; their almost invisible ears and eyes; the massive incisors and capacious furry cheek- pouches, opening outside the mouth; but in the constant and fearful damage they work in the field, orchard and nursery. Burroiving powers The pocket-gopher digs , y Burroiving powers. THE GRAY GOPHERS The pocket-gopher digs as long as he lives, and generally all winter, for he does not hibernate, even at the coldest. All THE GRAY GOPHERS 115 his life is passed underground, except when for an instant he emerges into the air to push a load of earth from a freshly opened hole. Ex- cept for one month of the year, the mating sea- son, all pocket-gophers live an entirely solitary life ; and like most other hermits, they are of an extremely surly disposition. They will fight viciously on all occasions, and they have for- midable weapons. In tunneling in the earth, they use their long and powerful front teeth as a pick to loosen the ground. At the same time the forefeet, which are armed with long curved claws, the sides of the toes being lined in turn with bristles which prevent the dirt from passing between them, are hard at work both in digging and in pressing the dirt back under the body. There the hind feet take it and push it farther back. When earth enough has been accumulated be- hind the gopher, he whirls about, and by bring- ing his wrists together under the chin, with the palms of the hands held vertically, he pushes the earth out in front. He will move backward as rapidly as forward, and can push dirt either ANIMAL COMPETITORS 116 way. His movement in digging often seems as rapid and automatic as that of a shuttle. Except in times of deep frost, the burrows are seldom more than a foot underground, and generally about six inches. At intervals, often within a few feet, the gopher comes to the sur- face to throw up a little hill of dirt; but the opening which he makes is closed by being DIAGRAM OF A GOPHER'S BURROWING. packed so full of dirt that no trace of the tunnel is visible except the little mound. The gopher goes on digging in winter as well as in summer ; but if the frost prevents him from coming to the surface, he uses a cross section of his tunnel into which to pack the earth which he has dug for his new excavations. These tightly packed cylinders of earth are often turned up by the plow. THE GRAY GOPHERS p y p Pocket-gophers apparently breed only once a THE GRAY GOPHERS 117 year, in the spring, when two to six young are produced in a litter in some roomy central chamber made comfortable with dry grass. chamber made comfortable with dry grass. Destructive to crops. "Throughout their range pocket-gophers are very destructive to crops. They eat the roots of fruit trees and in this way sometimes ruin whole orchards. They eat both roots and tops of clover, alfalfa, grasses, grains, and vegetables, and are espe- cially harmful to potatoes and other tuberous crops. Besides this, they throw up innumer- able mounds of earth in meadows, pastures, and grain fields, which cover and destroy far more of the crop than is eaten by the animals or killed by having the roots cut off. These mounds also prevent close mowing, so that much of the hay crop is lost, and the pebbles they contain often break or injure farm ma- chinery. The loss due to gopher mounds in the clover and alfalfa fields in some of the Western States has been conservatively estimated at one-tenth of the entire crop. In many of the fertile valleys where they abound the animals -are by far the most formidable of the farmer's mammalian enemies. In addition to all this, dry grass. Destructive to crops. "Throughout their range pocket-gophers are very destructive to crops. They eat the roots of fruit trees and in this way sometimes ruin whole orchards. They eat both roots and tops of clover, alfalfa, grasses, grains, and vegetables, and are espe- cially harmful to potatoes and other tuberous crops. Besides this, they throw up innumer- able mounds of earth in meadows, pastures, and grain fields, which cover and destroy far more of the crop than is eaten by the animals or killed by having the roots cut off. These mounds also prevent close mowing, so that much of the hay crop is lost, and the pebbles they contain often break or injure farm ma- chinery. The loss due to gopher mounds in the clover and alfalfa fields in some of the Western States has been conservatively estimated at one-tenth of the entire crop. In many of the fertile valleys where they abound the animals -are by far the most formidable of the farmer's mammalian enemies. THE GRAY GOPHERS In addition to all this, ANIMAL COMPETITORS 118 in the far West they burrow in the banks of irrigation ditches and thus cause extensive breaks, the repair of which results in the ex- penditure of much time and money." (Lantz.) An enemy to orchard and forest. Re- cently, attention has been especially called to the injury done to orchards and nursery stock, often before the owner becomes aware of the presence of the animal, and it is evident that great watchfulness should be maintained by tree planters in gopher-infested country. This watchfulness should be especially alert where the orchardist, in order to prepare the soil, first raises and turns down crops of alfalfa, clover or cowpeas, sweet potatoes or sugar-beets. Any of these attract the rodents, and make their attacks more than likely upon the newly planted saplings. A gopher which in tunneling comes to a tree root attacks and eats through it. If the root is relished, it is followed and eaten close up to the tree trunk. Then another root is destroyed, and so on until the entire root system is gnawed away, wood and bark alike, leaving the trunk loose in the ground. THE GRAY GOPHERS 119 The rapidity with which the animal works is amazing. In his rare monograph on the fam- ily Merriam assures us that a pocket-gopher can make two hundred complete strokes with his teeth in a minute. Its jaws are so arranged that thirty-eight distinct single cuts are made SOUTHERN POCKET GOPHER (GEOMYS BURSARITS). From a Painting by E. T. Seton. SOUTHERN POCKET GOPHER (GEOMYS BURSARITS). From a Painting by E. T. Seton. by the forward stroke of the jaw and twenty- eight by the backward stroke. Thus, the little creature's jaws may make a grand total of 13,200 cut a minute when in active opera- tion! Large trees are sometimes entirely girdled ANIMAL COMPETITORS 120 just below the ground, the gopher cutting deep into the wood, causing immediate death. The girdling of large roots is also common. In California the fig seems to suffer most, but orange, lemon, apricot and all other fruit-trees are attacked. Complaints from western nurserymen of injury to their stock by pocket-gophers are frequent. The trees in nursery-rows are set small and close together. Consequently a go- pher by following the rows can in a short time kill many trees. THE GRAY GOPHERS Such injury is usually done in late fall or winter, and the nurseryman is often unaware of it until spring. The gopher takes the entire root, not merely the bark, cuts it into short pieces, packs them into its enor- mous cheek-pouches, and carries them away to its food-caches, which sometimes contain half a bushel of such provender. Plantations of young trees for wind-breaks or ornament, or to afforest a district, are equally hurt; in fact the gophers are worse than rabbits, because they work unseen and almost invariably kill in- stead of merely injuring the trees. Wherever they abound orchards are almost an impossi- THE GRAY GOPHERS 121 Tapping irrigation-ditches. Another most serious mischief, in regions depending on irri- gation, is the destruction of ditch-banks. Some- times the animals are forced out of irrigated land and take up new quarters in the dry ditch- banks, or in course of the regular extension of their tunnels a ditch is encountered and the bank is followed in search of a crossing-place. In either case the burrow is almost sure sooner or later to penetrate below the water-line and start a leak that cuts out the bank and empties the ditch. Altogether, it has been estimated by the Biological Survey that the loss, due to go- phers in the western United States is not less than a million dollars a month. "No animals," the Survey declares, at the same time, "are more easily controlled on a small farm or along ditches than gophers. They are readily trapped or poisoned, and once cleared out of a field others do not come in at once. Their mode of travel, which is princi- pally by extending their burrows, is of ne- cessity slow; and if occasionally caught or poisoned around the edge of fields or along ditches, they can be effectually controlled." ANIMAL COMPETITORS 122 This implies that the damage done is largely the result of neglect on the farmer's part. The gopher as a soil-maker. THE GRAY GOPHERS In view of this record of harmfulness (due, of course, simply to mankind trying to modify nature for his own ends in the path of the animal's natural way of living, so that from nature's point of view the cultivator is the aggressor and the gopher merely defending himself and living off the enemy), it is only fair to point out how the animal, throughout the history of the species, has been laying the present farmer and ranch- man under his debt. "For unknown ages," declares Dr. C. Hart Merriam, in the monograph already referred to, "the gophers have been steadily at work plowing the ground, covering deeper and deeper the vegetable matter, loosening the soil, draining the land, and slowly but surely cul- tivating and enriching it." Ernest Thompson Seton illustrates this statement very forcibly by the example of Manitoba, one of the richest soil-areas in the world where, as elsewhere in northwestern THE GRAY GOPHERS 123 Canada, there are no earthworms to act as pre- historic cultivators. The black loam there is from one to two feet thick, and is a thoroughly mixed soil of both mineral and vegetable par- ticles. There is no doubt that, in the absence of earth worms, this mixing is done by burrow- ing animals, by far the most important of which is our subject. In his great work, Life His- tories of the Northern Animals, Seton shows by text and drawings what an astonishing number of active gophers there are (or were) over every square mile of that and other regions; and the still more astonishing bulk of soil brought to the surface from deep layers day by day. He cites a district in California with an estimated average of 6,000 hills to the acre, and enough soil heaved out each summer to cover the whole with an inch of new earth ; and other similar cases elsewhere. "If the fertility of tens of millions of acres of land in the North- west, and consequently their value, has been mainly the work of moles [pocket-gophers]," declares Dr. Robert Bell, the Canadian geol- ogist, after giving proof for his thesis, " these ANIMAL COMPETITORS 124 apparently insignificant little creatures may be regarded as the most important of the native animals of the country." If Mr. THE GRAY GOPHERS Gopher could speak he would probably remind the agriculturist of this, and ask whether the delayed fees he was now exacting were too large for the service. SQUIRRELS, GOOD AND BAD A view of our tree-squirrels. The badge of the true squirrel is his plume-like tail, which, though it seems to our eyes only an elegant ornament, is to him a balancing-pole assist- ing his agile bounds from branch to branch, an umbrella by day, and a blanket when he withdraws to his hole for the night. No better type of this delightful group can be found than the red squirrel, the genius of the American woods. He is exceedingly common, not at all shy, and recognized by almost everybody, yet few persons know really much about him. There is practically only one species on the continent, but local varieties differ much in size and colors, Those in the South are larger and redder, for example than those of Canada ; and on the Pacific coast, where they are called pine-squirrels, their coats are almost brown. Their colors vary also with the seasons, the 125 ANIMAL COMPETITORS 126 winter coat being paler and lacking the black side-stripe which so handsomely borders in summer the rufous mantle of the back. Red squirrels at home. The red squirrel's home is properly in some hollow of a tree or A NORTHWESTERN RED SQUIRREL. From a Photograph by James A. Donaghy, Elphinstone, Manitoba. A NORTHWESTERN RED SQUIRREL. From a Photograph by James A. Donaghy, Elphinst stump, sometimes low down and hence danger- ously exposed to foxes, weasels and snakes. Frequently he chooses to live in a hole beneath tree-roots or some old stone wall, adapting to his purpose an abandoned chipmunk burrow stump, sometimes low down and hence danger- ously exposed to foxes, weasels and snakes. Frequently he chooses to live in a hole beneath tree-roots or some old stone wall, adapting to his purpose an abandoned chipmunk burrow SQUIRRELS, GOOD AND BAD 127 or the hollow left by a rotting root, and extend- ing it into various connecting chambers. In the evergreen forests of northern New Eng- land and Canada, however, he often constructs a winter nest among the dense foliage of a spruce or cedar, which is a marvel of work- manship. SQUIRRELS, GOOD AND BAD "When convenient," to quote Cram's account of those familiar to him, "he chooses the nest of some large bird for a foun- dation, and in this builds a structure of moss, bark, pine-needles, and dead leaves, with walls several inches in thickness, and a soft nest of dry grass and feathers inside. The bark used is of two sorts, the rough outer bark of dif- ferent trees broken into small pieces, and what appears to be the inner bark of the red cedar, torn into narrow strips or ribbons to bind the whole together. It is put together with re- markable solidity, and usually freezes hard early in the winter, furnishing a thorough de- fense against the cold or any other enemy from without. The narrow opening at one side is provided with a hanging curtain. ' ' p g g These tree-houses are, however, abandoned in the spring, when they become soaked with ANIMAL COMPETITORS 128 rain, and a hollow tree is sought and furnished with clean bedding of moss, lichens, etc. Here the young are born rather early in the season, five or six of them, and there they remain together until fully grown. "The young squirrels," to quote again Mr. Cram's delightful history, "are most absurd looking little beasts at first, like miniature pug-dogs, blind and naked, with enormous heads. In a few days their fur begins to show like the down on a peach, and as a fringe of short hair along each side of the tail, which at length assumes something of the flattened aspect of that worn by their elders, but without displaying much of the fluffy, shadowy quality of the ideal squir- rel tail until late in the following autumn, . . . Although they do not remain long in the nest, they are seldom seen abroad until fully grown, or very nearly so, at least, which is rather remarkable when you come to consider the number that are brought Tip each summer in every pine grove or thicket where these squirrels are abundant. , . ." "The young squirrels," to quote again Mr. Cram's delightful history, "are most absurd looking little beasts at first, like miniature pug-dogs, blind and naked, with enormous heads. SQUIRRELS, GOOD AND BAD In a few days their fur begins to show like the down on a peach, and as a fringe of short hair along each side of the tail, which at length assumes something of the flattened aspect of that worn by their elders, but without displaying much of the fluffy, shadowy quality of the ideal squir- rel tail until late in the following autumn, . . . g Although they do not remain long in the nest, they are seldom seen abroad until fully grown, or very nearly so, at least, which is rather remarkable when you come to consider the number that are brought Tip each summer in every pine grove or thicket where these squirrels are abundant. , . ." How Ck red squirrel fares< The red squir- rel eats almost anything he c$n lay his teeth to, but his chief diet, of course, consists of berries, nuts, a.corns and similar hard fruits, especially the seeds found in the cones of ever- green trees rtlie mainstay of those living in SQUIRRELS, GOOD AND BAD 129 the far northern woods. In the early spring he must often content himself with buds, pre- ferring those of the maple and elm ; and it is a pretty sight to see him and his friends dangling from the tips of the swaying branches, peril- ously high, reaching for the bursting buds. In March he taps the maples for sap, cutting out little cups in the bark, in which the sugary liquid gathers and is lapped up, for he drinks like a cat. He climbs rotten stubs and, like the woodpecker, listens for noise made by in- sect larvaB, which are quickly dug out. He searches for haws of the rose and thorn-trees, and hunts through the orchard for old apples now thawed soft. A little later, I am sorry to say, he is on the lookout for birds' eggs and young, of which he destroys far more than any other squirrel. No nest is safe from his inquisitive eye and eager appetite, even the Baltimore oriole's, but he is often driven away by the owners. Nest- lings are more to his taste than eggs, even ; and now and then he is able to catch small birds, or even mice and little snakes, while grass- hoppers and fat larvae are a regular part of ANIMAL COMPETITOES 130 his bill of fare. SQUIRRELS, GOOD AND BAD Few animals, remarks Manly Hardy, are more fond of meat: ''They will eat any kind of meat or fish as quickly as a cat and will live on it days when a chance offers. I have often had them eat each other when one was in a trap. Around camps where provisions are stored they are great pests. Their sense of smell must be very acute, as I have seen where one gnawed a large hole through a new overcoat to get at a bottle of coffee which one of my men had rolled up inside to keep it warm. The squirrel must have smelled it through all the folds of the thick cloth. Where not troubled they soon become very tame, often coming into a camp and stealing biscuit or gingerbread from the table. I have seen those which certainly could tell one person from another, as they would let one who had never molested them come very near, while, when a person who had stoned them appeared, they would instantly dodge into a hole." As summer advances the red squirrel finds ripe berries and fruit to his taste, and in July begins, in the northern coniferous woods, to attack the green cones, especially of the white pine, cutting them off "and burying them, half a dozen in a place, under the pine needles, to be dug up in the winter and spring, and opened for the seeds they contain." At this season, SQUIRRELS, GOOD AND BAD 131 too, he bites into many mushrooms, especially those which grow upon old wood; and certain of these he stows away in dry places for future reference. Preparing for the winter. This squirrel is a hard worker at all times, the merriest sprite of the woods, yet always industrious and thrifty ; but his busiest time is in autumn when the ripening nuts must be harvested. In the forests of the southerly portions of his range, butternuts, hickorynuts, and those of the chestnut, pecan, hazel and beech, with acorns and chinkapins, are most important. Their substance is not very nourishing, but they supply in abundance the fat which is so neces- sary for animals to accumulate in the autumn as a fuel to keep the fires of life burning dur- ing the winter. 1 A full discussion of the meaning of this custom of storing food against a coming time of scarcity; and of its probable origin and development through the influence of natural selec- tion, will be found in the chapter entitled " A Squirrel's Thrift," in my Wit of the Wild, 2d edition, Dodd, Mead & Co., New York, 1911. SQUIRRELS, GOOD AND BAD In the Southern States the winters are so mild that there is not the need to lay up the large food-supply required in the North, and methods vary, too. Instead of having a single storehouse, as do most other provident rodents, the red squirrels bury a part of their gains, one or a few nuts in a place, and hide the rest in a variety of nooks ANIMAL COMPETITORS 132 and crannies. It is thus difficult to judge what this scattered accumulation amounts to in the aggregate, but it is probably a good deal more than one animal wants. The little rascals seem to recognize no property rights in these sav- ings, but during the winter seize anything they can find, so that fierce fights are always happen- ing, in which the thievish grays take a full share. With a short account by Mr. Hardy of the cone-saving squirrels of the northern woods I will conclude this part of the subject : * Storing pine-cones. "With us [in Maine] he lays up large stores of the cones of pine and spruce and knows the exact season when they are fit to cut for his use. If cut too early they will be sealed closely with pitch ; if cut too late the winged seeds will have escaped. The red squirrel cuts them by the hundreds the last of September, just when the sticky covering has hardened into drops of stiff pitch and just before the cones have opened. One who is in the pine woods then will hear the dull, heavy thud as they fall, and if he gets a close view of the squirrel, will see that his paws and face are smeared with pitch. . . . SQUIRRELS, GOOD AND BAD 133 "The squirrel knows exactly how to get the seed with the least labor. A squirrel wishing to eat a cone, sits up on his hind feet, standing the cone up before him on its small end. Then he cuts off the upper scale at the butt of the cone. These scales do not run in straight lines, but are arranged spirally, with a seed under each scale. The seeds in a white pine-cone are about the size and shape of a small apple-seed ; those of a spruce, about as large as seeds of turnip or mustard. SQUIRRELS, GOOD AND BAD Both kinds have a wing which serves to carry the seed often to long distances, when it falls naturally from the cone. The squirrel eats the first seed, then gives the cone a slight turn and cuts the next scale, and so keeps turning and eating until the central pith is in his way, when he cuts it off and continues eating until near the end of the cone, which he always leaves, as he knows that the seeds there are too small and poor to be of use to him." Gray squirrels and fox-squirrels. The red squirrel has been given so much space because his life is typical of that of the tribe, and be- cause he is not accurately known although so widespread and numerous. More familiar to most readers are the large "gray" and "fox" squirrels, both of which are very variable. Thus the northern gray squir- rels are at their best a clear silvery tint, while ANIMAL COMPETITORS 134 southward they become yellowish or rusty, and in some localities a black variety is prevalent. Well-grown specimens of this species are about 18 inches long, including the splendid feather of the tail. West of the Alleghenies, to the border of the Plains, and as far north as South Dakota, lives the northern fox- or cat-squir- rel, which is larger (23.5 to 25.5 in.), and in general tint foxy red; but the species is ex- tremely variable, one large southern variety being wholly black save the white nose and ears, and a good deal of black and orange are likely to appear on any specimen, north or south. It may be mentioned here that Mexico has among its many species and races of squirrels perhaps the most beautiful of any in America, the red-bellied. ''Its upper surface is pale grizzled gray, and its under parts bright rusty red; it inhabits the forests of eastern Mexico, ascending the high mountains to an elevation f f t " It is only in the Appalachian region that the gray and the fox-squirrels meet. They are much alike in habits, and both have become bold acquaintances of civilized man, and are SQUIRRELS, GOOD AND BAD 135 public pets in a thousand villages and urban parks. SQUIRRELS, GOOD AND BAD In some places, indeed, they are so numerous and bold as to injure gardens, and to work ruin in roofs and cornices by digging through them to make their nests inside. As pets in captivity they, like the reds, are not very desirable, since they grow cross with age, and if more than one is kept in a cage the strongest will probably kill or injure the others. If allowed the freedom of a room they will work havoc, and prove practically untamable. p p y It is as easy and much better, however, to domiciliate them in the trees about the house, by placing high among the branches cabins (short sections of hollow logs are best), and protecting and feeding their tenants. They will come to a window-sill where you place regularly cracked nuts, grains of corn or bits of cracker, and you will enjoy their society much more in their free shy activity than if they were immured in a small wire jail. A good plan, if you like their visits to your window- sill, is to provide them with a pole-bridge from the nearest tree, as they are shy of going upon the ground where dogs and cats may be. ANIMAL COMPETITORS 136 Peculiarities of these larger squirrels. In one or two respects the gray and the fox-squir- rels differ decidedly from their smaller rela- tives. Instead of retiring to holes under- ground, they dwell in winter in holes in trees, coming out nearly every day to hunt and gambol about. They make great summer nests of leafy twigs in which the mother and young reside while the male squirrels lead a bachelor ex- istence, often with far wanderings. Their food is substantially the same as that of the reds, but they rarely rob the nests of birds, or are thievish of meat ; and their only method of storing food is by burying it, one nut or acorn in a place. That months later, when wind-blown leaves and perhaps deep snow cover the ground, they can recover these treasures is truly remarkable ; but they seem to know pre- cisely where each nut is buried, and go directly to it, then dive down through the snow and presently reappear with the morsel in their teeth. SQUIRRELS, GOOD AND BAD It would seem improbable that this is an effort of memory, and more likely that a cer- tain amount of memory is aided by the faculty of smell Often after i i one buried nut Peculiarities of these larger squirrels. In one or two respects the gray and the fox-squir- rels differ decidedly from their smaller rela- tives. Instead of retiring to holes under- ground, they dwell in winter in holes in trees, coming out nearly every day to hunt and gambol about. They make great summer nests of leafy twigs in which the mother and young reside while the male squirrels lead a bachelor ex- istence, often with far wanderings. Their food is substantially the same as that of the reds, but they rarely rob the nests of birds, or are thievish of meat ; and their only method of storing food is by burying it, one nut or acorn in a place. That months later, when wind-blown leaves and perhaps deep snow cover the ground, they can recover these treasures is truly remarkable ; but they seem to know pre- cisely where each nut is buried, and go directly to it, then dive down through the snow and presently reappear with the morsel in their teeth. It would seem improbable that this is an effort of memory, and more likely that a cer- tain amount of memory is aided by the faculty of smell. Often after finding one buried nut SQUIRRELS, GOOD AND BAD 137 they bore their way beneath the snow here and there in search of others, and so get a whole nieal. These squirrels are so large and toothsome that they have always been reckoned among our game animals, and years ago were to be seen in every market in the land. Now this is less common, because they have become scarce in many parts of the country. No longer, then, are they accused by farmers of being a pest; but a century ago they certainly were so all along the frontier. That was the time when occasionally vast migrations descended upon the fields of corn in the milk, ruining the crop ; and for years great sums in bounties were paid for their destruction in Pennsylvania and Ohio. A very full account of this matter, and of the gray squirrel generally, may be found in the first chapter of my Wild Neighbors. SQUIRRELS, GOOD AND BAD p y g The gray squirrel of California is a separate species, larger and brighter than the eastern gray. Along the Mexican border occur several allied species, more or less marked with yellow and reddish, of which the handsomest is Abert's, which has a band of chestnut along the ANIMAL COMPETITORS 138 spine, side-stripes of black, white underparts and feet, and tufted ears. It is also found in the mountains of Colorado. The squirrel that flies. All squirrels are clever at falling. They often slip at great heights, and when they can not clutch a lower branch will turn in the air, spread out their legs and usually alight without harm. The skin is loose, and is pulled far out when the legs are widely stretched; and in one sort the side fold is so ample as to form a regular para- chute, enabling the animal to make long slides through the air; it becomes, in fact, a living aeroplane. This is the flying-squirrel, the prettiest fourfoot in the American woods. p There are two species. One dwells in north- ern Canada, measuring 14 inches in length, and is cinnamon-brown above (sooty in winter), with a black ring around the eye, and the fur of the whitish underparts gray near the roots. A smaller variety occurs in the St. Lawrence Valley. The other species is the common one of the eastern and southern half of the Union, which is only about 9.5 inches in length. Its fur is dense and exquisitely soft, with the tail SQUIRRELS, GOOD AND BAD 139 almost as flat as a feather; the color is drab above, irregularly tinged with russet, while the hair of the underparts is pure white to the roots. Cram notes the "protective" similarity of their clouded cream-buff colors, to the lichens on the trees to whose bark they often cling motionless for long periods. They are not much exposed to any but nocturnal enemies, such as owls and the weasel tribe, however, so that this similitude cannot have much practical importance. They are forest folk, haunting the hardwood groves, and few farmers suspect how many of these tenants profit by the old stubs left along the edges of their clearings. SQUIRRELS, GOOD AND BAD Really they are tenants of the woodpeckers, who are good enough not to occupy one of their care- fully dug nesting-holes twice, but to leave it to the occupancy, rent-free, of squirrels, chick- adees, little owls and other feebler neighbors. The squirrels are capable, however, of carving out a deep hole for themselves, or will take possession of some natural cavity, and in it arrange a luxurious bed of shredded bark, etc., mingled with the fur they shed plentifully in the fall Sometimes many will room together ANIMAL COMPETITORS 140 in a large cavity. Now and then a pair will form an outdoor ball-like home in some old bird's-nest; or will even invade the garret of the farmhouse. Charm of tlie flying-squirrel. Strike one of these tall stubs a smart blow with an ax or stone and the squirrels will come pouring out of their hole and go sailing away to neighboring trees like birds. They alight near the bases of the trunks and scamper upward to prepare for an- other glide, but unless sharply pursued will quickly turn to have a curious look at their dis- turber. Their " flights" are made upon the parachute of loose skin which extends in a furry fold down to the feet, and is further supported by a slender curved bone hinged to the back of the wrist, while the flat tail acts as both balancer and rudder as in a bird. When starting from a high perch, and going down hill, they may sail 200 or 300 yards; but have little or no power of deviating from the straight line of the intended leap, yet make a quick upward curve as they alight. Audubon and Bachman have given a delightful account of their gambols on SQUIRRELS, GOOD AND BAD 141 summer evenings near Philadelphia, about 1840: ' ' During the half-hour before sunset nature seemed to be in a state of silence and repose. The birds had retired to the shelter of the forest. The night-hawk had already commenced its low evening flight, and here and there the common red bat was on the wing ; still for some time not a flying-squirrel made its appearance. SQUIRRELS, GOOD AND BAD Suddenly, however, one emerged from its hole and ran up to the top of a tree ; another soon followed, and ere long dozens came forth and com- menced their graceful flights from some upper branch to a lower bough. . . . Crowds of these little creatures joined in these sportive gambols; there could not have been less than 200. Scores of them would leave each tree at the same moment, and cross each other, gliding like spirits through the air, seem- ing to have no other object in view than to indulge a playful propensity." ' ' During the half-hour before sunset nature seemed to be in a state of silence and repose. The birds had retired to the shelter of the forest. The night-hawk had already commenced its low evening flight, and here and there the common red bat was on the wing ; still for some time not a flying-squirrel made its appearance. Suddenly, however, one emerged from its hole and ran up to the top of a tree ; another soon followed, and ere long dozens came forth and com- menced their graceful flights from some upper branch to a lower bough. . . . Crowds of these little Family life of the flying-squirrel. Not very much is known of the winter life of the ordinary or southern flying-squirrels, but they seem to retire to their warm nests as soon as cold weather comes, and to stay there until spring. This would mean hibernation, or else the storing of food in their holes; and that the latter is their habit would seem indicated ANIMAL COMPETITORS 142 by the actions of captives to be mentioned presently; but if so it presents a curious anomaly to the rule, for it is certain that the large northern species, although dwelling in much colder regions, where proper food is ap- parently scarcer, does neither, but goes abroad every evening, no matter how severe may be the cold, to get its subsistence, and fares well. The young are born in early spring, and when about six weeks old begin to appear at the door of their house, playing about like kittens under the watchful care of their mother. "And what a lovely little mother she is! She takes the greatest care of them from the time they are born. SQUIRRELS, GOOD AND BAD She tucks them under her, pulls the cedar bark over them and blocks up the entrance on cold days to keep them warm. If you put your finger into the hole she will rake all the babies out of harm 's way with her front paws, and then with her nose she will make a determined effort to push your finger out of the hole again. Failing in that, she will not bite you, as a red squirrel would have done at the beginning, but she will probably take your finger gently in her teeth, as though to ask you please to be a gentleman and refrain from causing her any further annoyance. y ' ' Even if you remove the young ones from the nest she will not bite you, but she will come out after SQUIRRELS, GOOD AND BAD 143 them at once in evident distress. If they are near the mouth of the hole, so that she can reach them without leaving the nest entirely, she puts out her head, seizes the youngsters by the neck or back with her teeth and pulls them in after her, one by one. But if she has to leave the nest altogether she picks the children up, turns around and pushes them into the hole before her. A flying-squirrel once disturbed in this way is not likely to allow the matter to pass unheeded. She is almost sure to remove her family to a new home at the first opportunity." Taken young, and fed on milk and vegetables until they get their growth, they form delight- ful pets, though mischievous ones, unless their activity is curbed. It is from captive specimens, indeed, that we have learned most that we know as to the habits, tastes and dispo- sitions of these secretive little creatures. GROUND-SQUIRRELS AND PRAIRIE- DOGS We come now to the ground-squirrels, which are of small size, have flat and comparatively short tails, and keep near the earth, living be- neath it and deriving their food from weeds and low bushes for the most part. There are scores of species which fall into two groups, the striped chipmunks of the eastern and north- ern woods, and the unstriped spermophiles of the western plains. The chipmunks. Our familiar chipmunk is the only eastern representative of this large group, and is seen everywhere. In size and manners he is much like the saucy red squirrel, but the five black stripes alternating with two whitish ones on his chestnut coat (fading through yellowish on the sides into a white vest), distinguish him in an instant. His race extends clear across the continent and to Alaska, wherever timber grows, but the western 144 GROUND-SQUIRRELS 145 varieties differ so much in size and tint that the early naturalists made several species. p ''This squirrel," to quote the pleasant phrases of Dr. Godman, ''is most generally seen scudding along the lower rails of the com- mon zigzag or 'Virginia' fences, which afford him at once a pleasant and secure path, as, in a few turns, he finds a safe hiding place be- hind the projecting angles, or enters his burrow undiscovered. When . . . his retreat is cut off he . . . runs up the nearest tree, uttering a very shrill cry or whistle, indicative of his distress, and it is in this situation that he is most frequently made captive by his per- secuting enemies, the mischievous schoolboys. ' ' No animal is better prepared than the chip- munk to withstand the cold and hunger of a northern winter, for he has learned how to con- struct a model home and to i i it well struct a model home and to provision it well. The burrow and its furniture. This is a burrow which usually begins beside a stone or among the roots of a tree where it will not at- tract notice, moreover all the earth that is taken from the hole is scattered at a distance in order not to betray the excavation. GROUND-SQUIRRELS AND PRAIRIE- DOGS It is first carried ANIMAL COMPETITOES 146 straight down in a narrow shaft below the frost line, then turns and winds away horizontally, and as the tunnels are used year after year, with continual enlargements, old ones may reach to a great length, with branches and chambers accommodating several pairs, and secret exits. In the autumn one of the under- ground chambers is furnished with soft bedding and becomes the living-room of a family, while other chambers are stored with provender or set apart as receptacles for refuse. Now the chipmunk becomes exceedingly busy, fattening himself upon the ripening nuts and seeds, not only, but upon many tuberous roots, mushrooms and green corn. On each side of his mouth, separated from it by thin partitions of muscu- lar skin, are large cavities or pouches, opening behind the teeth, which are as useful to him as are our baskets and wheelbarrows to us. He brings to the surface in them the material ex- cavated from the distant ends of his burrow, and after packing them full of seeds or nuts he returns to empty their loads perhaps half a pint at a time in one of his storehouses. All the ground-squirrels have such cheek-pouches; GROUND-SQUIRRELS 147 and So they can speedily gather, while they are plenty, the large stores they need to preserve life during the long season of famine ahead; and snug in their warm nests deep under the sod, they doze away the winter, now and then emerging when the February sun tempts them out, but for the most part lying close, yet not in complete dormancy. p y Taking the freedom of the camp. These cheerful little fellows, and especially the four- striped Rocky Mountain kind, are extremely numerous in the rougher parts of the West, and are amusing visitors at every camp and cabin until they wear out their welcome by misbe- havior. In some of the national forests they have proved a great nuisance by digging up newly planted tree-seeds. ' ' In camp, ' ' writes an explorer of Mt. Shasta, ' ' they made frequent visits to the mess-box, which they clearly regarded as public property, approaching it boldly and without suspicion, and showing no concern at our presence in marked contrast to the golden- mantled squirrels, which approached silently, stealth- ily, and by a circuitous route, in constant fear of detection. GROUND-SQUIRRELS AND PRAIRIE- DOGS If disturbed while stuffing their cheek- pouches with bits of bread, pancake, or other eatables, ANIMAL COMPETITORS 148 each chipmunk usually seized a large piece in its mouth and scampered off, returning as soon as we withdrew. In fact they made themselves perfectly at home in camp." The striped gopher and spermophile. A variety of this species, the sage-chipmunk of TWO SPECIES OF ROCKY-MOUNTAIN CHIPMUNKS. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by H. W. Nash. TWO SPECIES OF ROCKY-MOUNTAIN CHIPMUNKS. From Warren's "Mammals of Colorado." By Permission of G. P. Putnam's Sons. Photo by H. W. Nash. the Great Basin, is the smallest and sprightliest of the race. It lives mainly in the sage-brush, scrambling about these diminutive bushes or scampering from one to the other, and often sitting on the top of a sage-bush eating the little seeds from its hands; but, like other GROUND-SQUIKRELS 149 squirrels they vary their seed-diet with insects. It is a relative of these chipmunks, marked by thirteen stripes, dark brown on rusty yellow, which is known throughout the Northwest, from Lake Michigan to Alberta, as the "striped gopher," and as a pest to farmers on account of the grain it steals and the runways for water its burrows make. Still worse are several other northwestern ground-squirrels which have plain yellowish-gray coats and are known as "gray gophers," though the term "gopher" should be restricted to the Geomys; the most familiar is Franklin's spermophUe. p p This graceful animal was originally abundant as far south as central Missouri and Illinois, but long ago disappeared before the civilizing of its prairie home, and now remains numerous only in the wilder districts of the Dakotas and northward. It is pretty and interesting, but too much of an impediment to good agriculture to permit the farmer to tolerate it ; yet the an- imal increases so rapidly under the protective and food-supplying conditions which the hu- man settlement of the country brings it, that its extermination will be a matter of great diffi- ANIMAL COMPETITORS 150 culty. Referring to this matter Dr. GROUND-SQUIRRELS AND PRAIRIE- DOGS Merriain made the following appealing remarks in a re- cent paper on these pests in California: p p p Striped spermophiles exist along the grassy eastern border of the plains right down to the Gulf of Mexico; and Texas has, besides, a beautiful little "sand-squirrel," spotted with white on a yellowish ground, relieved by black markings. It is a shy, inconspicuous little creature, rarely noticed until it attracts atten- tion by a fine trilling bird-like whistle. These and other spermophiles are most numerous where the mesquit grows, for its seeds afford them good food. They are fond, too, of the fruit of the small prickly pear, the sand-bur, and other shrubs and weds, and eat many grasshoppers and other insects. The graceful antelope-squirrel, taking its name from its col- ors, is another species conspicuous for its beauty, carrying its short, wide, white-lined tail curled over its back like a plume. All these burrow at the edge of thickets and cactus clumps and apparently hibernate. Sometimes they do much damage by boring through the banks of i i ti ditches Another south- Striped spermophiles exist along the grassy eastern border of the plains right down to the Gulf of Mexico; and Texas has, besides, a beautiful little "sand-squirrel," spotted with white on a yellowish ground, relieved by black markings. It is a shy, inconspicuous little creature, rarely noticed until it attracts atten- tion by a fine trilling bird-like whistle. These and other spermophiles are most numerous where the mesquit grows, for its seeds afford them good food. They are fond, too, of the fruit of the small prickly pear, the sand-bur, and other shrubs and weds, and eat many grasshoppers and other insects. The graceful antelope-squirrel, taking its name from its col- ors, is another species conspicuous for its beauty, carrying its short, wide, white-lined tail curled over its back like a plume. All these burrow at the edge of thickets and cactus clumps and apparently hibernate. Sometimes they do much damage by boring through the banks of irrigating ditches. Another south- GROUND-SQUIRRELS 151 western group includes the rock-squirrels, which are never seen far from cliffs or broken ledges. Bailey 'tells us that they climb the trees for acorns and berries, but when surprised al- ways rush to the ground and scamper away to the nearest rock-pile. They are extremely wary. GROUND-SQUIRRELS AND PRAIRIE- DOGS "Like most of the smaller ground- squirrels of the arid regions they usually bur- row under a cactus or some low thorny bush, where they obtain shade and the protection of thorny cover. They apparently do not hiber- nate, but during the cold weather have the un- squirrel-like habit of closing their burrows and remaining inside, as a protection against en- emies, and especially snakes. . . . Like other members of the genus, these ground- squirrels feed on seeds, grain, fruit, green foliage, lizards, and numerous insects, and often gather around gardens and green fields, where they do considerable damage in spring by digging up corn, melons, beans and various sprouting seeds, and, in summer and fall, by feeding on the ripening grain." Squirrels and bubonic plague. One of these g p g g Squirrels and bubonic plague. One of these ground-squirrels, that most common in central ANIMAL COMPETITORS 152 and southern California (Clitellus beecheyi) has special prominence in our list because it shares with the rat the bad distinction of being a dangerous carrier of plague-germs. It was observed as early as 1903, as we learn from Pro- fessor Doane's book previously alluded to, that an epidemic was killing these ground-squirrels in the neighborhood of San Francisco bay. The matter was at once investigated by Dr. Ru- pert Blue, of the U. S. Public Health and Marine Hospital Service, who speedily ascer- tained that the disease was bubonic plague, which had probably been caught from the town rats which at harvest time wander into the country in large numbers and make free use of the holes and runways of the field-squirrels. A single infected rat might sow the seeds, for its fleas, escaping, from its dead body, would readily attach themselves to a squirrel and multiply and spread among them. Among the tens of thousands killed and examined a con- siderable number of infected ones have been found; and several instances are recorded in which human cases of plague in California resulted from handling infected squirrels. PRAIRIE-DOGS 153 Whether the disease has been exterminated among these wild rodents, remains to be seen. The fact that the Beechey ground-squirrels have shown themselves receptive to the fleas which are peculiar to brown rats, and to the disease, led to observations and experiments as to other rodents. GROUND-SQUIRRELS AND PRAIRIE- DOGS It is found that rock-squir- rels are quite readily infected, mice and pouched gophers less so, but wood-rats and prairie-dogs succumbed at once. There seems no reason to suppose that any rodent may not carry the fleas about in its fur a short time, if not permanently ; or that any rodent is immune against the plague if punctured by an infected flea. A ray of light is shed upon this dark as- pect of the case by the announcement that along with the fleas goes a small staphylinid beetle which exists as a parasite on both rats and squirrels, and feeds ravenously on the fleas. q Importance of the prairie-dog. But of all the ground-squirrels none equals the prairie- dog in interest or importance. g p It is a denizen of the dry plains east of the Rockies, while two or three other species inhabit the mountains, the Utah basin, and ANIMAL COMPETITORS 154 southward into Mexico. This animal is some- times confused towards the north with the ''^"v'Sf? 1m :v -: A.M4, B. Funnel jhafifd entrance, to Uurrcnf C. McufVflttfsagt^ inch uvduurteter HjIrtjonM J. A l/jorbentmatter carryuta buutfJudt . of carbon. KjbsUipnof. DIAGRAM OF A PBAIBIE-DOG'S BURROW. larger gray gophers, especially the Columbian DIAGRAM OF A PBAIBIE-DOG'S BURROW. larger gray gophers, especially the Columbian and Franklin's, so that we wrongly hear of larger gray gophers, especially the Columbian and Franklin's, so that we wrongly hear of larger gray gophers, especially the Columbian and Franklin's, so that we wrongly hear of PRAIRIE-DOGS 155 "prairie-dogs" on the Canadian plains; it is to be distinguished by its slightly larger size, dis- tinctly brownish color, and very short tail (two inches), which is flat and black toward the end. The prairie-dog is about a foot long, and robust, with strong limbs and claws. It dwells in colonies, whose permanent ''towns" of bur- rows, each marked by a hillock of earth about the entrance, spread densely over many acres under the natural prehistoric conditions, but now sometimes cover hundreds of square miles. The burrows are deep and extensive, and at first go down at a very steep slope to a depth of twelve to fifteen feet, when they turn hori- zontally, and here and there branch into cham- bers, some of which are family rooms, while in others fodder is stored, or refuse and dung are deposited. GROUND-SQUIRRELS AND PRAIRIE- DOGS The mound about the hole is packed hard, not only by the tramping of the animals, but by crowding it down with their noses ; this hillock prevents water from running into the burrows when the plain is flooded by heavy rains, and also serves as a tower of ob- ANIMAL COMPETITORS 156 "The prairie dogs feed upon grass and herbage, which is soon exhausted near the burrows, compelling the animals to go farther and farther away for food. This they dislike to do, as it exposes them to attack from enemies; and after a time they prefer to dig a new burrow nearer a supply of food. Thus a 'town' is always spreading and contains many empty bur- rows. Like other animals habituated to desert re- gions, they do not drink at all. . . . The animals are diurnal and most active morning and evening. They come out daily during the winter, except when it is very stormy; but this practice varies with the latitude and climate. "They are prolific, especially in the southern half of their territory, and would multiply with excessive rapidity were it not for numerous enemies, especially rattlesnakes and other serpents. These are coura- geously resisted by the prairie dogs, who sound the alarm the moment a snake enters a hole, gather, and proceed to fill the entrance with earth, packing it down, thereby sometimes entombing the snake for- ever. Probably few snakes go down the passages, which are so steep they could with difficulty climb out, but depend upon lying hidden in the grass and striking down the young squirrels when out at play or in search of food. This is the method of the coyote, kit-fox, wildcat, hawks, and owls, who find the dog-towns a profitable hunting-ground. Badgers, however, can, if they will, easily dig up a burrow and devour the helpless family. The worst enemy is the black-footed ferret, a weasel of the plains, which PRAIRIE-DOGS 157 easily penetrates the burrows, and against whose ferocity and skill the squirrels can make little defense. GROUND-SQUIRRELS AND PRAIRIE- DOGS ' ' All these conditions together served in the natural state of things to hold the prairie-dogs in check, but the changes brought about by civilization have been so favorable to these little animals, by the reduction of their enemies on the one hand, and the augmentation on the other hand of their food supplies by the farm- ers' plantations of meadow grass, alfalfa, and grain, that they have increased into a very serious pest." A serious pest-problem. How serious this pest has become in the grazing regions of western Kansas, Oklahoma and Texas, may be inferred from the information furnished by Yernon Bailey in his report upon the condi- tions in Texas in 1905. "Usually," he states, "they are found in scattered colonies, or 'dog-towns,' varying in extent from a few acres to a few square miles, but over an extensive area lying just east of the Staked Plains they cover the country in an almost continuous and thickly in- habited dog-town, extending from San Angelo north to Clarendon in a strip approximately 100 miles wide by 250 miles long. Adding to this area of about 25,- 000 square miles the other areas covered by them, they cover approximately 90,000 square miles of the State, wholly within the grazing district. It has been roughly estimated that the 25,000-square-miles colony contains 400,000,000 prairie-dogs. If the remaining ANIMAL COMPETITOES 158 SCEJSES ITT PRAIBIE-DOG LITE. SCEJSES ITT PRAIBIE-DOG LITE. PBAIRIE-DOGS 159 65,000 square miles of their scattered range in the State contains, as seems probable, an equal number, the State of Texas supports 800,000,000 prairie-dogs. According to the formula for determining the relative amount of food consumed by animals of different sizes, this number of prairie-dogs would require as much grass as 3,125,000 cattle. "In many places the prairie-dogs are increasing and spreading over new territory, but on most of the ranches they are kept down by the use of poison, or bisulphid of carbon, or, better, by a combination of the two. As a Texas cattle ranch usually covers from 10,000 to 100,000 acres, the expense of destroying the prairie-dogs in the most economical manner often means an outlay of several thousand dollars to begin with and a considerable sum each year to keep them down. The increase of prairie-dogs is clearly due to the destruction of their natural enemies. . . . GROUND-SQUIRRELS AND PRAIRIE- DOGS "In autumn the prairie-dogs become fat, but in Texas they do not hibernate as they do to some extent in the North. If their fur should become fashionable, or roast prairie-dog an- epicurean dish, the problem of keeping them in check would be settled, and there is no reason, save their name, for not counting them, properly prepared and cooked, a delicacy. While owing their name to a chirping or 'barking' note of warning, they are in reality a big, plump, burrow- ing squirrel of irreproachable habits as regards food and cleanliness. An old stage driver expressed the idea in graphic words one day: 'If them things was called by their right name, there would not be one ANIMAL COMPETITORS 160 left in this country. They are just as good as squir- rel, and I don't believe they are any relation to dogs.' " Since the citation above was written the pub- lic suppressive measures taken in Kansas have reduced the pest to negligible proportions ex- cept in the remote northwestern counties. p The woodchucks. These large ground-squir- rels bridge the gap between the true squirrels and the marmots, as they are called in the Old World, known to us "woodchucks" or " ground- hogs." They are stout, short-legged, inactive animals, with short tails and closely appressed ears, whose dense fur is grizzled gray, in- clined to chestnut or blackish, and whose habits are distinctly terrestrial. Our eastern wood- chuck is found everywhere east of the Plains in all open woodlands, prairies or cultivated regions, for he thrives in the midst of civil- ization, whose cleared fields are to his liking, while he is furnished an abundance of food by the raising of field and garden crops. Few animals are so familiar to the country boy, who early becomes acquainted with its burrows in the hillsides, but rarely has the PKAIRIE-DOGS 161 courage to dig the owner out of their tortuous depth. In the West are other similar species; and all the high mountains from the Rockies westward, and far to the North, have a larger one which dwells near timber-line, and is known as the siffleur (whistler) on account of its clear sharp call. The food of all these marmots con- sists of herbage and succulent roots, and they do great damage in gardens where not re- strained. GROUND-SQUIRRELS AND PRAIRIE- DOGS They do not store any of this food, however, but in the early autumn retire to their burrows, very fat, and pass the winter in a state of complete torpor, during the continu- ance of which their fatness decreases, being ab- sorbed to sustain the trifling exertion of life caused by the continued slow beating of the heart. When, therefore, they emerge early in the spring, they are lean and very weak, but soon recuperate on the fresh grass and herbage. Their fur is of little value, and their flesh poor, so that as game they are attractive only to the boy and his dog, or, in the West, to hungry In- dians. Food-value of the porcupine. Here, if any- where, should be said a few words about the ANIMAL COMPETITOES 162 sluggish, stupid northern porcupine, whose re- lation to man is far more to the good than to the bad. It is true that he strips a few forest trees of their foliage, and occasionally inad- vertently girdles one; but his flesh is excellent eating and large in quantity. His nature and habits make it possible to approach him with- out difficulty and to kill him with a club. Hence he has always been a reliance of the forest-dwelling Indians and white fur-hunters for winter food, and now it is of so much impor- tance in the great forested regions of the Northern States, and in Canada, that timber- cruisers and others whose business takes them into the wilderness should be able to find a porcupine in such emergencies as are always likely to arise in their adventurous lives, espe- cially in winter, that the animal is protected by law; and this law is well lived up to by the frontier folks for they appreciate its impor- At the same time it must be confessed that porcupines make themselves very troublesome in camps or about houses in the woods which are left alone for a time, as often happens PKAIRIE-DOGS 163 among loggers and miners amid the western mountains. The porcupines at once make themselves at home, and if they can get inside will demolish nearly every wooden thing in their search for food, and their enjoyment of nib- bling anything salty. GROUND-SQUIRRELS AND PRAIRIE- DOGS As salt pork and bacon enter largely into the provisions of these people, their tables, cupboards and utensils are likely to be more or less spotted if not satu- rated with salty grease; and the porcupine knows no reason why he should not get to the last taste. BABBITS, USEFUL AND INJURIOUS RABBITS hold a prominent place among the obstacles to success met with by both the farmer and the orchardist. They number many species, and one or more is present in any habitable part of the continent to which you may refer. The East has in its middle and southern part the familiar and widely distrib- uted gray rabbit or Molly Cottontail, which ex- tends westward to the plains ; and the smaller, and redder swamp-rabbit of the South; while in our northeastern States and in eastern Canada the larger American or varying-hare, which turns white in winter, is present, and con- stitutes the principal winter fare of such worthy animals as the lynx, wolf, fox and vari- ous martens, and of some hawks and owls. In the West the great jack-rabbits abound, and in the far North the arctic white hares. Excellence of rabbit-flesh. To the native 164 BABBITS 165 people of our forests and plains rabbits were of the utmost importance as food, especially in winter. The early pioneers everywhere re- lied largely on them. Their thick fur too, was, and is, a precious resource to the Indians, es- pecially those of the Northwest, who make from it artistic as well as substantial garments and coverings. The smaller American rabbits have long been esteemed as game. While their flesh is less tender than that of the domesticated species it is of much finer flavor, and when properly prepared .for the table is much more desirable as food. With the same care in dressing and handling bestowed upon the rabbit in English markets, our cottontail rabbit would stand much higher in popular flavor. The jack-rab- bits are not so good, yet many reach the market. A strong prejudice against eating jack-rab- bits often exists because occasional individuals are infested by warbles and the tapeworm larva. Unless badly affected, however, the flesh is not injured by these parasites, and there is no good reason why the animals should not be ANIMAL COMPETITORS 166 extensiv.ely used as food. The half-grown or nearly full-grown young of the year are usually healthy and very good eating when properly cooked. Those not needed for the table may be fed to dogs and poultry, but should be cooked. The principal natural enemies of jack-rabbits are coyotes, foxes, bobcats, hawks, owls, and eagles. BABBITS, USEFUL AND INJURIOUS When rabbits become abun- dant these enemies gather to feast on them, and then at least should be afforded protection. p Rabbit fur is not in demand in this country except for trimmings, since it is brittle, has no underfur, and does not wear well ; but from one and a half to two million skins are bought an- nually to be made into felt for hats and similar purposes, and it might be well for farm-boys to enquire whether they could not profitably trap or shoot in their neighborhood for this and the flesh market. General breeding-habits. Our American rab- bits are not so prolific as the common Euro- pean species. Some of them produce three or four litters of young in a season, while others seem to breed but twice. The period of gesta- tion is about thirty days, and the breeding sea- EABBITS 167 son is from April to September or even later. The young are produced in natural depressions under rocks, stumps, or weeds, or in shallow burrows made by other animals. When these are lacking, the female scratches a shallow hole under a bunch of grass or weeds, makes a nest of leaves or grasses and lines it with fur from her own body. Here the young, averaging in most of our species about four, are produced; they are fully furred and have their eyes open when born. The female, while caring for her young, re- mains in the vicinity of the nest. If enemies approach, she runs away for a short distance; but when the young are attacked and cry out, she has been known to fight desperately in their defense, and even to vanquish such a formidable foe as a cat or a snake. When attacking, she jumps and strikes the enemy with her hind feet members capable of a powerful blow, as many a boy who has captured a live rabbit can^ testify y Young rabbits are attended and suckled in the nest for about three weeks, after which they are left to shift for themselves. Since sue- ANIMAL COMPETITORS 168 culent food is usually abundant, this is not a difficult task, and they soon adapt themselves to an independent life. Apparently the mother takes no further interest in the career of her offspring. The male parent is probably never concerned in the care of the young. i The bounty on rabbit ears paid by Gray County, recalls the bounty paid on gophers in Wallace and Greeley counties [Kansas] in 1894. The former county paid the bounty on scalps, while Greeley (Bounty paid it on gopher tails. The boys along the county line traded gopher tails for scalps, and realized 10 cents on each gopher. A five-cent bounty was paid on rabbit ears also, and rabbits and gophers were so plentiful that many families made their living from hunting. So much bounty money was claimed that Wallace County resorted to scrip payment, and this became so plentiful that its value dropped to 50 cents on the dollar. The county finally tried to repudiate the scrip, and in suits fought to the highest court, it succeeded after five or six years of ex- pensive litigation. Kansas telegram to New York Times, Jan. 2, 1911. BABBITS, USEFUL AND INJURIOUS y g Injury to gardens and orchards. The cot- tontail is fond of frequenting farms and planta- tions, and having taken up its residence in some chosen fence-corner or thicket remains near it, feeding upon the succulent vegetables in the farmer's garden, or the clover, turnips, or corn in his fields. In the fall it feasts upon apples, cabbages, turnips, and the like left exposed in garden and orchard, and in winter, when all else is frozen hard or covered with snow, it turns its attention to twigs and bark of woody plants. The other rabbits have similar habits, varying with the environment. In the West some of the smaller kinds live largely in the abandoned burrows of prairie-dogs, badgers, and other animals. Babbits feed upon nearly all growing crops, but the damage to small grains is usually so slight as to pass unnoticed. Clover and al- BABBITS 169 falfa are favorites. In the Southwest they are quick to seize upon garden-patches, and in parts of Texas cantaloupes cannot be grown unless well fenced. Eabbits, however, are most feared by tree- planters. They injure trees and shrubs in two ways by cutting off the ends of branches and twigs, and by tearing away the bark, often until the tree is entirely girdled. The differ- ence between the work of rabbits and that of field-mice may easily be detected by the large tooth-marks of the former, and by the height (16 to 18 inches above the ground) of the wound. Newly planted orchards are especially liable to injury from rabbits, and few are now set out without provisions for winter protection from these animals. The losses of orchard and nur- sery stock in one neighborhood in Arkansas during the mild winter of 1905-6 were reported at $50,000. , Laws protecting the rabbit. In New Eng- land and the Middle Atlantic States the rabbit is protected, while throughout most of the West and South no restrictions are placed on hunt- ANIMAL COMPETITORS 170 ing the animals. In some Western States they are regarded with such disfavor that bounties have been paid for their destruction. 1 In States where they are most abundant, protection is rarely afforded. In sections of the country where a close season on rabbits is accompanied by a strict enforcement of laws against tres- pass by hunters, rabbits have often become a nuisance. BABBITS, USEFUL AND INJURIOUS On the whole, in America shooting has been the most effective means for keeping down the number of rabbits. Ferreting usually is impracticable, since few of our native rabbits take refuge in burrows. Moreover, the use of ferrets is forbidden by BABBITS 171 law in some States which protect the rabbit. Coursing with greyhounds has many advocates and is popular in the West, where the swifter jack-rabbits abound. Smaller rabbits are often chased with fox-hounds, but the beagle is rap- idly taking precedence as a favorite for rabbit- hunting, the gun being depended upon for securing the game. All of these are most ex- hilarating sports. Where the country is sufficiently open for the purpose, one of the most successful methods of reducing the numbers of rabbits is the or- ganized hunt, known as the "drive." This method has been tried in many localities in the West and in Australia with satisfactory re- sults, the number of rabbits killed in a single drive reaching as high as 10,000 or even 20,000. , Complete extermination of rabbits in any part of the United States is not desirable, as has been remarked, even if it were possible. In most cases where protection seems neces- sary a rabbit-proof fence may be cheaply con- structed of wire netting, iy2 inch mesh, from 2 to 3 feet high, with the lower edge sunk a ANIMAL COMPETITORS 172 RABBITS 173 little, or a furrow plowed against it to prevent an occasional disposition to dig under. g Protecting trees against rabbits. The de- vices that have been recommended for protect- ing trees from rabbits are too numerous for separate mention. The majority consist of paints, washes, or smears supposed to be dis- tasteful to the animals. Unfortunately, those that are sufficiently permanent to afford pro- tection for an entire winter often injure or even kill the trees to which they are applied. Coal tar, pine tar, tarred paper, and various oils are likely to kill young trees. Blood and animal fats when freshly applied will protect from rabbits, but are objectionable, since they are highly attractive to the destructive short- tailed field-mice. Carbolic acid and other vol- atile substances afford only temporary protec- tion, and must be renewed too often to justify their use. Bitter substances, like commercial aloes, or quassia, are useless against rabbits. "Where protection from rabbits only is required, woven wire netting is recommended. This should be made of No. 20 galvanized wire, 1-inch mesh, such as is BABBITS, USEFUL AND INJURIOUS q , Among the most promising washes that have been recommended for tree protection is the "lime-and-sulplmr" wash, so effective in win- ter for the destruction of the San Jose scale. ANIMAL COMPETITORS 174 If this cheap method of controlling our worst insect pest of the orchard has further value in protecting trees from rodents, the fact can not AN APPLE-TREE KILLED DY RABBITS. AN APPLE-TREE KILLED DY RABBITS. be too widely advertised. The results of per- sonal observation by the Survey seem to fully warrant its recommendation, and its cheapness EABBITS 175 makes the method worthy of general trial by orchardists. The formula for the wash, reduced to the basis of the capacity of the ordinary kerosene barrel commonly used in the preparation, is : Unslaked lime 20 pounds Flowers of sulphur 15 pounds Water to make 45-50 gallons A little salt may be added to increase the adhesive property of the mixture. The lime, sulphur, and about a third of the water are boiled together for at least one hour, and, the full quantity of water is then added. For San Jose scale the wash in the form of a spray is applied to the entire surface of the trees. For protection from mice and rabbits the trunks only require treatment, and the wash may be applied with a brush. One application in No- vember should last the entire winter. Mechanical contrivances for protecting young orchard trees are many. The following are recommendations : ANIMAL COMPETITORS 176 often used for poultry netting. For cottontail rabbits rolls 18 inches wide are recommended, but as a protec- tion against jack rabbits wider material is safer. The wire is cut into 1-foot lengths, and one of these sec- tions is rolled into shape about the trunk of each tree, the ends being brought together and fastened at sev- eral places by means of the wire ends. No other fastening is needed. The wire is not in contact with the trunk and may be left on the tree permanently. It will probably last as long as the tree requires pro- tection, and the cost of material need not be over 1% cents for each tree. For young evergreens, material of the same kind 1 foot wide and cut in li/2-foot lengths will give excellent protection. BABBITS, USEFUL AND INJURIOUS g p "If trees are to be protected from both rabbits and mice, materials of closer mesh must be used. Wire window-screen netting is excellent for the purpose, and the cost, when permanence of protection is con- sidered, is not great. g 11 Veneer and other forms of wood protectors are popular and have several advantages. When left permanently upon the trees, however, they furnish retreats for insect pests. For this reason they should be removed each spring and laid away until cold weather. While the labor of removing and replacing them is considerable, they have the advantage, when pressed well into the soil, of protecting from both mice and rabbits. They cost from 60 cents per hun- dred upward, and are much superior to building paper or newspaper wrappings. The writer has known instances where rabbits tore wrappings of BABBITS 177 building paper from the apple trees and in a single night injured hundreds of them. 'Gunny-sack' and other cloth wrappings, well tied on, are effective pro- tectors. Cornstalks also furnish a cheap material for orchard protection. They are cut into lengths of 18 to 20 inches, split, and tied with the flat side against the tree, so as fully to cover the trunk." BABBITS AS PETS. It hardly falls within the scope of this book to treat the subject of rabbits as pets; but some brief directions for keeping them may not be amiss. All the various forms of pet rabbits, includ- ing what is called the Belgian hare, are varie- ties of the wild European rabbit brought about by selective breeding in domestication. That under favorable circumstances they would thrive and multiply wild here, or in any tem- perate part of the world, is shown by the hordes of them which became a formidable pest in Australia. A vast amount of time and work and money have been expended in trying to get rid of them, and it has been found that the best plan has been to fence them out of the farm-lands and sheep-pastures where they ANIMAL COMPETITORS 178 were so undesirable. Lately, they have been turned to account as food, enormous numbers of their frozen carcases being shipped to Eu- rope; and also great quantities of canned rab- bit-flesh. I am not aware that in this country any really wild colony has been permitted to grow except one near Belleville, Ontario, on a rocky point covered with cedars, which jutted into the -Bay of Quinte. The increase in only two years was astonishing. Undoubtedly an en- closed space of waste land, where the creatures might burrow easily, if devoted to a colony would shortly produce a large annual crop for market with a minimum of expense and trouble; but whether regular sale for them could be found is another question which would depend for its answer largely on local circum- stances. A few years ago an effort was made by the Department of Agriculture to arouse an in- terest in breeding and eating the large variety, Imown as Belgian or Dutch hare, which orig- inated in the Netherlands fifty or sixty years ago, but it did not succeed. These large rab- BABBITS 179 bits may still be purchased of dealers in pet stock, and are the best for practical rabbit- culture. Advisable for children. No pets are more interesting for young children than rabbits, and if attended to gently and properly they become very tame. BABBITS AS PETS. The ordinary scrub is good enough to begin with; and after the youngster has learned the lesson of regularity and care in keeping and breeding them, he will want and may be trusted with fancy varieties, such as the lop-ear or angora, which are the aristo- crats of their kind. Hutches. Although the hutches may be built out of rough materials, such as an old box, it is a mistake to suppose that any sort of a kennel will do. The hutch should be tight and warm, protecting the animals well from rain, cold drafts and a burning sun. Each hutch should consist of two parts, an inner and an outer compartment. The in- ner one, or bedroom, should be about 2y2 feet square and high, and have a solid floor. It should also have a hinged door in order to per- mit of cleaning it, and also a little low door, ANIMAL COMPETITORS 180 or hole, giving access to the outer, or dining- room. This outer apartment should be some- what larger and consist of a stout framework covered with chicken-wire on all sides, in- cluding the floor. Then, in fine weather, the hutch may be moved about the yard, and the rabbits will be able to eat the grass through the wire flooring; when the forage has been eaten in one spot, the affair may easily be lifted and set down in another. However, in wet or chilly weather, a board floor should be provided, either within or beneath the cage. Dampness and chill are always to be avoided; hence the roof ought to slant so as to shed water, and the hutch ordinarily be raised above wet ground by supports, such as a brick under each corner. A few gimlet holes should be bored in the floor of the bedroom for drainage, and also near its ceiling for ventilation. Other than this there should be no windows or cracks, as it is necessary that this room should be tight and dark, like a burrow. Its only furniture should be a little dry hay, changed as often as it becomes soiled. The feeding and water- EABBITS 181 ing pans should also be kept very clean; and these ought to be of pottery or iron, as wooden ones are ruined by nibbling. BABBITS AS PETS. The hutch should be cleaned regularly ; in fact, it is a good thing to make a practice of raking out the refuse every morning and giving the rabbits a bed of fresh straw. Babbits should be fed regularly twice a day. Almost anything in the way of fresh vegetables is good for them green grass, lettuce, cab- bage-leaves, roots, such as carrots, sweet apples, and vegetable parings from the kitchen. A regular daily ration, however, should be a small quantity of grain, half oats and half bran, or something similar, and in the winter good hay. Many recommend in winter, warm tea-leaves and dandelion as an occasional treat, good for their health, and boiled potato-parings given warm. One experienced man warns us against giving cabbage-leaves to the young; and also warns us that all green stuff offered must be quite dry or it is likely to "pad" the animals give them cholera morbus. They do not need much water when fed on juicy food, but drink a good deal when living on hay and _ fke three' rao&its together near the <op"tr the Silver Grey (dark), the Silve' Cream (raTd"dIe)Taad'"u Polish. Tie big rabbit with tab ears standing quite erect U the Flemish Claot, and tha one next ft, biding tiehind the t is a P t i Ne*t come the two black and white bblM which are Dutch The bt rabbit BABBITS 183 shorts; and a supply should always be kept within their reach. within their reach. Directions for breeding. Babbits will mate and breed every two months or so. When the doe desires the buck, as the male rabbit is called, she will announce it by stamping with her feet and behaving restlessly. The period of gestation is four weeks, and when the young are about to be born the mother will tear fur from her breast and form a soft bed for them. She should then be given a hutch quite to her- self, and left undisturbed. Your curiosity will be great to see the little ones, but it is better to restrain it until, a fortnight later, they be- gin to be brought out for air. They mature rapidly, but should be left with the mother five or six weeks, unless she shows by fighting them away that she is weaning them earlier. BABBITS AS PETS. The young should not be given as fresh, rich food as the old rabbits, but selected and somewhat dry and wilted food. The first litter is some- times lost, by bad mothering, but rarely a sec- ond or subsequent one. Directions for breeding. Babbits will mate and breed every two months or so. When the doe desires the buck, as the male rabbit is called, she will announce it by stamping with her feet and behaving restlessly. The period of gestation is four weeks, and when the young are about to be born the mother will tear fur from her breast and form a soft bed for them. She should then be given a hutch quite to her- self, and left undisturbed. Your curiosity will be great to see the little ones, but it is better to restrain it until, a fortnight later, they be- gin to be brought out for air. They mature rapidly, but should be left with the mother five or six weeks, unless she shows by fighting them away that she is weaning them earlier. The young should not be given as fresh, rich food as the old rabbits, but selected and somewhat dry and wilted food. The first litter is some- times lost, by bad mothering, but rarely a sec- ond or subsequent one. b q Babbits are subject to various diseases, but these can usually be prevented by good care. SUPPRESSION OF RODENTS AS PESTS INTERESTING and beautiful as most of the rodents may be regarded, and beneficial to mankind in much of their work, there are un- doubtedly many times and places when, owing to their excessive numbers and activity, they constitute a serious nuisance and must be sup- pressed. Foolish destruction of rodents' enemies. Prominent among the recognized causes for the great increase of rodent pests in recent years is the persistent destruction of the birds, mam- mals, and snakes that habitually prey upon them. Even farmers have joined in the war- fare against the so-called "vermin," and too often have sought to get ill-advised legislation against mammals and birds that are beneficial. Among the wild mammals of North Amer- ica known to feed upon field-mice and their burrowing relatives are bears, wolves, foxes, 184 SUPPRESSION OF RODENTS 185 wildcats, all the weasel-tribe, and the brown rat, one of his few commendable traits. Bears, the puma and the wildcats, foxes and wolves, all subsist largely on mice and ground- squirrels. Dogs frequently follow their mas- ters to the field at plowing and harvesting, ready to pounce upon every mouse or gopher uncovered; and they sometimes become very fond of both the prey and the sport and hunt independently. Some cats are good mousers, and in places live largely on wild game; but unfortunately they also learn to destroy song- birds and game-birds, and their nests. The ordinary farm cat is a terrifically destructive animal, and when it develops hunting pro- clivities it should either be reformed, or belled or killed. Rats kill many mice, in the fields, as well as about houses, forcing their way into their burrows. Even the ferocious little shrews may do so, dashing fearlessly at one twice the shrew's size. The weasel tribe the best police. The most efficient check upon the over-production of ro- dents, however, so far as mammals are con- cerned, is furnished by the tireless hunting of ANIMAL COMPETITORS 186 the skunks, badgers, minks, ferrets and similar animals of the weasel family, albeit they do other things which are less pleasing to the farmer. The smaller weasels easily traverse the surface-runways of the larger species of Microtus, and even follow them into under- ground burrows. The larger weasels feed upon pocket-gophers, prairie-dogs, ground- squirrels, and various kinds of mice and rats. SUPPRESSION OF RODENTS AS PESTS While occasionally they capture game- or song- birds, as well as poultry, their principal food consists of injurious rodents. This general statement applies to minks, skunks and badgers, as we shall see when we come to speak of these animals more particu- larly; and nothing could be more unwise than to kill these animals or allow anyone else to kill them upon farm or ranch, except in the oc- casional case where one is known to have ac- quired the habit of taking eggs or poultry. In most cases the blame is placed on the wrong head. Almost all skunks leave birds entirely alone, as also does the badger, which in the gopher country ought to be protected with the utmost solicitude. It has been repeatedly no- SUPPRESSION OF RODENTS 187 ticeable that when white weasel-skins are high-priced, and, consequently, many of these animals are trapped in winter, the following season will be one of excessive mischief by all the smaller rodents. L. C. Cummins, of Riverside, Cal., writing to the Biological Survey, February 12, 1892, says : At one nursery we were bothered with gophers ; all at once the gopher became scarce and from one to five weasels could be seen nearly every day running through the nursery stock and over an adjoining hill. They completely drove away and killed all the gophers. Useful aid by birds of prey. A similar ac- count might be made of the birds not only birds of prey, of which the owls and the marsh- hawk are to be put foremost, but shrikes (butcher-birds), crows, jays, roadrunners, gulls and several of the heron tribe. Bitterns, egrets, cranes and the like, search steadily for mead- ow-mice. In California the great blue heron is protected on many ranches in realization of valuable service. A letter to The Pacific Rural Press, Oct. 23, 1897, from W. M. Bistoe, re- lated that a neighbor found barn-owls had ANIMAL COMPETITORS 188 made their home in his pigeon-house. Think- ing they were after the pigeons he shot the male and the next day trapped the female. On investigation he found four young owls in the nest, together with the remains of ten pocket- gophers. He immediately released the cap- tured mother, with his apologies. This owl is so regular in its daily capture of these pests that it might well be named the gopher-owl. SUPPRESSION OF RODENTS AS PESTS g g p Serpents devour mice and gophers. Snakes must be included among the natural enemies of field-mice. The larger bull-snakes (Pituo- phis), blacksnakes (Callopeltis), and rattle- snakes (Crotalus) feed largely upon rabbits, prairie-dogs, pocket-gophers, and ground-squir- rels, as well as different species of rats and mice. Blacksnakes and bull-snakes probably kill more field-mice than the others ; but black- snakes destroy also a considerable number of nestling birds and birds' eggs, so that part of their beneficial work is offset by this injurious habit. A nurseryman in Pennsylvania reports that he secured immunity from mice in his nur- sery by turning loose in it 50 blacksnakes. y y g The Pacific bull-snake (Pituophis catenifer), SUPPRESSION OF RODENTS 189 because of its habit of- killing pocket-gophers, is quite generally called the gopher-snake. A writer in The Pacific Rural Press for May 12, 1888, says of the reptile : It is an act of insane folly to destroy them, for they are the most active and efficient allies of the nurseryman, farmer, and fruit raiser in the destruc- tion of those most pernicious pests, the gopher and the squirrel. They destroy more gophers than all the appliances that man can bring to bear in the shape of traps, poisons, and gases. On the whole, snakes, except the venomous species, are deserving of the farmers' protec- tion. Like the toad, the smaller kinds feed almost wholly upon insects; but an inherent prejudice induces thoughtless people at every opportunity to destroy these friends of agri- culture. Poisoning and fumigation. Undoubtedly the most effective methods of getting rid of ro- dent pests of all kinds is by poisoning them, and by trapping them. Directions for doing this applicable to the various kinds will be found in the last chapter, and need not be dwelt upon here. ANIMAL COMPETITOES 190 Fumigation is a method of despatch which has proven effective with prairie-dogs and ground-squirrels, but is of little use in the case of gophers or moles, because the latter dig so fast and so quickly close their tunnels against the fumes as to escape its effects. Machines have been invented for blowing the fumes of burning sulphur down the burrows. Better results, however, are gained with carbon disul- phide. SUPPRESSION OF RODENTS AS PESTS This is an antiseptic liquid which may be bought in quart-bottles, and is very useful in general for killing vermin, protecting stuffed birds, etc., in museum cases or in boxes, and similar purposes ; but must be kept away from fire as it is easily inflammable and explosive. It evaporates rapidly, making pungent fumes in a closed place. The method to be used is to carry it, corked, to the mouth of a burrow, pour a small quantity upon a little wad of rags or corn-husk, or a ball of horse-dung, and push it quickly as far as you can down a burrow and immediately close the entrance with packed earth. Unless the soil is,very dry and the bur- row extensive the animals at home will be smothered. In the case of prairie-dogs, the Fumigation is a method of despatch which has proven effective with prairie-dogs and ground-squirrels, but is of little use in the case of gophers or moles, because the latter dig so fast and so quickly close their tunnels against the fumes as to escape its effects. Machines have been invented for blowing the fumes of burning sulphur down the burrows. Better results, however, are gained with carbon disul- phide. This is an antiseptic liquid which may be bought in quart-bottles, and is very useful in general for killing vermin, protecting stuffed birds, etc., in museum cases or in boxes, and similar purposes ; but must be kept away from fire as it is easily inflammable and explosive. It evaporates rapidly, making pungent fumes in a closed place. The method to be used is to carry it, corked, to the mouth of a burrow, pour a small quantity upon a little wad of rags or corn-husk, or a ball of horse-dung, and push it quickly as far as you can down a burrow and immediately close the entrance with packed earth. Unless the soil is,very dry and the bur- row extensive the animals at home will be smothered. In the case of prairie-dogs, the rie dogs, SUPPRESSION OF RODENTS 191 field should first be gone over with poison, and the bulk of the animals thus destroyed. y Flooding the burrows. Where available, water is one of the best means of combatting pocket-gophers. SUPPRESSION OF RODENTS AS PESTS Flooding the land in winter is especially effective, as it wets the animals and drives them to the surface, where they soon succumb to the cold. In warm weather the method can be made effective if men and dogs are on hand to kill the animals as they seek refuge on the embankments. S. E. Piper, of the Biological Survey, reports that about the middle of April, 1909, at Modesto, Cal., he saw some boys killing pocket-gophers that had been driven from an alfalfa patch by flooding. A hundred gophers, more than half of them young of the year, were killed from a three-acre tract. y , Difficulties of extermination. Much hope has been entertained that a bacterial disease fatal to rodents, and particularly to rats, might be found, but thus far has been disappointed. It was thought that a bacillus (B. typliimur- ium) given to the field-mice which over-ran Thessaly in 1892-3 had put an end to the plague, but it is now thought it did little to ANIMAL COMPETITORS 192 hasten the end. Experiments with similar or- ganisms have been reported successful since in Eussia and in France; but none of the like at- tempts made during the mouse-plague in Ne- vada in 1908 had an appreciable effect. As Mr. Lantz says: "The destruction of noxious mammals is a more complicated problem than that of insect destruction. The farmer who fights these higher forms deals with instincts and intelligence well adapted to cope with his own in the struggle for existence. It is not enough that he place poisoned food or traps in the way of the creatures he desires to destroy; he must make the baits attractive and allay the natural suspicion of the animals by ridding traps of all suggestion of their real nature. He must know the traits of the animals and take advantage of any habit that will enable him to circumvent and destroy them." Furthermore : "In warfare against any rodent pest little per- manent good can be accomplished except by coop- erative effort. Although it always pays the indi- vidual farmer or fruit grower to exterminate pocket- gophers from his own lands, yet if he can not se- cure cooperation of the whole community he must constantly guard against a return of the pests and be ever ready to renew offensive operations against them. SUPPKESSION OF RODENTS 193 With united effort the animals can be completely exterminated over entire townships, or even counties, and when this is accomplished immunity from the pest will continue indefinitely." MOLES, SHREWS AND BATS UNDER this caption might be written a long list of American insectivores, animals which are to be thought of not merely as feeding upon insects, but as belonging to the Order In- sectivora. This order is a group of small, slender, plantigrade animals, having fine sharp teeth and digestive organs especially suited to a diet of worms and insects. They are found .all over the world, except in South America and Australasia; and are of particular interest to the zoologist because much evidence allies them with the earliest known type of mammal, so that the insectivores seem to represent, with little alteration, the most ancient mammalian stock. They are most nearly related to the lemurs and the bats. The moles under the laicn. Our insecti- vores are all small and inconspicuous, and pop- ular interest is attached only to the moles 194 MOLES, SHREWS AND BATS 195 which disfigure our lawns and flower-beds. It is commonly believed that the mole bites off roots and eats such things as lily-bulbs and sweet potatoes; but all the harm it does is now and then to upset a plant or disarrange a bit of grass-plot. It is in search of worms, grubs and burrowing insects, that the mole pushes his way beneath our feet ; and he devours a vast number of these, which do prey upon the roots and stems of grasses and other plants. The real mischief occasionally observed is due to the field-mice which sometimes follow his track. The mole has become extraordinarily well fitted for his underground work. His body is a loosely filled sack which will stand a lot of bending and squeezing, and his head is like a round wedge with a flexible point, really an exquisitely sensitive nose and a mouth filled with capable teeth. Within that sack are the most massive shoulders and forearms for their size in the animal kingdom, the latter terminat- ing in broad, strongly webbed hands, armed with long, sharp claws, like a shovel ending in five pick-blades. These great hands are twisted so that their palms are outward, thumbs down, ANIMAL COMPETITORS 196 and the thumb is braced by ail extra wrist- bone, and armed with a horny outgrowth of skin with a knife-like edge. The accompany- ing muscles are enormous. "Whenever," he says, "its course was impeded by the feet of the chairs, which were of large size, it would not go around them, but wedging itself be- tween them and the wall, pushed them with apparent ease far enough to obtain a free passage, and it thus continued to move several in succession. What was more astonishing, it passed in a similar manner be- hind the legs of a small mahogany breakfast table, and pushed it aside in the same way it had done the chairs, finally hiding itself behind a pile of quarto volumes, more than two feet high, which it also moved out from the wall." MOLES, SHREWS AND BATS As the creature goes through the soil he stretches these instru- ments ahead of his nose, drives the claws into the soil, then sweeps them outward, and so progresses by a swimming motion, kicking the loose soil behind him, and now and then throw- ing it out upon the surface in a " mole-hill. " g p The strength required for this is prodigious, as may be tested when a mole is placed among movable objects. Godman describes one which, after a fall from a mantel-piece, hurried to the wall and then began to travel around the room. MOLES, SHREWS AND BATS 197 Dr. Merriam worked out the dynamics of this last feat, and found it equivalent to a man's exerting a pressure of 12,000 pounds ! p , p Methods of the garden mole. In loose old cultivated ground, where earthworms and grubs are numerous, moles travel in every direc- tion just beneath the surface, and often never return on their tracks ; but many of their sub- terranean paths are regular galleries or run- ways, intersecting with others and centering in a home nest which seems to be occupied year after year, and often by several pairs or fam- ilies. This nest and the runways are kept in excellent repair. "When the shrew-mole en- counters a rock or an old log, or stump, in the course of his subterranean wanderings," re- marks Merriam, " instead of avoiding it he takes great pains to burrow beneath, making extensive excavations in contact with its under surface. The reason is obvious, for he knows . . . that in such places are to be found many slugs, ants with their eggs, and other tender insects." As winter comes on the mole sinks below the frost-line, as do the earthworms, and so pur- ANIMAL COMPETITORS 198 sues its prey at a safe depth. It does not hi- bernate hut is much less active than in summer, and doubtless is made so drowsy by the chill and the scarcity of air beneath the frozen top- soil that it sleeps most of the time. In sum- mer these little creatures have a curious habit of coming to the surface precisely at noon, and peering out, or even taking air and sun- shine in a little walk. MOLES, SHREWS AND BATS They are not blind, but their eyes are hardly larger than mustard seeds, so that vision must be restricted to little more than the perception of light. A closely related but smaller northeastern species is Brewer's or the hairy-tailed mole; and the Pacific coast has Townsend's and other species. The mole with the rosette. The star-nosed mole is a very interesting one, common in the Great Lakes region and on the Atlantic slope. It is larger than the garden mole, has a longer tail, a blackish-brown coat impervious to water, and particularly a rosette of pinkish fleshy feelers around the end of its pig-like proboscis. It lives by choice in swamps and wet meadows, where its burrows often open in some stream- MOLES, SHREWS AND BATS 199 bank below the water. It can swim and dive excellently, and no doubt adds to its fare of worms and grubs many small creatures and their eggs caught in the water and on the stream-bed. It seems to be more active in win- ter than the others, frequently moving about under the snow or on its surface. Moles are hardy, easily tamed and supported on shreds of meat, and exhibit intelligence as well as an ugly temper. When two or more are confined together the murder and eating of the weaker is likely to follow, until one cannibal is left. Shrews and their ways. The shrews are relatives of the moles, which do not tunnel, but are so small, secretive and nocturnal, that few persons suspect their presence, although they are numerous and of many sorts all over the country, even very far to the north. Our eastern long-tailed shrew is the smallest known mammal and could curl up in a walnut husk, yet it exists at the Arctic Circle and runs about in the snow of a Canadian winter. They are mouse-like animals, extremely swift and agile in their movements, but instantly separable ANIMAL COMPETITORS 200 by their long, flexible, trunk-like noses, be- whiskered noses, minute eyes and ears, and red-pointed teeth, which, of course, are not in the least like those of a mouse. MOLES, SHREWS AND BATS As I have written elsewhere: "All the shrews are ceaselessly active, wandering about underneath leaves, old grass, and logs, and bor- ing their way into loose loam or the punky wood of decayed stumps, in search of earthworms, grubs, beetles, slugs, and similar prey, including young mice and the fledglings of ground-nesting birds, and vary- ing this fare by bites from soft-shelled beechnuts, tuberous roots, etc. They are astonishingly quick of hearing; are bold, pugnacious, and fierce, often kill- ing and eating other shrews ; difficult to keep alive in captivity, utterly untamable, and easily frightened to death. All kinds exhale from glands near their armpits a musky odor which no doubt is protective, since most hawks, cats, foxes, etc., do not eat them unless excessively hungry; but owls and weasels ap- pear to be well pleased with such flavors, and catch and devour them in large numbers. ' ' We have a large number of shrews, some of which are aquatic, and the variety of color and size is considerable. Cuba and Hayti each also possesses a large sort of insectivore, called almiqui and looking like a shrew as big as a rat. MOLES, SHREWS AND BATS 201 Little is known of the habits of either, and they are chiefly notable because closely related to certain insectivores of Madagascar, the tenrecs, indicating descent from a common and ex- tremely primitive source, as the only explana- tion of their now living in two so widely sepa- rated regions. American bats. These insect-eating mam- mals of the air constitute a distinct group (Order Chiroptera), characterized by the pos- session of leathery wings, and other features which separate them from the terrestrial in- sectivores. North America has about twenty species, nearly all of the world-wide family Vespertilionidce, in which the nostrils are without those membranous appendages called a nose-leaf, and the ears are of moderate size and shape. On our southwestern border, however, occurs a true nose-leafed, fruit-eat- ing bat or two, representing families numerous in Mexico, Central America and the West In- dies, to one of which belongs the dreaded vampire, the blood-sucker of equatorial South America. Most of the North American bats are confined to the warm South, but half a ANIMAL COMPETITORS 202 dozen kinds are spread all over the Northern States and southern Canada, some regularly migrating southward in winter and returning in spring like birds. MOLES, SHREWS AND BATS The commonest ones are the little brown bat, which is glossy brown above and paler below; the very similar, but more southern pipistrelle ; the silvery bat, whose fur is dark brown with white tips ; and the rusty or foxy-gray red bat. All these are small (3.4 to 4.4 in. long). A larger dark brown kind, the Carolina bat (4.6 in.) is common in all the Southern States; while the North has the big hoary bat (5.4 in. long, grizzled above, white below), a long- winged, swift-flying denizen of forests, rarely seen and a winter migrant. Other species are locally well known in California and along tlie Mexican border, especially the little " free- tailed" Texan bat (Nyctinomus), which repre- sents a tropical group in which the tail is free from the membrane stretched between the hind legs All these bats are similar in habits, sleeping in some dark and sheltered place during the day, and hibernating more or less completely MOLES, SHREWS AND BATS 203 in winter. At twilight they come squeaking out to hunt in swift zigzag flight for small flying insects, thus destroying hordes of gnats and mosquitoes ; and in the early morning they take another meal before disappearing. In this business their sharp eyesight is aided by an inconceivably delicate sense of touch in their wings and elsewhere. Where caves or rocky crevices abound they often cluster on their walls in great numbers, or elsewhere throng in hollow trees ; but they are quick to resort to buildings, finding their way into barns, gar- rets, broken eaves, belfries and like places, and sometimes becoming a nuisance by their noise and dirt and abominable smell, but otherwise they are harmless. The superstitious fear of them felt by some persons is only a part of the nonsense that has come down to us from the Dark Ages, when all nocturnal animals were supposed to be somehow connected with the so- called "powers of darkness." in winter. At twilight they come squeaking out to hunt in swift zigzag flight for small flying insects, thus destroying hordes of gnats and mosquitoes ; and in the early morning they take another meal before disappearing. In this business their sharp eyesight is aided by an inconceivably delicate sense of touch in their wings and elsewhere. MOLES, SHREWS AND BATS Where caves or rocky crevices abound they often cluster on their walls in great numbers, or elsewhere throng in hollow trees ; but they are quick to resort to buildings, finding their way into barns, gar- rets, broken eaves, belfries and like places, and sometimes becoming a nuisance by their noise and dirt and abominable smell, but otherwise they are harmless. The superstitious fear of them felt by some persons is only a part of the nonsense that has come down to us from the Dark Ages, when all nocturnal animals were supposed to be somehow connected with the so- called "powers of darkness." p The bats breed annually, usually producing twins in early summer, which are born naked and cling to the mother's body, where they may be suckled wrapped in her wings as she hangs ANIMAL COMPETITORS 204 head downward in her dark retreat or carried safely as she flies abroad. Extreme care and affection are shown by the little mothers, and their babies are long dependent upon them. No animals seem to prey on our bats, but they are quarrelsome and pugnacious among them- selves, and captives are not easily tamed. Relations to humanity. Bats are not of much economic interest in this country not as much as in the eastern tropics, where it is quite impossible, in some places, to raise soft tree-fruits unless the trees are carefully and strongly screened against the big fruit-eating fox-bats. Our species, on the other hand are wholly beneficial in their feeding habits, from our point of view, because their fare consists wholly of flying insects, most of which are in some way injurious or annoying to us. It is delightful to watch their dancing flight in the twilight the more so when we try to count the number of mosquitoes they catch ; and it is per- fectly foolish for any one to be afraid of them as some women and girls pretend they are. It is also charged that they carry bedbugs and introduce them into houses. In common with MOLES, SHREWS AND BATS 205 most animals the bat acts as host to a few small insect parasites, one of which is a relative of the bedbug and somewhat resembles it. MOLES, SHREWS AND BATS It is possible that at rare intervals one may be found carrying a true bedbug, which is not one of its natural parasites, and might drop it in some garret; but the danger from this is slight. Bats are naturally cave-dwellers, and certain caves favorably situated in wild regions have been tenanted by them in enormous numbers and for an untold length of time. In such caverns, discovered in various parts of world, thick deposits of guano have been found, and have to some extent been utilized, being ex- ceedingly strong in nitrogenous elements and unimpaired, in their protected situation by the washing away of the soluble elements in which their virtue largely lies. ' l It is decidedly smaller and less conspicuous, being silver-gray, darker on the back, and tinged with rufous on the ears, sides of the neck, breast, and un- der parts, while the tips of the ears, top of the nose, FOXES AND FOX-FARMING WE have in North America several different sorts of foxes. The red fox is the most wide- spread and important of these. A large form of it dwells in southern Alaska, and another variety, or species, is peculiar to Newfound- land. Then, in the "West, are two small foxes, the kit or swift fox of the central plains, and the big-eared swift of southern California and Arizona. The kit-foxes of the west. These last are handsome grizzled-gray and yellowish little animals, with keen, interesting faces, tall, alert ears, and the ability to run like a streak. Each has a full measure of the cunning of its harried race, and makes a good living during all the warmer half of the year at the expense of mice, spermophiles and gophers, but in winter, when most of these are safe under ground, it must have a harder time, and resort to various foxy 206 FOXES AND FOX-FARMING 207 A CORNER, WITH STORES TO PREVENT THE FOXES DIGGING OUT. A CORNER, WITH STORES TO PREVENT THE FOXES DIGGING OUT. A CORNER, WITH STORES TO PREVENT THE FOXES DIGGING OUT. ANIMAL COMPETITORS 208 tricks to catch snow-birds and grouse, espe- cially in the far North. It is quite harmless to man, and would prob- ably increase rather than diminish as ranching and civilization gradually overspread its once lonely haunts, did it not so often fall a victim to the poisoned baits laid out for coyotes. This is unfortunate, especially in prairie-dog regions, for it is an indefatigable and skillful hunter of these troublesome burrowers. The gray fox. Throughout the Southern States occurs a rather small fox differing in color, for it is prevailingly gray and also in habits, from the northern red foxes. This gray fox seems to have less sharpness, adapta- bility and fearlessness than the red; and it is also less fecund, its young rarely exceeding four or five annually, whereas the litter of the red often numbers seven or eight. Otherwise the gray fox seems to have several advantages, as I have pointed out in my Life of Mammals: FOXES AND FOX-FARMING 209 chin, and feet are black. FOXES AND FOX-FARMING It is a woodlander, and seems incapable of adapting itself to the cleared dis- tricts in which the red fox so easily makes itself at home; climbs trees almost like a cat, and takes to them naturally for safety or to get grapes and per- simmons to eat. There, too, it makes its home in a hollow stump or log, not digging a burrow, for the weather of its southerly habitat, and the later date of its breeding, do not require for its young the warmth of an underground nursery ; and all the year round it can supply itself with food by its own cun- ning tricks, while the red fox must wander over many miles of country. The ground-breeding birds and waterfowl and their eggs form its principal fare, perhaps in summer, when hens or turkeys straying in the woods are likely to be seized; but rarely is the poultry disturbed on the home roost, nor can such worse depredations as killing young pigs, lambs, etc., be laid at its door. Audubon, whose account of this to him very familiar animal is circumstantial, speaks of it as a 'pilfering thief and of the red fox as a 'daring and cunning plunderer.' Gray foxes will run before hounds only a short distance, doubling constantly and for a short time, when they either 'hole' in a tree or climb one; while a red fox may run straight eight or ten miles away and then back in a parallel course. parallel "Extremely interesting is the arctic fox, of the polar regions right round the world. It is a shy, swift little beast with blunt nose, short rounded ears, a very long bushy tail, and the soles of its feet well ANIMAL COMPETITORS 210 shod with moccasins of hair, giving them a firm hold on the slippery rocks, snow, and ice, over which it leaves its tiny tracks from Labrador to the Lincoln Sea. Every arctic explorer from Steller down has had much to say of this animal, the accounts given by Richardson, Feilden, and Nelson being especially full and good. The most remarkable feature of its history relates to its varying phases of coloration. During the short arctic summer its dress is brown with the under parts lighter, often drab. FOXES AND FOX-FARMING In autumn this coat is replaced by one of pure white, beneath which is a fine wool; and this warm, white dress, invisible against the snow, is the normal winter hue of the great majority of arctic foxes. A small pro- portion, however, are never either white or dark brown, but are slate-gray all the year round. This double phase may occur anywhere, one or two, per- haps, arising from a litter that becomes white; but in some rather southerly places the 'blues' prevail, forming a local race. Such is the case in Greenland, Iceland, and in the Aleutian Islands, where blue foxes are now carefully preserved and cared for in a semidomestic condition, for the sake of their highly valuable fur, a certain number being killed an- nually." The American red fox. Returning now to the common or red fox, it appears that this is one of the most widely distributed of animals, for it is hard to distinguish more than such FOXES AND FOX-FARMING 211 local varieties as might easily arise in different climates and from local peculiarities of food throughout the whole northern world. It is convenient to name them as species, but it is practically the same "Reynard the Fox" right around the globe. "Our American form," to quote again from my Life of'.. .Mammals heretofore mentioned, "seems especially variable since its typical yellowish red, darkest on the back and shoulders, may be very bright or very pale ; or may have the markings on the spine and withers very dark and distinct, making it a 'cross fox,' or be totally black with a white-tipped tail ; or black, with the tips of most of the hairs white, giving the fur a frosted or 'silver' appearance. . . . "Our American form," to quote again from my Life of'.. .Mammals heretofore mentioned, "seems especially variable since its typical yellowish red, darkest on the back and shoulders, may be very bright or very pale ; or may have the markings on the spine and withers very dark and distinct, making it a 'cross fox,' or be totally black with a white-tipped tail ; or black, with the tips of most of the hairs white, giving the fur a frosted or 'silver' appearance. . . . FOXES AND FOX-FARMING "Foxes everywhere are naturally burrowers and nocturnal hunters of ground-nesting birds from ducks and geese to sparrows, and of their eggs; rodents of every sort, frogs, lizards, insects, and in summer and autumn fruit and berries. Some of the prey is got by running it down, for the fox is fleet; some by digging it out of its underground holes; some by stalking it with crafty caution; some by lying appar- ently dead until the victim approaches near enough to be seized by a catlike pounce. These are the es- sential tactics of its food getting in all lands, the fare and the method varying with the country; and end- less stratagems match the native precautions of the small quarry. All the larger cats and wolves are its giving appearance. "Foxes everywhere are naturally burrowers and nocturnal hunters of ground-nesting birds from ducks and geese to sparrows, and of their eggs; rodents of every sort, frogs, lizards, insects, and in summer and autumn fruit and berries. Some of the prey is got by running it down, for the fox is fleet; some by digging it out of its underground holes; some by stalking it with crafty caution; some by lying appar- ently dead until the victim approaches near enough to be seized by a catlike pounce. These are the es- sential tactics of its food getting in all lands, the fare and the method varying with the country; and end- less stratagems match the native precautions of the small quarry. All the larger cats and wolves are its ANIMAL COMPETITORS 212 enemies in the wilderness, and the skill in avoiding them inherited from innumerable ancestors serves it well when in civilized lands the fox finds troops of dogs set upon its track. g p "Standard works are supplemented by admirable essays on the American fox by Thoreau, Burroughs, Lottridge, Robinson, Seton, and others who know him well; none is more complete and intimate than the history given by Mr. Cram, who asserts that in New England, at least, the foxes in cultivated districts are far more highly developed in intellect than are those of the outlying parts, or than were the foxes of a century ago. FOXES AND FOX-FARMING They are the most bold, skillful, and inveterate of poultry thieves, and will sometimes take as many as 'thirty pullets in a single night'; and often half or more of the booty of such a raid will be found in a pile in some hiding-place, which goes to show that the foxes of all cold regions prob- ably store surplus food. In return for levying upon his chickens (or, in Europe, upon the pheasants and other treasures of the gamekeeper) the animal aids the farmer by destroying numberless rats, mice, gophers, and similar pests." Value of fox fur. Of all the products de- rived from wild animals furs are the most useful and valuable. Indispensable to primi- tive man, they are scarcely less important to the most civilized, for in warmth, beauty and durability, no manufactured fabrics excel them, FOXES AND FOX-FARMING 213 But expanding civilization is steadily diminish- ing the supply of furs, and the animals which bear them are proportionately decreasing, es- pecially those whose coats are of high quality. The growing demand may be met partly by stricter enforcement of the game laws, but it is evident that it is becoming necessary to propagate fur-bearers in confinement, and by this means an important and new industry will presently be developed. This has already been the subject of no little thought and experiment, but mainly in reference to the smaller and less valuable animals, such as skunks and minks, as we shall see. Eesults of considerable impor- tance have been obtained recently with Alaskan blue foxes, and a good deal has been done quietly in experimental cultivation of the Cana- dian silver fox. This last industry has recently been made the subject of official investigation by Mr. W. H. Osgood, of the United States Department of Agriculture, from whose report the present article is mainly compiled. More persons have engaged in it than the public generally is aware of, for they have been disposed to keep their ANIMAL COMPETITOES 214 experiments quiet and their results a secret; but a recent investigation pursued by W. H. Osgood of the Biological Survey has gathered many facts as to method, of which I shall avail myself liberally. Variability of the red fox. FOXES AND FOX-FARMING The common fox, as has been said, varies from red to black, and these extremes, with the gradations be- tween them, form four more or less distinct phases, respectively known as red, cross (or patch), silver, and black. In the red phase the animal is entirely rich fulvous, except restricted black markings on the feet and ears, a white area at the end of the tail, and certain white-tipped hairs on the back and rump. From this phase to the next the black increases in extent until, in the typical "cross" fox, the black predominates on the feet, legs and underparts, while fulvous over- lying black covers most of the head, shoulders and back. A gradual increase of the black and elimination of the fulvous, or its replacement by white, brings us to the next phase, the "sil- ver, " or " silver-gray, ' ' in which no fulvous ap- pears, the entire pelage being dark at the base FOXES AND FOX-FARMING 215 and heavily or lightly overlain with grayish- white. Silver foxes vary from those in which the color is entirely grizzled to those in which it is entirely black, except a few white-tipped hairs on the back and rump. Finally, in the black phase, the white is absent from all parts except the tip of the tail. The red phase is much more abundant than the others, but the three interbreed freely, and wherever one occurs occasional examples of the others also may be expected. In general, the cross fox is fairly common, the silver-gray is comparatively scarce, and the pure black is excessively rare. The prices usually paid for skins of the different phases vary according to the relative scarcity of the animals. Thus red skins command only a moderate price ($1.50 to $3.50), cross foxes are somewhat higher ($4 to $8), silver foxes are several times higher ($50 to $250), and pure black skins are exceedingly valuable, being higher priced than any other fur except sea-otter $1,000 to $2,000. except $ , Area suited for fox-farming. The natural habitat of this fox includes the greater part of North America, from the central United States ANIMAL COMPETITORS 216 northward to and including the border of the treeless tundra. The red phase inhabits nearly all this region, but the silver phase, although known in most parts of it, is very irregularly distributed. FOXES AND FOX-FARMING In general it is much more com- mon in northern localities than in southern, and seems especially numerous in Newfound- land and on the interior heights of Labrador. Altogether, it appears likely that the area suitable for rearing silver foxes successfully is confined to Canada, and a small strip of coun- try south of it, including the higher parts of the Alleghenies. Prince Edward Island has already about 100 breeders, and can supply good breeding-stock. g Arrangement of breeding-quarters. It is a mistake to suppose that a great space is re- quired for rearing silver foxes, or a rough area approximating natural conditions of fox-life. Indeed, this is disadvantageous, for it tends to keep the animals so wild as to be unmanage- able. The endeavor should always be to tame the captives as much as possible, and to do this a small and uniform area is necessary. Foxes thrive in enclosures not more than 40 feet FOXES AND FOX-FARMING 217 square. These may be but a few rods from a farm-house, or, if visitors are excluded, in a quiet place on the outskirts of a village. A half-acre will accommodate about six pairs of foxes, which is quite as many as a beginner should attempt to handle. The selection of ground may depend upon circumstances, but ef- fort should be made to include a few trees or small shrubs. These afford shade and a feel- ing of seclusion and security to the animals. Inclosures for foxes are made with woven- wire fencing, but the mesh should be not greater than 2-inch, for young foxes are able to wriggle through an opening three inches square. The fencing should be about 10 feet high and sunk into the ground two feet. The foxes try to escape, when first placed in the pen, by digging at the edge of the wire, and abandon the effort when they find themselves stopped near the surface. The top of the fence, however, must have an inward overhang of two feet to prevent the animals climbing out. g Form of enclosures. In the arrangement of sub-divisions the general plan should conform to that shown in the accompanying diagram. FOXES AND FOX-FARMING ANIMAL COMPETITOES 218 Here a wide outer court is provided, separating the smaller enclosures in which the foxes are actually kept from the unfenced area possibly FOXES AND FOX-FARMING 219 to give additional security as to prevent curi- ous visitors or stray dogs, etc., from annoying the foxes. Seclusion, indeed, is of prime im- portance, hence no one ought to be permitted inside it except the regular keeper, to whom the prisoners are accustomed. Any means which will effect the desired seclusion, as hedges or a high-board fence, may be used instead of this outer wire enclosure. The inner enclosures are of two kinds, most of them small and designed for single animals or pairs, but one or more are larger and in- tended to accommodate a number of foxes at one time. Every compartment should be pro- vided with doors so arranged that animals may be transferred from one to another readily. The beginner with only one pair of foxes may start with two of the small compartments, and add others as needed, keeping in mind a con- venient general plan. The small compart- ments should be at least 30 feet square. Those shown in the diagram are 30 by 40 feet, and the larger runs 75 by 40 feet. Passageways giving free access to all the compartments should be 4 to 6 feet wide. ANIMAL COMPETITORS 220 Each compartment should contain a small house or shelter-box, for, although the foxes often dig natural dens in the ground, they usu- ally accustom themselves readily to artificial shelters. A common form of these is much like a dog-kennel and about the same size. They are ordinarily made four or five feet square and two or three feet high, with an entrance about six inches square. No nesting material is needed inside the boxes, as the old foxes either do without or provide themselves from refuse in their enclosure. Foxes easy to keep. The mere keeping of foxes in confinement is a simple matter. They do not, as a rule, however, become very tame, even after several generations. They seem contented and happy in their cages, and rarely make determined efforts to escape. FOXES AND FOX-FARMING Several cases are recorded where captive silver foxes, having climbed out of their enclosures in win- ter, when high drifts of snow gave them a chance to reach the top of the fence, have re- turned voluntarily to their home. Although in general suspicious of mankind and inclined all their lives to snap at or bite FOXES AND FOX-FARMING 221 even their best known and kindest attendants, these foxes do not quarrel much among them- selves, especially where properly fed. So far as known, fatal disease has been so rare as to be negligible in any general con- sideration of fox-raising. Here and there an animal has died of some unknown internal complaint, but no particular disease has mani- fested itself. Nothing in the nature of an epi- demic has thus far appeared, and even minor diseases have been exceedingly few. Fleas occasionally have proved troublesome, and, no doubt, foxes may contract mange and other diseases to which dogs are subject, but if kept in cleanly quarters and fed properly they are reasonably safe. y Cold weather has no terrors for them, and they delight in snow, but should not be per- mitted to lie upon it when it is alternately freezing and thawing, as their fur, by freezing to the crust and then being torn loose, will be injured injured. Food and feeding rules. Wild foxes eat a great variety of food, including mice, rabbits, birds, and insects, such as grasshoppers, crick- ANIMAL COMPETITORS 222 ets and beetles. At certain seasons berries are eaten in large quantities. Meat, therefore, is only a part of their natural diet, and if fed exclusively is likely to have ultimately a bad effect. It is much better to supply the foxes with a mixed diet, including, besides meat, such food as bread, milk, table-scraps or dog-bis- cuits, all of which are relished. There is less danger in any particular food than in too large quantities at irregular intervals. Over-feed- ing produces fat, sluggish animals, that do not breed well, and it has been responsible for some expensive failures. The normal weight of a fox is from six to nine pounds, so animals weighing over ten pounds are too fat. A reg- ular daily ration is the proper method, both for the sake of their stomachs and because it tends to a more constant and friendly relation be- tween the keeper and his charge. FOXES AND FOX-FARMING It is a good plan, nevertheless, to give them bones with lit- tle meat on them, now and then, upon which they may gnaw indefinitely. Occasionally they may be regaled with tidbits consisting of small wild mammals, as rabbits, woodchucks, rats, mice and other animals likely to be captured FOXES AND FOX-FABMING 223 about the farm. Fresh drinking-water, of course, should be supplied regularly. If a spring or other natural supply can be included within the yards much labor is saved. A daily allowance for each fox, according to the experts consulted by Osgood, is one-fourth of a pound of meat and a small handful of miscellaneous scraps. One of the most success- ful breeders feeds a quarter of a pound of meat and a quart of skim milk daily. Another varies the meat-diet with a sort of hoecake made of corn meal and sour milk. The meat used is beef or mutton in the form of butchers' scraps, unsalable parts, and the like or, most com- monly, horse-meat procured especially for the purpose. p p In the producing season, November to March, feed must be restricted to just the right quantity and carefully chosen. In the summer less caution is required. Two eggs should be given daily to a nursing mother for a month after the pups are born; and fresh milk three times a day. When located on the seacoast near fishing settlements fox-raisers supply fish, lobsters, and other sea-foods to their foxes at ANIMAL COMPETITORS 224 little or no cost, and find them satisfactory. Take care that the stronger, quicker foxes do not rob and starve their fellows. Reproduction, and treatment of young. Foxes breed only once a year, beginning when a little less than a year old, but the first litter will be small. The mating or rutting season includes the months of February and March, ONE FORM OF BREEDING KENNEL. A barrel, with a similar elbowed entrance might easily take the place of this box. ONE FORM OF BREEDING KENNEL. A barrel, with a similar elbowed entrance might easily take the place of this box. and the young are born in April and May. The number of young in a litter varies from two to eight, the average number born to adult parents being five. In the wild state foxes are monogamous. The male has only one consort, at least only one in a season. FOXES AND FOX-FARMING In confinement, however, one FOXES AND FOX-FARMING 225 male sometimes has been mated successfully with two or even three females. In certain cases this may be desirable, and at an advanced stage of the business may offer no difficulties, but at first it is advisable to handle the animals in pairs. It is possible, also, as proved in a number of instances, to allow male and female to remain together throughout the year with- out bad results, but it is much better to keep them separate, except during the mating sea- son. They may be paired in December or Jan- uary and separated in March or April. The females should be kept in- the small enclosures continuously and the young removed when weaned. The separation of the sexes is not, as many suppose, to prevent the male from viciously killing the young; for, unless suffering from hunger, he usually is a model parent. But the presence of the male often results in injury to the female during pregnancy, resulting in abor- tion; or it excites her unduly after the young are born, leading to rougher treatment than they are able to stand. y Some foxes are much better breeders than ANIMAL COMPETITORS 226 others ; some can never be induced to mate, and others mate, but do not produce young. They are constantly in a state of fear, and this fear is probably the chief cause of the failure to breed regularly. It may cause the female to refuse the attentions of the male, or, having re- ceived them, she may prove infertile, or she may become excited so as to injure herself and give birth prematurely. But, worst of all, even after producing a litter of healthy young, she may be so solicitous for their safety that in her effort to get them out of imaginary harm's way she maltreats or kills them. y When born the young are small and weak, but if all is well they grow rapidly, and when about six weeks old begin to come out to play and to lap a little milk or to take an occasional bit of solid food. If allowed to do so, they will continue to nurse for nearly six months. y Importance of good care. Keeping the foxes in a secluded place free from visitors is not sufficient alone to overcome these difficulties. FOXES AND FOX-FARMING Although strangers should be kept away, a regular attendant should visit the animals daily and use every effort to gain their conn- FOXES AND FOX-FARMING 227 dence. This is not easy, and a great deal de- pends upon the personality of the man in charge. One not thoroughly interested or not naturally fond of animals, and therefore slow to understand their ways, is not likely to suc- ceed. Careful observation and a faculty of intuition enables a good keeper to anticipate the moods of the animals, and to interpret their actions at critical times, so as to act quickly and without violence. He knows just when the foxes are getting too much food, just when the sexes should be brought together or separated, when the female becomes pregnant, when the young should be born, when they need special attention, and when they may safely be left to the exclusive care of the mother. He is not over-inquisitive as to the number of young that are born, and seldom needs to disturb the anx- ious parent, relying on her actions to show whether the little ones are thriving. BREEDING FOB IMPROVED STOCK. Hope for increased profits in fox-raising lies almost entirety in improving the stock, and successively getting better and blacker coats, by ANIMAL COMPETITORS 228 selective breeding. The darker the animal the more valuable its pelt. Hence the object of every breeder should be to produce pure black foxes, or as nearly pure black as possible. To do this he must retain his darkest and most valuable animals for breeding, selling only the poorer ones. The temptation to sell animals of high value is often very great, but in the long run such animals are likely to be more profitable if kept for breeding. The possibil- ities of modification and improvement by selec- tion are fully as great with wild animals as with domestic, and already have been demon- strated in the case of foxes. Some of the high- est-priced fox-skins ever put on the market have been from animals reared in confinement and improved by selective breeding. p y g Breeding for disposition is perhaps fully as important as breeding for color. So far this has not been attempted to any extent, but in Mr. Osgood's opinion it may be of great im- portance in overcoming some of the princi- pal difficulties now encountered. By selecting those animals which show the least aversion to man, due regard being paid to other qualities, FOXES AND FOX-FARMING 229 such as prolificness, a strain may be obtained which will breed with the certainty of our do- mestic animals. This in time should produce a thoroughly domesticated race of foxes, a re- sult of inestimable value, amply justifying the utmost efforts. Although it may not be fully accomplished by those who begin it, every breeder should keep its importance in mind, for every slight improvement will be to his ad- vantage, and in the end the unqualified success of the business will be assured. Slight improvement of individual male an- imals not intended for breeding may be ob- Ained by castration. This has been tried with red foxes, and found to yield an animal of somewhat increased size. In buying a young breeding-pair of silver foxes great care is nec- essary. Try to see the parents of the proposed purchase. October is the best month for buy- ing, and examination should be made in bright sunshine. BREEDING FOB IMPROVED STOCK. An experienced breeder advises the use of a magnifying-glass in examining the fur to see if it is thick and glossy with a long dark under-fur and no trace of red, especially inside the ears ANIMAL COMPETITORS 230 Preparation of skins. The preparation of skins requires some care, but no special imple- ments or preservatives. The opening and only cut is made with a sharp-pointed knife, begin- ning on the bottom of one hind foot and extend- ing up to the hind side of the leg to the vent and thence down the other leg to the foot. The entire body is removed through this opening, using the knife to separate the skin when necessary, and proceeding down over the head to the lips, where the final cuts are made. Thus the skin is turned completely inside out. The -tail bone must be carefully withdrawn, prefer- ably by the use as a vise of two firmly-held sticks (or a split stick), through which the bone is passed. To facilitate this it may sometimes be necessary to slit the tail on the under-side. The skin is then carefully fleshed that is, all the fats and bits of flesh adhering to it are re- moved. To dry the skin it is slightly stretched on a long, narrow, somewhat tapering board with a blunt, rounded end. After slipping over the board (hair side in) it should be hung in a cool, dry place, and allowed to dry gradu- Preparation of skins. The preparation of skins requires some care, but no special imple- ments or preservatives. The opening and only cut is made with a sharp-pointed knife, begin- ning on the bottom of one hind foot and extend- ing up to the hind side of the leg to the vent and thence down the other leg to the foot. The entire body is removed through this opening, using the knife to separate the skin when necessary, and proceeding down over the head to the lips, where the final cuts are made. Thus the skin is turned completely inside out. The -tail bone must be carefully withdrawn, prefer- ably by the use as a vise of two firmly-held sticks (or a split stick), through which the bone is passed. To facilitate this it may sometimes be necessary to slit the tail on the under-side. BREEDING FOB IMPROVED STOCK. The skin is then carefully fleshed that is, all the fats and bits of flesh adhering to it are re- moved. To dry the skin it is slightly stretched on a long, narrow, somewhat tapering board with a blunt, rounded end. After slipping over the board (hair side in) it should be hung in a cool, dry place, and allowed to dry gradu- ally. Ordinarily no preservative is necessary, FOXES AND FOX-FAKMING 231 and the drying should not be hastened by ex- posure to the sun or artificial heat. p Expectation of profit. Every silver-fox raised is likely to yield a pelt having a market value of over $100. Even pale skins bring this figure, and darker ones much more. Pure black skins command prices ranging from $500 to $2,000. It is, therefore, evident that a moder- ate income may be derived by raising compara- tively few foxes. In the present stage of the business the sale of foxes for breeding-stock is very profitable, as the live animals in good con- dition often bring fully twice as much as their cured skins. In fact, good, live, silver foxes seldom can be obtained for less than $500 per pair, and much higher prices have been paid. p g p The high prices paid for silver-fox skins are due to the rarity of the animals, and the ex- tensive production of such skins would neces- sarily tend to a reduction in price. Increasing population and wealth, however, insure a large future demand for fine furs, and no great de- crease in prices is likely to occur until produc- tion reaches large proportions. GRAY WOLVES AND COYOTES THE time has gone by when the farmer in the eastern half of the United States has to guard his stock and perhaps his family against wolves as in the days of his forefathers. In the north- ern parts of Canada, however, in the forested parts of northern Michigan, Wisconsin, and; Minnesota, and in scattered localities through- out the whole Northwest, and thence northward to the Arctic regions, the great gray or timber wolf is still a menace to the ranchman if not to the cultivator of fields. It also causes the destruction of great numbers of game espe- cially deer, which can ill be spared; and now and then attacks travelers or their horses when picketed out at night. Consequently ranchmen are everywhere mak- ing determined cooperative efforts, aided by the government, to kill them off, by breaking up their dens and trapping and poisoning the 232 GRAY WOLVES AND COYOTES 233 old ones. The most effectual method is by searching out and destroying the dens and pups, of which six to ten are usually born in a litter to a pair of gray wolves. These are produced early in spring in some rocky niche or cave or sheltered hollow in the open country, and in a hollow log or stump when the region is forested. Both parents continue in company, caring for the young, until the latter are well-grown. y g g Character of the coyote. The smaller-red- dish prairie-wolves or coyotes (coy-yo-teh) are far more widespread, numerous and annoying, though rarely dangerous ; and if farming oper- ations, with their attending domestic animals and poultry, are to be carried on in the plains country or mountain valleys of the West ; and if sheep-farming is ever to be made productive there, these keen and pertinacious little wolves must be subdued. At the same time it must not be forgotten that they perform a most excellent service by killing a vast number of noxious mice, gophers, prairie-dogs, rabbits and other pests. If it were possible, then, to keep the coyote as a harmless ranger of the plains, a sort of Cossack that freed the fron- ANIMAL COMPETITORS 234 tiers of marauders while doing no damage to human arts and interests it would be a most advantageous arrangement; and it is toward this solution of the problem that efforts should be concentrated. GRAY WOLVES AND COYOTES The following general ac- count of the animal and its habits is by David E. Lantz: "While in general denizens of the higher open plains, coyotes are found also on the low tropical coasts of Mexico and Texas and in the higher moun- tain ranges of the interior. In the northern and northeastern parts of their range they inhabit par- tially wooded country, and even on the plains they are partial to broken and hilly sections. p "Coyotes breed once a year. The mating season is late in January or early in February. The period of gestation is about sixty-three days. The young are produced in dens and number from four to eight or even more. The dens are usually enlarged from those made by badgers or smaller animals, and are often among rocks or in washed-out places along banks of streams. Probably at times they are made entirely by the coyotes. They are rarely far below the surface, but sometimes of considerable extent, and with two or more openings. Little attempt is made to provide nests for the young. In the Central West these are born early in April, and usually may be heard in the dens during May. In June they come out to play around the mouths of the burrows, which GRAY WOLVES AND COYOTES 235 are finally deserted during July. By August 1 the young are left by the parents to shift for themselves. ' ' In the earlier descriptions the prairie wolves were usually said to hunt in packs. Lewis and Clark, Say, Richardson, and others so reported, but the Prince of Wied met them only singly. It is probable that they hunt in numbers only when the quarry is large, as in the case of deer and antelope, and as many as three have been known to pursue a single jack-rabbit. are finally deserted during July. By August 1 the young are left by the parents to shift for themselves. ' ' In the earlier descriptions the prairie wolves were usually said to hunt in packs. Lewis and Clark, Say, Richardson, and others so reported, but the Prince of Wied met them only singly. It is probable that they hunt in numbers only when the quarry is large, as in the case of deer and antelope, and as many as three have been known to pursue a single jack-rabbit. GRAY WOLVES AND COYOTES "Coyotes feed chiefly upon animal matter, but when such food is scarce they freely eat peaches, apricots, grapes, and other fruits, and even melons, usually destroying more than they eat. In certain areas they feed largely on juniper berries, manzanita berries, and the fruit of the prickly pear. . . . Horned toads and other lizards are eaten, and, on the low, tropical coast of eastern Mexico and Texas, coyotes have been seen searching the beach for crabs, fish and turtle-eggs. are finally deserted during July. By August 1 the young are left by the parents to shift for themselves. ' ' In the earlier descriptions the prairie wolves were usually said to hunt in packs. Lewis and Clark, Say, Richardson, and others so reported, but the Prince of Wied met them only singly. It is probable that they hunt in numbers only when the quarry is large, as in the case of deer and antelope, and as many as three have been known to pursue a single jack-rabbit. "Coyotes feed chiefly upon animal matter, but when such food is scarce they freely eat peaches, apricots, grapes, and other fruits, and even melons, usually destroying more than they eat. In certain areas they feed largely on juniper berries, manzanita berries, and the fruit of the prickly pear. . . . Horned toads and other lizards are eaten, and, on the low, tropical coast of eastern Mexico and Texas, coyotes have been seen searching the beach for crabs, fish and turtle-eggs. "Coyotes feed chiefly upon animal matter, but when such food is scarce they freely eat peaches, apricots, grapes, and other fruits, and even melons, usually destroying more than they eat. In certain areas they feed largely on juniper berries, manzanita berries, and the fruit of the prickly pear. . . . "Beneficial food-habits. Coyotes destroy many in- jurious species of mammals, and in this way are of positive benefit to farming interests. The various species of jack-rabbit are often included in their diet, and the smaller rabbits are habitually eaten. The constant warfare of the coyote upon these rodents has much influence in keeping down their numbers, and the growing abundance of rabbits in some sec- tions of the West has been attributed to the destruc- tion of coyotes as the result of high bounties offered for them. "Prairie-dogs also are a staple coyote food. GRAY WOLVES AND COYOTES The ANIMAL COMPETITORS 236 coyote usually captures them by hiding behind clumps of weeds or bunches of grass at some distance from the burrows, and when, in feeding, the unsus- pecting rodent approaches near enough a few leaps enable the coyote to capture it. It is probably the fear of the coyote that causes the prairie-dogs to crop off all the tall, growing grass and weeds near their burrows. "In addition to rabbits and prairie-dogs the food of the coyote includes rice-rats, kangaroo-rats, wood- rats, ground-squirrels, woodchucks, pocket-gophers, chipmunks, and pocket-mice. All of these are harm- ful to agriculture, and the coyote in preying upon them performs a valuable service to man. This service is not spasmodic, but lasts throughout the year and throughout the life of the coyote, and has an important influence in helping maintain the 'bal- ance of nature/ "The coyote is useful also as a scavenger. In the prairie country, especially in winter, it comes into towns at night searching for garbage. Here it finds remnants of meat from the table, offal, and similar prizes. "When hungry it rejects no animal food, not even carrion. The slaughter-houses near the towns are favorite feeding places, and the animals are often shot there. On the ranges they soon consume dead horses and cattle, leaving the bones clean. g "Injurious food-habits. Considerable game is de- stroyed by coyotes, including quail, grouse, and wild ducks, and their eggs. . . . hens, ducks, geese, and turkeys. Its usual method of capturing GRAY WOLVES AND COYOTES 237 them in daytime is to lurk behind weeds or bushes until the fowls are within reach. Turkeys, which range far afield in search of grasshoppers and other insects, are frequent victims. At night the coyote captures poultry from the roost unless care is taken to guard against its entrance. A correspondent of the Biological Survey wrote from Eexburg, Idaho, that one neighbor lost 60 chickens and another 30 in one night by coyotes. A correspondent in Mayer, Ariz., wrote that he had lost about a hundred chick- ens by coyotes, but that, although they destroyed poultry, he believed them to be beneficial, as they kept down the rabbit pest. ept pest. GRAY WOLVES AND COYOTES "In approaching ranch buildings, either by day or by night, the coyote comes from the leeward side and with great caution; but once satisfied that no danger lurks in the shadows, it becomes very bold." The coyote as a pest. Few of the mammals of the farm are exempt from the raids of this enterprising little wolf, whose record of mis- deeds includes the capture and death of young colts, calves, pigs, lambs and goats. The coyote watches until the little ones are left un- guarded a moment by their mothers, then rushes in. Under exceptional circumstances old ani- mals may be pulled down. It is especially, however, as an enemy of sheep that this hardy wolf becomes important in its relation to human ANIMAL COMPETITORS 238 industry. In many parts of the West the rais- ing of sheep has been greatly deterred on this account; and woeful stories of destruction are on record. It was this state of affairs that led the Biological Survey a few years ago to make special studies of the coyote situation. "It is evident that the wealth of any State can be materially increased if it is possible everywhere to keep small flocks of sheep. Flocks increase rapidly under favorable conditions and good management, and the cost of keeping is small when herders can be dispensed with. The double product, wool and mutton, usually places the profit of handling sheep above that of cattle or horses. The gains also come oftener, since sheep mature in a year, while cattle and horses require three. q "In the region about Seguin, Tex., according to Vernon Bailey, chief field-naturalist of the Biological Survey, no sheep are kept, because of the abundance of coyotes. The farmers admit the advantage of in- troducing sheep, but the fear of coyotes deters them from the experiment. Similar conditions prevail over large areas in many parts of the West. The number of sheep in the United States has been de- creasing during the past two years [1904-5], while the price of wool has been excellent and the demand for mutton steadily increasing. Montana, with an area of 146,000 square miles, leads the States in the number of sheep kept, which is 5,638,967. Yet Eng- GRAY WOLVES AND COYOTES 239 land, with an area of only 50,867 square miles, has about five times as many as Montana. GRAY WOLVES AND COYOTES In Montana sheep are herded in immense flocks ; in England every landowner and farmer keeps a small flock. "The advantage of sheep upon the farm as weed- destroyers is not usually appreciated in America. The Iowa Agricultural Experiment Station reports that out of 600 species of grasses and weeds, cattle are known to eat only 50, horses 82, while sheep eat 550. "With abundance of pasturage, favorable cli- mate, good prices for wool and mutton, and increased fertility and productiveness of the soil upon which sheep are grazed, there should be a decided advance in the sheep industry. p "The chief discouragement seems to lie in the dep- redations of worthless dogs and coyotes. The evil of worthless dogs can be best remedied by a resort to taxation. Dogs should be regarded as property and taxed sufficiently to put all of the dangerous and worthless curs out of existence." The methods which have and may be used to lessen this pest by traps and poison are dis- cussed in the final chapter. Here it may be said that none of them are as satisfactory as fenc- ing, in spite of the stimulation of bounties by States and counties, and of the encourage- ment of hunting coyotes as a sport and good sport it is when one rides to Russian wolf- hounds or greyhounds. ANIMAL COMPETITORS 240 Fencing against other wild animals. Fen- cing as a means of protection against wild an- imals has for several years been in use in the Australian colonies and in South Africa. In Australia wire nettings are used successfully to keep rabbits, dingoes, and some species of kan- garoos out of pastures and crops. In Cape Colony jackals are a great hindrance to sheep- and ostrich-farming, and have been successfully checked only by wire-fencing they could not get over, nor under nor through. The expense of such fencing -in our own plains country would probably be $250 a mile; but it would pay for itself, according to the South African experi- ence, in the increased number of lambs reared, fleece secured, health of the stock, improved pasturage and less cost of herding. p g To those interested these arguments do not need expanding nor expounding ; nor is it need- ful to explain and discuss the recommenda- tions for various designs of fence. CHAPTER XV CHAPTER XV GRAY WOLVES AND COYOTES The follow- ing summary of recommendations made by Special Agent Lantz, of the Biological Survey, in 1905, are good for to-day, and have stood the test of experience; GRAY WOLVES AND COYOTES 241 "Summary of Conclusions. (1) Prairie coyotes will not willingly jump over a fence above 30 inches in height g " (2) They will readily climb over fences built of horizontal rails or crossbars, especially in order to escape from captivity. p captivity. " (3) Barbed wires do not deter them from crawl- ing through a fence to escape. " p p y " (3) Barbed wires do not deter them from crawl- ing through a fence to escape. " g g p " (4) Woven-wire fences should have meshes, when rectangular, less than 6 by 6 inches to keep out coyotes. For such fences triangular meshes are much better than square ones. g g p " (4) Woven-wire fences should have meshes, when rectangular, less than 6 by 6 inches to keep out coyotes. For such fences triangular meshes are much better than square ones. q " (5) In fencing against coyotes with woven fences care must be used to see that there are no openings at the ground through which the animals can force themselves, since they are more likely to crawl under a fence than to jump over it. " q " (5) In fencing against coyotes with woven fences care must be used to see that there are no openings at the ground through which the animals can force themselves, since they are more likely to crawl under a fence than to jump over it. " j p " (6) It seems reasonably certain that a fence con- structed of woven wire with a triangular mesh not over 6 inches across and having a height of 28 to 42 inches, supplemented by two or three tightly stretched barbed wires, would prove to be coyote-proof. It is difficult to make exact estimates of the cost. Woven fences differ in weight, price, and durability, and freight charges on materials depend on the distance from distributing points. The cost of posts and labor varies much. An estimate based on so many variable factors is of little value, but an average of $200 per mile would probably allow the use of the best ma- terials,'" THE FUR-BEAKERS AND THEIR CULTURE THERE now present themselves a company of small carnivores of unusual interest in every aspect the martens, ermines, wolverines, bad- gers, skunks, etc., all of the weasel family Mustelidce. "They constitute," as I have written elsewhere, "an army of sharp-toothed, keen-witted, bloodthirsty devourers of the small life of the world, doing in the North the police work which in the Oriental tropics is committed to the civet-cats and mungooses. These are the animals whose coats, acquired to keep themselves warm amid arctic frosts, make our most beautiful furs, as sable, marten, mink, ermine, and the rest. The sable is Siberian, the marten is North-European, and its Ameri- can brother is the pine-marten, or ' sable ' of the Canadian forests. The three are scarcely dis- tinguishable, each averaging about eighteen in- ches in length, plus seven or eight inches of tail, 242 CULTUEE OF FUR-BEAKERS 243 and are brown, somewhat lighter below (throat and breast-spot orange in the Canadian sable), and variable according to age, sex, and season. The winter fur is thick, soft, an inch and a half deep, of richest hue, and has scattered through it coarse black hairs which the furrier pulls out; the tail is somewhat bushy. . . ." y Canadian fur-bearers. For two hundred and fifty years the Canadian marten has sup- plied, as had the sable for perhaps as many centuries, the most valuable furs sent to market, excepting a few rarities like sea- otter. North America also contains the giant of the tribe in Pennant's marten, named by the early French Canadians pekan, and by modern trap- pers "fisher," ''black cat," or "black fox," it being none of the three ! It is remarkable for its great size 24 inches, plus 13 inches of tail and for its dog-like head. A third still larger relative is the wolverine or "carcajou," an uncommonly large, clumsy, shaggy marten, of great strength, and displaying extreme per- severance and sagacity in procuring food where the supply is limited and precarious. ANIMAL COMPETITORS 244 All these, in early times common enough throughout all our northern forests, have been destroyed, or have retreated before civilization until now few are seen south of the wilds of northern Canada, where they still yield their furs to the Indian and the wandering trapper. This brings us to the more familiar weasels, minks and ferrets. THE FUR-BEAKERS AND THEIR CULTURE "Slender, lithe, perfectly toothed, sharp-clawed, secretively colored, and endowed with strength, speed, cleverness, and indomitable courage, the weasels are the scourge and terror of all the small ground-keep- ing animals, and do more than any other class of agents to restrain mice, gophers, and similar nui- sances. Some or all can climb, but their preference for the ground distinguishes them from the martens, as also do the comparatively short tail, close fur, three instead of four premolar teeth, and the pres- ence of anal glands whence they may discharge a fetid odor. This musky, nauseous secretion is most copious and evident in the large European polecat, but most distressing to human nostrils in an old mink ; and ordinarily it is not very noticeable in a weasel. Its emission is under control, and becomes perceivable mainly when the animal is excited or alarmed. Its service seems to be that of attracting the sexes; and trappers save it to put upon their bait as an addi- tional allurement." CULTURE OF FUR-BEAKERS 245 The ermine and his family. Naturalists distinguish over 20 species and subspecies of weasel in North America. Most of them, however, belong to the West and far North, and they differ little in general character; such 'peculiarities as belong to each Dr. C. Hart Merriam, their monographer, connects with their food. Thus he finds that the group represented by our common eastern species (Putorius cicognani) flourishes only in the country where the meadow-mice abound; the large western weasel (P. longicauda), does not range much outside of the region inhabited by the pocket-gophers ; the black-footed one (P. ni- gripes) frequents only the prairie-dog country southward; and . " in the far North, where the frozen tundras are inhabited by lemmings as well as voles, two weasels are present : the tiny, narrow-skulled 'rixosus,' which feeds mainly on mice, and the large, broad-skulled 'arcticus' (analogue of the true ermine) on lemmings and rabbits. ' ' With these fine points of classification we need not here concern our- selves. A weasel, in the Old World or in the The ermine and his family. Naturalists distinguish over 20 species and subspecies of weasel in North America. Most of them, however, belong to the West and far North, and they differ little in general character; such 'peculiarities as belong to each Dr. C. Hart Merriam, their monographer, connects with their food. THE FUR-BEAKERS AND THEIR CULTURE Thus he finds that the group represented by our common eastern species (Putorius cicognani) flourishes only in the country where the meadow-mice abound; the large western weasel (P. longicauda), does not range much outside of the region inhabited by the pocket-gophers ; the black-footed one (P. ni- gripes) frequents only the prairie-dog country southward; and . " in the far North, where the frozen tundras are inhabited by lemmings as well as voles, two weasels are present : the tiny, narrow-skulled 'rixosus,' which feeds mainly on mice, and the large, broad-skulled 'arcticus' (analogue of the true ermine) on lemmings and rabbits. ' ' With these fine points of classification we need not here concern our- selves. A weasel, in the Old World or in the ANIMAL COMPETITORS 246 CULTURE OF FUR-BEARERS 247 New, in Labrador, or Florida, or Mexico, on the Yukon as on the Hudson, is substantially the same, a keen, agile, relentless, indomitable hunter, within Ms powers a being of the high- est type of effectiveness. "The weasel's head is small and trim, An' he is little an' long an ; slim, An' quick of motion an' nimble of limb. An' ef you'll be advised by me, Keep wide awake when you're catchin' him." "The weasel's head is small and trim, "The weasel's head is small and trim, An' he is little an' long an ; slim, The weasel turns white in winter in all cold, snowy latitudes that is, when the brown sum- mer-coat is shed in the fall it is replaced by a white one, which in turn is lost in the spring and replaced by the soft brown again. The tip of the tail, however, always remains black. In this white winter dress with the black- tipped tail every weasel is an "ermine"; and it is only in this coat that his fur becomes valu- able in the market. It comes to market from Alaska, northern Canada and sub-arctic Rus- sia, and is used mainly for trimmings of garments. In old times it was reserved exclu- sively for the wearing of royalty, and of cer- tain officers of high rank. It was especially ANIMAL COMPETITORS 248 prominent in England in the regalia of judges ; whence our figurative expression "the ermine" as a symbol of the judicial office. "Whenever an ermine has taken up its residence the mice in its vicinity for half a mile around have been found rapidly to diminish in number. Their active little enemy is able to force its thin vermiform body into the burrows, it follows them to the end of their galleries, and destroys whole families. We have on several occasions, after a light snow, followed THE FUR-BEAKERS AND THEIR CULTURE y Large, purely white ermine skins are still valuable in the fur-market, so that the animal is well worth the attention of trappers; but it is chiefly in its other relations that it now interests us. Weasels and chickens. Weasels of one sort or another are more or less common in aJl parts of the country, even to the suburbs of the cities ; and they are so swift, secretive, alert and wise that they remain everywhere numer- ous in spite of the constant efforts of the aver- age countryman to kill them off. y This enmity is due mainly to the animal's delight in killing chickens, at which it is ex- ceedingly expert and bold, often invading a barn-yard in full daylight; and when one or more usually a pair has acquired the habit of chicken-killing it is likely to murder the whole flock in a short time if not prevented. This bloodthirst is only a natural outcome of its habit of preying on wild birds, especially those which keep to the ground; and in places CULTURE OF FUR-BEAEERS 249 where quail or game is preserved the animal often does great harm. It does not appear, however, that every weasel kills chickens, nor that the same weasel devotes its whole energies to this end, as a man-eating tiger will do when once it learns how easy it is to secure human prey. It seems, rather, that an occasional weasel now and then seizes a pullet or duck. The worst of it is, however, that when it has done so its ferocity is likely to be so fired by the taste or smell of blood that it goes on massacring the fowls after the manner of a wolf or a puma in a sheep-fold, as though in a rage of blood intoxi- cation. The weasel as a mouser. There is another side to the account, however, and that is the ceaseless and extensive beneficial work of these ferocious little creatures in pursuit of the ror dents which year by year destroy more grain and young trees than all the poultry loss of a year amounts to a hundred times over. In the West they are the determined and inde- fatigable enemies of ground-squirrels, gophers and all sorts of mice, which they follow to the ANIMAL COMPETITOKS 250 uttermost ends of their burrows if necessary. Mr. THE FUR-BEAKERS AND THEIR CULTURE Osgood says of the small Arizona weasels : "They are the most effective enemy of pocket- gophers. ... In a narrow valley where an old weasel had her young I found it impos- sible to secure a single pocket-gopher. A single weasel will effectually keep down the go- phers and meadow-mice on a field or small ranch. Except in very rare cases they should be protected with the greatest care." The black-footed "ferret" (a true weasel) is rarely found away from prairie-dog towns, where it plays the bandit unceasingly. The writings of naturalists abound in evidence of the same sort. A notable instance may be found in the great work on American mammals, The Quad- rupeds of North America, published half a century ago by Audubon and Bachman, a few paragraphs from which may well be quoted : CULTURE OF FUR-BEARERS 251 the trail of this weasel through fields and meadows, and witnessed the immense destruction which it occa- sioned in a single night. It enters every hole under stumps, logs, stone heaps and fences, and evidences of its bloody deeds are seen in the mutilated remains of the mice scattered on the snow. The little chip- ping or ground squirrel, Tamias Lysteri, takes up its residence in the vicinity of the grain-fields and is known to carry off in its cheek-pouches vast quanti- ties of wheat and buckwheat, to serve as winter stores. The ermine instinctively discovers these snug retreats, and in the space of a few minutes destroys a whole family of these beautiful little Tamice; without even resting awhile until it has consumed its now abundant food, its appetite craving for more blood, as if im- pelled by an irresistible destiny, it proceeds in search of other objects on which it may glut its insatiable vampire-like thirst. The Norway rat and the com- mon house mouse take possession of our barns, wheat stacks, and granaries, and destroy vast quantities of grain. In some instances the farmer is reluctantly compelled to pay even more than a tithe in contribu- tions towards the support of these pests. Let, how- ever, an ermine find its way into these barns and granaries, and there take up its winter residence, and the havoc which is made among the rats and mice will soon be observable. "Two years ago a pair of weasels took up their abode in our tree-cellar, breeding there last year. They kept most of the mice killed off. In the sum- mer we saw the old one quite often carrying mice to its young from outside the shed." THE FUR-BEAKERS AND THEIR CULTURE The ermine pursues them to their farthest retreats, and in a few weeks the premises are entirely free from their depredations. We once placed a half-domesticated ermine in an outhouse infested with rats, shutting up the holes on the ANIMAL COMPETITORS 252 outside to prevent their escape. The little animal soon commenced his work of destruction. The squeak- ing of the rats was heard throughout the day. In the evening, it came out licking its mouth, and seemed like a hound after a long chase, much fatigued. A board of the floor was raised to enable us to ascertain the result of our experiment, and an immense number of rats were observed, which, although they had been killed in different parts of the building, had been dragged together, forming a compact heap. gg g , g p p "The ermine is then of immense benefit to the farmer. We are of the opinion that it has been over- hated and too indiscriminately persecuted." Again, in another place, Dr. Bachman returns to the weasel's abilities as follows: "We have traced the footsteps of this bloodsucking little animal on the snow, pursuing the trail of the American rabbit, and although it could not overtake its prey by superior speed, yet the timid hare soon took refuge in the hollow of a tree, or in a hole dug by the marmot or skunk. Thither it was pursued by the ermine and destroyed, the skin and other re- mains at the mouth of the burrow bearing evidence of the fact. We observed an ermine, after having captured a hare of the above species, first behead it and then drag the body some twenty yards over the fresh fallen snow, beneath which it was concealed, and the snow lightly pressed down over it; the little prowler displaying thereby a habit of which we be- came aware for the first time on that occasion. To CULTURE OF FUR-BEARERS 253 avoid a dog that was in close pursuit, it mounted a tree and laid itself flat on a limb about twenty feet from the ground, from which it was finally shot. We have ascertained by successful experiments, repeated more than a hundred times, that the ermine can be employed, in the manner of the ferret of Europe, in driving our American rabbit from the burrow into which it has retreated. THE FUR-BEAKERS AND THEIR CULTURE In one instance the ermine employed had been captured only a few days before, and its canine teeth were filed, in order to prevent its destroying the rabbit; a cord was placed around its neck to secure its return. It pursued the hare through all the windings of its burrow, and forced it to the mouth, where it could be taken in a net, or by the hand. ' ' Seton, in his magnificent work on Northern Mammals, relates many instances of the wea- sel's work in Canada, mentioning among other facts its persistent preying upon rabbits. In Lantz's Economic Study of Field-Mice is given a letter from a farmer in Waukegan, 111., who writes : ANIMAL COMPETITORS 254 Weasels are extraordinarily fearless of man- kind, and will soon become so regardless of him as to be positively tame. In the West miners and hunters often welcome them to their cabins and establish very friendly relations with them, recognizing that they keep the premises free from the wild mice, which otherwise would in- fest the houses and play havoc with supplies brought in at great expense and labor. This is only a return to a very ancient practice, for, as I have shown in my Life of Mammals, the household mouser of the Greeks was. not a cat, but a weasel the European stone-marten. p Life of the mink. The mink is a semi- aquatic weasel. It inhabits the whole of the United States, excepting the arid regions, which are unfitted for its habits of life. It is a species of great economic importance, both on account of the value of its fur and on ac- count of its injurious habits. As an enemy to the poultry yard it ranks ahead of all other North-American mammals. Furthermore, it kills large numbers of fish, as it not only swims and dives with facility, but can remain long under water, pursuing and capturing its prey CULTURE OF FUR-BEARERS 255 by following it below the surface. Oftentimes its destructiveness in this respect renders it a serious obstacle to the industry of fish-culture. Though amphibious, and commonly inhabiting the borders of ponds and streams, it makes long excursions, and is frequently found in THE AMERICAN MINK. THE AMERICAN MINK. places remote from water-courses. It often takes up its abode in or near the poultry-yard or duck-pond, remaining there for weeks. THE FUR-BEAKERS AND THEIR CULTURE Its small size and nocturnal habits help to conceal its movements, and the daily loss of a fowl is commonly attributed to the skunk, fox, weasel, or owl. The mink is remarkably strong for so places remote from water-courses. It often takes up its abode in or near the poultry-yard or duck-pond, remaining there for weeks. Its small size and nocturnal habits help to conceal its movements, and the daily loss of a fowl is commonly attributed to the skunk, fox, weasel, or owl. The mink is remarkably strong for so ANIMAL COMPETITORS 256 small an animal, and has been known to drag a mallard duck more than a mile in order to get to its hole, where it was joined by its mate. Value of the mink. Such is the farmer's view of the mink, but the picture is not with- out a brighter side. His loss of chickens and eggs is largely due to his own slovenly way of keeping his property, or rather of trusting it to keep itself. The depredations of the mink are almost wholly made at night. A tight poultry-house will keep him out, even a wire- fence of small mesh around the yard will do so. If the chickens are allowed to roost in .* trees or in any old shed it is foolish to com- plain when they are seized by rats, weasels, minks, skunks or, 'coons. In its natural life the mink habitually feeds upon small mammals, birds and their eggs, fish, frogs, turtles' eggs, crayfish, earthworms and the like. It is one of the busiest hunters of injurious rodents, particularly muskrats and common rats and mice. Hence it is a pub- lic benefactor in localities where muskrats damage dikes, canals, irrigating ditches, and ponds ; and day by day it seeks out the runways CULTURE OF FUR-BEARERS 257 of the wild mice and devours their families. If you catch sight of a prowling mink, some day, along the brookside, squeak like a mouse, and see how interested he will at once become. Indeed, a mink or a weasel which takes up its abode under a barn will soon clear out the rats. Good mink-skins have always fetched a fair price, and this price is rising. THE FUR-BEAKERS AND THEIR CULTURE As they are abundant, and among the most easily trapped of our wild animals they have ever been one of the sources of pocket-money to the lads of the rural parts of the country. p y "The old-fashioned deadfall is the trap that should be used," Seton advises, "as it does not injure the fur and it kills the animal instantly, so that there is no unnecessary suffering. The box-trap is effectual and humane if visited regularly. It should have, at the back, a window covered with %-inch-mesh wire netting. It has the advantage of protecting its catch from passing marauders. The steel trap, if used, should be visited often. The less the creatures suffer the better the fur." The pelt should be stripped and cased in the same man- ner as that of the ermine or the muskrat. ANIMAL COMPETITORS 258 Mink cultivation for profit. The raising of captive mink for the sake of the fur has been tried very often, and succeeds well where one tried very often, and succeeds well where one HOME OF A MINK FAMILY IN A STUMP IN NORTHERN ONTARIO. pays proper attention to it, and the expenses can be kept low enough to ensure a profit from CULTURE OF FUR-BEARERS 259 the results. Such establishments are known as "minkeries," and have been described in many publications. It frequently happens that a pair of young minks are caught; or a full-grown male and one or more females may be bought from deal- ers who advertise their wares in journals de- voted to sport or to trapping. One male will suffice for five or six females. The breeding season is February, when the females all come into heat, and for three or four weeks the male should be allowed to associate with them freely. Their behavior will indicate when they no longer need his attentions. The period of gestation is 42 days, so that births will occur early in April. They number five or six, as a rule, sometimes ten, are blind, almost naked, and remain hidden in the nest for five or six weeks, when they begin to come out; but they stay with the mother, and are the object of her tender solicitude and brave defense until the end of the summer. THE FUR-BEAKERS AND THEIR CULTURE If taken in hand when they first appear they will become as tame, gentle and playful as kittens, provided they are kept entirely away from the mother. Res- It frequently happens that a pair of young minks are caught; or a full-grown male and one or more females may be bought from deal- ers who advertise their wares in journals de- voted to sport or to trapping. One male will suffice for five or six females. The breeding season is February, when the females all come into heat, and for three or four weeks the male should be allowed to associate with them freely. Their behavior will indicate when they no longer need his attentions. The period of gestation is 42 days, so that births will occur early in April. They number five or six, as a rule, sometimes ten, are blind, almost naked, and remain hidden in the nest for five or six weeks, when they begin to come out; but they stay with the mother, and are the object of her tender solicitude and brave defense until the end of the summer. If taken in hand when they first appear they will become as tame, gentle and playful as kittens, provided they are kept entirely away from the mother. Res- ANIMAL COMPETITORS 260 seque, a mink-breeder whose experience and methods are described particularly in Elliot Cones 's Fur-bearing Animals, admits, how- ever, that they become exceedingly mischiev- ous, prying into all sorts of food receptacles, etc., and can hardly be recommended as house- hold pets. Merriam trained some in his pos- session to be excellent ratters, following the rats into their holes and soon clearing all the premises of this pest. p p Care of captive minks. In planning a minkery a yard say 50 feet square should be set apart and enclosed by a tight board fence 7 or 8 feet high, which should rest upon a stone or cement foundation sunk 2 feet into the ground ; or else a close and strong wire netting must be deeply sunk along the bottom, for minks are good diggers. The top of the fence should have an inward overhang of tin, zinc or galvanized iron at least 2 feet wide, and still broader at the corners, or else the animals will climb out. THE FUR-BEAKERS AND THEIR CULTURE Of course the best way would be to pave the whole interior with, and base the fence upon, concrete, but this is costly; if it is CULTURE OF FUR-BEARERS 261 done the cement floor should be covered with a deep layer of earth. At one end of this enclosure should be built of strong, close meshed wire netting, a series of cages, resting on a tight wooden floor, each about 3 feet high, 8 or 10 feet long and about 4 feet wide. These are for the use of the breeding females, one to each cage. In the front of each should be a door large enough to pass in and out a nesting-box about the size of a raisin-box, with a small hole for admittance of the animal, closed by a sliding door so that she may be shut in if desirable. y The remainder of the enclosure should be divided in the middle by a fence of close wire netting, guarded from digging and climbing like the outer fence, forming two courts or yards for exercise, etc. A small low door will afford communication between these yards. Water must be supplied in abundance run- ning water if possible. There should be a trough for each breeding-cage and a large swimming tank which may be accessible from both yards. The cost of such an outfit will ANIMAL COMPETITORS 262 vary with circumstances, but may easily be cal- culated. "Mink," says Seton, "may be fed exactly as one would feed a house-cat table-scraps va- ried with meat two or three times a week. Fish is very much to their liking, and may be given nearly every day if other things are used. Bread and milk, johnny-cake, etc., should be added for variety ; even raw liver may be given sparingly at intervals, but cooked food, as a rule, is safer. Two light meals, morning and night, or one substantial meal, late in the day, is sufficient; and at all times an abundance of clean water. ... A fast-day once in two weeks is a good thing for fat animals." Each old animal should have a cage to itself and be kept in it except in the breeding month (February), when a male and four, five or six females may be turned out together in one of the yards; but persistently bad-tempered ones should be taken away from the band. THE FUR-BEAKERS AND THEIR CULTURE No bad smell will be made if general cleanliness is maintained, the yard-soil being frequently raked and overturned to remove droppings and air it. If their cages are kept clean the ani- CULTURE OF FUR-BEARERS 263 mals are likely to remain perfectly neat and healthy and they will breed regularly and grow fine coats. The best should always be kept for breeding, and so the stock will be steadily improved. The beautiful otter. The noble and beauti- ful otter has become so rare south of the north- ern wilderness, or outside of large tracts of southern swamp-lands, that it has little claim to inclusion in a book devoted to the industrial aspects of our wild quadrupeds. The food of otters is mainly fish ; and in a preserved stream they may do vast damage to the angler's treasures by devouring numberless trout. They also catch and kill many muskrats. Merriam, in his natural history of the Adiron- dacks, and Seton in his Northern Mammals, give extensive biographies of this most inter- esting and most intelligent of the mustelid race. This brings us to the related group of fur- bearers which includes the badgers and skunks. The misunderstood badger. Our badger is very similar to the European one, and formerly occurred wherever west of the Alleghanies un- This brings us to the related group of fur- bearers which includes the badgers and skunks. The misunderstood badger. Our badger is very similar to the European one, and formerly occurred wherever west of the Alleghanies un- ANIMAL COMPETITORS 264 forested country gave it the conditions it liked. Its general habits, so far as they can txe ob- served in so shy, secretive and well-hidden a creature, are interesting, as I have shown in the chapter "A Badger and his Kin" in my Wild Neighbors. These cannot be dwelt upon here, but they show that none of our small mammals has been more misunderstood or mistakenly and wastefully persecuted. As a result the badger is now restricted in its dis- tribution to the arid region, although scattered pairs linger here and there even in Wisconsin and Minnesota. And yet, its disappearance has not been wholly due to reckless destruction, for it seems unable to endure the forestation and cultivation of lands as they are settled. THE FUR-BEAKERS AND THEIR CULTURE This may be due to the sedentary nature of the little beast, which is by no means a wanderer or even a traveler. Seton remarks that prob- ably a badger never in his whole life goes a mile from the home in which he was born. The consequence is that when a family has been ex- terminated another is not likely to take its place. It is for this reason that a man should be careful how he wastes the life of a badger CULTURE OF FUR-BEARERS 265 on his property, for not only is he putting out of business a most useful ally in his contest with nature, but is destroying one which will not easily be replaced. Apart from that wanton, thoughtless disposi- tion to kill any and every wild creature met with, which possesses the ruder sort of men, and most boys who have not been taught to restrain the innate savagery of the human an- imal, the excuses made for killing badgers are usually either that its fur is wanted or that it digs bad holes in the ground. g As to the pelt it is now of no great value, and its use is mainly to furnish hairs for art- ists' pencils and for the making of shaving- brushes. The hide is strong, however, and good overcoats and carriage-robes may be made of it. Badger-holes as man-traps. The second ex- cuse is that it digs holes in the land which may be dangerous pitfalls for horses and cattle, and which furnish runways for water that some- times, after heavy storms, develop into bad gullies. g That this charge was originally well-founded ANIMAL COMPETITORS 266 I know by my own observation in the North- west, as well as by reading. In the buffalo-coun- try, where gophers and spermophiles dwelt in countless numbers, badger-holes were in old times extraordinarily numerous. "I do not see how they could well be more numerous any- where," wrote Dr. Elliott Coues of the region of the Upper Missouri as he saw it in 1875. "In some favorite stretches of sandy, sterile soil, their burrows are everywhere, together with those of kit-foxes, prairie-dogs and spermophiles, and, as already stated, these holes are a source of annoyance and even danger to the traveler. THE FUR-BEAKERS AND THEIR CULTURE In ordinary jour- neying one has to keep constant lookout lest his horse suddenly goes down under him, with a foreleg deep in a badger-hole; and part of the training of the western horse is to make him look out for and avoid these pitfalls. In the buffalo country particularly, badgers live in extraordinary numbers, attracted and retained by the surety of abundant food-supply. y y pp y ' ' The burrows of the badger are known from those of the prairie-dog and other spermophiles by their greater dimensions; besides, they differ from the for- mer in never being built up around the entrance into the regular mound or circular buttress which usually surmounts the well-kept domicile of Cynomys. From the holes of kit-foxes and coyotes they are not distinguishable with any certainty; in fact it is prob- able that these animals frequently or almost habit- CULTURE OF FUR-BEARERS 267 ually occupy deserted burrows of the badger, re- modeled, if need be, to suit their convenience. ually occupy deserted burrows of the badger, re- modeled, if need be, to suit their convenience. "But it must not be supposed that all the in- numerable badger-diggings are the residences of these animals. The badger, too slow of foot to capture the nimble rodents which form its principal food, perpetually seeks them in their own retreats; and it is the work of a few minutes for this vigorous miner to so far enlarge their burrows that it can enter and reach the deepest recesses. In places where the badgers and spermophiles most abound, the continual excavation of the soil by these animals fairly under- mines and honeycombs the ground." modeled, if need be, to suit their convenience. "But it must not be supposed that all the in- numerable badger-diggings are the residences of these animals. The badger, too slow of foot to capture the nimble rodents which form its principal food, perpetually seeks them in their own retreats; and it is the work of a few minutes for this vigorous miner to so far enlarge their burrows that it can enter and reach the deepest recesses. In places where the badgers and spermophiles most abound, the continual excavation of the soil by these animals fairly under- mines and honeycombs the ground." Prehistoric plowing. THE FUR-BEAKERS AND THEIR CULTURE The conditions above described existed mainly in regions of little use for agriculture, and as fast as civilization was extended into the badger country the animals lessened rapidly, for one reason or another, and their holes became filled up. This is illustrated by experience in Manitoba, where, as Seton in- formed us, "the'work of the badger is now con- fined largely to the strips of prairie that exist along the road-allowances, where it can do but little harm." As an offset to these troublesome habits (from man's point of view) it must be remem- bered that by the incessant and multitudinous ANIMAL COMPETITORS 268 upheaval of the ground in his digging opera- tions, the badger has been for centuries assist- ing in that work of preparing the soil for man 's cultivation which is the valuable heritage of the present from the small plains-animals of the past. Ever since the glaciers of the great Ice Cap left the surface of northern America an expanse of smooth rock and lifeless gravels they have been fallowing the soil of that great field which stretches from the Ohio to the Co- lumbia, and from the Bio Grande to the Sas- katchewan, and rendering it fruitful for the pasturage first of large game, next of the ranch- man 's herds and finally for the farmer's fat stock and for the planting of his grain and fruits. How the badger aids the farmer. It must be remembered that the badger's food consists almost wholly of those insects and animals which prey upon crops and young orchards, and in this his services have been and are of im- mense and constant value. Wherever he goes he picks up insects, and in Kansas is noted to have lived very largely on the dreaded grass- hoppers. Beetles and their grubs are taken, CULTURE OF FUR-BEARERS 269 and now and then a snake or frog, a nest of ground-building birds, or even a settler's young chickens when they wandered too far afield ; but he rarely if ever raids a poultry-yard. These, however, are incidents of his carnivorous pur- suits, which are mainly nocturnal. The bulk of his food is found in the small and always mis- chievous rodents. As Osgood says: "Almost the whole life of the badger is spent in digging out the various rodents that constitute its food. THE FUR-BEAKERS AND THEIR CULTURE It requires two or three fat ground squirrels a day, or a few gophers and a dozen mice, to keep a badger in good condition. ... In case of pocket- gophers the badger digs down in several places along the line of the burrow and sometimes succeeds in cornering and capturing the occupant. Mice are easily unearthed, and a nest of young mice is a special delicacy. . . . When in pursuit of a gopher, a badger may dig into and endanger ditch-banks, but in most cases the gopher, if left alone, would do far more mischief. ' ' Practically the only enemy of the badger is man, and it seems incomprehensible that men of intelli- gence should wantonly destroy on every possible oc- casion the most useful and least harmful of all our native mammals. So generally, however, are badgers killed that after a valley has been settled for some time they become extremely scarce, and are really in danger of local extermination. As a result one of ANIMAL COMPETITORS 270 the most important checks on the increase of ground squirrels, mice, and gophers is removed, and these animals occasionally surprise the farmer by taking his whole crop." Skunks and skunk-farming. A similar plea may be made for skunks. These animals are far more widespread, equally harmless and quite as beneficial; and, like badgers, should everywhere be protected in country districts except in special cases. As for chicken-steal- ing, there is none of his race so little to be feared. The skunk is not as a rule a chicken- thief ; he is too large to creep through the small crevices that admit rats or minks, and he can't climb well. It is only needful to have a fairly well-fenced yard and tight coop to be quite safe from him. I have many times been asked to advise as to skunk-farming, and my advice has almost al- ways been No. This was due to the fact first that with wild skunks so numerous and easily trapped, and consequently skunk-pelts so cheap as is the case at present, little or no profit could be hoped for. Yet if done intelligently and on a large scale several hundreds of skunks sys- CULTURE OF FUR-BEARERS 271 tematically looked after the enterprise would probably turn out a fair investment. THE FUR-BEAKERS AND THEIR CULTURE The general directions as to housing, feeding, cleanliness and health would be substantially the same as those given for a mink-farm. Skunks, however, need much more room for A DOG-PROOF FENCE. A DOG-PROOF FENCE. wandering about, picking up insects, etc., so that several yards of an acre or so each should be provided for a large systematic skunkery, with 50 or so animals assigned to each. They are capable diggers, and the fences must be made secure at the bottom, but need be tall wandering about, picking up insects, etc., so that several yards of an acre or so each should be provided for a large systematic skunkery, with 50 or so animals assigned to each. They are capable diggers, and the fences must be made secure at the bottom, but need be tall ANIMAL COMPETITORS 272 enough only to keep out dogs and other dis- turbers. The breeding-cages should have hol- low logs or underground vaults of stone or con- crete as nesting-dens; and all retiring-quarters must be roofed with a stout wire mesh to pro- tect the occupants from great horned owls the skunk's worst enemy big dogs and other ma- rauders. If the skunks are not frightened by rough treatment there is little or no danger from the musk-gun an instrument kept as a last resort ; in fact captive skunks easily become docile and often affectionate and amusing pets. The best way to kill them, when that sad necessity arises, is by smothering with illuminating gas or some similar agent, or by drowning. The finest, blackest specimens should be selected for keep- ing as breeders, the less white on a pelt the more valuable it is. It might be possible, in a few generations, to get a strain that should be wholly black. RAISING DEER FOR PROFIT A CHAPTEB remains to be written on the wild hoofed animals of North America in their re- lation to the farmer and stock-breeder. This group includes the various deer, the almost extinct bison (buffalo), the antelope, the wild sheep (bighorn), the white goat, the peccary and the herds of escaped horses. Of these the most important, from our point of view, are the deer, the rearing of which, for profit, may be- come an industry in which the first-comers will reap a great harvest. p g Native American deer. Many forms of deer are native to the United States and Can- ada. The moose (substantially identical with the European elk) ; the caribou in several forms identical with or very similar to the European reindeer; the wapiti, which we erroneously call "elk," and which is the American form of the red deer of Europe and its Asiatic analogues 273 ANIMAL COMPETITORS 274 from the Caucasus to Kamchatka; the widely spread Virginia or white-tailed deer; the mule or jumping deer of the West; the blacktail of the Pacific coast, and many intermediate sub- species. All these are now protected by law as the only means of saving the several species from total extinction by men who, to say the least, are utterly selfish; and there seems no good reason why some of them should not be cul- tivated, so as to -form herds whose flesh and skins may be regularly sold as are those of cattle and sheep. This is doubly desirable as an economic movement, since it will not only add to the food-resources of the nation but may be a means of utilizing tracts of rough sterile land which otherwise will continue to lie idle. That venison is good food needs no proof, but it is not generally understood that besides having a most attractive flavor, and lending itself well to various methods of cooking, it is in composition and nutrient quality very sim- ilar to beef, and exceeds mutton in food-value, while quite as easily digestible. Venison is in constant demand far greater demand than RAISING DEER FOR PROFIT 275 can be supplied; and this will always be true; while the price is correspondingly high. The skins and horns of deer are also steadily salable at remunerative rates. Capability of domestication. RAISING DEER FOR PROFIT It has been shown by centuries of experience in parks that deer of all kinds are susceptible of cultiva- tion, thriving and breeding readily in captivity under reasonable conditions, yet few attempts have been made to rear or domesticate them un- der intelligent management. Foremost among the exceptions to this negligence must be men- tioned the work of the Duke of Bedford in his park at Woburn Abbey, England, where a large number of species are assembled under the most favorable arrangement for their in- crease. ''But raising deer for profit does not neces- sarily imply their complete domestication," as Mr. Lantz remarks in a Bulletin on this subject issued by the U. S. Department of Agriculture in 1908. "They may be kept in large preserves with surroundings as nearly natural as pos- sible and their domestication entirely ignored. Thus the breeder may reap nearly all the profit ANIMAL COMPETITORS 276 that could be expected from a domestic herd, while the animals escape most of the dangers incident to close captivity. But the breeder who aims at the ultimate domestication of the animals, and whose herd approaches nearest to true domesticity, will in the end be most suc- cessful." Rearing wapiti for profit. Of the various kinds of deer, native and foreign, with which one might stock a range or park, the most prof- itable, at any rate in the eastern or central parts of the country, would be elk (wapiti) and white-tailed deer. Breeding-stock of both these species is most easy to get, both are hardy in various climates and each has been tried by ob- servant experimenters. p The best ground for an enterprise of this kind is precisely that of least value otherwise, a rough tract, well watered and having some high nut-bearing timber and much brush with grassy spaces among it. Deer are both brows- ers and grazers the elk eating grass much more freely than the whitetail. (Of course the two would not be herded together.) "Where deep snow does not cover the ground for long EAISING DEEE FOE PEOFIT 277 periods both deer will subsist very well in win- ter, the whitetail on browse, and the elk by pawing away light snow and eating the grass; but even where not required by the severity of the season regular winter feeding is advised. RAISING DEER FOR PROFIT "Hay and cornfodder are excellent winter forage; but alfalfa hay has proved to be the best dry food for both elk and deer. A little oats or corn whole or chopped may be fed each day. Elk are fond of corn, and feeding it affords excellent opportunities for winning their confidence and taming them. The same may be said of salt, which should be furnished liberally to all deer kept in enclosures. Sun- ning water, although not essential, is of great importance in maintaining elk in good condi- tion." If not closely confined elk do not gnaw the bark from trees nor eat evergreen foliage ; but, like goats, they do clear out the thickets. , g , y The providing of a buck and a few does to start with is not very expensive; but a rather costly item of preparation is the fencing. Or- dinarily a five-foot fence is sufficient, especially in the case of elk, which are less inclined to jump than are other deer. Old bucks, and ANIMAL COMPETITOES 278 even the young ones in the rutting season, are highly excitable, if not really vicious, and for these must be provided small and strong en- closures. It is, indeed, unsafe to let a male elk over four years old run at large, especially if he has once shown viciousness. The remedy for this is castration, which not only makes him docile but improves the venison; and all except the small breeding stud, frequently changed, should be so treated when young. "We find from long experience," writes a man who has made a business of deer-farming in the Ozark Mountains of" Arkansas, "that cattle, sheep and goats can be grazed in the same lots with elk, providing, however, that the lots or enclosures are not small; the larger the area the better. . . . An elk is the natural enemy of dogs and wolves. We suffered great losses to our flocks until we learned this fact; since then we have had no loss. A few elk in a thou- sand-acre pasture will absolutely protect the flocks therein. The whitetail as an investment. Virginia deer are even better adapted than elk to rocky forested places, such as are so numerous in the Alleghenies. RAISING DEER FOR PROFIT "Advocates of the Angora-goat industry state that within the United States RAISING DEER FOR PROFIT 279 there are 250,000,000 acres of land not suited to tillage or to the pasture of cattle, horses or sheep, which are well adapted to goats." Much of this land, it has been well suggested, is suited also to deer, and can be utilized for these animals with less injury to the forest- cover than would result from the browsing of goats. g A herd of whitetail favorably located and properly cared for will increase in a steady and rapid ratio. The rutting season is in Novem- ber, and the fawns are born in May and June. A doe will breed when 18 months old, and at first produces only a single fawn, but after- ward usually two fawns. Certain diseases, es- pecially "black tongue," are to be feared, but an annual increase of 60 or 70 per cent, may be confidently looked for. Like elk the whitetails require a certain amount of winter feed; and also should have shelter provided against the winter storms. A stack of clover or pea hay to which they may have free access in winter is recommended; and they need plenty of water a stream or pond being especially good for h ANIMAL COMPETITORS 280 In selecting herd-bucks an intelligent owner will naturally seek the best in all respects ; and if this were carried out generally a greatly im- proved breed of deer would presently be de- veloped. The great hindrance in the way of instituting profitable enterprises in this much-to-be de- sired direction is the present condition of the laws relating to deer and venison, which have been made wholly with reference to sportsmen and pot-hunters. These laws vary locally, but in every state, probably, must be modified to admit of deer-farming and the sale of its prod- ucts. Such modifications will probably take the form of licensing private deer-parks and breeding-farms, with a method of tagging the venison so that it can be easily identified. Some progress in this direction has already been made, and more will follow as the industry grows. A pest of wild horses. In some parts of the West bands of wild horses, derived from es- caped stock, roam over the thinly settled plains and hills, and threaten to become a serious nui- sance. RAISING DEER FOR PROFIT The United States Forest Service re- RAISING DEER FOR PROFIT 281 cently stated that there are fifteen thousand of these untamed beasts upon the Toiyabe, To- quina and Monitor forest reserves in Landor County, Nevada, alone, and many more in neighboring districts. No fence is strong enough to stop these horses, and when they ap- pear in force they have even been known to knock down and kill cows and calves. After each visitation from a, herd the ranchman is likely to mourn the loss of his domestic horses, and it requires only a few days' association with their new companions for the best-broken animals to become as wild as their nomadic comrades. The Legislature of Nevada passed a law many years ago allowing hunters to shoot wild horses and to sell their hides. The law opened the way to a new and profitable industry, and one which had the ?dded zest of sport. Pres- ently, however, hide-hunters began to kill also branded and shod horses, and this caused a quick repeal of the law. This left the animals free to increase, and now they have become a serious pest. p On the ranges of many of the national forests ANIMAL COMPETITORS 282 the supervisors have been trying for several years to devise a method to meet the difficulty in the face of the prohibitions enacted by local laws. The solution of the problem remains to be worked out. Texan musk-kogs. The small wild pig of western Texas, known in books as collared peccary, and to Texans as javelin or musk- hog, is more interesting than important. Once spread as far north as Arkansas, these pigs now abound only in the sandy, rocky districts along the lower Rio Grande, where they are extremely wary, hiding during the day in swamps, thorny thickets or among rocks, so that it is difficult to get near them without the aid of dogs and horses. Occasionally sports- men attempt to utilize this game for "pig-stick- ing" after East-Indian methods, but the sport usually involves severe injury to the horses before the tough little boar succumbs. They have formidable tusks in both jaws, those of the upper jaw being turned downward instead of upward as in typical swine, and all four as keen as knives. RAISING DEER FOR PROFIT They run, when chased in open ground, with great fleetness, but will RAISING DEER FOR PROFIT 283 squat and dodge like a rabbit, and it is at these quick turns that the horses get gashed. g g At night the javelins sally forth to dig roots, mushrooms, etc., and to feed upon nuts and acorns, the latter now forming their chief fare in Texas. They also kill and eat snakes, liz- ards, frogs, and any other small animals they can catch. Near settlements they are, or used to be, a nuisance by entering and damaging gardens and planted fields. These animals are to be seen in most zoological gardens where they thrive and grow tame ; but they have few qualities to recommend them in a practical way. Bighorn and mountain goat. The bighorn is easily domesticated and would doubtless flourish in any dry and elevated part of the country, but it appears to be of no practical value except for its flesh, and the gamy quality of that, which now recommends it, would prob- ably disappear in animals raised in a corral and fed an unvaried pasture or lowland fodder. The same may be said of the pronghorn ante- lope. Its flesh is a better "venison" than that of the sheep; but is difficult to adapt to cap- ANIMAL COMPETITOES 284 tivity, and would make more expense and trouble for its owner than the returns would be worth in the present state of the meat-market. The beautiful Eocky Mountain white goat, like the bighorn, is chiefly interesting and of value as an object of sport. Its flesh is very poor eating, and its pelt worthless, but its thick white hair and wool have been used by the Pacific Coast Indians from time immemorial for making robes, etc., and could enough of this fine soft coat be obtained doubtless civilized skill would transform it into beautiful fabrics. Perhaps the rearing of these goats may at some future time become an industry of Alaska and the Yukon Territory, where a large fixed population will slowly but surely be accumu- lated. Pronghorned antelope and bison. Anything for the preservation of this beautiful prong- horn, once so abundant in the West, must be done quickly. RAISING DEER FOR PROFIT Late in 1910 eight head of this antelope, selected from the National herd in Yellowstone Park, were placed in the Wichita National Game Refuge in Oklahoma, through the generosity of members of the RAISING DEEE FOR PROFIT 285 Boone and Crockett Club of New York, who bore the expenses of the transfer. bore the expenses of the transfer. The pronghorn is unlike any other antelope on the face of the earth, and has so many pe- culiarities that naturalists class it in a family by itself. Formerly existing by thousands on our open western plains, it has now been so reduced in numbers that its absolute extinction is certain in the very near future unless pro- tection is given to the few remaining. It does not do well east of the Mississippi river, and can not be successfully bred in captivity; but it thrives if allowed to roam practically free within large enclosures and under conditions closely approaching the natural ones. In 1908 the Biological Survey estimated that the total number of antelope in the United States had been reduced to 17,000. Of these about 10,000 were in Montana, Wyoming, and the Yellow- stone National Park, and the remaining 7,000 were distributed in 12 other states. On the Wichita Game Refuge it is hoped that the ante- lope will find themselves in surroundings suited to their increase. The pronghorn is unlike any other antelope on the face of the earth, and has so many pe- culiarities that naturalists class it in a family by itself. Formerly existing by thousands on our open western plains, it has now been so reduced in numbers that its absolute extinction is certain in the very near future unless pro- tection is given to the few remaining. It does not do well east of the Mississippi river, and can not be successfully bred in captivity; but it thrives if allowed to roam practically free within large enclosures and under conditions closely approaching the natural ones. In 1908 the Biological Survey estimated that the total number of antelope in the United States had been reduced to 17,000. Of these about 10,000 were in Montana, Wyoming, and the Yellow- stone National Park, and the remaining 7,000 were distributed in 12 other states. On the Wichita Game Refuge it is hoped that the ante- lope will find themselves in surroundings suited to their increase. RAISING DEER FOR PROFIT The Wichita is really a National Forest, but ANIMAL COMPETITORS 286 was set aside by Act of Congress as one of the two National Game Eefuges because of its especial suitability as a breeding-place for the wild creatures of the plains. This does not mean that outside of these refuges the game on the National Forests is not protected, for the officers of the Forest Service are uniformly instructed to see that the game laws of the various states are observed on their forests, in so far as they can do this in justice to their other duties. Many of the Forest officers are regularly appointed Deputy State Game Wardens, and it is the policy of the Depart- ment of Agriculture to encourage their acting in this capacity when they can do so without prejudice to their work as Forest officers. In the Wichita and the Grand Canyon Game Refuges the Government has not left the mat- ter of caring for game protection wholly to the States, but has established national reser- vations on which an attempt will be made to g The Wichita Refuge is further notable for the fact that it has a small herd of buffalo upon it, donated by the American Bison So- RAISING DEER FOR PROFIT 287 ciety and shipped from the New York Zoolog- ical Garden in 1908. They then numbered 15, and in two years had been increased by the addition of 10 calves; but two of the original herd had died. The bison seems destined to remain with us only as an object of curious interest, and a reminder of the vast herds which so short a time ago pastured on our western plains. "In- teresting as have been the experiments made by Mr. C. J. Jones and others, in the cross- breeding of buffaloes with domestic cattle," de- clares W. T. Hornaday, than whom none is more fitted to render a verdict, "it is now quite time that all such experiments should cease. It has been proven conclusively that it is im- possible to introduce and maintain a tangible strain of buffalo blood into the mass of western range-cattle. ' ' DIRECTIONS FOR POISONING AND TRAPPING THE most effective method of warfare against the multitude of rodent pests of the granary, garden, field and orchard, and the wolf-enemies of the sheepfold, is undoubtedly by the use of poison. The employment of this agent requires knowledge and care, however, since it is equally dangerous to animals that the farmer has no desire to kill, and to himself and his family. Fortunately for us, therefore, we can now avail ourselves of the results of much experience and of carefully formulated advice prepared by experts. p p y Waste of effort. We are met at the outset by the startling statement from the Biological Survey that "at present fully half the expendi- ture in the United States for rodent poisons is wasted." As it is added that "in the West the people of a single county sometimes expend $25,000 to $30,000 a year for poisons for de- 288 POISONING AND TRAPPING 289 stroying rodent pests," and as insecticides and other poisons for the entire country cost many millions of dollars annually, the saving of waste in this item of agricultural expense is seen to be important. Probably the buyer of proprietary poisons has the greatest cause for complaint. Often one or two cents' worth of material is retailed at from 25 cents to a dollar. The difference be- tween the cost of the material and the selling price represents the manufacturer's profit and the retailer's profit. Such large returns enable proprietors to spend much money in advertis- ing or otherwise exploiting their wares, which, if not worthless, are never so good as an intel- ligent man may compound for himself at a small fraction of their price. It is to enable the readers of this book to do so that the in- structions which follow are here given. They are formulas which have been dictated by scien- tific knowledge and approved by experience; and the methods of application recommended are those which have been found to involve the least possible danger to man, to domestic stock, and to valuable wild birds and mammals. It ANIMAL COMPETITORS 290 should be remembered, however, that in most states laws exist relative to the putting out of poisons ; and every man should inform himself as to these laws in his locality before beginning operations. Phosphorus and arsenic. i Essence of turpentine is said to be a positive antidote for phosphorus poison, and a cure for external burns by this element. DIRECTIONS FOR POISONING AND TRAPPING The poisons most commonly used to destroy mammal pests in America are phosphorus, arsenic, and strych- nine. Yellow phosphorus seems to be the one most in use for the destruction of rats and other rodents, but there are several serious ob- jections to it. First, a fourth of a grain is a dangerous and sometimes fatal dose for a human being. 1 Second, its slow, irritant ac- tion causes needless torture to the animals killed something we have no right to do. Third, it is very likely to cause disastrous fires. This substance is kept and cut under water and should not be touched with the hands. Its efficiency depends upon the fineness of its divi- sion, which is accomplished by first dissolving POISONING AND TRAPPING 291 the phosphorus in carbon disulphide, after which the substance is mixed with any suitable material, as flour or meal or glucose in the rat and roach pastes (from 2 to 4 per cent, strong), or is formed into the waxy sticks offered for sale. Now its instability in contact with the oxygen of the air is so great that it is liable when dry, and has been known, to burst into flame, setting fire to everything it touched. Cases have occurred in the West where it has thus caused fires which destroyed entire fields of ripe wheat and barley, and buildings in which prepared phosphorus was stored. Some hazard attends the use even of carefully prepared phos- phorus pastes. Arsenic, in the form of Paris green or Lon- don purple is widely employed as an insecticide. It is comparatively cheap, but is by no means as deadly as phosphorus or strychnine. The smallest quantity known to have been fatal to a human being is 2.5 grains. Its action on ro- dents is exceedingly variable, and there is ample proof that rats after taking small doses frequently become entirely immune to its further effects. Moreover, it is likely to sour ANIMAL COMPETITORS 292 baits, and so prevent their being readily eaten by the creatures to be got rid of. y g Virtues of strychnine. Strychnine is one of four alkaloids obtained from nux vomica, the seed of a tree known to botanists as Strychnos nux vomica. The chief supply comes from the Malabar coast, India. 1 "In case of poisoning by strychnine an emetic should be promptly given a teaspoonful of mustard in a glass of water (warm, if available). Another excellent emetic is zinc sul- phate (10 to 60 grains in tepid water) or apomorphine (4 drops by hypodermic injection). A stomach pump cannot be used after the first few minutes. As soon as the emetic has acted, the patient should be put slightly under the influence of chloroform or ether, and kept so for several hours. He should be kept in a darkened room and away from noise of all kinds. Further treatment may be left to the physician, who should be summoned as soon as the poisoning is dis- covered." U. S. Dept. Agr., Yearbook, 1909. DIRECTIONS FOR POISONING AND TRAPPING It consists of colorless crystals or white powder, and of several salts, chiefly the sulphate and the nitrate, in needle- like crystals. On account of its solubility the sulphate is most convenient for poisoning small animals, and is the one which should always be used. Strychnine is very bitter, and to disguise this sugar, honey, or an equivalent of its weight in saccharine, is mixed with the powdered poison; but this is not required for rabbits and field-mice, which are accustomed to bitter foods. As a poison for noxious animals strychnine has several advantages over the others com- monly in use. It kills quickly, without the long tortures of corrosive poisons. It spite of its bitterness, baits containing it are rejected less often than those containing arsenic. If strych- nine is properly labeled and kept from children, it is less dangerous to have on the premises POISONING AND TRAPPING 293 than most other poisons. Should strychnine be accidentally swallowed by an adult, antidotes are usually available, and by prompt action a fatal result may be prevented. 1 Experiments by the Biological Survey show that strychnine, all things considered, is a cheaper poison than arsenic. Strychnia sul- phate may be purchased in bulk at about 75 cents an ounce; white arsenic costs about 15 cents a pound. An ounce of strychnine will thoroughly poison 60 pounds of wheat intended for field-mice; a pound of arsenic will poison only 10 to 12 pounds of the grain for the same purpose. The cost of preparing the 60 pounds of wheat, therefore, will be about the same with either poison; but more of that containing ANIMAL COMPETITORS 294 arsenic is required to kill. Actual field experi- ments show that an ounce of strychnine, if properly distributed, is enough to kill 4,500 prairie-dogs or large ground-squirrels, or 9,000 field-mice. Various other poisons, both mineral and vegetable, have been used, but none is to be recommended as compared with strychnine, with the possible exception of barium carbonate as an agent for killing rats and mice about buildings. This mineral, which is cheap, has the advantage of being without taste or smell, and also that in the small doses fed to rats and mice it would be harmless to domestic ani- mals. Its action upon rats is slow, and if exit is possible, they usually leave the premises in search of water. Poisoning rats and house-mite. DIRECTIONS FOR POISONING AND TRAPPING For the reasons above given phosphorus and arsenic are not recommended in poisoning rats and mice; although powdered white arsenic mixed with oatmeal and sugar, or made into a paste of 12 parts of cornmeal and one part of arsenic with whites of eggs, is often effective. gg , Barium carbonate may be fed in the form of POISONING AND TRAPPING 295 dough composed of four parts of meal or flour and one part of the mineral ; or of oatmeal with about one-eighth of its bulk of the mineral; or the barium may be spread upon toasted bread, etc. A small quantity say a teaspoonful should be placed in the rat-runs, and repeated with change of bait until all the rats disappear. This is probably the best poison for use in dwelling houses. Strychnia sulphate is too rapid in action to make it advisable for use in our houses, since the animals die and decay in their holes in the walls and foundations, but for barns, ware- houses and outer premises generally it is the most effective agent. The dry crystals may be inserted in small pieces of raw meat, or toasted cheese, and these placed in the runs or burrows ; or oatmeal may be moistened with a strychnine sirup, and small quantities laid in the same way. y Strychnine sirup is prepared as follows: Dissolve a half ounce of strychnia sulphate in a pint of boiling water; add a pint of thick sugar sirup and stir thoroughly. A smaller quantity of the poison may be prepared with ANIMAL COMPETITORS 296 a proportional quantity of water. In prepar- ing the bait it is necessary that all the oatmeal should be moistened with the sirup. Wheat and corn are excellent alternative baits, but must be soaked in the sirup over night. To use this poison in places occupied by poultry a good plan is to provide two boxes, one considerably larger than the other and each having two or more holes in the side, large enough to admit rats. The poisoned bait should be placed on the bottom and near the middle of the smaller box and the larger box inverted over it. Then fowls cannot get at it if they try. It must be remembered that old rats become very wise and wary, and much caution has to be used if you are to succeed. Poisoning field-mice. DIRECTIONS FOR POISONING AND TRAPPING The most effective poison for the short-tailed field-mice is strych- nine. In the outbreak of these pests in Ne- vada, the best baits proved to be alfalfa and crushed wheat. An ounce of strychnia sulphate dissolved in 5 or 6 gallons of water will effectu- ally prepare 30 pounds of chopped dry alfalfa hay ; or, with iy2 gallons of water, will prepare 45 pounds of green alfalfa cut into short POISONING AND TRAPPING 297 lengths. The poisoned food is distributed near or in the mouth of burrows, a small pinch at a place, especially in cold weather, when the an- imals do not feed in the open. Green alfalfa bait should not be put out when the sun is hot. In the absence of alfalfa, crushed wheat, oat- meal and corn, among the grains, and seeds of various plants, as the tomato, dandelion, sun- flower and others, may be substituted. The bait should be soaked over night in a poisoned sirup, a quart of which is enough to poison half a bushel of grain. If after thorough mixing the solution is not sufficient to wet all the grain, add a little water. After standing over night, if the grain is too wet, a little dry corn-meal will take up the excess of moisture. If oat- meal is used as a bait, when the mass is wet throughout with the sirup, it may be used im- lengths. The poisoned food is distributed near or in the mouth of burrows, a small pinch at a place, especially in cold weather, when the an- imals do not feed in the open. Green alfalfa bait should not be put out when the sun is hot. p In the absence of alfalfa, crushed wheat, oat- meal and corn, among the grains, and seeds of various plants, as the tomato, dandelion, sun- flower and others, may be substituted. The bait should be soaked over night in a poisoned sirup, a quart of which is enough to poison half a bushel of grain. If after thorough mixing the solution is not sufficient to wet all the grain, add a little water. After standing over night, if the grain is too wet, a little dry corn-meal will take up the excess of moisture. If oat- meal is used as a bait, when the mass is wet throughout with the sirup, it may be used im- mediately. DIRECTIONS FOR POISONING AND TRAPPING y Because of the danger of destroying native birds, such as quail, the poisoned bait should be placed only under shelters which will admit mice but exclude birds. Wide boards lying upon thin cross-pieces of wood are excellent for the purpose. For pine mice baits may be ANIMAL COMPETITORS 298 placed in the underground tunnels. For other mice pieces of drain tile may be laid along the trails, and the baits inserted into the tiles with a long knife or spoon ; old tin cans with flattened ends or small openings are excellent substitutes for tiles. One trick of wolf-poisoners in Col- orado, was to make their strychnine pellets chiefly of lard so that if not eaten at night they would melt and soak into the ground, out of danger, in the next day's warm sun. g y Winter is the most favorable season for poi- soning field-mice, and the best time to set the poison is in the evening of a mild day. At that season cut small twigs or suckers from apple- trees, and either dip them in the strychnine sirup or apply the sirup to them with a brush. Scatter the poisoned twigs near the trees to be protected. This plan is excellent for either field-mice or rabbits, and it entirely obviates the danger of poisoning birds or domestic animals. Poisoning rabbits. Winter has proved to be the best time for poisoning rabbits, especially the western jack-rabbits, since there is no green food to attract them from the prepared titbits. Pieces of apple, carrot, sweet potato, Winter is the most favorable season for poi- soning field-mice, and the best time to set the poison is in the evening of a mild day. At that season cut small twigs or suckers from apple- trees, and either dip them in the strychnine sirup or apply the sirup to them with a brush. Scatter the poisoned twigs near the trees to be protected. This plan is excellent for either field-mice or rabbits, and it entirely obviates the danger of poisoning birds or domestic animals. g p g Poisoning rabbits. Winter has proved to be the best time for poisoning rabbits, especially the western jack-rabbits, since there is no green food to attract them from the prepared titbits. Pieces of apple, carrot, sweet potato, POISONING AND TRAPPING 299 melon-rind and the like are favorite baits. DIRECTIONS FOR POISONING AND TRAPPING Crystals of strychnine sulphate are inserted in them and they are left along rabbit-runs, either on the ground or elevated on short sticks. Arti- ficial runs may be made in orchards with a drag or a one-horse scraper. Another excellent bait is oatmeal soaked in strychnine sirup. In any case the rabbits must be carefully fenced away from haystacks, or they may not eat the poi- soned bait. Poisoning prairie-dogs and ground-squir- rels. A few years ago the State of Kansas carried on extensive operations against prairie- dogs, destroying them almost completely over nearly 2,000,000 acres of thickly infested land. The poison was prepared at the State Agricul- tural College, and was sold to townships and individuals at cost, or the formula for prepar- ing it was given to citizens who asked for it. A modification was found necessary, however, when the mixture was to be used in large quantities and this was perfected by David E. Lantz, with the following result : For 1 gallon poisoned sirup, use ANIMAL COMPETITORS 300 4 ounces powdered strychnia sulphate; 4 ounces potassium cyanid; 4 ounces green coffee; g 6 ounces alcohol; 4 eggs (whites only) ; 1/2 gallon thick sugar sirup. Mix the coffee and whites of eggs, and let the mix- ture stand over night. Dissolve the cyanid of potas- sium in a little less than a quart of hot water, and let it cool before using. Prepare the sugar sirup previously, so that it is not hot when used. Pour the cyanid of potassium solution over the coffee-and-egg mixture, stir, and then strain into the mixing vessel through a sieve fine enough to hold the coffee, which is rejected. Add the sugar sirup and stir thoroughly. Dissolve the strychnia in a little less than a quart of boiling water. Pour the alcohol into this solution and stir. Then add the mixture of strychnine, alcohol and water to the contents of the mixing vessel and stir thoroughly. The strychnine will be precipitated by the cyanid, and when the poison is placed in a can and allowed to stand will settle at the bottom. The poison should be kept closely corked until used. A gallon of this poisoned sirup is enough to poison two bushels of wheat. DIRECTIONS FOR POISONING AND TRAPPING Before it is mixed with the wheat it should be thoroughly stirred or shaken, and a few pounds of corn- rneal added to make the sirup adhere to the POISONING AND TRAPPING 301 grain. This preparation may be used immedi- ately. Another way is to add more water and leave the wheat over night to absorb the strych- nine. grain. This preparation may be used immedi- ately. Another way is to add more water and leave the wheat over night to absorb the strych- nine. Green alfalfa or alfalfa hay for poisoning prairie-dogs should be chopped into short lengths and sprinkled with strychnine water or sirup until thoroughly wet. A large metal washtub should be used as a mixing vessel. An ounce of strychnia sulphate dissolved in a half gallon of water will prepare 30 pounds of green alfalfa; or the same quantity of strych- nine dissolved in 3 or 4 gallons of water will prepare 20 pounds of alfalfa hay. p p p y For the smaller ground-squirrels, use 1 ounce strychnia sulphate, 35 pounds clean wheat, 2 gallons water. Dissolve the strychnine in the water in a large mix- ing vessel. Then pour in the wheat and allow all to simmer for an hour, the vessel being covered. Stir occasionally. The water will probably be entirely ab- sorbed by the grain, but if not, a little corn-meal will take up the extra moisture. If preferred, the strych- nine may first be dissolved in a pint of boiling water, the ingredients then mixed in a large vessel, and all ANIMAL COMPETITORS 302 left over night to absorb the poison. Distribute the poisoned wheat, a half teaspoonful at a place, at the mouth of the squirrel-burrows; do not scatter broad- cast on account of the danger of killing birds. For the larger ground-squirrels reduce the quantity of wheat to 25 pounds and the water in proportion. Experiments in California in destroying the digger ground-squirrel (Clitellus bcecheyi) with pieces of sugar-beets into which crystals of strychnia sulphate had been inserted with a knife gave good results ; and even better success has followed the use of poisoned barley-heads. Poisoning pocket-gophers. The pocket-go- phers are readily poisoned by strychnine, espe- cially in the late fall and early winter. Crys- tals may be inserted into pieces of potato, carrot, prunes or raisins, and inserted into the gopher tunnels several feet from fresh mounds. DIRECTIONS FOR POISONING AND TRAPPING Any strong prod will answer to make the holes into which the baits may be dropped. These holes need not be closed. When the animals are in ditch-banks the tunnel should be fol- lowed by digging with a garden trowel from the freshest mound to the main runway, the bait left there, and the opening closed. Car- bon bisulphide is successful with gophers only when the soil is moist and packed. POISONING AND TBAPPING 303 Poisoning wolves. The proper dose for a wolf is four grains of strychnia sulphate; for a coyote, two grains. Two sizes of gelatin capsules may be bought at drug-stores. Fill, cap, and carefully wipe each capsule to remove every trace of the drug from its outside. In- sert this filled capsule into a piece of beef- suet the size of a walnut and close the cavity. The baits should be carried in a can or pail, and not handled except with gloved hands or with forceps. They should be dropped from horseback along trails followed regularly by wolves, or along an artificial trail made by dragging an old bone or piece of hide well saturated with some fetid scent. Or they may be placed about a carcass on which wolves feed Trapping rats and mice. The best of all traps for this purpose, are the cheap snapping wire traps called guillotine traps, because they catch the animal by the neck and choke the life out of it in a moment. Those made entirely of metal (see illustration on page 75) are bet- ter than those with a wooden base. (jruillotine traps, according to Lantz, should ANIMAL COMPETITORS 304 be baited with small pieces of German sausage (Wienerwurst) or fried bacon. A small sec- tion of an ear of corn is an excellent bait if other grain is not present. The trigger wire should be bent inward to bring the bait into proper position to permit the fall to strike the rat across the neck. Other excellent baits for rats are oatmeal, toasted cheese, toasted bread (buttered), fish, fish offal, fresh liver, raw meat, pine nuts, apples, carrots, corn, and sunflower, squash, or pumpkin seeds. Broken fresh eggs are good bait at all seasons, and ripe tomatoes, green cucumbers, and other fresh vegetables are very tempting to the animals in winter. DIRECTIONS FOR POISONING AND TRAPPING When seed, grain, or meal is used with a guil- lotine trap, it is placed on the trigger plate, or the trigger wire may be bent outward and the bait sprinkled under it. p The old-fashioned barrel-trap may often be used to advantage. For several nights rats are tolled to food placed on the tops of barrels, or a barrel, covered with stiff brown paper tied over the top so as to not be displaced or break under their weight. Then two cross- POISONING AND TRAPPING 305 slits are cut in the paper, through which the rats fall, while the corners spring back making a deceptive surface for the next visitor. One of many variations of this, is a barrel with a FORMS OF THE BARREL-TRAP FOR RATS. The one on the left is covered with stiff paper, sprinkled with bait (b), and so slit that the rats fall through, while the corners of the paper fly back into, place. The one on the right has a tipping cover, resting on a cleat (a) at one side. FORMS OF THE BARREL-TRAP FOR RATS. FORMS OF THE BARREL-TRAP FOR RATS. The one on the left is covered with stiff paper, sprinkled with bait (b), and so slit that the rats fall through, while the corners of the paper fly back into, place. The one on the right has a tipping cover, resting on a cleat (a) at one side. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. When a great many rats are known to have gathered in a certain place, as under an old corn-shock (perhaps left and baited for the very purpose) a wire fence may be thrown around \t, the shock overturned, and the rats killed ANIMAL COMPETITORS 306 by men and dogs as they come out into the enclosure. One man reports that he has made great catches with a wire-cage trap, which he sets inside of a wooden box having a hole in one end against which the hole in the baited cage- trap is fitted. The box is then covered with trash and left. Sometimes several get in at once; especially if a single rat is left in it, whose squealing attracts others. g Large cage-traps, another advises, should be baited and left open for several nights until the rats are accustomed to enter them to obtain food. They should then be closed and freshly baited, when a large catch may be expected, especially of young rats. y p , p y y g Trapping is a simple way to destroy field- mice, but it needs to be steadily and systemat- ically continued to be of much service. It has special advantages for small areas such as lawns, gardens, and vegetable or nursery pits and packing houses, where a limited number of mice are present, and wherever, for any rea- son, there are objections to the laying out of poison. As voles do not readily enter cage- POISONING AND TRAPPING 307 traps, simple wire traps of the guillotine order, in which mice are instantly killed, are the most effective. Traps without bait may be set across the runs of the mice, where the animals spring them by coming in contact with the trigger, or they may be baited with oat or corn meal. METHOD OF CONSTRUCTING A TIP-UP TRAP WITH A BOX. METHOD OF CONSTRUCTING A TIP-UP TRAP WITH A BOX. For trapping pine mice an opening should be made in the underground tunnel large enough to receive the trap, which should be set across the bottom of the runway. The traps may be baited or not, but the opening should be cov- ered. Traps for gophers. Trapping is a success- ful method when followed intelligently and persistently. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. It is especially adapted to small ANIMAL COMPETITORS 308 fields, orchards, gardens, and irrigation em- bankments, where only a few gophers are pres- ent; but in the case of large areas that are badly infested, the method involves too much labor. An ordinary No. steel trap may be employed with success, but there are on the market several special gopher-traps which are better adapted for general use. ONE FORM OF GOPHER-TRAP. ONE FORM OF GOPHER-TRAP. In using the ordinary steel trap, the first step is to make an opening into the main go- pher tunnel. The trap should then be sunk so that the jaws are level with the bottom of the runway and lightly covered with green clo- ver, alfalfa grass, or even loose soil, care being taken that these do not clog under the pan, or trigger. No bait is required. The hole should POISONING AND TRAPPING 309 be just large enough to receive the trap and should be covered so as almost to exclude the light. Scalding the trap frequently to remove the animal odor is important. "A few days' experience will teach one more about setting traps for gophers than pages of directions could. He must not be discouraged by failure at first, but vary the method of setting the trap until he learns the best way for his locality. While the method is somewhat slow, persistent trapping steadily decreases the pests until the last gopher on a farm may be captured. A correspondent of the Biological Survey writes that he caught 1,332 of the animals within 2 miles of his home. A friend of the writer in Kansas trapped 350 gophers on a 40-acre clover field in four months. A California newspaper stated that in the spring of 1901 a man near Watsonville, by using 52 traps, caught 233 in twenty-four and one-half hours. William Burniece, of Bowbells, N. Dak., trapped more than 1,500 gophers on his quar- ter section during a single year." Snares and Traps for Rabbits. Babbits are easily trapped or snared, and few farmer-boys need instruction how to do it. An improve- ment upon the figure-four and similar traps is widely used in the West, and known by the name of its inventor, Fred Wellhouse, of To- ANIMAL COMPETITORS 310 peka, Kansas. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. This trap is a box of old six- inch fencing-boards, two feet or less long and 10 inches square, closed at the back by a door, but in front by a wire door only, which hangs from the top and swings inward. The trap is set and the wire door is kept open by a wire trigger-rod, held in place by two staples fas- tened to the top of the box. This trigger is bent downward near the rear of the trap and formed into a loop or a figure eight. As the rabbit enters the trap and crowds into the back part, it pushes upon the loop, moves the trigger wire backward, and releases the wire door. This falls and makes the rabbit a pris- oner. Bait may be used, but is not necessary, since the cottontail is constantly looking for dark places to hide from enemies or cold winds. Mr. Wellhouse uses about three traps per acre in young orchards and many among the bear- ing trees. The materials needed are: Four boards 1 by 6, 21 inches long; one piece 1 by 6, 8 inches long for the back ; a short cleat for the door stop ; 2Sy2 inches of wire to serve for the door; 22 inches of wire for the trigger; four ll d peka, Kansas. This trap is a box of old six- inch fencing-boards, two feet or less long and 10 inches square, closed at the back by a door, but in front by a wire door only, which hangs from the top and swings inward. The trap is set and the wire door is kept open by a wire trigger-rod, held in place by two staples fas- tened to the top of the box. This trigger is bent downward near the rear of the trap and formed into a loop or a figure eight. As the rabbit enters the trap and crowds into the back part, it pushes upon the loop, moves the trigger wire backward, and releases the wire door. This falls and makes the rabbit a pris- oner. Bait may be used, but is not necessary, since the cottontail is constantly looking for dark places to hide from enemies or cold winds. Mr. Wellhouse uses about three traps per acre in young orchards and many among the bear- ing trees. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. The materials needed are: Four boards 1 by 6, 21 inches long; one piece 1 by 6, 8 inches long for the back ; a short cleat for the door stop ; 2Sy2 inches of wire to serve for the door; 22 inches of wire for the trigger; four small and nails POISONING AND TRAPPING 311 Trapping the wary coyote. All agree that coyotes are not easily trapped, and what is said here can be regarded as only a suggestion. These pestiferous little wolves travel in pretty well-defined paths and usually hunt against the wind. Having a keen sense of smell, they easily detect the tracks of man, and if they have had previous experience of traps or guns they are suspicious of danger. g y g The best No. 4 double-spring trap, with a heavy, welded wolf-chain, should be used. If the trap is to be fastened to a stationary ob- ject the chain should have a swivel at each end; if to a drag, such as a forty-pound stone (to which it must be attached with telegraph wire), one swivel next the trap is enough. Where it can be done it is a good plan to bind the trap- chain to a heavy pole lying on the ground, be- ing careful not to change its natural position. If the trap is anchored to a bush or small tree the chain must be securely fastened with snap or wire. A stout stake over which the ring will not slip, driven out of sight into the ground, is better. Every part of the trap and chain is covered, and the ground left in as ANIMAL COMPETITORS 312 natural and undisturbed condition as pos- sible. Any kind of fresh meat will do for bait rab- bits and other small rodents are often used, but larger baits seem to be more attractive. It is also of advantage after setting the trap to make a "drag" of the bait for a quarter to a half mile, at the end of a rope from the saddle horn, and finally to fasten it to a bush or stake close to the trap, or cut it in bits and scatter all around the trap, so that not all can be reached by the coyote without walking over the trap. The skill of the trapper and the situation of the trap will determine the best arrangement. top swinging on pivots which dumps a rat that leaps upon it, then rises level again. The suspicion of the coyote is lessened apparently after following the bloody trail of a well-planned drag. Before setting the traps many trappers rub their feet and hands on a skin or some strong- smelling meat or carcass to conceal the human odor. Oil of anise or rhodium is sometimes used for the same purpose. Any strong odor is likely to attract the attention of the coyote and allay suspicion. Care must be taken not to spit on the ground or kneel or throw POISONING AND TRAPPING 313 down any clothing in the vicinity of the trap. A good plan is to set a line of traps and leave them for a day or two, and then go the rounds with a horse and drag and bait the traps with- out dismounting. American voles, 50. INDEX American voles, 50. Antelope, 273 ; pronghorned, 284. Canadian fur-bearers, 243. Carcajou, 243. Caribou, 273. Chase, A. W., 103. Arsenic, use of in destroying pests, 290. Arsenic, use of in destroying pests, 290. Bachman, Dr., 252. Marten, 242; Canadian, 243; Pennant's, 243 ; stone-mar- ten, 254. Bachman, Dr., 252. Bachman, Dr., 252. Chase, A. W., 103. Badger, 263; badger burrows, 265 ; prehistoric plowing, 267; aids the farmer, 268. Chipmunk, 144 et seq.; the burrow and its furniture, 145; familiarity, 147. Construction, rat-proof, 28. Cotton-rat, 68, 105; habits, 106. Chipmunk, 144 et seq.; the burrow and its furniture, 145; familiarity, 147. 145; familiarity, Construction, rat-proof, 28. ; , Bailey, Vernon, 96, 99, 157, 238. ; , Bailey, Vernon, 96, 99, 157, 238. p , Cotton-rat, 68, 105; habits, 106. p Cotton-rat, 68, 105; habits, 106. Barabaschi, Dr. P., 42. Barabaschi, Dr. P., 42. Coues, Dr. Elliott, 46, 260, 266, 267. Coues, Dr. Elliott, 46, 260, 266, 267. Barium carbonate as a poison, 294. Barium carbonate as a poison, 294. Bats, 201; species of, 202; economic relations, 204. Bats, 201; species of, 202; economic relations, 204. Beaver, 94 et seq. Belgian hare, 177. Bell, Dr. Robert, 123. Bighorn, 273, 383. Bison, 284. Black fox, 243. Blanford, W. T., 7. Bowen, George T., 82. Breeding foxes, 227; mink, 259; skunks, 271. Bristoe, W. M., 187. Buffalo, 273. 3 Coyote, 232 et seq.; character of, 233; beneficial food hab- its, 235; injurious food habits, 236; as a pest, 237; fencing against, 241 ; traps for, 311. Bats, 201; species of, 202; economic relations, 204. Beaver, 94 et seq. Belgian hare, 177. Bell, Dr. Robert, 123. Bighorn, 273, 383. Bison, 284. Black fox, 243. Blanford, W. T., 7. Bowen, George T., 82. Breeding foxes, 227; mink, 259; skunks, 271. Bristoe, W. M., 187. Buffalo, 273. 3 Coyote, 232 et seq.; character of, 233; beneficial food hab- its, 235; injurious food habits, 236; as a pest, 237; fencing against, 241 ; traps for, 311. Cultivation of muskrats, 89; of the mink, 258; of skunks, 270. Cultivation of muskrats, 89; of the mink, 258; of skunks, 270. , Cummins, L. C., 187. ; Bristoe, W. M., 187. , Buffalo, 273. 315 316 INDEX Fumigation of rodents, 190. Fumigation of rodents, 190. Fur-bearing animals, 242 et seq.; Canadian, 243. of domestication, 275; rear- ing wapiti for profit, 276; rearing vvhitetail deer, 278. Deer-mice, 68. Doane, Prof. R. W., 23, 152. of domestication, 275; rear- ing wapiti for profit, 276; rearing vvhitetail deer, 278. Deer-mice, 68. g Fur-bearing animals, 242 et seq.; Canadian, 243. , Doane, Prof. R. W., 23, 152. , Doane, Prof. R. W., 23, 152. Enemies of rodents, 185. Elk, 273. Bachman, Dr., 252. Enemies of rodents, 185. Elk, 273. Ermine, 242; his family, 245; habits, 250 et seq. Exterminating rodents, 191. Gopher, 112 et seq.; burrow- ing powers, 114; destruc- tive to crops, 117; enemies to orchards and forests, 118; tap irrigating ditches, 121; as soil makers, 122; traps for, 307. , Ermine, 242; his family, 245; habits, 250 et seq. , seq. Exterminating rodents, 191. Exterminating rodents, 191. Exterminating rodents, 191. Fencing against wild animals, 240. t aps , Gopher-snake, 189. t aps Gopher-snake, 189. p Gopher-snake, 189. Ferrets, 32. Gray squirrel, 133; peculiari- ties of, 136. , Ground-hog, 160. , Ground-hog, 160. Flying-squirrel, 138 ; charm of, 140; family life of, 141. Flying-squirrel, 138 ; charm of, 140; family life of, 141. Fox, 206 et seq.; the kit-fox, 206; gray fox, 208; arctic fox, 209; red fox, 210; value of fox fur, 212; vari- ability of the red fox, 214; fox farming, 214; area suited for, 215; arrange- ment of breeding quarters, 216; form of enclosures, 217; easy to keep, 220; food and feeding rules, 221; re- production and treatment of young, 224; importance of good care, 226; breeding for improved stock, 227; preparation of skins, 230; expectation of profit, 231. Flying squirrel, ; of, 140; family life of, 141. Fox, 206 et seq.; the kit-fox, 206; gray fox, 208; arctic fox, 209; red fox, 210; value of fox fur, 212; vari- ability of the red fox, 214; fox farming, 214; area suited for, 215; arrange- ment of breeding quarters, 216; form of enclosures, 217; easy to keep, 220; food and feeding rules, 221; re- production and treatment of young, 224; importance of good care, 226; breeding for improved stock, 227; preparation of skins, 230; expectation of profit, 231. Hardy, Manly, 130, 132. Harvest-mice, 68. Hornaday, W. T., 287. Horses, wild, pest of, 280. Horses, wild, pest of, 280. p House of the wood-rat, 99. Japanese jumping mice, 38, 107. Jones, C. T., 287. Jones, C. T., 287. Jones, T., Kangaroo rats, 107, 109. Kangaroo rats, 107, 109. Lantz, David E., 0, 9, 10, 15, 18, 48, 58, 80, 118, 192, 234, 240, 253, 275, 299. Marten, 242; Canadian, 243; Pennant's, 243 ; stone-mar- ten, 254. q , ties of, 136. Pine-mouse, 51, 53; food hab- its, 55. Piper, Stanley E., 60. g Pine-marten, 242. Pocket-mouse, 68. Musk hog, , Muskrat, 76 et seq.; mischief done by, 76; value of, 78; as a food, 80; recipe for 68; , Musk-hog, Texan, 282. Bachman, Dr., 252. INDEX 317 Meadow-mouse, 48 et seq.; common, 51; general habits, 56; plague of, 58; preven- tion of plagues, 62 ; food of, 65; damage from, 66; pre- vention measures, 68; pro- tection of orchards against, 70. cooking, 82; trapping, 84; preparation of pelt, 88; cultivation of, 89; possibil- ities, 90; suitable places and proper care, 91. Nelson, E. W., 66. cooking, 82; trapping, 84; preparation of pelt, 88; cultivation of, 89; possibil- ities, 90; suitable places and proper care, 91. 70. Orchards, protection of against mice, 70; against rabbits, 173. Osgood, W. H., 213, 250, 269. Otter, 263. Merriam, Dr. C. Hart, 119, 122, 197, 245. Mink, 242; life of, 254; value of, 256; trapping, 257; cul- tivation for profit, 258; care of, 260. , Minkeries, 259. Mole-gopher, 113. y, Pack-rat, 101 et seq.; thiev- ing propensities, 102. Pekan, 243. Pennant's marten, 243. Pantry-mouse, 37 et seq. Pest of wild horses, 280. Phosphorus, use of in destroy- ing pests, 290. Moles, 194; under the lawn, 194; strength of, 196; methods of, 197; star-nosed mole, 198. Moles, 194; under the lawn, 194; strength of, 196; methods of, 197; star-nosed mole, 198. , Montgomery, F. U., 9. , Montgomery, F. U., 9. Montgomery, F. U., 9. Moose, 273. Mountain goat, 282. Mountain goat, 282. Mountain goat, 282. Mouse, 37 et seq.; pantry- mouse, 37 ; meadow-mouse, 48 ; Japanese dancing mouse, 38; prevalence of, 40; carry bacilli, 41; musi- cal, 43; prairie mouse, 53; food habits, 55 ; wild habits, 56; pine mouse, 53; deer mouse, 68; pocket mouse, 68; harvest mouse, 68. Musk-hog, Texan, 282. Muskrat, 76 et seq.; mischief done by, 76; value of, 78; as a food, 80; recipe for g Mouse, 37 et seq.; pantry- mouse, 37 ; meadow-mouse, 48 ; Japanese dancing mouse, 38; prevalence of, 40; carry bacilli, 41; musi- cal, 43; prairie mouse, 53; food habits, 55 ; wild habits, 56; pine mouse, 53; deer mouse, 68; pocket mouse, 68; harvest mouse 68. Pine-mouse, 51, 53; food hab- its, 55. Pine-mouse, 51, 53; food hab- its, 55. Poisoning and trapping, direc- tions, 288 et seq.; waste of effort, 288; phosphorus and arsenic, 290; virtues of strychnine, 292 ; poisoning rats and house mice, 294; poisoning field-mice, 296 ; poisoning rabbits, 29H ; poisoning prairie dogs, 299; Serpents as destroyers of ro- dents, 188. Salamander, 112. p g , Spermophile, 148. p , Vermin, directions for exter- mination, 288 et seq. Virginia deer, 278. Voles, American, 50. Bachman, Dr., 252. Poisoning and trapping, direc- tions, 288 et seq.; waste of effort, 288; phosphorus and arsenic, 290; virtues of strychnine, 292 ; poisoning rats and house mice, 294; poisoning field-mice, 296 ; poisoning rabbits, 29H ; poisoning prairie dogs, 299; Musk hog, , Muskrat, 76 et seq.; mischief done by, 76; value of, 78; as a food, 80; recipe for 318 INDEX Recipe for cooking muskrat, 82. Red-backed mouse, 53. poisoning pocket-gophers, 302; poisoning wolves, 303. Porcupine, 161. p 82. Red-backed mouse, 53. Red squirrel, 126; home of, p Prairie-dog, 153; dwellings, 156; a serious pest prob- lem, 157; poison for, 299. squirrel, 126; home of, 127; food of, 128; prepara- tions for winter, 131. Remedies for injured trees, 175. ; p Prairie-mouse, 51, 53. 175. Preparation of fox skins, 230. Preparation of fox skins, 230. Rodents, suppression of as pests, 184; foolish destruc- tion of their enemies, 184; weasel tribe best police, 185; useful aid by birds, 187; aid from serpents, 188; poisoning and fumiga- tion, 189; flooding burrows, 191; difficulty of extermina- tion, 191. Rabbit, 164 et seq.; excellence of flesh, 164; breeding hab- its, 166; injurious to gar- dens, 168; laws protecting, 169; bounty on, 170; hunting, 171; protections against, 173; as pets, 177; hutches, 179; feeding, 181; directions for breeding, 183 ; traps for, 301. p Rat, 3 et seq.; brown house- rat, 3; black rat, 4; habits, 5; history, 6; fecundity, 8; cost, 10; destructiveness in fields, 12 ; destructiveness to poultry, 15; damage to buildings and stored goods, 17; carriers of disease, 19; responsible for the plague, 21; methods of suppression, 26 ; rat-proof construction, 28; keeping food from, 30; disposal of dead, 31; four- footed enemies, 31; co-oper- ation in subduing, 34; traps for, 303. Serpents as destroyers of ro- dents, 188. Serpents as destroyers of ro- dents, 188. Seton, Ernest Thompson, 47, 110, 122, 253, 262. Seton, Ernest Thompson, 47, 110, 122, 253, 262. , Shrews, 199. Skunks, 270; skunk farming, Skunks, 270; skunk farming, 271. 271. Squirrel, 125 et seq.; tree- squirrel, 125; red-squirrel, 126; gray and fox squir- rels, 133; flying squirrel, 138; ground squirrel, 144; squirrels and bubonic plague, 151. Squirrel, 125 et seq.; tree- squirrel, 125; red-squirrel, 126; gray and fox squir- rels, 133; flying squirrel, 138; ground squirrel, 144; squirrels and bubonic plague, 151. Rat, cotton, 68. Rat, cotton, 68. Bachman, Dr., 252. p p , Star-nosed mole, 198, INDEX 319 Striped gopher, 148, 150. Wapiti, 273; raising for profit, 276. Water-rat, 76. Wapiti, 273; raising for profit, 276. Water-rat, 76. Wash lor protection against mice, 73; against rabbits, 175. Traps, for mink, 257 ; for rats and mice, 303; barrel trap, 305; for gophers, 307; for rabbits, 309; tor coyotes, 311. Weasel, 247; and chickens, 248; as a mouser, 249. ; Weasels, enemies of rats, 32; and rodents, 242. White tail deer, 279. Wichita National Game Ref- uge, 286. Woodchuck, 160. Wood-mouse, 105. Wood-rats, 98. Wolverine, 243. Wolves, 232 et seq ; poison for 303. Weasels, enemies of rats, 32; and rodents, 242. White tail deer, 279. Wichita National Game Ref- uge, 286. Woodchuck, 160. Wood-mouse, 105. Wood-rats, 98. Wolverine, 243. Wolves, 232 et seq ; poison for, 303. Tree-protectors and washes, 71. Tree-protectors and washes, 71. Tree-protectors and washes, 71. Trees, to protect from mice, 70; from rabbits, 173; rem- edies for injured, 74. Trees, to protect from mice, 70; from rabbits, 173; rem- edies for injured, 74. j , Texan mush-hog, 282. j , Texan mush-hog, 282. Wolverine, 243. Wolves, 232 et seq ; poison for, 303. p , Vermin, directions for exter- mination, 288 et seq. Virginia deer, 278. Voles, American, 50. Yerkes, Prof. Robert M., 39. t* i- .- c).lte stamped below II III I I I I I II 3 1158 00133 0314 II III I I I I I II 3 1158 00133 0314 00, rigg-jgg, 00, rigg-jgg, .NGEtES LIBRARY
https://openalex.org/W2465670720
https://rua.ua.es/dspace/bitstream/10045/50262/1/2015_De-la-Cuesta_TextoContextoEnferm_eng.pdf
English
null
A QUALIDADE DA INVESTIGAÇÃO QUALITATIVA: DA AVALIAÇÃO À CONCRETIZAÇÃO
null
2,015
cc-by
5,797
Reflection - 883 - Reflection - 883 - http://dx.doi.org/10.1590/0104-070720150001150015 LA CALIDAD DE LA INVESTIGACIÓN CUALITATIVA: DE EVALUARLA A LOGRARLA RESUMEN: El objetivo de este artículo es enfatizar la importancia de la calidad en el proceso de investigación y no en su valoración después de ella, algo a lo que la bibliografía se ha dedicado extensamente. Así mismo, reflexiona sobre el debate de calidad de la investigación cualitativa y muestra la valoración de la calidad como una práctica situada. La reflexividad se presenta no como un criterio para evaluar la calidad, sino como un instrumento para lograrla. Tres son las características de la investigación cualitativa a las que los investigadores deben prestar una atención reflexiva. La primera es que los estudios cualitativos versan sobre experiencias humanas; la segunda que estas experiencias son de carácter subjetivo; y la tercera, que el conocimiento cualitativo es ideográfico y construido durante la investigación. Alrededor de estas características, en el artículo se señalan cuestiones que se repiten constantemente en los estudios e inadvertidamente amenazan su calidad. DESCRIPTORES: Control de calidad. Investigación cualitativa. Validez social de la investigación. THE QUALITY OF QUALITATIVE RESEARCH: FROM EVALUATION TO ATTAINMENT Carmen de la Cuesta Benjumea1 Carmen de la Cuesta Benjumea1 1 Ph.D. Full Professor, Departamento de Psicología de la Salud, Facultad de Ciencias de la Salud, Universidad de Alicante. Alicante, Spain. E-mail: ccuesta@ua.es ABSTRACT: The objective of this paper is to emphasize the importance of quality in the research process instead of its valuation afterwards, an issue the literature has given extensive attention to. In addition, it is a reflection on the debate about the quality of qualitative research and presents the assessment of quality as a situated practice. Reflexivity is presented not as a criterion to assess the research quality but as an instrument to achieve it. There are three characteristics of qualitative research that researchers need to pay reflexive attention to. The first is that qualitative studies deal with human experiences; the second that these experiences are subjective; and the third that qualitative knowledge is ideographic and constructed during the study. Beyond these characteristics, issues are signaled that are constantly repeated in the studies and that unknowingly are a threat to their quality are addressed in this paper. DESCRIPTORS: Quality control. Qualitative research. Social validity. INTRODUCTION INTRODUCTION to assess it.5 Then, several criteria and evaluation models were proposed, which opened the door for what has been called the quality debate.6 “Methods are not procedures to be followed in any standardized way, but rather are created anew in every research project by researchers who hold their work to a standart.”1 The issue about the rigor of qualitative re- search can be overwhelming to young researchers in view of the large number of publications and different positions about it. In a recent literature review, four approaches were identified: 1) those who think that no specific criteria are needed as there is only one kind of research; 2) those who defend specific criteria for qualitative research; 3) those who consider that each qualitative method should contain specific criteria; and 4) those who use an overlapping criterion that comprises the general and particular factors of the qualitative methods.7 The most common position nowadays are researchers who defend specific guidelines for qualitative researcher,2 but adjusted to the differ- ent qualitative methods. Since the end of the past century, the quality of qualitative research has been a theme of inter- est, debate and, in some cases, dispute. The need to establish the legitimacy of qualitative research in the scientific community and to provide tools for reviewers external to the research process have played a decisive role in the proliferation of articles, texts and the design of evaluation instruments. In addition, the boom in systematic reviews that include qualitative studies and meta- syntheses has sharpened the need for agreements on the most appropriate way to value the scientific merit of qualitative studies. To date, proposals of criteria to assess the merit of qualitative research are endless and in some cases repetitive, with authors indicating different terms for the same thing.7 With his controversy comes the proliferation of technical language. Assessing the quality of a study is not a tech- nical, aseptic exercise, but is contextualized and, in addition, has an educational impact, as the as- sessment criteria serve as pedagogies of practice.2 It is the educational aspect of quality this article is focused on. I will emphasize the importance of quality in the research process instead of its evaluation afterwards, something the bibliography has paid extensive attention to. INTRODUCTION I agree with the belief that rigor is an aspect constructed during the study and that it is the researcher’s responsi- bility to achieve it.3-4 Instead of writing for future evaluators external to the research process, I write for researchers and mainly beginning researchers, doctoral students and junior researchers. I intend to alert them to issues that interfere in the research quality and I raise themes they should pay reflex- ive attention to. The core of the quality debate has been the impossibility to assess quality by applying stan- dardized criteria in view of the flexible nature of qualitative research, besides the danger that these criteria will serve as straightjackets.6 Neverthe- less, there exists a generalized consensus today that flexible criteria are needed, in view of great disagreements in the bibliography about the most appropriate criterion to assess research quality. What is paradoxical about this undertaking is to underwrite the subjectivity and, at the same time, establish objective criteria that demonstrate the merit of a qualitative study.6,8 Hence, what seems to underline the whole quality issue is the tension about positivist and interpretative positions. In this paper, I link reflexivity with quality. I present reflexivity not as an indicator of quality, but as a tool to achieve it and with an educational potential. I support my arguments on my research and teaching experience, share my own and other researchers’ errors. First, I will briefly contextual- ize the issue of the quality of qualitative research. These tensions are shown in proposals for evaluation criteria of qualitative studies as well as in its assessment tools. To give an example, it has been appointed that the evaluation models are based on the rationality of biomedical sciences and that some tools contain soft criteria from quantita- tive research applied to qualitative research.2 In ad- dition, it has been raised the question about what counts as evidence. It has been pointed out to the fact that, in the health area, evidence is considered as what adjusts to certain positivist standards.3 The consequence of this is that qualitative evidence is discarded. A QUALIDADE DA INVESTIGAÇÃO QUALITATIVA: DA AVALIAÇÃO À CONCRETIZAÇÃO RESUMO: O objetivo deste artigo é enfatizar a importância da qualidade no processo de investigação e não sua valorização depois de concluído, assunto sobre o qual a bibliografia tem se dedicado largamente. Reflete sobre o debate de qualidade da investigação qualitativa e mostra o valor da qualidade como uma prática em contexto. A reflexividade apresenta-se não como um critério para avaliar a qualidade, mas sim como um instrumento para atingi-la. São três as características da investigação qualitativa a que os investigadores devem prestar uma atenção reflexiva. A primeira é que os estudos qualitativos dizem respeito às experiências humanas; a segunda é que as experiências têm caráter subjetivo; e a terceira, que o conhecimento qualitativo é ideográfico e construído durante a investigação. Para além destas características, assinalam-se questões que se repetem constantemente nos estudos e que, inadvertidamente, ameaçam a sua qualidade. DESCRITORES: Controle de qualidade. Pesquisa qualitativa. Validade social em pesquisa. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. - 884 - De la Cuesta Benjumea C Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. THE ASSESSMENT OF QUALITY: A CONTEXTUALIZED PRACTICE To assess the merit of a qualitative study, are available closed tools, like the checklists that con- tain a large number of items and predetermined score sheets; and open tools with a small number of criteria for the reviewers to use them as guide- lines. While, in the first case, the reviewer does not need to be familiar with qualitative research, in the second case, it is absolutely necessary to know it well. In fact, many of the closed score sheets were elaborated to help people who were not familiar with the qualitative method.2 Their use has en- tailed negative consequences though. It has been appointed that the blind application of criteria weakens qualitative research,4 and that some crite- ria have encouraged superficial qualitative analy- sis.3 On the other hand, the closed score sheets tend to present the research as a set of procedures to comply with and can promote the elaboration of reports with quality jargon but void of contents. I have received plenty of research reports that men- tion terms like: theoretical sampling, saturation or negative case, without evidence that this actually took place in practice. The quality of qualitative research should be considered as an ongoing debate6,13-14 instead of a problem that needs solving.15 In the last 30 years, the debate has led to the improvement and devel- opment of its quality. Approaches have moved from that of evaluating the studies to proposing a less punitive approach of assessing them; from emphasizing the difference between qualitative and quantitative criteria to accepting a shared terminology; from considering assessment as a technical topic to highlighting its ethical and politi- cal component; from seeking a single standard for all qualitative studies to recognizing that studies should be multiple; from attending to method- ological issues to also attending to paradigmatic issues; from focusing on the scientific aspects of the study to also focusing on the creative aspects of the study; from focusing on the content of the study to also focusing on the form.2,6,8,11,13-14,16 For those who are starting their qualitative research journey, a review using an open tool that comments on the strengths and weaknesses of their work is undoubtedly more useful than a report in which it is indicated what was complied with and what was not. THE ISSUE ABOUT THE QUALITY OF QUALITATIVE RESEARCH The contemporary criteria for qualitative research assessment have been comprehensively developed, despite the lack of a full consensus. One might say that everything starts in 1985, when specific language and criteria are proposed Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. The quality of qualitative research: from evaluation to attainment - 885 - Where the emphasis of the evaluation is put should also be taken into account, whether it is on the results of a study or it is on the methodologi- cal process through which it is developed. In that sense, the emphasis on the assessment of aspects of the research process has been criticized and at- tributed to positivist positions.9 The importance of critically examining the results of qualitative studies was highlighted in a metasynthesis about women with HIV/AIDS.10 These authors detected an important number of studies whose findings were not qualitative, although the studies figured as such in the methodological sense. In addition, methods like the grounded theory are ruled by criteria that apply to the study results, in which the credibility, originality, resonance and utility of the theory produced are valued.11 The concern with the methodological rigor of qualitative research is a positive legacy.12 arguments. Three is the rigor of the study, which refers to the methodological validity.6 These prin- ciples are balanced and respond to the cannons of qualitative research. Not only the reviewers, but whoever engages in qualitative research should be familiar with them. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. THE ASSESSMENT OF QUALITY: A CONTEXTUALIZED PRACTICE Much of the research that is learned as a novice researcher comes from the reviewers who attract attention to the weaknesses of the reports. Nowadays, a generalized consensus exists that the quality criteria should act as guidelines and should adjust to each qualitative method. Nevertheless, the difficulty to assess the merit of a qualitative study is acknowledged.6,8,14 Authors of qualitative research texts are increasingly inclined to present some guiding principles, together with a series of questions that guide the assessment of a study’s quality.6,11,17 Three recurring principles are present in the bibliography. One is the study’s contribution to the research. This contribution re- fers to the value and relevance of the evidence the study presents. The second criterion is the likeli- hood or credibility of the research findings. This principle relates to the plausibility or solidity of the But assessing a qualitative study is a situated activity. It always implies a professional judg- ment.6,11 It demands that the evaluator discerns between hardly important errors or omissions to interpretative or fatal ones.18 In addition, evalu- ators belong to interpretative communities that influence what they read and how they read it; not only a collective, but also a personal and esthetic judgment is made.18 Hence, it should cause no estrangement that, although the same criteria are Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. De la Cuesta Benjumea C - 886 - is difficult to achieve the former. Traditionally, reflexivity has been understood as the process of looking at oneself to critically examine the effect produced in the development of the research.21 In this paper, I will present it as the awareness that the researcher should have about the quality of his/her study. used by evaluators, different conclusions can be reached. Hence, it is common practice in system- atic reviews that the studies be assessed by at least two persons, with a third one to solve discrepan- cies. The subjective component of the assessment is thus tacitly acknowledged here. Assessing is a situated act in which not only the reviewer, but also what is reviewed intervenes; that is: if it is a manuscript for publication in a certain journal, if it is a report from a doctoral dissertation or a funding project. THE ASSESSMENT OF QUALITY: A CONTEXTUALIZED PRACTICE In addition, in this assessment is considered how scientific merit is defined in each discipline, that is: what counts as evidence.3,11 To give an example, as qualitative evidence tends to be considered as mere opinion in the health area, the criteria of what is considered as evidence should be expanded to integrate the findings from qualitative studies.3 One might say that we qualitative research- ers need to achieve that quality by naturally integrate it into our know-how, that is, that our research actions flow without any need to reflect on their coherence and pertinence. Given the emerging nature of qualitative studies, however, we should also maintain an alert and questioning attitude about their quality. Hence, we are judges and stakeholders here, objects and subjects of our actions at the same time, reflexive beings who in the action of talking and questioning ourselves, we mold our research beings. Therefore, reflexivity is something more than an item on a checklist to verify the rigor of a study, it is key to the achieve- ment of a habitus,22 of what we might call that of an investigative quality. That is how to achieve the standard referred to in the epigraph of this paper.1 Finally, in the assessment of the research quality there has been a trend to mix up the report itself with the research it represents.18 The report is designed more to persuade the reader of the merits of the study than to reflect it. The emphasis is on the writing, on producing a convincing, evocative text. In the report, linguistic resources are used to persuade the reader about the validity of the study.18 It is what is called literary technology that turns readers into “virtual witness(es)” to what they have never seen: namely, the conduct of the project itself”.18:6. In my experience, epistemological coherence is the key to the quality of qualitative studies and, at the same time, its Achilles heel. The strong pres- ence of positivist research in the health area and the students’ socialization in that perspective leads to misunderstandings in my opinion. Qualitative research has three characteristics, deriving from its epistemology, which are a cause of errors, mainly among novice researchers, and which threaten the quality of their studies. The first is that qualitative research deals with human experiences, the second that these experiences are subjective, and the third that qualitative knowledge is ideographic and constructed. ACHIEVING QUALITY: A REFLEXIVE ACT The emphasis on assessing the quality of a study after the study itself has left the role and responsibility of the investigator in the back- ground.3-4 More than 25 years ago, it was alerted that the quality of qualitative research rests on the researcher.19 Other authors1,4 emphasize this when they affirm that the research is as good as the researcher is, this is not strange as (s)he is the research tool.20 Similarly, quality cannot be sepa- rated from a reflexive act as, without the latter, it THE ASSESSMENT OF QUALITY: A CONTEXTUALIZED PRACTICE These characteristics are well-known but their profound implications tend to go by unnoticed. Making them explicit intends to alert young researchers so as to enable them to correct them before it is too late. Let us examine them. Quality judgments are based on the way the study is presented instead of the actual study.8,18 Understanding this clearly, helps to receive the criticism of a manuscript. If it is late to correct the errors as they are mistakes committed during the study, it will prepare the researcher for the next one. One learns research practice through mis- takes. Therefore, reflexive attention to the quality is needed. An issue that I will discuss next. Qualitative studies take interest in the subjectivity of a human experience: asking In a qualitative study, there are at least three classes of questions, all different but sharing the objective of capturing the subjectivity of what is being studied. These questions are the research question, the questions asked to participants and the questions asked to the data during analysis. The research question indicates the experi- ence or phenomenon being studies and, in general, the answer to this question will invite a narration i.e. a description. Thus, the researcher will examine whether the research question addresses a subjec- tive phenomenon, is about something external to the people or is about people themselves. That is of paramount importance, as the research question puts the research in motion and a proper start is needed. The researcher needs to pay attention so as not to raise concealed hypotheses or to relate variables. The term, “if” in a conditional mode, in a research question or objectives, indicates rela- tionships among variables, that the nature of the phenomenon is known beforehand, that a test will be developed. All of these are positivist positions the researcher should detect and avoid. A positiv- ist research question cannot be answered appro- priately using a qualitative method, but makes the research incoherent and gives the researcher a headache in the course of the study. Participants in a qualitative study do not constitute a sample in the positivist sense, as the goal is not to represent a population or universe; the subjects in a qualitative study are called infor- mants,26 as they share an experience that provides specific information. Here, the reflexive attention should focus on sampling, seeking different infor- mation with a view to a rich description of what is under study27 and, I repeat, not about who is being studied. If the researcher asks whether the number of participants is sufficient instead of the quality and diversity of the information, (s)he will be asking positivist questions and that will devi- ate the attention from the study, moving, without even noticing it, from what to whom. Similarly, if one asks about aspects like age and education level without any indication in the data that these topics are relevant, one will be looking for relations among variables, developing a positivist analy- sis. At this point, one should stop, inspect one’s research practice and remember what is being studied and what one wants to know. The quality of qualitative research: from evaluation to attainment The quality of qualitative research: from evaluation to attainment - 887 - this is not bad, as it can be correct during the study, the nature of qualitative research allows this, but one should notice it. The researcher should assess whether the writing of analytic memos and the advances in the results are centered on the participants instead of the description of the research phenomenon. The use of terms in the writing like some, others, most, alert the researcher about a positivist turn, indicating that the analysis is centered on the study informants and not on describing their experience. In this situation, the researcher will find that managing negative cases will be complicated, as these will be about people itself and categories will not saturate. Above all, it is fundamental for researchers to pay attention to and reflect on what his/her study is about, about how to formulate the research theme and not mix it up with a population group. To give an example, there is a difference between a study on pregnant adolescents or informal care- givers and another about adolescent pregnancy or family care. In the former, the theme is related to people, to a population group; in the latter, to a human phenomenon, i.e. to a theme that affects a group of people. Qualitative research is about what and not about whom. Therefore, the choice of the partici- pants will be based on their experience instead of some demographic or social variables. They par- ticipate in an experience and are therefore selected for the study.25 Introducing variables here and applying stratified sampling, that is: taking a bit of everything, introduces bias and threatens the saturation of the categories. Similarly, choosing the participants based on an experience they do not have experienced themselves and which they witness at most, will provide data that which is not qualitative, no matter how descriptive this data are or how openly it were obtained. Qualita- tive research is interested in the subjectivity of an experience and data should reflect this. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Qualitative studies focus on human phenomena or experiences instead of populations: making the study proposal Qualitative studies are interested in human experiences.23-24 That is a key characteristic from which questions about the research theme, sam- pling, data analysis and the scope of the research derive. Issues that if neglected, invalidate a study or lead to the rejection of qualitative research.18 Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Qualitative studies take interest in the subjectivity of a human experience: asking Similarly, here should be clear to the re- searcher that the place of study is not the object of the study,28 although the former evidently conditions the latter.29 If the theme under study includes the place of study, the researcher will need to reflect on what makes the place special and clearly indicate its particularities. The place of study is not invariably equal to the research The researcher will also focus on how the data analysis is being developed, on the axis of data analysis. If the analysis is more of a recount of people instead of reporting their experiences, - 888 - De la Cuesta Benjumea C context either, the place of the study is the physical support of the action-interaction.17,20 The context will constitute people’s relationships and indicates the conditions under which that action-interaction takes place. If the researcher does not take this into account, it will be difficult to contextualize this study and it is highly possible that its findings will lose explanatory power. One of the most clarifying things in a qualitative research study is precisely to discover that context, as it will give meaning to the experience and will explain the participants’ actions-reactions in understandable terms. data. If software is used to manage the analysis, special caution is due. These programs are very efficient and, if not operated correctly, they pro- mote a mechanical, fragmented and superficial analysis. It is increasingly clearer that findings in a qualitative study are constructed, that they emerge from the interaction between the researchers, the participants and the data.11 The reflexive diary should report these interactions and, if that is the case, re orient the analysis to achieve the quality of the study. Consequently, the questions that are posed to participants in fieldwork will reflect the re- searchers’ interest in capturing their subjective experience. There will be few open, exploratory questions with a descriptive aim. These questions will invite study participants to report on the uniqueness of their history. What is of interest here is to obtain details of the experience instead of generalities, this is a crucial characteristic of data that is qualitative. Researchers will be aware that the qualitative data are obtained through the relationships established with the participants and will pay attention to what is asked and on how it is done. Qualitative studies take interest in the subjectivity of a human experience: asking It is fundamental that the researcher critically analyzes the interview guide and his/ her style to obtain data. Nevertheless, it will not be rare for the participants to opine on something external, not experienced, that they report on other people’s experiences, that they offer inter- pretations about facts or people and that they use technical jargon. Therefore, in data analysis, the researcher will consider in the first place whether the information constitutes data and, in the second place, will distinguish between the descriptive and the interpreted data, so as to avoid inconsistent analysis.30 Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Qualitative knowledge is ideographic and constructed: knowing The knowledge produced in qualitative studies is ideographic, as they search for gener- alizations in the case and not in populations.10 Therefore, not generalizing one’s findings to other groups is not a limitation, as affirmed in some research reports and papers. Qualitative research has its own limitations, many of which derive from the sampling instead of the sample. The limitations imposed in the fieldwork mold the study and the researcher should be aware of these limitations and acknowledge them in the reflexive diary. The methodological decisions taken during the fieldwork are not so much related to objective motives of rigor, but to negotiations and ethical decisions about what data to obtain and how to do it. Quality of practice demands ethical reflection. Similarly, qualitative knowledge is con- structed in the action of research itself. It is not something that it is found, that it is “there”, outside, waiting to be discovered. The data are not collected, although this is the way that is commonly mentioned, but data are obtained or, better, constructed.11 Data triangulation, used frequently as a technique to ensure the validity of a study, can invalidate it when the researcher who uses this technique presupposes the existence of external, objective and verifiable knowledge through multiple sources. In qualitative studies, triangulation does not serve to seek a single real- ity or an objective fact, but to enrich the data, that is: to add characteristics that turn the description into something saturated. Finally, there are the questions the researcher asks the data with a view to generate analysis. These questions are asked to discover what is actually being studied and tuning the researcher with the insiders’ point of view. As example, the following questions will be asked to data: What happens in the data? What is the study about? What are participants concerns?30 This type of questions puts the researcher in a position of em- pathy and openness to discovery. Hence, during the analysis, the researcher will reflect on how (s) he interacts with the data, realize if (s)he mechani- cally undertakes the procedures and passively await the concepts “to appear”, or whether she/he engages in an active and interactive dialogue with Knowledge construction is a slow process developed in phases. It is not a whim, but follows the principle of building subjective and saturated knowledge. REFERENCES 1. Sandelowski M. Foreword. In: Thorne S, organizador. Interpretative description. Walnut Creek (US): The Left Coast Press; 2008. p.11-4. 2. Denzin NK. The politics of evidence. In: Denzin NK, Lincoln YS, organizadores. The SAGE handbook of qualitative research. Los Angeles (US): SAGE; 2011. p.645-57. Qualitative studies offer new knowledge or new ways of seeing the world. They are a personal construction of the researcher that takes place in interaction with study participants. This construc- tion is a product of who the researcher is, his/ her biography and way of thinking. As research instrument, she/he put all this into play to achieve a good study, but needs to be aware of who (s)he is, how (s)he thinks and does things. In other words, (s)he needs reflexivity for everything to favor the study quality. 3. Morse JM. Qualitative health research. Walnut Creek (US): Left Coast Press; 2012. 4. Morse JM, Barrett M, Mayan M, Olson K, Spiers J. Verification strategies for establishing reliability and validity in qualitative research. Int J Qual Methods [online]. 2002 [acceso 2014 Dic 15]; 1(2):. Disponible en: https://www.ualberta.ca/~iiqm/ backissues/1_2Final/pdf/morseetal.pdf 5. Lincoln YS, Guba EG. Naturalistic inquiry. Newbury Park (US): SAGE; 1985. 6. Spencer L, Ritchie J. In pursuit of quality. In: Harper D, Thompson AR, organizadores. Qualitative research methods in mental health and psychotherapy. Chichester (UK): Wiley-Blackwell, 2012. p.227-42. Qualitative knowledge is ideographic and constructed: knowing Qualitative analysis is concurrent with obtaining data, as it will end when catego- Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. - 889 - The quality of qualitative research: from evaluation to attainment ries are theoretically saturated or complete.11,17,20,30 way of thinking, they will rectify those that impede the development of a study of quality. This reflec- tion will tuned them as research instruments and will turn quality into a research act. This saturation permits generalizations about the research phenomenon, that is, the ideographic knowledge, and the researcher should distinguish it from information redundancy. In order to have saturation, theoretical sampling should take place, this means in practice, changing the questions the researcher asks to informants. Hence, if no new information is obtained in the fieldwork, the researcher will consider whether this happens because (s)he is always asking the same questions or because different information is sought related to a given category. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. CONCLUSION 7. Ravenek MJ, Rudman DL. Bridging conceptions of quality in moments of qualitative research. Int J Qual Methods [online]. 2013 [acceso 2014 Dic 05];12: 436- 56. Disponible en: http://ejournals.library.ualberta. ca/index.php/IJQM/article/view/11192/15687 To ensure the quality in the research process has tended to pass unnoticed in the literature, which has centered on how to assess it instead of how to achieve it. The assessment of qualitative studies has been immersed in positivist logic to the detriment of qualitative research itself. Despite criticisms, the quality debate is needed as, without rigor, qualitative research loses its utility. 8. Savin BM, Major CH. Qualitative research: the essential guide to theory and any practice. London (UK): Routledge; 2013. 9. Koch T. A review of nursing quality assurance. J Adv Nurs. 1992; 17(7):785-94. In this paper, I have centered my attention on reflexivity. I have presented it not as a quality criterion, but as a tool to achieve it, with a learning potential. I have proposed neither criteria nor new models or schemes, instead I exposed issues that are constantly and inadvertently repeated in some research studies and that threaten their quality. 10. Sandelowski M, Barraso J. Handbook for synthesizing qualitative research. New York (US): Springer; 2007. 11. Charmaz K. Constructing Grounded Theory. Los Angeles (US): SAGE; 2014. 12. Koch T, Harrington A. Reconceptualizing rigour: the case for reflexivity. J Adv Nurs. 1998; 28(4):882-90. 13. Calderón C. Researcher reflections. In: Savin BM, Major CH, organizadores. Qualitative research: the essential guide to theory and any practice. London (UK): Routledge; 2013. p.481-2. Qualitative studies are flexible and permit rectifying mistakes and learning from failures. In this lays to a larger extent its educative nature. I have turned reflexivity into the key element for this to happen. Researchers will assess their paradig- matic position, the way in which they constructs the research problem and the way they relate to others in fieldwork and with data. In the dialogue they establish with themselves, they will gradually construct a particular perspective and when they realize the positivist ideas that might slip into this 14. Calderón C. Assessing the quality of qualitative health research: criteria, process and writing. Forum Qual Soc Res [online]. 2009 [acceso 2014 Dic 01];10 (2):. Disponible en: http://www. qualitative-research.net/index.php/fqs/article/ view/1294/2807 15. Flilck U. Introducción a la investigación cualitativa. Madrid (ES): Morata; 2004. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. - 890 - De la Cuesta Benjumea C 16. Received: March 09, 2015 Approved: July 30, 2015 30. Glaser B. Theoretical sensitivity. Mill Valley (US): The Sociology Press; 1968. CONCLUSION Thorne S. Interpretative description. Walnut Creek (US): The Left Coast Press; 2008. ciencia aplicada. Texto Contexto Enferm [online]. 2012 [acceso 2015 Ene 21]; 21(3):675-83. Disponible en: http://www.scielo.br/scielo.php?pid=S0104- 07072012000300024&script=sci_arttext 17. Corbin J, Strauss A. Basics of qualitative research. Los Angeles (US): SAGE; 2008. 25. Morse J. Biased reflections: principles of sampling and analysis in qualitative inquiry. In: Popay J, organizador. Moving beyond effectiveness in evidence synthesis. London (UK): National Institute for Health and Clinical Excellence; 2006. p.53-60. 18. Sandelowski M, Barroso J. Reading qualitative studies. Int J Qual Methods [online]. 2002 [acceso 2014 Dic 15]. 1(1):74-108. Disponible en: http:// ejournals.library.ualberta.ca/index.php/IJQM/ article/view/4615/3764 26. Field PA, Morse JM. Nursing research: the application of qualitative approaches. London (UK): Croom Helm; 1985. 19. Strauss A. Qualitative analysis for social scientists. Cambridge (UK): Cambridge University Press; 1987. 20. De la Cuesta-Benjumea C. El investigador como instrumento flexible de la indagación. Int J Qual Methods [online]. 2003 [accesso 2015 Ene 21]; 2(4):. Disponible en: http://www.ualberta.ca/~iiqm/ backissues/2_4/pdf/delacuesta.pdf 27. Mayan MM. Essentials of qualitative inquiry. Walnutt Creek (US): Left Coast Press; 2009. 28. Geertz C. Descripción densa: hacia una teoría interpretativa. In: Bohannan P, Glazer M, organizadores. Antropología. 2ª ed. Madrid (ES): MacGraw-Hill; 1993. p.547-68. 21. Davies AC. Reflexive ethnography. A guide to researching selves and others. London (UK): Routledge; 1999. 29. De la Cuesta C. “Aquí cuidamos todos”: asuntos de individualidad versus colectividad en un estudio sobre cuidado en la casa de pacientes con demencia avanzada. Forum Qual Soc Res [online]. 2006 [acceso 2010 Dic 05];7(4):. Disponible en: http://www. qualitative-research.net/index.php/fqs/article/ view/170/381 22. Bourdieu P. El oficio de científico. Barcelona (ES): Anagrama; 2003. 23. Denzin K, Linclon YS. Introduction: entering the field of qualitative research. In: Denzin K, Lincoln YS, organizadores. Handbook of qualitative research. Thousand Oaks (US): SAGE; 1994.p.1-17. 24. Amezcua M, Hernández SM. Investigación sobre el cotidiano del sujeto: oportunidades para una 30. Glaser B. Theoretical sensitivity. Mill Valley (US): The Sociology Press; 1968. Received: March 09, 2015 Approved: July 30, 2015 Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90. Text Context Nursing, 2015 Jul-Sep; 24(3): 883-90.
https://openalex.org/W2329982730
http://www.zurnalai.vu.lt/sociologija-mintis-ir-veiksmas/article/download/9858/7680
Lithuanian
null
Fenomenologiškai grindžiamos socialinės teorijos sampratos klausimu. III. Relevantiškumo problema
Sociologija. Mintis ir veiksmas
2,016
cc-by
69,136
Fenomenologiškai grindžiamos socialinės teorijos sampratos klausimu. III. Relevantiškumo problema Santrauka. Trečioje esė dalyje nagrinėjama relevantiškumo problema. Pirma, relevantiškumas eks­ plikuojamas kaip gebėjimas nuskaidrinti „pamatinę metodinę įžvalgą“, vadovaujantis nuostata, kad ana­ lizė, įgyvendinta transcendentalinėje sferoje, taikytina ir natūraliosios laikysenos sferoje. Antra, tiriamos relevantiškumo problemos filosofinės ištakos, o tiksliau, aki-vaizdos problema. Trečia, relevantiškumo eks­ plikaciniai principai taikomi fenomenologiškai skaidrinant Tocqueville’io tezę (kad lygybė turi polinkį iš esmės pažeidinėti laisvės teises), skiriant keturis bendruosius laisvės nepakantumo nelaisvei ir nelaisvės artimumo laisvei postulatus ir tris konrečios analizės lygmenis. Kritiškai svarstoma skirtis tarp fenome­ nologijos ir liberalizmo. Pagrindiniai žodžiai: relevantiškumas, akivaizda, laisvė, lygybė, liberalizmas, natūralizmas, feno­ menologija. Key words: relevance, evidence, freedom, equality, liberalism, naturalism, phenomenology. Metodologija Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Algimantas Valantiejus Algimantas Valantiejus III. 1. Relevantiškumo problema: jei tik ir kai tik į dienos šviesą iškeliamos inter-subjektyvios sąsajos „Relevantiškumo problemos akiratyje – atranka iš pasaulio, priešduoto gyvenimui, kaip ir mąstymui, viseto.“ (SCHUTZ 1996; 4) „Relevantiškumo problemos akiratyje – atranka iš pasaulio, priešduoto gyvenimui, kaip ir mąstymui, viseto.“ (SCHUTZ 1996; 4) „Kiekvienas reikšmingas sprendinys gyvenimo-pasaulyje priešstato žmogų veidu į veidą su hipotetinės kilmės teminių relevancijų aibėmis, kurios turi būti interpretuojamos ir užklausiamos jų motyvacinės įterpties į gyvenimo- planą atžvilgiu.“ (SCHUTZ1966; 131) „Esminė neokantininkų mokyklos klaida yra jų įsitikinimas, kad metodas – kad ir ką šis terminas galėtų reikšti – „sukuria“ tyrimo dalyką. Metodologija – ir jos išdavos praktiniame lygmenyje, o tiksliau, receptai ir veiksmo maksimos, elgsenos būdai ir pan. – teikia nuorodą tik į tinkamą apibrėžimą to, kas esti interpretaciškai relevantiška anksčiau 57 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 vyravusių temų atžvilgiu. Savitos metodologijos sritys – tai moksliškai ir praktiškai korektiški tipizacijų vartojimo būdai, gilių tyrimo lygmenų eksplikavimas, parankinių tikslų apibrėžimas, ir panašiai. Metodologija niekada negali nustatyti, kokia tematika mums yra relevantiška; operacinės taisyklės negali teikti motyvacinių interesų židinių.“ (SCHUTZ 2011C; 167) „Mes galėtume traktuoti kiekvieną išraišką, esančią už skliaustų, kaip „referencijų sistemą“ tiems, kurie lieka skliaustuose. O tai, ką mes ketiname suskliausti, ir tai, ko suskliausti neketiname, yra funkcijos dalyko, kurį savo žodyne vadinu „relevancija“.“ (IŠ ALFREDO SCHUTZO LAIŠKO ARONUI GURWITSCH’IUI; SCHUTZ 1989; 114 [NIUJORKAS, SAUSIO 25, 1952].) „Kad ir kaip būtų, aš nemanau, jog bendroji relevantiškumo teorija gali apsiriboti nustatydama tipologinę sistemą, tipų katalogą, – arba susijusį, arba nesusijusį su bendrąja dorybių ar vertybių sistema. Be abejo, tokia tipologija yra bendrosios relevantiškumo teorijos dalis, ir aš visiškai sutinku su Tavimi dėl to, kad tokia tipologija turi remtis filosofine antropologija. Bet visos šios problemos yra tik bendrosios relevantiškumo teorijos – objektyvių problemų statiškame lygmenyje – srities dalis. Akivaizdu, kad taip pat galima ir teorinė dinamika, t.y. istorinė motyvacijų formų nuskaidrinimo problema. Pavyzdžiui, vartojant Tavo kalbą, kaip motyvacijų raidos teorija: nuo ritualo per mitą prie simbolio.“ „ g j , y 2 Integralių kultūros mokslų (Geisteswissenschaften; cultural sciences) sampratą reikia aiškiai skirti nuo dvi-nario hibrido, – dirbtinio, „netikro asmens, atstovaujančio kito žodžiams ar veiksmams“ 1 „Teleologija, tai yra ateities-muzika“. (IŠ ALFREDO SCHUTZO LAIŠKO ERICUI VOEGELINUI; SCHUTZ 1989; 114 [1952-ŲJŲ GRUODIS].) „Aš paprašiau Husserlio pasakyti keletą žodžių apie Teleologie. „Teleologie, das ist Zukunftsmusik“,1 – atsakė jis. Jis darkart aprašė įvairius konstituacijos lygmenis, kuriuos randa fenomenologinė analizė, pabrėždamas, kad nors analizė grindžiama abstrakcija, fenomenologo prieš-akyje visuomet esti konkretus visetas. Nors svarbiausias fenomenologo rūpestis yra suprasti bendresnes ir formalesnes struktūras, jis visada gali sugrįžti prie to, kas yra duota, ir, lygindamas, išsiaiškinti, kas buvo pražiūrėta. Jis gali, pavyzdžiui, įžiūrėti, kad tuomet, kai aprašė sąmonę tik kaip jos struktūrą, paliko aukštesnįjį sąmonei duoto tikslingumo lygmenį. Visi konstituacijos lygmenys turi šios aukštesnės teleologijos žymes, ir mes galime apibūdinti ją kaip kryptingumą (vok. Tendenz, angl. tendency), kurio mes laikomės įgyvendindami aktus. Tai reiškia: viena vertus, saistančio pasaulio konstituaciją (būtiškumo, vertybių, tikslų), kita vertus, asmens, įgyvendinančio aktus, „savi-saugą“.“ (CAIRNS 1976; 51–2) Relevantiškumas (Relevanz; relevance), arba su(si)saistymas, yra pagrindinė, kaip tinkamai mus orientuoja šio dalyko-problemos pirmtakas Alfredas Schutzas, sociologijos ir kultūros mokslų (Geisteswissenschaften; cultural sciences) tema.2 Tai socialinių mokslų, autentiška šio žodžio prasme, tema, kurią nuskaidrinti gali ir netgi privalo (dėl ypatingo metodo, lygiagrečiai ap­ 58 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija socialinių mokslų ir fenomenologinių tyrimų tėvonijoje – jau randame (dar prieš konstruoda­ mi antrinius idealiuosius tipus) praktinius tipus. Tiksliau, žmonių konstituotus ap-linkinius sąryšius: įpročius, polinkius, prie-tarus, kitų asmenų veiksmo kryptis, motyvus, tikslus, jų veiklos sociokultūrinių išdavų tipizacijas, o taip pat ir savitipizacijas (biografinės aplinkos saitų su socialiniu bei gamtiniu pasauliu įsivertini­ imančio ir paprastesnę empirinio patyrimo, ir eidetinę transcendentalinės patirties sferų analizę) fenomenologija. Esminius fenomenologijos ir socialinių mokslų ryšius tinkamai perteikia du iš pažiūros paprasti, bet santykiškai painūs, žings­ niai: 1) tipizacijų, 2) relevancijų eksplikavimo. Svarbu nuo pačios pradžios neišleisti iš akių to, ko tradicinėse metodologijose buvo nepaisoma. Kad pirminėje socialinėje tikrovėje – (Hobbes 1999; 170; Hobbesas sukuria visiškai naują mokslą ir visiškai naują „moralę“; Strauss 1963), – susidedančio iš susvetimėjusių viena kitos atžvilgiu – socialinės ir humanitarinės – dalių. Radęsis griežtai diferencijuojančios mokslo biurokratijos dirbtinio potvarkio paliepimu, dvigalvis socialinių ir humanitarinių mokslų kentauras, sudarytas iš dviejų skirtingų rūšių – procedūrinės administracinės ir bendruomeninės orientacinės – interesų, yra šiomis dienomis labai įtakingas skai­ dančio biurokratijos potvarkio Padarinys. Išties dailus klasifikaciniuose piešiniuose (dailiuose gra­ fikuose ir gražiose lentelėse), bet tikrovėje darantis prie-vartą (vartantis žodžius, sakinius ir teiginius aukštyn kojomis ir laikantis juos ore už kojų, kabant jiems aukštyn galva). Už šeimininko pamalo­ ninimą matoma, bet nepastebima prie-vartos kalba, Padariniui atlygina iš gausių medžiaginių (ide­ alieji – jau suvartoti) išteklių. Šokli, daininga ir mįslinga prie-vartos kalba (policinės dramaturgijos kalba: „Vaidinti kaip teatre“, „Šokti ir dainuoti ratelyje“, „spėlioti mįsles“; Wittgenstein 1995; 130 [23]) ekspertų perteikiama vadinamosiomis pasiūlos-paklausos kreivėmis, – darniai augant abiem kūnams, ir gražiai vienas kitą atvaizduojant. Padarinys yra grynoji geometrinė forma, be pra­ eities, dabarties, ateities sąsajų, be teleologijos, be socialinio politinio intencionalumo. Devyniolik­ tojo amžiaus antrosios pusės pasaulėžiūra, kaip tikslina Edmundas Husserlis, apakinta pozityviųjų mokslų klestėjimo („prosperity“; Edmundas Husserlis vartoja anglišką terminą; Husserl 1976b; 3), nusigręžia nuo klausimų, esminių žmogaus egzistencijai (žmogiškam čiabūtiškumui; menschliches Dasein; ten pat). „Gręžtis nuo“ drauge reiškia „gręžtis į“: jei Descartes’as apodiktinį akivaizdumą, episteminę garantiją, gauna iš Dievo, tai naujoji pasaulėžiūra apodiktinį akivaizduma gauna iš savęs, globaliai augančio technologinio kūno, Be-soties-padaro (valgo ir vis nori). (CAIRNS 1976; 51–2) Gręžiantis nuo ir gręžiantis į randasi dvi tarp savęs beveik nesusijusios kūno dalys. Pirmoji dalis, „humanitari­ nių“ mokslų, yra „minkšta“ (tai mirštanti modernybės siela), tačiau apgailėtinai silpna, stinganti „griežto“ metodo. Antroji dalis, „socialinių“ mokslų, „kietesnė“, atskirta nuo „minkšto“ savo už­ nugario. Tai reiškia: sudaryta iš ne visai tobulo „operacinio“ įskiepio, gauto iš gamtotyros metodo. Kietesnės dalies paskirtis (gręžiantis nuo gyvenimo ir gręžiantis į grynąją formą) – užvaldyti bendrą turtą, paversti jį privačios nuosavybės – privat-žmogių (private men; Hobbes 1998) – savisklaida, kaip parašyta vienoje pirmųjų dirbtinio liberalizmo konstitucijų. Plg.: socialiniai saitai yra dirbti­ niai, visuomenė – dirbtinis Žmogus, arba bendraturtis (žr. T. Hobbeso dvidešimt pirmo Leviatano skyriaus posk. „Artificial bonds, or covenants“; „an artificial man, which we call a commonwealth“; ten pat; 141). Tikrovėje yra kitaip: net ir gręžiantis nuo ko nors, galva ne atitrūksta, o apdairiai sukiojasi ir į šalis, ir atgalios. Tikrovėje socialinė (supratimo) ir humanitarinė (akivaizdumo) dalys jei ne puikiai, tai bent iš dalies dera gyvenimo-pasaulyje. Ir vis tik pridurtina: šioje maždaug dermė­ je – esant prie-vartos išlygai – trūksta tarpusavio sąsajų. Sąsajų stoka yra primestos padarinio veikos ir įtakos rezultatas: tik faktinių mokslų dėka randasi tik „faktiniai-žmonės“ (Tatsachenmenschen; Husserl 1976b; 4). 59 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 mus). Tipizacijos, pirmtakų ir amžininkų, tam tikra, t.y. mūsų įmesties pasaulin, prasme mums esti pri-mestos (kaip matomų, bet nepastebimų relevancijų sistemos). Šioji konkreti ontologinė pasauliškumo struktūra apima ir problemines, ir atviras galimybes, susijusias su motyvacinių, teminių, interpretacinių relevancijų konstituaci­ jomis gyvenimo-pasaulyje. patyrime, todėl yra akivaizdi): tinkamai laikytis reiškia laikytis relevantiškai (o tai yra viena ir tas pat). Tinkamų turinių mano galvoje ir tinkamų polinkių mano kūne sutapimas giliai įsišaknijęs man priešduotų ontologinių ir mano spontaniš­ kų motyvų bei interesų su-tapimų, su-buvoji­ mų – sąmonės aktų ir patyrimo – regionuose: „Visas patyrimas ir visi aktai grindžiami re­ levancijų struktūromis. Bet koks sprendimas daugiau ar mažiau aiškiai įtraukia, be veikiančio asmens, relevancijų aibes“ (Schutz, Luckmann 1973; 183). Darkart verta akcentuoti du relevantiškus – fenomenologinį ir sociologinį – žingsnius. Pirma. Edmundas Husserlis atskleidžia, kad jau nuo pa­ čios pradžios ikipredikatinis gyvenimo-pasaulio patyrimas konstituojamas pagal tipus. Antra. Tai, ko Husserlis nenuskadrina, „savo publikuotuose tekstuose“ (Schutz 1966; 125), yra fenome­ nologijos papildymas natūraliosios laikysenos fenomenologija prasminėse gyvenimo-pasaulio struktūrose: „<...> kad tipizacija įgyvendinama kaip savitų relevantiškumo struktūrų sklaida“ (ten pat). (CAIRNS 1976; 51–2) Relevantiškumo problemą dera susieti, kaip mums rekomenduoja Edmundo Husserlio Karteziškųjų meditacijų § 34, su principiniu fenomenologiškai grindžiamos metodologijos įsižiūrėjimu į metodologiją; dalyką, pirmesnį už socialinių mokslų metodą, priešduotiškumą laikantį savaime suprantama duotimi. Tiksliau, kai 1) eidetinė intuicija ir 2) transcendentalinė redukcija (Husserl 2005; 91) stoja greta viena kitos, turime mokymą (ir „turime“ ne tik galvo­ je, o ir tikrovėje) apie grynąsias galimybes. Įžiūrėti relevantiškumą – tolygu sugriebti „pamatinę metodinę įžvalgą“ (ten pat; 86): „Tik tam, kad būtų lengviau įeiti į fenomenologiją, prabylame apie tai taip vėlai“ (ten pat). Nors Husserlis čia kalba apie transcendentalinę analizę kaip eidetinę analizę, o mums visų pirma rūpi gyvosios gy­ venimo-pasaulio sąsajos, relevancijos (simbolinių reikšmių sistemų prasminės „kraujagyslės“, o ne anonimiški socialinių sistemų „siūlai“ arba „tinklai“), tačiau čia vadovaujamės nuostata, kad analizė, įgyvendinta transcendentalinėje sferoje, taikytina ir natūraliosios laikysenos sferoje. Klausiant orientuotai (tai, sakytume, yra relevantiškumo esmė): kas yra relevantiškumas? Pradžioje galbūt prabilkime visai paprastai: tai man ką nors reiškia. O jei neaišku, ką tai reiškia (ką „turime“ galvoje), nedarykime klaidinančios išvados, kad „tai nieko nereiškia“, bet klauskime etnometodologiškai (mandagiai, bet su fenome­ nologiniu įskiepiu lūpose): ar gerai laikomės mes, ir ar gerai laikotės jūs? Po taktiško pasikeitimo savo sąmonės turiniais (ką mudu gero „turi­ me“ savo galvose), klauskime dar prasmingiau (reikšmė tikslinama, o prasmė gilinama): ar gyvendami laikomės tinkamai? Eidami vis gilyn ir laikydamiesi abipusiš­ kumo reikšmių (ką abudu štai-dabar turime galvoje) prieiname prie išdavos (išvada – užbai­ giama samprotaujant, o išdava – priešduodama Nepaprastai turtinga naujos rūšies atradimų ir problemų įvairovė, kaip pažymi Husserlis, iš 60 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija akiratyje. Kita vertus, aš, metodologas, privalau skirti išgyvenamą patyrimą ir refleksiją. O tai reiškia, kad brėždamas šią skirtį (net iki mano paties relevantiško patyrimo suskliautimo!) aš neturiu iškreipti reflektuojamo patyrimo turi­ nio, nors ir galiu tai padaryti (patyrimo turinys suskliaučiant ištuštinamas, tačiau patyrimo forma gundo ją tuoj pat kuo nors užpildyti). O tai vėlgi rodo gyvenimo-pasaulio, eidetinės intuicijos ir transcendentalinės redukcijos susietu­ mą. Metodas veda kiekvieną mūsų atgal į mūsų transcendentalinį-aš: pradžių geriau įžiūrima aprengiant ją „papras­ tesniu paprasto empirinio aprašymo rūbu“ (ten pat). Šis teiginys puikiai dera su sociologiniu požiūriu: kad patyrimas natūralioje laikysenoje esti pirminis. Tačiau šio teiginio papildymas – kad empirinis aprašymas įgyvendinamas „vien transcendentalinės patirties sferoje“ (ten pat; kursyvas mano – A. V.) – trikdys, kaip matysi­ me, natūraliosios laikysenos fenomenologijos pirmtaką Alfredą Schutzą (paprastas klausimas yra tas: kaip pirminį patyrimą galima pamatyti transcendentalinėje sferoje?). (CAIRNS 1976; 51–2) Vėlgi pakartosime principinę mūsų nuostatą: žingsnis po žingsnio išskleisdami fenomenologiškai grindžiamos meto­ dologijos – ir kaip mokymo apie tikrovę, ir kaip mokymo apie grynąsias galimybes – aspektus, turėsime ne tarp savęs nesusijusius, bet glaudžiai susijusius sąmonės gyvenimo pilnumoje – gyve­ nimo-pasaulyje, eidetinėje intuicijoje ir transcen­ dentalinėje redukcijoje – indus. Kalbant trumpai, zigzaginė kelionė nurodytomis kryptimis yra fenomenologinė. „Aš pats, kaip būtent toks ego, medituoda­ mas toliau aptinku deskriptyviai pagaunamus ir intencionaliai išplėtojamus tipus, žingsnis po žingsnio intencionaliai atskleisdamas savąją monadą, galiu eiti toliau čia atsiveriančiomis pa­ grindinėmis kryptimis.“ (Husserl 2005; 86–7) Relevantiškumas, kaip motyvacinė, teminė, interpretacinė problema, ne tik diferencijuoja, ne tik skiria, bet ir saisto. Ir sąlygoja, ir savitai atveria saitų išsklaidą bet kokioje situacijoje: kad ir štai šio stalo jutiminio suvokimo drauge su laisvomis ir bet kuriomis kryptimis links­ tančiomis variacijomis (ten pat; 87). To, kas žinoma, pažįstama ir kuo tikima, ir to, kas dar nepažįstama, turinys yra visada santykiškas: tiek kalbant apie asmens biografinę situaciją, tiek apie bendruomenės istorinę situaciją. O jei žinojimas yra santykiškas, tai pa-linkęs. Taigi moja mums vos pastebimu galvos mostu: ženk atgal temiškai įsižiūrėdamas į relevantiškas žinojimo pradžias. Nuskaidrindamas siauresnius ir platesnius mūsų motyvacijų bei interesų išsišakojimus, relevan­ tiškumas „<...> persmelkia mūsų egzistavimą, mūsų gyvenimą, mūsų pažįstamą patyrimą (Schutz 1996; 3). Taigi svarbu – pati šio dalyko- problemos pradžia. Drauge svarbu pažymėti: principingai įsižiū­ rėdami į relevantiškumą kaip problemą, rodos, pasiekiame filosofijos ir sociologijos tarpusavio santykių – galimo jų susikalbėjimo arba galimo nesusikalbėjimo – slenkstį. Šis vos įžiūrimas slenkstis skiria patyrimą ir refleksiją, bet ne­ būtinai turi skirti socialinius mokslus (tiesiogiai tiriančius patyrimą) ir filosofiją (netiesiogiai reflektuojančią apie patyrimą). Šis slenkstis svarbus ir metodologinei laikysenai. Viena vertus, aš, metodologas, gyve­ nu-natūraliai-laikydamasis-relevancijų, kurios veikiau nepasirodo, nei pasirodo aktualiame mano suvokimo lauke, o jei ir pasirodo, tai dažniau potencialiame intuityvaus kryptingumo 61 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Relevantiškumo pradžia yra esminė sąsa­ ja: žmogiškų motyvacijų stygas užgaunantis tęstinis, teleologinis momento santykis tarp gyvenimo ir racionalių planų: „Teleologie, das ist Zukunftsmusik“. Šį santykį reikia skirti nuo natūralizmui būdingo atkarpos santykio tarp priemonių ir priemonių (po kąsnelį pro­ cedūriškai atkerpamų „užbaigtų“ vienetinių faktų). Antruoju atveju objekto suvokimo aktas atskiriamas nuo jo objekto, o atkirptas objektas jau neturi tęstinės refleksijos pagrin­ dų. Pirmuoju atveju galioja tęstinis refleksijos aktas kaip jo objekto pagrindas. 3 Fenomenologiškai grindžiamos metodologijos požiūriu, šioji skirtis neįkainojama (neįperkama net galingiausių ir turtingiausių pasaulio valdovų, projektinių užsakymų „ir t.t.“ vadovų, suvoki­ mo aktų ekspertų). Natūralistinė / materialistinė laikysena, kurią Husserlis vadina „<...> grubesne plačiai paplitusia pokarteziškąja nuostata, ignoruojančia karteziškąjį epoche (Husserl 2005; 104), daiktus traktuoja kaip pagrindus, kuriais remdamasi sąmonė grindžia savo aktus kaip paskirus sudedamuosius momentus, priklausomus nuo šių daiktų. O fenomenologinė laikysena grynajame reflektyviame suvokime apima daiktus ne kaip pagrindus, implikuojančius refleksijos veiką, o kaip sąsajas sąmonės veikos atžvilgiu. Būtina, kaip pabrėžia Husserlis, skirti tiesiog įgyvendinamą (CAIRNS 1976; 51–2) Atsižvelgiant į tai, kad „relevantiškumas“ visada yra „pro­ blemos-relevantiškumas“ (Schutz 1964; 235) ir akcentuojant refleksijos momento, skiriamo nuo paskiros suvokimo akto atkarpos, svarbą, verta pasitelkti saistantį Roberto Sokolows­ ki’o refleksijos apibrėžimą: „REFLEKSIJA YRA NE-NEPRIKLAUSOMAS MOMENTAS, kuris randasi visete tarp refleksijos akto ir jo objekto. Refleksija turi savo kaip jos objekto pagrindą“ (Sokolowski 1974; 188). kimi tam, ką matome, – o ne tam, kad politiškai „laimėtume dangų“, o filosofiškai „priverstume mažiau išmanančius tavimi gėrėtis“ (Descartes 1978; 104)? Relevantiškumo problema yra svarbi, reikš­ minga, prasminga (o tai yra viena ir tas pat). Tik veidu-į-veidą sąsajų, arba porininkų, pasaulyje mes galime senti drauge (Schutz 1962; 220), šitaip atsakingai ir atskaitingai tikrindami dar sudėtingesnes – amžininkų, pirmtakų, įpėdinių pasaulių supratimo – galimybes. Tarp jų – savo aktualius pasirinkimus drauge su galimomis „ir t.t.“ modifikacijomis: „Kiekvienas reikšmingas sprendinys gyvenimo-pasaulyje priešstato žmo­ gų veidu į veidą su hipotetinės kilmės teminių relevancijų aibėmis, kurios turi būti interpretuo­ jamos ir užklausiamos jų motyvacinės įterpties į gyvenimo-planą atžvilgiu“ (Schutz 1966; 131). Taigi problema – visad šiuo-laikinė: kuriose gyvenimo-pasaulio provincijose mes esti? Ku­ riose motyvacinės įterpties į gyvenimo-planą kryptingumo provincijose esti kiti? Nebus per daug pakartoti. Gyvoji dabartis, jei tik ir kai tik joje išlaikoma praeitis ir ateitis, orientuodama į pilnai išsiskleidžiantį sąmonės gyvenimo srautą, reikalauja – tai relevantiškumo nuskaidrinimo sąlyga – aiškiai skirti refleksiją ir daikto suvokimo aktą (Sokolowski 1974; 189–204; § 70; taip pat žr. „Transcendentalinė ir natūrali refleksija“; Husserl 2005; 43–8 § 15).3 Jei su-si-saistymas kaip teleologinė tęsti­ numo muzika matomais, bet nepastebimais brūkšneliais persmelkia mūsų egzistavimą, mūsų gyvenimą, pažįstamą patyrimą, kaip galime – filosofiškai, metodologiškai, moksliškai, poli­ tiškai – atsisieti nuo šių sąsajų: tam, kad geriau matytume tai, ką matome, tačiau išliktume išti­ 62 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija vimo tvarkos. Kalbant etnometodologiškai, tai tipizavimo, arba aksiologinio rangavimo „pagal ką nors“ tvarkos: „klasifikacinės statistinės“ („tiek ir tiek“), „tipiškų mainų“ („tu man, aš tau“), „tipiškos lupikiškos“ („tu man, ir tu man, ir tu man...“), „tipiškos tarybinės“ („sprendžiu ne aš, sprendžia taryba“), „tipiško akivaizdu­ mo“ („tai faktas“) „tipiškos asociacinės“ (man geriau „jau matytas“, nei „visai nepažįstamas“), „tipiškai draugiškos“ („draugą“ skiriančios nuo „priešo“), „tipiškai priešiškos“ („šiaušiančios plauką prieš bet ką“, reikalui esant, „net ir prieš draugą“), „tipiškai abejingos“ („man vis vien“), „tipiškai susitaikstančios“ („man geriau kaip kiti“). Santykiškai natūralios pasaulė-žiūros yra prasmingos socialinio pasaulio struktūros. Pasaulė-žiūros apima mūsų patikrintas, įti­ krintas, „ir t.t.“ tikrinamas (plg.: „Žr. tokį arba tokį empirinį tyrimą.“) tipizacijų ir relevancijų sistemas. pagaunantį juslinį suvokimą, prisiminimą, sprendimą, vertinimą, tikslo pasirinkimą, ir refleksijos aktus, kuriuose tik ir atsiskleidžia pirmieji aktiniai išgyvenimai: „Suvokdami tiesiog, pagauname, tarkime, namą, o ne to namo suvokimą. Tik refleksijoje atsigręžiame į tą suvokimą ir į jam būdin­ gą, juslinio suvokimo pobūdį turintį nukreiptumą į namą“ (Husserl 2005; 43–4). (CAIRNS 1976; 51–2) Šios žiūros, arba subjektyvios pasau­ liškumo struktūros, randasi ikipredikatiniame patyrime ir laikomos savaime suprantamomis bet kuriuo konkrečiu laiko – iš čia randasi natūralioji laikysena – momentu. Būtent čia, santykiškume, randasi socialinės „tvarkos“ užuomazgos. Įprastas patyrimo, pažįstamumo, žinojimo atsargų turėjimas (habitus) apima įvairius lygmenis, rodančius, kad gyvenimo- pasaulis struktūrizuotas į „tam tikras“, „mums relevantiškas“, provincijas. Taigi tvarka – tai in­ terpretacinės prasmės struktūros, kurias didžiąja dalimi sudaro dviejų idealiųjų tipų – asmeniškų idealiųjų tipų (įgyvendinamos arba įgyvendintos individualios raiškos) ir veiksmo krypčių tipų (simbolinių raiškos procesų) – konstituacijos, varijuojančios nuo didesnio ar mažesnio kon­ krečios aplinkos pažįstamumo iki beveik visiško santykių anonimiškumo. O drauge tai estetinio ringavimo – arba „į taktą“, arba „ne į taktą“ – tvarkos. Kai klasifi­ kaciniai, praktiniai, moraliniai daiktų „vaizdai“ pagaunami pritariančiais, ritmiškai konstituoja­ mais, kūno judesiais: akių „perbėgimais“ viena ar kita kryptimi, galvos „pasukimais“ betarpiš­ koje sąveikoje („vos aukštyn“, vos „žemyn“, vos „atgal“ ir vos „į šonus“: „Taip, tebeklausau“, „Na, ir kas iš to?“, „Gal jau užteks“, „Žinai, man laikas“), pagreitintais ištarimais arba sulaikymais („Ar akcentą pagavai?“), sąveikos aplinkoje pro šalį praplaukiančiomis veido išraiškomis („Nors tave matau, tačiau nepastebiu“), žvairuojan­ čiu – ką nors nežinomą gaudančiu – akiračiu („Matau daugiau, nei pastebiu“). Čia, ne kur kitur, atrodymų su-tapimuose, randasi tikrumo, pagrįstumo, patikimumo – daiktų pasirodymų mums kaip tikrų – aibės. Tvarka yra dinamiška sąvoka, kurią įpras­ mina veiksmažodžiai tvarkyti, organizuoti, ranguoti, ringuoti. Tvarka – tai socialinės figū­ racijos, sociokultūrinėse aplinkose suranguotos savaime suprantamai. Įprasčiau būtų sakyti: tai savaime suprantamų vertybių, normų, įpročių, papročių, taisyklių, prie-linksnių (sąmonės polinkių ko nors link) ir prie-tarų (pačių įvai­ riausių praktinių ir mokslinių tipizacijų) ranga­ 63 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Fenomenologiškai grindžiamos metodolo­ gijos požiūriu, relevantiškumas, sudarantis su­ bjektyvių sąsajų atitinkamose žinojimo formose pagrindus, apima dvi pagrindines tirtinas sferas: metodologinę ir ontologinę. Pirma. Natūralio­ sios pasaulėžiūros yra santykiškos biografinės asmens laikysenos arba istorinės bendruomeniš­ kumo laikysenos atžvilgiais; jos ne objektyviai vientisos, o prasmingai struktūrizuotos.4 Kaip tik todėl idealiųjų tipų formavimas turi atitikti relevantiškumo principą, kuris reiškia, kad problema, socialinės srities mokslininko kartą pasirinkta, suponuoja referencijų schemą ir konstituoja diapozono, kuriame gali būti for­ muluojami relevantiški idealieji tipai, apimtį (Schutz 1964; 18). mūsų galvose, kiek mūsų veiksmai tam tikrose laikysenose. Veiksmo laikysenos – tai mūsų in­ teresų kompleksai, problemų kompleksai, planų kompleksai, galimybių kompleksai. Ne tik ir ne tiek fragmentiškų (faktinių), o visų pirma gyve­ nimo-planų ir gyvenimo-galimybių. 4 Nebus per daug pakartoti, kad tipizacija (fenomenologinė ir ikipredikatinio, ir predikatinio ly­ gmenų samprata) skirtina nuo klasifikacijos (analitinės-predikatinės kategorijos). Klasifikacinės kilmės tyrimai, kaip įprasta, orientuojami į vadinamosios „betarpiškos tikrovės“ („juslinių fak­ tų“ žvejybos plotų) sritį. Dirbtinis, dar daugiau, galimai subjektyvistinis (dėl tik tyrėjui relevantiš­ kų atrankos principų taikymo) klasifikacijų pobūdis įprastai užpildomas, – kad neliktų „tarpų“ (nežinomųjų), – gausiu laimikiu, „šviežiais faktais“. Reikia aiškiai pasakyti: šis natūralizmui bū­ dingas pavėluotas „dedukcijos“ tūptelėjimas „indukcijai“ (maždaug: atleisk, drauguže, už grubią pirmumo teisę ir trasimachišką teisingumo sampratą) klaidina. Betarpiškas patyrimas (unmittelbare Erfahrung), kaip pažymi Husserlis, dažnai skiria daiktus remdamasis kokiais nors „aiškiais“ (dar­ kart: „Žr. tokius ir tokius empirinius tyrimus.“) skirtumais, kurie gali slėpti aktualiai egzistuojančią vidinę vienovę (innere Zusammengehörigkeit). Vadinasi, reikia skirti, kaip rekomenduoja Husserlis, neesminius ir esminius tipus (Husserl 1939; 402–403). Klasifikuojantis racionalizmas niekad, – šį konservatyvų racionalizmo bruožą kritiškai tinkamai, kaip pabrėžia Husserlis, įvertina Kantas, – negrįžta į subjektyviąsias mūsų pasauliškumo-sąmonės – dar prieš mokslinį pažinimą – struktūras (Husserl 1976b; 105). Taigi didieji kritiniai Kanto atradimai nėra už-baigti. Esamos klasifikacijos reikalauja visų pirma kritinės analizės (kritische Analyse),– a) interpretacijos eksplicitiniame saviap­ mąstyme supratimo ir b) paslėpto prasminių-figūracijų (Sinngestalt) šeimininkavimo įsisąmoninimo (ten pat; 106). (CAIRNS 1976; 51–2) Vadinasi, relevantiškumas užkabina žinojimo (plačiąja prasme) paskirstymo socialiniame pasaulyje pagrindus; „žinojimo nelygybės“ (!) – tiek tarp asmenų, tiek tarp socialinių grupių ir pačiose grupėse – pagrindus. Šie elementai – ne „empiriniai faktai“, o „sintetinės struktūros faktai“ (Husserl 2005; 53), kurie iš prigimties (sintetinis – lygu sais­ tantis) esti aksiologiniai. Mes esti vertinami ir, dar daugiau, patys trokštame vertinti (daug aistringiau už visus kitus mus vertinančius drauge paėmus). Tai pirminis sintetinės struktūros fak­ tas, kartais netinkamai redukuojamas į siaurai suprantamą „interesą“. Jį motyvuoja žmogiška, pernelyg žmogiška aistra: ne tik vertinti, bet ir įvertinti, t.y. pasitenkinti užbaigiant (vertinimą). Antra. Mūsų kalbiniai žaidimai (ypač tasai saldus žodis mūsų, kaip ir žodis laisvė, perteikia begalę matomų, tačiau nepastebimų struktūrinių bruožų) įtikrina daugybę žinojimo formų. Šios formos, arba interpretacinės relevancijos, – ne tik ir ne tiek interpretuotų prasmių „turėjimas“ 64 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ontologijos aktualus ir potencialus susidūrimas esti ypatingas dėl to, kad čia susiliečia socialinio erdviškumo (intersubjektyvumo, arba supratumo) ir socialinio laikiškumo (politiškumo, arba tele­ ologijos, ateities-muzikos) sandai. Juos privalu nagrinėti ne-formaliems socialiniams kultūros mokslams, tarp jų – sociologijai ir politikos filosofijai. Nes jos savo eidetiniame akiratyje ir nepriklauso nuo patyrimo faktų, ir drauge visad gręžiasi į juos. Tiksliau, gręžiasi į trilypį – savas­ ties, erdviškumo ir laikiškumo – santykį, rodantį idealiųjų santykių galimybių struktūrų link. Išorinis rangavimas pagal funkciją, vaidme­ nį, statusą, padėtį ir šį socialinės struktūros mo­ tyvą papildantis vidinis ringavimas – kinestezinis pritarimas (tarkim „kinkčiojant“ arba „knap­ sint“ galva“) arba nepritarimas („purtant“ galvą, „žybsint“ akimis, netgi jas „išverčiant“) – yra vie­ nu metu ir „aukštesnės“ / „žemesnės“ padėčių, ir jų vertinimų (drauge su pritarimų / nepritarimų figūromis) aibių kasdienis rikiavimas, kartotinis surikiavimas, proginis išrikiavimas, struktūrinis perrikiavimas gyvenimo-pasaulio struktūrose. Rangavimas kaip galios veiksnys eina drauge su ringavimu kaip psichologiniu veiksniu. Verti­ nimu, o ypač vertinimų užbaigimu („pagaliau pasitenkinimu“) sociokultūrinėse aksiologinėse situacijose, kuriose, be ranguojamų išorinių fi­ gūrų (vaidmenų, padėčių, statusų), ringuojamos vidinės psichologinių žemėlapių – „galios“ ir „negalės“, „pilnavertiškumo“ ir „nepilnavertiš­ kumo“, „stiprumo“ ir „silpnumo“, „atitikimo“ ir „neatitikimo“, „normalumo“ ir „keistumo“, „gero atrodymo“ ir „blogo atrodymo“, „tinka­ mos elgsenos“ ir „netinkamos elgsenos“, „mūsų“ ir „jų“ – figūros: Eidetiniai predikatai skirtini nuo faktinių. Jei pirmieji iš jų yra asociaciniai (fenomenolo­ giniai), tai antrieji – disociaciniai (analitiniai). (CAIRNS 1976; 51–2) Pirmuoju atveju rūpi santykių konstituacija: tikrovės, o taip pat subjektyvumo, nuskaidrinimas, tiksliau, filosofinis supratimas (Sokolowski 1970; 1), įtraukiant ypač transcendentalinės konstitu­ acijos sąvokos tikslinimą „proto“ ir „ne-proto“ rubrikų, – kurios savo ruožtu yra tokių atžvilgių kaip egzistavimas ir neegzistavimas, galimybė ir negalimybė sąsajos, – atžvilgiu (Husserl 2005; 70–1). Metodologiniu požiūriu, esminis dalykas, svarstant „faktiškumo“ klausimus, yra tas: nors santykis tarp nuorodos į objektą ir prasmės yra labai glaudus (prasmės akto struktūra įtraukia ir nuorodą į objektą, ir prasmę), tačiau prasmė niekada nesutampa su objektu. Nurodomas objektas nebūtinai turi būti tikrai egzistuojantis daiktas. O tai liudija, kad nurodymas į objektą yra problema, susijusi su intencionalumo sąvoka. Sąmonė nėra savaiminė uždara sala, bet esmin­ gai įtraukia nuorodas į daiktus: sąmonė visada rodo į daikto pateikimą, Vorstellung, presentation. Čia svarbios dvi tarp savęs susijusios pusės: „Taigi relevancijų sferos pačios struktūrizuo­ jasi pagal aukštesnės-vertės ir žemesnės-vertės tvarkas (angl.: „themselves arranged in an order of superiority and of inferiority“); o jų tvarkos skir­ tingose grupėse kinta“ (Schutz; 1964; 241). Taigi: konkrečioji metodologija ir konkrečioji ontologija yra – viena kitos atžvilgiu – relevan­ tiškos. Tai reiškia, kad jų susidūrimų (aktualių bei galimų) laukuose randasi platus dar netyri­ nėtų probleminių klausimų visetas. Konkrečių konkrečios visuomenės aksiologinės ir politinės kilmės – tipizacijų priešduotiškumo – klausi­ mų. Konkrečiosios metodologijos ir konkrečiosios 65 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 intencionalumas rodo, kad sąmonė yra susijusi su daiktais, bet taip pat – kad ji pateikia daiktus sau pačiai (Sokolowski 1970; 45–6). motyvai, interesai, tikslai, subjektyvios prasmės, gyvenimo-planai). Paprasčiausioji konstituacinės (žingsnis po žingsnio skaidrinančios) analizės forma, individualaus subjektyvumo (Sokolowski 1970; 2), stimuliuoja kitų regionų, turinčių dar neapibrėžtus vidinius ir išorinius horizontus, išsklaidą. Nebus per daug pakartoti, kad feno­ menologinė, „materialiosios geometrijos“, analizė visų pirma apima konkrečiosios ontologijos sferas: vėlyvuoju laikotarpiu Husserlis dažnai vartoja sąvoką gyvenimas, nusakydamas aš-sąmonę kaip laikinės trukmės tėkmę, emanuojančią iš dabarties momento (ten pat; 207). Kitaip tariant, sąmonė nėra nuo-šali. Ji saisto. Fenomenologiškai grindžiamos meto­ dologijos požiūriu, tai įpareigoja (privalėjimo atskaitingumui požiūriu) tirti kasdienio sąmonės patyrimo aktus ne kaip nuo-šalius ir tik mecha­ niškai-susumuojamus faktus, o kaip sąmonės pateikimo (Vorstellung, presentation) funkcijas, sudedamąsias intersubjektyvių relevancijų siste­ mų dalis. Šios reikšmingos, – konstituojančios prasmes sąmonės ko nors atžvilgiais, – funkcijos tradicinėse socialinių mokslų metodologijose tiesiog „praleidžiamos“ arba suplakamos su procedūriškai gaunamomis „reprezentacijomis“ (be intencionalių sąmonės pateikimo nuorodų). 5 Plg.: „Taigi tai, kas teisinga, numato bent keturis dalykus, nes yra du žmonės, kuriems jis yra tuo, kas teisinga, ir du dalykai, kurių atžvilgiu pasireiškia tai, kas teisinga. Lygybė bus ta pati ir žmonėms, ir dalykams, nes kaip tarpusavyje susiję dalykai, taip bus susiję ir žmonės. Juk jeigu žmonės bus nelygūs, jie ir negaus po lygiai, – dėl to kyla ginčai ir vaidai, kai lygūs žmonės gauna nelygiai arba nelygūs – po lygiai. Tai aišku ir iš vertumo sąvokos. Mat visi sutinka, kad teisingai dalijant būtina atsižvelgti į tam tikrą vertumą, bet ne visi vertu laiko tą patį dalyką: demokratijos šalininkai juo laiko laisvę, oligarchijos šalininkai – turtą arba kilmingumą, aristokratai – dorybę.“ (Aristotelis 1990; 150 [1131a 27–40]) (CAIRNS 1976; 51–2) Analitinėje, arba liberalioje (o tai yra viena ir tas pat) metodologijoje paprasčiausias vienetas yra elementarus analitinis punktyras, susijęs su idealia teorine analizės schema, bet nesusijęs su gyvenimu (kalbant analitiškai, arba liberaliai, gyvenimas yra „metafizinė“, „neverifikuojama“ sąvoka). Pirmuoju, konservatyviosios meto­ dologijos (saugančios užnugarį kaip priešakio sąlygą ir galimybių modifikacijas) atveju turime intersubjektyvumo geometriją, o antruoju, libera­ liosios metodologijos (saugančios priešakį kaip atskirties galimybę), atveju – interobjektyvumo geometriją. ų ą p ų Šiais atvejais relevantiškumo problema pakei­ čiama izoliuotų faktų kaupimo metodikomis. Statistinėmis-metodinėmis: iš ko susideda; kiek iš viso; kiek gauname sudėję, o kiek – atėmę (kiek per penketą metų įsisavinta projektinių lėšų; kiek mūsų, vidurkių, liko per keletą dešimčių metų). Procedūrinėmis-kartotinėmis: konstitu­ cinės, bet ne konstituacinės, rinkimų-politikos, jaudulingai teikiančios lygybės sąlygų regimy­ bę, – teisės dalyvauti sulaukus tiek-ir-tiek-metų kas ketverių metų rinkimuose. Kitaip nei meto­ dologiškai netematizuojami, todėl anonimiški, socialinio ir politinio intencionalumo fenomenai, statistiniai-metodiniai ir procedūriniai-kartoti­ niai reiškiniai yra mums-pažįstami (nebus per stipru pasakyti: „kaip tikrai socialiniai ir tikrai politiniai“, arba „tikrai iki kaulų smegenų“). Fenomenologiškai grindžiamos metodolo­ gijos akiratyje – ne pavieniai mąstymo aktai, ne patys objektai, skutami kaip „tik bulvės“ (nors švieži bulvė, daiktas, „baltas ovalas su juodais taškeliais“, turi habitus, – „Viską ir netgi daugiau negu Viską“; Šliogeris 2010; 7) parankiniais – nelygu tyrėjo interesai – operacionalizacijos skustukais. Fenomenologijai svarbu santykio konstituacija, supratingumo nuskaidrinimas: santykių, sintetinių procesų ir objektinės prasmės (įskaitant, kaip minėta, egzistavimo ir neegzista­ Fenomenologiškai grindžiamoje metodo­ logijoje paprasčiausias vienetas yra gyvoji da­ bartis (gyvenimo-pasaulyje veikiančių asmenų 66 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija vimo, galimybių ir negalimybių predikatus bei jų modalines atmainas). O tai reiškia (juk kalbame apie metodologi­ jos ir konkrečiosios ontologijos sąsajas): neišleisk iš akių vidinių intencionalių horizontų, kurie adekvačiai, – užkabindami praktinių tipizacijų tvarkas, – orientuoja į tam tikras matomas, bet nepastebimas žiūrų veiksmo laikysenose, relevan­ cijų, praktinio racionalumo sistemas. Darkart: šios žiūros veiksmo laikysenose yra aki-vaizdžios (matomos, bet nepastebimos). Visas mūsų paty­ rimas ir visi sąmonės aktai skleidžiasi relevancijų pri-mestyse: sąsajų-struktūrose, kuriose mes-bu­ vojame-įmesti. Relevancijos – tai akivaizdžios subjektyvumo-duotys, dinamiški – žinojimo ko nors atžvilgiu – faktai, kuriuos visad supa intersubjektyviai struktūrizuojamų – „teisingų“, „neteisingų“, „gerų“, blogų“, „gražių“, „negra­ žių“ „tinkamų“, „netinkamų“ ir pan. – atžvilgių ko nors atžvilgiais sistemos. modalines atmainas). Darkart pakartotina, kad eidetiniai predika­ tai siejasi ne su pačiu objektu ir objektyviai ap­ dorotais faktais ir net ne su aktualiai akivaizda, o su objektine prasme, kviečiančia nuskaidrinti esminių galimybių ir negalimybių perskyras. 6 Schutzo konstituacinės natūraliosios laikysenos fenomenologijos židinyje – įprasto žinojimo kasdie­ niame pasaulyje, sveiko protavimo savaime suprantamumas, dėl kurio natūralumo visos problemos čia be didesnio vargo išsprendžiamos praktiškai. Gamtos ir sociokultūrinio pasaulių patyrimo intersubjektyvumo natūralioje laikysenoje problema, kaip pažymi Schutzas, „netgi išvengė filosofų dėmesio“ (Schutz 1962; 57). Drauge Schutzas nurodo ypatingą filosofų – Jameso, Bergsono, De­ wey, Husserlio, Whiteheado – įnašą į sritį, vadinamą „įprasto protavimo kasdieniame gyvenime“ pagrindais; Lebenswelt, kuriame randasi visos, netgi loginės, sąvokos; socialinę aplinką, kurioje pasirodo neapibrėžtos situacijos, žingsnis po žingsnio apibrėžiamos ir tampančios į-tikrintomis: „Visi šie mąstytojas sutaria, kad bet koks žinojimas apie pasaulį įprastame protavime, o taip pat ir moksle, įtraukia mentalines konstrukcijas, sintezes, apibendrinimus, formalizacijas, idealizacijas, būdingas atitinkamiems mąstymo organizavimo lygmenims“ (ten pat; 58). (CAIRNS 1976; 51–2) Ypač konkrečiose ontologinėse gyvenimo- pasaulio relevancijose: kaip mes esti politiškai gyvenimo-pasaulio struktūrose? Ne šiuolaikine, savaime suprantama, o priešduota socialumo ir politiškumo prasme. Jei relevancijų regionai surikiuojami pagal galios ir negalės – paskirs­ tymo, išsidėstymo, įtikrinimo ir įsitikrinimo, vertinimo, įvertinimo, nuvertinimo, perverti­ nimo – tvarkas, tai mums, šiuo-laikiniams, itin vertinga yra Aristotelio ar Platono proporcijų ir atžvilgių analizė, orientuojanti į relevantiškų struktūrų heterogeniškose aplinkose tyrimus (Schutz 1964; 240–2).5 Ne mažiau svarbu ir „fenomenologinis“ Aristotelio požiūris į prieš­ duotiškumo ir duotiškumo modusų tarpusavio santykį. Tiksliau, kad ikipredikatinė ir predika­ tinė tvarkos turi viena kitą atliepti, kaip pabrėžia Filosofas: „Aišku tik tai, kad įstatymai turi ati­ tikti santvarką“ (Aristotelis 1997; 157; [1282b 10–11]). O san-tvarka yra bendra-buvojimo santykių atžvilgiu sąlyga: „<...> juk sakoma, kad teisingumas yra tam tikras dalykas tam tikriems asmenims ir kad lygiems jis turi būti lygus“ (ten Tik ištyrę šių intersubjektyvios pri-mesties kilmės sistemų sandaras, relevancijų buvoji­ mo štai čia ir dabar (čiabūtiškumo) sąlygas, tipizacijų pasyviose tapatumų ir panašumų sintezėse ypatumus ir jų galimybių apimtį, kurią Husserlis ir Schutzas sieja su problemi­ nių galimybių tikėtinumo laipsnių aprašymais (Schutz, Luckmann 1973; 186), galime tikėtis (atitikimas – tik tikėtinas) adekvatumo tarp teo­ rinio racionalumo ir praktinio racionalumo tipų, arba, tariant Aristotelio žodžiais, tarp įstatymų 67 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 privalo saugoti kaip savo akį, – jei tik ji siekia apginti esminę skirtį tarp fenomenologijos ir natūralizmo. Galimybė ištrinti šią skirtį, gali­ ma sakyti, suponuota pačioje Alfredo Schutzo dalykinėje laikysenoje: orientacijoje į kasdienį, įprastą patyrimą gyvenimo-pasaulyje, kuris Schutzo laikomas (tai labai svarbus akcentas) pirmine prasmių provincija.6 Klausimas yra tas: ne kad reikia, bet kaip ir kokia apimtimi reikia išplėtoti konstituacinę relevantiškumo, pa-grin­ džiančio ir gyvenimo-pasaulio prasmines linijas, ir esmingiausius, eidetinius sąmonės lygmenis, analizę? Tai fenomenologinės pilnumo metodolo­ gijos, susidedančios ir filosofinės ir sociologinės metodologijų, klausimas. 7 Savo aplinkas patiriame tipizuotais būdais. Įprastai organizuojamais ir įprastai atpažįstamais. Tam tikros daiktų rūšys pažįstamos ir atpažįstamos kaip tokios, o ne kitokios. Tai didžiąja dalimi pri­ klauso nuo turimo habitus: tipinių patyrimo schemų savitoje tipinio relevantiškumo visuomenėje, žinojimo atsargų saugykloje, bendroje kalboje (Schutz, Luckmann 1973; 233–5). At-renkame to­ kias, o ne kitokias tipizacijas, nes esame relevantiški, saistomi a) situacijų ir b) patys susiedami prasminius atžvilgius motyvų, interesų, tikslų visetuose. Atrenkame relevantiškus bruožus, kitus at-žvilgių bruožus laikydami nerelevantiškais. Būtina pridurti (nurodant būdingą fenomenologinį komponentą ir skiriant jį nuo natūralistinio), kad socialinio veiksmo prasmės nėra būdingos pa­ čioms „sąveikoms“, patiems „veiksmams“. Prasmės randasi interpretuojant sąveikas, – tipizuotas sąveikas „pagaunančioje“, jas „sugriebiančioje“ refleksijoje; pasitelkiant numanomas perspektyvų abipusiškumo idealizacijas, kurios savo ruožtu priklauso nuo relevancijų. Pagrindinė konstitua­ cinės natūraliosios laikysenos fenomenologijos užduotis yra eksplikuoti sąsajas tarp relevancijų sistemų ir prasminių gyvenimo-pasaulio provincijų. ir santvarkos. Atskirų stebinių grandinėse, sąsajose, rele­ vancijose, išplėtojamos tam objektui būdingos vidinės – savaime ir sau – apibrėžtys (Husserl 2005; 125–6). Tačiau tyrimas negali apsiriboti tik empiriškai-faktinių lygmenų aprašymais. Kiekvienas individualiai pasirinktas tipas, kuris iškeliamas iš savo aplinkos, – pabrėžia Husser­ lis, – turi būti perkeliamas į grynųjų esmių sferą, nors tai nereiškia, kad išnyksta vidiniai intencio­ nalūs horizontai: jie tik patys tampa eidetiniais horizontais, kurie, – karteziškai medituojančio aš ribose,– gali būti perkeliami atgal į empirinės tipizacijos pavidalus (Husserl 2005; 89). Rele­ vancijų sistemos potencialiai išsaugo esmiškai apibrėžtą galiojimą visko, kas gali būti sukurta laisvame variavime, kuris gali tapti moksliškai autentišku atskleidimu tik grįždamas prie apo­ diktinių principų, prie esmei būdingų bendrybių ir būtinybių, kurios garantuoja fakto susiejimą su racionaliais šio fakto pagrindais: „Taigi mokymas apie grynąsias galimybes „savaime“ yra pirmesnis už mokymą apie tikrovę ir tik jis daro pastarąją apskritai galimą kaip mokslą“ (ten pat; 90–1). Santykį tarp prasmės ir relevantiškumo galima ir reikia apibrėžti kaip pagrindinę fenomenolo­ giškai grindžiamos metodologijos problemą, apimančią „savyje“ ir subjektyvumo ir racio­ nalumo problemas. Socialinės tikrovės, žmogaus veiksmo ir socialinės sąveikos tarpusavio sąryšių prie-žiūra konstituojama būtent relevantišku­ mo – gręžiantis į ką nors ir gręžiantis nuo ko nors – sąsajose. Tai pa-grindžiantysis fenomenas. Relevancijų fenomeną aptinkame elementaria­ me bendra-subjektyvumo – galėjimo suprasti vienas kitą bendro veiksmo laikysenoje – akte: Fenomenologiškai grindžiama metodolo­ gija mokymo apie grynąsias galimybes pirmumą 68 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija transcendentalinę sintezę, pėdomis,8 bet žymiai radikaliau, remdamasis dinamiškesniu „judriu orientyru“ (Husserl 2005; 68), iš esmės atitin­ kančiu Schutzo „abipusiškumo perspektyvų“ natūralioje laikysenoje (Schutz 1962; 315–29), gairę, Husserlis, sugriebia santykį tarp dviejų pažinimo polių kaip relevantiškumo įgyvendini­ mo santykį tarp intencionalumo aktų ir intuicijos aktų: noetinėje-noeminėje vienumoje, sisteminėje struktūrinėje tipikoje, universalioje konstituty­ vioje sintezėje žingsnis po žingsnio, laiptas po abipusiškas supratimas galimas tik atsiremiant į relevancijų sistemas, kurios mūsų išgyvenamame patyrime sudaro savitas „atramas“.7 Pilnumo metodologijos požiūriu, verta darkart nurodyti skirtį tarp psichologinio aš ir transcendentalinio aš (Husserl 2005; 33–5; [§ 11]) kaip pamatinę analogiją pereinant nuo subjektyvumo problemos prie relevantiškumo problemos. Tai perskyra tarp empirinio paži­ nimo ir grynojo pažinimo. Eidamas iš principo Kanto, eksplikavusio figūrinę vaizduotės 7 Savo aplinkas patiriame tipizuotais būdais. Įprastai organizuojamais ir įprastai atpažįstamais. Tam tikros daiktų rūšys pažįstamos ir atpažįstamos kaip tokios, o ne kitokios. Tai didžiąja dalimi pri­ klauso nuo turimo habitus: tipinių patyrimo schemų savitoje tipinio relevantiškumo visuomenėje, žinojimo atsargų saugykloje, bendroje kalboje (Schutz, Luckmann 1973; 233–5). 9 Plg.: „Visa tai nurodo į universalią konstitutyvią sintezę, kurioje tam tikru sutvarkytu būdu funk­ cionuoja visos sintezės ir kuri aprėpia visas realias ir galimas sąmonės atmainas. Taip pat galime sakyti, kad čia ima ryškėti grandiozinis uždavinys, kuris ir yra transcendentalinės fenomenolo­ gijos uždavinys. Šio uždavinio esmę sudaro tai, kad visi fenomenologiniai tyrimai, realizuojami sisteminės ir visa apimančios tvarkos vienybėje, remiantis judriu laiptas po laipto plėtojamos visų galimo suvokimo objektų sistemos (taip pat ir formalių bei materialių kategorijų sistemos) orien­ tyru, būtų vykdomi kaip atitinkami konstitutyviniai tyrimai, tyrimai, kurie šitaip remtųsi griežtai sisteminiu būdu vienas su kitu ir būtų vienas su kitu susiję.“ (Husserl 2005; 68) ir santvarkos. At-renkame to­ kias, o ne kitokias tipizacijas, nes esame relevantiški, saistomi a) situacijų ir b) patys susiedami prasminius atžvilgius motyvų, interesų, tikslų visetuose. Atrenkame relevantiškus bruožus, kitus at-žvilgių bruožus laikydami nerelevantiškais. Būtina pridurti (nurodant būdingą fenomenologinį komponentą ir skiriant jį nuo natūralistinio), kad socialinio veiksmo prasmės nėra būdingos pa­ čioms „sąveikoms“, patiems „veiksmams“. Prasmės randasi interpretuojant sąveikas, – tipizuotas sąveikas „pagaunančioje“, jas „sugriebiančioje“ refleksijoje; pasitelkiant numanomas perspektyvų abipusiškumo idealizacijas, kurios savo ruožtu priklauso nuo relevancijų. Pagrindinė konstitua­ cinės natūraliosios laikysenos fenomenologijos užduotis yra eksplikuoti sąsajas tarp relevancijų sistemų ir prasminių gyvenimo-pasaulio provincijų. ų p ų gy p p jų Žr. Grynojo proto kritikos Įvadą: „Apie skirtumą tarp grynojo ir empirinio pažinimo“ (Kantas 1982; 55–72). Grįsdamas dviejų minėtų pažinimo pusių „tiltą“ Kantas aiškiai skiria, viena vertus, figū­ rinę, „synthesis speciosa“, vaizduotės transcendentalinę sintezę, ir, kita vertus, intelekto sintezę, „synt­ hesis intellectualis“: „Vaizduotė yra sugebėjimas įsivaizduoti objektą ir jo nestebint.“ <...> Būdama figūrinė ši sintezė skiriasi nuo intelektinės sintezės (atliekamos tik intelekto, be jokios vaizduotės). Kadangi vaizduotė yra spontaniškumas, tai aš ją kartais dar vadinu kuriančiąja vaizduote ir skiriu ją nuo atkuriančiosios vaizduotės, kurios sintezė paklūsta išimtinai empiriniams dėsniams, būtent asociacijos dėsniams, ir kuri dėl to nė kiek nepadeda paaiškinti apriorinio pažinimo galimybės ir todėl yra ne transcendentalinės filosofijos, bet psichologijos sritis“ (ten pat; 144–5). „Tiltas“, kurį Kantas, pasitelkdamas kuriančiąją vaizduotę, grindžia tarp transcendentalinės ir patyrimo pusių, jo vadinamas „schema“: „Mes galime tik tiek pasakyti: vaizdas yra kuriančiosios vaizduotės empiri­ nio sugebėjimo produktas, o juslinių sąvokų (kaip figūrų erdvėje) schema yra grynosios apriorinės vaizduotės produktas ir tarsi monograma; pirmiausia dėl schemos ir pagal ją tampa galimi vaizdai, bet su sąvoka jie visada turi būti susiję tik per schemą, kurią žymi, ir patys savaime jie ne visiškai sutampa su sąvoka. Tuo tarpu grynosios intelektinės sąvokos schema yra kažkas, ko negalima laikyti vaizdu, ji yra tik grynoji sintezė, atliekama pagal vienumo taisyklę remiantis sąvokomis apskritai, kurią išreiškia kategorija [<...>]; ji yra transcendentalinės vaizduotės produktas, liečiantis vidinio jutimo apskritai apibrėžimą pagal jo formos (laiko) sąlygas visų vaizdinių atžvilgiu, kiek jie turi būti a priori susieti viena sąvoka pagal apercepcijos vienumą.“ (Ten pat; 161.) 69 Metodologija Sociologija. ir santvarkos. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 laipto išskleisdamas sintetinės struktūros faktus (Husserl 2005; 53, 65).9 tai, įsižiūrėti į a) kultūros ir b) socialinę aplinką, o tiksliau, į elementariąsias tiek a) pirmtakų aplinkos (darkart: prasmė gali būti nuosekliai atskleista, tačiau niekad negali būti pakeista; Husserl 2005; 187), tiek b) am-žininkų (mįslin­ giausios socialinių mokslų problemos) tipizacijų ir relevancijų aibes. Taigi santykis tarp idealiųjų transcendenta­ linių ir tikrovės patyrimo aspektų privalo išlikti fenomenologiškai grindžiamos metodologijos dėmesio centre (net ir tuomet, kai aprašoma, analizuojama ir nuskaidrinama vien natūralioji laikysena gyvenimo-pasaulyje). Tačiau šis san­ tykis yra ne statiškas, o dinamiškas, todėl kie­ kvienas konkretus taikymas turi savo specifiką. Tariant Richardo Zanerio žodžiais, kiekvienas pažinimo teiginys yra kontekstualus dviguba prasme: a) turi vidinį horizontą, susijusį su savita „rūpesčių“, arba „baigtinės prasmės provincijų“, sfera, atsiremiančią į po ranka turimas žinojimo atsargas, ir b) turi išorinį horizontą, struktūri­ zuotus kalbinių figūrų, tipizacijų laukus (Zaner 1970; 17). Kadangi kalbame apie proto ir įpročio (įstatymų ir santvarkos) tarpusavio atliepimo principą, galime ir turime atidžiau įsižiūrėti į relevancijų-problemą: tematizuoti ir kultūrinės, ir socialinės aplinkos elementų (motyvų, inte­ resų, tikslų, gyvenimo-planų, jų įgyvendinimo probleminių galimybių ir atvirų galimybių) porininkų, pirmtakų, amžininkų ir netgi įpėdinių (teleologija esti sąsajos) pasauliuose sferas. O santykiai tarp prasmių ir relevancijų skleidžiasi ne natūraliame (objektyviame), bet intersubjektyviame gyvenimo-pasaulyje, kuris yra prasmingai struktūrizuotas. Prasmingu­ mas priklauso nuo tipizuotų aktų ir objektų interpretavimo, o interpretavimas savo ruožtu priklauso nuo relevantiškumo – pagal santykį su jau esamomis kalbos figūromis, o taip pat biografiniais motyvais, interesais ir tikslais konkrečiose aplinkose – sistemų. „Grandiozinis transcendentalinio lygmens uždavinys“ (Husserl 2005; 68) natūralioje laikysenoje taip pat „gran­ diozinis“: kadangi visų mokslų ir menų tikslas yra gėris (Aristotelis 1997; 157; [1282b 10–11]). Tai reiškia, kad dinamiškas („judraus orientyro“) santykis tarp intencionalaus akto ir intuityvaus, eidetinio akto turi būti tinkamai ekstrapoliuojamas į empirinį – prasmės akto ir relevantiškumo akto santykio – lygmenį. Prasmės, kurias žmonės, bendraudami sieja su kalbos figūromis (sąvokomis) įprasto konkrečių socialinių grupių sąveikų aplinkose, kaip pabrė­ žia Schutzas, susijusios su „jų pačių patvirtintų“ (Schutz 1964; 226) tipizacijų ir relevancijų sistemomis. Jei tik ir kai tik atsižvelgiame rele­ vantiškumo, principą, turime atidžiau, nei prieš 70 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija yra gyvenimas, o gyvenimas yra veikla, ne tik gaminimas: „Gyvenimas yra veikla, o ne gami­ nimas <...>“ (Aristotelis 1997; 69 [1254a 7–8]). Metodologui privalu (tai minėta atskaitingumo ir atsakingumo dorybė) aiškiai skirti gyvenimą ir gaminimą. Tai rūšinis, esminis skirtumas. 10 Plg.: „Kai vaikas mokosi tokios kalbos, jis turi atmintinai išmokti eilę „skaitvardžių“: a, b, c... Jis taip pat turi išmokti juos vartoti.“ (Wittgenstein 1995; 122 [9]). ir santvarkos. Pir­ masis sietinas su relevantiška veiksmo laikysena, o antrasis – su tik gaminimu, tik naudojimu, tik gaminių atgaminimu (idealiųjų tipų, vaidmenų, funkcijų), kurie „savo kojomis“, kaip Hefaisto trikojai (ten pat; 1253b 41–42), – ne pirmojo asmens, o trečiojo asmens gaminiai, – ateina į dievų susirinkimus (mokslines teorijas, kompromi­ sines sutartis, politines partijas, administracinius potvarkius). O jei pavyksta prasiskverbti (žengiant griežto subjektyvumo mokslo, kurį būtina skirti nuo subjektyvaus mokslo, laiptatakiais) į priešduotas prasmines socialinio pasaulio struktūras, ku­ rias sudaro elementariausi bendrieji sąmonės požymiai, galime tikėtis įžiūrėti esminę skirtį tarp neskubraus mokslo (relevantiško gyvenimui ir geram gyvenimui mokslo: nuo Aristotelio iki Husserlio) ir skubraus mokslo (relevantiško ištariamiems žodžiams, vardams, ypač „skaitvar­ džiams“, mokslo: nuo Hobbeso iki šiuolaikinio natūralizmo). Neskubrus mokslas, priėjęs net prie ele­ mentariausio išgyvenamo patyrimo santykio (aš-su-porininku, arba mes-santykio), laikosi santūrumo, – skubriųjų metodikų pasauliuose nykstančios dorybės (atskaitingumo drauge su atsakingumu). Netgi betarpiškoje sąveikoje, ku­ rioje aš, buvodamas drauge su porininku, galiu daugiau ar mažiau aiškiai – „esant labai žemam anonimiškumo ir labai aukštam pilnumo laips­ niams“ (Schutz 1962; 25) – numanyti jos ar jo motyvus ir interesus, aš vis dėlto „tik konstruo­ ju“ personalinį porininko tipą ir numanomos veiksmo krypties tipą. Taigi aš privalau būti santūrus: susilaikyti nuo skubriam metodologui būdingo polinkio „sugriebti kitą savyje“. Juk netgi betarpiškame bendrabuvojime aš netampu Kitu. Todėl privalau skirti Kitą ir jos ar jo tipą (socialinį vaidmenį, statusą, funkciją), kurį aš „tik konstruoju“. Metodologinis bendrabuvojimas – tai ne abs­ traktus geometrinis erdvės ir laiko užvaldymas remiantis „a priori“, „sudėjimu“ ir „atėmimu“ (t.y. „reasoneth“; žr. Hobbeso Leviatano penktą sk. „Apie protą ir mokslą“), o adekvatus patyri­ mo priešduotose tipizacijų formose aprašymas, analizė ir nuskaidrinimas. Naujųjų laikų nesu­ laikomas „skaitvardžių“10 mokslas (kai žmogus samprotauja, reasoneth, jis nedaro nieko kito, o vien suvokia visą sumą...; Hobbes 1999; 60), nukreipiamas prieš Aristotelio esmių mokslą. Skaitvardžių natūralizmas kaip išorinių daiktų spaudimo geometro sąmonei („išorinio daiktų spaudimo, nukreipto į mūsų akis, ausis ir kitus tam skirtus organus“; Hobbes 1999; 36) pada­ rinys randasi iš reasoneth (verifikacijos) metodo, kuris nedaro nieko kito (kitaip sakant, nesivargi­ na ieškoti prasminių sąmonės intencionalumo laukų), o vien tik „sudeda“, „atima“ ir „skubriai Metodologo metodą kaip jos ar jo nuo- savybę (są-monę) reikia skirti nuo metodo kaip įrankio (metodinio gaminimo). Pirmasis 71 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija po-linkių – savitų formų ir turinių – radimosi priežiūra. Gyvenimą ir galimai gerą gyvenimą tvarkančių principų konstituavimo priežiūra. 11 Plg.: „Nes žodžiai yra išmintingų žmonių ženkleliai, kuriuos jie vartoja tik skaičiuodami, tačiau jie yra tikri kvailių pinigai, kuriuos jie vertina, remdamiesi Aristotelio, Cicerono, Tomo ar kurio nors kito daktaro ar šiaip žmogaus autoritetu.“; „For words are wise men’s counters, they do but reckon by them: but they are the money of fools, that value them by the authority of an Aristotle, a Cicero, or a Thomas, or any other doctor whatsoever, if but a man.“ (Hobbes 1999; 56; Hobbes 1998; 24.) ir santvarkos. net „Kierkegaardo šuolius“: per-eiti, – visiškai nesinaudodama įtikrinančiomis Hobbeso „su­ dėjimo“ ir „atėmimo“ lygtimis, – iš suderinamų prasmių laukų baigtinėse prasmių provincijose (re­ levancijų sistemose) į kitas, vidujai suderinamas, bet tarp savęs nesuderinamas, prasmių sistemas. Aš-galiu: jei susilaikau (o būdamas laisvas, galiu tai daryti, ir darau) nuo bet kokio patyri­ mu grįsto tikėjimo, bet laikausi drauge su visu patiriančio gyvenimo srautu (Husserl 2005; 26). Kitaip tariant, aš ne dirbtinai išpučiu idea­ liuosius paskirų psichologinio patyrimo „faktų“ tipus, o spontaniškai, savo kūnišku buvojimu įgyvendinu vaizduotės transcendentalinę sinte­ zę, kuri neprieštaringai gali apimti tarpusavio ryšius tarp, formaliu požiūriu, nesuderinamų – kasdienybės, darbo aplinkos, svajonių, muzikos, vaizduotės (vaizdų judėjimo vandenyje, stebinių žvaigždėtame danguje, ateities praeityje, praeities ateityje), ribų, teatro, kito pirmo karto – prasmi­ nių tikrovių. Aš galiu iš-laikyti šias struktūriškai relevantiškų faktų akivaizdas erdvės-ir-laikišku­ mo rišlume. Gera veiksmo laikysena – visad šiuo-laikiška. Tad klauskime, kaip mes laikomės šiuo-laiki­ niame pasaulyje? Pa-saulyje, kurį išgyvename patirdami ir kuris esti mums priešduotas įsta­ bioje įmestyje, arba gyvenime (o tai yra viena ir tas pat). Šiuo-laikiniame pasaulyje, kurio ideali struktūrinė galimybė tinkamai apibrėžta Alexio de Tocqueville’io. Mūsų šio pasaulio žinoji­ mas, – taria Tocqueville’is, – yra dar ne mūsų rankose, o tik po ranka (tik – lygu be galo atran­ kos galimybių modusas). Pa-saulis visad yra ko nors atžvilgiu. Štai kodėl svarbu pirmojo asmens, mūsų pačių, pagava. Kaip pra-rasto patyrimo at-radimas mažų mažiausiai dviem esminiais atžvilgiais: 1) politinio intencionalumo (laisvės lygybėje laikysenos bruožų ir galimų modifika­ cijų nuskaidrinimo), 2) socialumo (am-žininkų supratimo aplinkybių eksplikavimo). Pagauti, sugriebti, atrasti prarastą patyrimą – tolygu susieti, surišti, suprasti ne tik partnerius ir pori­ ninkus, bet ir amžininkus, pirmtakus, įpėdinius socialinės politinės laikysenos aplinkoje: kai laisvė stiprina lygybę, o lygybė – laisvę (Tocque­ ville 1996) (žr. priedą). Ranka ir atranka, laikančios tinkamus darbo įrankius (o ypač vaizduotės įrankius), yra atranka iš relevantiškų pasaulio, mūsų gyvenimui prieš­ duoto, elementų, kurie esti savitai žmogiški, „iš­ skiriantys žmones iš visų kitų gyvūnų“ (Aristote­ lis 1997; 66 [1253a 19]). Relevantiškumas – tai laikysena, kuri, viena vertus, gali būti atskaitinga (ko nors atžvilgiu), bet nebūtinai atsakinga (gėrio ir blogio, teisingumo ir neteisingumo atžvilgiais), kita vertus, gali būti ir atskaitinga, ir atsakinga. Laikanti ir išlaikanti (išlaikyti galima tik aš-ga­ liu savigrąžoje) gero gyvenimo labui. ir santvarkos. Žiūra, kuri gali būti ir netematizuojama, savai­ me suprantama, ir tematizuojama, – atsklei­ džiant subjektyvių prasmės-kontekstų objektyvius prasmės-kontekstus. Jei tik ir kai tik sugriebiame skirtį tarp gyvenimo ir refleksijos (savigrąžos), pradedame suprasti griežtos fenomenologinės metodologijos ir mūsų laikymosi pasaulyje (gyvenimo ir galimai gero gyvenimo) abipusiš­ kumo perspektyvas. Tuomet jau galime klausti šiuo-laikiškai: kaip mes, socialiai relevantiško veiksmo požiūriu, laikomės ikipredikatinio patyrimo pasaulyje? reprezentuoja“. Tai reprezentacinių apklausų, reprezentacinių scenų, reprezentacinių žmonių požiūris, polinkis, veiksmo laikysena, kreipiama tik į šviesią sceną, o scenos šviesa – į ateitį. Ir dėl šviesos greičio nepakanti pirmtakams: Aristote­ lio ir „kitų daktarų“11 „absurdas“ (ten pat; 63) šviesuolių pakeičiamas neginčytinos geometrijos metodu (ten pat; 64). O neskubrus mokslas, tariant Edmundo Husserlio žodžiais, symphilosophein (žr. Schutz 1996; 155), įskaitant ypač socialinių mokslus, reikalaujančius pilnumo metodologijos, nepalieka be dėmesio gyvenimo ir gero gyvenimo (tikrovės sąlygų ir galimų jos modifikacijų refleksijų). Tai žinojimo galimybių, t.y. būklės, kuri dar-ne- įgyvendinta, sfera. Kitaip sakant, tai relevantiš­ kumo struktūrų – kaip probleminių galimybių ir atvirų galimybių – sfera. Atsižvelgdami į tai, kad socialinių mokslų tyrimo dalykas yra objektyvūs subjektyvių prasmių kontekstai, turime formuluoti pirmąjį pirmosios metodologijos principą: adekvati mokslinė analizė objektyvų mokslinių tipų konstravimą privalo grįsti žinoji­ mo formomis, patyrimo figūromis, gautomis iš gyvenimo-pasaulio prasminių struktūrų, prieš­ duotų žmogaus patyrime tipiniais būdais, kurie savo ruožtu randasi iš interpretacinės (supratumo) veiklos, būdingos ikipredikatiniam patyrimui. Mes laikomės – visada – metodologiškai, tačiau nebūtinai laikyseną tematizuojame. Juk relevantiškumas – vėlgi kalbant banaliai – tai atranka. Dažniau to, kas po ranka, bet nebū­ tinai. Vienaip ar kitaip, relevantiškumas yra po-linkių, – mūsų sąmonės ir kūnų buvojimo kinestezėje, – visetas. Prie-žiūra, arba ranka, at­ renkanti priešduoto pasaulio elementus ir iš esa­ mo gyvenimo, ir iš galimai gero gyvenimo (kiek čiabūtiškume esama rankos judesių refleksijos). Kasdienybės pasaulyje mano atranka (są­ monė) – įprasta, netematizuojanti. Gyveni­ mo-pasaulio prasminėse transcendencijose, pereigose, o ir moksliniuose pasauliuose at-ran­ kos – neįprastos, tematizuojančios. Atranka, arba sąmonė-drauge-su ranka, gali įgyvendinti Šiek tiek rizikuodami  – tardami išties banaliai – pažymėsime: relevantiškumas yra prie-žiūra. Visų pirma tikrovės, tiksliau, jos 72 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija įgyvendinimuose. Reikalaujančiuose skirti pedantiškumą (ekspertinį faktiškumą) ir gerumą (prie-žiūrą): geras žmogus „<...> pasirenka ir daro tai, kas yra teisinga, bet nėra pedantas, kuris smulkmeniškai tol siekia teisingumo, kol pagaliau teisingumas virsta neteisingumu <...>“ (Aristotelis 1990b; 167 [1138a 1–3]). ir santvarkos. Išlaikanti tikrovę ne tik tikrumo gaminiuose, bet ir tikrumo galimybių įgyvendinimuose, eidetikos sąlygų Taigi darkart šiuo-laikiškai: ar mes esti objektyviai tįsiame geometriniame pasaulyje, ar štai-šiame, laikiškame ir vietiškame, pasaulyje: dar iki mūsų-gyvenimo-pasaulio įtraukimo į jų- projektų-pasaulį (fenomenologijai, kaip ne kartą pabrėžta, svarbiau už bet ką kita esti vaizduotė, 73 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija t.y. galimybių sritis)? Kažkuris vienas iš šių dviejų labai skirtingų pasaulių mums esti relevantiškas, o kitas – mums nerelevantiškas, nors kam nors galbūt relevantiškas, kaip teigia relevantiškumo būtinybės, arba atrankos, principas: „Relevantiš­ kumo problemos akiratyje – atranka iš pasaulio, priešduoto gyvenimui, kaip ir mąstymui, viseto“ (Schutz 1996; 4). Tą patį relevantiškai sako ir Filosofas, Aristotelis: „Tačiau reikia atsižvelgti į tai, kokių dalykų atžvilgiu lygybė ir kokių – nelygybė <...>“ (Aristotelis 1997; 1282b 24) nas konkretus suvokimo lauko momentas – tiek pasyvioje, tiek aktyvioje sintezėje – teikia nuo­ rodas į dalykinių, motyvacinių, interpretacinių relevancijų sistemas, tipizacijas, pažįstamumo ir nepažįstamumo laipsnius, racionalumo ir, vadinasi, pačios tikrovės apibrėžimus. „Šis pasau­ lis kaip visetas esti iš principo nepermatomas; kaip visetas jis nei suprastas, nei suprantamas“ (Schutz 1966; 130). Štai kodėl (tai vadintume pačia relevancijų pradžia, pačiomis jų ištakomis) „tikrą tikrovę kaip ją pačią“ galime gauti tik iš aki-vaizdos (Husserl 2005; 74–5). Iš pažiūros relevantiškumo problema, tei­ kianti gana mįslingą nuorodą į mūsų žinojimo atsargų genezę, yra „tik“ filosofinė (plg. su II-dalyje nagrinėtu natūralistiniu požiūriu į subjektyvumą: tai „tik“ subjektyvumas). Tačiau dėmesingesnis įsižiūrėjimas į „tik“ filosofinį relevantiškumo lygmenį papildomai atskleidžia „tik“ politinį sąmonės gyvenimo lygmenį. Čia matomai, bet nepastebimai susieina du indai – metodologinis ir politinis: taisyklingumo-ir- teisingumo. Objektai mums esti „plačiausia“, todėl ir „tikriausia“, prasme dėl tam tikro tipiško „taisyklingumo“ (politikos čia tarsi nėra: tik loginė forma), kursai „valdo sąmonės gyvenimą apskritai“, kursai „vienintelis padaro“, kad tiesa ir tikrovė – tikri daiktai, išgyvenimai, skaičiai, dalykų padėtys, dėsniai, teorijos – mums turi ir gali turėti ir t.t. prasmę (Husserl 2005; 74). O aki-vaizda (epistemologinės kilmės brūkš­ nelio svarba išsamiau aptariama III. 2 poskyryje „Relevantiškumo problemos filosofinės ištakos: aki-vaizdos problema“) reikalauja ne izoliuotų nuo tikrovės „faktų-atkarpų“, o „sintetinės struktūros faktų“, ypač dar netyrinėtų. Aki- vaizdos problema, skirtina nuo neproblemiškai atkirpo „akivaizdaus empirinio fakto“, reika­ lauja dalyko, kurį minėtas analitinis, griežtai diferencijuojantis mokslo biurokratijos potvarkis griežtai draudžia (totali taisyklingumo politika visad randa jai relevantiškus saugos būdus). Tai dalykas-problema, ne „susidedantis“ iš filosofi­ jos ir sociologijos dalykinių intarpų, o tęstinai buvojantis filosofine sociologija (Schutz 1966; 132). 12 Čia kalbama apie idealųjį realistinio materializmo / natūralizmo tipą „Ir t.t.“ Kalbant visai papras­ tai, fragmentacija, reliatyvizacija ir „neapibrėžtumas“ (naujas protagoriškas šiuo-laikinių sofistų išradimas) eina „ir t.t.“. O kur? – klausite jūs. Tik pats niekas žino: „Nesėkmingos sociologi­ j g j j 13 Tarp gausių abstrakcijų, persisotinusių subjektyvistiniais faktais-atraižomis, išskirtume įtakingiau­ sią (sparčiausiai sėjančią fragmentiškumą šiuo-laikiškume). Tai dedukcinė abstrakcija (vadinamoji procedūrinė sąvokų „operacionalizacija“, kitaip sakant, turiningų tikrovės atžvilgių naikinimas griežtai formalizuotoje „mano suvokimo lauko“ atrankoje), grindžiama grynosios indukcijos (faktų- atraižų prišliejimo prie jau stabilizuoto „mano suvokimo lauko“ modelio) regimybe. „Aš žinau“ – lygu: „Aš turiu faktų-atraižų“, arba „Padariau empirinį tyrimą“ (o tai yra viena ir tas pat). Plg.: „Įsivaizduok ką nors sakantį: „Bet aš žinau, koks mano ūgis“ ir įrodymui dedantį ranką sau ant viršugalvio!“ (Wittgenstein 1995; 234 [279]). 14 Žr. Hobbeso „samprotavimo apibrėžimą“: „Remdamiesi visu tuo, mes galime apibrėžti, t.y. deter­ minuoti, kas yra tai, kas turima omeny, vartojant žodį samprotavimas (reason), kai priskiriame jį mąstymo (mind) sugebėjimams. Juk SAMPROTAVIMAS šia prasme yra ne kas kita, o tik skaičia­ vimas (t.y. sudėjimas ir atėmimas) bendrųjų vardų sekų, sutartų ir pripažintų žymėti (marking) ir ženklinti (signifying) mūsų mintis.“ (Hobbes 1999; 61)ii ir santvarkos. Relevantiškumo, griežta šio žodžio prasme, filosofinės sociologijos, problema glaudžiai susijusi su pasaulio, sąmonės ir daiktų tarpusavio san­ tykio nuskaidrinimu globalėjančiame, veikiau plyštančiame į fragmentiškas atkarpas – pagal politiškai korektiškus geometrinio fizikalizmo maldos „ir t.t.“ žodžius – gyvenimo-pasaulyje.12 Kitaip tariant, metodologija yra priešduota politiškai: štai-šis mūsų žinojimas turi savitas, in­ dividualias figūracijas. Metodologijos priešduo­ tyje matome akivaizdą, tampančia rišlia „ir t.t.“ politika. Mūsų gyvenimo-planų visete kiekvie­ 74 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija zontų (ten pat; 187). Tai, ką tęstinai įgyvendina fenomenologinė laikysena, yra viseto konkretu­ mas. Fenomenologija – ir patyrimo sulaikymo, ir drauge išlaikymo būdas. Būdas, susaistantis kiekvieną nesusietą faktinę-atkarpą ir ginantis įprastą gyvosios dabarties patyrimą kaip prasmę, kuri niekad negali būti pakeista (ten pat), tar­ kim, remiantis, moksliškai racionalesniais, for­ maliais ir abstrakčiais, dirbtinai atribotais nuo sąmonės gyvenimo srauto viseto, procedūriniais vertybinio neutralumo sofistikatais.13 Kad įžiūrėtume gyvybiškai svarbias skirtis gyvenimo-pasaulyje, dar prieš tai turime nu­ skaidrinti fenomenologiškai savitos laikysenos – sąmonės atsietumo nuo daiktų – ypatumus. Esminis dalykas yra tas, kad fenomenologinė atsietumo laikysena, tęstinio pirmojo asmens fe­ nomenologinės redukcijos taikymo natūralioje laikysenoje požiūriu, nėra abstrakti. (Plg. su pozityvistine „vertybiškai neutralia nuostata“, siejama su formaliomis, todėl abstrakčiomis trečiojo asmens metodinėmis procedūromis.) Fenomenologinė laikysena nėra tik viena tarp daugelio kitų tolygių laikysenų, sofistiškai skel­ biančių visuotinę tarsi-lygybės apoteozę: ašai žvelgiu iš neutralios trečiojo asmens padėties, ištuštinusios laikiškumo refleksiją, – „Mes esti niekur“. Tik pilnai išsiskleidžiančioje refleksijoje gali­ ma ir radikaliai atsisieti nuo daikto, ir drauge sąmonės dėmesingumo židinyje tęstinai išlaikyti refleksijos akto tematiką. Ir priešingai, refleksija natūralioje laikysenoje yra pertraukta: suskilusi į a) nuomonę, susijusią su daiktu (tarkim, infor­ manto informacija), ir b) stebinčios sąmonės aktą, kuris laikomas „priklausomu“ nuo daikto, dar daugiau, netgi „pagrindžiamu“ tik pačiame objekte. Fenomenologinėje laikysenoje transcenden­ tinis objektas išsaugomas sąmonės konstituoja­ mų – nukreiptumo daiktų link – aktų sąsajose. Tačiau drauge įdėmiai įsižiūrint į natūralias „naivaus galiojimo“ (Husserl 2005; 25) natūra­ lioje laikysenoje „atkarpas“ ir nuo jų „radikaliai atsisiejant“, tačiau tik tam, kad būtų galima tirti pasauliškumo patyrimo akivaizdumo galiojimą ir apimtį (ten pat; 24). Prasmė pasirodo kiekvie­ name aktualiame patyrime, apsupta, t.y. saistoma, principinio nuskaidrinimo reikalaujančių hori­ Pilnai išsiskleidžianti refleksija pertraukiama naujojo mokslo naujaisiais laikais. Kontrastas tarp empirizmo ir racionalizmo neturėtų klaidinti, kaip pažymi Husserlis: Johno Locke psichikos natūralizacija žengia koja kojon su Thomas Hobbeso natūralistiniu fizinio-racionalumo modeliu. Tie patys procesai vyksta ir kituose mo­ 75 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija derniojo laikotarpio moksluose (Husserl 1976b; 63–6; § 11). ( g y g) ų ( ) 15 Filosofija, kaip pažymi Robertas Sokolowski’s saugo įprastą patyrimą savitai atsietu būdu. Sofis­ ir santvarkos. Kalbant glaustai, nustatomas „tie­ sinis“ funkcinis ryšys tarp sam-protavimo kaip skaičiavimo (sudėjimo ir atėmimo) ir proto kaip su-sumavimo (ką gauname sudėję ir atėmę).14 vertus, tikras mokslas, nustatantis tikrus faktus. Aiškus ir ryškus psichologstinis dualizmas išlieka iki XX a. pozityvistų mokslinės ir nemokslinės (metafizinės) metodologijų perskyros, o paskui išsiskaido į paskiras empiristines-reliatyvistines metodologijas. Šiuo-laikinis psichologizmas yra vis labiau stiprėjanti tik faktinių mokslų ir tik faktinių įsitikinimų tendencija. Tendencija, kuri sąmonę kaip nepagrindžiamą ir nepagrindžian­ čią tik refleksiją ir ypač saviapmąstymą (pažink save, kad galėtum pažinti kitą) atsieja nuo daiktų. Naujasis mokslas, pertraukdamas senąją graikišką ir Viduramžių proto kaip ir intuity­ vaus, ir racionalaus tradiciją, protą susiaurina tik į „skaičiavimą“, kuris būtinai yra pertrauktas, sudarytas iš faktinių dalių (kad būtų ką su kuo sudėti ir ką iš ko atimti), neturinčių tarpusavio intencionalumo sąsajų. Stebinčios sąmonės aktas laikomas priklausomu nuo daikto ir pagrindžia­ mu tik pačiame objekte: „Jutimų priežastis yra išorinis kūnas <...>“ (Hobbes 1999; 35); daiktų pasirodymo būdai – „tik“ regimybė (seeming), „tik“ įsivaizdavimas (fancy) (ten pat; 36). „Ti­ krovė“ į naująjį, šiandieną vadinamą moderniu, pasaulį ateina kaip naujų „empirinių mokslų“ tikrovė, t.y. „tikresnė“ tikrovė ir už „metafizikų išprotautą“, ir netgi už „natūraliąją“ tikroves. „Tikrumo“ garantas – empirinis pagrindimas, kuris randasi iš „pagrindžiamojo“ psichologiz­ mo įsitikinimų, kad štai-šis pagrindimas ir yra jau patikrinta tikrovė. Kitaip sakant, pagrindi­ mas įgyvendinamas „visiškai naujomis“: jau ne metafizikos, o tikrųjų mokslų teikiamomis empirinėmis priemonėmis. Naivu manyti, kad metodologija, kaip įpras­ tai teigiama, rūpinasi tik metodų ir taisyklių, procedūrinių kriterijų artikuliavimu. Šiuolaiki­ nės tik faktinės metodologijos paskirtis – empiriš­ kai pateisinti savo įsitikinimus. Kad įsitikinimai, juos atsijojus vertybinio neutralumo sieteliu, taptų tikri. Tai, ką įgyvendina tik faktinių mokslų ideologinis, politinis, ekonominis sąjūdis, yra procedūrinių taisyklių, visiškai atsietų nuo sąmonės, pateisinimas. Ypač žalingas politinės filosofijos – politinį bendruomeniškumo inten­ cionalumą konstituojančio dalyko – ir socialinių mokslų – socialinį intencionalumą konstituojan­ čio dalyko – raidai. Sunyksta mokslininko kaip žmogaus, gebančio buvoti ir ištikimu kultūros priešduočiai, ir atskaitingai ją nagrinėti, dorybės. Žr. ypač Roberto Sokolowski’o Husserliškų medi­ tacijų § 70 („Filosofinis sąmonės atsietumas nuo daiktų“): „Sokratas buvo ir ištikimas, ir atsietas“ (Sokolowski 1974; 192).15 Šiuo-laikinis psichologizmas – iš esmės skai­ do. Ilgainiui atsiranda trivialus, bet grėsmingas dualizmas: viena vertus, „tik“ metafizika, kita 76 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Metodologija (!!!) priklauso (užtektų ir vieno šauktuko: tik ne šį kartą) – tai yra jos priešduotis, kurią privalu gerbti drauge ieškant laisvo abipu­ siško santykio – epistemologijai ir ontologijai. tinių antpuolių apsuptyje tai vienintelė galima (! – šauktukas mūsų) įprasto patyrimo gynyba. Tai savo ruožtu reikalauja filosofiją sugrąžinti į politinį kontekstą. Jei atėnietis, – klausia Sokolows­ ki’s, – nori išsaugoti, išlaikyti savo pasaulį kaip pasaulį, kurį savo rankose ir intersubjektyviame akiratyje laiko tik atėniečiai, kodėl ji ar jis turėtų svyruoti norėdami tai įgyvendinti? Juk jei jis mąsto, kaip turi mąstyti geri atėniečiai, tai turi baimintis, kad laikydamas atėniečių pasaulį kaip tik vieną tarp daugelio pasaulių, jis gali prarasti žmogiškumą, kurį įgijo būdamas atėniečiu. Atė­ nietis, sukirsdamas rankomis su Sofistu, – kad Atėnų pasaulis yra tik vienas tarp daugelio pasaulių, taptų panašus į sofistus. Jis liktų be ištikimybės bet kokiam miestui. Tol, kol negebėtų filosofiškai suprasti Atėnų pasaulį kaip Atėnų pasaulį: arba per prigimties suteiktas malonės dovanas, arba per ką nors kaip Sokratas: „Sokratas buvo ir ištikimas, ir atsietas“ (ten pat; 192). Laikytis susijus, tačiau mąstyti atsijus, – tokia yra metodologo prievolė ir jo kaip žmogaus egzistavimo sąlyga: „Jei mūsų tauta staiga išnyktų, ir tai, ką ji laiko susiejusi, pradingtų, kiekvienas mūsų akimirksniu pavirstų pabaisa, kurio išsigąstų pats.“ (Manent 2008; 8) p p g p 16 „Simbolį preliminariai apibrėšime kaip aprezentacinę aukštesnės tvarkos nuorodą: kai teikiamas poros narys yra objektas, faktas, įvykis mūsų kasdienio gyvenimo tikrovėje, o kitas pateiktas poros narys nurodo į idėją, kuri transcenduoja kasdienio gyvenimo patyrimą“ (Schutz 1962; 331). tinių antpuolių apsuptyje tai vienintelė galima (! – šauktukas mūsų) įprasto patyrimo gynyba. Tai savo ruožtu reikalauja filosofiją sugrąžinti į politinį kontekstą. Jei atėnietis, – klausia Sokolows­ ki’s, – nori išsaugoti, išlaikyti savo pasaulį kaip pasaulį, kurį savo rankose ir intersubjektyviame akiratyje laiko tik atėniečiai, kodėl ji ar jis turėtų svyruoti norėdami tai įgyvendinti? Juk jei jis mąsto, kaip turi mąstyti geri atėniečiai, tai turi baimintis, kad laikydamas atėniečių pasaulį kaip tik vieną tarp daugelio pasaulių, jis gali prarasti žmogiškumą, kurį įgijo būdamas atėniečiu. Atė­ nietis, sukirsdamas rankomis su Sofistu, – kad Atėnų pasaulis yra tik vienas tarp daugelio pasaulių, taptų panašus į sofistus. Jis liktų be ištikimybės bet kokiam miestui. Tol, kol negebėtų filosofiškai suprasti Atėnų pasaulį kaip Atėnų pasaulį: arba per prigimties suteiktas malonės dovanas, arba per ką nors kaip Sokratas: „Sokratas buvo ir ištikimas, ir atsietas“ (ten pat; 192). Laikytis susijus, tačiau mąstyti atsijus, – tokia yra metodologo prievolė ir jo kaip žmogaus egzistavimo sąlyga: „Jei mūsų tauta staiga išnyktų, ir tai, ką ji laiko susiejusi, pradingtų, kiekvienas mūsų akimirksniu pavirstų pabaisa, kurio išsigąstų pats.“ (Manent 2008; 8) ir santvarkos. Tai reiškia: bet koks socialinių mokslų metodas pri­ klauso filosofijai. Filosofija yra bet kokių metodų priešduotis, kad ir ką kalbėtų „gamtotyrininkai“: „Bendrai priimta tezė yra ta, kad mokslas ir me­ tafizika yra atskiros intelektinės veiklos. Ši tezė yra nauja ir nėra laikoma savaime suprantama filosofinėse klasikinės antikos sistemose, vidu­ ramžiais, ankstyvaisiais moderniosios filosofijos raidos amžiais. Ši tezė suponuoja mokslų ir jų metodologijų egzistavimą“ (Seebohm 2010; 7). trečia, kad santykių tarp įvairių lygmenų visetas suprantamas tik patyrimo konkretume: pasiro­ dymo-priešais, buvojimo gyvosios dabarties prieš- akyje. Tikriausiai dėl šios priežasties Alfredas Schutzas – itin įžvalgiai ir subtiliai – kartoja, kad metodologija nėra mokslininko auklėtoja arba globėja, o visada esti mokslininko mokinė, t.y. tik patiriančios akivaizdą metodologijos. Konkrečiosios ontologijos požiūriu, relevan­ tiškumas yra mūsų patyrimo organizavimas tiek ikipredikatiniame, tiek predikatiniame lygme­ nyse; nu-kreiptumo susidarant tikrovei principas. Relevantiškumas yra motyvaciškai suinteresuotas sąlygotumas. Dar kitaip: su(si)saistymas esti mūsų įmestis mums pri-mestyse. Čia pri-mestys – lygu tipizuotos priešduotys gyvenimo-pasaulyje. Jei natūralioje laikysenoje žmogus gyvena pačioje su(si)saistymų tėkmėje, turėdamas re­ levancijas kaip į-rankius savo rankose, savo natūraliame akiratyje, tai fenomenologinėje laikysenoje relevancijos tampa „temomis“. Tai ap-imčių, kurias reikia skirti nuo socialinių gamtamokslų imčių, problemų aibės. Tiksliau: Filosofinės sociologijos tyrimo dalykas yra istorinis, apimantis a) betarpiško mes-santykio, b) amžininkų, c) pirmtakų ir d) įpėdinių sąsajas simbolinių aprezentacijų konstituacijose.16 Tai reiškia, pirma, kad motyvacinių, teminių ir interpretacinių su(si)saistymų laukai yra socialiai ir kultūriškai sąlygoti; antra, kad tarp įvairių minėtų intersubjektyvumo lygmenų išsikristalizuoja būdingi tarpusavio santykiai; Jei natūralioje laikysenoje žmogus gyvena pačioje su(si)saistymų tėkmėje, turėdamas re­ levancijas kaip į-rankius savo rankose, savo natūraliame akiratyje, tai fenomenologinėje laikysenoje relevancijos tampa „temomis“. Tai ap-imčių, kurias reikia skirti nuo socialinių gamtamokslų imčių, problemų aibės. Tiksliau: tinių antpuolių apsuptyje tai vienintelė galima (! – šauktukas mūsų) įprasto patyrimo gynyba. Tai savo ruožtu reikalauja filosofiją sugrąžinti į politinį kontekstą. Jei atėnietis, – klausia Sokolows­ ki’s, – nori išsaugoti, išlaikyti savo pasaulį kaip pasaulį, kurį savo rankose ir intersubjektyviame akiratyje laiko tik atėniečiai, kodėl ji ar jis turėtų svyruoti norėdami tai įgyvendinti? Juk jei jis mąsto, kaip turi mąstyti geri atėniečiai, tai turi baimintis, kad laikydamas atėniečių pasaulį kaip tik vieną tarp daugelio pasaulių, jis gali prarasti žmogiškumą, kurį įgijo būdamas atėniečiu. Atė­ nietis, sukirsdamas rankomis su Sofistu, – kad Atėnų pasaulis yra tik vienas tarp daugelio pasaulių, taptų panašus į sofistus. Jis liktų be ištikimybės bet kokiam miestui. 17 Grindžiamoji „ir t.t.“ („Und so weiter“), „iteracinės begalybės“, forma, kurios, kaip pabrėžia Hus­ serlis, niekada netyrė logikai, turi savo subjektyvią sąsają: „Visada galima darkart“ („man kann immer wieder“). Tai idealizacija, nes de facto niekas negali „visada darkart“. Ši forma įgyvendina prasmę steigiantį vaidmenį (sinnbestimmende Rolle) logikoje: visada galima darkart grįžti prie idealą perteikiančios vienovės arba prie kitos idealios vienovės. Čia susiduriame su paslėptu konstrukci­ jų metodu, kuris formuojamas ir performuojamas kaip norma: „ir t.t.“ Šioji norma, „akivaizda“ drauge su jos savitomis formacijomis, turi tapti, fenomenologiniu požiūriu, tema. (Husserl 1974; 195–6). ir santvarkos. Tol, kol negebėtų filosofiškai suprasti Atėnų pasaulį kaip Atėnų pasaulį: arba per prigimties suteiktas malonės dovanas, arba per ką nors kaip Sokratas: „Sokratas buvo ir ištikimas, ir atsietas“ (ten pat; 192). Laikytis susijus, tačiau mąstyti atsijus, – tokia yra metodologo prievolė ir jo kaip žmogaus egzistavimo sąlyga: „Jei mūsų tauta staiga išnyktų, ir tai, ką ji laiko susiejusi, pradingtų, kiekvienas mūsų akimirksniu pavirstų pabaisa, kurio išsigąstų pats.“ (Manent 2008; 8) 16 77 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija kokia ap-imtimi jau turimos tipizuotos žinojimo atsargos mūsų laikomos savaime suprantamomis? Kokia apimtimi turimų tipizacijų (habitus), taigi ir interpretacinių susaistymų, visetai yra mūsų pa-tikrinti, mūsų pa-grįsti: priešduoti be jokių klausimų (iki paskesnių pastebėjimų)? savo nepažįstamumo faktu: „Mes nedarome šio patyrimo teminiu savo valingais aktais, – štai kodėl vadiname šią relevancijų rūšį primestomis relevancijomis“ (ten pat). Tačiau nepažįstamas patyrimas nėra vienintelė mums „primetama“ tematika: Kokia apimtimi tipizacijos sudaro mums priešduotų susaistymų, arba jau esamų relevan­ cijų, sąlygas? Kokia ap-imtimi mūsų vidiniai ir išoriniai horizontai figūruoti socio-kultūriškai? Kokia yra mūsų varto-sena (senų-dalykų – san­ tykių, įrankių, knygų, metų laikų, medžių lapų vartymas): ne tik ir ne tiek žodyno, kiek bendros šnekamosios kalbos sintaksinės struktūros. Pil­ numo struktūros, kurią reikia aiškiai skirti nuo pliuralistnių „kultūros struktūrų“: Wilhelmas von Humboldtas ją vadino vidine-šnekos-forma (innere Sprachform), apimančia tipiškas orien­ tacijų ir gyvenimo-pasaulio įgūdžių (Schutz 1966; 132), – papročių, procedūrų, socialinių institutų, įpročių, pėdsakų, prie-tarų, prie-žo­ džių, aiškių ir numanomų ženklinimų, – aibes? „Yra daug kitų primestų dalykinių relevanci­ jų rūšių. Pavyzdžiui, šoko patyrimas, kuris, kaip matome, yra būdingas bet kokiam sąmonės dė­ mesingumo pokyčiui kaip šuoliui iš vienos pras­ mės provincijos į kitą, primetančiam naujas da­ lykines relevancijas; tą patį galime pasakyti apie bet kokį nevalingą pokytį mūsų asmenybiniame lygmenyje, ypač santykiško artimumo pakeiti­ mo į santykišką anonimiškumą. Be to, bet koks staigus pokytis laiko, kuriame kiekvienas mūsų gyvename vienu ir tuo pačiu metu, dimensijo­ se, primeta mums kitas dalykines relevancijas. Kalbant apibendrintai, bet koks nuo-trūkis arba modifikacija, nutraukiantys idealizacijų „ir t.t.“, „vėl ir vėl“, kurios esti viso mūsų patyrimo iš­ takose,17 tęstinumą, sukuria mums primetamas dalykines relevancijas. Galiausiai, kaip parody­ sime paskui, dalykinės relevancijos yra mums primetamos remiantis socialinės sąveikos prie­ monėmis, kurias apibrėžia mūsų porininkai arba mes patys kaip asmenys arba socialinių grupių dalyviai.“ (Schutz 2011c; 108–9) Tai, ką jau turime, yra mums „primestos relevancijos“ („imposed relevances“; Schutz 2011c; 108–9). 18 Inter-esse – tai ne tik bendrojo pobūdžio žmogaus polinkis eiti daikto link, bet tęstinis po-lin­ kis savita („vis einant dar arčiau“; „immer näher zu kommen“) prasme, laukiant naujų daikto „savasties“ praturtinimo galimybių. Tema, tikslia šio žodžio reikšme, kaip pažymi Husserlis, ir aš-kryptingumas daikto link, ne visada sutampa. Aš galiu temiškai įsitraukti į mokslinį darbą, kurį pertraukia gatvės triukšmas. Kai triukšmas mane pasiekia, aš tam tikru momentu atsigręžiu į jį, tačiau ankstesnė mano tema ne apleidžiama, o tik nugrimzta užnugaryje, potencialiame mano akiratyje (vok. žodis Hintergrund čia verčiamas kaip užnugaris, nes žodžiai „fonas“, „užkulisiai“, „antras planas“, mūsų nuomone, tik iš dalies atliepia sąmonės-kūno patyrimo išgyvenant vieno­ vę). Ji tebelieka mano tema, prie kurios aš betarpiškai sugrįžtu, kai tik trukdžiai baigiasi. Šiuo požiūriu, platesnė domesio sąvoka yra domesio aktai. Tai kiekvienas aš-kryptingumo-link aktas (tiek pertrauktas, tiek tęstinis). Aš būtiškumo-su [inter-esse]. („<...> des Dabeiseins [inter-esse] des Ich). (Husserl 1939; 175.) 19 Galbūt, o ir veikiausiai, dėl to, kad grindžiamųjų iteracinės begalybės „ir t.t.“ („Und so weiter“) formų niekada netiria ne tik logikai, bet ir įprasto protavimo žmonės. Čia susiduriame su pa­ slėptais konstrukcijų metodais, kurie formuojami ir performuojami kaip šiuo-laikinės socialinio konstrukcionizmo (objektyvistinių metodologijų, manipuliuojančių „subjektyvumo“ vardu) nor­ mos. Šios normos randasi atrenkant iš pasaulio, kuris mums priešduotas, tam tikrus elementus, nukreipiančius tikrovę begalinėse idealizacijose: „visada galima darkart...“. ir santvarkos. Mes esti įmesti pri-mestyse, arba priešduotose relevancijose, iš kurių randasi kitos – motyvacinių, dalykinių, interpretacinių – su(si) saistymų rūšys. Nepažįstamas patyrimas, kaip pabrėžia Schutzas, primeta mums save pačiu Jei racionalumo ir subjektyvumo temų nagri­ nėjimas pirmose dviejose šios esė dalyse buvo iš principo siejamas su epistemologinėmis ir metodologinėmis problemomis, tai relevantišku­ mo temos eksplikavimas sietinas, kaip bandėme 78 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija mingai surikiuotos ypatinga prasme (Husserl 2005; 193). parodyti šios dalies pradžioje, dar ir su konkrečio­ sios ontologijos problemomis. Fenomenologija, kitaip negu natūralistiniai analitiniai mokslai, faktą ir esmę ne atskiria, o su-saisto: faktas ir esmė esti neperskiriami. Nebus per daug pakartoti. Susisaistymo problema atgręžia mus ne tik į prasmės forma­ cijas socialinio veiksmo analizės – subjektyvumo ir racionalumo sankirtų – lygmenyje, bet ir į konkrečios ontologinės pirminės tikrovės, gyve­ nimo-pasaulio, formacijas. Socialinio veiksmo ir gyvenimo-pasaulio struktūrinio susisaistymo tyrimai yra relevantiškumo istoriniame socio­ kultūrinio priešduotiškumo horizonte tyrimai. Relevantiškumo dalyko tematizavimas impli­ kuoja konkrečius prasmingų pasauliškumo struktūrų praktinio konstituavimo (tikrovės pasirodymo reikšmingais ko nors ir kam nors atžvilgiais gyvenimo-pasaulio struktūrose) tyrimus. Tai filosofinės sociologijos tyrimų sritis: „Filosofinės sociologijos požiūriu, fenomenolo­ ginė gyvenimo-pasaulio struktūrų analizė, turi išsaugoti būtinąsias priešsąlygas („the necessary preconditions“)“ (Schutz 1966; 132). Filosofi­ niu antropologiniu požiūriu, relevantiškumo problema sietina su žmogaus inter-esų plačiąja prasme (inter-esse) tematizacija.18 Pagrindžiantieji patyrimo aktai tikrovę stei­ gia individualiai: kaip tai, kas randasi štai-šiame laikiniame sulaikyme, turinčiame savo trukmę ir savo tikrovės-turinį (Realitätsgehalt), kuris gali – savo esmės / būtiškumo (Wesen) atžvilgiu – buvoti ir kitame laikiniame sulaikyme. Tikrovės faktas, be to, steigiamas štai-šioje vietoje, ir gali – savo esmės / būtiškumo atžvilgiu – buvoti kitoje vie­ toje ir turėti kitą pavidalą. Kalbant universaliai, individualus bet kokios rūšies buvojimas esti “atsitiktinis“ („zufällig“). Bet šio atsitiktinumo, vadinamo faktiškumu, prasmė yra ribojama sąsajos su būtinybe (Notwendigkeit), susijusia su ne tik de facto egzistavimu pagal erdvės-laiko faktų koordinavimo taisykles, o su eidetinio universalumo sąsajomis (žr. Husserl 1976a; 12–3; § 2). Faktinės monados sferoje ir kiekvienoje sferoje, kurią sudaro galimybė kaip įmanoma ideali esmė, –pažymi Husserlis, – randasi visos problemos, susijusios su atsitiktiniu faktiškumu, mirtimi, likimu, žmogaus autentiško gyvenimo galimybėmis, kurios numanomos kaip pras­ Aš-nukreiptumas, motyvacija, domesys (inter- esse) ir dėmesingumas esti keturi pagrindiniai sudedamieji relevantiškumo elementai. Kalbant 79 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 mes turime skverbtis į atvirą tipiškumo horizon­ tą.“ (Schutz 1962; 9–10) glaustai, mes matome, – sintetinės struktūros faktus, suteikiančius noetinį-noeminį vienumą (Husserl 2005; 53, – ieškodami ko nors. ir santvarkos. „In­ tencionalaus išgyvenimo esmė yra „būti ko nors suvokimu““ (ten pat). Esti, kaip pažymi Schutas tęsdamas fenomenologines Edmundo Husserlio ir Williamo Jameso nuorodas, įvairūs prasminio susietumo, „baigtinių prasmės provin­ cijų“, regionai, kuriuos saisto relevantiškumo, kylančio iš „motyvų“ ir „interesų“ (plačiąja prasme), sistemos: Matome ko nors ieškodami, o ieškome kuo nors domėdamiesi. Ne tik aktyvioje, o ir pa­ syvioje sintezėje. Kaip tik todėl panašų (nors ir ne šį čia esantį) daiktą esame jau anksčiau matę (Husserl 2005; 138). Taigi: domėdamiesi fenomenologijos ir socialinių mokslų tarpusavio ryšiais, ieškome dviejų jau nagrinėtų dimensijų – racionalumo ir subjektyvumo – papildinių. Vėl gręždamiesi (fenomenologija yra Zickzack; Cairns 1976), – ir drauge buvodami mąstymo-laikyse­ noje, kurios esmė apima galimybę kaip teigimo gebėjimą „Ich kann“ (Husserl 1974; 314), – į jau iš dalies išskleistas racionalumo ir subjektyvumo temas, įžiūrime panašų daiktą, kurį esame jau anksčiau matę, – relevantiškumą. „Žmogus randa save bet kuriuo kasdienio gyvenimo momentu biografiškai apibrėžtoje si­ tuacijoje, t.y. jo paties apibrėžtoje fizinėje ir so­ ciokultūrinėje aplinkoje, kurioje šis žmogus turi savo padėtį; ir ne tik turi padėtį fizinėje erdvėje ir išoriniame laike, ne tik turi statusą ir vaidme­ nį socialinėje sistemoje, bet turi ir savo moralinę bei ideologinę padėtį. Sakyti, kad šis situacijos apibrėžimas yra biografiškai apibrėžtas, – tolygu sakyti, kad šis apibrėžtumas turi savo istoriją. Tai yra ankstesnio žmogaus patyrimo, organizuoto įprastomis parankinio žinojimo atsargų turėji­ mo formomis, sedimentacija, – tai unikalus tu­ rėjimas, duotas šiam žmogui ir tik šiam žmogui. Šioji biografiškai apibrėžta situacija apima tam tikras paskesnes praktinės arba teorinės veiklos, trumpai apibūdintinos „parankinių tikslų“ var­ du, galimybes. Būtent šie parankiniai tikslai apibrėžia tuos elementus tarp visų kitų (kuriuos apima šioji situacija), kurie yra relevantiški šių tikslų atžvilgiu. Relevancijų sistema savo ruožtu apibrėžia, kokie elementai turi būti generalizuo­ jančios tipizacijos substratu, kokie šių elementų bruožai ir kokie individualūs bei unikalūs kiti turi būti atrinkti kaip būdingi tipiniai elemen­ tai; t.y. relevancijų sistema apibrėžia, kaip toli Relevantiškumas, – šitaip paprastai apibrė­ šime čia skleidžiamą temą, – yra subjektyvus racionalumo „šešėlis“: jau anksčiau matytas subjektyvumo ir racionalumo dimensijų papildinys. Relevantiškumo struktūra yra pa­ radoksali. Viena vertus, relevantiškumas esti kaip esminė subjektyviai racionalaus polinkio – buvoti subjektu-pasauliui ir drauge buvoti objek­ tu-pasaulyje – struktūra. Kita vertus, esminės struktūros gyvenimo-pasaulyje yra objektyviai racionalizuojamos. Subjektyvios struktūros esti mūsų pačių nuo mūsų pačių savitai ati-tolinamos / už-slepiamos.19 Prasmė „objektyvus pasaulis“, – atkreipia mūsų dėmesį Edmundas Husserlis, – 80 Sociologija. 20 Žr. Husserlio Krizės trečiosios dalies A pavadinimą: „Kelias į fenomenologinę transcendentali­ nę filosofiją užklausiant iš priešduoto gyvenimo-pasaulio“ („Der Weg in die phänomenologische Transzendentalphilosophie in der Rückfrage von der vorgegebenen Lebenswelt aus“). ir santvarkos. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija konstituojasi daugeliu pakopų (Husserl 2005; 132). drąsiai galime nuimti kabutes. Nes tai ne tik graži metafora, bet ir subjektyvus mokslinio racionalumo įgyvendinimas šiuo-laikiškume. Subjektyvus racionalumo šešėlis yra tamsi (dar- ne-įsisąmoninta) šviesios (jau-įsisąmonintos) logikos pusė. Štai kodėl nuo mūsų tolsta, o ypač formuo­ jantis šiuo-laikinam objektyvumo kultui, pati pirmoji objektyvumo forma, inter-subjektyvumas kaip pirmoji bendruomeninė tikrovė, kurią susieja relevancijos, „matomais, bet nepastebimais“ ryšiais grupuojamos pagal esminio rūpesčio, – žmogaus baigtinumo ir mirties neišvengiamu­ mo, – saistomas linijas. Juo labiau pirminio objektyvumo linijos nuo mūsų tolsta, tuo labiau primirštame, kad bet koks objektyvus logiškumas turi „subjektyvumo“ sąsajas kaip intencionalumo konstituacijas (Husserl 1974; 38), kad relevantiškumas iš esmės yra „subjektyvi“ sąsaja („subjektives“ Korrelat). O tai reiškia es­ minį ati-tolimą nuo mums priešduotų baigtinių tikrovės provincijų ir vienpusišką objektyvumo sušiuolaikinimą. Dvi, subjektyvią ir objektyvią, puses turinti, tačiau šio dvilypio aspekto dar neįsisąmoninusi, neaiški (unklar) mokslų idė­ ja randasi, kaip atkreipia dėmesį Edmundas Husserlis, šiuo-laiku: kaip teorijos, vienpusiškai nukreiptos griežto objektyvumo link, pavidalas (ten pat; 40). Anksčiau minėti keturi kompleksiški isto­ riniai intersubjektyvumo – mes-santykių, amži­ ninkų, pirmtakų, įpėdinių pasaulių – lygmenys sudaro antropologinius žmogiškos būklės pa­ grindus. Šiuose konstituaciniuose pa-grinduose išsiskiria mes-santykių pirmumas: lytėjimo, arba intersubjektyvumo patyrimo, dar prieš filosofinį ir mokslinį palytėjimą, pirmumas. Nebus per daug įkyru pakartoti: mus gerai pažįstamos prasmių figūros tipiškai struktūrizuojamos priešduotoje socialinėje-ir-gamtinėje, arba pažįstamumo-ir- anonomiškumo, aplinkoje.20 Sąryšiai yra intersubjektyvūs ap-linkoje, pa-saulyje, gyvenimo-pasaulyje, (Lebenswelt; environment, surrounding world, world; Schutz 1996; 189). Tai, ką turime po ranka (pasaulį po saule!), – „Ar mūsų filosofinės situacijos beviltiškumas galiausiai neišplaukia iš fakto, kad šių meditacijų spinduliuojami impulsai prarado savąjį pirmapradį gyvastingumą, prarado jį dėl to, kad išnyko filosofinės saviatsakomybės (Selbstverantwortlichkeit) radikalizmo dvasia?“ (Husserl 2005; 12), – yra atpažįstamas, – mūsų išgyvenamu patyrimu, – habitus. Spinduliuo­ jantis (asocijuojantis, tipizuojantis, saistantis) pa-saulis: daugis pagal santykį steigiamoje sąmo­ nėje (Husserl 1939; 174). Taigi relevantiškumo Logika kaip visų mokslų mokslas (ten pat; 36) ir mokslinio „racionalumo ir t.t.“ įgyven­ dinimas apima dvi kryptis – subjektyvią ir objektyvią. Priešinga logikos tematizavimo veikla yra subjektyvi, susijusi su giliai paslėptomis su­ bjektyviomis formomis (ten pat; 38). Štai dabar nuo ankstesnio apibrėžimo – relevantiškumas esti subjektyvus racionalumo „šešėlis“ – jau 81 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija problema yra štai-čia po ranka, o štai-dabar jau mano rankose. 21 „Kollektives Zusammennehmen und beziehendes Betrachten“. Husserlis pasitelkia būdingą pa-sauliš­ kumo atžvilgių pavyzdį: mano žvilgsnis (Blick) „slysta daiktais“ ant stalo, – rašaline-knyga-pypke- plunksnakočiu ir t.t. (ten pat). 22 ir santvarkos. Bendruomeniška atodaira ir atžvilginis įsižiūrėjimas21 į šį daugį pagal santykį steigiamoje sąmonėje jokiu būdu nereiškia, kad pakanka tik jungti vienus objektus su kitais, o paskui – juos susumuoti (kaip marionečių raci­ onalisto rankose visetą). Tai, ko reikia, Husserlis vadina interesais plačiąja prasme (ten pat; 175). kad viskas, – mėnulis, net ir saulė, taigi ir in­ ter-esse, – meta savo šešėlius, relevancijas, kurios neturi savaiminės mokslinės vertės. Kalbant universaliai, kam nors mesti šešė­ lį – lygu ką nors užtemdyti tam tikroje vietoje ir tam tikru laiku. Kalbant šiuo-laikiškai, kam nors mesti šešėlį – lygu „mesti iššūkį“: ką nors suviskam patraukti iš savo sąmonės lauko dėme­ singumo židinio. Tiek vienu, tiek kitu atveju tai reiškia relevantiškai gręžtis nuo gręžiantis į. Štai-čia – dėmesio kam nors ir nedėmesingumo kažkam nors – perskyrose randasi struktūrinės relevantiškumo, arba domesio, linijos. Randasi kaip po ranka mūsų turimos, parankinės žino­ jimo atsargų – pasyviosios genezės ir aktyviosios genezės laukų sandūrose – istorijos. Tarp jų – fenomenologiškai grindžiamos metodologijos istorijos.22 p j p p Viskas, taigi ir gyvenimas (kas žino, gal net pa-saulis?), randasi iš motyvų, arba to, ką Edmundas Husserlis vadina inter-esse (do­ mesiu). Domesys yra tai, ką matematizuotas teorinis objektyvumas – vadinamieji pozityvūs mokslai – griežtai pašalina iš mokslinio pasaulio kaip tik „subjektyvumą“, kaip tik „jausmines kokybes“ („sinnlichen Qualitate“; ten pat; 41). Vadovaudamiesi dar natūralioje laikysenoje atrastu, o paskui „ir t.t.“ ištobulintu principu: 21 „Kollektives Zusammennehmen und beziehendes Betrachten“. Husserlis pasitelkia būdingą pa-sauliš­ kumo atžvilgių pavyzdį: mano žvilgsnis (Blick) „slysta daiktais“ ant stalo, – rašaline-knyga-pypke- plunksnakočiu ir t.t. (ten pat). 22 2 Relevantiškumas yra ir fenomenologiškai grindžiamos metodologijos susietumo galimybių – regi­ oninių laukų universaliai konkrečioje ontologijoje tarpusavio išsišakojimo (Husserl 2005; 192) – patvirtinimų išbandymas. Taip pat ir formaliųjų metodologijos galimybių  – juo metodologija apibrėžtesnė, tuo tinkamesnė (relevantiškesnė) – išbandymas. Tai kūniškas (kalbant ne tik me­ taforiškai) subjektyvumo ir racionalumo sąmonės struktūrų išbandymas. (Racionalus vaizdas są- monėje randasi relevantiškame – subjektyvaus kinestezinio kūniškumo – patyrime. Išgyvenant savi­ duodamą aki-vaizdą.) Skirtini trys istoriški fenomenologiškai grindžiamos metodologijos išbandymo momentai. Pirma. Maxas Weberis savo supratumo metodologiją, tikslindamas jo aiškiai skiriamą subjektyviai numanomų ir objektyviai pagrindžiamų prasmių skirtį, kurios, anot jo, neartikuliuoja Georgas Simmelis (Weber 1922; 14), ieško griežtesnės metodologijos, kurios paskirtis – „priežas­ tingai aiškinti“ („ursächlich erklären“; ten pat; 15) supratingo socialinio veiksmo kryptis. Antra. 22 Relevantiškumas yra ir fenomenologiškai grindžiamos metodologijos susietumo galimybių – regi­ oninių laukų universaliai konkrečioje ontologijoje tarpusavio išsišakojimo (Husserl 2005; 192) – patvirtinimų išbandymas. Taip pat ir formaliųjų metodologijos galimybių  – juo metodologija apibrėžtesnė, tuo tinkamesnė (relevantiškesnė) – išbandymas. Tai kūniškas (kalbant ne tik me­ taforiškai) subjektyvumo ir racionalumo sąmonės struktūrų išbandymas. (Racionalus vaizdas są- monėje randasi relevantiškame – subjektyvaus kinestezinio kūniškumo – patyrime. Išgyvenant savi­ duodamą aki-vaizdą.) Skirtini trys istoriški fenomenologiškai grindžiamos metodologijos išbandymo momentai. Pirma. Maxas Weberis savo supratumo metodologiją, tikslindamas jo aiškiai skiriamą subjektyviai numanomų ir objektyviai pagrindžiamų prasmių skirtį, kurios, anot jo, neartikuliuoja Georgas Simmelis (Weber 1922; 14), ieško griežtesnės metodologijos, kurios paskirtis – „priežas­ tingai aiškinti“ („ursächlich erklären“; ten pat; 15) supratingo socialinio veiksmo kryptis. Antra. Alfredas Schutzas, gręždamasis į Husserlio fenomenologinių aprašymų išteklius, bando koreguoti, anot jo, pernelyg stiprų Weberio polinkį „priežastiniam aiškinimui“, paliekančiam nenuskaidrin­ tą, neaiškią numanomo supratumo (Schutz 1932; 247) sąvoką. Savo ankstyvojo veikalo pačioje pa­ baigoje Schutzas pažymi, kad Husserlio Formalioji ir transcendentalinė logika ir ypač Karteziškosios meditacijos iš dalies išpildo pažadą, siejamą su intersubjektyvaus pasauliškumo patyrimo pagrįstumo tyrimais (ten pat; 285). Tačiau paskesniuose savo tekstuose (Schutz 1962; 99–117; 140–9; Schutz 1966; 92–115) jis suabejoja eidetinės redukcijos metodo taikymo pirminėje natūraliosios laikysenos sferoje galimybėmis sutelkdamas dėmesį į intersubjektyvumo betarpiškumą ikipredikatiniame gy­ venimo-pasaulio patyrime. Atkreipdamas dėmesį į tai, kad Husserlis nebuvo susipažinęs su kon­ p g į y y j ( ) 23 Čia laikomės nuomonės (kad ir ypač žavėdamiesi Georgo Simmelio „impresionistine“ metodolo­ gija, kurią kritikuoja Maxas Weberis žengdamas supratumo ir aiškinimo galimo derinimo link), jog griežtą Maxo Weberio skirtį tarp subjektyviai numanomų prasmių ir objektyviai pagrįstų prasmių reikia saugoti (skirtį, kuri gyvybiškai svarbi pačios metodologijos, orientuotos į teorinių prin­ cipų išskleidimą, galimybei). Ne teorinis duotiškumas, o teorinis relevantiškumas: šitaip glaustai nusakytinas svarstomas dalykas. Teorinė refleksija į dienos šviesą kelia ne dalyką-kaip-duotybę, o dalyką-kaip-problemą, kurią reikia tęstinai skaidrinti: priešklausiant visus duotus, savaime supran­ tamus dalykus, kurie įprastai priimami kaip akivaizdos jų – aki-vaizdų – neužklausiant. Žr. 1951- ųjų Schutzo laišką Ericui Voegelinui: „Vasario mėnesį Leo Straussas perskaitė paskaitą, pavadintą „Maxo Weberio įnašą peržiūrint“, bendrame seminare Naujojoje mokykloje. Aš manau, kad jo požiūris sutampa su Tavo požiūriu. Jis aiškino, kad objektyvumas socialinių mokslų srityje yra neįgyvendinamas, kadangi vertybės implikuotos pačioje problemos atrankoje, pačioje analizuo­ jamoje medžiagoje ir taikomuose metoduose. Paskesnėje diskusijoje aš teigiau, kad privalu skirti relevantiškumą ir vertybes, kaip tai apibrėžė Weberis, – priešingu atveju probleminė diskusija virsta netiriamais klaidinančiais samprotavimais“ (Schutz 2011e; 136). ir santvarkos. Alfredas Schutzas, gręždamasis į Husserlio fenomenologinių aprašymų išteklius, bando koreguoti, anot jo, pernelyg stiprų Weberio polinkį „priežastiniam aiškinimui“, paliekančiam nenuskaidrin­ tą, neaiškią numanomo supratumo (Schutz 1932; 247) sąvoką. Savo ankstyvojo veikalo pačioje pa­ baigoje Schutzas pažymi, kad Husserlio Formalioji ir transcendentalinė logika ir ypač Karteziškosios meditacijos iš dalies išpildo pažadą, siejamą su intersubjektyvaus pasauliškumo patyrimo pagrįstumo tyrimais (ten pat; 285). Tačiau paskesniuose savo tekstuose (Schutz 1962; 99–117; 140–9; Schutz 1966; 92–115) jis suabejoja eidetinės redukcijos metodo taikymo pirminėje natūraliosios laikysenos sferoje galimybėmis sutelkdamas dėmesį į intersubjektyvumo betarpiškumą ikipredikatiniame gy­ venimo-pasaulio patyrime. Atkreipdamas dėmesį į tai, kad Husserlis nebuvo susipažinęs su kon­ 82 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija srityje: „Tu esi visiškai teisus, – rašo Alfredas Schutzas Ericui Voegelinui, – teigdamas, kad pirmojo elemento subordinacija antrajam yra vienas pražūtingiausių pozityvistinio požiūrio rezultatų; tai ypač taikytina požiūriui, kuris me­ todą traktuoja kaip „modelį-metodą“ (Schutz 2011e; 136). Antra, tai painiava mokslinių principų ir vertybių tarpusavio santykių srityje; painiava, kuri randasi dėl nesugebėjimo aiškiai skirti relevantiškumą ir vertybes bei jų implikaci­ jas socialinių mokslų metodologijai.23 Subjektyvumo ir racionalumo dimensijų eksplikavimas reikalauja ne tik apibrėžti savitus kiekvienos jų bruožus, bet ir aprašyti tarpusavio sąsajas. Žinoma, kad prieš-klausiant: kaip, t.y. kokiais būdais, galima aprašyti santykius tarp įvairių intersubjektyvumo lygmenų jau struktū­ rizuotame gyvenimo-pasaulyje? Mūsų patyrimo žinojimo atsargų – nuo-monių, nuo-sėdų, nuo-tykių – istorijos susideda iš priešistorijų: „Rodos, kad bet kokios tematizacijos prielaida yra tai, kad patyrimas, sudarantis šią temą, turi savo istoriją, susidedančią iš sedimentacijų“ (Schutz 2011c; 100). Ką dar svarbu suprasti aiškinantis painią relevantiškumo problemą? Kad relevantišku­ mas, – dalyko-kaip-problemos požiūriu, – galioja ne tik moksliniame, bet ir praktiniame pasau­ liuose. Relevantiškumas yra prieš-duotiškumo, susijusio su motyvais ir interesais, pasirodymas pa-tyrime kaip „tema“, reikalaujanti paskesnio, Ką visų pirma reikėtų suprasti aiškinantis painią relevantiškumo problemą? Visų pirma derėtų pripažinti, kad painiava jau yra. Pirma, tai painiava subordinacijos, – svarstant teorinio relevantiškumo ir metodo tarpusavio santykį, – krečiomis socialinių mokslų problemomis (Schutz 1962; 140), Schutzas pareiškia, kad abejotina, jog galima eidetiškai sugriebti („to grasp eidetically“) materialas sferas, arba būtiškumo regionus: „Ar galima, pasitelkiant tik laisvas variacijas vaizduotėje, sugriebti konkrečių rūšių eidosus <...>?“ Schutzas, mūsų nuomone, pernelyg akcentuoja „nesėkmingus“ Husserlio mokinių pavyzdžius: Edith’os Stein ir Gerdos Walther „<...> naivų eidetinio metodo panaudojimą analizuojant soci­ alinių santykių, bendruomenės ir valstybės problemas <...>“ (ten pat; 140–1). krečiomis socialinių mokslų problemomis (Schutz 1962; 140), Schutzas pareiškia, kad abejotina, jog galima eidetiškai sugriebti („to grasp eidetically“) materialas sferas, arba būtiškumo regionus: „Ar galima, pasitelkiant tik laisvas variacijas vaizduotėje, sugriebti konkrečių rūšių eidosus <...>?“ Schutzas, mūsų nuomone, pernelyg akcentuoja „nesėkmingus“ Husserlio mokinių pavyzdžius: Edith’os Stein ir Gerdos Walther „<...> naivų eidetinio metodo panaudojimą analizuojant soci­ alinių santykių, bendruomenės ir valstybės problemas <...>“ (ten pat; 140–1). Čia laikomės po­ žiūrio, – beje, drauge su Alfredu Schutzu, – kad analizė, įgyvendinta transcendentalinėje sferoje, pagrįstai taikytina natūraliosios laikysenos sferoje (Schutz 1962; 149). ir santvarkos. Čia laikomės po­ žiūrio, – beje, drauge su Alfredu Schutzu, – kad analizė, įgyvendinta transcendentalinėje sferoje, pagrįstai taikytina natūraliosios laikysenos sferoje (Schutz 1962; 149). p g į y y j ( ; ) 23 Čia laikomės nuomonės (kad ir ypač žavėdamiesi Georgo Simmelio „impresionistine“ metodolo­ gija, kurią kritikuoja Maxas Weberis žengdamas supratumo ir aiškinimo galimo derinimo link), jog griežtą Maxo Weberio skirtį tarp subjektyviai numanomų prasmių ir objektyviai pagrįstų prasmių reikia saugoti (skirtį, kuri gyvybiškai svarbi pačios metodologijos, orientuotos į teorinių prin­ cipų išskleidimą, galimybei). Ne teorinis duotiškumas, o teorinis relevantiškumas: šitaip glaustai nusakytinas svarstomas dalykas. Teorinė refleksija į dienos šviesą kelia ne dalyką-kaip-duotybę, o dalyką-kaip-problemą, kurią reikia tęstinai skaidrinti: priešklausiant visus duotus, savaime supran­ tamus dalykus, kurie įprastai priimami kaip akivaizdos jų – aki-vaizdų – neužklausiant. Žr. 1951- ųjų Schutzo laišką Ericui Voegelinui: „Vasario mėnesį Leo Straussas perskaitė paskaitą, pavadintą „Maxo Weberio įnašą peržiūrint“, bendrame seminare Naujojoje mokykloje. Aš manau, kad jo požiūris sutampa su Tavo požiūriu. Jis aiškino, kad objektyvumas socialinių mokslų srityje yra neįgyvendinamas, kadangi vertybės implikuotos pačioje problemos atrankoje, pačioje analizuo­ jamoje medžiagoje ir taikomuose metoduose. Paskesnėje diskusijoje aš teigiau, kad privalu skirti relevantiškumą ir vertybes, kaip tai apibrėžė Weberis, – priešingu atveju probleminė diskusija virsta netiriamais klaidinančiais samprotavimais“ (Schutz 2011e; 136). 83 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 tęstinio pa-tyrimo. „Aš galiu“, „ir t.t.“ (ir t.t. iki paskesnio pastebėjimo) būdais, turinčiais pa-tvirtinamų sintezių pavidalus; sintezių, priklausančių sferai, kurią apibrėžia „Aš galiu“ (Husserl 2005; 72). tematizacijose – praktinio ir prasminio pasaulio patyrimo regionus.24 Štai kodėl dar prieš kon­ krečiau aptariant relevantiškumo tipus, verta su­ konkretinti skirtį tarp paskiro „analitinio fakto“ (užbaigto fakto pozityviose metodologijose) ir integralaus „fenomenologinio fakto“ (neužbaig­ to ir tęstinio fenomenologiškai grindžiamose metodologijose). Fenomenologinis faktas yra pa-tiriamas (tiriamas išgyvenant tiek kasdie­ niame, tiek moksliniame pasauliuose) tęstinai. Numanomas įvairiais būdais: fenomenologinis metodas reikalauja dėmesingumo ne objektams „patiems-sau“ (ne santykiškiems objektams), o objektams-kaip-numanomiems; tiesioginis apra­ šymas yra objektų per se aprašymas, o fenome­ nologinis aprašymas yra intencionalių objektų aprašymas (Cairns 1940; 9). Plg.: Darkart: relevantiškumas buvoja pasaulyje, kursai jau duotas, – turi savo pradžią bei pabaigą, ir kurio patį buvojimo būdą jau apibrėžia pasau­ lio duotiškumas (Gegebenheit der Welt; being-at- hand; Schutz 2011d; 53). Pasaulio buvojimo būdas mums relevantiškas paradoksaliai: tik galimai-mums relevantiški pasaulio „elementai“ didžiąja dalimi netampa problemiškais-mums dalykais, tačiau jie nuolat buvoja sąmonės suvokimo lauko „pakraščiuose“. Tai nėra laten­ tiškas, „giliai nugrimzdęs“, pa-tyrimas. 24 „Motyvo“ apibrėžimas orientuoja į dvi skirtingas situacijas. Viena vertus, kalbama apie dalykų, kurių siekiama, būsenas, numanomus tikslus. Tai „idėjos“, kurios skatina mus veikti, eiti priekin, įgyvendinti atitinkamus veiksmus: sakykim, „kad“ patikrintume pavojaus tikėtinumo – „gyvatės“ arba „susirangiusios virvės“ tamsiame kampe (Schutz 2011c; 102) – laipsnį. Šias situacijas Schut­ zas vadina „kad“ („in-order-to“) motyvais, kurie turi „pirminio relevantiškumo“ statusą paskesnių žingsnių atžvilgiu. Kita vertus, jau pradėjus įgyvendinti veiksmą, tą pačią situaciją galima perteikti remiantis „kadangi“ motyvų grandine: tarkim, man reikia lazdos, „kadangi“ noriu patikrinti štai tą tamsoje gulintį įtartiną daiktą. „Kad“ ir „kadangi“ motyvai – tai tik kalbiškai skirtingi būdai pasakyti vieną ir tą patį dalyką: „Pirmoji „kad“ sakinių grandinė logiškai ekvivalentiška antrajai „kadangi“ sakinių grandinei, nes abiem minėtais atvejais pirminis ketinimas („the paramount pro­ ject“) esti motyvaciškai relevantiškas numatant konkrečius žingsnius; tačiau konkrečių žingsnių įgyvendinimas yra „priežastingai relevantiškas“ siekiant norimų rezultatų“ (Schutz 2011c; 121). ir santvarkos. Galimi relevantiškumo „elementai“ – visur esantys Leibnizo „mažieji suvokiniai“ (Schutz 2011c; 100) – jau yra po ranka mūsų sąmonės pagavos laukuose. Jie „pasirengę būti mūsų sugriebti“: išnirti priešais mus dalyko-problemos formomis (ten pat; 107). Kitaip tariant, relevantiškumo „elementai“ pasirengę tapti esminiais motyvais formuojantis tipizacijoms, kurios savo ruožtu struktūrizuoja mūsų žinojimo atsargas. „<...> visos problemos yra susijusios viena su kita, visos teminės relevancijos formuoja sistemas, ka­ dangi visos motyvacinės relevancijos yra subjek­ tyviai patiriamos kaip planų sistemos, subordi­ nuotos konkretaus asmens gyvenimo-planui. Kadangi šios motyvacinių relevancijų sistemos apibrėžia ne tik teminių relevancijų sistemas, bet ir atitinkamas interpretacines relevancijas, pas­ tarosios irgi laikosi sisteminiame kontekste; tas pat taikytina tipizacijoms, kurios čia randasi iš atitinkamų mūsų žinojimo atsargų pažįstamumo laipsnių.“ (Schutz 1966 [1975]; 130) Relevancijos susieja „kad“ ir „kadangi“ mo­ tyvus, steigiančius – vidinio ir išorinio horizontų 84 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Kitaip sakant, relevancijos,  – Alfredo Schutzo čia skaidomos į motyvacines, temines ir interpretacines, – esti „šaknys“, iš kurių randasi konkretaus asmens gyvenimo-planai ir tikslingi veiksmų kasdienio gyvenimo pasaulyje pasirin­ kimai, – jie liktų mums ne-pažįstami (savaime suprantami), jei į dienos šviesą nekeltume relevantiškumo problemų. O kelti į dienos šviesą relevantiškumo problemas galime tik tuomet, jei tik ir kai tik įsižiūrime į gyvenimo-pasaulio struktūras. Įsižiūrėti (kiek čia galioja sangrąžinė dalelytė) – lygu pastebėti anksčiau tik neapi­ brėžtai matomas pasyvumo-aktyvumo skirtis, o drauge – priešduotas pradines aktyvaus forma­ vimo medžiagas, asociacijas, kaip pamatinius pa­ syviosios genezės principus, pagal esmės dėsnius įgyvendinamus sferoje, kurią Edmundas Husser­ lis vadina ego konstituacijos įgimtu a priori (žr.: Husserl 2005; 100–1; Husserl 1974; 314–26). ties, skausmo, netgi „skausmo pojūčio šešėlio“; Wittgenstein 1995; 277 [448]) atžvilgiu esti dvejopa, – a) daiktai daro mums poveikį, b) mes darome poveikį daiktams. Tai galima perteikti primityviomis, bet kaip tik todėl užtikrintomis „formulėmis“, sklandžiai galiojančiomis pasy­ vumo-aktyvumo skirčių paribiuose. Pirma. „Ir t.t.“: tai, kas mūsų pa-tyrime pagrįsta, išlieka pagrįsta ir paskui, at-eityje (galiojant ir tam tikrai atsitiktinumo išlygos galimybei: „iki paskesnio pastebėjimo“). Antra. „Aš vėl tai galiu padaryti“: tai, ką įgyvendinu, darydamas poveikį daiktams, galiu padaryti ir paskui, at-eityje.25 Pasyvumo-aktyvumo skirtis kviečia tirti, pir­ ma, pasyvų patyrimą, atskleidžiantį daugeriopus pasyviųjų sintezių (jokiu būdu ne „empirinių dėsningumų“, siejamų su „psichikos viduje veikiančiais gravitacijos įvaizdžiais“; Husserl 2005; 101) lygmenis, antra, nuskaidrinti aki- vaizdų saviduotiškumą, arba tikrovės-fenome­ nus, – daiktų-kaip-numanomų saviduotišumą arba nesaviduotiškumą (Cairns 1940; 9). O tai reikalauja refleksijos, o ne „tiesioginio“ stebėji­ mo. 25 Plg.: aktyvūs sprendiniai sukuria „mąstymo dalykus“ (Denkgegenständen), kategorinius darinius (Gebilden). Jų esmė apima galimybę kaip subjektyvų gebėjimą: „Aš galiu“ („Ich kann“). Aš galiu eiti ir eiti vis aukštesniais galimybių lygmenimis (kalbant idealiai, iki kartojimosi in infinitum). Nors aktyvus sprendinys nėra vienintelė sprendinio forma, tačiau tai yra originali forma, kurioje „sprendinys“ sau-duodamas originaliai (Husserl 1974; 275). 26 Žr., pavyzdžiui, metodologinę A. Schutzo relevantiškumo postulato traktuotę. Idealiųjų tipų for­ mavimas turi atliepti relevantiškumo principą, kuris reiškia: kartą socialinės srities mokslininko pasirinkta problema suponuoja referencijų schemą ir konstituoja diapozono, kuriame eksplikuojami relevantiški idealieji tipai, apimtį. (Schutz 1964; 18) 27 „Tikrovės-fenomeną“ Herbertas Spiegelbergas apibrėžia kaip ne tik „bet kokio daikto-savęsp-laiky­ seną“ (angl. „standing-on-its-own-account“ of any object) drauge su apibrėžta savęsp-vieta (a definite place of its own), bet ir kaip pilną fenomenalų objektą, pretenduojantį būti tikru (the full phenomenal object which claims to be real). Taigi: tikrovės-fenomenas yra fenomenas, kuris vienu ir tuo pačiu metu yra numanomai tikras (supposedly real). Tikrovės-fenomeną reikia skirti nuo „tik“ fenomeno, kuris nuo pradžios nepretenduoja būti tikru. Epistemologija turi skirti autentiškus tikrovės-feno­ menus (genuine reality-phenomena), aktualiai implikuojančius tikrovę, ir tik atrodančius, arba apgaulingus (sham) tikrovės-fenomenus, kurie yra tik tariamai tikri (Spiegelberg 1940; 86–7). ir santvarkos. Refleksijos, kurios akiratyje – santykis tarp „pasyvaus suvokimo“ ir „predikatinio sponta­ niškumo“ (Schutz 1966; 113). Tai „santykių įsižiūrėjimas“ (ten pat), kviečiantis nuskaidrinti vidinį suvokimo lauko horizontą ir išorinio horizonto tematizavimo galimybę.26 Bandyti intuityviai sugriebti gyvenimo- pasaulio struktūrą – lygu vienu ir tuo pačiu metu išlaikyti savo aktualiame ir potencialiame akiratyje šiuos relevantiškus (žr. Schutz 1966; 116) aspektus: 1) kad natūralioje laikysenoje mes buvojame tarp kitų, mūsų-porininkų, savo patyrimu išgyvendami kultūros ir visuomenės fenomenus, 2) kad mūsų laikysena kitų daiktų (plačiąja prasme: nuo jūros mūšos, akmens, medžio šakos, debesies krašto iki vilties, nevil­ 85 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Relevancijų regionų daugis sudaro heteroge­ nišką gyvenimo-pasaulio aplinką. Dar daugiau: relevancijų regionai savo ruožtu esti struktū­ rizuoti tam tikrais reikšmingumo, tiksliau, vertybių tvarkos lygmenų, atžvilgiais. Kaip tik todėl pati relevantiškumo analizės pradžia turi būti pakankamai aiški: susiejama su motyvacijų ir inter-esų (plačiąja prasme) eksplikacija. Orien­ tuojantis į dėmesingumo spindulio konstituavi­ mo – „gręžimosi į“ ir „gręžimosi nuo“ (Schutz 2011c; 95) – bruožus. Descartes’as, – atkreipia dėmesį Husserlis (1974; 288), – drauge su išorinio patyrimo kritika at­ veria kelią transcendentalinei fenomenologijai, tačiau klaidingai atkerta savo požiūrį nuo esmi­ nės patyrimo prasmės, – patyrimo kaip pirminio jau-saviduotiškumo (der Erfahrung als einer originalen Selbstgebung verdeckt). Fenomenolo­ gija kaip vienovė yra nekas kita, kaip mokslinė transcendentalinio subjektyvumo savimonė, kuri prasideda paprastais žingsniais, tačiau žingsnis po žingsnio atsigręžia į savo pačios „logines“ ištakas (ten pat; 280). Šį iš prigimties žmogišką, esminį atžvilgį Edmundas Husserlis (1939; § 22) apibrėžia kaip suvokimo-interesų kryptingumą (die Tendenz des Wahrnehmungsinteresses). Alfredas Schutzas, – orientuodamasis į Husserlio dėmesingumo spindulio funkcijų tyrimus (Schutz 2011c; 94), – atitinkamai gręžiasi į sąmonės lauką, struktūrizuotą į teminį branduolį, jo aplinkos horizontą ir vidinės trukmės „dabar“ (ten pat). Aiškinimas (Klärung) visada reiškia darymo akivaizdžiu modusą, implikuojantį sintetinį ke­ lią, vedantį nuo neaiškaus manymo į atitinkamą prieš-įvaizdinantį stebinį (Husserl 2005; 73). Kalbant glaustai, svarbu – trijų pagrindinių lygmenų analizė: 1) dėmesinga įžiūra (die be­ trachtende Anschauung), 2) eksplikacinis peržiū­ rėjimas (explizierende Betrachtung), 3) tam tikras papildomas lygmuo (Eine weitere Stufe), kurio interesai reikalauja ne tik eksplikuoti vidinį objekto horizontą, bet ir iškelti į dienos šviesą objektų bendra-buvojimą išoriniame horizonte (Husserl 1939; 112–6 ). Apibrėžimas ne nukentėtų, o, suvokimo požiūriu, galbūt nuskaidrėtų, jei šį esminį atžvilgį fenomeną vadintume tiesiog domesio kryptingumu. 28 Tai reiškia klausti: kokiais atžvilgiais nuo pačios pradžios yra aki-vaizdu, kad visi predikatai, ku­ riuos mes priskiriame daiktams, vadindami juos maloniais, gražiais, naudingais, praktiškai tinka­ mais, tobulais, gamtos mokslininko visiškai nesvarstomi (kaip vertybės, dorybės, tikslo-objektai, instrumentai, gėris-kam-nors ir pan.)? Kadangi viešpataujanti „apercepcija“ iš anksto determi­ nuoja tai, kas yra ir kas nėra natūralus-mokslinis objektas, gamta-mokslininkui nerūpi fenomeno­ loginė vienovė, universalios akivaizdų sąsajos, – jos „nepriklauso“ gamtai jo paties duota prasme (Ten pat; 2.) ir santvarkos. Plačiausia prasme šiame esminia­ me atžvilgyje skirtini du pradžios momentai: 1) nu-mananti sąmonė, kuri a priori gali būti tuščia, mananti-iš-anksto, ir 2) akivaizda kaip intencionalaus gyvenimo prieš-fenomenas, kuriame „daiktai“ pasirodo betarpiškame są­ monės saviduotiškume (Husserl 2005; 71–2). Šie trys lygmenys, kviečia gręžtis į akivaizdos priešduotį, aki-vaizdą, – tarp savęs susijusiose suvokimo-intereso kryptingumo sąsajose. Tiks­ liau, kviečia gręžtis ir į sąmonės saviduotiškumą kaip tikrovės-fenomeną,27 ir į vienu ir tuo pačiu 86 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija muzikos kūrinio sandaros pavyzdžiuose, yra kontrapunkto santykis (Schutz 2011c; 99): ke­ leto drauge-skambančių daugiabalsio kūrinio melodinių linijų dermė. Kontrapunkto santykis taikytinas visoms tikrovės kaip darnos galimybių tikslinguose intersubjektyvumo sąryšiuose for­ moms, įskaitant ir erdvinę-laikinę „visatą“ (uni­ versalaus „viseto“ porūšį), vadinama „gamta“. metu įgyvendinamas saviduotiškumo funkcijas. Remdamiesi principine fenomenologiškai grin­ džiamos metodologijos nuostata, – kad analizė, įgyvendinta transcendentalinėje sferoje, taikyti­ na ir natūraliosios laikysenos sferoje, – pačioje pradžioje glaustai pažymėsime: relevantiškumo problema atliepia Edmundo Husserlio univer­ salios teleologinės sąmonės struktūros funkcijų tyrimus. g Visų pirma, kaip pabrėžia Husserlis, reikia pažymėti, kad „gamta“ yra galimo patyrimo sfera: pasakymai „gamtos-mokslas“ (Naturwissenschaft) ir „patyrimo-mokslas“ (Erfahrungswissenschaft) esti sinonimai (Husserl 1952; 1). Gamtos- mokslo paskirtis – jokiu būdu ne ginčytinai apsiriboti savųjų „objektų“ arba predikatų, nurodančių į šiuos objektus, pasirinkimu. Tai, kas pagrindžia gamtos-mokslą, yra gamtos esmės idėja, kad ir numanoma. Atitinkamai ir sąmonė, kuri funkcionuoja ir kaip gamta-mokslinis paty­ rimas, ir kaip mąstymas, susijęs su gamta-moksli­ niu patyrimu, implikuoja esminę fenomenologinę vienovę: štai-ši sąmonė gamtai turi esmines sąsajas (wesentliches Korrelat). Už-davinys yra nuskai­ drinti šias sąsajas, – ir būtinai fenomenologinėje vienovėje.28 Fenomenologinis už-davinys – tarsi pasauliškumo valties subjektyvus kylis, kuris natūralistinėje socialinio gamta-mokslininko laikysenoje ne-egzistuoja: Sąmonės saviduotiškumo – aki-vaizdoje (vaizdo-saviduotiškume sąmonei) – funkcijos, kaip pabrėžia Edmundas Husserlis, yra visa apimančios, funkcionuojančios universaliose sąmoningumo-sąsajose (Husserl 1974; 168). Pavienė saviduotiškumo įgyvendinimo akivaiz­ da – dėl savo intencionalumo – „reikalauja“ („fordern“) paskesnių sąmonės gyvenimo visa­ pusiškumo sąryšių, „universalios teleologinės struktūros“ (universale teleologische Struktur; ten pat; 168–9), esmingai susijusios su vidi­ nių ir išorinių horizontų išsklaidos galimybių aibėmis. Pagrindinė epistemologinė problema, kurią reikia nuskaidrinti eksplikuojant šią gi­ luminę struktūrą, yra apodiktiškumo ir klaidos santykio problema (žr.: Husserl 2005; § 5–§ 7§; § 24–26). Kalbant apie relevantiškumą kaip teleologinį įsižiūrėjimą į socialinį tikrovės-fenomeną, inten­ cionalią intersubjektyvumo struktūrą, tinkamiau­ sia metafora, Alfredo Schutzo rasta tipiškuose „Kalbant metaforiškai, subjektyvumas yra esminis pasauliui kaip tikrumas (angl. as real) 87 Metodologija Sociologija. 29 „Tikrovė yra esminiai sąryšiai (Wesenszusammenhang; essential interconnection)“(Cairns 1976; 5). 30 Kalbant paprastai lietuviškai, relevantiškumas (vok. Relevanz; angl. relevance, arba pertinence; Schutz 1989b; 226) – tai to, kas po ranka (to, ką matau, bet dar nepastebiu) perėmimas į rankas (to, kas man dabar reikšminga). Man svarbu, kaip rašo Alfredas Schutzas Aronui Gurwitsch’iui, skirti, viena vertus, planingą pasaulį (kitaip sakant, planų, motyvų, uždavinių, tikslų tarpusavio sąsajas, – nes vien tik darymas, veikla, working, yra pernelyg siauras terminas, neįtraukiantis planų; Gurwitsch 2011; 224) ir, kita vertus, tiesiog kasdienio gyvenimo pasaulį (ten pat). „World of wor­ king“ (čia: „planingas pa-saulis“), – pabrėžia Schutzas, – yra netinkamas vokiško žodžio „Wirkwelt“ vertimas: „Tu, žinoma, visiškai teisus, kad tai neišverčiamo Wirkwelt surogatas“ (ten pat). Dau­ giareikšmis, prasmingai struktūriškas žodis Wirkwelt čia verčiamas kaip turiningas ir prasmingas lietuviškas žodis, tariamas – prieš tai giliai įkvėpus – lėtai iškvepiant: pa-saulis. ir santvarkos. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 religijos apibrėžimu, tačiau galėtų juo baigtis“ (Weber 2000; 7); „Galėsime steigti tikslią ter­ mino „racionalus“ reikšmę tik paskesnėse šio tyrimo dalyse“ (Schutz 1996; 123). Vis dėlto – kad ir dar neturėdami eksplikuojančio įsižiūrė­ jimo (explizierende Betrachtung) – ir galime, ir turime api-brėžti tai, ką Edmundas Husserlis vadina įsižiūrėjimu-į-vaizdą (die betrachtende Anschauung), arba intuityvia-žiūra, prieš-kie­ kvieną-eksplikaciją (vor jeder Explikation). Tai įsižiūrėjimas, nukreiptas daikto „kaip viseto“ (Gegenstand „im Ganzen“) link. Šis paprastas su-griebimas (Erfassung), arba su-vokimas (akies mirksnių visetas) ir įsižiūrėjimas (Betrachtung) esti žemiausias bendrojo, objektyvuojančio domesio-suvokimo įgyvendinimo (Auswirkung des Wahrnehmungsinteresses) krypties lygmuo (Husserl 1939; 114). tuo pačiu būdu, kuriuo valties kylis yra būtina pačios valties sąlyga. Valtis be kylio neatsiranda; tačiau pats kylis nėra valtis; kylis nėra ir vienin­ telė valties „priežastis“. Tai yra paprastoji sąlyga, kuri yra būtina pasi-rodant laivui.“ (Sokolowski 1970; 137) Esmingas teleologinis objektyvuojančio domesio suvokimo įgyvendinimas pa-saulyje, susietumo įžiūrėjimas po-saule, – šitaip lituani­ zuotume relevantiškumą kaip dalyką-problemą.29 Darkart: minėtų subjektyvumo ir racionalumo dimensijų skaidrinimas reikalauja įsižiūrėti ne tik į tyrimo dalyką, o į dalyką-problemą, for­ maliai susidedančią iš trijų esminių są-sajų (žr.: Schutz 2011e; 136): 1) už-duoties (Aufgegeben), 2) prieš-duoties (Vorgegebenen), 3) už-davinio (Fragestellung). Kitaip sakant, relevantiškumas yra domesio (motyvų ir inter-esse) per-davimo iš prieš-duoties į už-davinį problema: to, kas pasi­ rodo man kaip tema mano domesio akiratyje, kviečianti nuskaidrinti už-davinį.30 Relevantiškumas žemiausiame – domesio- suvokimo įgyvendinimo – lygmenyje yra paprasta ir nedalijama daikto įžiūra šio daikto tęstinių, tarpregimybinių pasirodymų jungčių vienovėje. Kalbant esmingai glaustai: polinkių aibė aš-esmi- link laikysenoje. Relevantiškumas – tai linksnių- visetas. Esminiai žmogaus polinkiai daugyje: buvojant-subjektu pasauliui ir drauge buvojant objektu pasaulyje. Ne tik polinkiai naudinin­ Štai-šis tyrimas negali prasidėti relevantiš­ kumo apibrėžimu, tačiau galėtų juo baigtis. Galėsime steigti tikslią termino „relevantiškas“ reikšmę tik paskesnėse šio tyrimo dalyse. Šiuo supratumo metodologijos reikalavimu privalome kaskart pradėti (kad tinkamai užbaigtume) savo tyrimus. Plg.: „Šis tyrimas negali prasidėti 88 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ko linksniui (utilitarizmo-liberalizmo gairės, arba materializmo esmės), o esminis polinkis inter-subjektyvumo linksniui, tariant tiksliu Edmundo Husserlio žodžiu, inter-esse (Husserl 1939; 93). Susietumas, arba relevantiškumas, yra būdingas socialinės tikrovės žodis. Štai-šio būdingo fenomenologinio žodžio, – rašo Aronas Gurwitsch’ius savo draugui ir metodologiniam relevantiškumo analizės porininkui Alfredui Schutzui, – ieškojau išsamiuose žodynuose ir „<...> atradau, kad aštuonioliktojo amžiaus pradžioje žodis relevance vartotas būtent mūsų vartojama prasme, o paskui išnyksta iš apyvar­ tos“ (Gurwitsch 1989; 225). 31 Dėl šio „kad“ motyvo („kad“ įsigytume griežtų ir tikslių „faktų sprendinių“) esame linkę užglais­ tyti tampančios tikrovės ir jos regimybių (linksnių viseto) tarpusavio santykį, o drauge – tampančių prasmių ir jų relevancijų tarpusavio santykį. „Griežtas“ metodas randa tikrą, arba „tikslią“, tikrovę, o atžvilgiai, arba regimybės, yra praleidžiami kaip „netikri“. Randantis griežtumui ir tikslumui randasi tai, kas neegzistuoja. „Faktų sprendiniai“ neegzistuoja (Schutz 2011e; 136). g j ų p g j ( ) 32 Neužbaigtas Schutzo tekstas „Refleksijos relevantiškumo problemos klausimu“, rašytas (nuo 1947-ųjų rugpjūčio iki 1951-ųjų rugpjūčio mėnesių) angliškai, rastas tarp kitų nepublikuotų filosofo ir sociologo tekstų. Tai I-oji suplanuoto, bet, deja, neužbaigto penkių dalių tyrimo, pava­ dinto Pasaulis kaip savaime suprantamas: natūraliosios laikysenos fenomenologijos link, dalis (Schutz 2011c; 93). ir santvarkos. sąsajų laukus, tačiau visus inter-subjektyvumo brūkšnelius esame linkę užglaistyti griežtais „faktų-sprendiniais“. Idealizuoti, tipologizuoti, katalogizuoti: žinau, kad žinau, kad žinau...31 Aiškumo dėlei relevantiškumo problemą suskaidykime į dvi tarp savęs susijusias dalis: formalią ir neformalią. Nors relevantiškumas natūralioje laikysenoje yra ne-formali problema, tačiau, analizės požiūriu, pilna relevantiškumo dimensija apima dvi – formalią metodologinę ir neformalią antropologinę – sudedamąsias. Kalbant formaliai, relevantiškumo dalykas- problema kreipia mus į dviejų horizontų – iš­ orinio ir vidinio – link. Jųdviejų neapibrėžtume turime įžiūrėti ir eksplikuoti apibrėžtumo struktūras (Husserl 2005; 58). Tariant Alfredo Schuzto, relevantiškumo problemos išskleidi­ mo artikuliuota forma pirmtako,32 žodžiais, šis dalykas-problema – tai sąmonės dėmesingumo spindulio funkcijų tyrimas: Galima spėti: galbūt relevantiškumas iš­ nyksta iš apyvartos, viena vertus, traukiantis į paribius filosofinei antropologijai, o, kita vertus randantis socialiniams mokslams, jų tipologi­ nėms sistemoms ir tipų katalogams, kalbant tiksliau, randantis pozityvistiniams modeliams- metodams (dažniausiai girdima klišė, – rašo Ericas Voegelinas, – yra „griežti metodai“, nors visiškai neaišku, ką reiškia griežtas žodis „griežtas“; Voegelin 2011; 143)? Nors „kasdie­ nio gyvenimo pasaulis nuo pačios pradžios yra inter-subjektyvus“ (Schutz 1962; 312), nors parankines problemas tik perėmę į savo rankas, jau steigiame relevantiškumo ir nerelevantiškumo „Pirmasis mūsų analizės objektas yra sąmonės laukas, kiek šis laukas struktūrizuotas į tema­ tinį branduolį, kuris laikosi priešais aplinkinį horizontą, ir yra duotas kiekvieno „dabar“ vi­ dinėje trukmėje. Husserlis tiria „dėmesingumo spindulio“ funkcijas, atskleidžiančias tematinio branduolio konstituaciją ir viso lauko struktūri­ zaciją.“ (Schutz 2011c; 95) 31 Dėl šio „kad“ motyvo („kad“ įsigytume griežtų ir tikslių „faktų sprendinių“) esame linkę užglais­ tyti tampančios tikrovės ir jos regimybių (linksnių viseto) tarpusavio santykį, o drauge – tampančių prasmių ir jų relevancijų tarpusavio santykį. „Griežtas“ metodas randa tikrą, arba „tikslią“, tikrovę, o atžvilgiai, arba regimybės, yra praleidžiami kaip „netikri“. Randantis griežtumui ir tikslumui randasi tai, kas neegzistuoja. „Faktų sprendiniai“ neegzistuoja (Schutz 2011e; 136).l g j g j 32 Neužbaigtas Schutzo tekstas „Refleksijos relevantiškumo problemos klausimu“, rašytas (nuo 1947-ųjų rugpjūčio iki 1951-ųjų rugpjūčio mėnesių) angliškai, rastas tarp kitų nepublikuotų filosofo ir sociologo tekstų. Tai I-oji suplanuoto, bet, deja, neužbaigto penkių dalių tyrimo, pava­ dinto Pasaulis kaip savaime suprantamas: natūraliosios laikysenos fenomenologijos link, dalis (Schutz 2011c; 93). 89 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Yra ir kita – neformalioji antropologinė – relevantiškumo problemos pusė. 33 Žr. ypač Husserlio Erfahrung und Urteil § 83 a) dalį: „Die Gewinnung der empirischen Begriffe aus der Typik der natürlichen Erfahrungsapperzeption“ (Husserl 1939; 331–3). 34 Plg.: Ich und Umwelt; ego and surrounding world. Ir dar: tiksliau vartoti sąvoką natūralioji laikysena (vok. Einstellung, angl. attitude), o ne sąvoką natūralioji nuostata (kaip tik psichologinę nuostatą, siejamą su vadinamuoju „vidiniu patyrimu“). 33 Žr. ypač Husserlio Erfahrung und Urteil § 83 a) dalį: „Die Gewinnung der empirischen Begriffe aus der Typik der natürlichen Erfahrungsapperzeption“ (Husserl 1939; 331–3). 34 Plg.: Ich und Umwelt; ego and surrounding world. Ir dar: tiksliau vartoti sąvoką natūralioji laikysena (vok Einstellung angl attitude) o ne sąvoką natūralioji nuostata (kaip tik psichologinę nuostatą 33 Žr. ypač Husserlio Erfahrung und Urteil § 83 a) dalį: „Die Gewinnung der empirischen Begriffe aus der Typik der natürlichen Erfahrungsapperzeption“ (Husserl 1939; 331–3). aus der Typik der natürlichen Erfahrungsapperzeption (Husserl 1939; 331–3). 34 Plg.: Ich und Umwelt; ego and surrounding world. Ir dar: tiksliau vartoti sąvoką natūralioji laikysena (vok. Einstellung, angl. attitude), o ne sąvoką natūralioji nuostata (kaip tik psichologinę nuostatą, siejamą su vadinamuoju „vidiniu patyrimu“). 35 Schutzo relevantiškumo supratimą tinkamai papildo ontologinis Erico Voegelino požiūris į teoriją: „teorija“ – tai metodas, galintis tinkamai aprašyti supratingas transcendencijų patyrimo sferas. Teo­ rija turi būti pagrįsta. Tai reiškia, kad visos tipinės socialinių mokslų sąvokos turi būti grindžiamos ontologiškai: Maxo Weberio tipai stokoja būtent tokio pagrindo: „Aš įsižiūrėjau į Weberio sąvokų struktūrą, o taip pat į jo proginius prisipažinimus, sakykim, kad jis esąs „religiškai nemuzika­ lus“, – nors aš tiksliai nežinau, ką tai reiškia.“ (Voegelin 2011; 141–2) Weberio tipologija, anot Voegelino, neleidžia įtraukti be galo daug moksliškai „įdomios“ medžiagos. Būti teoriškai relevan­ tišku – tolygu būti susijusiu su mokymu apie žmogaus esmę (Wesenslehre), aristotelišku – episteme politike – žmogaus ir visuomenės mokslu (ten pat). p g p 36 Plg.: „Tipizacija priklauso nuo mano parankinės problemos, – apibrėžiant ir sprendžiant apie tai, kuris tipas turi būti formuojamas. Atitinkamai galima parodyti, kad bent vienas biografiškai ir situaciškai apibrėžtų interesų ir relevancijų aspektas yra subjektyviai patiriamas įprastose kasdie­ nio gyvenimo nuovokose, – veiksmų, daromų pasirinkimų, įgyvendinamų tikslų bei uždavinių motyvų sistemose.“ (Schutz 1962; 60) ir santvarkos. Schutzą domina savita socialinė Husserlio ateities-mu­ zikos – teleologijos – dalis, arba tai, ką jis vadina sudedamąja perspektyvų abipusiškumo teze: a) laikysenų tarpusavio-pasikeitimų idealizacija („The idealization of the interchangeability of staindpoints“), b) relevancijų sistemų suderi­ namumo idealizacija („The idealization of the congruency of the systems of relevances“) (Schutz 1962; 315–6). Relevantiškos žiūros židinyje – intersubjektyvumas, abipusiškas gręžimasis Tavęsp. Drauge su Husserlio fenomenologija. Kartu su Maxo Schelerio antropologija: joms kaip klausa akla spalvai, pagavos. Jausmi­ niai proto aspektai nėra grindžiami samprota­ vimu; juos suranda etika, nepriklausomai nuo logikos.“ (Schutz 1996; 137) Darkart verta pakartoti: relevantiškumas yra ne-formali problema, kurią sudaro dvi dalys: antropologinė (pilnesnė aprašomoji) ir metodologinė (formalesnė analitinė). Nors re­ levantiškumas, kalbant analitiškai, yra formalus metodologinis dalykas, tačiau fenomenologiškai skleidžiant problemą, ryškėja glaudūs relevan­ tiškumo, racionalumo ir subjektyvumo sąryšiai. Kalbant ne formaliai, o gyvenimo kaip gyve­ nimo sąsajų požiūriu, svarbiausias dalykas yra metodologinis gebėjimas nuskaidrinti empirinio supratumo radimosi iš tipizacijos natūralaus paty­ rimo-apercepcijose sąsajas.33 Dėmesingumo kaip aš-kryptingumo, aš-kryptingumo kaip „intereso“ (Aufmerksamkeit als Ichtendenz; Ichtendenz als „Interesse“; Husserl 1939; 84, 86) natūralioje laikysenoje sąsajas: aš ir ap-linkos, aš ir pa-saulio, aš ir po-saule (o tai yra viena ir tas pat).34 „Pasak Schelerio, Asmuo ir aktas priklau­ so neišskiriamai vienovei. Asmuo nėra tuščias veiksmo pradžios taškas. Asmuo egzistuoja ir gy­ vena tik įgyvendindamas intencionalius aktus. Tarp šių aktų esti jausmai, pirmenybės, meilė, neapykanta, žavėjimasis, pritarimas, atmetimas ir pan., kurie susiję su vertybėmis. Šie aktai yra ne tik vertybes-jaučiantys bet ir vertybes-at­ skleidžiantys aktai. Husserlis atskleidė, kad yra idealūs žinojimo objektai, kurie yra tokie pat nepriklausomi nuo pažįstančiojo subjekto, kaip kad realūs objektai. Scheleris pasitelkė šią įžvalgą kurdamas naują požiūrį toje srityje, kuri buvo neprieinama tiek Nietzsche’s reliatyvizmui, tiek Kanto formalizmui, t.y. atskleisdamas konkre­ čiųjų vertybių sritį. Vertybės, Schelerio požiūriu, yra intencionalių jausenų objektai (intentiona­ le Gegenstände des Fühlens), bet, kaip vertybės, visiškai skirtinos nuo pačių jausenų. Jos yra a priori, objektyvios, amžinos ir nekintančios. Jos yra duotos mums vaizdinyje ir per vaizdinio su­ vokimą. Suvokimo, kurio objektai yra vertybės, būdas ir amžina jų tvarka yra už intelekto, aklo Relevantiškumas yra laik-ysena. Laikiška, taigi subjektyvi pasauliškumo aprėptis dabar­ tiškumo galimybinių sąsajų akivaizdoje. Filoso­ finės saviatsakomybės (Selbstverantwortlichkeit) aprėptis (Husserl 2005; 12), kurios sociologinis idealas yra bendroji relevantiškumo teorija (Schutz 2011f; 155–69), apima a) įvairius eti­ kos dalykus, nagrinėjančius dorybės klausimus, b) įvairias santykiškai natūralias pasaulė-žiūras, buvojančias kasdieniame prieš-moksliname 90 Sociologija. 37 Pasaulis, kaip pažymi Husserlis, yra galimo patyrimo (möglicher Erfahrung) ir patyrimo-suvokimo (Erfahrungserkenntnis) daiktų pilnuma; daiktų, kurie atpažįstami remiantis aktualaus patyrimo pa­ ir santvarkos. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Teorija turėtų idealiai apimti kiekvieną lygme­ nį, išskleidžiamą nagrinėjant intersubjektyvu­ mo problemą, o taip pat idealiai jungti šiuos lygmenis ir eksplikuoti juos remiantis termi­ nais, kurie yra relevantiški socialiai veikiančiam asmeniui. Kiek relevancijų teorija svarsto šiuos įvairius intersubjektyvumo lygmenis jų svarbos veikiantiems asmenims atžvilgiu, galima sakyti, kad tai yra motyvacijų fenomenologija. Ji svars­ to įvairias intersubjektyvumo sritis remdamasi betarpiškais veikiančio asmens, įgyvendinančio savo veiksmus socialiniame pasaulyje, interesais ir motyvais.“ (Vaitkus 1991; 94). Teorija turėtų idealiai apimti kiekvieną lygme­ nį, išskleidžiamą nagrinėjant intersubjektyvu­ mo problemą, o taip pat idealiai jungti šiuos lygmenis ir eksplikuoti juos remiantis termi­ nais, kurie yra relevantiški socialiai veikiančiam asmeniui. Kiek relevancijų teorija svarsto šiuos įvairius intersubjektyvumo lygmenis jų svarbos veikiantiems asmenims atžvilgiu, galima sakyti, kad tai yra motyvacijų fenomenologija. Ji svars­ to įvairias intersubjektyvumo sritis remdamasi betarpiškais veikiančio asmens, įgyvendinančio savo veiksmus socialiniame pasaulyje, interesais ir motyvais.“ (Vaitkus 1991; 94). patyrime, c) pirmenybinio polinkio eksplikaciją, d) žmogaus veiksmo bendrąją motyvacijų teoriją (ten pat; 155–6).35 Relevantiškumo dalyko-problemos analizę pradėti dera intencionalaus ir tipizuojančio aš-akto horizontu (o ne anonimizuojančio tre­ čiojo asmens socialinio veiksmo priežastingumu, grindžiamu a priori tipologija): tai, kas patiria­ ma suvokiant objektą patyrimo procese, tuoj pat „apercepciškai“ („apperzeptiv“) priskiriama kiekvienam suvokiamam objektui, turinčiam panašių pirminio suvokimo (eigentlicher Wahrnehmung) komponentų (Husserl 1939; 399). Pradžia, kaip teigia Husserlio veikalo Erfahrung und Urteil (Patyrimas ir sprendinys) § 83, yra empirinis-tipiškas bendrumas ir pasyvi jo prieškonstituacija („Die empirisch-typische Allgemeinheit und ihre passive Vorkonstitution“). Domesys yra aš-kryptingumas. Štai kodėl rele­ vancijų teoriją Stevenas Vaitkus pagrįstai vadina motyvacijų fenomenologija36: Pradėti reikia pa-tyrimu, išgyvenamo pa­ tyrimo tyrimu, o ne „empiriniu tyrimu“, t.y. idealiųjų tipų tyrimu. Einant abiem vienalaikiš­ komis nuo-savybės intencionalumo kryptimis: ir link savęs, ir nuo savęs, ir savęsp, ir Tavęsp. Nes svarbu atidžiai tirti kelią, kuris nuo pasyviai prieškonstituotų tipizacijų mus veda empirinio supratumo, o paskui ir empirinių sąvokų link. Ir ne tik kasdienių sąvokų, bet ir aukštesniojo lygmens empirinių mokslų sąvokų (empirischen Wissenschaften) link.37 Pradėti dera pa-tyrimu, rodančiu ir savęsp, ir už savęs, todėl turinčio „Relevancijų teorija iš esmės yra pastanga sukurti integruotą intersubjektyvumo teoriją. 37 Pasaulis, kaip pažymi Husserlis, yra galimo patyrimo (möglicher Erfahrung) ir patyrimo-suvokimo (Erfahrungserkenntnis) daiktų pilnuma; daiktų, kurie atpažįstami remiantis aktualaus patyrimo pa­ 91 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 savitą habitus: „tuščio pavidalo intereso“ kryp­ tingumą, užpildomą paskesniuose „aš-darybos“ būduose to, kas esti patiriama (Husserl 1939; 85–6). grindu (die auf Grund aktueller Erfahrungen). Natūralioje laikysenoje visi mokslai, – tiek mate­ rialaus, tiek psichofizinio pobūdžio (fiziologija, psichologija ir pan.), yra vadinamieji natūralieji mokslai (Naturwissenschaften), įskaitant istoriją, kultūros mokslus, įvairių rūšių sociologines disci­ plinas (Husserl 1976a; 11). Ir universalus mokslų susietumas natūralioje laikysenoje, ir fenome­ nologinių mokslų susietumas (Husserl 2005; 190) rodo, kad skuboti yra tie teiginiai, kurie teigia, kad filosofija, įskaitant ypač fenomenologiją, „sąmonėja“ tuomet, kai „atsiskiria nuo kitų mokslų, pavyzdžiui, sociologijos“ (žr.: Sverdiolas, Kačerauskas 2008; 18). grindu (die auf Grund aktueller Erfahrungen). Natūralioje laikysenoje visi mokslai, – tiek mate­ rialaus, tiek psichofizinio pobūdžio (fiziologija, psichologija ir pan.), yra vadinamieji natūralieji mokslai (Naturwissenschaften), įskaitant istoriją, kultūros mokslus, įvairių rūšių sociologines disci­ plinas (Husserl 1976a; 11). Ir universalus mokslų susietumas natūralioje laikysenoje, ir fenome­ nologinių mokslų susietumas (Husserl 2005; 190) rodo, kad skuboti yra tie teiginiai, kurie teigia, kad filosofija, įskaitant ypač fenomenologiją, „sąmonėja“ tuomet, kai „atsiskiria nuo kitų mokslų, pavyzdžiui, sociologijos“ (žr.: Sverdiolas, Kačerauskas 2008; 18). ir santvarkos. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 taip formuluojamas: jokia nuomonė negali būti priimama kaip filosofinis žinojimas tol, kol šis žinojimas neįmatomas kaip adekvačiai steigtas stebinyje to, kas matoma kaip saviduotiškumas „asmenyje“. Bet koks įsitikinimas, kuris nesu­ derinamas su tuo, kas matoma saviduotiškume, turi būti atmetamas.“ (Cairns 1940; 4) „Mes galėtume traktuoti kiekvieną išraišką, esančią už skliaustų, kaip „referencijų sistemą“ tiems, kurie lieka skliaustuose. O tai, ką mes ketiname suskliausti, ir tai, ko suskliausti neke­ tiname, yra funkcijos dalyko, kurį savo žodyne vadinu „relevancija“.“ (Schutz 1989; 114) Tiksliau, galėtume siekti tiesiogiai „įmatyti“ motyvacijų ir interesų sąsajų gyvenimo-pasauly­ je, arba relevancijų, esmiškus bruožus. Irrealių (irrealer) daiktų, t.y. daiktų, kurie yra idealūs plačiausia prasme, akivaizda, kaip pabrėžia Husserlis, esti analogiška įprasto vidinio ir išorinio patyrimo akivaizdai. Patyrimo, kuris, gręždamasis į ne kokius nors kitus, o prie-tarų (Vorurteils) pagrindus, įgyvendina pirminę objektyvaciją. Ko nors idealaus identitetas, o todėl ir objektyvumas, gali būti tiesiogiai įmatomas, tiesiogiai išgyvenamas patiriant (jei norime suteikti šiam žodžiui tinkamą išplėstinę prasmę: direkt zu erfahren; Husserl 1974; 163). Taip pat žr.: Šie Husserlio ir Cairnso apibrėžimai labai svarbūs siekiant per-eiti iš I-oje šios esė dalyje svarstytos racionalumo, o II-oje – subjektyvumo, problematikos prie filosofinės relevantiškumo problemos ištakų. Ten, kur aki-vaizda pasirodo „susidvejinusi“: kaip tikrovė ir tikrovės fenome­ nas. Juk vienas dalykas yra matyti, išgyventi, patirti, turėti ką nors suvokimo lauke, bet visai kitas dalykas yra dėmesingai įgyvendinti suvo­ kimo aktą specifine prasme, „gyventi“ („leben“) matant specializuota „tikėjimo“ („glaubend“) prasme ir įgyvendinti sprendimo aktą (Husserl 1952; 5). Vienas dalykas yra apskritai nutuokti, kad dangus yra mėlynas, o kitas dalykas yra įgyvendinti teiginį, kad dangus – dabar mėlynas (ten pat). „Esminis metodologinis fenomenologijos principas, manau, pačioje pradžioje gali būti ši­ ir santvarkos. atsigręžimu į daiktą, bet daro jį tematiniu: kai aš tematiškai linkstu daiktan kiekvienas aš-akto polinkis – baigtinis arba tęstinis aš-atžvilgiu – buvoja inter-esse (Husserl 1939; 93). Kaip aš- esmi-link laikysena. Plg.: Šį savitą habitus vadintume aš-domesio- Tavęsp-esme (plg.: inter-esse; Husserl 1939; 93), relevantiško gręžimosi-į ir nerelevantiško gręžimosi-nuo laikysena. Tai laikysena, kurią Edmundas Husserlis vadina dėmesingu doksiniu po-linkiu (doxische Zuwendung), dėmesingu­ mu kaip aš-kryptingumu (Aufmerksamkeit als Ichtendenz), objektyvuojančiai išgyvenamu patyrimu, kuriame sąmonei pateikiamos „esmės“ (Seiendes). Esmės-domesys yra aš-kryptingumas intencionalaus daikto link. Po-linkis vienovei, kuri pasirodo tęstinai, – jos duotiškumo modusų išsklaidose (relevancijose), ir priklauso esminėms savito aš-akto (Ichaktes) tikslia šio žodžio prasme struktūroms (Husserl 1939; 85). „Husserlis tiria funkcijas to, ką jis vadina „dėmesingumo spinduliu“,  – konstituojančiu tematinį branduolį ir šitaip struktūrizuojančiu visą lauką. Bet kuriuo momentu esti daugelis patyrimų, besitęsiančių vienalaikiškai. Tai, kas sudaro šių laikiškai besitęsiančių ir vienalaikiškų patyrimų vieną (tiksliau, vieną įtemptą) temati­ nį lauką, yra faktas, kad aš laisvai atsigręžiu į juos arba apmąstau juos (vadinasi, tai yra aš-veika: tiek, kiek aš esu visų mano sąmoningo gyvenimo veikų šaltinis).“ (Schutz 2011c; 95) Relevantiškumas pasyviai glūdi toje „ma­ tomoje, bet nepastebimoje“ natūralioje laiky­ senoje: intencionalumo įvairovės neįmanoma tematiškai aprėpti nagrinėjant cogitata vien kaip aktualius išgyvenimus, nes kiekvienas išgyvenimas turi išgyvenimų horizontą, kuris modifikuojasi kintant sąmonės kontekstui (Husserl 2005; 56). Kitaip sakant, kiekvienas išgyvenimas turi „vidinį“ rūpesčių kontekstą, vienus daiktus tematiškai įtraukiantį į dėme­ singumo centrą, o kitus susiejantį su motyvų ir interesų „pakraščiais“. Ar galėtume ir kaip ga­ lėtume adekvačiai sugriebti šiuos horizontiškai kintančius „relevantiškumo“ ir „nerevantišku­ mo“ laukus? Darkart: Aš-kryptingumas patyrime įgyvendinamas kaip „interesas“ siauresne ir platesne sąvokos interesas prasme. Pirma, iš dalies kaip paprasta intuityvi trukmė suvokime, iš dalies kaip dar neapibrėžtų horizontų, susijusių su naujų praturtinimų galimybėmis ir lūkesčiais, žadi­ nimas (Pirmoji interesų samprata, kaip tikslina Schutzas, atliepia Leibnizo sąmonės kaip kryp­ tingų pastangų tęsti įgyjant naują patyrimą, apibrėžimą; Schutz 1996; 98). Antra, platesnė interesų samprata neapsiriboja tik paprasčiausiu grindu (die auf Grund aktueller Erfahrungen). Natūralioje laikysenoje visi mokslai, – tiek mate­ rialaus, tiek psichofizinio pobūdžio (fiziologija, psichologija ir pan.), yra vadinamieji natūralieji mokslai (Naturwissenschaften), įskaitant istoriją, kultūros mokslus, įvairių rūšių sociologines disci­ plinas (Husserl 1976a; 11). Ir universalus mokslų susietumas natūralioje laikysenoje, ir fenome­ nologinių mokslų susietumas (Husserl 2005; 190) rodo, kad skuboti yra tie teiginiai, kurie teigia, kad filosofija, įskaitant ypač fenomenologiją, „sąmonėja“ tuomet, kai „atsiskiria nuo kitų mokslų, pavyzdžiui, sociologijos“ (žr.: Sverdiolas, Kačerauskas 2008; 18). 92 Metodologija Sociologija. 38 Euklido geometrija ir antikinė matematika, kaip pažymi Husserlis, žino tik baigtinius uždavinius, baigtinai uždarus a priori. O modernioji matematika sugriebia šio uždavinio begalinę galimybę, kuri kaip savaime suprantamas dalykas susiejama su geometrinės erdvės samprata ir geometrijos kaip mokslo, priklausančio šiai erdvei, sąvoka. Šiai idealiai erdvei priklauso universalus, siste­ miškai koherentiškas a priori ir begaliniai savaime uždara – dėl jos begalybės – sisteminė teori­ III. 2. Relevantiškumo problemos filosofinės ištakos: aki-vaizdos problema „Dvasiškai būti su pačia būtybe veidu į veidą“ (Es-selbst-geistig-zu-Gesicht-bekommen). „Viską, kas gali būti filosofinės pradžios taškas, turime išsikovoti patys.“ (HUSSERL 2005; 18–9) „Pirminiai ir esminiai fenomenologijos ir epistemologijos uždaviniai, žinoma, nesutampa. Ir tai reikia pabrėžti vengiant fenomenologizmo, visą filosofiją ištirpdančio į fenomenologiją. Vis dėlto tarp šių dviejų sričių esti labai glaudūs ryšiai. <...> Pagrindinė problema – santykis tarp „tikrovės-fenomeno“ ir aktualios tikrovės.“ (SPIEGELBERG 1940; 84) (SPIEGELBERG 1940; 84) 93 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 „Eidetiniai tyrimai nagrinėja ne konkrečius realius daiktus, bet galimai vaizduojamus daiktus.“ „Mes vėlgi matome, kad eidetinis požiūris yra tik metodologinis įrankis, padedantis išspręsti specialų uždavinį. Fenomenologas, galima sakyti, tiria ne pačius daiktus; jis domisi jų prasme, – kaip šią prasmę konstituoja mūsų sąmonės aktai.“ (SCHUTZ 1962; 114-5; KURSYVAS AUTORIAUS) (SCHUTZ 1962; 114-5; KURSYVAS AUTORIAUS) „Nėra pakankamai aiškiai įsisąmoninta, kad kai kurios pagrindinės Europos minties sąvokos formavosi esant įtakingam, bet klaidinančiam įsitikinimui, kurį praėjusio amžiaus mokslinė pažanga pakoregavo tik iš dalies. Klaidingą įsitikinimą sudaro painiava tarp galimybės (potentiality) ir tikrovės (actuality). Tęstinumas susijęs su galimybe; o tikrovė – tik atomiška.“ (WHITEHEAD 1978; 61) Kalbant metodologiškai, dvi René Descartes’o aiškių, ryškių ir skaidrių minčių lygiagretės, tinkamai greti­ namos Samprotavime apie metodą, ir šiandieną tebereikalauja papildomų sprendinių: 1) „Vis dėlto neužtenka turėti sveiką protą (l’esprit bon), svarbiausia – mokėti jį gerai panaudoti (appliquer).“; 2) „Ypač didelį malonumą turėjau, domėdamasis matematikos mokslais, nes čia viskas tikra ir akivaizdu, tačiau tada dar nesuvokiau jų tikro panaudojimo, manydamas, kad jie padeda tik mechanikai. Aš stebėjausi, kad ant tokių stiprių ir solidžių pamatų nėra sukurta nieko išties didaus.“ ( SCA T S ) (DESCARTES 1978; 101–5). (DESCARTES 1978; 101–5). (DESCARTES 1978; 101–5). Artimai, ir netgi iki visiško tapatumo su­ gretinę ryškią sveiko proto aki-vaizdą ir aiškią sveiko mokslo aki-vaizdą, patiriame didelį – ir mažų mažiausiomis pastangomis – malonumą: akivaizdos be brūkšnelio. Išgyvename pa-tirdami (tirdami kūnu, o spręsdami protu: štai kaip dalyvaujama stebint!) grynąjį sugretinimą. Tik įžengiame pro pa-tyrimo vartus, o jaučiamės galį „<...> priversti mažiau išmanančius tavimi gėrėtis“ (ten pat; 104). 103) – gali išnykti. Jei akivaizdą poruojame su metodu arba akivaizdą ištęsiame apodiktiškai, apie tai, kas „egzistuoja“ tįsioje tiesėje, galime spręsti iš anksto, ir be akivaizdos malonumo. Modernusis Galileischen metodas yra ne kas kita, kaip akivaizdos (įsitikrinimo geometrinės tikrovės racionalistiniu vaizdiniu) ištęsimas. Be-atodairiškas ištęsimas, metafizinis tikrovės transcendavimas į „naująją matematinio natū­ ralistinio mokslo, galilėjiško, idėją („<...> neue Idee der mathematischen Naturwissenschaft: der Galileischen“; Husserl 1976b; 21).38i Tačiau, kaip tinkamai pažymi Descartes’as, akivaizdos be brūkšnelio malonumas – štai-da­ bar-VISKAS-AIŠKU „lyg paveiksle“ (ten pat; Paprastai (gaudant vaizdą akimis) arba rafi­ nuotai (užpildant vaizdą formulėmis) ištęsdami 94 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija faktus kaip socialinių mokslų objektus; priski­ riamų prasmių aktus arba interpretacines pras­ mes; žmogaus veiksmus arba žmogaus pastangų produktus. Bet kuriuo atveju vienas dalykas yra aiškus: visa tai, kas gali būti socialinio-mokslinio samprotavimo, siekiančio optimalaus aiškumo ir skaidrumo, objektu, jau yra protavimo kasdienia­ me pasaulyje aktai arba turiniai socialiniame pa­ saulyje, taigi santykiškai neaiškaus ir neskaidraus protavimo aktai arba turiniai. Kitaip sakant, žalia socialinio-mokslinio samprotavimo medžiaga yra būtinai, prasmės požiūriu, priešformuota, – remiantis prasmių formavimo aktais kasdie­ niame socialinio pasaulio gyvenime. Ir visa tai yra priešformuota neaiškiais būdais. Priešingai, mokslinis samprotavimas jokių priešsąlygų arba priešduočių negali priimti kaip paprastai duotų, kaip duočių, kurios nereikalautų paskesnio nu­ skaidrinimo. Todėl pirmasis socialinių mokslų tikslas yra maksimalus nuskaidrinimas ir ekspli­ kacija to, ką įprastai asmenys, gyvenantys sociali­ niame pasaulyje, mano apie šį pasaulį.“ (Ten pat.) tikėjimą akivaizda („Juk kodėl aš neturėčiau pa­ tikrinti savo akių, kad pažiūrėčiau, ar matau abi rankas?“; Wittgenstein 2009; 51 [125]; kursyvas autoriaus), mokomės tinkamai frazuoti kalbines iš-raiškas be patyrimo akivaizdos. Beatodairiškai netikrindami savo akių, matome, kad tuomet, kai akivaizdą taikome, – ir netgi grakščiai pran­ cūziškai (appliquer), – akivaizda, bent jau nuo Prancūzijos geometrinės revoliucijos laikų, „padeda tik mechanikai“, arba naudininko – utilitarizmo-liberalizmo – linksniui. Ir vėlgi, grįždami atgalios, t.y. saugodami kūno, kurį galimai veikia sąmonė, pusiaus­ vyrą, darkart patiriame malonumą teigdami (formaliai neprieštaringai), kad samprotauti moksliškai – lygu būtinai protauti aiškiai ir skaidriai. ja. Ši teorija, pradedama nuo aksiominių teiginių ir sąvokų, leidžia dedukciškai nedviprasmiškai konstruoti bet kokį suvokiamą pavidalą, brėžiamą erdvėje. Tai, kas geometrinėje erdvėje idealiai „egzistuoja“, iš anksto nedviprasmiškai nuspręsta visose jos determinacijose. Apodiktinis sampro­ tavimas, tįsdamas – per sąvokas, teiginius, išvadas, įrodinėjimus – begalybės link, tik „atranda“ tai, kas jau savaime egzistuoja kaip tiesa (Husserl 1976b; 21–2). (DESCARTES 1978; 101–5). Vis dėlto, – kai tik imame svarstyti, kokiu laipsniu štai ši akivaizda, įgyvendindama aiškumo ir skaidrumo funkciją, „<...> gali pre­ tenduoti į apodiktinį pobūdį, – mums tuojau kyla abejonės“ (Husserl 2005; 24). Bet kokio sąmonės gyvenimo-proceso (inten­ cionalumo) ir akivaizdos, t.y. sąmonės ko nors saviduotiškumo proceso, sąryšis, kaip tai žingsnis po žingsnio išskleidžia Edmundas Husserlis, yra esminis. Akivaizda – lygu intencionalus saviduo­ tiškumo įgyvendinimas (die intentionale Leistung der Selbstgebung; Husserl 1974; 166); buvojimas duotu betarpiškai akivaizdžiai – originaliter – modusu (Husserl 2005; 72). O tai reiškia, kad būtent čia, „šioje lemiamoje pradžios vietoje“ (ten pat; 21), turime medituodami skverbtis giliau, ir kelti esminį epistemologinį klausi­ mą, – ką reiškia kalbėti apie absoliučią garantiją, absoliutų neabejotinumą (ten pat)? Plg.: „Bet ką gi čia reiškia aiškumas (clarity) ir skaidrumas (distinctness)? (Schutz 1996; 122) Tai reiškia, kad mes visų pirma turime įsižiūrėti į socialinių mokslų dalyko – socialumo kaip intersubjektyvumo – sandarą, kuri „radikaliai skiriasi nuo gamtos mokslų dalyko sandaros“ (ten pat; 124). Kalbant trumpai, čia minimas radikalus skirtumas reiškia, kad socialinės srities mokslininkui, kitaip nei gamtinės sferos moks­ lininkui, duotis esti tik kelias į priešduotį: „Kalbant konkrečiai, galime turėti polinkį api­ brėžti žmogaus veiksmus arba žmogaus sąmonės 95 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Fenomenologijos kaip tikrai gyvybingo, nes ilgesingo (tikrovės galimų modifikacijų pasiro­ dymo tęstinės sintezės vienovėje) mokslo pradžia mūsų lūkuriuoja subjektyvumo paradokso – buvoti subjektu-pasauliui ir drauge buvoti objek­ tu-pasaulyje – sąsajose. Kaip tinkamai apibrėžia Josephas Kockelmans: „Fenomenologijos kaip subjektyvios grynosios logikos sąsajos pradžia“ (Kockelmans 1994; 101). O tai labai skiriasi nuo hipotezinių mokslų apie faktus ir apriorinių mokslų pradžios. Fenomenologijoje, kritiškai reflektuojančioje akivaizdos kaip akivaizdos įgy­ vendinimą (Leistung), priešduotas (vorgegeben) jutiminis patyrimas negali „lyg niekur nieko“ (!) būti pripažintas kaip apodiktinis akivaizdu­ mas; negali turėti absoliučiai pirmos akivaizdos privilegijos (Husserl 2005; 24). Atitinkamai natūralus įvykis, kurį reikia paaiš­ kinti, neįgyjamas mano privačiame pasaulyje, o įgyjamas intersubjektyviame pasaulyje, kuris prieinamas visiems mums.“ (Schutz 1996; 125) Trūkstant, kaip pažymi Husserlis, nuose­ klios fenomenologinės intencionalaus saviduo­ tiškumo įgyvendinimo formų analizės, „akivaiz­ da“ įprastai suvokiama kaip absoliutus apodik­ tiškumas (absoluten Apodiktizität), absoliutus užtikrintumas (absoluten Sicherheit). „Akivaizda“ susiejama su paskirais sąmonės aktais, atplėštais nuo konkrečios subjektyviai išgyvenamo (ne fragmentiškai patiriamo, o išgyvenamo tęstinai) konteksto vienovės (Husserl 1974; 165). Taigi čia, gūdžiausiuose tikrovės kaip tikro­ vės kampuose (tamsiuose kampuose, kuriuose daug aklų sąmonės aktų, vienišų vilkų, niauriai urzgiančių tampančioje regim-ybių tikr-ovėje), pasirodo bent dvi parankinės aš-kryptingumo galimybės: natūralistinė ir fenomenologinė. 39 Plg.: „Kasdienis gyvenimas  – įprasta tikrovė – yra „mūsų“ nuo pačios pradžios, t.y. visi kasdienio egzistavimo pasaulio elementai laikomi „tikrais“ Tau, kaip ir man, ir kiekvienam, įžengiančiam (DESCARTES 1978; 101–5). Pirma, akivaizda, – net ir toji, kuri pradžioje pasirodo kaip įtartinas netiesos krislas mano akyje, – gali tapti neabejotina, užtikrinta, netgi absoliučiai apodiktine (aiškiu faktu, tiksliu duo­ menimi, užtikrintu skaičiumi, įtikinama tiese, įsitikrinamu buvojimu) darbščiose mano ranko­ se: „<...> jei elgsena implikuoja kūno judesius, orientuotus į išorinį pasaulį, pavadinsime ją, atsižvelgdami į mūsų tikslus, „planinga veikla“ (angl. „working“)“ (Schutz 1996; 28). Antra, aš galiu išlaikyti dėmesingame akiratyje galimybę „dvasiškai būti su pačia būtybe veidu į veidą“, – tematizuotą filosofinę (nors filosofas – nelygu filosofui) tradiciją: „Atsiprašau, aš nežinau tiksliai, kas yra tikrovė“ (ten pat; 24).39 Įprastas suvokimas – įprastas, bet ne papras­ tas, o sudėtinis. Tai, rodos, paprastas, bet nelen­ gvai suvokiamas apibrėžimas: ir mūsų į-protis sudarytas iš daugelio kito pirmo samprotavimo įgūdžių (šių įgūdžių, – įskaitant ypač matymą, lytėjimą ir girdėjimą, – vienovėje daiktus pa­ gauname „protu“); ir su-vokimas susideda iš daugelio aki-vaizdžių akies vokų mirksnių. Kai tik pradedame detaliau išskleisti akivaizdos tobulumą („lyg vientisame paveiksle“), „šis ima diferencijuotis“ (ten pat; 21). Plg.: „Natūralus įvykis aiškinamas jį nedviprasmiš­ kai integruojant į visuminį patyrimo kontekstą. Čia „patyrimas“ reiškia ne tik mano asmenišką patyrimą, bet ir Tavo, ir mūsų visų, patyrimą. Galiausiai tai reiškia aiškaus ir skaidraus sampro­ tavimo patyrimą, siejamą su „dėsniais“ ir bendrai vadinamą „gamtos mokslu“. Patyrimas  – lygu intersubjektyvus patyrimas nuo pačios pradžios. 96 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Aš, suvokdamas savo patyrimo kontekstu­ alumo sandarą, arba sąmonės suvokimo lauko struktūrą (branduolio, ribų, paribių, prasmingų sąsajų ir figūracijų, išorinių ir vidinių horizontų kaip suvokimo lauko regionų man-tikrovišku­ mą) tampu ap-dairesnis. Galiu – bet kuriuo vidinio laiko momentu – arba plėsti, arba siau­ rinti sąmonės suvokimo laukų įtampas. Galiu nusigręžti nuo arba atsigręžti į. Vadinasi, galiu artėti prie są-monės, arba savasties, branduolio (dar kitaip: domesio). Pirmuoju atveju aš – dar prieš tyrimą – įgyju nuo-savybę ir už-tikrinu savo tapatybę. Kaip kryptingas natūralistas, aktyviai kreipiantis buvojimo židinį objektyvumo link, imu į savo rankas atitinkamai atrinktas – pagal gręžimosi pasaulin kryptį – akivaizdas (tarsi tikroviškas reprezentacijas), kurios apsaugo mane nuo bet kokios subjektyvios apgaulės ir nuo paties pikto. Akivaizdos tampa absoliučiu priešnuodžiu nuo pikto, kartą ir visiems laikams kryptingai įgyven­ dinant polinkį – ir „tikint“, ir „sprendžiant“ – objektyvumo link.40 Antruoju atveju aš, kaip kryptingas fenomenologas, tariu: „Atsiprašau, aš nežinau tiksliai, kas yra tikrovė.“ Ir „imuosi iš naujo grįsti reikšmes“ (Husserl 2005; 20). 40 Reikia skirti, kaip pabrėžia Husserlis, viena vertus, „akivaizdą“, kita vertus, „akivaizdos-sprendi­ nį“. Vienas dalykas yra į-matyti, iš-gyventi, turėti ką nors suvokimo lauke; visai kas kita yra dėme­ singai įgyvendinti – „tikint“ ir „sprendžiant“ – matymo aktą, buvoti aktyviai kreipiant buvojimo židinį objektyvumo link (Husserl 1952; 3). į žmonių aplinką. „Laikymo tikru“ dalykas – tai ne išvada arba formalus predikatas, o pirminis įmatymas ir sugriebimas; daikto arba įvykio kaip tikro, ir kaip tikro mums visiems, pagava.“ (Na­ tanson 1970; 103) 40 j y 41 Žr. Husserlio Idėjų § 82: „Tęstinumas. Trinaris išgyvenimo-horizontas, drauge kaip išgyvenamos- refleksijos horizontas“ („Fortsetzung. Der dreifache Erlebnishorizont, zugleich als Horizont der Erlebnisreflexion“). Pirma, kiekvienas išgyvenimas-dabar (Erlebnisjetzt) būtinai turi savo ką-tik (DESCARTES 1978; 101–5). Jei tik ir kai tik aš imuosi iš naujo tikrinti reikšmes, tai, kas matoma, darosi man-pačiam (!) pastebima: prieš-duota. „Aš galiu“ (ten pat; 72). Artėjimas savęsp (prie sąmonės branduo­ lio) – tolygu senasis gerasis neokartezianizmas. Aš domiuosi, taigi esu-Tavęsp, arba Aš-galiu (o tai yra vena ir tas pat). Aš galiu net tiek, kiek, kaip anksčiau atrodė, neįmanoma: netolti nuo savęs; nesigręžti nuo dėmesingumo-gyveni­ mui pirmapradėje natūraliosios laikysenos tikrovėje. Aš galiu skirti tikrovę ir tarsi-tikrovę (Husserl 2005; § 25). Aš galiu prieiti arčiau savęs, įsižiūrėti į savo paties pasirodymų būdus transcendencijas išgyvenančioje subjektyvioje vienovėje: „Nuodugnesnis tyrimas, be kita ko, rodo, kad aš, šioji psicho-fiziologinė vienovė, vienu ir tuo pačiu metu gyvenu keleriopuose tikrovės regionuose“ (Schutz 2011c; 96). Aš galiu tyrinėti ne vien tik kitus kaip neklys­ tantis (nebent su menkos paklaidos išlygomis) natūralistas. Galiu tirti patį save. Pirmasis vai­ kiškas mano „žingsnis“ apibrėžia patį ar pačią (nelygu aš) sąmonės lauką, įskaitant teminį branduolį ir aplinkinį horizontą, priešduotą bet kuriuo mano vidinio laiko momentu. Antrasis, jau brandesnis, mano „žingsnis“ atskleidžia įvairių tikrovės provincijų tarpusavio susietumą mano paties sąmonės vienovėje. Jei tik ir kai tik – ir būtinai trinariame išgyvenimo-refleksijos-horizonte41 – pagau­ 97 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 name, užlaikome ir ištęsiame atskaitingumą patyrimui, arba relevantiškumą (o tai yra viena ir tas pat), šias subjektyvumo ir racionalumo polinkių pasauliui sąlygas, kurių ištakose – savasties patyrime išgyvenama akivaizda kaip universalus intencionalumo būdas, susijęs su visais sąmonės gyvenimo-procesais, randasi daiktiškumas, pasauliškumas, objektiškumas. Nebus per daug pakartoti: „Objektiškumo kategorija ir akivaizdos kategorija yra sąsajos“ (Husserl 1974; 169).42 Manyti, kad ji randasi kažkur toli už mūsų, būtų klaida. Husserlis, kaip tikslina Dorionas Cairnsas, ragina gilinti kinestezės prasmę, įskaitant ir „atsigręžimą į“ objektą at-mintyje kaip įtraukiantį kinestezę (Cains 1976; 5).44 Jei tik ir kai tik pagauname tikrovę kaip esmines sąsajas (Wesenszusammenhang), sugriebiame ir teiginių „aš galiu“ arba „aš negaliu“ prasmę. Kad aktyvumas randasi iš tam tikro pasyvumo, tam tikro duotiškumo, iš tam tikros kineste­ zinės sąsajų sandaros: drauge su pradmeninio pasyvumo (ursprünglicher Passivität) buvoja ne tik aktyvumo (Aktiviytät) sfera, bet ir antrinio pasyvumo sfera, rodanti, kas būtinai randasi mums aktyviai atsigręžiant į tam tikrą situaciją. O „gręžimasis į“ vidinio laiko-sąmonėje, trinario išgyvenimo horizonte, mums saviduoda ne tik ribą tarp praeities ir ateities, bet ir relevantišką vienalaikiškumo figūrą, formą, tipą. Laiko-są­ monėje randasi praeitis ir aktualizuojama ateitis: „Įdomu, kad pastarajame procese randasi kažkas panašaus į habitus: ateitis įgyvendinama pagal pavyzdį, apibrėžtą praeities“ (ten pat; 17). horizontą (Horizont des Vorhin). Antra, kiekvienas išgyvenimas-dabar būtinai turi savo po-to ho­ rizontą (Horizont des Nachher). Trečia, išgyvenimas-dabar esti ne tik laikiškumo seka, o ir viena­ laikiškumo forma. Veikiau būtinai apima išgyvenimo-sąryšių (Erlebniszusammenhang) formų, arba tipų, įvairovę vienalaikiškumo požiūryje („<...> sondern auch unter dem Gesichtpunkt der Glei­ chzeitigkeit“; Husserl 1976a; 174). Visos trys – ką-tik horizonto, po-to horizonto, vienalaikiškumo horizonto – dimensijos yra būtinos sąsajos (ten pat; 185). 43 Husserlis, – atkreipia dėmesį Richardas Zaneris, – daug kartų akcentuoja esminę tradicinės filoso­ fijos klaidą: kad ji neįstengia išplėtoti tinkamos ir adekvačios akivaizdos (angl. evidence) sampratos (Zaner 1973; 184). 44 „Objekto suvokimas įtraukia kūno kaip organizmo buvojimą. Tik todėl, kad aš kaip kūnas esu daiktas pasaulyje, aš galiu turėti pasaulį“ (ten pat; 6). j y ą j p 42 „Kategorie der Gegenständlichkeit und Kategorie der Evidenz sind Korrelate“ (ten pat). 4 (DESCARTES 1978; 101–5). Štai-dabar galima kalbėti formaliau: savi­ duotiškumas, kaip ir bet koks kitas intencio­ nalumo procesas, yra visa apimančių sąmonės tarpusavio sąsajų funkcija (Funktion; ten pat; 168). Tai nuoroda į esminį intencionalumo dėsnį: bet kokia ko nors sąmonė a priori priklauso atvirai begaliniam galimų sąmonės būsenų daugybiškumui, galinčiam sintetiškai pasirodyti sąsajų bendra-buvojimo vienovėje kaip sąmonės „tas-pat“ („demselben“; ten pat; 168). Štai kodėl akivaizda (Evidenz) yra universalus intencio­ nalumo būdas, susijęs su visapusišku sąmonės gyvenimu:43 akivaizdumo dėka, sąsajų relevan­ tiškumo – bendrabuvojimo con-posito – dėka, sąmonės gyvenimas turi universalią teleologinę struktūrą (ten pat). Šiame at-pažinimo sąsajų svarstymo kon­ tekste aiškėja paties pažinimo kontekstualumo svarba, tiksliau, dviejų – išorinio ir vidinio – pažinimo kontekstualumo horizontų. Bet 98 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija drauge su kitomis laikysenomis, – vertinimo laikysena (plačiąja prasme, įskaitant grožio ir gėrio vertinimą) ir praktine laikysena. koks mūsų teigimas, numanymas, numatymas, tvirtinimas, pareiškimas, ieškinys randasi „spe­ cifiniuose kontekstuose“, „baigtinėse prasmės provincijose“ (Schutz 1962; 207–59), kurios viena kitos atžvilgiu esti „išoriškos“. Drauge bet koks mūsų teiginys teikiamas, įteikiamas, perteikiamas kalba: „Be to, kiekviena kalba turi steigti skirtis, randamas natūralioje laikysenoje (Einstellung, attitude), subjekto ir objekto, Ich und Umwelt (ego ir aplinkinio pasaulio), koky­ bės, santykio ir panašiai“ (Cairns 1976; 1). Tikslinančios Husserlio nuorodos kviečia įsi­ žiūrėti, pirma, į ato-trūkį: natūralizmo-istorizmo atplyšimą nuo gyvenimo-pasaulio, antra, nuskai­ drinti metafizikos-mokslo tarpusavio susietumą. Metodologiškai svarstyti galimybes: ne ar gali­ ma, o kaip galima sugrįžti iš istorizmo į istoriją? Sugriebti jau nebematomą, nepastebimą, natū­ ralistinių ženklų užtušuotą brūkšnelį. Brūkšnelį, kuris, – mums gyvenant progreso, atskirto nuo gyvenimo-pasaulio, iliuzijoje „pliuralaus istoriz­ mo“ sąlygomis, – kviečia refleksijoje sugriebti istoriškumą: „Fenomenologiją Husserlis steigė daugiau nei prieš šimtą metų, ir ji iš jo gimtosios vietos paplito į visus pasaulio kampelius ir prigijo kaip „reflektyvios analizės“ metodas, taikomas konkrečiam patyrimui“ (Toru 2010; 103) Metodologiniu požiūriu, tai reiškia, jog kalba yra tipizuojančių patyrimo schemų, grindžiamų betarpiško subjektyvaus patyrimo idealizacijomis ir anonimizacijomis, sistema. Alfredas Schutzas ir Thomas Luckmannas atkreipia dėmesį į „paprastą“ (įprastai mūsų matomą, bet nepastebimą) dalyką. Kad nors tipiškumo-struktūros ir netgi „prieškalbinis“ patyrimas gali būti suvokiami ir be kalbos, tačiau pagrindžiantieji (relevantiškumo / nerele­ vantiškumo) santykiai yra tokie, kad tipizaciją implikuoja kalbos struktūra (Schutz, Luckmann 1973; 233), o ne atvirkščiai: Socialinių mokslų metodologijos požiūriu, tai reiškia ne ką kita, kaip nuskaidrinti daugia­ narį domesio objektą: 1) socialinio pasaulio stebė­ tojo, 2) veikiančio asmens, 3) asmens porininkų, o taip pat atitinkamų a) interpretacinių schemų ir b) relevancijų sistemų. 45 Jitendra Nath Mohanty’s tinkamai nuskaidrina esminę skirtį tarp „savi-akivaizdos1“ ir savi-aki­ vaizdos2“ („self-evidence1“, self-evidence2“). Pirmoji savi-akivaizda neatskiriamai (angl. inalienably) priklauso sąmonei. Ji skirtina nuo saviduotiškumo, suprantamo kaip tiesos patyrimas, ir vadinamo savi-akivaizda2“. Jei šios skirties nepaisoma, tuomet neretai daroma prielaida, kad „savi-akivaiz­ da1“ yra savi-akivaizdos2“ priežastis: „Pavyzdžiui, įrodinėjama, kad teorija, kuri priskiria savi-aki­ vaizdumą sąmonei, negali atsižvelgti į klaidos galimybę, – o tai yra tas pat, kaip įrodinėti, kad visiškai savi-akivaizdi sąmonė eo ipso yra tiesos suvokimas. Tačiau nebūtinai šitaip turi būti. Kad „savi-akivaizda1“ nėra savi-akivaizdos2“ priežastis galime suprasti svarstydami tai, kad sąmonė gali būti ir tiesos, ir klaidos sąmonė, ir kad bet kuriuo atveju sąmonė kaip sąmonė yra savi-akivaizdi.“ (Mohanty 1973; 209–10). (DESCARTES 1978; 101–5). Nuskaidrinti derinant pirminio dėmesingumo (ko nors kaip domesio objekto sugriebimo gyvoje dabartyje, prieš-akyje) ir antrinio dėmesingumo (santykiškų – gyvosios dabarties atžvilgiu – reikšmingumo bruožų ir struktūrinių linijų gyvenimo-pasaulio būtišku­ me) modusus. Išlaikant ato-dairoje konkrečius betarpiško nukreiptumo dabartin aspektus, tačiau drauge abstrakčiai sugriebiant – dėmesingumo ontizuojamiems santykiams būdais – jungiančią­ sias sąsajas. Adekvačiai objektyvuojant subjekty­ vaus buvojimo pasauliui ir drauge objektyvaus buvojimo pasaulyje sąryšius. „Patyrimo tipizacijos, atsietos nuo subjekty­ vumo, yra socialiai objektyvuojamos, tapdamos socialinio a priori, prieš-duoto subjektui, kom­ ponentu. Normaliai bręstančiam asmeniui natū­ ralioje laikysenoje tipizacija glaudžiai susijusi su kalba.“ (ten pat; 233–4) Štai kodėl Husserlis teorinę laikyseną (the­ oretische Einstellung) apibūdina kaip doksinę teorinę (doxisch-theoretische). Tai, kas vadinama „gamta“, tiksliau, „natūra“ (Natur) yra būtent patyrimo, įgyvendinamo šioje laikysenoje, intencionali sąsaja (intentionale Korrelat): te­ minė natūralaus mokslinio natūros patyrimo ir patyrimo tyrimo laikysena yra doksinė-teorinė, 99 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Jungli teorinė pirminio dėmesingumo ir antrinio dėmesingumo forma vadintina pa­ prastu vardu: dairumu. Dairumas yra teleologija, daugiabalsė domesio laikiškumui muzika, konsti­ tuojanti relevantiškų prasminių atžvilgių visumą: vieniems jų tampant („gręžiantis į“) atskaitinga tikrove, įprastu turėjimu, habitus, tikrai tikra tikrove, o kitiems („gręžiantis nuo“) tik regimybe, t.y. santykiškai atitolusia, atsijusia, susvetimėjusia, anonimiška, tariamai tikrove. „<...> buvo prarastos miglose, kylančiose visų pirma iš nesugebėjimo sugriebti akivaizdos ir, atitinkamai, paties patyrimo, esmę“ (Zaner 1973; 189). Štai kodėl pirminis bet kokios fi­ losofijos reikalavimas – tai gebėjimas atsižvelgti į savo pačios galimybes (ten pat; 190). „Savi-akivaizda1“ būdinga kiekvienam baig­ tiniam žmogui, įsisąmoninančiam (sangrąžinė dalelytė „-si-“ – lygu savęsp) aki-vaizdos mįslę kaip išgyvenamo patyrimo ir objektyvaus vaizdo radimąsi gyvoje dabartyje. Savi-akivaizda2“ – tai žmogaus po-linkis ieškoti tiesos, įskaitant įvairias ko nors tiesos formas. Antrosios akivaizdos (įpras­ tai be skiriamojo brūkšnelio) forma randasi savitai su-tampant relevantiškiems ir nerelevan­ tiškiems tikrovės ir regimybės atžvilgiams, konsti­ tuojamiems intersubjektyviame lauke, kuriame kiekviena monada implikuoja ir suponuoja kitas monadas. Relevantiškumas – lygu „visuotinis prasmės susisluoksniavimas“ (Husserl 2005; 132), kurio ištakose – savaime pirmos svetimy­ bės (pirmojo Ne-Aš) kaip Kito-Aš – patyrimas. Tai pirmoji, taigi esminė inter-subjektyvumo sąlyga: pirmojo Ne-Aš kaip Kito-Aš prasmės, Tavęsp-laikysenos (Dueinstellung; Schutz 1932; 182) įgyvendinimas.46 Ir nors prasmės suvokimas saviduotiškume (saviapmąstyme) nėra vienintelė prasmės suvoki­ mo forma, tačiau ji, kaip buvo tikslinama II-oje šios esė dalyje („Subjektyvumo problema“) yra pirminė akivaizdos vieta. Tai reiškia, kad sąmonė savo aki-vaizdoje implikuoja savo buvojimą, neatskiriamą nuo jos pačios. 46 Čia kalbama apie ikipredikatinio patyrimo, pasyviosios genezės ir sintezės sferą. Tavęsp-laikysena apibrėžiama kaip ypatingas aktų, kuriuose aš sugriebia kito asmens egzistavimą originaliu būdu (im Modus des originalen), intencionalumas. Šis originalumas, žinoma, nėra „pirminis“, – pridu­ (DESCARTES 1978; 101–5). Sąmonės savi- duotiškumo modusą reikia skirti nuo sąmonės intencionalumo (nukreiptumo ko nors atžvilgiu) moduso.45 Skirtis tarp sąmonės savi-duotiškumo ir sąmonės intencionalumo orientuoja į platesnę ir gilesnę patyrimo laikyseną: ne tik „vidujai išgyvenamos tikrovės“ (psichologinio patyrimo aprašymų), bet ir idealiųjų loginių a priori, pati­ riamų imlesne, nei įprasta, prasme: šios idealybės 46 Čia kalbama apie ikipredikatinio patyrimo, pasyviosios genezės ir sintezės sferą. Tavęsp-laikysena apibrėžiama kaip ypatingas aktų, kuriuose aš sugriebia kito asmens egzistavimą originaliu būdu (im Modus des originalen), intencionalumas. Šis originalumas, žinoma, nėra „pirminis“, – pridu­ 100 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Štai kodėl svarbu skirti, viena vertus, są­ monės ko nors intencionalumą ir, kita vertus, akivaizdos savasties saviduotiškume patyrimą. Taigi pradėti svarbu – nes jokia akivaizda nėra apsaugota nuo galimos apgaulės – nuo radi­ kalaus saviapmąstymo. Nuskaidrinant tai, ką saugiai apeina tradicinė empirinė epistemologija ir, atitinkamai, metodologija: kad bet kokio a) intencionalumo samprata (bet kokio sąmonės ko nors gyvenimo proceso) ir b) akivaizdos samprata (intencionalumo kaip ko nors saviduo­ tiškumo; Intentionalität der Selbstgebung) yra iš esmės susijusios (Husserl 1974; 168). Tai reiškia veikiau daug, nei mažai: tiesiogiai „įmatyti“ (vok. „sehen“; ten pat; 163) ko nors idealaus objektyvumą. damas, kad ši aplinkybė vertė jį ilgai abejoti dėl galimybės parašyti tinkamą įvadą. Dalykas tas, kad fenomenologinių tyrimai turi zig-zag formą („labai primityviai“ išskleistą Husserlio veikalo Logische Untersuchungen įvade): 1) pradedama, 2) laikomasi tam tikro atstumo, 3) paskui grįž­ tama į pradžią ir 4) tai, kas eksplikuota, taikoma skaidrinant pradžią. Į Cairnso pastabą, kad mokslo idealas „iš­ gyvenimo per“ aspektu Prancūziškųjų meditacijų (Husserlis aptariamuoju laiku rašo neužbaigtų Vokiškųjų meditacijų variantą) pradžioje per­ teikiamas neadekvačiai, ir kad jis norįs išsiaiš­ kinti, kaip turėtų būti įgyvendinamas idealas, arba tikslas, Husserlis atsako aiškiai skirdamas lygmenis. Fenomenologiniu požiūriu, išties natūralu norėti turėti „paskesnį aiškinimą“. Tačiau iki įvado į fenomenologinę redukciją fe­ nomenologijos prošvaisčių Meditacijose, pasak Husserlio, dar nėra. Husserlis siekia išskleisti būdus, konstituojamus pačiame kasdieniame gyvenime. Būdus, kurie motyvuotų pereigą į transcendentalinę sferą. Todėl jis tikslų steigimo pradžioje 1) kalba visiškai naiviai: kaip apie ką nors, duotą natūralioje laikysenoje. Fenomeno­ loginis metodas, įveikęs šį pirmąjį natūraliosios laikysenos naivumą, 2) sugrąžina mus į naują naivumą: paprastai aprašant sąmonės aktų pro­ cesus. ria Alfredas Schutzas trumpoje išnašoje: kadangi sąmoningas kito asmens gyvenimas tiesiogiai iš principo man nepasiekiamas („<...> in direkter Wahrnehmung prinzipiell nicht zuganglich ist <...>“). Vartojant Husserlio terminus, tai yra „antrinis“ originalumas (ten pat). (DESCARTES 1978; 101–5). Tai savo ruožtu įveikiama žengiant prie 3) gilesnės konstituacinės analizės: Pačioje pradžioje verta įsiklausyti (kad gebėtume tiesiogiai „matyti“ ne tik paskirus svarstomos problemos fragmentus, bet ir trijų dimensijų sąryšių visetą) bent į kai kurias įdo­ maus pokalbio, vykusio 1931-ųjų rugpjūčio 28-tą dieną tarp Edmundo Husserlio ir Doriono Cairnso, detales (Cairns 1976; 27–8). Eksplika­ cijos lygmenys, kuriuos įvardija Husserlis, svarbūs ne tik nagrinėjant relevantiškumo problemą, bet ir siekiant suprasti ryšius tarp fenomenologinės filosofijos ir socialinių mokslų metodologijos. „Labai sunku suprasti, kur pradėti, nes vis­ kas, kas ateina paskui, rodos, turi atramą ten, kas randasi prieš tai“, – šią giliai probleminę Cairnso giją, intencionaliu skambesiu liudijan­ čią fenomenologinės pradžios daugiabalsiškumo sandarą, Husserlis netiesiogiai patvirtina atsaky­ „Konstituacinę analizę reikia skirti nuo ap­ rašomosios analizės. Antrojoje eksplikuojami objektyvūs objekto momentai, objekto kokybės, santykiai su kitais objektais. Konstituacinėje 101 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 analizėje eksplikuojami aktai, kuriuose yra duoti objektai ir objektyvūs momentai. Husserlis, skaidrindamas metodologiją, kaip pažymi Dorionas Cairnsas, aiškiai skiria socialinių-kultūros mokslų ir gamtos mokslų konstituojamus objektus. Jis sutelkia dėmesį į kasdienio gyvenimo natūralaus pasaulio konsti­ tuavimą, kuriame fizikas randa motyvus, kuriais remdamasis jis konstituoja „savo“ pasaulį. Štai kad ir tokį „savo“ pasaulį: „Husserlis, be to, svarsto tokias problemas kaip kasdienio gyve­ nimo geometrizavimo, pavyzdžiui, geometrinio trikampio steigimo vietoj matomo trikampio“ (Cairns 1976; 26). Pridurtina, kad tikrumo modalizacijos įgyvendinamos nesulaikomu (ungehemmt) ir sulaikomu (gehemmt) būdais (Husserl 1939; 93). O tai reiškia, kad kryptys, aš-kryptingumo pasauliui ir pasaulyje laikysenų būdai, – tai ne tik akli vis naujų duotiškumo būdų siekiniai, bet eina žingsnis po žingsnio drauge su lūkesčių-intencijomis (Erwartungsin­ tentionen), protenciniais lūkesčiais (protentionalen Erwartungen) (ten pat). Pavyzdžiui, aptarkime veikimą, veiksmą (Han­ deln). Galime analizuoti jį naiviai: skirti kiekvie­ ną tarpsnį kaip susijusį su tikslu, šio darbo raido­ je rasti tokius požymius, kaip „pusiau užbaigtas tarpsnis“, turintis objektyvius požymius. Šiuose ryšiuose išskleidžiamos entelechijos ir dynamis sąvokos. Konstituacinė analizė šiuos aprašymus, šias kokybes daro suprantamomis pasitelkdama sąryšius su sąmonės aktų sintezėmis, kuriose jos konstituojamos. Toks naivumas, kaip jį išskleidžia gamtos mokslai, ir tokia ontologija esti bloga ontolo­ gija, jei ji mano, kad yra filosofija. Vadinasi, ir logika gali būti naivi. Savo veikale Formale und Transzendentale Logik Husserlis siekia atskleisti būdus, leidžiančius pereiti iš naivios logikos į fe­ nomenologiją.“ (Cairns 1974; 27–8) Kitaip tariant, svarbu – konstituacinė anali­ zė, kuri dėmesinga 1) pirminei savasties-akivaiz­ dos aplinkai, 2) sąsajoms, 3) radikaliai savigrąžai (jei epistemologijai rūpi visos galimos žinojimo formos, jai turi rūpėti ir savi-žinojimas). 47 Husserlio teiginį, kad „blogiausi tarp visų prietarų yra susiję su akivaizda“ („<...> die schlimmsten aller Vorurteile sind hier die die Evidenz betreffenden“; ten pat) reikėtų suprasti kaip teigimą, kad „natūraliausi prietarai yra atkakliausi“. Ir Descartes’as nurodo įprastą, taigi be galo tvirtą aki­ vaizdos atkaklumą: sveika nuovoka – gebėjimas spręsti ir atskirti tikra nuo netikra – yra natūralus dalykas visiems žmonėms, ir niekas šio dalyko (išties elegantiškai perteiktas etnometodologinis Descartes’o atradimas) „paprastai netrokšta daugiau, negu turi“ (Descartes 1978; 101). (DESCARTES 1978; 101–5). Tai galima pavadinti dvinare relevantiškumo ir sa­ vi-relevantiškumo problema. Kiekvienas mūsų teiginys įgyvendinamas esant tam tikroms sąlygoms, – tiriant, veikiant, samprotaujant, įsitikinant kuo nors. Šie – tyrimo, veikimo, sam­ protavimo, įsitikinimo – regionai savo ruožtu susiję su mūsų biografine aplinka ir kultūros (autentiška šio žodžio prasme: skirtingų pras­ minių jos provincijų, savitų kalbinių žaidimų) aplinka. Vadinasi, teiginiai negali būti atsieti, kaip teigia „mokslinės“ vertybinio neutralumo koncepcijos, nuo savitų kalbinių žaidimų. Tik suprasdami šių ap-linkų (prasminių „linksnių“ įvairovių) tipizacijas ir relevancijų sistemas, mes pradedame suprasti arba galime tikėtis pradėti suprasti ir „išorinę“ aplinką. Legitimuotas tikėjimas faktais (remiantis gamtos mokslų metodo analogijos „pavyzdžiu“) yra pragaištingas socialinės rities metodologi­ joms. Griežti ir tikslūs faktai yra anonimiški, – jų nerevantiškumo, t.y. be-prasmiškų sąsajų su socialiniu pasauliu požiūriu. O kai tik šiems sudaiktintos dabarties faktams šypsosi politikos politika (reikia skirti nuo gyvenimo politikos), tai randasi tįsi tikslių, griežtų ir grynų geometrinių figūrų, kurių niekur nerastume gyvenimo- pasaulyje, dabarties struktūra. Reikia aiškiai pažymėti, kad faktiškumų aibės – ne socialinių, o gamtos mokslų metodo daromos prievartos (be kabučių) išdava. Nors pasaulis yra visko, kas apskritai egzistuoja, visumos tikrintinas vardas (Husserl 2005; 25), nuo pradžios absoliučiai 102 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija pirminį pa-tyrimą – sąmonės aktų ir procesų. Būtent čia išryškėja metodologinio tęstinumo – tarp sveiko protavimo ir mokslinio samprota­ vimo komponentų – problema, reikalaujanti išskleisti ikipredikatinių ir predikatinių lygmenų sąveiką, atsigręžti į tarp-ininko (vertėjo) dė­ mesingumą ir atidumą taikant vadinamąsias „transformacijos lygtis“ kasdienybės pasaulyje (konstituojamas tipizacijas verčiant į mokslines idealizacijas). garantuotos duotys kryptingai geometrizuo­ jamos. O geometrizacija yra kryptinga stilis­ tika, – randantis ir gausėjant faktinių ženklų, faktinių žmonių tipams, anonimiškai tikslių faktų tipams, tiesių kreivių, užpildytų lentelių, funkcijų grafikų tipams. Viena aišku: mūsų, sekuliarizuotų atomistų, tikėjimas „faktais“, apdorotais technopatolo­ ginių (kantiškąja šio žodžio reikšme) metodų, yra griežtas, tikslus ir grynas, kitaip sakant, pseudo-religinis: „<...> „realitas objectiva“, iliustruojantis tik įvairių dalių, atribotų viena nuo kitos juslinių duomenų skirtumais, tįsumą“ (Whitehead 1978; 61). Objektyvi tikrovė, arba „išorinė gamta“, – jei tik ir kai tik ji atplėšiama nuo gamtos supratumo natūralioje laikysenoje, yra tik natūralistinio atomizuoto kontinuumo vardas. Skirtis tarp natūralistinės laikysenos ir fenomenologinės laikysenos liudija: 1) gamta čia – gamtininkų pasaulyje – yra teorinis objektas (ir ne tik teorinis objektas); 2) „gamta“ čia – socialiniame pasaulyje – yra teorinis subjektas; ji priklauso savo sąsajų-sferai. (DESCARTES 1978; 101–5). Relevantiškumo linijų atpažinimo kelias driekiasi iš ikipredikatinės pasyviosios genezės sferos, – kurioje pasirodo analoginė apercepcija (Husserl 2005; § 50) kaip poravimo (Paarung, pairing) procesų, tarp jų ir tipizacijų, išdava, – į aukštesniąsias Asmens ir socialinių santykių struktūras. Nėra lengva tiesiogiai „įmatyti“ būtinus ryšius, tačiau, žengiant nuosekliai kons­ tituacinės analizės kryptimi, ir galima, ir reikia rasti formalias intersubjektyvumo konstituacijų struktūras. Šiuo aspektu relevancijos problema, kad ir siekianti būti motyvacijų fenomenologija, yra „formali“. Relevantiškumas yra būdinga fenomenolo­ giškai grindžiamos, arba daugiabalsiai kompo­ nuojamos, metodologijos problema, rodanti į ikipredikatinę socialinės tikrovės prigimtį. Būtent čia susiduriame su naivia aki-vaizdos (per brūkšnelį) prigimtimi: 1) tuo, kas pasirodo (tiksliau, kaip pasirodo), 2) tuo, kam pasirodo, 3) tuo, kas įgyvendinama – per pirminį są-lytį, Logika, ypač modernioji logika (nuo Loc­ ke’o Esė; Husserl 1974; 283), rodos, taip pat siekia nuskaidrinti ikipredikatines pradžias, gręždamasi į šaltinius, kuriuos sudaro vadi­ namasis „vidinis patyrimas“. Problema yra ta, kad į pirminius šaltinius gręžiamasi drauge su prie-tarais (Vorurteil), įskaitant tarp jų „pačius blogiausius“, susijusius su akivaizda (ten pat).47 103 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Atimti naivų galiojimą iš universalaus „naivybės“ (patyrimo pasaulio) pagrindo be fenomenologi­ nės redukcijos yra sunku (Husserl 2005; 25). Yra vienintelis, kraštutinai radikalus būdas, leidžiantis sulaikyti prie-tarus – radikalus savia­ pmąstymas / įsisąmoninimas (Selbstbesinnung). Tik saviapmąstymas geba pernelyg greitai nenu­ grimzti į naivų pozityvumą, o lieka visiškai tuo pačiu, kuo ir buvo pradžioje, – saviapmąstymu (Husserl 1974; 282). pirminį ir visa persmelkiantį (tam tikrais lai­ kotarpiais) filosofijos uždavinį, kaip tai didžią­ ja dalimi daroma kalbos analizėje ir kritikoje. Reikšmių ir nuomonių,  – įprasto protavimo arba labiau rafinuoto samprotavimo,  – anali­ zė nėra pirmasis filosofijos uždavinys. Tai, nuo ko turi pradėti filosofija, yra patys fenomenai ir problemos; teorijų tyrimai, kad ir kokie jie būtų svarbūs, yra tik šalutinis dalykas.“ (Ten pat; 122.) Akivaizda, arba struktūriškai išskleidžiamas sąmonės procesas, yra intencionalus saviduotišku­ mo įgyvendinimas (die intentionale Leistung der Selbstgebung; Husserl 1974; 166). Tradicinių akivaizdos teorijų, tampančių, – matomai, bet nepastebimai, – saugiomis metodologijomis, esminė klaida yra ta, kad akivaizda laikoma absoliučiu apodiktiškumu, absoliučiu saugumu be apgaulės. Tai saugi tiesa-pati-sau (Wahrheit- an-sich; ten pat; 283). Tik įsisąmoninimas gali sugriebti akivaiz­ dą ir tęstinai išlaikyti ją savo aktualiame ir potencialiame aki-ratyje. Sugriebti ir išlaikyti akivaizdą prieš-akyje. 48 Santykį tarp logikos, – kuri Platono laikais randasi kaip atsakas esantiems, buvusiems ir būsian­ tiems „sofistams“, neigiantiems „filosofiją“ kaip mokslą, – ir mokslo, galima atkurti kaip principinę galimybę (die prinzipielle Möglichkeit; Husserl 1974; 6), steigiamą kritikos. Dalykas tas, kad pir­ minis logikos ir mokslo ryšys moderniaisiais laikas pertraukiamas: mokslai tampa nepriklausomais. Jie steigia kraštutinai diferencijuotus metodus. Šių metodų vaisiai yra praktiniai, tačiau pasiekimai (Leistung) nenuskaidrinami iš esmės. Metodai steigiami pasitelkiant ne kasdienio žmogaus naivumą, bet esminio laipsnio naivumą (Naivität höherer Stufe), kuris nusigręžia nuo grynosios idėjos ir po­ (DESCARTES 1978; 101–5). Fenomenologinis tyrimas įgyvendinamas zig-zag forma: saviapmąstant aki-vaizdą, tęstinai – kitą pirmą kartą – su­ griebiamą ir išlaikomą konkrečiose relevancijų sistemose: „Husserlis daug kartų pabrėžia, kad fundamentali tradicinės filosofijos klaida yra jos nesugebėjimas eksplikuoti tinkamą ir adekvačią akivaizdos sampratą“ (Zaner 1973; 184). Tik saviapmąstymas, sugriebdamas akivaizdą ir tęstinai išlaikydamas ją savo aktualiame ir poten­ cialiame akiratyje, gali atsilaikyti prieš visuotinį reliatyvizmą ir skepticizmą (Kockelmans 1994; 121), o kvietimą grįžti atgal prie pačių daiktų atskirti nuo ankstesnių šūkių (renesansininkų kvietimo: „Atgal prie šaltinių“; arba neokanti­ ninkų: „Atgal prie Kanto“): Tai ypatinga mokslinio tikėjimo – indivi­ dualių, izoliuotų faktų metodiško stebėjimo saugumu – forma: „Atlikau empirinius tyrimus!“ Empiristas pripažįsta Kito žinojimą tik tada, kai šį žinojimą patvirtina stebimi, tiksliau, metodo patvirtinami individualūs faktai: „Empiristui tik nuomonės, kurios adekvačiai įrodomos remiantis „juslėmis“, arba jusliniu suvokimu, yra oficialiai patvirtintas, „mokslinis“ žinojimas“ (Cairns 1940; 5). O kas, jei tiesa, – klausia Husserlis, – yra beribė pagrindžiančioji idėja (Unendlichen liegende Idee; ten pat; 284)?48 „Ši frazė visų pirma yra atsisakymas formu­ luoti filosofijos teorijas ir šių teorijų kritiką kaip 104 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija reikio pateisinti metodus remiantis grynaisiais principais: pagal galutines a priori galimybes ir bū­ tinybes („<...> nach letzten apriorischen Möglichkeiten und Notwendigkeiten zu rechtfertigen“; ten pat; 6). Fundamentali fenomenologijos maksima, kaip pažymi Dorionas Cairnsas, reikalauja atmesti empiristinio metodo apribojimus, nes jie verčia nepaisyti griežtai saviduotų ir intersu­ bjektyviai tikrinamų „pačių daiktų“. Fenomenologas atmeta empiristinį metodą ne dėl kokio nors „nematomo absoliuto“ ar netgi tariamai būtinų patyrimo galimybės sąlygų, o dėl to, kad empiris­ tas „nemato“ (overlooks) arba oficialiai nepaiso saviduotiškumo dalykų (Cairns 1940; 6). Teiginys, kad mentaliniai fenomenai yra vidinio suvokimo objektai, gali būti tam tikra prasme pagrįstas. Tačiau šis požiūris „nemato“ pirminio sąmonės saviduotiškumo būdo. Dalykas tas, kad linksta­ ma, kaip pažymi Jitendra Nath Mohanty’s, traktuoti šį „vidinį suvokimą“ kaip specialų sąmonės intencionalumo atvejį: kai sąmonės aktas intencionalų objektą sugriebia kitu aktu: „Nors tai yra galima, pavyzdžiui, pirminės retencijos, Wiedererinnerung, refleksijos formomis, tačiau šioji galimy­ bė neatsižvelgia į esminį sąmonės saviduotiškumo būdą, kurio nedera redukuoti į intencionalumo funkciją. Sąmonės aktas yra saviduotas jam nebūnant objektu: arba paties savęs (o tai yra absurdas), arba kito akto, vienalaikiško arba paeiliško“ (Mohanty 1973; 208). Taigi tai, ką Alfredas Schutzas vadina pilnumo metodologija, reikalauja pradėti nuo pradžios: subjektyvaus akto ir objektyvumo steigties sąsajų paralelizmo (Kockelmans 1994; 42). Teleologija yra dairumas, o ne faktinis saugių tiesų-pačių-sau (nustatytų empiriniais tyrimais) kaupimas. (DESCARTES 1978; 101–5). Plg.: „Husserlis, užuot tiesiogiai atsakęs į mano klausimą dėl prisiminimų akivaizdos, išskleidė pirminę, – ko nors suvokimo, ego kaip dėmesingumo kam nors dabartyje (Gegenwart) esant doxa modusui, – fenomenologinę situaciją“ (plačiau žr.: Cairns 1976; 30–1). Kitaip sakant, Husserlio eksplikacija pradedama saviapmąstan­ čio aš dėmesingumu aki-vaizdai (pirmojo asmens susidūrimui su tampančia pirmine tikrove). Dėmesingumas kam nors dabartyje – lygu dėmesingumas kam nors prieš-akyje (Gegen-wart). Kal­ bant apie minėtas savitas prisiminimų akivaizdos rūšis, verta patikslinti skirtį tarp įsiminimo ir prisiminimo. Erinnerung ir Wiedererinnerung skirtį čia perteikiame kaip įsiminimo ir prisiminimo, arba prisiminimo ir pri-si-minimo perskyrą. Pavyzdžiui, Nijolė Keršytė šią skirtį perteikia kaip perskyrą – atitinkamai – tarp retencijos ir prisiminimo. Plg.: „Tai, ką Husserlis vadina „antriniu prisiminimu“, toliau vadinsiu taip, kaip kasdienėje kalboje įprasta jį vadinti – tiesiog „prisimi­ nimu“, o jo „pirminį prisiminimą“ vadinsiu vien „retencija“ (Keršytė 2012; 199). Nurodomas motyvas: reikia skirti atmintį kaip kartojimą („Daugybę šimtmečių atmintis buvo suvokiama kaip sugrįžimas prie to paties dalyko, tos pačios patirties pakartotinis išgyvenimas.“) ir atmintį kaip retencijos ir prisiminimo skirtumą. Kitaip sakant, saugant skirtumą geriau vartoti labai skirtingų žodžių porą, nei labai panašių žodžių porą: „Polinkį trinti skirtumą rodo ir to paties žodžio „pri­ siminimas“ vartojimas abiem atvejais: „pirminis prisiminimas“, „antrinis prisiminimas“ (ten pat). Verta pakartoti, kad prisiminimas – tolydus, o pri-si-minimas – netolydus. Ištrinant netolydumo, taigi ir skirtumų, momentus, – neatmeta tokios galimybės N. Keršytė, – galima pavojingai artėti prie atminties kaip kartotės idėjos: 1) „Antai Davidas Hume’as, atmintį lygindamas su vaizduote, sako: „Gebėjimas pakartoti savo įspūdžius (...) vadinamas atmintimi. (...) Akivaizdu, kad atmintis išsaugo pirmapradę formą, kuria buvo duoti objektai“ (ten pat); 2) „Ir vis dėlto Husserlis linkęs ne ryškinti retencijos ir prisiminimo skirtumą, bet jį slopinti, trinti“ (ten pat). Antrasis teiginys, sa­ kytume, yra skubotas teiginys. Husserlis linkęs ne ryškinti ir ne slopinti, bet veikiau „išskleisti pras­ mę, kuri gali būti filosofiškai atskleista, tačiau niekad negali būti pakeista“ (Husserl 2005; 187). Fenomenologinėje Husserlio ir Schutzo metodologijoje pri-si-minimas (kartotinis prisiminimas) niekada nėra kartotė, o veikiau „ankstesnio patyrimo interpretacija žvelgiant iš esamo Dabar su reflektyvia nuostata“ (Schutz 1962; 210). Husserlis ne slopina, ne trina, o skaidrina, eksplikuoja: „Jis taip pat kalbėjo apie laikinį horizontą ir Anschaulichkeit (įžvalgumo/intuityvumo; intuitiveness) praradimą, kuris kiekvienu momentu pergyvenamas jam grimztant į retenciją (už-laikymą). Pa­ 105 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Pasak Husserlio, akivaizda (Evidenz) yra ypatinga sąmonės rūšis, kurioje daiktas, daly­ kų padėtis, bendrybė, vertybė ir t.t. j 49 Fenomenologiškai grindžiama transcendentalinė estetika praplečia Kanto siaurai apibrėžtą trans­ cendentalinę estetiką, „logos“ atgręždama į sąmonės patyrimo „istorijas“ netgi elementariame – iki­ predikatiniame – lygmenyje. Kitaip tariant, sugrįždami prie savęsp-čia pateikiančių elementariųjų patyrimo modusų, randame jau konstituotas persiklojančių elementų „istorijas“. Pa-tyrime, arba transcendentalinėje estetikoje, nuskaidrinama bet kokio galimo pasaulio, duoto „grynajame pa­ tyrime“ ir todėl esančio anksčiau už visus mokslus „aukštesne“ prasme eidetinė problema („Sie behandelt das eidetische Problem einer möglichen Welt überhaupt als Welt „reiner Erfahrung“, als wie sie aller Wissenschaft im „höheren“ Sinne vorangeht <...>“ (Husserl 1974; 279). našus Anschaulichkeit praradimas gali būti Wiedererinnerung (pri-si-minimo; recollection) atveju. Šiais atvejais mes galime kalbėti apie Klarheit (skaidrumą, clarity) kai objektas lieka deutlich (ryškus, aiškus; distinct, plain) kaip visuomet: struktūra lieka akivaizdi, nors turinys darosi „tuščias“. Tačiau čia gali būti Weckung (pabudimas; awakening) iš aktyviai numanomo prisiminto objekto tarpsnio į ankstesnį periferinį tarpsnį, tarkim, tarpsnį, kuris, netgi kai objektas buvo lebendige Gegenwart (gyvoje dabartyje; living present), niekada nebuvo kitas, negu periferinis, niekada nebuvo aktyvaus suvokimo objektas (Cairns 1976; 54). 50 Nors pirmoje Descartes’o Samprotavimo apie metodą taisyklėje formuluojama tinkama nuostata („rūpestingai vengti skubotumo ir išankstinio nusistatymo“; „einantys palengva nueina daug to­ liau“; Descartes 1978; 112, 102), tačiau Descartes’as teikia šansą apgaulės galimybėms, apodiktinį akivaizdumą painiojančioms su tik psichologine-empirine akivaizda, neskiriant antrosios nuo pir­ mosios, t.y. filosofinės, epistemologinės akivaizdos fenomenologinės-kritinės akivaizdos prasme (žr. toliau). (DESCARTES 1978; 101–5). pasirodo ir pateikia save „galutiniu“, arba „pirminiu“ (fenomenologiškai grindžiamos metodologijos požiūriu, tai yra viena ir tas pat), buvojimo savęsp-čia (selbst da) modusu, – betarpiškai akivaizdžiai (Husserl 2005; 71–2). O tai rodo, kad radikalus fenomenologinis metodas, viena vertus, išsaugodamas nenykstamos vertės kar­ tezinių apmąstymų motyvus, ir, kita vertus, nurodydamas paskesnius pakeitimus ir pertvar­ kymus (Husserl 2005; 7), ypač subjektyvumo ir racionalumo santykių lygmenyje, lygiai tą patį gali įgyvendinti nenykstamos vertės kantiškųjų relevantiškumo tyrimų – kritiškų saviduotišku­ mo prasmių apmąstymų – srityje: „Tik kritinis kelias dar lieka atviras“ (Kantas 1982; 582). Tik būtina pridurti: išlaikant akivaizdos kritikos ir patyrimo kritikos paraleliškumą, t.y. neišleidžiant iš akių, kad aki-vaizdos suvokimas visuomet yra patyrimo patyrimas. gmenų, galiausiai sugrąžina prie saviapmąstan­ čio susidūrimo su patyrime suvokiamu vaizdu, aki-vaizda. Susidūrimas saviduotiškumo aki- vaizdoje yra relevantiškumo problemos ištakos tikrąja šio žodžio prasme (juk čia randasi vienoks arba kitoks tikrovės ir regimybės santykis ir šio santykio išdava, vaizdinys). Relevantiškumo problema, kalbant griežtai metodologiškai, reikalauja predikatinę akivaizdą genetiškai sugrąžinti prie ikipredikatinės akivaizdos, vadintinos pa-tyrimu (saviapmąstančio aš ir išgyvenamo patyrimo susidūrimu). Šis genetinis sugrįžimas prie bet kokio galimo pasaulio pa- tyrimo (aktualaus patyrimo galimybių sąlygų pa-tyrimo, skirtino nuo natūralistiškai naivių „vidinių psichologinių būsenų“ tyrimų) vadinti­ nas pa-tyrimu, įgyvendinamu, – vartojant tikslų Edmundo Husserlio terminą, – „transcendenta­ linės estetikos“ sferoje.49 Vadinasi, relevantiškumas, tinkamai pa­ pildydamas subjektyvumo ir racionalumo dimensijų ir jų tarpusavio santykio išsklaidas, padeda adekvačiai (ne subjektyvistiškai ir ne objektyvistiškai) sugrįžti prie patyrimo kaip Tai reiškia, kad relevantiškumo problema, susidedanti iš įvairių probleminių tyrimo ly­ 106 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija kumas subjektyvumo ir racionalumo tarpusavio santykį papildo adekvatumo-pasauliui (plg.: buvoti subjektu pasauliui ir drauge buvoti objektu pasaulyje) kriterijumi. Subjektyvumo ir racionalumo santykį postuluojanti sąmonė yra nu-manoma (daugiau ar mažiau hipotetinė), ir gali būti tuščia, mananti-iš-anksto, netiesioginė. Tiesioginę dimensiją – įsižiūrėjimo (einsehen) į akivaizdą – implikuoja relevantiškumo nu­ skaidrinimo galimybė.51 Tiesioginę dimensiją implikuoja relevantiškų sintezių galimybė kaip tikslas, teleologija, kaip esminis intencionalaus gyvenimo bruožas: pa-tyrimo: patyrimo ir žinojimo tarpusavio santykio, aki-vaizdos. Pirminis-ir-galutinis (aukščiausiasis) metodologinis kriterijus sugrąži­ na į kompleksiškus transcendentalinei estetikai būdingų sąsajų – relevancijų – lygmenis. Svarbu metodologiškai skirti du tarp savęs susijusios momentus – neokartezinį ir neokantiš­ ką. Pirma. Kad akivaizda ikipredikatiniame ly­ gmenyje tiesiogiai susijusi su patyrimu: pirminiu intencionalaus gyvenimo fenomenu (Husserl 2005; 71). Antra. 51 Tai, be kita ko, reiškia, kad reikia tinkamai įvertinti savitą fenomenologinio „radikalumo“, kurio nedera painioti su kasdienėmis reikšmėmis, reikalavimą. Tai, kas teigiama Husserlio Karteziškųjų meditacijų § 2 („Radikalaus filosofijos atnaujinimo būtinybė“), neturėtų būti suprantama ties­ mukai. Visos naujos teorijos siekia mažesnio arba didesnio radikalumo, o ypač „naujų prielaidų“ postulavimo srityje. Fenomenologinio saviapmąstymo kriterijus, „bent jau Husserlio požiūriu“ (Schutz 2011e; 52), rodo, kad mes nestovime pačiose filosofinės nuostabos pradžiose, susidūrę su nauju pasauliu, kurį reikėtų atrasti bei interpretuoti. Yra jau kitaip: filosofinių problemų pasaulis jau atrastas ir interpretuotas. Mes nebesame filosofavimo „pradininkai“: „Tipiškos problemų for­ mos ir tipiškos jų sprendimų galimybės ne tik esti kaip tradiciniai mūsų supratimo turiniai, bet ir rodo į raidos, kuri glūdi mūsų aprėpiamose galimybėse ir uždaviniuose, linijas“ (ten pat). Nebus per daug pakartoti: rodo į „relevancijas“. Husserlio problema – ne istoriko problema, – priduria Schutzas. Husserlis atnaujina ir tęsia Descartes’o laikysenos kryptį apodiktinio transcendentalinės filosofijos pagrindimo link. Kita Descartes’o filosofijos dalis – šio klausimo atžvilgiu – nerelevantiška (ten pat; 59). Nebus per daug pakartoti, kad Descartes’as, teikdamas mums ypatingą saviapmąsty­ mo pamoką (Husserl 2005; 10), kartu kreipia dar ir pirminio saviduotiškumo apgaulės-galimybės (Täuschungsmöglichkeiten) link (Husserl 1974; 288). (DESCARTES 1978; 101–5). Kad nepakanka apsiriboti akivaizda kaip ypatinga dekartiškojo „aiškumo“ ir „ryškumo“ rūšimi, kuri teikia tik „paskirų“ mentalinių procesų duomenis.50 „Norėdami prieiti prie ikiduoto pirminio mūsų gyvenimo pasaulio (Lebenswelt) pirminio patyrimo, turime grįžti prie suvokimo lauko, duoto mums bet kuriuo momentu; suvokimo lauko, struktūrizuoto skirtingais atžvilgiais, tu­ rinčio tematinį branduolį ir jo išorinį bei vidinį horizontus; suvokimo lauko, kuris nėra patiria­ Fenomenologiškai grindžiamos metodolo­ gijos užduotis – ne rinkti paskiras akivaizdas ir sumuoti jas užbaigtose „reprezentacijose“, o ade­ kvačiai išskleisti subjektyvumo ir racionalumo sąveikų gyvenimo-pasaulyje sąsajas. Relevantiš­ 51 Tai, be kita ko, reiškia, kad reikia tinkamai įvertinti savitą fenomenologinio „radikalumo“, kurio nedera painioti su kasdienėmis reikšmėmis, reikalavimą. Tai, kas teigiama Husserlio Karteziškųjų meditacijų § 2 („Radikalaus filosofijos atnaujinimo būtinybė“), neturėtų būti suprantama ties­ mukai. Visos naujos teorijos siekia mažesnio arba didesnio radikalumo, o ypač „naujų prielaidų“ postulavimo srityje. Fenomenologinio saviapmąstymo kriterijus, „bent jau Husserlio požiūriu“ (Schutz 2011e; 52), rodo, kad mes nestovime pačiose filosofinės nuostabos pradžiose, susidūrę su nauju pasauliu, kurį reikėtų atrasti bei interpretuoti. Yra jau kitaip: filosofinių problemų pasaulis jau atrastas ir interpretuotas. Mes nebesame filosofavimo „pradininkai“: „Tipiškos problemų for­ mos ir tipiškos jų sprendimų galimybės ne tik esti kaip tradiciniai mūsų supratimo turiniai, bet ir rodo į raidos, kuri glūdi mūsų aprėpiamose galimybėse ir uždaviniuose, linijas“ (ten pat). Nebus per daug pakartoti: rodo į „relevancijas“. Husserlio problema – ne istoriko problema, – priduria Schutzas. Husserlis atnaujina ir tęsia Descartes’o laikysenos kryptį apodiktinio transcendentalinės filosofijos pagrindimo link. Kita Descartes’o filosofijos dalis – šio klausimo atžvilgiu – nerelevantiška (ten pat; 59). Nebus per daug pakartoti, kad Descartes’as, teikdamas mums ypatingą saviapmąsty­ mo pamoką (Husserl 2005; 10), kartu kreipia dar ir pirminio saviduotiškumo apgaulės-galimybės (Täuschungsmöglichkeiten) link (Husserl 1974; 288). 107 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 mas kaip aiškus loginių sprendinių posluoksnis, ir dar nėra susklaidytas į loginius subjektus bei jų predikatus. Norėdami prieiti prie šio pirminio mūsų patyrimo lygmens, turime eliminuoti visas idealizacijų ir generalizacijų rūšis, implikuotas loginėje mūsų veikloje.“ (Schutz 1962; 278). sis savo žinojimo atsargomis ir panašių (tipiškų) kitų stebėtojų tyrimų panašiose (tipiškose) aplinkybėse struktūrine analize. Be to, netgi paprasčiausiame ko nors patyri­ mo išgyvenime esminė konstituojančios tėkmės patyrimo forma yra tokia, kad akivaizdos – keičiančios viena kitą tęstinėse modifikacijose, transcendentaliniame arba psichologiniame vidiniame gyvenime – funkcionuoja drauge: įvairiopos save konstituojančios objekto-kate­ gorijos (die mannigfaltigen, sich konstituierenden Gegenstands-kategorien) jungiasi viena su kita (ten pat; 293). (DESCARTES 1978; 101–5). Norėdami prieiti prie pirminio mūsų paty­ rimo lygmens (konvenciniai empiriniai ty rimai dažniau eina prie metodo patyrimo, o ne išgyve­ nimo patyrimo, lygmens), turime nuosekliai, t.y. adekvačiai, eiti racionalumo ir subjektyvumo santykio, kalbant dar paprasčiau, interesų ir mo­ tyvų, santykio link. Turime eiti kaip pėdsekiai, tiksliau, relevantiškai – nei pernelyg į dešinę, nei į kairę, nei pernelyg į priekį, nei atgal – lai­ kytis atstumo. Relevantiškumas yra atskaitinga laikysena, reikalaujanti visų pirma nuskaidrinti akivaizdos duomenis, įgyvendinamus vidinio laiko sintezėse. Įsižiūrėti į imanentinius objektus kaip imanentinio patyrimo sąsajų objektus, t.y. imanentinio laiko objektus, yra pirmoji užduo­ tis, nuo kurios turėtų pradėti pradedantysis fenomenologijoje (Husserl 1974; 294). O tai reiškia, kad nėra taip, kaip bando įteigti mokslinė epistemologija: kad kiekvieną objektą „atitinka“ specifinė akivaizda. Objekto akivaizda ir pats objektas kaip akivaizda įgyven­ dina funkcijas, kurios persidengia su kitomis akivaizdomis ir objektais (ten pat; 294). Štai kodėl pamatinė akivaizdos kritika gręžia į radi­ kalią („galutinę“) kritiką, kritiką tų akivaizdų, kurią fenomenologija pirminiame ir vis dar naiviame lygmenyje įgyvendina tiesiogiai. O tai reiškia, kad būdinga pirmoji supratumo, kuriuo remiasi visi kiti, kritika yra transcendentalinė savikritika, susijusi su pačiu fenomenologiniu supratumu („Die an sich erste Erkenntniskritik, in der alle andere wurzelt, ist die transzendentale Selbstkritik der phänomenologischen Erkenntnis selbst.“; ten pat; 295). Tai reiškia, kad visus betarpiškai suvoktus objektus, suvokinius, suvokimo fenomenus turime tikrinti. Turime tikrinti jų sąsajas su tikrove, – vengiant psichologistinio požiūrio, kad akivaizda yra privilegijuotas, specialusis „faktinis“ sąmonės mentalinio gyvenimo momentas. Svarbu ne paskira akivaizda, o akivaizdos kritika. Bet koks „žinojimas“ (laiky­ sena, padėtis, patvirtinimas), bet koks žinojimo modalumas (tikrumas, tikėtinumas, netikrumas, abejotinumas) rodo trimis kryptimis: 1) to, kas (tariamai) žinoma, 2) pažįstančiojo, 3) kito-aš (subjekto ar subjektų, kuriems aš, po ilgesnių ar trumpesnių apmąstymų, tyrimų, stebėjimų, galiausiai priskiriu „tam tikrą turinį“, remdama­ Vadinasi, tikrovės ir vaizdo lyginimas (ly­ ginamoji analizė), kuri reikalautų tiesioginio – fenomeno, kuris mums pasirodo ir „tikrosios“ tikrovės – lyginimo, negali būti įgyvendinamas atskirai nuo pasirodymo fenomeno, tiksliau, šio fenomeno tęstinumo ir „neapibrėžtumo“: „Bet kokia epistemologija, siekianti būti išties 108 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija laikomos savaime suprantamomis. Metodolo­ ginis relevantiškumo kriterijus kaip tik ir rodo į dar paprastesnę paprastos laikysenos-gyvenant- relevancijose prigimtį: kad objektyvūs ženklai randasi iš subjektyvių. Nei daugiau, nei mažiau: nei kad šių relevancijų tikrovė yra „objektyvi“, nei kad mes negalime suprasti įvairiais kasdie­ nybės būdais idealizuojamų, anonimizuojamų, objektyvizuojamų figūrinių sąsajų. kritiška, turi pripažinti šią esminę dilemą. 52 Politiškai radikalaus ir rečiau nei dažniau atsakingo kalbos liberalizavimo (lietuvių kalbos, o drau­ ge ir bendruomeninio patyrimo atsargų sąmonės) laikais žodį „išdava“ jau galima vartoti (žr.: Dabar­ tinės lietuvių kalbos žodyną, 2006). Tačiau čia mums rūpi ne išdava kaip „rezultatas“, o „priėjimas prie“ rezultato, taigi priėjimas prie duoties, ikiduotiškumo tapsmas. Čia domimės žodžiu „iš-dava“ kaip naujos reikšmės – iki-duoties – žodžio radimosi sąlygomis, atviromis žodžio duotis darybos ir kaitybos galimybėmis. Ikiduoties daryba čia pabrėžia ne statišką, o procesinį – duotybės radimosi patyrime – būdą. Iškeliant priešdėlį „iki-“ prieš šaknį „duot-“, siekiama ne tiek reikšminio taisy­ klingumo, kiek prasminio nuovokumo ir apibrėžtumo. Pridurtina, kad žodis „išdava“ jau varto­ jamas filosofinėje lietuvių kalboje (žr. ypač prof. Rolando Pavilionio L. Wittgensteino Filosofinių tyrimų vertimą). 53 Intencionalus saviduotiškumo įgyvendinimas („die intentionale Leistung der Selbstgebung“; Husserl 1974; 166) į lietuvių kalbą kartais verčiamas kaip išsipildanti saviduotybė, tačiau „saviduotybė“ išsipildo ne savaime (kaip trečiojo asmens dovana pirmajam asmeniui: išsipildanti malonė), o yra įgyvendinama, steigiama. „Išsipildymo“ atveju trūksta pirmojo asmens intencionalumo – sąmo­ nės ko nors – sąsajų. (DESCARTES 1978; 101–5). Epistemologija negali norėti ištraukti savo už savo plaukų iš subjektyvumo liūno dar iki aukštesniojo idealaus tarpininko lygmens. Šis pamatinis bet kokio pažinimo keblumas liudija, kad mes galime tik sugriebti fenomeną, ir nieko daugiau.“ (Spiegelberg 1940; 85). Kad ir koks „paprastas“ atrodytų lyginamasis metodas, jį sunku įgyvendinti dėl skirtingų subjektyvumo lygmenų – 1) to, kas (tariamai) žinoma, 2) pažįstančiojo, 3) kito-aš – įsitraukimo. Šioje, pažinimo požiūriu, išties šokiruojančioje episte­ mologinėje situacijoje išryškėja fenomenologiškai grindžiamos metodologijos svarba. Relevantiškumas konstituojamas tipiškumo radimosi, – sąmonės nukreiptumo ko nors link sąsajų, – ištakose. Ir vidiniame, ir išoriniame tematinio lauko horizonte, kuriame vienu metu randame suvokimo patyrimą dabartinėje erdvi­ nėje padėtyje, ir savo autobiografines situacijas dabarties momentais, kurie konstituojami kaip inter-subjektyvios patyrimo sedimentacijų išdavos.52 Kaip intencionalaus saviduotiškumo įgyvendinimas.53 Metodologijai svarbu – paprasti, arba tipiški, akivaizdos figūravimo – akivaizdos pa-vaizdavi­ mo – procesai mūsų kalboje. Kalba yra tipinių patyrimo, – kuris betarpišką subjektyvų patyri­ mą objektyvuoja, – schemų sistema. Aiškėja galimybė, leidžianti artėti prie patikimesnio pažinimo. Kasdieniame pasaulyje laikysenos- gyvenant-relevancijose turi tam tikrus pasikar­ tojančius bruožus, – nors pačios relevancijos šiose laikysenose-relevancijose gyvenančųjų Relevantiškumo dimensija kreipia į kon­ trapunktinių santykių, – taigi ir mūsų sąmonės srauto, ką nors tematizuojančio, o todėl at- renkančio, – prigimtį (Schutz 2011c; 99). Prie pačios tikrovės slenksčio veidu į veidą ir netgi akis 109 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija į akį susiduriame su ypatingu Santykiu: žmo­ giškos tikrovės Tarp radimosi didžiąja, o paskui banalesne mažąja raidėmis. Santykiu tarp savęsp patyrimo išgyvenimo (imanentiškų arba realių sa­ viduotiškumo duomenų suvokimo) ir am-žininkų patyrimo randantis panašioms, arba tipinėms, perspektyvų abipusiškumo figūroms. ir atvirkščiai. Pirmoji balsė neatskiriama nuo antrosios, kaip ir antroji – nuo pirmosios. Jos jau yra (dar prieš pranešimą, teigimą, įrodymą, empirinį pagrindimą): 1) „Apgaulės galimybė esti patyrimo akivaizdoje ir neanuliuoja nei esminio jos pobūdžio, nei jos rezultatų <...>“; 2) „Patyrimo akivaizda atitinkamai visada jau implikuota sąlygų.“ (Husserl 1974; 164)54 Kalbant metaforiškai (o šitaip pradžioje ir reikėtų: juk nuo akivaizdos, o ne miglotos reprezentacijos pradedame), prie pačios tikrovės slenksčio mūsų laukia susidvejinusi patyrimo ir žinojimo santykio Regimybė. Scilė ir Charibdė gražiais lietuviškais – Apgaulės ir Akivaizdos – vardais. Regimybė tikrovėje, arba tikrovė regimy­ bėje (o tai yra viena ir tas pat). Pereigos iš vienos į kitą tiltas vadinamas įvairiais – pranešimo, teigimo, įrodymo, pagrindimo, empirinių duomenų – vardais. Ã-Ã esti sklindančios, – dar prieš garso išgir­ dimą, o ką jau kalbėti apie ištarimą, – bangos. 54 „Die Möglichkeit der Täuschung gehört mit zur Evidenz der Erfahrung und hebt ihren Grund­ charakter und ihre Leistung nicht auf <...>; Evidenz der Erfahrung ist also hierbei immer schon vorausgesetzt“ (ten pat). Atranka: tokį vardą tikrovės ir regimybės („matomo, bet nepastebimo“ fenomeno, kurį pernelyg nuvertina tradicinė epistemologija) santykiui suteikia Alfredas Schutzas, nerdamas į gilius vandenis, – irkluodamas relevantiško skeptiko (reikia skirti nuo pomodernaus nerelevantiško: „Jei Nieko nėra, tai viskas galima“) Karneado link: „Williamo Jameso Psicholo­ gijos principų ir Bergsono ankstyvųjų tekstų publikavimas suteikė daugelio pripažintą galimybę kalbėti apie sąmonei būdingą atranką. Bet abu šie didieji filosofai grindė atranką beveik vien tik remdamiesi praktiniu motyvu. Pasak Bergsono, tai mūsų praktinis interesas, apibrėžiantis veiksmą išoriniame pasaulyje, apribojantis sąmonės lauko elementus, kuriuos sugriebia mūsų intelektas. Apibrėždamas ir apribodamas mūsų patyrimo pasaulyje segmentus kalboje, tampančioje tematiš­ ka, intelektas iki-apibrėžia linijas paskesniam veiksmui, – tuo būdu, kuriuo skulptorius apibrėžia kontūrus marmuro medžiagoje formuodamas juos savo kirtiklio mostais. (Ši metafora, be kita ko, teikia nuorodą į Leibnizą, kuris ją vartojo aprašydamas sampratų ir sąvokų kilmę be nuorodų į praktinius interesus. Metafora atgręžia netgi į Karneadą, kuris, pasak Cicerono aprašymų [De divinatione, I, XIII, XXIII], vartoja ją kaip pavyzdį, svarstydamas santykį tarp tikėtinumo ir atsi­ tiktinumo.)“ (Schutz 2011c; 95) 55 „Ich weiß“ hat nur Sinn, wenn eine Person es äußert. Dann aber ist es gleichgültig, ob die Äußerung ist „Ich weiß ...“ oder „Das ist ...“.“ (Wittgenstein 1970; 49). (DESCARTES 1978; 101–5). Todėl tikrovei svarbu ne tik mūsų ištartis, bet ir tartis. Kaip savo pagavia klausa garsą intuityviai sugriebia Ludwigas Wittgensteinas: „Aš žinau“ turi prasmę tik tuomet, kai tai ištaria asmuo. Tačiau tada būna nesvarbu, ar ištartis yra „Aš žinau...“, ar „Tai yra...“ (Wittgenstein 2009; 127 [588]).55 „Tai“ (žr. santykio tarp Aš ir Tai aptarimą II-oje šio teksto dalyje) rodo, kad mes visi, – ir netgi gamtos mokslininkų „pagalbiniai darbi­ ninkai“, natūralizmo filosofai, – susiduriame Dar tiksliau: akivaizdos garsais. Visad atviro­ mis balsėmis – A-A – sklindančiomis ir tyliomis, ir garsiomis bangomis: iš apgaulės į akivaizdą, 55 110 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija bar, momentai, kurie neapriboja nuolat atviros galimybės, kad ši ar kuri kita laisvė bus užblo­ kuota“ (Husserl 2005; 57–8). Viena vertus, mes turime žinojimo atsargas, jau teikiančias mums interpretacines struktūras (matomas, bet nepastebimas „referencijų schemas“), kita vertus, turime galimybę steigti skirtingas, netgi alterna­ tyvias interpretacines struktūras (matomas, bet nepastebimas „relevancijų sistemas“). su patyrimo akivaizdos regimybėmis ir ap­ gaulės galimybėmis. „Tai“ rodo, kad filosofas, mokslininkas, kasdienybės gyventojas ir netgi ekonomistas (asmuo, modeliuojantis priežas­ ties-padarinio ir priemonių-tikslų figūras, iš kurių mes visi kartais pešame naudos) randasi atvirame relevantiškumo problemos horizonte ir visada turi nepilną, neužbaigtą supratimą apie štai-šią problemą. Būtent šiuo atveju inter-subjektyvumas ir gyvenimo-pasaulis yra tinkamiausi žodžiai (per brūkšnelius). Šias dvi matomas, bet nepastebimas gyve­ nimo-pasaulio prigimtis – referencijų schemas ir relevancijų sistemas – struktūrizuotai susieja subjektyvumo, racionalumo, relevantišku­ mo dimensijos. Svarstant subjektyvumo ir racionalumo tarpusavio santykio problemą, į dienos šviesą iškyla artimumo ir anonimiškumo sąvokos, o nagrinėjant relevantiškumo proble­ mą – reikšmingumo (susietumo, prasmingumo, tęstinumo) ir atsainumo (atsietumo, dėmesin­ gumo spindulio išcentrinimo, nerevantiškumo) sąvokos. Štai kodėl ir fenomenologinės sąvokos, ir pati fenomenologija visuomet pasirodo ne kaip naujų, o jau kažkur kažkada matytų sąvokų ištakos. Matytų išorinių ir vidinių inter- subjektyvumo horizontų neapibrėžtume, dar nepažįstamume.56 „Per brūkšnelius“ reiškia: pa-saulis nuo pačios pradžios yra su-bendrintas, su-žmogintas drauge: konstituojant daiktiškumą, objek­ tyvumą, pasauliškumą. Gyvenimo-pasaulis yra savitai – „Patyrimo akivaizdos visada jau implikuotos sąlygų“ – struktūrizuojamas per brūkšnelius, per relevantiškumų funkcijas. Net tai, ką skubotai vadiname „tik subjektyviu požiūriu“, mums jau duota socialiniuose sąly­ čiuose, subjektyviai žingsniuojant dabar jau „tik objektyvaus požiūrio“ apskritimu: nuo nuo- monės , arba jau esamų žinojimo atsargų, prie atsargaus manęs (nes ne savęs), o paskui – nuo užbaigtos sąmonės prie užbaigtos nuomonės. 56 Akivaizda visada yra patyrimas, kurį privalau reflektuoti ir tikrinti jos saviduotybės (Selbstgebung) laipsnį (Husserl 2005; 19–20). 57 „<...> stebėdamas pats save, esu linkęs daugiau nepasitikėti savimi, negu pasikliauti <...>“ (Des­ cartes 1978; 102). Drąsią, nesibaiminančią tamsių kampų, kartezinę maksimą puikiai atliepia lie­ tuviškais prie-žodis, linkintis mums, ne tik prancūzams, nepaliaujamai tikrinti aki-vaizdą: „Kiek akys norės, tiek niekas neturės.“ 56 Akivaizda visada yra patyrimas, kurį privalau reflektuoti ir tikrinti jos saviduotybės (Selbstgebung) laipsnį (Husserl 2005; 19–20). 57 b d l k d k kl “ (D 56 Akivaizda visada yra patyrimas, kurį privalau reflektuoti ir tikrinti jos saviduotybės (Selbstgebung) laipsnį (Husserl 2005; 19–20). 57 „<...> stebėdamas pats save, esu linkęs daugiau nepasitikėti savimi, negu pasikliauti <...>“ (Des­ cartes 1978; 102). Drąsią, nesibaiminančią tamsių kampų, kartezinę maksimą puikiai atliepia lie­ 57 „<...> stebėdamas pats save, esu linkęs daugiau nepasitikėti savimi, negu pasikliauti <...>“ (Des­ cartes 1978; 102). Drąsią, nesibaiminančią tamsių kampų, kartezinę maksimą puikiai atliepia lie­ tuviškais prie-žodis, linkintis mums, ne tik prancūzams, nepaliaujamai tikrinti aki-vaizdą: „Kiek akys norės, tiek niekas neturės.“ (DESCARTES 1978; 101–5). Relevantiškumas kaip problema yra ben­ drabūtiškumo patyrimo horizontinio konteks­ tualizavimo tradicijoje paslaptis: „Čia visur su tomis galimybėmis sąveikauja momentai iš tokios sferos kaip Aš galiu ir Aš darau, arba, atitinkamai, Galiu daryti kitaip, negu darau da­ Mes judame tarp mūsų pačių konstituojamų relevantiškumo-nerelevantiškumo linijų ir kreivių; lemtingai, bet ne neišvengiamai randamės aki­ vaizdaus-ir-apgaulingo tikrumo įmestyje (žinau, kad nežinau, kad nežinau...57 56 Akivaizda visada yra patyrimas, kurį privalau reflektuoti ir tikrinti jos saviduotybės (Selbstgebung) laipsnį (Husserl 2005; 19–20). 57 bėd li k d i i ikė i i i ikli i “ (D 111 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Štai kodėl fenomenologiškai grindžiama metodologija vadintina supratumo teorija (Er­ kenntnistheorie), kurios paskirtis yra reflektuoti akivaizdos fenomenus integraliuose subjektyvu­ mo, racionalumo, relevantiškumo sąryšiuose. Kalbant glaustai: išskleisti prasminių tikrovių susietumo fenomenus. akivaizdos (ten pat), taip ir metodologinis są­ monės gyvenimas mokslinėje laikysenoje negali egzistuoti neįtraukdamas sąsajų, Alfredo Schutzo vadinamų relevancijomis, akivaizdos. Pasakymas „Tai akivaizdu“ reikalauja, pir­ ma, gręžtis ne į „tai“, o į „aš“, antra, patyrimo iš-gyvenime ir per-gyvenime tikrinti aki-vaizdą. Kad akivaizda atsivertų kaip savi-akivaizdi. Kad akivaizda – jau kaip savi-akivaizdi – gręžtųsi ne į iki-pasaulį ir ne už-pasaulį, o į gyvenimo- pasaulį, kursai esti man, kaip socialinės srities mokslininkui, prieš-duotas bendrojo-gėrio laikysenoje. Kadangi, kaip teigia Filosofas, visų mokslų ir menų tikslas yra gėris (Aristotelis 1997; 157; [1282b 10–11]). O tai labai skiriasi nuo mokslinės episte­ mologijos ir empiristinės psichologijos bandymų indukciniais arba priežastiniais empiriniais būdais „išgauti“ vidinio psichinio gyvenimo „empirinius elementus“, „nenatūralius specduo­ menis“ („absonderliches Spezialdatum“; Husserl 1974; 295). Kaip kad sąmonės gyvenimas natūralioje laikysenoje negali egzistuoti neįtraukdamas LITERATŪRA Aristotelis. 1997. Politika (iš senosios graikų kalbos vertė Mindaugas Strockis). Vilnius: ALK Pradai. Ardrey, Robert. 1970. The Social Contract. A Personal Inquiry into Evolutionary Sources of Order and Disorder. New York: Atheneum. Arendt, Hannah. 1968. Men in Dark Times. New York: A Harvest Book Harcourt, Brace & World Inc. Audi, Robert. 2004. „Theoretical Rationality. Its Sources, Structure, and Scope“ in Alfred R. Mele (ed.) The Oxford Handbook of Rationality: 17–34. Arendt, Hannah. 1969. On Violence. San Die­ Bacon, Francis. 2001. The Essays. www. Mo­ zambook.net go, New York, London: Harcourt Brace Jovanovich. Barton, Tamsyn S. 1994. Power and Knowledge. Astrology, Physiognomies, and Medicine under the Ro­ man Empire. Ann Arbor: The University of Michi­ gan Press. Arendt, Hannah. 1995. Tarp praeities ir ateities (iš anglų kalbos vertė Arvydas Šliogeris). Vilnius: Aidai. Arendt, Hannah. 2001. Totalitarizmo ištakos (iš anglų kalbos vertė Arvydas Šliogeris). Vilnius: Tyto alba. Bauman, Zygmunt. 1996. „On Communitar­ ians and Human Freedom: Or, How to Square the Circle“, Theory, Culture, and Society 13 (2): 79–90. Aristotelis. 1990a. Kategorijos (iš senosios grai­ kų kalbos vertė Jonas Dumčius, Marcelinas Ročka, Vosylius Sezemanas) kn. Aristotelis. Rinktiniai raštai. Vilnius: Mintis. h Bellamy, Richard. 1999. Liberalism and Plural­ ism. Towards a politics of compromise. London and New York: Routledge. Aristotelis. 1990b. Nikomacho etika (iš seno­ sios graikų kalbos vertė Jonas Dumčius, Marcelinas Ročka, Vosylius Sezemanas) kn. Aristotelis. Rinkti­ niai raštai. Vilnius: Mintis. Berger, Peter. 1984. „On the Obsolence of the Concept of Honour“ in Michael J. Sandel (ed.) Lib­ eralism and Its Critics. New York: New York Univer­ sity Press: 149–58. 112 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Berger, Peter L.; Luckmann, Thomas. 1995. Modernity,Pluralism and the Crisis of Meaning. The Orientation of Modern Man. Gütersloh: Bertels­ mann Foundation Publishers. Gurwitsch, Aron. 1989. „As late as the begining of the eighteen century there was a word „relevance“ in roughly our sense“ in Richard Grathoff (ed.) Philosophers in Exile. The Correspondence of Alfred Schutz and Aron Gurwitsch, 1939–1959. (Trans. by J. Claude Evans.) Bloomington and Indianapolis: Indiana University Press: 224–5. Berlin, Isaiah. 1995. Vienovė ir įvairovė (iš anglų kalbos vertė Alvydas Jokubaitis). Vilnius: Amžius. Cairns, Dorion. 1940. „An Approach to Phe­ nomenology“ in Marvin Farber (ed.) Philosophical Essays. In Memory of Edmund Husserl. Cambridge, Mass.: Harvard University Press. Hayek, Friedrich A. 1948. Individualism and Economic Order. Chicago: University of Chicago Press. Cairns, Dorion. 1976. Conversations with Hus­ serl and Fink (ed. LITERATŪRA By Husserl-Archives in Louvain; with a Foreward by Richard M. Zaner). The Hague: Martinus Nijhoff. Hayek, Friedrich A. 1978. New Studies in Phi­ losophy, Politics, Economics and the History of Ideas. London: Routledge. Hayek, Friedrich A. 1979. Law Legislation and Liberty, vol 3: The Political Order of a Free People. Chicago: University of Chicago Press. Camus, Albert. 2007a. „Absurdas ir savižudy­ bė“ kn. Albertas Camus, Sizifo mitas (iš prancūzų kalbos vertė Violeta Tauragienė). Vilnius Baltos lankos: 10–6. Hayek, Friedrich A. 1991. Kelias į vergovę (iš an­ glų kalbos vertė Algirdas Degutis). Vilnius: Mintis. Camus, Albert. 2007b. „Absurdo sienos“ kn. Albertas Camus, Sizifo mitas: 17–33. Hayek, Friedrich A. 2002. Individualizmas ir ekonominė tvarka (iš anglų kalbos vertė Algirdas De­ gutis). Vilnius: ALK / Eugrimas. Albertas Camus, Sizifo mitas: 17–33. Camus, Albert. 2007c. „Absurdo laisvė“ kn. Al­ bertas Camus, Sizifo mitas: 55–69. Hayek, Friedrich A. 2008. Free Market Mone­ tary System. Auburn, Alabam: Ludwig von Mises Institute. Camus, Albert. 2007d. „Sizifo mitas“ kn. Al­ bertas Camus, Sizifo mitas: 123–7. Canetti, Elias. 2007. Apakimas (iš vokiečių kalbos vertė Teodoras Četrauskas). Vilnius: Lietu­ vos rytas. Hegel, Georg Wilhelm Friedrich. 2000. Teisės filosofijos apmatai, arba Prigimtinės teisės ir valstybės mokslo metmenys (vertė Loreta Anilionytė). Vilnius: Mintis. Curtis. Neal. 2004. „Nihilism, Liberalism and Terror“, Theory, Culture & Society 21 (3): 141–157. Hobbes, Thomas. 1998. Leviathan, or the Mat­ ter, Forme, & Power of a Common-wealth Ecclesiasti­ cal and Civil (ed. With an Introduction and Notes by C. A. Gaskin). Oxford, New York: Oxford Uni­ versity Press. Dawson, Christopher. 1931. „Introduction“ in Carl Schmitt, The Necessity of Politics. An Essay on the Representative Idea in the Church and Modern Europe. London: Sheed & Ward: 9–28. Descartes, René. 1978a. Samprotavimas apie metodą. Kn.: Descartes, René. Rinktiniai raštai (iš prancūzų k. vertė Gvidonas Bartkus, Petras Račius). Vilnius: Mintis. Hobbes, Thomas. 1983. De Cive. Philosophical Rudiments Concerning Government and Society (Eng­ lish Version). Oxford, New York: Oxford University Press. Descartes, René. 1978b. Metafiziniai apmąs­ tymai. Kn.: Descartes, René. Rinktiniai raštai (iš prancūzų k. vertė Gvidonas Bartkus, Petras Račius). Vilnius: Mintis. Hobbes, Thomas. 1999. Leviatanas (iš anglų kalbos vertė Kęstutis Rastenis). Vilnius: Pradai. Husserl, Edmund. 1939. Erfahrung und Urteil. Untersuchungen. Zur Genealogie Der Logik. Prag: Academia / Verlagsbuchhandlung. Gray, John. 1993. Post-liberalism. Studies in Po­ litical Thought. London and New York: Routledge. Gray, John. 1996. „What Liberalism Cannot Do“, New Statement & Society (September 20): 18–21. Husserl, Edmund. 1952. LITERATŪRA Ideen Zu Einer Reinen Phänomenologie und Phänomenologischen Philoso­ phie. Zweites Buch. Phänomenologishe Untersuchun­ 113 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 gen Zur Konstitution. (Hrsg. H. L. von Marly Bie­ mel.) Band IV. Haag: Martinus Nijhoff. Manent, Pierre. 2008. Demokratija be tautų. Apie savivaldos pabaigą Europoje (iš prancūzų kal­ bos vertė Jūratė Navakauskienė). Vilnius: VERSUS AUREUS. f Husserl, Edmund. 1974. Formale Und Trans­ zendentale Logik: Versuch einer Kritik der logischen jetzt kaufen. (Hrsg. von H. L. Van Breda.) Husser­ liana. Edmund Husserl. Gesammelte Werke. Band XVII. Haag: Martinus Nijhoff. Mohanty, Jitendra Nath. 1973. „Towards a Phenomenology of Self-Evidence“ in David Carr and Edward S. Casey (eds.) Explorations in Phenom­ enology. The Hague: Martinus Nijhoff: 208–29. f Husserl, Edmund. 1976a. Ideen zu einer reinen Phänomenologie und phänomenologischen Philo­ sophie. Buch 1, Allgemeine Einführung in die reine Phänomenologie. (Hrsg. von Erstes Buch.) Edmund Husserl. Husserliana. Gesammelte Werke. Band III/1. Haag: Martinus Nijhoff. hf Montesquieu, Charles. 2004. Apie įstatymų dva­ sią (iš prancūzų kalbos vertė Vita Malinauskienė). Vilnius: Mintis. Natanson, Maurice. 1970. “Alfred Schutz on Social Reality and Social Science” in Maurice Na­ tanson (ed.) Phenomenology and Social Reality. The Hague: Martinus Nijhoff: 101–21. f Husserl, Edmund. 1976b. Die Krisis der europä­ ischen Wissenschaften und die transzendentale Phäno­ menologie. Eine Einleitung in die phänomenologische Philosophie. (Hrsg. von Walter Biemel.) Husserlia­ na. Edmund Husserl. Gesammelte Werke. Band VI. Haag: Martinus Nijhoff. g jf Nicolacopoulos, Toula. 2008. The Radical Critique of Liberalism: In Memory of a Vision. Mel­ bourne: e.press. Norkus, Zenonas. 2008. Kokia demokratija, koks kapitalizmas? Pokomunistinė transformacija Lietuvoje lyginamosios istorinės sociologijos požiūriu. Vilnius: Vilniaus universiteto leidykla. f Joxe, Alain. 2002. Empire of Disorder. Los Ange­ les, CA and New York: Semiotext(e). Jokubaitis, Alvydas. 2012. Vertybių tironija ir politika. Vilnius: Vilniaus universiteto leidykla. Nussbaum, Martha. 2010. Not for profit : why democracy needs the humanities. Princeton, New Jer­ sey: Princeton University Press. Kantas, Immanuelis. 1982. Grynojo proto kri­ tika (iš vokiečių kalbos vertė Romanas Plečkaitis). Vilnius: Mintis. Oakeshott, Michael. 1975. Hobbes on Civil As­ sociation. Oxford: Basil Blackwell. Kantas, Immanuelis. 1987. Praktinio proto kri­ tika (iš vokiečių kalbos vertė Romanas Plečkaitis). Vilnius: Mintis. Ortega y Gasset, José. 1993. Masių sukilimas (iš ispanų kalbos vertė Elena Treinienė). Vilnius: Vaga. Kantas, Immanuelis. 1996. „Atsakymas į klau­ simą, kas yra Švietimas“ kn. Politiniai traktatai (iš vokiečių kalbos vertė Antanas Gailius ir Gediminas Žukas). Vilnius: Aidai: 4 Ortega y Gasset, José. 1999. „Mūsų laikų tema“ kn. LITERATŪRA José.Ortega y Gasset, Mūsų laikų tema ir kitos esė (iš ispanų kalbos vertė Rūta Samuolytė). Vilnius: Vaga. Keršytė, Nijolė. 2012. „Atmintis ir kartotė vakarietiško mąstymo tradicijoje“, ATHENA 8: 198–226. Parsons, Talcott. 1949. The Structure of the So­ cial Action. A Study in Social Theory with Special Ref­ erente to a Group of Recent European Writers. Glen­ coe, Ill.: The Free Press. Kymlicka, Will. 1989. Liberalism, Community and Culture. Oxford: Clarendon Press. h Parsons, Talcott. 1951. The Social System. Lon­ don: Routledge. La Mettrie, Julien Offray de. 2011. Žmogus- Mašina (iš prancūzų kalbos vertė Petras Raščius). Vilnius: Vaga. Rawls, John. 2001. Justice as Fairness: A Restate­ Rawls, John. 2001. Justice as Fairness: A Restate­ ment. Cambridge, MA: Harvard University Press. Sandel, Michael J. 1998. Liberalism and the Limits of Justice (second ed.). Cambridge: Cam­ bridge University Press. ment. Cambridge, MA: Harvard University Press. Lorenz, Konrad. 1974. Civilized man’s eight deadly sins: Harcourt Brace Jovanovich. g Sandel, Michael J. 1998. Liberalism and the Limits of Justice (second ed.). Cambridge: Cam­ bridge University Press. MacIntyre, Alasdair. 1988. Whose Justine? Which Rationality? Notre Dame: Notre Dame Uni­ versity Press. Sartre, Jean-Paul. 2002. Šleikštulys (iš prancūzų kalbos vertė Alina Kiliesaitė). Vilnius: Vaga. 114 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Correspondence of Alfred Schutz and Aron Gurwitsch, 1939–1959. (Trans. by J. Claude Evans.) Bloom­ ington and Indianapolis: Indiana University Press: 226–7. Schmitt, Carl. 2014. Politinė teologija. Politinė teologija II (iš vokiečių kalbos vertė Antanas Gail­ ius).Vilnius: Versus Aureus. Schütz, Alfred. 1932. Der Sinnhafte Aufbau der sozialen Welt. Eine Einleitung in die verstehende Sozi­ ologie. Wien: Verlag von Julius Springer. Schutz, Alfred. 1996. Collected Papers IV (ed. by Helmut Wagner and George Psathas in collobo­ ration with Fred Kersten). The Hague / Boston / London: Martinus Nijhoff. Schutz, Alfred. 1962. Collected Papers I. The Problem of Social Reality (ed. by M. Natanson). The Hague / Boston / London: Martinus Nijhoff. Schutz, Alfred. 2011a. “Husserl and His Influ­ ence on Me” in Lester Embree (ed.) Collected Papers V. Phenomenology and the Social Sciences. Dordrecht, H id lb L d N Y k S i 1 4 Hague / Boston / London: Martinus Nijhoff. f Schutz, Alfred. 1964. Collected Papers II. Studies in Social Theory (ed. by A. Brodersen). The Hague / f Heidelberg, London, New York: Springer: 1–4. Boston / London: Martinus Nijhoff. Schutz, Alfred. 1966 [1975]. Collected Papers Schutz, Alfred. 2011b. LITERATŪRA “The Theory of Social Action and Letters with Talcott Parsons” in Lester Embree (ed.) Collected Papers V. Phenomenology and the Social Sciences. Dordrecht, Heidelberg, London, New York: Springer: 5–74. III. Studies in Phenomenological Philosophy (ed. h M. Natanson). The Hague / Boston / London: M rtin Nijh ff M. Natanson). The Hague / Boston / London: f Martinus Nijhoff. Schutz, Alfred. 1967. The Phenomenology of the Social World (trans. by George Walsh and Frederick Lehnert). Evanston, IL: Northwestern University Press. Schutz, Alfred. 2011c. “Reflections on the Problem of Relevance” in Lester Embree (ed.) Col­ lected Papers V. Phenomenology and the Social Sci­ ences. Dordrecht, Heidelberg, London, New York: Springer: 93–199. Schutz, Alfred; Luckmann, Thomas. 1973. The Structures of the Life-World (trans. by Richard M. Zaner and H. Tristram Engelhardt, Jr.), vol. 1. Ev­ anston, IL: Northwestern University Press. Schutz, Alfred. 2011d: „Schütz to Voegelin“ (November 11, 1943) in Gerhard Wagner and Gil­ bert Weiss (eds.) A Friendship That Lasted a Lifetime (trans. by William Petropulos). Columbia and Lon­ don: University of Missouri Press: 49–59. Schutz, Alfred. 1978a. „Parson’s Theory of So­ cial Action: A Critical Review” in R. Grathoff (ed.) The Theory of Social Action: The Correspondence of Alfred Schutz and Talcott Parsons (Bloomington / London: Indiana University Press: 8–60. Schutz, Alfred. 2011e: „Schütz to Voegelin“ (April 22, 1951) in Gerhard Wagner and Gilbert Weiss (eds.) A Friendship That Lasted a Lifetime (trans. by William Petropulos). Columbia and Lon­ don: University of Missouri Press:135–9. Schutz, Alfred. 1978b. “You have to go a few steps further in radicalizing your theory: March 17. 1941.” in R. Grathoff (ed.) The Theory of Social Ac­ tion: The Correspondence of Alfred Schutz and Talcott Parsons (Bloomington / London: Indiana University Press: 95–106. Schutz, Alfred. 2011f: „Schütz to Voegelin“ (November 1952) in Gerhard Wagner and Gilbert Weiss (eds.) A Friendship That Lasted a Lifetime (trans. by William Petropulos). Columbia and Lon­ don: University of Missouri Press:155–69. Schutz, Alfred. 1989a. „A Letter to Aron Gur­ witsch, January 25, 1952“ in Richard Grathoff (ed.) Philosophers in Exile. The Correspondence of Alfred Schutz and Aron Gurwitsch, 1939–1959. (Trans. by J. Claude Evans.) Bloomington and Indianapolis: Indiana University Press: 153–60. Schrag Calvin O. 1983. Existence and Freedom Towards an Ontology of Human Finitude. Albany: State University of New York Press. Seebohm, Thomas M. 2010. „Naturalism, His­ torism, and Phenomenology“ in Thomas Nenon and Philip Blosser (eds.) Advancing Phenomenology. LITERATŪRA Essays in Honor of Lester Embree. Dordrecht, Heidel­ berg, London, New York: Springer: 7–34. Schutz, Alfred. 1989b. „It has become impor­ tant to me to contrast the „world of working“ and the „world of everyday life“, New York, June, 1954“ in Richard Grathoff (ed.) Philosophers in Exile. The 115 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 London, Thousand Oaks, New Delhi: SAGE Pub­ lications. Seidman, Steven. 1992. „Postmodern Social Theory as Narrative with a Moral lntent“ in Steven Seidman and David Wagner (eds.) Postmodernism and Social Theory. Oxford: Blackwell: 47–81. Unger, Roberto Mangabeira. 2001. False Neces­ sity. Anti-Necessitarian Social Theory in the Service of Radical Democracy. London, New York: Verso. Ši Radical Democracy. London, New York: Verso.i Simmel, Georg. 2007. Sociologija ir kultūros filosofija. Rinktinė (iš vokiečių kalbos vertė Albinas Lozuraitis). Vilnius: Margi raštai. Šliogeris, Arvydas. 2010. Bulvės metafizika. Iš filosofo dienoraščių. Vilnius: Apostrofa. Vaitkus, Steven. 1991. How Is Society Possibble? Intersubjectivity and the Fiduciary Attitude as Prob­ lems of the Social Group in Mead, Gurwitsch, and Schutz. Dordrecht, Boston, London: Kluwer Aca­ demic Publishers. Sokolowski, Robert. 1970. The Formation of Husserl’s Concept of Constitution. The Hague, Ni­ jhoff. Evanston: Northwestern University Press. Sokolowski, Robert. 1974. Husserlian Medita­ tions. How Words Present Things. Evanston: North­ western University Press. Whitehead, Alfred North. 1978. Process and Reality. An Essay in Cosmology. New York: The Free Press. Spiegelberg, Herbert. 1940. „The „Reality- Phenomenon“ and Reality“ in Marvin Farber (ed.) Philosophical Essays. In Memory of Edmund Hus­ serl. Cambridge, Mass.: Harvard University Press: 84–105. Wittgenstein, Ludwig. 1970. Über Gewißheit (Herausgegeben von G. E. M. Anscombe und G. Wittgenstein, Ludwig. 1970. Über Gewißheit (Herausgegeben von G. E. M. Anscombe und G. H. von Wright.) Frankfurt am Main: Suhrkamp Verlag.i g g H. von Wright.) Frankfurt am Main: Suhrkamp Verlag.i Strauss, Leo. 1963. The Political Philosophy of Hobbes. Its Basis and Its Genesis. Chicago, London: The University of Chicago Press. Wittgenstein, Ludwig. 1995. Filosofiniai tyrinėjimai kn. Liudvikas Vitgenšteinas Rinktiniai raštai (iš vokiečių ir anglų kalbų vertė Rolandas Pa­ vilionis). Vilnius: Mintis. Sverdiolas, Arūnas; Kačerauskas, Tomas. 2008. „Filosofija Lietuvoje“, Problemos 74: 16–26. Wittgenstein, Ludwig. 2009. Apie tikrumą (iš vok. kalbos vertė Saulenė Pučiliauskaitė). Vilnius: Baltos lankos. Taylor, Charles. 1979. „What’s Wrong with Negative Liberty“ in Alan Ryan (ed.) The Idea of Freedom. Essays in Honour of Isaiah Berlin. New York, Toronto, Melbourne: Oxford University Press: 175–94. Voegelin, Eric. 2011. 140–3. Trinarė išnaša X, Y, Z: Tocqueville’io tezės – kad lygybė turi polinkį iš esmės pažeidinėti laisvės teises – fenomenologinis skaidrinimas Trinarė išnaša X, Y, Z: Tocqueville’io tezės – kad lygybė turi polinkį iš esmės pažeidinėti laisvės teises – fenomenologinis skaidrinimas TOWARDS A PHENOMENOLOGICALLY GROUNDED UNDERSTANDING OF SOCIAL THEORY. III. THE PROBLEM OF RELEVANCE The third part of the article aims to specify the phenomenological aspects of the problem of relevance. The paper focus upon four major areas of concern: investigation of basic concepts such as social reality, human action, and social interaction (I); investigation of the philophical roots of the problem by focus­ ing reflectively on the perceptual field, and attempting to describe the primary locus of self-evidence (II); analyses of fundamental matters of political philosophy as state, community, society, individual (III); and finally, investigation of the relationship between the freedom and equality, and of various aspects of this fundamental relationship in contemporary Lithuanian society. Sociologijos katedra, Filosofijos fakultetas Vilniaus universitetas, Universiteto 9/1; LT-01513 Vilnius LITERATŪRA „Voegelin to Schütz“ (April 30, 1951) in Gerhard Wagner and Gilbert. Walzer, Michael. 1990. „The Communitarian Critique of Liberalism“, Political Theory 18 (1): 6–23. Taylor, Charles. 1994. „The Politics of Recog­ nition“ in Amy Gutmann (ed.) Multiculturalism. Examining the Politics of Recognition. Princeton: Princeton University Press: 25–74. Weiss (eds.) A Friendship That Lasted a Lifetime (trans. by William Petropulos). Columbia and Lon­ don: University of Missouri Press: 140–3 . Tocqueville, Alexis de. 1996. Apie demokratiją Amerikoje (iš prancūzų kalbos vertė Valdas Petraus­ kas). Vilnius: Amžius. Wagner, Gerhard; Weiss, Gilbert (eds.) 2011. A Friendship That Lasted a Lifetime (trans. by William Petropulos). Columbia and London: University of Missouri Press. Toru, Tani. 2010. „Quo Vadis, Phenomenol­ ogy?“ in Thomas Nenon and Philip Blosser (eds.) Advancing Phenomenology. Essays in Honor of Lester Embree. Dordrecht, Heidelberg, London, New York: Springer: 103–118. Petropulos). Columbia and London: University of Missouri Press. Weber, Max. 2000. Religijos sociologija (iš vokiečių kalbos vertė Zenonas Norkus). Vilnius: Pradai. Turner, Bryan S. 1986. Equality. London and New York: Tavistock Publications. Weber, Max. 1922. Wirtschaft und Gesellschaft (Grundriß der Sozialökonomik. Abt. III). Tübingen: J.C.B Mohr. Turner, Bryan S.; Rojek, Chris. 2001. Soci­ ety and Culture. Principles of Scarcity and Solidarity. Wolin, Sheldon S. 2008. Democracy Incorporat­ Wolin, Sheldon S. 2008. Democracy Incorporat­ 116 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 ed: Managed Democracy and the Specter of Inverted Totalitarianism. Princeton:  Princeton University Press. ed: Managed Democracy and the Specter of Inverted Totalitarianism. Princeton:  Princeton University Press. ed: Managed Democracy and the Specter of Inverted Totalitarianism. Princeton:  Princeton University Press. Zaner, Richard M. 1973. „Reflections on Evi­ dence and Criticism in the Theory of Conscious­ ness“ in David Carr and Edward S. Casey (eds.) Ex­ plorations in Phenomenology. The Hague: Martinus Nijhoff: 184–207. Zaner, Richard M. 1970. „The Phenomenol­ ogy of Epistemic Claims: And Its Bearing on the Essence of Philosophy“ in Maurice Natanson (ed.) Phenomenology and Social Reality. Essays in Memory of Alfred Schutz. The Hague: Martinus Nijhoff: 17–34. Достоевский Ф. М. 1985. Записки из подполъя. В кн: Достоевский Ф. М. Собр. соч., тт. 2, 3. М.: Правда. Keturi bendrieji laisvės nepakantumo nelais­ vei ir nelaisvės artimumo laisvei postulatai. 2. Laisvė yra dar iki prie-vartos, dar prie- vartų, už kurių – bereikšmių, todėl be­ prasmiškų žodžių vartojimo perteklius. 2. Laisvė yra dar iki prie-vartos, dar prie- vartų, už kurių – bereikšmių, todėl be­ prasmiškų žodžių vartojimo perteklius. Nelaisvė – už vartų: savaime supran­ tamoje beprasmiškų žodžių vartojimo prievartoje. 1. Laisvę nuo ne-laisvės skiria vos mažas brūkšnelis. Nelaisvę nuo laisvės neskiria niekas. Niekas yra matomas, bet nepas­ tebimas savaime suprantamumas. Nelaisvė – už vartų: savaime supran­ tamoje beprasmiškų žodžių vartojimo prievartoje. 117 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 3. Laisvė gimsta iš savigrąžos prie-varton, artimų-žodžių-vartojiman: tebesilaiky­ damas savaime suprantamų galvojimo ir elgsenos būdų, aš artėju prie kalbos vartų, už kurių prasideda veiksmo na­ tūralizmas, žodžių vartojimas savaime suprantamumo pertekliuje. vyros (įsisąmonintos lygybės negalimybės, laisvės būtinybės ir teisingumo tikimybės); sakinys „Nepriklausomybėje aš net nesapnavau laisvės, nors ilgai, – ne tiek saldžiai, kiek neramiai, – miegojau“, tai­ komas faktiškoje – politinės savivartos – situacijoje, yra klaidingas ir bereikšmis, tačiau ne beprasmis, nes aš turiu žinoti, kur ieškoti sapno, laisvės įsikūnijimo priešduotiškume požiūriu; 4. Žodžių varta kartotiniame veiksme yra prie-varta, tenkinimasis-lemtimi, bet dar ne išgyvenama arba įgyvendinama prie­ varta: tik antrojoje susiliečia įsisąmonintas prievartos laukimas ir išsipildymas, pasiro­ dant – arba artėjant, arba tolstant – laisvei. d) ir atvirkščiai: juo daugiau vidurkių tiesų, atspindžių atspindžių atspindžių..., nepri­ klausomai reikšmingų valdžios ataskaitų ir apie jas kasvalandinių diskusijų, juo daugiau gyvenimo-pasaulį ardančių žiniaskaidų, ir kuo mažiau saistančio intersubjektyvumo, tuo visuomenėje daugiau agresyvių lygybės norų, prigim­ tinės nelygybės baimės ir natūralėjančios netvarkos, arba liberalizmo tvarkos (o tai yra viena ir tas pat). d) ir atvirkščiai: juo daugiau vidurkių tiesų, atspindžių atspindžių atspindžių..., nepri­ klausomai reikšmingų valdžios ataskaitų ir apie jas kasvalandinių diskusijų, juo daugiau gyvenimo-pasaulį ardančių žiniaskaidų, ir kuo mažiau saistančio intersubjektyvumo, tuo visuomenėje daugiau agresyvių lygybės norų, prigim­ tinės nelygybės baimės ir natūralėjančios netvarkos, arba liberalizmo tvarkos (o tai yra viena ir tas pat). X dalies tezės: a) juo mažiau „nepriklausomo individo“ ir panašių „sandorin judančių valingų individų“ prigimtinio suvereniteto iliu­ zijų, tuo daugiau neabejingų požiūrių į pradžios – savasties ir laisvės santykio – patyrimą (aš-matau, aš-suvokiu, aš-gir­ džiu, aš-užuodžiu, aš-lyčiu, aš-suprantu, aš-išgyvenu santykį, originalą); Darant prielaidą, kad upė, įtekanti į jūrą, o šioji – dargi į vandenyną, ir upė, įtekanti į ežerą, negali būti vienodai lygios, bet gali būti vienodai laisvos, galima klausti, kokių reikia sąlygų, kad rastųsi laisvės tikrovė? b) ir priešingai: juo daugiau „nepriklauso­ mo individo suvereniteto“ iliuzijų, tuo lengviau laisvę – asmens politinėje visuo­ menėje ir visuomenės politiniame asmens intencionalume – paleisti iš savo akiračio ir netgi iš rankų, – artėjant ne-laisvei ir alsuojant į veidą nelaisvės šleikštuliui; Kad rastųsi čiabūtiškumo sandara. Laikiš­ kumo san-dara: gyveniman linkmė, polinkio tėkmė ir laiko prasmė. Ir egologinė vidinio laiko sąmonė, ir intersubjektyvi socialinio politinio intencionalumo relevancija. c) juo daugiau socialiai saistančio melo (tęstinio laikiškumo pasakojimų), dailių pasakojimų, taurios gyvenimo poezijos, in­ tersubjektyvumo filosofijos ir kuo mažiau vidurkių tiesų (santraukų, visuomenės nuomonės apklausų, žinia-skaidos), tuo visuomenėje daugiau laisvės ir pusiaus­ c) juo daugiau socialiai saistančio melo (tęstinio laikiškumo pasakojimų), dailių pasakojimų, taurios gyvenimo poezijos, in­ tersubjektyvumo filosofijos ir kuo mažiau vidurkių tiesų (santraukų, visuomenės nuomonės apklausų, žinia-skaidos), tuo visuomenėje daugiau laisvės ir pusiaus­ Ir dar. Kad „kartą gyvenime“ (Husserl 2005; 8) įgyčiau drąsą, arba supratimą (o tai yra viena ir tas pat), pirma, apie savo paties iš-minties genetiką ir sandarą, antra, apie įgimto polinkio savęsp (solipsizmo) nepakankamumą, savo paties stokos ir drauge savo galimybių supratimą. Kad 118 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija mano išmintis (be saistančio priešdėlio) netaptų vien privačiu mano reikalu. Kai keturios didžiosios visuomenės institu­ cijos – politika, religija, ekonomika, teisė – iš­ sivaduoja (plevėsuojant tikrojo individualizmo idėjai) nuo visa persmelkiančios vertybių sistemos, socialinio politinio intencionalumo viseto, bendrosios tautos valios, jos tampa tik funkciniais institutais: tik administruoja konkre­ čias sferas. Plevėsuojant tikrojo individualizmo idėjai (jau nebe skliausteliuose), į priekį išsiveržia trys institucijos (eilės tvarka) – ekonomikos, politikos, teisės. Visos jos administruoja ins­ trumentiškai-racionalų žmogaus veiksmą ir atitinkamus objektyvių prasmių laukus. Ir kad šis tarsi privatumas netaptų savipatai­ kavimu šiuo-laikiniam racionalumui, natūraliz­ mui, tarsi-laisvumo-teisių automatizmui, teisių infliacijos nelaisvei. Kad ne nepriklausomybės, o priklausomybės nelaisvėje nesijausčiau giliai vienišas savo paties puikybės papėdėje. Kartas nuo karto gailiai suinkšdamas, ir vis prašydamas šeimininko daugiau išlygų. X dalies tezės: It visiškai išlaisvėjęs ir nepriklausomas net nuo savęs vilkinis šuo, gaunantis tikslias – vienas ir tas pačias – naujųjų liberalizmo dresiruotojų komandas: „Pulti!“ ir „Sėdėti!“ O gavęs išlygą dėkingai suloti: „Au!“ (Wittgenstein 1995; 131 [27]). Ir neužmiršti pirmosios ne-laisvės taisyklės, – nuolat vizginti uodegos. Ir vėl prašyti iš-lygų. Subjektyvių prasmių laukai ne išnyksta, o tik sunyksta. Centrinėse ekonominėse, politinėse ir teisinėse pasiūlos-paklausos kreivėse jos ma­ tomos, bet nepastebimos, – tik „pakraščiuose“. Trys didieji institutai, pasidarę plastines savo sąmonės ir kūno operacijas (pakeitę savo for­ mas, lytis, ir atitinkamus jų turinius), išplečia savo galių laukus: siūlo naujai visuomenei tas pačias instrumentiškai-racionalias gaires jau kaip vertybiškai-racionalias. Dargi „reikalauti“ („Čia laisva šalis“) – vis naujų – svetiminančių mano savastį (trūkstant vidinio laikiškumo sąmonės pasipriešinimo) – teisių laisvėms klasifikacijų, pigių ne-laisvių. Štai-dabar kylančių verčių vienetų kiekybinių teisių: noro turėti ne mažiau, nei greta eilėje stovintis, ir tuoj pat (pinigų, valdžios, dėmesio, nuotykių, malonumų, mane lydinčių garbės kortežų, – policinių švyturėlių, ilgųjų mašinų, piktųjų šunelių, švelniųjų katyčių, išmaniųjų telefonų...), skonio teisių, madų teisių, sąmonės arba kūno tapatybės keitimo operacijų teisių, lygių galimybių ir lygių negalimybių teisių, laisvesnių, nei iki šiol, skundų teisių, vienodų galimybių pasirinkti patarėjus ir patarėjas teisių ir, žinoma, išlaisvintų, o ne suvaržytų karinės visuomenės etoso (o ne tik formalių kasdienių kareivos teisių: teisių laisvai nešiotis ginklą kiše­ nėje arba laikyti jį po pagalve) teisių ... Kai primygtinai siūlo, anksčiau ar vėliau perka: toks yra nematomos rankos, laisvumo prekių prie-vartos, dėsnis. Instrumentiškai-ra­ cionalios gairės darosi vertybiškai paklausios, taigi natūraliai racionalios. Tačiau religija, kaip mus įspėja religijos fenomenologai, šiuolaiki­ nėje visuomenėje ne išnyksta, o neatpažįstamai sunyksta: ji – dėl centrinių ekonomikos, politi­ kos ir teisės institutų pasiūlos-paklausos kreivių prie-vartos (kartotinių ciklų) – tegali „pasiūlyti“ vieną prasmių tvarką, beveik neišvengiamai orientuotą į centrinių institutų virš-tvarką (Berger, Luckmann 1995; 25). 119 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Milžinas ant molinių kojų  – politika, ekonomika, teisė – įgauna tarsi-religines funk­ cijas, kurios beveik sutampa su šių funkcijų administruojama naująja visuomene. Beasmenė tai – lygu visuomenė. Dar kitaip ji vadinama darbine sutartimi. Sutartimi dėl legitimaus, nors ne legalaus tautos laisvės perėmimo į privačias savo rankas ir atitinkamų laisvių išpardavimo aukcionų liaudies valdžios liberalizmo mugėse. Šiuo aukščiausiuoju įsaku perdavus visas teises įgaliotinių tarybai, laisvė, arba laisvo žmogaus gimtis, kilmė, privatizuojama. Laisvė dresiruo­ jama, jaukinama, mokoma komandų. Keičiasi šiuolaikinio žmogaus prigimtis, o juk prigimtis, kitaip sakant, kilmė, yra esminė kilnumo sąlyga. „Darbinė sutartis“ žmogaus kilmę ir kilnumą privatizuoja. X dalies tezės: O kas bus tuomet, jei, – pasiremkime laisvo, taigi abejojančio žmogaus prielaida, – žmonės bus ne visiškai laisvi ir visiškai lygūs, o visiškai išlaisvėję ir visiškai standartizuoti, it putpelės, dedančios santraukas (tobulai apskritus kiauši­ nius) utopinio pliuralizmo narve? kažkodėl gelia, skauda ir skelia galvą. Dar dau­ giau: gręždamasis pakraštin, gyvenimo-pasaulin jaučiu koktulį, šleikštulį, pykinimą... Juk galiu „tai“ užuosti... Galiu nejučia „tai“ palytėti ranka arba akimis... Ir nuvertėti: tapti neberacionaliu, nešiuolaikišku ir pažemintu objektyvaus laiko akyse. visi bus visiškai lygūs, ir visi bus visiškai lygūs, nes bus visiškai laisvi. Štai šito idealo ir siekia demokratinės tautos“ (Tocqueville 1996; 555). O kas bus tuomet, jei, – pasiremkime laisvo, taigi abejojančio žmogaus prielaida, – žmonės bus ne visiškai laisvi ir visiškai lygūs, o visiškai išlaisvėję ir visiškai standartizuoti, it putpelės, dedančios santraukas (tobulai apskritus kiauši­ nius) utopinio pliuralizmo narve? Galiu palytėti – tik pasaulį, štai-tokią mir­ tingą tušt-umą, tik atsitiktinumą – neapdairiai: priartėjęs prie visuomenės nuomonės apklausų pakraščių. Gręždamasis nuo šiuolaikiškų verčių vertės ir atsisukdamas į bevertį – matomą, bet nepastebimą – savo savasties, senųjų filosofų vadinamos siela, egzistavimą. O besigręžiodamas galiu išleisti iš savo rankų pranašios tiesos pagrin­ dą, o iš akiračio – valdžią masių rankose, ryškų kaip amžina saulė – viskas yra politika – spin­ dulį... Galiu likti vien su paprasto, pakraštinio, nepakylėto savo egzistavimo istorija. Tuomet visi tupėsime narve, kurio vieninte­ lis privalumas bus komandinis darbas, o nepri­ valumų netgi nebus. Visi dirbsime absoliučioje lygybėje ir absoliučioje nelaisvėje: „Pagalbos!“ (Wittgenstein1995; 131 [27]). Kokių reikia sąlygų, kad rastųsi laisvesnis kalbinis žaidimas, bent jau teisingos tikrovės siekinys: „Puiku!“ (ten pat; [27])? Kad laisvė ir teisingumas netaptų – atitin­ kamai – privačia ekonomikos ir teisės ekspertų (bankų ir teismų) nuosavybe? Kad laisvė ir teisingumas netaptų tam tikra baimės buvoti savasčiai iš prigimties būdingame polinkyje pasauliui atmaina. Randantis uždaroje – savęsp veidrodžių, veidrodžių ir vien tik veidrodžių – bu­ veinėje, privačiai atspindinčioje viešąjį reikalą, šiuolaikinio gyvenimo natūralizmą. Randantis grynajame potraukio atspindžiui pasaulyje. Taigi visiškame atotrūkyje nuo gyvenimo-pasaulio. Didybės, kvailybės ir puikybės laikysenoje: veidrodžių epocha „nekeliaklupsčiauja prieš praeitį, o teikia pirmenybę ateičiai“ (Ortega y Gasset 1993; 45). Pradžia visuomet turi pabaigą, o pabai­ ga – pradžią. Tarp jų – tikrumo ir netikrumo istorijų: pirmoji gręžiasi į, o antroji – gręžiasi nuo. Karingos mūsų epochos, savęsp-veidrodžio, istorija yra „ne tai, kas dabar baigiasi, bet tai, kas dabar prasideda“ (Ortega y Gasset 1999; 157). O prasideda ji maždaug nuo T. Hobbeso, galbūt F. X dalies tezės: tuo daugiau natūralaus pavydo; juo daugiau natūralaus pavydo, tuo daugiau ekonominio ir teisinio judėjimo dar neužkariauton išorinėn erdvėn... Amebos laisvė – tai primityvus socialinės ir individualios pusiausvyros nepaisantis vien judėjimas, natūralizmas. Žmogaus prigimtį ir socialinę kilmę, vadinasi, kilnumą sistemingai bukinanti ekonominio liberalizmo tiesa („Apsau­ gok mus nuo vidutiniškų vidurkių ir santraukų tiesų, ir duok mums daugiau socialiai teisingų melo formų: tikėjimų, pasakų, pasakojimų, filosofijos, literatūros, meno...“). ii Tai nepriklausomo žmogaus, „dirbančio ant savęs“ – individo ne-tiesa, liberalo puikybė (o tai yra viena ir tas pat). Kad drauge su su­ permarketų rojumi (kur žvėrys ir žmonės savo iltis laiko saugiose tikrojo individualizmo, kaip jį vadina gajausio – anglų liberalizmo – riteris F. Hayekas, įdėklėse) randasi politinio egalita­ rizmo, vidutiniųjų, arba lygiųjų, visuomenė, abstraktaus-ekonominio-teisinio-nihilizmo- pusiausvyros-tendencija. Lygybės negalimybės iliuzijų ir laisvės bei teisingumo eidosų išpar­ davimo aukcionas. Kultūra už-miršta, – kad teisinga visuomenė yra įgyvendinamas tikslas: „Nors lygiųjų visuomenė – babuinų arba kuosų, liūtų arba žmonių – yra natūrali negalimybė, teisinga visuomenė yra įgyvendinamas tikslas“ (Ardrey 1970; 1–2). Šiuo-laikinio žmogaus pradžiose užkoduota individualistinė, – atpalaiduoto nuo sąmonės kūno laisvumo, kurį lengviau dresiruoti, – laisvė. Laisvos sąmonės galimybę praradusios amebos, kuri gali judėti tik keisdama bendrąjį socialinės laisvės pavidalą, „laisvė“: pirma, laisvumą aš suprantu kaip pasipriešinimo ne­ buvimą, antra, priešinimąsi aš suprantu kaip išorines judėjimo kliūtis (Hobbes 1999; 217). Liberalios amebos laisvė yra vien tik išorinėn erdvėn judantis laisvumo atkerėjimas, sekulia­ rizacija ir privaloma „skirtumų“, kurie darosi vienas į kitą panašūs kaip du vandens lašai, tolerantizacija. Kūno – jau be sąmonės – ma­ lonumų tolerantizacija, vietą keičianti erdve, o vidinį laiką – išoriniu laiku. Karinės kilmės – pa­ rankinis žinojimas yra jėga – laisvumas. Prievarta išlaisvinantis iš vietos, – kai ten nebelieka vidinio laiko, – milžiniškas mases žmonių. Dirbtinio rojaus laisvė: juo daugiau dirbtinių skirtumų, Teisinga visuomenė reikalauja socialumo ir individualumo pusiausvyros. Pusiausvyros pačioje tikrovėje, o ne pusiausvyros normos: „Tarkim, jog visi piliečiai dalyvauja valstybės valdyme ir visi turi lygias teises jame dalyvauti. O kadangi visi yra lygiateisiai, niekas iš jų ne­ galės tapti tironu; žmonės bus visiškai laisvi, nes 120 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija visi bus visiškai lygūs, ir visi bus visiškai lygūs, nes bus visiškai laisvi. Štai šito idealo ir siekia demokratinės tautos“ (Tocqueville 1996; 555). X dalies tezės: Bacono: „<...> nėra nieko malonesnio, kaip stovėti ant pranašaus tiesos pagrindo [the vantage ground of truth] (nepriklausomos kalvos, kur oras visada skaidrus ir giedras), ir matyti klaidas ir klystkelius, rūkus ir vėtras slėnyje, apačioje...“ (Bacon 2001; 12). Arogancija, kurią rodo šiuo-laikinis žmogus, Thomas Hobbesas, su koktuliu, šleikštuliu, py­ kinimu, aversija (neutralios negatyviosios laisvės pergalingoje liberalizmo istorijoje ištakomis), gręždamasis nuo „Aristotelio, Cicerono, Tomo Sukantis vienu ir tuo pačiu ratu dirbtiniame pasaulyje be ribų, be pakraščių. Privataus žmo­ gaus gyvenimo pasaulyje, be brūkšnelio: nuo kurio tolstant grįžta malonumas, džiaugsmas ir savimi pasitikėjimas, o prie kurio artėjant 121 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ar kurio nors kito daktaro“ (Hobbes 1999; 56) socialinio ir politinio intencionalumo („kvailių pinigų“; ten pat), dovanoja šiuo-laikiniam žmo­ gui erdvės, šiandieną vadinamos globalizacija, tuštumą. Intencionalumą – sąmonės ko nors – pakeičia „dirbtinis natūralizmas“, „dirbtinio gyvūno“, „automatų“, „spyruoklių ir ratų“, „dirbtinės sielos“ menas (ten pat; 29). Pradžioje ne natūralus, o paskui natūralus. Bet kokia kaina siekiantis trečiojo asmens „anonimiškumo“, arba „panašumo į socialinius gamtos mokslus“. Štai kodėl šiuo-laikinis žmogus, stovintis natū­ ralia poza ir kalbantis liberalia proza, gali šitaip užsklęsti ir užsisklęsti: „Dirbu ant sàvęs.“ kysenoje: visuomenės nuomonės apklausų link. O fenomenologinėje ir vėl, ir vėl laikysenoje: aš laikausi intuityviai apodiktinio žinojimo link. Kad atsirastų politinio ir socialinio in­ tencionalumo aki-vaizdos. Ne tik štai-dabar patiriamo pasaulio, o ir idealių atskaitomybių ir atsakomybių – esmės. Kad pasirodytų politikos saviduotiškumo principų pa-tyrimas: betarpiš­ kai juos suvokiant jų-man-teigime, tačiau juos įsisąmoninant empiriškai absurdiško laisvės ir lygybės susidūrimo refleksijoje. Idealių daiktų saviduotiškumą išgyvenant drąsiame intencio­ nalaus są-lyčio įgyvendinime. Kad rastųsi aki-vaizda kaip pa-tyrimo būdas. Ne tik mano vidinio patyrimo: tik psichologinės dabartiškumo akivaizdos patyrimo jausenos. O radikalaus, kritiško, drąsaus, egzistenciškai absurdiško ir gyvenimiškai būtiško tamsaus kampo – gimties-mirties-išsklaidon – patyrimo suvokimo sąlyčio refleksijoje. Darant prielaidą, kad upės negali būti vienodai lygios, bet gali būti vienodai laisvos, galima klausti, kokių reikia sąlygų, kad rastųsi laisvės tikrovė šiuo-laikinio gyvenimo lygume, natūralizme? Kad rastųsi gyvenimo-pasaulis, į kurį gręžčiausi vidiniu savo sąmonės horizontu. Kad rastųsi daiktas, kurį konstituoja man prieš­ duotas, bet mano dar neapibrėžtas laikiškumo horizontas, relevantiškas šio daikto pasirodymo būdui. Kad prieš mano akis, veikiau mano akims, išnirtų tęstinumo ne savęsp-link, o Tavęsp-link horizontas. Mano sąmonės ir numanomo tikslo gyvenimo-pasaulin są-lytis. Kad akyse raibuliuotų ne tik amžinosios regimybės: metodinės grynųjų ekonomikos ir politikos faktų – amžinumo ir t.t. – garantijos. X dalies tezės: O kad pasirodytų fenomenologinio laiko durų – pravirų ir neužveriamų – prilaikomos, užlaiko­ mos, išlaikomos gyvosios dabarties relevancijos. Ne liberalūs amebiško dabartiškumo – prie­ monių-priemonių – faktai, o tikrovės esminėse laiko-erdvės sąsajose fenomenai. Prasmingi kūno ir sąmonės są-lyčiai su tęstiniu pa-tyrimu. Sąlytis, kviečiantis, veikiau raginantis, „ir vėl, ir vėl“ gyventi, išgyventi (gyvenant patirti) ir pergyventi aki-vaizdos lauko tęstiniame hori­ zonte pakraščius. Naivius, natūralius, savaime suprantamus gyveniman įkvepiančio patyrimo pakraščius. Kad nuskaidrėtų nebūtinai visiškai skai­ draus, bet būtinai darnaus pa-tyrimo esmė, idėja, eidos. O ne tik raibuliuotų tuščia erdvės ontologija, geometrinė biurokratinio liberaliz­ mo faktų regimybė, animuojama spontaniškose privačių liaudies valdžios interesų nedarnose. Kad pasirodytų ne tik liberalaus pasirinkimo Kad gyventum ne ir t.t., o ir vėl, ir vėl... Nuo gimties lig mirties, nuo priešduoties lig jos pakraš­ čių: ir vėl, ir vėl stebėtumeisi. Kad gyventum ne doksinėje ir t.t. dabartiškumo (liberalizmo) lai­ 122 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija pliuralizmo-egalitarizmo – provincijose būtinai baigiasi mirtimi, vienodumu arba – visuomet galima – kita pirma įvairove. Taip jau yra: buvojame vienose, o ne kitose – ontologinėse, supratingai vertinančiose, matomai vertinančiose, girdimai darniose arba nedarniose, užuodžiamai lengvo arba sunkaus kvapo, gyvenimo-pasaulio provincijose. tarp visiems gyvūnams būdingų natūralistinės kilmės potraukių dabartis, o visų pirma žmo­ giško pasirinkimo – tikresnių, geresnių dalykų atžvilgiais – lūkesčiai. Kad raibuliuotų ne vienovės regimybės (fa­ šizmo arba komunizmo) arba įvairovės regimybės (liberalizmo) – bendra-telkšojimo nykuma. Ny­ kuma, neturinti saviduotiškumo tikrovės ir gali­ mybės modusų. Toli gražu ne lemtis, o veikiau nelemtis: „Nėra tokios lemties, kurios nebūtų galima įveikti panieka“ (Camus 2007d; 125). Kokių reikia sąlygų, kad rastųsi laisvės tikrovė: ontologinė, supratingai vertinanti, aki­ vaizdžiai matoma, gerai girdima ir laikiškai už­ uodžiama intuityvioje vaizduotėje? Kokių reikia sąlygų ir aplinkybių, kad išgirstume socialinio politinio intencionalumo melodiką, o tikrovę atpažintume ne kaip „išrinktųjų“ brandžiose balsavimo procedūrų aritmetikose tapatybę, o kaip tebeskambančio laiko prasmę. Kad akis badytų ne teisiškai apibrėžta ir po­ liciškai dar neįgyvendinta tarsi-duotis. Ne gyve­ nimo-pasaulio sandara, susidedanti iš daugybės procedūrinių užbaigtų nutarimų ir užbaigtų bylų labirintų. Ne primesta tarsi-dabartis kaip amžina užmestis į ateitį. Ir ne įkalinto kvapo, „už orą sun­ kesnio kvapo“ (Sartre 2002; 176), tikrovė, kurios jau neišgainios jokie vėjai, – nebent elektroninės teisės, globalaus Leviatano skersvėjai. Pažįstamumo-anonimiškumo skalėse visų pirma turime pažinti ir pripažinti mums prieš­ duotų tipizacijų ir relevancijų prie-vartos teisę. Prigimtinio Įstatymo teisę. Teisėtą tradicijos valdžią. Pažinti ją ir pripažinti: tam, kad mūsų įmestis, arba lemtis, netaptų aktualia prievarta. X dalies tezės: Gimties-mirties, arba tradicijos-naujumo poliai, jei tik ir kai tik tarp jų randasi socialinis politinis intencionalumas, yra sąmonės ko nors fenome­ nas. Mūsump – intencionalaus saviduotiškumo įgyvendinimo – akustika. Ne tiek skaidri (tik pliuralistai ieško, bet niekaip neranda vadina­ mojo skaidrumo, tikslumo, faktiškumo jų pačių užduotame neskaidrume), o skambi, laikiška. Kad rastųsi ne nešiuolaikiška miesto valsty­ bės – esminio panašumo politinėje Mes-tikro­ vėje – arba virš-šiuolaikiško globalaus vienodu­ mo – mes vartojame veidu-į-veidą ir balsuojame petys-į-petį – sandara. Ir net ne epistemologinė sandara: mes senstame supratumo-Tavęsp tikrovėje. Juk di­ džiąja dalimi esame – gręžimosi į ir gręžimosi nuo atžvilgiais – tarsi-mirę vieni kitiems. Grę­ žimasis į, kuris vienu ir tuo pačiu metu reiškia gręžimąsi nuo, sudaro menką galimų gyvenimo atsisukimų, nusisukimų, apsisukimų, įsisukimų ir, deja, persisukimų dalį. Atsisukimas, arba pažįstamumas, panašiose – veidu-į-veidą – pro­ vincijoje įprastai baigiasi gimtimi, draugyste arba priešiškumu. O nusisukimas, arba nepažįstamu­ mas, anonimiškose – kad ir liberalaus politinio Ne vertybiškai neutrali (privatizuojanti laiką erdvėje), o įsiklausanti ir įsižiūrinti į muzikavimo drauge vertę (žr.: „Making music together“; Schutz 1964; 159–78). Kaip taria laisvės metodologas Alfredas Schutzas: „<...> socialinių santykių tarp klausančiųjų muzikos tyrimai yra bet kokio ty­ 123 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 rimo šioje srityje prielaida <...>“ (Schutz 1964; 159; išskirta mano – A. V.). grąžindama gyvenimo-pasauliui 1) prasmių įvairovę ir 2) atrasdama subjektyvumo polin­ kius čiabūtiškume, o ne transcendentinio proto regimybėse (Camus 2007b; 32). Reikia ne tiek skaidrumo (pozityvistinių skaičių, grafikų, lentelių, rodiklių...), o skam­ bumo. Kad rastųsi ne aplinkos mums primestis, o egzistenciškai pajaustos pri-mesties sąsajos. O jos pasirodo ne pačioje tėkmėje, – tik refleksijos akiratyje: vienu ir tuo pačiu metu ir są-monės savęsp, ir sąmonės ko nors. Iš vidinių ir išorinių sąmonės horizontų neapibrėžtumo eidamos intersubjektyvaus apibrėžtumo link: kad pasi­ rodytų gyvosios tikrovės bendra-tėkmė, arba laiko-prasmė. Kad nuskaidrėtų dabartiškumo refleksijos praeitis, sutampanti ne su betarpiš­ kais minčių ir veiksmų srautais, o su laikišku savigrąžos sąsajų tęstinumu. Savasties-sąmonė, kaip teigia fenomenologiškai grindžiama me­ todologija, net negali būti dabartiška. Kalbant drąsiai fenomenologiškai, savasties-sąmonė gali būti išgyvenama ją patiriant tik būtajame laike. Kitaip sakant, fenomenologija kviečia išvaikščioti ne racionalaus proto ir galimybės san­ tykio regionus, o racionalios tikrovės ir galimybės santykio horizontus. Paties gyvenimo-pasaulio sąsajas, išorinius ir vidinius jo horizontus, atvirus šių horizontų pakraščius, relevancijas, tipizacijas, – ne transcenduojant, o tikrinant aki-vaizdas ir susiejant jas tarp savęs. X dalies tezės: Kad patekčiau į gyvenimo-pasaulio brūkšne­ lio valdas, laisvės upokšnio tikrovių-ir-galimybių aplinkas, privalau išvaikščioti tarpines praktinio veiksmo erdves ir laikus, nes kiekvienas prasmin­ gas veiksmas turi būti atpažįstamas objektyviose gyvenimo-pasaulio struktūrose: kiekviena tikro­ vė implikuoja savo galimybes, kurios nėra tuščios galimybės, o galimybės, iš anksto intencionaliai apibrėžtos kiekvieno aktualaus išgyvenimo turi­ nio požiūriu (Husserl 2005; 56–7). Darant prielaidą, kad vandenynai, jūros, upės, upokšniai pagal apimtį negali būti vienodai lygūs, bet gali būti vienodai laisvi, galima klausti, kokių reikia sąlygų, kad rastųsi tarpsubjektyvus laisvės upokšnio šniokštimas: iš pagarbos ir pareigos tekėjimo dėsniui, vardu res publica? Privalau išvaikščioti pa-rankinį, aki-rati­ nį, tarpinį, intersubjektyvų, mūsump-pasaulį. Ne visa apimančios tėkmės, meilės vienodumo dėsniui, policiniam egalitarizmui pasaulį, kurį, rymodami nešališkoje transcendencijoje, renčia pagal nuomonę, bet ne pagal padėtį lygūs laisvės vardo savininkai, liberalai, o gyvenimo-pasaulį. Taigi: kokių trūksta sąlygų, kad rastųsi tik du paprasti dalykai, kurie negali kilti nei iš spontaniškų individų minčių bei veiksmų srautų vienodinančios tėkmės (demokratinės daugumos laisvųjų diskusijų), nei iš įgaliotinių valdymo keistenybių tėkmės (liberalių saugios mažumos rūpesčių)? Laisvė – ne dalijama į paskiras dalis: teisių laisvėms sąrašus. Ja dalijamasi: pradedant kultū­ ros priešduotimi ir drauge senstant inter-esse. Li­ beralizmas, priešingai, pradeda dirbtinai išrasta individualia duotimi ir skaidančiu samprotavimu (sudėtimi ir atimtimi). Liberali laisvė nuo pačios pradžios – dualistinė: spontaniškame apatiniame lygmenyje „pasipriešinimo nebuvimas“ (Hob­ Kaip primena laisvės filosofas Albertas Ca­ mus, šiuodu paprastus daiktus dar dvidešimtojo amžiaus pradžioje nuskaidrina fenomenologija, 124 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija bes 1999; 217), o racionaliame viršutiniame lygmenyje – didžiulė jėga ir galia, panaudojama baimės ir laisvės simetrijai dirbtinėje visuome­ nėje išlaikyti (ten pat; 183–4). šumo ir privatumo kategorijos atlieka lemiamą vaidmenį apibrėžiant konceptualius liberalių tyrimo būdų parametrus“ (Nicolacopoulos 2008; 255). Laisvė kyla ne iš abstrakčių kategorijų, kurias šiomis dienomis vainikuoja dviejų individų – pliuralizmo ir liberalizmo – lyties operacija: iš dviejų tapatumų randasi viena, abstrakti, formalistinė, egalitarinė „ne laisvės tapatybė“. Laisvė pasirodo ne iš rūpestingai arba atsainiai perrašomų laisvių teisėms ir teisių laisvėms sąra­ šų vieno labai didelio ir labai vienodo pasaulio laisvės konstitucijoje. Hayekas teisus: absoliuti laisvė kaip „globali“ laisvė (Hayek 1991; 260–1) yra tokia tiesmuka, bergždžia ir grėsminga, kaip ir belytis Kelias į vergovę (ten pat); kelias į absoliučią žmogaus mirtį, anonimizaciją vie­ nalyčiame liberalizmo-pliuralizmo kosminiame mėgintuvėlyje. Šis dualizmas liberalizmo teorijoms yra bū­ dingas iš prigimties, – ir netgi spontaniškosioms liberalaus relevantiškumo teorijoms, o ką jau kalbėti apie racionalistines. X dalies tezės: Laisvė – tai liberalių žmonių privačių interesų bendra-telkšojimas: visiems vienodų vertybių, normų, taisyklių ir jas įtikrinančių visuomenės nuomonės apklausų pliuralizmas. kai jį maitinantis – iš dangaus ar iš žemės – lie­ tus, suteikia jam gyvybę, tai drauge – ir laisvę. Upokšnio laisvės esmė yra lietaus savastis. Jiedu, upokšnis ir lietus, gyvena, išgyvena ir pergyve­ na – netgi tėkmę – drauge, jiedu sensta-drauge! įteka į prasmingo adekvatumo jūrą. Laikiškumo, istoriškumo, laisvės tvarka, kaip sako laisvės filosofas Alexis de Tocqueville’is yra ta: kai laisvė stiprina lygybę, lygybė – laisvę. Tikėjimas laisve ir abejojimas laisve... Šiedu trapūs socialinės tikrovės patyrimo, gyvenimo, išgyvenimo, tikėjimo ir pergyvenimo (tikėjimo ir abejojimo ribų) poliai jungia prasminių relevan­ cijų aibes paslaptingiausiame, koks tik gali būti, tvarkingo chaoso, racionalaus iracionalumo, lukšte, „visuomenėje“: „štai-čia“ gyvename sa­ vaime suprantamai, kasdien šlovindami techni­ ką ir komerciją, kritikuodami politikos policiją ir kartais prisimindami meną... Aiškiai, ryškiai ir akivaizdžiai matydami tai, ką matome. Iki pas­ kesnio visuomenės kaip ankstesnio už mus visus viseto pastebėjimo. Tarsi nė nežinotume apie mums priešduotą viseto – politiškai intenciona­ liai konstituojančios galimybių struktūros – esmę. Tik mes, žmonės, racionaliai – sodriu so­ cialinio gamtos mokslininko balsu – jiedviem paaiškiname: tai tik hidrologinis ciklas. Vandens garai natūralioje aplinkoje – tiek po žeme, tiek pažemiui – sunkiasi arba vėsta, kaupiasi arba tanksta, išteka arba krenta į paviršių, telkšo-sau, paskui tiesiog išgaruoja... „Pakui tiesiog...“, – priduriame mes, – čia, laikiškume, nereikia ieškoti jokios prasmės ir jokios laisvės, senėjimo drauge. Laisvė – tai liberalių žmonių privačių interesų bendra-telkšojimas: visiems vienodų vertybių, normų, taisyklių ir jas įtikrinančių visuomenės nuomonės apklausų pliuralizmas. Socialinių gamtos mokslininkų mums pa­ aiškintą laisvės ciklą apibrėžkime kaip tipišką liberalią priežastinio adekvatumo koncepciją. Drugelio ir upokšnio laisvės patyrimą – senėjimą, arba laiko išgyvenimą, drauge – apibrėžkime kaip tipišką fenomenologinę prasmingo adekvatumo koncepciją. Pirmoji koncepcija pranešama tre­ čiojo asmens – natūraliosios būtinybės – balsu. Antroji – teigiama, atsiremiant į pirmojo asmens patyrimą, bet teiginio dar neužbaigiant: tikint laisvės esme, bet abejojant įgyvendinta laisvės tikrove. Tikrovę konstituoja – ir tik paskui orga­ nizuoja, formuoja, struktūrizuoja – kasdienis mūsų visų, šlovinančiųjų ir abejojančiųjų, patyrimas. Įvairuojantis laisvės, lygybės, teisingu­ mo patyrimas, grindžiamas fenomenologiniais kasdienybės elementais, tipizacijomis. Tipizacijos, įskaitant laisvės ir lygybės idėjas, yra bendrieji principai, kuriais remdamiesi konstituojame – daugiau mažiau tikėdami, daugiau mažiau abejodami – tikrovę. Reikia, nors ir sunku, skirti tipizaciją ir tipizacijų tyrimą. Visi mes, šlovinantieji ir abejojantieji, tipizuojame. X dalies tezės: Friedrichas Hayekas, skirdamas „spontanišką anglišką“ ir „konstrukty­ vų žemyninį“ liberalizmo tipus, pažymi: „Kad ir kokie dideli būtų Descartes’o, Hobbeso, Leibnizo pasiekimai kitose srityse, jų įnašai į socialinio vystymosi procesus buvo tiesiog pragaištingi (disastrous)“ (Hayek 1978; 255). „Totalitarinės-pozityvistinės Fransis Bacono ir Thomas Hobbeso koncepcijos“ (ten pat; 158) suteikia išrinktųjų atstovų asamblėjai neribotą valdžią. Hobbesas sukuria „teisinio pozityvizmo formą“, pagal kurią „kiekviena įstatymo taisyklė turi būti dedukuojama iš sąmoningo įstatymų leidybos akto“ (ten pat; 15). Santykiška laisvė – ne tiesi, o tik tikėtina. Santykiška laisvė ragina skaidrinti gyvenimo- planą kiekvieną gyvenimo-valandą. Savigrąžus (tik ne savigražus) polinkis riboton laisvėn, pasauliškumo-santykin esti ir visad apribotas, ir visad santykiškai atviras. Čiabūtiška (tebūnie: čiabuviška!) laisvė konstituojama ne proto-gali­ mybės, o tikrovės-galimybės brūkšnelio akiratyje. Ir nors antiracionalistinė Hayeko laikysena turi paviršinių panašumų su fenomenologiškai grindžiamos fenomenologijos postulatais (žr. toliau), tačiau esminis skirtumas yra tas, kad Hayekas, pabrėždamas individų daugybiškumo ir jų žinių fragmentų spontaniškumą, vis dėlto, kitaip nei fenomenologai, postuluoja „abs­ traktumo“ ir „klasifikacinės virš-primesties“ pirmumą spontaniškojo lygmens atžvilgiu (Hayek 1978; 35–6). Politinio liberalizmo filosofo Johno Rawlso skirtis tarp, viena vertus, „protingo pliuralizmo fakto“, kita vertus, racio­ nalios „politinės filosofijos proto veiklos“ (Rawls 2001; 4, 2) – taip pat dualistinė ir abstrakti, į ką tinkamai atkreipia dėmesį radikalūs, taigi laisvi liberalizmo kritikai: „<...> abstrakčios vie­ Kad ir drugelio, plevenančio veikiau die­ ną, nei kitą (ir netgi tik vieną naktį!) laisvė. Drugelio – ir ne tik sklandūno machaono ar puošniojo vaivadrugio, bet ir nespalvoto bal­ tuko ar naktinio pelėdgalvio – laisvės esmė yra ypatingas jautrumas konstituacinėms savigrąžos „Jei..., tai...“, „Kaip..., taip...“, „Nes jei..., tai ir paskui...“ sąlygoms. Kad ir štai-šio upokšnio, dėkingai žvelgian­ čio į mus, kai dovanojame jam laivę, laisvė. Juk 125 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 kai jį maitinantis – iš dangaus ar iš žemės – lie­ tus, suteikia jam gyvybę, tai drauge – ir laisvę. Upokšnio laisvės esmė yra lietaus savastis. Jiedu, upokšnis ir lietus, gyvena, išgyvena ir pergyve­ na – netgi tėkmę – drauge, jiedu sensta-drauge! Tik mes, žmonės, racionaliai – sodriu so­ cialinio gamtos mokslininko balsu – jiedviem paaiškiname: tai tik hidrologinis ciklas. Vandens garai natūralioje aplinkoje – tiek po žeme, tiek pažemiui – sunkiasi arba vėsta, kaupiasi arba tanksta, išteka arba krenta į paviršių, telkšo-sau, paskui tiesiog išgaruoja... „Pakui tiesiog...“, – priduriame mes, – čia, laikiškume, nereikia ieškoti jokios prasmės ir jokios laisvės, senėjimo drauge. X dalies tezės: Kaip tik todėl tipizacijos – tiek teoretikams, tiek biurokratams (o tai yra viena ir tas pat) – yra tamsus baimės kampas. Gyvename taip (savaime suprantamai), tarsi nė nežinotume, kad štai- šiame kampe kaupiamos priešduotys, žinojimo atsargos: laisvės, lygybės, teisingumo ... tipai. Absurdo tiesa, arba laisvės tiesa (o tai yra viena ir tas pat), yra ta: abi koncepcijos įteka viena į kitą, nors šioje abipusiškumo dialekti­ koje dar nėra jokio absurdo. Tačiau (gal kartą pradėsime gerbti šį „tik“ žodį!) ne prasmingo adekvatumo jūra įteka į priežastinio adekvatumo upę, o priešingai, priežastinio adekvatumo upė 126 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija * * * Numanomas arba realus „šokas“, kurį išgyvena pergyvendamas savaime suprantamas ribas teo­ retikas, praktinis humanitaro-biurokrato tipas, yra visa persmelkiantis. Turėta laisvė aki-vaizdos nušviesto tamsaus kampo patyrime pasirodo pradžioje kaip ne-laisvė, o paskui kaip nelaisvė, niekas, nebūtis... Šioje trumpoje (turint mintyse nagrinėjamo daikto, laisvės, tematizavimo galimybių sandarą) išnašoje, vargu ar rasiančioje skaitytoją dėl ilgos sąmoningo neskubrumo laikysenos ir atitinka­ mos sandaros, aptariami trys tarp savęs susiję relevantiški santykiai: 1) laisvės ir lygybės, 2) lais­ vės ir laisvumo, 3) fenomenologinio ir liberalaus požiūrių į šiuos du santykius. Pradėsime ne nuo substancinio, pirmojo, bet nuo terminologinio, antrojo. Racionalios laisvės tipų, įskaitant komer­ cinius ir konstitucinius laisvių-teisių sąrašus, formos są-monėje su-yra... Tačiau kūnas (!) nepasiduoda, savyje atrasdamas kažkur kadais jau matytus vaizdus, kažką stipresnio už turėtą laisvę, arba prarastą nelaisvę, kuri inerciškai srūva sąmonės, arba visuomenės nuomonės apklausų, vidurkių būtinybės, upėmis: „Kūnas sprendžia ne blogiau už protą ir traukiasi nuo nebūties“ (Camus 2007a; 14). Susidūrus su nelaisvės ciniška „šypsena, kurios dantys išpuvę“ (Sartre 2002; 32–3), grįžta pasidygėjimas (Ca­ mus 2007a; 19), o drauge – mes visgi senstame drauge! – gyvybė, gyvoji dabartis. Nors rūsčiojo liberalizmo pirmtako T. Hob­ beso terminai laisvė ir laisvumas (Hobbes 1999; 217) vartoti sinonimiškai kaip terminai freedom ir liberty (Hobbes 1998; 139), šiuos du terminus būtina skirti. Laisvę, skirdami ją nuo liberalizmo ilgainiui mitologizuotos individualios valios, apibrėžkime kaip žmogaus veiksmo kokybę (įgy­ vendinamą transcendentalinio-aš metodinėje savigrąžoje, o ne vien empirinio-aš veiksmo nuo ko nors savigražoje), o laisvumą – kaip žmogaus veiksmo kiekybę (kalbant konkrečiau, liberalių teisių laisvėms sąrašus). Paskutinioji mašininio gyvenimo aki-mirka nugarma į nebūtį. Sugrįžta – ne iš liberalios ateities, o iš bendros praeities – laisvė. X dalies tezės: Lais­ vė – lygu draugėje senstančios pusiausvyros laivė: didis būtinybės ir laisvės susidūrimo ribų slinktelėjimas, grakštus subjektyvumo polinkis objektyvumui: „Tą akimirką absurdas, sykiu toks akivaizdus ir toks nepažabojamas, sugrįžta į žmogaus gyvenimą ir randa ten tėvynę“ (Ca­ mus 2007b; 56). Pusiausvyros laivė pažaboja nebūtį. Paisydama ne liberalių ir t.t. diskusijų. O iš pareigos ir pagarbos dėsniui (Kantas 1987; 101). Įstatymui, ankstesniam už įstatymą, dygaus menedžmento formos laisvumą. Laisvė yra as-mens (aš-manęs, są-monės savi-grąžos) ir ap-linkos (kultūros priešduoties) santykis. Laisvumas yra išorinio „pasipriešinimo nebuvimas“ (Hobbes 1999; 217), t.y. dirbtinis liberalizmo, pertvarkiusio laisvės prigimties san­ tykį į laisvumo prigimties – dalies-viseto – santykį, išradimas. Laisvumo – valingo-asmens-ateitin- judėjimo – kryptis grindžiama trivialia perskyra, turinčia šaknis kasdienybėje (tarp noro judėti be pasipriešinimo ir galimų judėjimo trukdžių): tarp būtinybės ir laisvės, tarp lemties ir atsitiktinumo. Pirmoji šių skirčių pusė yra nerangi, vangi, kartotinė – it molinėmis kojomis žengiančios amžinos tradicijos. Antroji pusė – vikri ir suma­ 127 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ni, atsinaujinanti, nors nebūtinai atnaujinanti: išsilaisvinančios nuo ko nors individualios valios. „Pozityviosios laisvės“ (tradicijos būtinybės) ir „negatyviosios laisvės“ (atsitiktinio judėjimo be pasipriešinimo) atskyrimas viena nuo kitos sustiprina valingo individo įsitikinimą savo pašaukimu: individuali valia ir gali, ir privalo visiškai atsiskirti nuo aplinkos, arba tradicijos, trukdžių. Negatyvioji laisvė („laisvumas“), atskyrusi tradiciją ir modernybę, laisvai veržiasi, vaduodamasi iš žmogaus, kol kas dar gimstan­ čio iš motinos, aplinkos, į absoliutų laisvumą, taigi į visišką laisvumo susvetimėjimą ne tik nuo aplinkos, bet ir nuo savęs paties. ni, atsinaujinanti, nors nebūtinai atnaujinanti: išsilaisvinančios nuo ko nors individualios valios. pasaulį jungiančių dimensijų – racionalumo, subjektyvumo ir relevantiškumo, arba „iracio­ nalumo, žmogaus ilgesio ir absurdo“ (ten pat; 33) – sąsajose. Štai kodėl laisvė neatskiriama nuo prieraišumo, pareigos ir pagarbos (Kantas 1987; 101) pilnumo metodologijos dėsniui: „Žmogaus prieraišume prie gyvenimo esama kažko stipresnio už visus pasaulio vargus“ (Ca­ mus 2007a; 14). Fenomenologinis santykis tarp laisvės ir lygybės, – visuomeninio gėrio (reikia skirti nuo viešojo gėrio, common-wealth, akcentuojančio ekonominių laisvių rodiklių aibes), įprastai vadinamo tauta, aplinkoje, kurios skiriamasis bruožas yra politinis intencionalumas, – apibrėž­ tinas kaip santykis tarp subjektyvių intencijų ir intersubjektyvių jų padarinių randantis abipusiš­ ko veiksmo prasmei, intersubjektyviai socialinei politinei laikysenai. Fenomenologiškai grindžiamoje metodolo­ gijoje laisvė suprantama kaip savigrąžus laisvės apibrėžtumas ap-linkoje, kaip inter-subjektyvu­ mas, kaip tikrovės-galimybės santykis, kaip tikro­ vės-vaizduotės santykio susietumas. Kaip sąmonės prie-raišumas prie gyvenimo-pasaulio: pasaulis esti prieš gyvenimą, o gyvenimas – link ir su pasauliu. X dalies tezės: Kaip santūri laikysena: buvo, yra ir tu­ rėtų būti faktinėje daiktų vertėje, nesuardančioje faktų ir verčių vienovės, neprarandančioje laisvės verčių dėl spontaniško faktinių mainų laisvumo. Skirtinas liberalus ir fenomenologinis požiū­ riai į politinę socialinę laikyseną, o ypač į bendro­ jo gėrio sandarą. Liberalus požiūris visuomenę traktuoja kaip dabarties eksperimentinę laborato­ riją, visiškai nepaisydamas arba paisydamas tik nominaliai abipusiškų perspektyvų laikysenos: kad tikrovė nuo pradžios yra intersubjektyvus kul­ tūros pasaulis (Schutz 1962; 10). Nebus per daug pakartoti esminio fenomenologinio akcento: tikrovė esti priešduota, – dar prieš įgyvendinant liberalius, arba efektyvius-racionalistinius, sudėties ir atimties veiksmus (žr. „Apie protą ir mokslą“; Hobbes 1999; 60–8). Mes nesame pasmerkti laisvumui, tiksliau, nesame pasmerkti liberalios valios nepaliauja­ mai kovai su politinės bendruomenės sąsajomis (plg.: „Politinės visuomenės gėris – be politinės bendruomenės“; Rawls 2001; 198–9). Esame pasmerkti ne laisvumo neapibrėžtumui, o laisvės ribų apibrėžtumui dviejuose neperskiriamuo­ se – vidiniame ir išoriniame – horizontuose, dviejose neperskiriamose – laikiškumo ir erdviš­ kumo – dimensijose. Liberalus požiūris nuo pradžios yra efek­ tyvus, arba pozityvistinis (o tai yra viena ir tas pat), nematantis jokio reikalo dar prieš privatų racionalumo apibrėžimą (laisvumo teisių sąrašo sudarymą) eksplikuoti bendros socialinės ir Laisvė „nustato ryšį su laiku“ ir „užima jame savo vietą“ (Camus 2007b; 20) trijų gyvenimo- 128 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija mis ir relevancijomis. Kai laisvė stiprina lygybę, stiprėja sąmonės subjektyvaus polinkio ko nors atžvilgiu orientacijos. Kai lygybė stiprina laisvę, stiprėja abipusiškų tipizacijų įtikrinamos objek­ tyvios struktūros, institucijos. Kai lygybė stiprina laisvę silpnėjant steigiančiajai subjektyvumo dimensijai, stiprėja išoriniai „socialinės struk­ tūros“ – „padėčių“, „vaidmenų“, „statusų“, „funkcijų“ – komponentai, tačiau šioje erdvėje įprastai nepaliekama vietos vidinėms, laikišku­ mo, laisvės dimensijoms. Kai minimi procesai, kuriuos steigia subjektyvumo orientacijos, yra abipusiški, visuomenė konstituojama kaip in­ tersubjektyvi tikrovė. politinės tikrovės priešduotiškumo sandaros, įprastai vadinamos tradicija. Efektyvus-pozi­ tyvistinis požiūris susiaurina intersubjektyvaus kultūros pasaulio galimybių struktūros sampratą į aktualaus eksperimento sampratą. Liberalizmo požiūriu, „socialinė struktūra“ yra tik „vardas“, nurodantis bendrąjį-turtą, kurį veiksmingai panaudoja laisvi ir racionalūs individai, indi­ ferentiški socialinėms ir bendruomeniškoms gėrio sampratoms, tolerantiški bet kokioms, tarp jų – ypač privačioms, nuomonėms apie skirtingas gėrio sampratas. g „Socialinė struktūra, liberaliu požiūriu, susideda iš konstituciškai apibrėžtų ir aktualiai konkretinamų procedūrinių galimybių (prieina­ mų visiems lygiems ir laisviems šios struktūros individams, kuriems universali liberalizmo struktūra steigia teises ir laisves): „vaidmenų“, „statusų“, „padėčių“, „funkcijų“ ir kitų aktualių ir potencialių gėrybių, susijusių su visiems ben­ drais-ištekliais (T. Hobbeso vadinamais common- wealth). X dalies tezės: Šioji simetriška „socialinės struktūros“ lygybė, arba politinis egalitarizmas, iš kurio kyla visos kitos egalitarizmo tikrovės ir regimybės, liberalizmo požiūriu, iš esmės stiprina laisvę, nereikalaudama politinės bendruomenės įsipa­ reigojimo bendrojo gėrio sampratai (darkart: būtinumo – iš pareigos ir iš pagarbos – dėsniui; Kantas 1987; 101), iš kurios, fenomenologiniu požiūriu, randasi laisvo ir racionalaus, intersu­ bjektyvaus, asmens ir būtinų sąlygų politinei ir socialinei bendruomenės savi-valdai (reikia skirti nuo administracinės savivaldos) apibrėžimai. Esminiai – žmogaus egzistavimo pasaulyje, o drauge intencionalaus polinkio pasauliui (atitinkamai: žmogaus egzistavimo visuomenėje, o drauge žmogaus polinkio visuomenei, ir kitų esmės vedinių) – klausimai yra socialiniai ir inter­ subjektyvūs: „Kaip galima visuomenė?“ (Simmel 2007; 25–39), „Kaip žmogus gali suprasti savo porininką?“, „būtent Kitą“ (Schutz 1964; 20; Husserl 2005; 112). Kalbant trivialiai, bet ne atsainiai, matomiausia, bet nepastebimiausia tikrovė – tikrovė-mums – susidaro (bet ne susi­ deda) iš trijų vienas nuo kito priklausančių fe­ nomenologinių „zigzagų“: a) socialinio veiksmo, b) socialinės sąveikos, 3) socialinės tikrovės radi­ mosi bruožų, įtikrinamų kartotinėse sąveikose. Socialinis tikrumas randasi ikipredikatiniuose, o ne loginio samprotavimo lygmenyse. Šie trys lin­ kmių zigzagai rodo, kad tai, ką vadiname pa-sau­ liu, – ir gamtiniu, ir socialiniu kultūriniu, – mes išgyvename drauge patirdami, arba įtikrindami (o tai yra viena ir tas pat). Svarbu priešdėliai: iš-gyvename į-tikrindami. Įtikrindami tam tikroje, metodologiškai skaidrintinoje, skalėje: Socialinis tikrumas randasi ikipredikatiniuose, o ne loginio samprotavimo lygmenyse. Šie trys lin­ kmių zigzagai rodo, kad tai, ką vadiname pa-sau­ liu, – ir gamtiniu, ir socialiniu kultūriniu, – mes išgyvename drauge patirdami, arba įtikrindami (o tai yra viena ir tas pat). Svarbu priešdėliai: iš-gyvename į-tikrindami. Įtikrindami tam tikroje, metodologiškai skaidrintinoje, skalėje: Fenomenologiniu požiūriu, visuomenė yra intersubjektyvi tikrovė (o ne teisinė-adminis­ tracinė „socialinė struktūra“) su priešduotomis socialinėmis ir politinėmis sąsajomis, tipizacijo­ 129 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 tipizacijų – pažįstamesnių (artimesnių) arba mažiau pažįstamų (anonimiškesnių) – aibėse. empirinis apibendrinimas, reikalaujantis papil­ domai skaidrinti veiksmo-laikysenų-relevancijas. Juk gimstame, griežta šio žodžio prasme, ne vien gamtinėje, o visų pirma socialiai-natūralioje aplinkoje, savitoje kalbos sistemoje, istoriškai sudarytoje iš tipizuotų betarpiško ir anonimiško patyrimo figūrų skirtingose prasminėse gyveni­ mo-pasaulio provincijose. tipizacijų – pažįstamesnių (artimesnių) arba mažiau pažįstamų (anonimiškesnių) – aibėse. Štai dabar galime brėžti metodologinę – pačių socialinės srities mokslininkų įprastai matomą, bet nepastebimą – skirtį tarp gimtosios kalbos ir negimtosios kalbos. Pirmoji kalba mums priešduota mūsų išgyvenamame patyrime. X dalies tezės: Pir­ ma, tipų-konstituacijos ir netgi tipizacijų-struk­ tūros gali būti iš principo suvoktos be kalbos, „ikikalbiniame patyrime“: „Empirines-genetines tipizacijos schemas pozityviai atskleidžia vaikai, kurie dar nekalba“ (Schutz, Luckmann 1973; 233); ankstyvos vaikystės mokymosi matyti daiktus patyrimai – genetiškai pirmesni už bet kokius kitus daiktų suvokimo būdus (Husserl 2005; 98–9). Antra, visi mes gimstame savitoje istorinio socialinio kalbos pasaulio aplinkoje, gi­ liąja prasme, gimtojoje kalboje: kalbos struktūra suponuoja tipizacijas, o ne atvirkščiai (ten pat). Šis socialinis-kalbinis gimties faktas metodolo­ giškai reikalauja skirti gimtą kalbą, t.y. tipizacijų ir jas sąlygojančių relevancijų sistemas, ir negimtą kalbą, t.y. anonimišką socialinių mokslų žodyną (standartizuotų sąvokų: nuo vaidmens, statuso iki institucionalizacijos ir socialinės politinės sistemos). Savasties-buvojimo-pasaulyje-su-kitais-ir- kitiems relevancijos laikytinos savaime-supran­ tamų-daiktų regionais. Nors mes artikuliuotai neįsisąmoniname aiškių ir ryškių daiktų vaizdų ir „neturime su savimi“ gilesnio jų sąryšių supratimo, tačiau dėl to „nesukame galvos“: laikome aplinką po ranka, savo akiratyje, savai­ me suprantamai manome, kad mums nereikia papildomų šios sandaros tyrimų. Šiuose regio­ nuose, – baigtinėse prasmių provincijose, ribos tarp kurių įveikiamos tik „transcendenciniais pagavos šuoliais“, o ne pozityviomis „aiškinimų lygtimis“, – viskas esti savaime suprantama. Esant numanomai išlygai: iki paskesnio matomų dalykų pastebėjimo, – kintant mūsų motyvacijų ir inter-esmių (inter-esų) sandarai. Pastebėdami matomus daiktus aplinkoje (kai po ranka bu­ vojančius daiktus sugriebiame savo rankomis ir tęstinai išlaikome savo aki-ratyje), atpažįstame „į kažką panašius“, „jau kažkur matytus“ aplinkos bruožus. Savaime suprantamas gimtosios kalbos pa­ saulis yra verčių, tarp jų – ne tik realių, bet ypač idealių žinojimo objektų, nepriklausomų nuo pažįstančio subjekto (Schutz 1966; 163), saugy­ kla. Verčių, kurios gerokai rečiau, nei dažniau, sutampa su mokslinio racionalumo – logikos su­ bjektų, jų predikatų ir metodologinių sprendinių – vertėmis. Esminė fenomenologiškai grindžiamos metodologijos nuoroda yra ta, kad gimtos kalbos vardai, – kiekvienas vardas, nurodantis reiškinį arba įvykį, – įtraukia tipizacijas ir esti neesminis Šie matyti, bet nepastebėti bruožai konstituo­ jami gyvenimo-pasaulyje kaip tipizacijos, kurias mes tematizuojame kaip tipus. Mes galime metodologiškai tiksliai aprašyti juos dėl, veikiau dėka mūsų kalbos, gimtos kalbos, – laikydami ir iš-laikydami relevantiškas tipizacijas jų tikrovės namuose, bendrinėje kalboje. Tikrus, arba aki- vaizdžius, sąmonės ko nors sąlyčio su kuo nors 130 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija fenomenus galime – turime unikalią galimybę – sugriebti ir išlaikyti gimtuose tikrovės namuose: „Kalba yra tipizuojančių patyrimo schemų sistema, grindžiama betarpiško subjektyvaus patyrimo idealizacijomis ir anonimizacijomis“ (Schutz, Luckmann 1973; 233). X dalies tezės: tiesioginę ir išvirkščią kalbamojo teiginio for­ mas: laisvė stiprina lygybę, o lygybė – laisvę, ir atvirkščiai, lygybė stiprina laisvę, o laisvė – lygybę. Šiuo atveju, kaip tartų Wittgensteinas, „turime nepriekaištingą tapatumo paradigmą daikto tapatume sau“ (ten pat; 219 [215]), t.y. turime nepriekaištingą demokratijos paradigmą. Turime gražų, dargi savigražų, taigi tobulą, pavyzdį, kuris šiuolaikinėje konstitucijoje (baltame ra­ cionalistinio-empiristinio popieriaus lape) skamba dar gražiau: liberalioji demokratija. „Nėra gražesnio pavyzdžio, – tarkime drauge su Wittgensteinu, – iliustruojančio bevertį sakinį, kuris tačiau susijęs su tam tikru vaizduotės žaidi­ mu: „Daiktas yra tapatus sau pačiam“ (ten pat; [216]). Kitaip sakant, kelkime klausimą: galbūt ir nėra gražesnio ir beprasmiškesnio pavyzdžio, iliustruojančio bevertį tapatumo žaidimą, – už savigražų „liberaliosios demokratijos“ žaidimą? Tai reiškia, kad šiose veiksmo laikysenose, ti­ pizuojančiose patyrimo schemose, konstituojamos štai-šios linkmės, o vartojant tikslesnį Edmundo Husserlio terminą, – inter-esse. Inter-esse moty­ vuoja mūsų galvojimą, gyvenimo-planus, tikslus, veiksmų kryptis ir steigia problemas, kurios, gręždamos ir lenkdamos mus vienais, bet ne kitais atžvilgiais, kviečia įsižiūrėti į tam tikrus savaime suprantamo prieš-žinojimo regionus. Šių in­ ter-esmių (domesio gyvenimui) ir motyvacijų aplinkose ir gali, ir turi būti nagrinėjamas Alexio de Tocqueville’io idealusis socialinės politinės laikysenos tipas: abipusiškų perspektyvų – kai laisvė stiprina lygybę, o lygybė laisvę – postulatas (Tocqueville 1996; 555). Esant empiriškai atpa­ žįstamai demokratinio gyvenimo šiuolaikinėse politinėse visuomenėse tendencijai, – kad lygybė nuolat pažeidinėja laisvės principus dėl vis stiprė­ jančio, vadinasi, natūralėjančio, polinkio, kuris ilgainiui, kaip intuityviai įžvelgia Tocqueville’is, virsta lygybės meilės tikėjimu (ten pat). Įsižiūrėdami į tikrovę mūsų akiratyje, tiks­ linkime, žingsnis po žingsnio artėdami prie antrosios, supratumo, laikysenos. Tikslinkime ne „tikrovę“, vaizduojamą konstitucijoje (raci­ onalistinėje dalyje, konstruotoje be sistemingų viseto tyrimų), o pačioje tikrovėje. Tikslinkime ne politikos mokslo (realių ar tariamų valdžių perskyrų ir jų pozityvistinių dedukcijų) teoriją ir praktiką, o tikrovės-fenomeną. Ne politikos mokslų fenomenologizmą, o tikrovės-feno­ menologiją. Tikrovės-fenomenas, kaip mums pataria Herbertas Spiegelbergas, yra įsižiūrėjimas į esamą tikrovę (Spiegelberg 1940; 84). Centrinį tikrovės regioną, kurį mes siekiame pažinti, sudaro betarpiškai suvokiami daiktai, t.y. suvo­ kimo fenomenai (ten pat). Kitaip sakant, turime palyginti tikrovę ir mūsų betarpiškai suvokiamą šios tikrovės vaizdą. Be abejo, turėdami mintyse Kad būtų aiškiau, – politinio intencionalumo, o ne lygybės meilės tikėjimo, – polinkyje, skirki­ me, vadovaudamiesi Wittgensteino Filosofinių tyrinėjimų 146-uoju fragmentu, supratumo laikysenos ir supratumo taikymo laikysenos krite­ rijus: a) pats supratimas yra būsena, iš kurios kyla taisyklingas vartojimas; b) pritaikymas išlieka su­ pratimo kriterijumi (Wittgenstein 1995; 187). X dalies tezės: Pirmoji laikysena (supratimo kaip išanksti­ nės veiksmo prielaidos) neprieštaringai apima 131 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija tikrovės pažinimo „absurdą“, arba „tarpiškumo“ fenomeną: kad tikrovės tiesiogiai mes nepažįs­ tame, o pažįstame tik fenomeną (ten pat; 85), ir nieko daugiau! rusios su politinio intencionalumo fenomenu, atatupstos gręžiasi nuo savo sąmonės: „Argi ne tiesa, kad, netikėtai išvydęs prarają ir išsigandęs, kūnas mechaniškai traukiasi atatupstas?“ (La Mettrie 2011; 83). Jei tariame, kad politinės tikrovės tiesiogiai pažinti negalime ir kad, ieškodami politinio intencionalumo, turime pradėti pirmojo asmens patyrimu, taip turime ir daryti. Įsižiūrėdami į tikrovę, pradžioje matome tik psichologinį „visur ir visada – politika“ fenomenalizmą: laisvąsias diskusijas, įskaitant brandžiausios po­ litinės aktualybės, sulaukusiųjų aštuoniolikos, rinkimų šurmus, ekonominį skaičiavimą su tam tikrų apskaičiavimų išlygomis, biurokratinę inži­ neriją su policiniais švyturėliais. Turime policinę politiką: interesų paskirstymą, pasiskirstymą ir kitą pirmą perskirstymą valdžios perskyrų – jei tokios realiai egzistuoja – struktūrose. Turime dar ir politiką kaip instrumentinio veiksmo rūšį, kurios paskirtis – surinkti mokesčius (juk ekspertų nuomone, „vis geriau sekasi surinkti mokesčius...“) ir parodyti liberalius metodus aktualioje kovoje dėl valdžios. Gręžtis nuo ko nors reikia tuo pačiu metu gręžtis į ką nors: šiuokart į valdžios personų pasirodymą – „šviesiaisiais liberalizmo laikais“ – policinio fenomenalizmo scenose. Tačiau tai, – kaip pabrėžia Hannah Arendt, – kad asmuo tiesiog pasirodo viešai, ir kad publika priima ir pritaria jai ar jam, jokiu būdu nėra savaime su­ prantamas dalykas“ (Arendt 1968; 3–4). Politi­ nio intencionalumo fenomenas mums duodamas be tarpininkų, ne viešai, – tik kaip šis fenomenas mums pasirodo. Politika – ne paviršiaus-fenome­ nas ir ne auditorijos įspūdžių valdymo-fenome­ nas. Fenomenologiniu požiūriu, apimančiu ir esmines Aristotelio bei Kanto nuorodas, politika yra įsipareigojimas veiksmo-bendra-buvojimo- laikysenai. Liberaliu požiūriu, – tik fizinių, arba psichologinių, objektų tam tikroje geometrinėje erdvėje valdymas: valdančiųjų ir valdomųjų hi­ erarchijos, dominavimo ir pavaldumo santykių įtikrinimas. Tai spyruoklinis liberalizmas. Visų nešališkų pusių – skirtingų pagal plauką, bet lygių pagal ideologiją politinių partijų – su­ tarimu, tai liberalioji demokratija su išlygomis, tik dabartinis pasaulis (plg.: „senasis pasaulis, dabartinis pasaulis ir būsimasis pasaulis“; Hobbes 1999; 444–5), dar ne „milžinų susirinkimas“ (ten pat; 436). Spyruoklinis liberalizmas, kuris kol kas randasi dar ne-vietoje... Tai ne politika, o policinis fenomenalizmas, ir nieko daugiau! Turime ne demokratijos (tautos inter-esse), o liberalizmo (liberalizmo prekių – negatyviosios laisvės ir aministracinių-policinių interesų – importo) pavidalą. X dalies tezės: Atsinaujinantį tik aktualioje kovoje dėl valdžios teikiamų išteklių, o ne dėl politikos kaip pašaukimo (Maxo We­ berio politinio intencionalumo) ir jokiu būdu ne dėl žmogaus orumo politinėje bendruomenėje užtikrinimo sąlygų (Hannah’os Arendt politinio intencionalumo: ir netgi „tamsiaisiais laikais“; Arendt 1968; 3–32). Juo atidžiau įsižiūri į štai- šį policinį fenomenalizmą, tuo ryškiau matosi žmogaus-mašinos „spyruoklės“, kurios, susidū­ Jei teisingumas, aprašytas didžiosiomis konstitucinėmis raidėmis, visiškai skiriasi nuo teisingumo, konstituojamo mažosiomis raidė­ mis kasdienybėje, turime būti dėmesingesni 132 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija teorijos. Tai tik privačiai-viešasis, išlaisvintas nuo bendrumo, ekonominis politikos vartoji­ mas (common-wealth), kurį, reikalui esant, labai tiksliai paaiškina ekonomikos ir teisės ekspertai, liberalizmo gamtos mokslininkai (socialumas – liberalizmui nežinomas žodis, nors ir mėgstamas vartoti kaip policinių žaidimų „prieskonis“). Pir­ ma, mūsų laisvės atstovai, laisvi ir lygūs asmenys, perėmę jiems perduotas asmens teises, sutaria dėl socialinės kooperacijos, grindžiamos teisingumo principais (Rawls 2001; 80). Antra, mūsų atsto­ vai, laisvi ir lygūs asmenys, vertybinio nesvaru­ mo, arba indiferentiškumo, būklėje, dūmodami apie su-tampančio konsenso pliuralistinėje visuo­ menėje teikiamas gėrybes, išleidžia liberalizmo relevancijas: vieniems teisingumo principams pritaria, o kitiems – nepritaria (ten pat). visetui, o ne kuriai nors paskirai jo daliai. Visuo­ menė, arba tikrovė, randasi ne iš jos „supratimo“ konstitucinėse knygose, o iš „paties taikymo“: laisvės, lygybės, teisingumo žodžių „vartojimo“ laisvės veiksmų kokybės ir laisvumo veiksmų kiekybinėse praktikose. Ne simetriškose valdžių atskyrimo regimybėse (kalbiniuose žaidimuose), o konkrečiose ne-laisvės, ne-lygybės, ne-teisin­ gumo ne-vietose. Reikia skirti, viena vertus, laisvę kaip galimybę savo aplinkoje patyrimu išgyventi ir pergyventi (transcenduoti) prasmę modifikuojant primygtinai siūlomą, itin paklau­ sią, liberalią laisvės reikšmę, ir, antra vertus, „laisvę“ kaip didžiųjų knygos raidžių taikymo sekos, arba laisvumo, patyrimo dėsnį: „Kai aš sakau, kad suprantu sekos dėsnį, vis dėlto tai sakau ne dėl to, kad patyriau, jog iki šiol taikiau algebros formulę būtent taip!“ (Wittgenstein 2005; 187–8 [147]; išskirta autoriaus). Jei tik ir kai tik gręžiamės į aki-vaizdą, patiriamą išgyvenant „vis per nauja, vis su įtampa“ (Camus 2007c; 56), matome kitą mūsų atstovų, laisvų ir lygių asmenų veiksmų, „dievų vaikų poravimosi su žmonių vaikais“ (Hobbes 1999; 436), padarinį: simetriškai suporuotą demokratijos idėją ir laisvosios rankos tikrovę. Kadangi šiam padariniui iš esmės trūksta vidinio sąmonės horizonto struktūros, tai jis, gręždamasis nuo prasminių gyvenimo-pasaulio, arba praktinio racionalumo, struktūrų, gręžiasi į „išorinės esaties santykių“ (Hegel 2000; 344) racionalizmą, visiškai neturintį „<...> ribos savyje, nurodančios, kas yra žalinga ar nežalin­ ga...“ (ten pat; išskirta autoriaus). X dalies tezės: Padarinys, arba procedūrinis racionalizmas, aiškiai linksta ne tautos valion, o vertybinio nesvarumo, arba potencialios neteisės pusėn, kurios paskirtis yra a) sudaryti iš anksto demokratinių laisvosios rankos teisių nevaržomoms laisvėms sąrašus ir Kitaip tarant, aš negaliu, kaip, tarkim, liberalizmo teoretikas, teigti, kad dabar, kai aš viską supratau ir visiems racionaliai paaiškinau, viskas turi vykti pagal mano atrastus būtinybės dėsnius, šįkart procedūrinius principus: „<...> visiško tikslumo būklės, ir lyg tatai būtų tikrasis mūsų tyrinėjimo tikslas“ (ten pat; 169 [91]). Tik savigrąžoje, linkmėje pasauliui, politinio sociali­ nio veiksmo laikysenoje aš galiu išskleisti įpročio prasmę, kuri niekada negali būti pakeista, nes prasmes pasaulis – mums visiems – turi anksčiau, negu pradedame kokiu nors būdu filosofuoti (Husserl 2005; 187). Tai, ką aki-vaizdžiai matau savigrąžoje, nėra nei Johno Rawlso teisingumo kaip nešališkumo, nei racionalistinės knygos įstatymo, kurį reikia aiškiai skirti nuo bendrojo gėrio, bendrosios valios Įstatymo (prasminių esmės sąsajų, relevancijų), 133 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 b) apibrėžti „galutinius policinio baudžiamojo teisingumo“ (ten pat; 343) punktus. ir švietimo sistemoje. Kai liberalizmas reiškia ir „viską“, ir drauge „nieko“, jis tampa „privataus skonio“ fenomenu, o todėl lengvai prasismel­ kia į kiekvieną visuomenės gyvenimo kertelę. Santykį tarp demokratijos ir liberalizmo esame linkę – visų pirma dėl mus apėmusios meilės ly­ gybei natūralizacijos, o visų antra dėl savivaldžių laisvumo padėčių nepriklausomybėje asimetri­ jos – išleisti iš akių. Juo daugiau liberalizmo, tuo mažiau demokratijos. Juo daugiau liberalų, tuo mažiau demokratų. Juo liberalizmas natūralesnis, tuo ne natūralesni antiliberalai (demokratai). Laikas sudėti tai, ką turime, ir iš gautos su­ mos atimti tai, ko neturime. (Bent vaizduotėje palyginti su sąmonės ko nors: metodo esmė, kaip pabrėžia fenomenologai ir egzistencialistai, yra nuoseklumas, drąsumas ir atkaklumas.) Turime a) parankinius išteklius ir b) itin profesionalias liberalų, arba pliuralistų, rankas, bendrąjį-turtą, ir procedūrines šių rankų operacijas, grindžiamas neprilygstamo laisvumo, būdingo spyruokli­ niam liberalizmui, principais: „netrukdyk!“, „nesikišk!“ ir „nelįsk!“ Turime dvi demokratijas: 1) „demokratiją pačią savaime“ ir 2) „demokra­ tiją liaudžiai“; 1) „elito kulto“ noumeną ir 2) „liaudies valdžios“ fenomeną. Turime simetrišką demokratijos idėjos ir laisvosios rankos santykį ir asimetrišką laisvės ir lygybės santykį. (Lietuviška liberali tvarka, simetrijos ir asimetrijos darna, atliepia, o gal net ir pranoksta įsibėgėjančią neoliberalizmo ne-tvarką: „<...> naująją impe­ rinio chaoso formą, kurioje viešpatauja, nors ir nekontroliuodama šio chaoso, JAV“ ; Joxe 2002; 104–9.) Neturėjimo santykį tinkamiau nu­ skaidrina antroji Wittgensteino nurodoma laikysena (supratimo taikymo), kuri išvirkščią teiginio dalį – lygybė stiprina laisvę, o laisvė lygy­ bę – laiko klaidinančia. X dalies tezės: Dėl tos priežasties, kad pirmoji, simetriška, „nepriekaištinga“ tapatumo paradigma sudaro sąlygas ne laisvei (pasirinkimo galimybei), o veikiau nelaisvei, – lygybės ir t.t. predikatui (aš laikausi taisyklės aklai): „Kai lai­ kausi taisyklės, nesirenku“ (ten pat; 220 [219]). Dalykas tas, kad laisvė yra egzistencinė sąvoka, o lygybė – tik teorinė samprata. Laisvė yra ribos sąvoka: į-pročio. Vos vos įžiūrimame brūkšnelyje vienu ir tuo pačiu metu užsimezga arba neužsimezga egzistencinė pirmo­ jo asmens ir politinė bendrojo gyvenimo drama. Vidinio laiko, arba subjektyvumo, drama. As­ mens ir aplinkos. Polinkio ir laikysenos. Są-mo­ nės savigrąžos ir sąmonės ko nors, pergyvenančios savastį, rodančios už savęs, aplinkos, socialinio, intersubjektyvaus pasaulio link. Drama, tariant Kanto žodžiais, tarp atitikimo (subjektyvumo ir objektyvumo, išgyvenimo ir pergyvenimo modusų abipusiškos grąžos) ir pagarbos bendro gyvenimo dėsniui, o ne iš meilės bet kokiam turėtų būti O neturime to, ko ir nenorime turėti, – mūsų pačių įkūnytos laisvės bendrojoje valioje, arba bendro laiko prasmės tęstinume. Neturime politinio laiko, politiškumo, intencionalumo, įsipareigojimo tikrovės. Kitaip sakant, politikos kaip tikrovės. Turime tik politikos fenomena­ lizmą, – laisvai ir nepaliaujamai kalbančią spy­ ruoklę, lėlę sūpuoklę, kuri akylai saugo esmines valdžios ir valdomųjų asimetrijas. Turime abso­ liučią nepriklausomųjų, o ne santykišką bendrai buvojančiųjų laisvę. Turime intensyvėjantį liberalių žodžių var­ tojimą praktikoje, ypač naujojo asmens sąmonėje 134 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija (Kantas 1987; 100). Tai ne tik drama, bet ir politinio intencionalumo – asmens valios ir bendros lemties, į-pročio ir įpročio akistata. Įmes­ ties (lemties, kurios negaliu pakeisti, o galiu tik modifikuoti) ir pri-mesties (savigrąžos politinio veiksmo laikysenoje) sąveika aktualiame ir po­ tencialiame įpročio akiratyje. (Kantas 1987; 100). Tai ne tik drama, bet ir politinio intencionalumo – asmens valios ir bendros lemties, į-pročio ir įpročio akistata. Įmes­ ties (lemties, kurios negaliu pakeisti, o galiu tik modifikuoti) ir pri-mesties (savigrąžos politinio veiksmo laikysenoje) sąveika aktualiame ir po­ tencialiame įpročio akiratyje. Savigrąža yra ne tiek racionali (samprota­ vimas – nuo Hobbeso – įprastai apibrėžiamas kaip vien sudėjimas ir atėmimas, kaip grynoji analitika), kiek supratinga. Gręždamasis savęsp aš atpažįstu savo socialines pri-mestis, t.y. „skir­ tingus priklausomybės ratus“ (Simmel 2007; 138). Eidamas dar arčiau savęs, prieinu ir prie socialinės kilmės laisvės paradokso: „Tai, ką mes jaučiame kaip laisvę, faktiškai dažnai yra tiktai prievolių kaita <...>“ (ten pat; 380). Įprotis – tai pirminis a) relevancijos savęsp ir b) relevancijos mūsump šaltinis. Tai: a) savigrąža lemtin, b) savigrąža sutartin. X dalies tezės: Pirmuoju atveju lemties – man gręžiantis savęsp – leidžiama pasiekti egzistencinį laisvės ir būtinybės santy­ kį: išgyvenant patirti absurdą, kad laisvė – tai sąžininga savigrąža būtinybėn. Antruoju atveju gimties, buvojimo įmesties galimybėje, kurią teikia autonomiška tautos valia, man leidžiama siekti bendro politinio veiksmo – įpročio, suti­ kimo, sutarties – laikysenos. Aš paklūstu daiktų prigimčiai, įpročiui, politinio veiksmo – lemties ir valios – dermėje: įsipareigojimo kaip moralinės atsakomybės ir įpareigojimo kaip lemties savivokos. Dabar, jei tik ir kai tik aš išgyvendamas patiriu šį paradoksą, jaučiu ne tik malonumą laisvai judėti – „išorinių judėjimo kliūčių nebuvimą“ (Hobbes 1999; 217) – tariamai išorinėje aplinkoje, bet ir vidinę laisvės galimy­ bių struktūrą. Natūralistinės kilmės potraukius (appettite; ten pat; 69) ir jais įprastai grindžiamas ekonomines pasiūlos-paklausos operacijas galiu pakeisti supratingų norų (daiktų, kurių trūksta ir kurie galbūt dar neegzistuoja), susijusių su nutolusiais tikslais, eksplikacija. Galiu suvokti, suprasti ir įsivaizduoti laisvės tikrovės ir laisvės galimybės santykio įvairovę, nesiaurindamas šios įvairovės į tik keletą galimų pasirinkimų, įprastai arba formalaus ekonominio, arba formalaus teisinio pobūdžio. Laisvė randasi drauge su įsisąmoninamomis tikrumo galimybių aibėmis, socialiai ir politiškai intencionaliais – tikrumo bendrabuvojimo atžvilgiu – bruožais. Tik savigrąžoje suvokiu egzistencinį laisvės absurdą: kad įprotis mano protui esti prieš­ duotas ir kad įprotis (jei be brūkšnelio) man siūlo laisvės, ir netgi laisvės prasmės, praradimo galimybę, aklą paklusimą „natūraliai“ daiktų tvarkai. Tik savigrąžoje man skleidžiasi rele­ vantiškas laisvės (subjektyvumo) ir racionalumo (pasauliškumo) santykis. O be savigrąžos aš netgi negaliu žinoti, ar esu racionalus, ir ar aš stoviu ant savo kojų. Ar esu supratingas, ar tik gebu tik derinti priemones ir tikslus (kai antrieji galbūt yra tik priemonės)? Ar esu racionalus vien todėl, kad galiu „numatyti“ tikėtinus instrumentinio veiks­ mo padarinius, ar todėl, kad galiu supratingai orientuotis, derindamas savo jutimų, vaizduotės ir proto gebėjimus socialinio pasaulio aplinkoje? Kai aš gręžiuosi savastin, privalau tikslinti tikrumo ir netikrumo skirtį. Ne tik tikrovę, pasirodančią mano akiratyje, bet ir platesnes, įvairesnes šio pasirodymo fenomeno galimybes; gręžtis į man priešduotą erdvės-sandarą ir laiko- sandarą. Tirti racionalumo formas, būdingas štai-šioms sandaroms. Nuskaidrinti teorines racionalumo formas: juk „racionalumas“ ir 135 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 „racionalus veiksmas“ predikatinėje struktūro­ je – tiek metodologijoje, tiek epistemologijoje – vartojamas skirtingomis prasmėmis (Schutz 1964; 64). Svarstant socialinę epistemologinę tikrumo ir netikrumo santykio problemą verta pasitelkti skirtį tarp a) bendros erdvės ir b) bendros laiko- erdvės. Bendros erdvės koncepciją čia apibūdin­ kime kaip liberalią, o bendros laiko-erdvės – kaip fenomenologinę. X dalies tezės: Liberalus akcentas yra tas: mes, orientuodamiesi socialinėje geometrinėje erdvėje, gręžiamės tik į mūsų veikos objektus, vadovaudamiesi sudėties ir atimties (samprota­ vimo) kriterijais, kurie galiausiai kyla iš dviejų elementariųjų (Hobbes 1999; 68–9) „valingų judesių“: 1) potraukio ko nors link ir 2) nusigrę­ žimo nuo ko nors (indiferentišku atsitraukimu arba pasibjaurėjimu). Fenomenologinė gairė yra ta: mes, orientuodamiesi į savo veiklos objektus, gręžiamės visų pirma į savo veiksmus sugriebda­ mi juos savi-refleksija, są-monės aktais. Galbūt aš, gręždamasis nuo priešreflektyvaus patyrimo į tik reflektyvų, remiuosi pernelyg siau­ romis, vienpusiškomis, svetinančiomis „trečiojo asmens“ (užsieniečio, kartografo, metodologo par excellence ir t.t.) racionalumo analogijomis? Plg.: „Miestas yra vienas ir tas pat trims minimiems asmenims: čionykščiui, užsieniečiui ir karto­ grafui. Tačiau čionykščiui miestas turi savitą prasmę: „mano gimtasis miestas“; užsieniečiui tai yra vieta, kurioje jis tam tikrą laikotarpį turi gyventi ir dirbti; kartografui miestas yra mokslo objektas, – jo tikslas yra nubraižyti žemėlapį. Vadinasi, tas pats objektas siejamas su trimis skirtingais svarstymo lygmenimis“ (Schutz 1964; 67). j fl j Liberalus akcentas yra betarpiška dabartis ir orientacija į kitą pirmą, paskesnį betarpiškumą. Liberalizmas – tai ateities suvaržyta dabartis, siejama tik su veiksmo laikysena ir t.t. Libe­ ralizmas yra suerdvintas laikas. Globalizacijos, arba laiko standartizavimo absoliutinėje erdvėje, lūkesčiai, – jau be laikiškumo vietose. Liberaliz­ mas – tik išorinis, objektyvus, didelis ir visiems vienodas laikas. Susvetimėjęs spyruoklinis La Mettrie pamėklės, žmogaus-mašinos-spyruoklės, laikas. Orientacija į kitą pirmą, paskesnį betar­ piškumą, kaip įprasta, yra instrumentinio veiksmo laikysena ir atitinkami lūkesčiai. Liberali veiks­ mo laikysena, orientuota į betarpiškos dabarties objektus, nelinkusi reflektuoti savo savasties ir savo minčių srauto, nes jaučia antipatiją laikiškumo dimensijai, bet kokiam galimam tradicijos – subjektyvios objektyvaus laiko trau­ kos – poveikiui. Liberalizmo apetitai, arba norai, jaučiasi – kaip namie – tik dabartyje. Minties Galbūt aš vertybiškai neutraliai, kaip kar­ tografuojantis pozityvistas, gręždamasis nuo tipizacijų ir relevancijų gyvenimo-pasaulyje, gręžiuosi į mano paties čia pat išrandamą „iš­ orinį pasaulį“, vadovaudamasis subjektyvistine nominalizmo taisykle, – kad juslinių įspūdžių aibės sutampa su mano duodamais joms – opera­ cionalizuojant sąvokas – vardais. O jei taip, – jei duotus vardus nerelevantiškai (gyvenimo-pa­ saulio atžvilgiu) tapatinu su esmėmis, – tai kyla klausimas, susijęs ir su racionaliu mano paties gyvenimo-planu: ar aš esu relevantiškas savo paties gyvenimo-plano sandaros atžvilgiu, ar tiesiog aklai, kaip marionetė, paklūstu taisyklei (laikausi jos gręždamasis įprotin), aklai laikausi man kitų teikiamos laisvės iliuzijos, tobulos simetrijos tarp kartotinio įpročio ir patyrimu neatnaujinamo proto? 136 Sociologija. X dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija pasirinkimą priklausyti vienai ar kitai socialinei grupei, pasirinkti vieną ar kitą socialinį vaidme­ nį, rinktis kitą pirmą įspūdžių rinkinį pagal skonį arba pagal madą (tai du pagrindiniai liberalios pozityviosios laisvės orientyrai) atliepia liberalus laisvumo kriterijus. Aš tiesiog racionaliai pasi­ renku arba vieną, arba kitą relevanciją mano privačiam racionalumui sutampant su tikrumu. Labai gerai orientuodamasis „progreso“, suda­ ryto iš dviejų vienas kitą keičiančių žingsnių – priežasties ir padarinio – relevancijoje. ir veiksmo „buvojimas praeityje“, liberalizmo požiūriu, yra laisvo kūnų judėjimo įkalinimas. p y ų j j į Fenomenologijos akiratyje – visų pirma laikiškumo analizė. Dabartis į mūsų refleksijos akiratį patenka kaip laikiškai relevantiška, tęstinė praeitis: tai, ką sugriebiame refleksija, niekada nėra mūsų minčių srauto dabartis; tai nėra ne­ priklausoma dabartis; tai, kas randasi refleksijos akiratyje – visad praeitis (Schutz 1962; 172). Fenomenologinės refleksijos veiksmo laikysenoje nedera painioti su liberaliu – tęstiniu tik dabar­ ties priemonių-priemonių grandinėje – veiksmu. Fenomenologinis požiūris atskleidžia liberalaus požiūrio ribotumą, uždarumą, fragmentiškumą, ir nemenka dalimi iš čia kylantį „priemonių-prie­ monių pažangos“ optimizmą, kuris, gręždamasis nuo tęstinės laiko refleksijos, gręžiasi ir nuo gimties-mirties, pažįstamumo-anonimiškumo, esminio žmogaus rūpesčio sąsajų. Tiksliau, nuolat virsdamas šiandienoje, arba virdamas šiandienoje, kurioje vertės atnaujinamos nepaliaujamai. Priežastis (liberali lemtis, kurios aš nežinau ir nenoriu žinoti) suponuoja padarinį (li­ beralią šiandieną). Priežastis (vakardiena, kurios aš nežinau ir nenoriu žinoti) visad yra nuvertėjusi prekė (pastovumas, vertybės, šeima, tėvynė, o ką jau kalbėti apie tokį seną reikalą kaip tautą). Ji visada jau keičiama dabartimi (pasekme), kuri tuoj virsta priežastimi (vakardiena), suponuo­ jančia naują padarinį (šiandieną)... „Vakar“ ir „rytoj“ – tik tuščios iliuzijos – tarnauja kaip liberalui būtino judėjimo dabartyje, – kuri išties jau nebegali judėti (be praeities ir ateities), – po­ liciniai, taigi progresyvūs švyturėliai. Fenomenologinis požiūris pabrėžia, kad gręžimasis į bent kokią „atskaitos pradžią“ reiš­ kia tęstinę savigrąžą ir tęstinę refleksiją: „Todėl visapusiška dabartis ir gyva mūsų savasties dabartis – neprieinamos mūsų refleksijai. Mes galime tik atsigręžti į mūsų minčių srautą tarsi jį būtų sustabdęs paskutinis mūsų sugriebtas patyrimas. Kitaip sakant, savasties-sąmonė gali būti patiriama tik modo praeterito, būtajame laike“ (ten pat; 173). Tačiau tai nereiškia, kad prasmingi yra tik įgyvendinti aktai, o ne tebesitęsiantys veiksmai: „Mes turime turėti mintyse, kad veiksmas pagal apibrėžimą visada grindžiamas priešsuvoktu planu, ir būtent šioji nuoroda į ankstesnį planą daro prasmingais tiek veiksmą, tiek jo refleksiją“ (ten pat; 214). Fenomenologinis požiūris skiriasi. X dalies tezės: Aš nesu tik mano paties pasirinktų „socialinių vaidme­ nų“, tam tikrų tuščių „dabarties laiko atkarpų“, užpildymas faktais, gaunamais iš spontaniškų mano juslinių reakcijų, – potraukių kam nors ir nusigręžimų nuo ko nors. Mano laisvės ap-linka – lygu dar neapibrėžti vidiniai ir išoriniai sąmonės ko nors atžvilgiu orientacijos horizontai. Mes senstame drauge ne izoliuotai (kaip individualūs nepriklausomi vienetai), o pripažindami vienas kito neapibrėžtumo laisvės ribas ir apibrėžtumo Liberalaus ir fenomenologinio požiūrių skirtį galima trumpai aptarti ir kitu atžvilgiu. Mano 137 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 galimybės horizontus. Darkart: pripažindami ne sutampantį tapatumą (tebūnie: net ne moralinį imperatyvą), o suvokdami mūsų neapibrėžtų ir tik abipusiškai apibrėžtinų sąsajų bendra-ho­ rizontą, girdėdami vidinę bendra-sąskambio – laiko tęstinume – darną. galimybės horizontus. Darkart: pripažindami ne sutampantį tapatumą (tebūnie: net ne moralinį imperatyvą), o suvokdami mūsų neapibrėžtų ir tik abipusiškai apibrėžtinų sąsajų bendra-ho­ rizontą, girdėdami vidinę bendra-sąskambio – laiko tęstinume – darną. Abu laisvės filosofai sutaria, kad žmogaus veiksmas kyla iš spontaniškos raiškos (Schutz 1962; 209; Hayek 2002; 58). Dar daugiau: abu filosofai sutaria dėl to, kad spontaniškumo raiškos (nebūtinai susijusios su veiksmu) paty­ rimas apima skirtingas formas. Filosofinėse ir socialinių mokslų metodologijose skirtingos spontaniškumo patyrimo formos artikuliuo­ jamos ir nenoriai, ir neaiškiai. Natūralistinėje biheiviorizmo metodologijoje tuščiai ieškotu­ me atsakymų į šį laisvės ištakų klausimą: kaip skirtingos spontaniškumo formos patiriamos sąmonėje, iš kurios jos ir randasi? Biheiviorizmas sutelkia dėmesį tik į stimulo-atsako, organizmo- aplinkos santykį, skirdamas a) aiškią, b) numa­ nomą ir (kartais) c) numanomą-aiškią elgsenos formas (Schutz 1962; 210–1). Jei tik ir kai tik aš gręžiuosi savastin, visų pirma privalau nuskaidrinti savasties-tipizaci­ jas: savo subjektyvaus skambesio darną arba nedarną aplinkos, kuri turi ne tik gyvą dabartį (duotį), bet ir priešduotą erdvės sandarą ir laiko- sandarą, atžvilgiu. Aš pats tikrinu „tikrumo“ ir „netikrumo“ skirtį, gręždamasis ir tęstinai reflektuodamas priešduotą socialinę aplinką kaip esminę laikiškumo ir erdviškumo sąryšių sąlygą. Tai visad egzistencinis santykis: įmesties kaip būtinybės ir laisvės santykio įsisąmonini­ mas (o ne valingo, arba racionalaus, pasirin­ kimo, kaip liberalizmo atveju). Kitaip sakant, aš-ir-mano-aplinkos relevancija yra egzistencinė savi-tipizacija bendro laiko dermės arba ne­ dermės atžvilgiais, o ne laisvai pasirenkama tipizacija dabarties išteklių vartojimo atžvilgiu. Aš-ir-mano-aplinkos relevancija – ne sekli, ne paviršinė „socialinių vaidmenų“ tipizacija, o gili, nes tęstinė, prasminių sąsajų savi-tipizacija. Abu minimi laisvės filosofai sutelkia dėmesį į prasmę, žmogaus veiksmo laikyseną inter-su­ bjektyvumo relevancijose. X dalies tezės: Juk tai, kas stebėtojui, stebinčiam konkrečių žmonių elgseną, pasirodo kaip objektyviai „ta pati elgsena“, šios elgsenos subjektams gali turėti visiškai skirtingas pras­ mes arba neturėti jokių prasmių (Schutz 1962; 210). Fenomenologinę supratingumo sampratą Hayeko koncepcijoje iš dalies atliepia draugiško veido metafora: „Tai, ką aš turiu omenyje kalbė­ damas apie „draugišką veidą“, nepriklauso nuo skirtingų konkrečių atvejų fizinių savybių, nes tie atvejai gali neturėti nieko bendra. Tačiau aš išmokstu juos atpažinti kaip tos pačios klasės narius – tai, kas juos daro tos pačios klasės na­ riais, yra ne kokios nors jų fizinės savybės, bet priskirtoji prasmė“ (Hayek 2002; 68). Kad minėtoji skirtis tarp a) bendros erdvės ir b) bendro laiko-erdvės ir tarp a) liberalaus spon­ taniško relevantiškumo ir b) politiškai intencio­ nalaus relevantiškumo dimensijų būtų aiškesnė, pasitelkime kitą sąvokų – atviros spontaniškos visuomenės (liberaliųjų mokslų idealo) ir atviros supratingos visuomenės (politinės filosofijos ide­ alo) – porą. Pirmąją koncepciją pavadinkime spontaniškumo filosofijos metodologo Friedricho Hayeko garbei, o antrąją – supratingumo filosofi­ jos metodologo Alfredo Schutzo garbei. Bet kokio veiksmo prasmė gali būti re­ levantiška dviem pagrindiniais atžvilgiais: susijusi arba su asmenišku, arba su socialiniu 138 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija mas, kad visas sąmoningas patyrimas, kurį mes laikome santykiškai konkrečiu ir pirminiu, yra daugelio įvykių, suvokiamų pagal jų svarbą įvairiais atžvilgiais, „klasifikacinės“ virš-primesties („a super-imposition of many „classifications““) rezultatai. Išnarstyti šių „klasifikacijų“ neįma­ noma, kadangi jos randasi vienu ir tuo pačiu metu, ir yra turtingesnio patyrimo, kuris randasi iš šių abstrakčių elementų, sudedamosios dalys (ten pat; 36). lygmenimis. Fenomenologiškai grindžiamos metodologijos požiūriu, pasaulis nėra mano privatus pasaulis, o, priešingai, nuo pradžios intersubjektyvus kultūros pasaulis, prasmių teks­ tūra, kurią mes turime interpretuoti, – tam, kad rastume jame mūsų atramas („our bearings“) ir savo veiksmo laikysenas šio pasaulio atžvil­ giu, abipusiškumo perspektyvas (Schutz 1962; 10–3). Pirminė socialumo, dar tiksliau, intersu­ bjektyvumo, tikrovė yra ir 1) priešduota, ir 2) struktūrizuota į skirtingus socialinius lygmenis, baigtines prasmės provincijas, kurių skiriamieji bruožai – savita sąmonės įtampa, specifiškas abejonės sulaikymas, būdinga spontaniškumo forma, ypatinga savasties patyrimo forma, savita socialumo forma, būdinga laiko-perspektyva (Schutz 1962; 230). Pažymėtina, kad dvinarė Hayeko relevan­ tiškumo koncepcija yra apgaulingai antiraci­ onalistinė. Viena vertus, gręžiamasi nuo bet kokios filosofinio racionalizmo formos. Kita vertus, sąmoningai bėgant nuo konservatyvios meškos (bendrumo, tradicijos, bendrosios valios pirmumo), šokama tiesiai į vilko (abstraktumo ir netgi virš-primesties, o ne pri-mesties, pir­ mumo) glėbį. Virš-primestis – lygu liber-lemtis, ir kerštinga, ir gailestinga „nematomoji ranka“, „socialinis protas“, „pusiausvyros tendencija“. X dalies tezės: Jei fenomenologiškai grindžiama metodo­ logija pradeda tirti skirtingus stebimo socialinio pasaulio lygmenis, siekdama suprasti, kaip subjektyvumo, racionalumo, relevantiškumo dimensijas pilnai išskleidžia patys socialinės tikrovės dalyviai, tai spontaniškoji liberalizmo metodologija pasitiki vien siauresne šių dalyvių racionalumo (priemonių-tikslų) samprata, o „ti­ krąjį racionalumą“ sieja su visumine dviejų spon­ taniškų racionalumo lygmenų pereigos iš vieno į kitą – iš spontaniškų individualių „pliuralumo aktų“ į spontaniškai racionalią „pusiausvyros tendenciją“ – sistema. Liberali Hayeko metodologija „socialumą“ pliuralizuoja, išskaido į individualių vienetinių judėjimų spontaniškas tvarkas. Tačiau klaida būtų manyti, kad šis spontaniškumas laikomas pirminiu aprašymo ir interpretacijos lauku, kaip kad fenomenologijoje: „Socialinės srities mokslininko konstruojami minties objektai ir rodo į ir yra grindžiami minties objektais, kuriuos konstruoja įprastai protaujantis žmo­ gus, gyvenantis kasdienį gyvenimą tarp kitų žmonių“ (Schutz 1962; 6). Hayeko „bendrosios erdvės“ koncepcijoje pirmoji spontaniškumo tvarka yra tik stebima visapusiškesnės antrosios tvarkos forma. „Socialumą“ Hayekas susieja, – siekdamas šokiruoti (galėčiau kalbėti, pažymi Hayekas, apie „bendrumo pirmumą“, bet tai nesukeltų šoko efekto; Hayek 1978; 35), – su „abstraktumo pirmumu“. Tai ne abstrahavimas, grindžiamas „konkretesniu“ patyrimu, o, priešingai, teigi­ Virš-primestis yra aukštesniojo sponta­ niškojo racionalumo forma (juk virš-primestis savaime tvarko). Kad ir kaip aptariamoji 139 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija koncepcija skirtųsi nuo liberalių racionalizmo teorijų, visos liberalizmo koncepcijos nepasitiki kultūra, priešduotiškumu, todėl orientuojasi arba į pusiausvyros tendenciją (savaiminio racionalu­ mo formą), arba remiasi gamtos mokslo metodo teise pradžioje apibrėžti racionalumą, o paskui savaime suprantamai taikyti jį sveiko protavimo lygmenyje: „Kadangi mokslas yra racionalus įgyvendinimas par excellence, čia apibrėžiamas požiūris remiasi analogija tarp mokslininko tyrėjo ir praktiškai kasdienybėje veikiančio asmens“ (Parsons 1949; 58). socialinio įpročio tyrimais, o liberalizmas – soci­ alinio proto. „Socialinis protas“ („social mind“; Hayek 1948; 54) yra pusiausvyros tendencijos – iš anksto neplanuotos, todėl nematomos planuo­ jančios teisingumo rankos – stebukladarys: „tarsi būtų kuriama vadovaujantis vieningu planu“ (Hayek 2002; 58). X dalies tezės: Pakartotina: „tendencija į pusiausvyrą“ (ten pat; 59) yra šios pradinės duoties individualių elementų sponta­ niškos veikos padarinys, savo ruožtu priklauso­ mas nuo „abstrakčių“ klasifikacinių primesčių. vio atitikimo ir neatitikimo, suderinamumo ir nesuderinamumo kategorijomis“ (ten pat; 43). Problema, – kad Hayekas neįžvelgia siste­ miškumo Schutzo koncepcijoje, – yra jųdviejų skirtingi požiūriai į pradžios metodologiją. Ki­ taip nei Hayeko pliuralistinėje erdvės teorijoje, Schutzo teorijoje ap-linka (Schutzo atitinkamai vartojamas terminas – „situation“) nėra tik išorinė faktiškumo tvarka, susidedanti iš įvairių objektyvių dimensijų. Fenomenologiškai grindžiama atviros su­ pratingos visuomenės koncepcija orientuoja į bendro laiko-erdvės patyrimo dimensijas, kurios laikomos socialinio žinojimo viseto (reikia skirti nuo žinių fragmentų) priešduotimis: „Tik labai maža mano šio pasaulio žinojimo dalis randasi iš mano asmeniško patyrimo. Didesnė dalis yra atėjusi socialiai, perduota man mano draugų, mano tėvų, mano mokytojų ir mano mokytojų mokytojų. <...> Gimtas kasdienis gyvenimas yra visų pirma įvardintų daiktų ir įvykių kalba, ir bet koks vardas įtraukia tipizaciją ir viseto-sąsajas, nurodančias į relevancijų sistemą, būdingą kal­ binei bendruomenei, kuri pagrindžia įvardytą daiktą, pakankamai svarbų, kad rastųsi atskiras vardas“ (Schutz 1962; 13–4). Schutzo teorijoje ap-linka yra savasčiai vidujai relevantiška tęstinės praktinės veiklos dimensija: „Kaip teigėme, šis pasaulis, mūsų natūraliosios laikysenos požiūriu, nėra visų pir­ ma mūsų mąstymo objektas, bet dominavimo laukas (a field of domination). Mes esame visų pirma praktiškai juo suinteresuoti, įtakojami būtinybės laikytis pagrindinių mūsų gyvenimo reikalavimų. Bet mes nesame vienodai suin­ teresuoti visais darybos pasaulio (the world of working) sluoksniais. Atrankinė mūsų interesų funkcija organizuoja pasaulį abiem – erdvės ir laiko – aspektais pagrindinių ir šalutinių relevancijų sluoksniuose. Mano aktualaus ir potencialaus akiračių pasaulyje šie objektai yra atrenkami kaip visų pirma reikšmingi, kaip ak­ tualiai esantys arba ateityje tapsiantys galimais tikslais arba priemonėmis, kurios leis įgyvendin­ ti mano planus, trukdys, bus man palankios (ar dar kokios nors kitokios) relevancijos“ (Schutz 1962; 227). X dalies tezės: Socialiniai mokslai „su­ tampa“ su tobulu ekonominiu spontaniškumo (žemesnės tvarkos) ir racionalumo (aukštesnės savaiminės tvarkos) atitikimo aiškinimu: „Nors esame praleidę esminę mūsų argu­ mentacijos grandį, vis dėlto manau, kad bent jau implicitiniu savo argumentacijos turiniu ekonomikos teorija labiau nei bet kuris kitas socialinis mokslas priartėjo prie atsakymo į svarbiausią visų socialinių mokslų klausimą: „Kaip gali žinių fragmentų, kuriais disponuoja skirtingi protai, junginys sukurti rezultatus, kurie, jeigu jų būtų siekiama sąmoningai, iš vadovaujančio proto reikalautų tokių žinių, kuriomis negali disponuoti joks atskiras pro­ tas?“ Sugebėjimas parodyti, kad spontaniniai individų veiksmai (tam tikromis sąlygomis, kurias galime apibrėžti) sukuria tokią išteklių pasiskirstymo tvarką, kuri atrodo taip, tarsi būtų kuriama vadovaujantis vieningu planu, nors jos niekas neplanavo, mano požiūriu, išties būtų at­ sakymas į tą problemą, kuri kartais metaforiškai apibūdinama kaip „socialinio proto“ problema“ (Hayek 2002; 58–9). Fenomenologiškai grindžiamos metodologijos požiūris yra tas: tik po preliminarių socialinio pa­ saulio prasminių lygmenų – tipizacijų, relevanci­ jų – aprašymo, galima tirti socialinį pasaulį taip, 1) kaip jis pasirodo stebinčiam mokslininkui, ir 2) kaip tarp savęs dera mokslinės ir kasdienybės interpretacijos (Schutz 1964; 65). Liberalios natūralistinės metodologijos požiūris yra tas: dvi tvarkos – žemesnė individualistinė spontaniškoji ir aukštesnė klasifikacinė spontaniškoji – pereina viena į kitą kaip dvi tarp savęs susisiekiančios pliuralumo tikrovės. Liberali tvarka randasi natūraliai: kūnams laisvai judant į priekį, atgal ir į šalis išoriniame jų racionalių pasirinkimų – priemonių-tiks­ lų – horizonte. Be jokių vidinių horizontų, be teleologinių laikiškumo prasmės struktūrų. Laisvė įsisąmoninama kaip nematomos rankos būtinybė: normos, taisyklės, institutai yra daugy­ bės individualių elgsenų nenumatyti padariniai. Abstrakčios klasifikacinės pusiausvyros primestys būtinai lenkia mus visus nenumatytų padarinių pusiausvyron. Minėta skirtis tarp bendros erdvės ir bendro laiko-erdvės dimensijų darosi aiškesnė. Libera­ lizmas rodo į „duotį“, esmingai fragmentuotą, pliuralistinę bendrą erdvę, sudarytą iš trijų pagrindinių elementų, – daugelio individų, jų spontaniškumo, jų žinių fragmentų („fragments of knowledge“; Hayek 1948; 54; ne „žinojimo-fra­ Čia randame esminį skirtumą tarp liberaliz­ mo ir fenomenologijos. Fenomenologija pradeda 140 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija vio atitikimo ir neatitikimo, suderinamumo ir nesuderinamumo kategorijomis“ (ten pat; 43). Problema, – kad Hayekas neįžvelgia siste­ miškumo Schutzo koncepcijoje, – yra jųdviejų skirtingi požiūriai į pradžios metodologiją. Ki­ taip nei Hayeko pliuralistinėje erdvės teorijoje, Schutzo teorijoje ap-linka (Schutzo atitinkamai vartojamas terminas – „situation“) nėra tik išorinė faktiškumo tvarka, susidedanti iš įvairių objektyvių dimensijų. gmentų“, o žinių fragmentų,– Algirdas Degutis verčia tiksliai; Hayek 2002; 58). X dalies tezės: Pripažindamas, kad „žinių pasidalijimo“ (Hayek 2002; 55) problema rodo bendresnio žinojimo sąsajų link („<...> mes faktiškai nusi­ ritame atgal prie prielaidos, kad kiekvienas žino viską, ir todėl net nemėginame ieškoti realaus problemos sprendimo“; ten pat) ir kad nereikia gėdytis, kad ekonomikos mokslas yra „tik“ tam tikra logikos atmaina, o tiesiog atvirai pripažinti šį faktą (ten pat; 70), rodantį intersubjektyvumo link, Hayekas (puikiai asmeniškai pažindamas ir Alfredą Schutzą, ir saistančią jo socialinių mokslų metodologiją) sausai pareiškia: „Mane nuo seno stebina tai, kad, mano žiniomis, sociologijoje nėra jokių sisteminių mėginimų individų siekius ir norus analizuoti jų tarpusa­ Filosofiją į ekonomiką redukuojantys fi­ losofai mato, tačiau nepastebi spontaniškumo patyrimo sąsajų įvairovės gyvenimo-pasaulio struktūrose (kurios labai skiriasi nuo abstrakčios metaforos, „socialinio proto“; Hayek 2002; 59). Tikra tiesa, kaip nustato didysis Hobbesas, kad 141 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija individualūs siekiai ir norai susiję su mūsų ape­ titais ir aversijomis, – o ne tik panašiais žodžiais, „atėjusiais iš lotynų kalbos“ (Hobbes 1999; 69). Tačiau elementariosios, instinktyviosios gyvūnų, taigi ir žmonių, relevancijos nėra pavieniui izoliuotos. Tik vulgariu liberaliu požiūriu, nesve­ timu ir sveikam protui, potraukiai, atotrūkiai, pertrūkiai prasideda drauge su individualių kūnų „norų“ ir „pasibjaurėjimų“ – išorinės aplinkos atžvilgiu – judėjimu: ir tik horizonta­ lioje geometrinėje dabartiškumo erdvėje. arba tradicijos, saviduotiškumo, todėl tarp savęs nesusieja subjektyvių norų ir subjektyvių vertybių formų, išlavintų bendro politinio intenciona­ lumo patyrimo, vaizduotės ir proto įgūdžiuose. Kad ir kaip tarp savęs skirtųsi liberalizmo formos, visos jos – atkakliai natūralistinės. „Juk pati gamta dažnai įdiegia tas tiesas, ant kurių žmonės vėliau suklumpa, kai ieško ko nors šalia gamtos“ (Hobbes 1999; 69). Kitaip sakant, nėra ko daugiau ieškoti samprotavime kaip vien „elementų sudėties ir atimties“. Arba šian­ dieną: „paklausos ir pasiūlos kreivių“. Ir netgi kritikuojantys abstrakcijas filosofai ekonomistai sukasi vienoje ir toje pačioje natūralizmo orbi­ toje (bando kritikuoti „ankstesnį“ savo požiūrį, tačiau neįsileidžia į „naujesnį“ požiūrį socialinių mokslų metodologijos): „Man turbūt derėtų prisipažinti, – rašo Friedrichas Hayekas, – kad pradėdamas studijuoti savo discipliną buvau visiškai įsitikinęs gamtos mokslų metodų uni­ versalumu“ (Hayek 2002; 61). Esti skirtingos spontaniško patyrimo žmo­ gaus sąmonėje ir veiksmo laikysenoje formos, neabejotinai susijusios su dalykais, kuriuos Hobbesas, socialinių gamtos mokslų pirm­ takas, vadina potraukiu (appettite), arba noru (desire), ir pasibjaurėjimu (aversion). X dalies tezės: Tačiau net elementariausi po-traukiai arba ato-trūkiai susiję ir su spontaniško patyrimo atsaku daiktų atžvilgiu, ir su vaizduotės (tikėtinumo, galimybių, alternatyvų numanymo) bei proto (sąmonės ir daikto santykio ribų nuskaidrinimu ir suprati­ mu) gebėjimais, kurie savo ruožtu tobulinami prasmingo žmonių bendra-buvojimo laike ir erdvėje, veiksmo, laisvės ir atsakomybės sąsajų skambesyje. Šis įsitikinimas, veikiau naivus scientistinis tikėjimas, gyvenimo-pasauliui yra pragaištingas. Scientistinis požiūris yra nemokslinis tikrąja šio žodžio prasme, nes mechaniškai ir nekritiškai taiko savo srityje suformuotus mąstymo įpro­ čius visų kitų mokslų sritims (Hayek 1978; 30–1). Dar daugiau, anot Hayeko, galima kalbėti apie „didįjį pokytį“ („the great change“), kuris suformuoja tvarką, žmogui vis labiau nesuprantamą, – atvirą abstrakčią visuomenę, kurios nebesieja į draugę bendri konkretūs tikslai, o tik paklusimas toms pačioms abstrak­ čioms taisyklėms (Hayek 1979; 164). Tai ilgos pliuralizmo – bauginimo praeitimi – diegimo raidos, kelio į beprasmišką abstrakcijų tikrovę ir vergovę, padarinys. Klaidinga laikyti aplinką mums-primestimi. Mūsų aplinka yra pri-mestis, kuri mums rele­ vantiška vidujai, o ne tik išoriškai. Relevancijos tęsiasi: jos išgyvenamos erdvėje ir išgyvenamos laike. Fenomenologiškai grindžiama metodo­ logija, kitaip nei liberali, sutelkia dėmesį ne į grynąją vaizduotę (tarkim, „socialinio proto“ metaforą), o į konkrečias prasmingas praktinio veiksmo tipizacijas ir relevancijų struktūras. Li­ beralizmo tradicija – nuo Hobbeso iki Hayeko ir Rawlso – primygtinai nepaiso priešduočių, 142 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija vertybiškam-anonimiškam individualių libera­ lizmo „marionečių“ („racionalių“ ekonomi­ kos veikėjų) modeliui. Tikrasis liberalizmas (suprask: tikrasis individualizmas) jau gali būti nebesiejamas su „apibrėžta politine doktrina“ (ideologine liberalizmo filosofija), o tik su „bendrąja sąmoningumo laikysena“ („general mental attitude“), reikalaujančia „išsilaisvinti nuo visų prietarų“ ir visų įsitikinimų, kurie negali būti „racionaliai pateisinami“ („rationally justified“). Racionalumas – lygu pliuralizmas: tai, ko racionaliai nepateisina pliuralizmas, turi būti „atmetama“ kaip „metafizika“. „Heterogeniškų principų ir faktų dariniai“ („istorinio atsitikti­ numo“ padariniai), Johno Rawlso supratingai vadinami „supratingu pliuralizmu“ yra natūra­ listinis tikėjimas natūralioje laikysenoje, įtariai žvelgiantis į bet kokį prie-raišumą, bet kokią tradiciją, ir siekiantis „supratingai rekonstruoti visą visuomenę“ („deliberate reconstruction of the whole of society“). Nors liberalai nostalgiškai sapnuoja „drau­ giškus veidus“, tačiau priešais juos styro tik aklos technologinės abstrakčių taisyklių akys. Nors bet kokios abstrakčios taisyklės turi būti derinamos, – jei tik ir kai tik laikomasi rele­ vantiškumo postulato, – su jau interpretuotais faktais, atitinkamomis tipizacijomis ir relevan­ cijomis gyvenimo-pasaulyje, tačiau „tikrajam individualistui“ (Hayek 2002; 12), tradicijos struktūros yra paprasčiausiai – pagal šios tradi­ cijos prielaidų logiką –nerelevantiškos. X dalies tezės: Kitaip sakant, tikras liberalas adekvatumo postulato nesilaiko dėl vertybinės (vadinamosios neutralios) laikysenos, kurios psichologines sąlygas apibrėžė didysis Hobbesas, su pasibjaurėjimu gręždamasis nuo praeities. Tikras individualistas, akivaizdžiai maty­ damas abstrakcijų viešpatavimą po-politikos pasaulyje, atsakymų ieško ne praktinių rele­ vancijų, išgyvenamų laike ir erdvėje, pasaulyje, o abstrakčios lemties – istorinio atsitiktinumo (priežasties) ir heterogeniškų principų (padari­ nio) – logikoje: „Tokie terminai kaip „liberaliz­ mas“ arba „demokratija“, „kapitalizmas“ arba „socializmas“ šiandien jau neteikia nuorodos į jokias nuoseklias idėjines sistemas. Jie nusako visiškai heterogeniškų principų ir faktų darinius, kuriuos istorinis atsitiktinumas susiejo su šiais žodžiais, nors jie neturi beveik nieko bendra, išskyrus nebent tai, kad skirtingais laikais šias idėjas arba vien tik jų pavadinimus skelbė tie patys žmonės“ (ten pat; 11; išskirta mano – A. V.). Vyraujantis natūralistinis pliuralizmas yra „britų liberalizmo forma“ (Hayek 1978; 119–20), skiriama nuo „konstrukcionistinio žemyninio liberalizmo“. Ne tik skiriama, bet ir atskiriama. Kaip ir pozityvizmo istorijoje: pradžioje atsiskiriama nuo tradicijos (religijos ir metafizikos, filosofijos), o paskui – vėl nuo tradicijos (naujos pozityvizmo brolijos atsiskiria nuo kitų, tradicinių). Taip ir liberalizmo isto­ rijoje: tikrasis vertybinis individualizmas, arba pliuralizmas, „matomais, bet nepastebimais“ ry­ šiais susijęs su beasmene rinkos disciplina žygiuoja per šiuo-laikinę istoriją, atskirdamas save nuo bet kokios tradicijos, kol galiausiai sutampa pats su savimi ir virsta politinio egalitarizmo, tebe­ vadinamo jam priešingu – pliuralizmo – vardu, Tikras liberalas šiandieną kalba giliai vertybiniu – vertybiškai neutraliu – priežasties (istorinio atsitiktinumo) ir padarinio (pliura­ lizmo) – vardu. Pasaulyje absoliučiai vyraujant 143 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 forma. Kad ir kokios įmantrios būtų liberalizmo teorijos, visų jų esmė – karinga ne teorinė, ne socialinė metodologinė ir ne filosofinė laikyse­ na: „Nėra jokios kitos galimybės: arba tvarka, pasiekiama per beasmenę rinkos discipliną, arba tvarka, nustatoma keleto individų valia <...> (Hayek 1991; 278). Ekonominio liberalo požiūriu, nėra ir negali būti politinio intencionalumo priešduotyje: tai esą nuo pradžios užakęs vergovės kelias, tradicija. Juk tikrojo individualizmo laisvė – pasipriešinimo ne­ buvimas. O kai intencionalumo tradicija visiškai nebesipriešina, visi tampa laisvi, arba išlaisvėję. Kai „tradicija“ jau išpasakota ir atkerėta, kai pereinama „<...> iš teologinės ir filosofinės tiesos į civilinio biznio tiesą“ (the truth of civil business; Bacon 2001; 13), ateina amžina dabarties – civi­ linio biznio – laisvė, ir galima nebebijoti mirties: „Žmonės bijo mirties, kaip vaikai bijo tamsos; ir kaip natūrali baimė vaikuose išauga drauge su pasakomis, taip pat ir mirties baimė“ (ten pat; 14). X dalies tezės: Pirma, neigia proto ir vaizduotės darną (kaip dar neatke­ rėtą, prietaringą tradicinį prieraišumą prie „esą egzistuojančios sielos“), antra, santykius tarp žmonių susiaurina į 2) „vartojimo“ ir 3) „ma­ nipuliacinius“ santykius (Nussbaum 2010; 6). Nesunkiai galime įžvelgti, kad pagal šiuos tris bruožus vertinami ne tik smulkieji laisvumo laikysenos pernešėjai-fariziejai dabartinėse inter­ netinėse žiniaskaidose ar vidutiniokai – nepri­ klausomų-TV šurmuose, bet ir pomoderni dok­ sinė valdžia (kuriai socialiniai santykiai – lygu valdžios santykiai), kuri, pagal parengtus stan­ dartinius kriterijus, ir vertinama ne pagal sveiką nuovoką, teorinį protą ir strateginę vaizduotę, o būtent pagal paraidiškumą (procedūrinę raidę), paradiškumą ir įspūdį auditorijai (reitingus). Tai integrali „kultūrinio“ liberalizmo projekto – ir kraštutinai abstraktaus, ir pedantiškai empiristi­ nio – pranešimas pro Kitą ir patį save. Kai „pilietiniai įstatymai“ prilyginami „dirbtinėms grandinėms“ (Hobbes 1999; 219), darosi visai neaišku (stingant gyvosios tikrovės) Paraidiškumas liberalizme tobulai dera su atitinkama šiuolaikine uniforma, kurią dėvi pagrindiniai laisvumo laikysenos pernešėjai fa­ riziejai, kurie, pasitelkiant Martha’os Nussbaum trumpą ir tikslų apibūdinimą, turi tris darbinių santykių su žmonėmis – vieną negatyviosios lais­ vės ir dvi pozityviosios laisvės – bruožus. Pirma, neigia proto ir vaizduotės darną (kaip dar neatke­ rėtą, prietaringą tradicinį prieraišumą prie „esą egzistuojančios sielos“), antra, santykius tarp žmonių susiaurina į 2) „vartojimo“ ir 3) „ma­ nipuliacinius“ santykius (Nussbaum 2010; 6). Antra, tapatumas komponuojamas iš vidaus. Suskliaučiant save savyje vidinio papriešinimo stokos, – „laisvės nelaisvėje“, arba „nelaisvės laisvėje“, – kabutėmis. (Tikrajam liberalui prie­ šinimasis – tik iš išorės: „priešinimąsi aš suprantu kaip išorines judėjimo kliūtis; Hobbes 1999; 217.) Suskliaučiant visus iš išorės ateinančius tradicinius pasakojimus, – ypač apie praeitį, mirtį ir absurdą, – dar prieš jiems pasirodant. Laukiant ir tikintis vidinio spontaniškai laisvo pasyvumo kaip imanentiško išganymo. Laukiant nieko kaip privalomos vidinio nesipriešinimo idėjos malonumo. Laukiant vidinio nesiprie­ šinimo savo iš išorės steigtai nepriklausomo individo vaidmens – laisvųjų vertybių pranešėjo, laisvės kelio ženklų reguliuotojo, laisvės pareigūno, autoritarinės asmenybės, komandinio žaidėjo, režimui tarnaujančio fariziejaus – figūracijai. Nesunkiai galime įžvelgti, kad pagal šiuos tris bruožus vertinami ne tik smulkieji laisvumo laikysenos pernešėjai-fariziejai dabartinėse inter­ netinėse žiniaskaidose ar vidutiniokai – nepri­ klausomų-TV šurmuose, bet ir pomoderni dok­ sinė valdžia (kuriai socialiniai santykiai – lygu valdžios santykiai), kuri, pagal parengtus stan­ dartinius kriterijus, ir vertinama ne pagal sveiką nuovoką, teorinį protą ir strateginę vaizduotę, o būtent pagal paraidiškumą (procedūrinę raidę), paradiškumą ir įspūdį auditorijai (reitingus). Tai integrali „kultūrinio“ liberalizmo projekto – ir kraštutinai abstraktaus, ir pedantiškai empiristi­ nio – pranešimas pro Kitą ir patį save. X dalies tezės: Štai kodėl liberalas gręžiasi nuo pasakų, pasakojimų ir tradicijų, sukdamasis vaikiško ne­ kaltumo link, įmesties-dar-neatsakomybėn, – čia ji ar jis nebegirdi baisių „tradicinių pasakų“ apie mirtį. Amžina dabarties – civilinio biznio – laisvė yra vidujai ištuštinta, išlaisvinta nuo sąmonės ko nors, tik fiziškai judančių kūnų erdvė. Tvarka, kurią nustato „beasmenė“ rinkos disciplina, turi aiškių tarsi-egalitarizmo, rea­ listinių-faktų-totalitarizmo bruožų: realistas „negali logiškai pripažinti kito vertybių mato, kaip tik faktiškumo matą“ (ten pat; 260–1). Tačiau pagrindas, kuriuo remiantis būtų galima bent jau kritikuoti šiuos funkcinius realistinių faktų totalaus pliuralizmo bruožus, yra ta pati abstrakti ekonominio samprotavimo „Jei..., tai...“, – ieškant „priežasčių“ ir „padarinių“ beasmenėje globalios rankos formoje, – logika. Ekonomistas – neprietaringas: instrumentinių priemonių-tikslų sąryšių niekad neieško nera­ cionaliuose arba iracionaliuose kultūros, arba tradicijos, sluoksniuose. Kategoriškas filosofų ekonomistų (liberalų) įsitikinimas kyla iš būdingos veikiau sveikam protavimui, o ne supratingam teoriniam požiūriui, laikysenos „Tebūnie...“. Šioje erdvėje tapatumas kuriamas dviem pagrindiniais būdais. Pirma, iš išorės. Netei­ singumo visuomenėje arba individualioje savi­ vokoje klausimai susiejami ne su struktūrinių bendro egzistavimo sąlygų (o ypač kokybiškai reikšmingu laisvės intencionalumo sąlygų) nuskaidrinimu, o vien kiekybiškai reikšmingu lygybės priemonių – madų ir skonių sferose – užtikrinimu. Nėra kito pasirinkimo, nei tas, kurs yra. Tai gręžtis vaikiško nekaltumo link: įmesties-dar-ne­ atsakomybėn. Pasirinkimo nėra. Yra tebūnie... Tai totalus ir totalitarinis – „<...> juk jei egzistuoja toks dalykas kaip totalitarinė asmenybė ar to­ talitarinė mąstysena, tai išskirtinis jos bruožas, be abejonės, yra toks išskirtinis sugebėjimas prisitaikyti ir nuoseklios pozicijos neturėjimas“ (Arendt 2001; 306) – politinio intencionalumo marinimas prisitaikymu, – ekonominio tebūnie intencionalumo užmestyje. Reikalaujant ne laisvės sąlygų, o teisių laisvėms sąrašų papildymų: pripažinimo, dė­ mesio, patrauklumo identifikavimo ir būtino individualaus grožio demonstravimo, nepaliau­ jamų ginčų dėl ko nors, privalomos institucijų restruktūrizacijos, palankių konkursų dėl kam nors nepalankių konkursinių sąlygų, visiems 144 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija privalo atitikti ne tik dėsnio raidę, bet ir jo dvasią“ (ten pat) – liberalizme ilgainiui tobulai suderinami. vienodo ir lygaus prisitaikymo prie naujų pi­ nigų išlaisvinimo aplinkybių ir sąlygų, visiems vienodo ir lygaus prisitaikymo prie naujų sek­ sualinių nuotykių išlaisvinimo atsitiktinumų, ne atsitiktinių, o būtinų skundų dėl nepakankamų išsilaisvinimo sąlygų ... suderinami. Paraidiškumas liberalizme tobulai dera su atitinkama šiuolaikine uniforma, kurią dėvi pagrindiniai laisvumo laikysenos pernešėjai fa­ riziejai, kurie, pasitelkiant Martha’os Nussbaum trumpą ir tikslų apibūdinimą, turi tris darbinių santykių su žmonėmis – vieną negatyviosios lais­ vės ir dvi pozityviosios laisvės – bruožus. X dalies tezės: Nudėvint ir nudrengiant „laisvės“ sąvokos turinį iki visiškos jos priešybės, „snūdulio“: nudėvėtų sąvokų galia tampa juo tironiškesnė, juo labiau tradicija praranda savo gyvybinę jėgą ir juo labiau silpsta jos pradžios prisiminimas (Arendt 1995; 32–3). Plg. su Kanto moralės dėsnio sąvokos nudėvėjimu liberaliai juslinėse naudos, arba laisvumo verslo, pratybose: „<...> net rizikinga manyti šalia moralės dėsnio esant dar kelias kitas paskatas (pavyzdžiu, naudos)“; su juslėmis ar netgi jausmu siejamas moralumas vadintinas tik legalumu (Kantas 1987; 90–1). Nauda ir legalumas paraidiškame – „poelgis Kai „pilietiniai įstatymai“ prilyginami „dirbtinėms grandinėms“ (Hobbes 1999; 219), darosi visai neaišku (stingant gyvosios tikrovės), kas yra laisvė, o kas – laisvumas (Hobbes 1999; 217; liberty or freedom; Hobbes 1998; 139). Grandinių liberalizme, keistai persipina išorinė policija ir vidinė policija. Akis pilna laisvų norų, bet širdis – pripildyta baimės, formuodama hie­ 145 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija rarchinį vergo ir šeimininko santykį: juo daugiau laisvumo, tuo daugiau galimų suvaržymų; juo daugiau galimų suvaržymų, tuo daugiau teisių laisvėms reikalavimų... skaičiavimo komisijomis ir ne mažiau šurmulin­ gomis laisvojo eterio diskusijomis? Nesant jokio pasirinkimo – „Arba liberalizmas, arba vergovė“, grūmoja arba „naivios nepriklausomybės“ arba „išvirkščio totalitarizmo“ (Wolin 2008) pirštas – leidžiama laisvai diskutuoti „atviroje abstrakčio­ je visuomenėje, kurios nebesieja į draugę bendri konkretūs tikslai, o tik paklusimas toms pačioms abstrakčioms taisyklėms“ (Hayek 1979; 164). Juk dirbtinės grandinės, tik formaliai pri­ lygintos pilietiniams įstatymams ir visuomenei, negali turėti nei politinio, nei socialinio inten­ cionalumo. Šios grandinės, kaip sužinome iš Hobbeso skaitymų, sudarytos iš dviejų „galų“: lūpų ir apatinio galo. Pirmasis yra racionalus, sudarytas iš gražių „vertybinio neutralumo“ žodžių, o antrasis – iracionalus, sudarytas vien tik iš romantiškų norų, išlaisvinančių iš nelaisvės nuo kūno nelaisvės. Abi liberalizmo dalys atme­ ta viena kitą: kaip kad lūpos atmeta apatinį galą, kaip kad racionalizmas atmeta iracionalizmą, kaip kad viršus atmeta apačią, o apačia – viršų. Tikrojo liberalizmo, pliuralizmo, modelis sutampa su tikrojo individualizmo, raciona­ laus homo economicus (grynosios racionalumo marionetės), modeliu. Abstraktus teisinis (o ne įstatymiškas, grindžiamas santykiu tarp proto ir papročio) modelis sutampa su abstrakčiu ekonominiu. X dalies tezės: Šio šalto ledo gabalas, legitimuo­ jantis kultūros esmei ir politiniam žmogaus intencionalumui priešišką ir svetimą vertybių komercializaciją, yra, kaip aiškiai apibrėžia Carlas Schmittas, galios pasisavinimas: „Tas, kas šiandien nori pabrėžti, kad jis turi teisę ir kad jo pretenzijos yra pagrįstos, tam nusakyti dažniau­ siai vartoja žodį legitimus, o ne legalus, – net ir tada, kai savo teisės pagrindą susikuria savo iš­ leistu įstatymu ir valdo visas įstatymo galimybės sąlygas – konsensą, viešąją nuomonę, įstatymo leidimo proceso faktorių valdymą, – taigi kai jo galios įgijimas ir moksliškai gali būti vadinamas galios pasisavinimu“ (Schmitt 2014; 170). Tipiškas individualistas tipiškose aplinkybė­ se Fiodoro Dostojevskio „nesveikas“, „piktas“, „nepatrauklus“ žmogus (Достоевский 1985; 229), irgi sudarytas iš dviejų „galų“: lūpų ir ap­ atinio galo. Abu – šeimininko ir vergo – „galai“, tarp savęs susiję tiesiogiai proporcingai: lūpos laukia meduolio, o apatinis galas – rykščių. Galiausiai jie abu laisvę randa savo paties sau pačiam paraidiniame nesipriešinime (aukščiau­ sios prabos liberalizme!), savęs paties pažeminimo malonume: „Bet argi galima, argi galima nors kiek gerbti savyje žmogų, kuris netgi pačiame savęs pažeminimo jausme prisiekia rasti malo­ numą?“ (ten pat; 239). Trys didieji liberalūs institutai (eilės tvar­ ka) – ekonomika, politika ir teisė okupuoja, t.y. visiškai formalizuoja, socialumo sferą, privatizuo­ ja ir paverčia visuomenės nuomonės apklausomis esminę bendro gyvenimo priešduotį, kultūrą, arba religiją (platesne šio žodžio prasme: pras­ mingumo sąsajas). Kultūrai nebelaikant vis labiau išlaisvėjančių individų, jiems belieka Argi galima, argi galima nors kiek gerbti savyje žmogų, kuris, atgavęs nepriklausomybę, pačiame laivės pažeminimo jausme, – perdavime įgaliotinių tarybos logikai laisvo laisvės išparda­ vimo sąlygomis, – prisiekia rasti malonumą: estetinį žavesį laisvų rinkimų šurmais, balsų 146 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija F. Hayekas, kalbant lietuviškai, taigi papras­ tai, „pyksta ant Hobbeso“. Viena vertus, ne be reikalo: išlenda kaip Pilypas (teisinis pozityvis­ tas) iš kanapių pačioje „angliško liberalizmo“ tė­ vynėje. Kita vertus, pyksta be reikalo: Hobbeso įgaliotinių pliuralizmas nurodo kelią į nacionali­ nę liberalizmo formą, o Hayeko spontaniškasis – į globalią liberalizmo formą. Esminis panašumas tarp jų – vienas ir tas pat pliuralistinis vienetas: šiuo-laikinis individas, kuris nebegali gyventi be pliuralizmo tarsi-religijos. Šios tarsi-religijos esmė – nepaliaujama individualizmo vertybių migracija, dar likusių kitokių vertybių reliaty­ vizacija ir žmogaus susvetinimas ir praeities, ir savęs paties atžvilgiu. skubėti į priekį, nes kelio atgal nebėra: „Šių dienų skubėjimas nepalieka žmonėms laiko mąstyti dar prieš jiems ką nors darant. X dalies tezės: Jie yra išdidūs būdami „darytojais“, neįtardami, kad jie tampa ardytojais, – ir gamtos ir savęs“ (Lorenz 1974; 19). Kai niekas nelaiko, laikas gena už mąstančio laiko: „Laikas yra pinigai.“ „Dabartiniai įvykiai skiriasi nuo istorijos tuo, kad mes nežinome padarinių, kuriuos jie sukels“, – savo pirmąjį sakinį – aklai pasitikėda­ mas „angliško liberalizmo“ genetika – įrašo Frie­ drichas Hayekas savo Kelią į vergovę, ją skirdamas nesusipratėliams, „Visų partijų socialistams“, o knygos pabaigoje pridurdamas: „Jeigu pirmasis mūsų mėginimas sukurti laisvų žmonių pasaulį nepavyko, turime mėginti iš naujo“ (Hayek 1991; 330). Kitaip tariant, privalome iš naujo išbandyti šaltą ir aklą tikrųjų individualistų kalkuliaciją „Homo homini lupus“. Turint pagreičio tikslą: kad iš baimės neatsigręžtum į tai, ko jau nėra; kad iš baimės neatsigręžtum į kultūrą, į mirtį. Y dalies tezės: a) juo labiau atskiriame savasties tapatumo ir laisvės patyrimo dimensijas, tuo labiau aiškėja, kad laisvė virsta tik negatyviąja kaive, – laisvumo laikysena utopinio realizmo, arba niekuo netikinčio pliu­ ralizmo, laikysenoje; „Visuomenė“ – tik paklusimas toms pa­ čioms abstrakčioms taisyklėms (Hayek 1979; 164). Trys didieji liberalūs institutai – ekonomi­ ka, politika ir teisė – pagaliau įgyvendina „didįjį pokytį“ („the great change“). Dabar drąsiai galime įsitraukti į liberalias diskusijas: „Aš manau taip, o aš – ne...“; „aš manau, kad scientistinis požiūris yra nemokslinis tikrąja šio žodžio prasme, nes mechaniškai ir nekritiškai taiko savo srityje su­ formuotus mąstymo įpročius visų kitų mokslų sritims (Hayek 1978; 30–1), „O aš manau, kad scientistinis požiūris yra mokslinis tikrąja šio žodžio prasme... b) juo sparčiau gyvenimo-pasaulyje įsi­ tvirtina abstraktusis racionalizmas, atskiriantis asmens tapatumą ir asmens laisvę (laisvės patyrimo aktą), tuo labiau išryškėja išvirkščios negatyviosios laisvės vertybės ir tuo greičiau lietuvis virsta „lithuanian“; c) juo artimiau susiejame savasties tapatu­ mo ir laisvės patyrimo dimensijas, tuo labiau aiškėja, kad laisvės patyrimas yra originalo suvokimas, kai Kitas stovi prieš mus kūniškai ir inter-subjektyviai baigti­ nio laikiškumo ir tęstinio istoriškumo laikysenoje. O aš manau, kad scientistinis požiūris yra visiškai sekuliarizuoto (be pasakų) šiuo-laikinio žmogaus tarsi-religija, pliuralizmas. 147 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Lygūs – su lygiais, nelygūs – su nelygiais, dideli – su dideliais, mažesni – su mažesniais, – šitaip surikiuokime (tik štai čia) žodžių tvarką; tam, kad parodytume, jog didžiojo Leviatano tiesos – mums per didelės (tuščia jų, tų šven­ tųjų): „Leviatanas yra didžiausias, galbūt vie­ nintelis, politinės filosofijos šedevras, parašytas anglų kalba“ (Oakeshott 1975; 3). Buvodami štai-čia, – ne iš racionalistinių aukštumų, o iš čiabūtiškumo, lytinčio aktualius ir galimus lais­ vės upės tėkmės atsitiktinumus, – suabejokime natūralistine didžiojo Leviatano tiesa: kad laisvė ir būtinybė yra lengvai suderinamos kaip vanduo ir upės vaga (Hobbes 1999; 218–9). rutinos vidurio, suviskam panirti laisvo kūnų judėjimo (be transcendentalinio-aš sąlygų sa­ viapmąstymo galimybės) būtinybėn, tėkmėn. Be galimybės prarasti galbūt klaidinančią ne-laisvės būtinybę. Be galimybės atsigręžti į subjektyvis­ tinius tėkmės laisvumo labirintus. Be galimybės išgyventi – ją asmeniškai patiriant – laisvės ir būtinybės santykio esmę. Būtinybė ir laisvė teoriniame liberalizme postuluojamos kaip du kintamieji dydžiai, kuriems aiškiai trūksta institucinės būtinybės – pozityvistinio mokslo ir liberalizmo politikos. Y dalies tezės: Kitaip tariant, naujo individualizmo reiš­ kinio plitimui naujaisiais laikais (Tocqueville 1996; Oakeshott 1975) trūksta ir pozityvaus įtikrinimo savaime suprantamame šiuo-laikiš­ kume, – tarkim, Talcotto Parsonso „empirinių faktų“ arba Johno Rawlso „pliuralizmo faktų“, – ir naujųjų institucinių dirbtinės visuomenės sąlygų – diferenciacijos, pliuralizacijos, reliatyvi­ zacijos – įtvirtinimo politikos, kurios šalutiniai padariniai yra naujojo žmogaus psichologinių bruožų – indiferentiškumo, narcisizmo, egoiz­ mo – normalizavimas. Naujas žmogus naujoje dirbtinėje visuomenėje nepripažįsta objektyvaus santykio tarp vidinio aš-gyvenimo ir bendrojo visuomenės gyvenimo. Šį pozityvų santykį Hob­ besas analitiškai išskaido į daugelį subjektyvių santykių, – daugybę negatyviosios kūniškos laisvės norų judėti be pasipriešinimo. Tiksliau: judėti inertiškai laisvosios rankos subjektyvizmo labirinte, „klaidinančioje būtinybėje“ (Unger 2001; xvii). Mūsų čia-tezė yra ta: savitoje metaforoje, kurią vartoja ankstyvasis liberalizmo (vieniems – vulgaraus materialistinio, kitiems – didaus idealistinio) pirmtakas Hobbesas, įrodinėdamas tezę, kad laisvė ir būtinybė yra suderinamos, glūdi natūralistinis procedūrinės laisvės-lygy­ bės įprotis, kuris, akcentuojant išorinių kūnų judėjimo tvarką be vidinio sąmonės akiračio ir horizonto, virsta – jau po daugelio šventų metų – procedūrinės kilmės ne-laisvės inercija, arba empirinių faktų kaupimo arogancija (o tai yra viena ir tas pat). „Laisvė ir būtinybė yra suderinamos, kaip, pavyzdžiui, upės vanduo, kuris turi ne tik laisvę, bet ir būtinybę tekėti savo vaga <...>“, – rašo Hobbesas (ten pat), gręždamasis nuo upės ištakų, būtinybės ir laisvės esminio santykio, ir gręžda­ masis į paskirus sudedamuosius – tinkamus judėjimo laisvei – tekančio vandens elementus. Laisvė ir būtinybė čia srūva savo vaga, be gali­ mybės atsigręžti į šios tėkmės būtinumo sąlygas. Kaip rašo anglų kultūros istorikas Chris­ topheris Dawsonas, anglosaksų politinė mintis, viena vertus, turi savų privalumų, kita vertus, praranda esminį sąlytį su socialiniu gyvenimu: Tai judančių kūnų laisvumas, turintis tik aktualų empirinio-aš šansą, – priėjus prie pačios 148 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija į socialinę politinę tikrovę, o į Kantą, tikslinda­ mas Kanto kategorinio ir hipotetinio imperatyvų atitikmenis savo teorijoje (ten pat). Nesvarstant galimų Kanto formaliosios etikos ydų, tiesiog šli­ fuojamos parankinės teorinės sąvokos. Ištrinama skirtis tarp gamtos mokslų ir socialinių mokslų. bendrieji dalykai (kultūros esmės, įskaitant visų pirma religiją) anglo sąmonėje laikomi tik priva­ čiais „asmeniškos nuomonės klausimais“ (Daw­ son 1931; 9–10). „Privatumo“ tendencija, – kiek ji nusigręžia nuo socialinio gyvenimo, daro tam tikrą poveikį pačiam teorizavimui, kuris savo ruožtu darosi nerelevantiškas priešduotoms prasminėms gyvenimo-pasaulio relevancijoms. Esminę šiuolaikinio – nuo T. Hobbeso iki J. Rawlso – liberalizmo metafizikos šerdį sudaro „socialinė geometrija“. Y dalies tezės: Johnas Rawlsas, pradėdamas sveika Hobbeso Leviatano, „tikrai didžiausio politinės filosofijos veikalo anglų kalba“ (Rawls 2001; 1), prielaida, o veikiau sveiko proto išvada, kilusia iš patyrimo, – kad geriau yra taika, negu karas (nors, reikia pripa­ žinti, „vienas tarp kitų liberalizmo šaltinių yra religijos karai šešioliktame ir septynioliktame amžiuose, įtakoti Reformacijos“ (ten pat), eina pirmtako išmintais kraštutinės geometrinės abstrakcijos, legitimaus, nors nebūtinai legalaus, Leviatano, takais. Socialinių mokslų „konstrukcijų“, kitaip nei gamtos mokslų „konstrukcijų“, paskirtis yra atskleisti būdingas, vidines, prasmingas socialinio gyvenimo struktūras, o ne tik apibrėžti tariamai „natūralų“ socialinės gamtos visatos regioną, atrinkti tam tikrų faktų ir įvykių aibes, faktų aspektus, interpretaciškai relevantiškus specifiniams tikslams. Socialinis pasaulis nėra iš esmės nestruktūriškas. Faktai, įvykiai ir duo­ menys čia turi prasmingai konstituotą sandarą, būdingas relevancijų struktūras, savitą prasmę čia gyvenantiems, mąstantiems, veikiantiems žmonėms (Schutz 1962; 5–6). Hobbesas parodo, kad individualizmą galima, – būtinai be išlygų paneigiant tradicinę politikos filosofiją, – paversti visiškai naujos, dirbtinės teorijos kertiniu komponentu. Jei tik sukonstruojame, tariant Hobbeso žodžiais, visuomenę, dirbtinę grandinę. Vienoje jos pu­ sėje individai laisvai juda be pasipriešinimo, o kitoje – perduoda teises įgaliotiniams, gaudami iš jų sceninius vaidmenis paprastame gyvenime: „Taigi asmuo yra tas pats kaip ir aktorius ir sce­ noje, ir kasdieniame gyvenime <...>“ (ten pat; 171). Dirbtinę politikos teritoriją sudaro dau­ gybė laisvų (judėjimo be pasipriešinimo požiūriu) individų ir lygiai tiek pat dirbtinių (prigimtinio ne socialumo požiūriu) asmenų. Vienoje scenos pusėje – laisvumas, o kitoje – laisvumo apribo­ jimo akys ir ausys (policinė valdžia ir legitimi „Privataus“ teorizavimo intuicija, kaip ir „socialinio“ teorizavimo intuicija, turi ir pri­ valumų, ir trūkumų. Visi supratingi socialiniai teoretikai, taigi ir liberalai, – nuo Hobbeso iki Johno Rawlso, – gerai žino, kad bet kokia teorija turi esminių ydų. Šios ydos dažnu atveju kore­ guojamos sugrįžtant į socialinę tikrovę, tikrinant jos praktinio racionalumo apibrėžimus. Jei socialumas laikomas „nuliniu“ dydžiu, belieka gręžtis tik savęsp: į privatų protą, į teorinį raci­ onalumą. Johnas Rawlsas, koreguodamas savo teisingumo kaip bešališkumo teorijos tikėtinai pernelyg racionalistinius principus („racionalumo sampratą kaip išskirtinę normatyvinę sampra­ tą“, kuri gali būti klaidingai suprasta kaip „nuo pradžios hobsiška“; Rawls 2001; 82), gręžiasi ne 149 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 teisė). Pirmoje pusėje – racionalus pasirinkimas (lengva nuo ko nors laisvė), o kitoje – pasirinkimo stoka (sunkus laisvumo apribojimo akiratis, – jei tik esi nepakankamai racionalus). li – jo kertinė dorybė, pliuralizmas. Kraštutinai racionalistinės (utopinės) filosofijos turėtų-būti principas (pliuralizmas) siekia pagrįsti dorybę gyvenimo-pasaulyje. Plg. Y dalies tezės: Kitaip sakant, neigia objektyvaus įstatymo, arba dorybių (o tai yra esmingai viena ir tas pat; ten pat), kaip viseto pirmumą. Drąsa „hobsizmo“ koncepcijoje – tik pajauta (passion) tarp kitų laisvai judančio nepriklauso­ mo individo pajautų: „Tas pats kartu su viltimi išvengti žalos priešinantis yra drąsa“ (Hobbes 1999; 73). „Hobsizmas“ kaip naujas moralės ir politikos mokslas, – panaudodamas analogijos pobūdžio lygybės ženklą tarp gėrio ir blogio (!), ypač pabrėžia, kad nėra jokios bendros gėrio ir blogio taisyklės, kurią būtų galima paimti iš pa­ čių objektų prigimties, nes ji gali būti įgyta tik iš žmogaus asmenybės (ten pat; 71). „Hobsizmas“, kuriuo drąsiai gali didžiuotis pozityvistiniai naiviosios indukcijos metodai, reikalauja tik empiriškai stebėti nustatant „tikras“ koreliaci­ jas: „Ženklas (sign) yra prieš einantis įvykis po einančio; ir priešingai, po einantis prieš einantį, jei panašūs padariniai buvo pastebėti anksčiau; ir kuo dažniau jie buvo pastebėti, tuo tikresnis yra ženklas“ (ten pat; 48). Hobbesas 1) visiškai nutraukia ryšius su tradicija, – ir ne dėl mokslinių argumentų, o dėl jam nepriimtinos moralinės laikysenos, būdin­ gos politinės filosofijos tradicijai; 2) pagrindžia naują moralinę laikyseną, – mokslinis metodas yra tik paskesnis „papildinys“; taigi Hobbeso poli­ tikos filosofija, kitaip nei Spinoza’os, nėra natū­ ralizmo filosofija; 3) politikos teoriją formuoja kaip progresyvią tam tikrų pažinimo normų seką, kuri užsibaigia Euklido geometrijos ele­ mentų ir Galileo mokslinio metodo atradimais; 4) padaro esminę įtaką paskesnei moralinei ir politinei minčiai, kuri aiškiai arba numanomai grindžiama „lūžio su tradicija“ linija, prigimties įstatymą pakeičiančia prigimtinėmis teisėmis kaip atskaitos pagrindu aiškinant pilietinių institutų ir suvereniteto radimąsi (Strauss 1963; taip pat žr.: Oakeshott 1975; 143–4). Einant prieš einantį ir einant po einančio ... ženklai darosi vis tikresni: „Šiandieną mes visi – liberalai“ (Gray 1996; 18). Modernus pliuralizmas, arba vertybinio neutralumo turėtų būti laikysena, reliatyvizuoja ir neatpažįstamai sumaišo gyvenimo, pasaulio, savasties, tarpusavio supratimo elementus į „netikro žinojimo regi­ mybes“ (tikras žinojimas – visad priešduotas), kuriose kiekvienas paskiras individas apibrėžia savo požiūrį į galimas veiksmo kryptis tarsi- automatiškai. Hobbesas išranda naują geometrinį politikos mokslą, „hobsizmą“, kurio pagrindas yra ma­ terialistinė-deterministinė laikysena, prie kurios istoriškai šliejasi visi tie, kurie nenori pripažinti subjektyvumo, intencionalumo, sąmonės, intersubjektyvumo tikrovių. Kitaip tariant, visi tie, kurie eina tik į priekį ir nenori gręžtis atga­ lios. Savigrąža reikalauja drąsos, nes tradicija, įskaitant ir naująją-liberalizmo, niekada niekur neišnyksta. Tradicija – visad brūkšnelyje tarp gimties-mirties. Y dalies tezės: su fenomenologine Maxo Schelerio ir Alfredo Schutzo laikysena: bet kokį turėtų-būti pagrindžia dorybė, o ne dorybę pagrindžia turėtų-būti (Schutz 1966; 169). Fenomenologiniu požiūriu, ne liberaliz­ mo politinės filosofijos vertybė kaip turėtų-būti (pliuralizmas) turi pagrįsti dorybę (skirtingas moralines laikysenas gyvenimo-pasaulyje), o priešingai, dorybė ir gali, ir turi grįsti vertybę kaip turėtų-būti. Derėtų pridurti: laisvės ir būtinybės santykis apibrėžtinas kaip dorybės (savasties savigrąžos esmėn) ir vertybės (turėtų-būti) san­ tykis. Moralinės dorybės yra asmeniškos dorybės: aš teigiu (žr. II-ąją šios esė dalį „Subjektyvumo problema“).ii Rawlsas, po daugelio šventų metų, savo ruožtu įtikrina gležną psichologinį individu­ alizmą tarptautiniu mastu: jau kaip teorinį pliuralizmo faktą. Psichologinis individualizmas (vertybė: konkretaus žmogaus laikysena) pra­ nyksta vertybiškai neutraliame pliuralizmo fakte (politinėje liberalizmo filosofijoje). Pliuralizmo faktas – lygu tikrojo individualizmo faktas. Tai „homo noumenon“, skirtinas nuo konkretaus žmogaus laikysenos, „homo phenomenon“. Pirmasis terminas yra absoliučiai nežinoma konstanta, daiktas-pats-savaime, taikoma tikro­ jo individualisto idėjai. Darkart: idėjai, o ne „homo phenomenon“. Nežinomoji konstanta, pasitelkiant Schutzo palyginimą, taip pat rodo į kiekvieną augalą, kiekvieną akmenį: „Kaip tuomet šioji samprata gali suteikti žmogui vertę, kuri skirtųsi nuo objektų vertės?“ (Schutz 1966; 161) Liberalus atsakymas į šį klausimą yra gana paprastas: tikrojo individualisto idėjos samprata negali suteikti žmogui vertės, kuri skirtųsi nuo objektų vertės. Politinė liberalizmo filosofija ne gręžiasi į tikrovę, o siekia daryti įtaką tikrovei: politinės filosofijos įnašas sietinas su poveikiu, kurį ji gali daryti žmonių galvojimui apie po­ litinius ir socialinius institutus kaip visumą; politikos filosofija, viena vertus, yra realistiškai utopinė, kita vertus, tikrinanti praktines politinės galimybės ribas (Rawls 2001; 2, 4). Plg. su labai panašiu T. Parsonso analitinio realizmo, arba socialinės utopijos, modeliu (Parsons 1949). ii Liberalizmo politikos filosofija, priešingai nei teigiama, nuo pradžios iki šių dienų yra moralės filosofija: turėtų-būti principas (pliuralizmas) siekia pagrįsti dorybę gyvenimo-pasaulyje. Plg. su Leo Strausso apibrėžimu: nauja Hobbeso politikos filosofija sutampa su nauja Hobbeso moralės filosofija (Strauss 1963; vii–xiv): randasi nauja „teisių“ („right“) kaip moralės ir politikos principų koncepcija, kuri neigia „įstatymo“ („law“), arba „dorybių“ („virtue“), pirmumą (ten pat; viii). Darkart: pirmumo teisė, išlaisvinant ją iš tradicinės politikos filosofijos vergovės, teikiama turėtų-būti principui (pliuralizmui). Taigi Hobbesas – pirmasis iškilus politikos mokslo nominalistas ir subjektyvistas: pradeda ne nuo objektyvaus viseto, įstatymo natūralioje laikysenoje, intersubjektyvaus įsipareigojimo natūraliai daiktų prigimčiai, o nuo dalies, – netikro asmens (Hobbes 1999; 170), arba tikro Nors politinis filosofinis liberalizmas bet ko­ kių vertybių atžvilgiu esąs neutralus, ne neutra­ 150 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija liberalo, nepriklausomybės „teisių“. Y dalies tezės: Šį brūkšnelį kviečia skaidrinti fenomenologinio metodo – „nebijoki tamsaus kampo“ – metafora, o dar prieš tai – Kanto apibrėžimas: „Turėk drąsos pats naudotis savo protu!“ (Kantas 1996; 48). Kai liberali laisvė, laisvumas, pasiekia perviršį (laisvo pasirinkimo negalimumą suvaržytoje – įvairiais atžvilgiais – pliuralizmo virtuozų, politikos, ekonomikos, teisės, žiniaskaidos ekspertų, apsuptyje) iš laisvės gimsta nelaisvės, arba pastovumo (tebūnie: susvetimėjusio!) lū­ kesčiai. Vargani lūkesčiai, vadintini „apatijos“, 151 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija arba „pliuralaus vienišumo“, būkle. Vargani šiuo-laikinio vergo lūkesčiai, kuriuos – silps­ tant sąmonei, bet atsigaunant kūnui, – visiškai atliepia aukštyn kylančios pasiūlos-paklausos kreivės. Vieninteliai trys galimi liberalizmo kaip pliuralizmo modusai – komfortas, greitis ir triukšmas – atlygina mums už mūsų priklau­ somybę nuo šeimininko, nepriklausomybės. Priklausomybė nuo technologinės kilmės lūkesčių – greičio ir triukšmo – yra tikrojo individualisto nepriklausomybė. Iš šiųdviejų nepriklausomybės modusų ir turi būti „išvedamos“ visos kitos priklausomybės. vartojant – gelme. Ji neįteka niekur, veikiau teka į niekur. Tikrojo individualizmo rikiuotė, arba pliuralizmas, ne leidžia, o verčia netrukdomai norėti to, ko galbūt niekada negalėsiu turėti dėl nuolatinio buvojimo viduryje tarp kažkur-link einančių kūnų... Tai betikslė – priemonių dėl priemonių, valdžios dėl valdžios, vartojimo dėl vartoji­ mo... – dirbtinių grandžių schema, nesusijusi nei su praeitimi, nei su ateitimi, nei su saistančia dabartimi, nebent tik su išorinio horizonto „reiškinių skiemenavimu“ (Kantas 1982; 276). Jei valia (Wille), vartojant Kanto terminus, perteikia mūsų gebėjimą steigti aukštesnius poreikius, pažinti sintetiniame vienume (ten pat; 276), steigiančiame teleologiją, „laisvę kaip tikslingą vienumą pagal proto principus“ (ten pat; 560), tai savivalė (Willkür) apima tik momentinius norus, paviršinius empiristinius „reiškinių skiemenavimus“ vulgarioje negaty­ viosios laisvės sampratos sekoje be vidinių ribų refleksijos (Taylor 1979; 192–3), o dar tiksliau, be intencionalumo įvairovės: „Intencionalumo įvairovė yra būdinga kiekvienam į pasaulį atsi­ gręžusiam cogito jau vien dėl to, kad šis cogito ne tik suvokia pasaulio objektus, bet ir pats kaip cogito yra suvokiamas vidiniame laiko suvokime, ir šios įvairovės neįmanoma tematiškai aprėpti, nagrinėjant cogitata vien kaip aktualius išgyve­ nimus“ (Husserl 2005; 56). Kai einant prieš einantį ir einant po einančio tarp einančiųjų nepriklausomybėje nepaliekama nė menkiausio tarpo, išryškėja moralinės panikos simptomai (ten pat), ir liberalizmas „visų mūsų“, liberalų, akyse virsta „fundamentalizmu“, ieško­ damas, – „bailiai“, kaip griežtai įvertina liberalas Johnas Grey’us, – „moralinio tikrumo“ ištakų (ten pat; 19–20). Šiame judančių išorinių kūnų patiklumo ir vidinių ribų paslėptumo liberalizme trūksta būtinybės, ilgimasi neišvengiamybės. Y dalies tezės: Vėlyvosios modernybės, arba pliuralizmo (o tai yra viena ir tas pat), skiriamasis bruožas – „perduodama“, „parduodama“ ir „išparduoda­ ma“ laisvė (kaip ir hobsizmo koncepcijoje). Ne laisvė, o laisvumas, gimstantis iš dviejų susveti­ mėjusių karinės rikiuotės – „kairė-ir-dešinė“ – žingsnių: baimės ir palaimos (saugiai ir smagiai kiūtinti po einančio), įtarumo ir patiklumo (kai įsitikini, kad einantis prieš tave įdėmiai stebi karinės rikiuotės tvarką, o ne tavo nugarą). Liberalizmo rikiuotė gąsdina tik gręžiantis atgalios (ar ilgai ištvertum atsukęs sprandą atgalios?): komforto, greičio ir triukšmo siekių ir norų nesuvartojamumo – vis intensyviau juos Be intencionalumo įvairovės visos Hobbeso moralinės ir politinės filosofijos sąvokos virsta tuščiomis formomis, susijusiomis tik su juslinio pobūdžio paskatomis: netgi jei Hobbeso mora­ lės filosofiją galima pavadinti utilitarizmu, tai tik su išlyga, kaip pabrėžia Leo Straussas (1963; 116). Tai moralė grindžiama smurtinės mirties 152 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Dar prieš šį argumentą, reikia gerai įsi­ žiūrėti į pamatinį Hobbeso apibrėžimą, kuris leidžia jam vertybių ir dorybių teoriją, būdingą tradicinei filosofijai (gręždamasis nuo filosofijos, Hobbesas gręžiasi į istorijos mokslą; Strauss 1963; 108), paversti paviršutiniška kūniškų aistrų teorija, – tiesiog ištuštinant sąvokų prasmes iki baimės pajautos grėsmės keliamų „moralinių at­ sakų“. Vartodamas politinės filosofijos sąvokas, tarkim, įstatymo (law), teisės (right), prigimtinio įstatymo (a law of nature; Hobbes 1998; 86), Hobbesas dalį (teisę kaip individo laisves) ir kitą dalį (teisę kaip policinę įstatymų priežiūrą) iškelia į pirmą vietą, o valstybės prigimtį, teleologiją, politinį intencionalumą, įstatymą, vertybines ir dorovines sąsajas, kurios yra pirmesnės už bet kurią paskirą dalį (Aristotelis 1997; 66 [1253a]), lengvai priskiria tradicinei politikos filosofijai (iki Hobbeso) būdingoms „klaidoms“ (Hobbes 1999; 642–3). baime, arba pajauta, kuri savaime nėra supratin­ ga, o veikiau apdairi (plg. dvi žodžio „prudent“ reikšmės kryptis: supratingas ir apdairus), nes ją apdairia padaro baimės pajauta. Tiek Hobbeso, tiek ypač liberalizmo praktikos „apdairumas“ kyla iš tų pačių juslinio pobūdžio paskatų, ne tiek naivių, kiek taktinių: svarbu apdairi sutartis, kad ir kokia vulgari ji būtų: geriau stipri valdžia, negu liaudies valdžia (nors ir čia, reikia pripa­ žinti, yra nemažai tiesos). baime, arba pajauta, kuri savaime nėra supratin­ ga, o veikiau apdairi (plg. dvi žodžio „prudent“ reikšmės kryptis: supratingas ir apdairus), nes ją apdairia padaro baimės pajauta. Y dalies tezės: Tiek Hobbeso, tiek ypač liberalizmo praktikos „apdairumas“ kyla iš tų pačių juslinio pobūdžio paskatų, ne tiek naivių, kiek taktinių: svarbu apdairi sutartis, kad ir kokia vulgari ji būtų: geriau stipri valdžia, negu liaudies valdžia (nors ir čia, reikia pripa­ žinti, yra nemažai tiesos). Už nelaisvą išlygą, – kaktomušon einančią valdžią, kuri laiko žmones baimėje grasindama bausme (ten pat; 179), – pasiūloma jauki do­ vana, veikiau dovanojamas jaukas. Šios išlygos gyvenimišką kainą Hobbesas, rodos, puikiai supranta, todėl ir paguodžia: „Aukščiausioji valdžia nėra tokia žalinga, kaip jos nebuvimas <...>“ (ten pat; 194). Teoriškai išardęs dau­ giau ar mažiau geras „oro sąlygas“ natūralioje laikysenoje, teleologiją bendruomenėje (politinį intencionalumą natūralioje laikysenoje) ir praktiškai įspėjęs moderniu pirštu (šiukštu neužklyskit, vaikai, tradicijon: čia vyksta nuož­ mus visų karas su visais), rūsčią racionalistinę artėjančio biurokratinio liberalizmo teologiją Hobbesas lengvai suderina su liaudies valdžios regimybe, laisvumu, noru netrukdomai daryti tai, ką noriu. Hobbeso teorinė schema išties paprasta (kitaip ir negali būti, kai teorija randasi tik iš dviejų kūniškų pajautų, baimės ir palaimos): prigimtinėje būklėje vyrauja aiškus nusiteikimas kautynėms visą laiką (visų karas su visais), o sutarties būklėje prižiūrimi ir kontroliuojami šio karingo nusiteikimo aktualūs padariniai, – kam reikia ir kam nereikia prispaudžiant uodegą grėsmingaisiais teisės diskursais. Ta pati schema sąmoningai ar nejučia perkeliama ir į palaimingą sutarties būklę, nes teisė yra laisvė ką nors daryti ar ko nedaryti, o įstatymas apibrėžia ir įparei­ goja laikytis vieno kurio nors iš šių elgesio būdų (ten pat; 141). Kitaip sakant, teisė yra aiškus išlaisvintų individų nusiteikimas kautynėms visą laiką, o įstatymas – įpareigojimas (reikia skirti nuo įsipareigojimo) ir griežta policinė kontrolė. Gal ir nujausdamas, kad „biurokratinis libe­ ralizmas“, „pilietinių įstatymų dirbtinės grandi­ nės“ (ten pat; 219), žvanga šiek tiek grėsmingai, Hobbesas šią numanomą ne liberalaus žmogaus (tik dabar „visi mes – liberalai“) klausos ydą kompensuoja remdamasis išties aukšto lygmens argumentu (galbūt įsižiūrėdamas į racionalisto Descartes’o Dievo buvimo įrodymo argumentą; Descartes 1978b; 202–9). 153 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Šioje dviejų dalių dirbtinėje grandinėje puikiai dera dvi (atitinkamai: jėga ir apgaulė) dorybės: „Jėga ir apgaulė kare yra svarbiausios dorybės“ (Hobbes 1999; 140). Šioje dviejų dalių dirbtinėje grandinėje puikiai dera dvi (atitinkamai: jėga ir apgaulė) dorybės: „Jėga ir apgaulė kare yra svarbiausios dorybės“ (Hobbes 1999; 140). drauge su pačiais kūnais. Žmogaus vertę, t.y. jos ar jo kainą (Hobbes 1999; 103), nustato ne pardavėjas, o pirkėjas (ten pat; 104). Visa galintis pirkėjas, orusis Leviatanas (žr. sk. Y dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 c) dirbtinės visuomenės grandinės (liberalizmo loginio Organono, Leviatano, ir tėviškos jo priežiūros: „bandyk“, „klysk“ ir „gerink“). Kad ir kaip skirtingai vadintųsi laisvės filosofija, tarkim, Aristotelio politine socialine filosofija arba liberalia Kanto politikos filosofija, svarbu visų pirma eksplikuoti santykių, relevan­ tiškumo, suderinamumo (laisvės pagal įstatymus, dėl kurių kiekvieno laisvė tampa suderinama su visų kitų laisve; Kantas 1982; 277) idėjas, o ne „<...> grubias sąvokas, kurios sužlugdo visus ketinimus kaip tik dėl to, kad paimamos iš patyrimo“ (ten pat). Laisvė, egzistencine prasme, yra nepriklausomybė nuo gamtos dėsnių, o praktine prasme, išsilaisvinimas (vienu ir tuo pačiu metu!) – nuo prievartos ir nuo bet kurių taisyklių kaip kelrodžio (ten pat; 335; išskirta autoriaus). O jei laisvei pritrūksta būtent kelrodžio, jai trūksta ne išsilaisvinimo, o inercijos, vidurio būtinybės, laisvumo nelaisvės. Štai kodėl nepriklausomo individo laisvumas (reikia skirti nuo savigrąžaus asmens laisvės) ir būtinybė liberalizmo aukščiausiame yra natūraliai suderinami: upės vanduo turi ne tik laisvumą, bet ir būtinybę tekėti savo vaga (liberalia rinka be jokių kitokios rūšies suderinamumų, be jokių vertybinės prigimties sąsajų). Liberalizmo aukščiausiasis pradeda ne objek­ tyvia priešduotimi (visetu), bet subjektyvistine solipsistinio aš – statistinio vidurkio – prielaida. Hobbesas visas moralines kategorijas redukuoja į „natūralaus apetito“ žadinamas reakcijas, tarp kurių dvi svarbiausios yra „norai“ ir „pasibjau­ rėjimas“, kuriuos žmogaus protas, susidedantis iš jutimų, greitos reakcijos, geros galvos, suma­ numo, nuovokumo, gudrumo, teisingo kalbos vartojimo (Hobbes 1999; 85–90), geba ištęsti į begalybę, FELICITY. Ištirpdęs iki nulinio taško moralinių sąvokų esmes, Hobbesas gali nepaisyti to, kad laisvė – greičiau nelygu, nei lygu (vienu ar kitu atžvilgiu). Nors žmonės yra įvairūs, skirtingi, turintys nevienodas ver­ tybių ir dorybių sampratas, Hobbesas pradeda abstrakčia vienatinio aš laisvės ir klaidinančia homogeniškos visuomenės vaizdo sampratomis: individas privalo turėti tiek laisvės kitų žmonių atžvilgiu, kiek jis leistų kitiems žmonėms turėti jo atžvilgiu (ten pat; 142). Laisvę Hobbesas susieja su statistine viduti­ nio žmogaus samprata, kurią taikosi pertvarkyti į patogią lygtį, prieš tai suskaidydamas laisvę į nepriklausomas dalis, paversdamas laisvę elementų – daugybės mažesnių ar didesnių norų – aibėmis. Vidutinis žmogus homoge­ niškoje (liberaliai atomizuotoje) visuomenėje klausia: ne kaip ir kodėl galima visuomenė, o „kiek jis leistų“ (ten pat) laisvės sau ir „kiek jis leistų“ laisvės kitiems? Laisvė materializuoja­ ma: susiejama su teisėmis į daiktus ir apdairiais paskaičiavimais. Elementarieji teisių į daiktus atomai, judantys tuštumoje iki kam nors pasi­ Saugodamasis nuo „grėsmių“ (tai šiuolai­ kiškas negatyviosios laisvės apibrėžimas), t.y. Y dalies tezės: „Orumas“), pirkdamas žmogaus vertę, nustato visuomeninius įkainius (įsidėmėtina: valstybė – lygu visuomenė; ten pat), tarp kurių – trys pamatiniai-policiniai: valdžios, teismo ir valstybinių įstaigų (ten pat). Tokiai aukštai vertei išskirti pasitelkiami ati­ tinkami vardai ir titulai, kurių stabilumą savo ruožtu užtikrina aukščiausiasis, priežastinis teo­ loginis lygmuo: „Todėl tam, kuris galėtų matyti šių priežasčių ryšį, visų žmogaus laisvų veiksmų būtinybė taptų akivaizdi (Hobbes 1999; 219; išskirta – rūstaus liberalizmo klasiko). Karo prigimtis, kuri apibrėžiama kaip aiškus nusiteikimas kautynėms visą laiką (ten pat; 138), ir kuri Hobbeso teorijoje priskiriama natūralia­ jai žmogaus laikysenai, perkeliama ir į dirbtinės sutarties visuomenę kaip karinė dorybė, tinka­ ma rūsčiai biurokratinio liberalizmo prigimčiai, sudarytai iš laisvumo-ir-baimės. Aiškus nusiteiki­ mas kautynėms, arba varžymuisi, visą laiką – tai pamatinė kasdienio kapitalizmo, kasdienio liberalizmo ir kasdienių socialinių mokslų meto­ dologijų, grindžiamų konflikto principais, gairė. Šią gairę naujasis moralinis požiūris išaukština kaip „prigimtinę“ nepriklausomo individo teisę: „PRIGIMTINĖ TEISĖ, kurią rašytojai dažniausiai vadina jus naturale, yra kiekvieno žmogaus laisvė naudoti savo paties jėgas, kaip jis pats nori, savo paties prigimčiai, t.y. savo paties gyvenimui, išsaugoti, taigi ir laisvė daryti viską, kas jo paties sprendimu ir supratimu yra tam tinkamiausia“ (ten pat; 141). Šioje aukščiausiojo priežastingumo grandi­ nėje, arba liberalioje biurokratinėje visuome­ nėje, dominuoja vidurinis – valdžios, teismo ir valstybinių įstaigų – lygmuo, pagal prigimtinį įstatymą kylantis iš nepriklausomo individo proto („sudėties“ ir „atimties“ operacijų). Priešduotas antrajam – prasmingų kultūros sąry­ šių – lygmuo natūralioje laikysenoje sunyksta, o viršduotas teologinis – išnyksta. Hobbesas, viena vertus, iki kraštutinumo racionalizuoja individualumo idiomą, sparčiai (madingai) plintančią septynioliktajame amžiuje Vakarų Europoje (Oakeshott 1975; 84), tiesiog per­ imdamas grubias sąvokas iš patyrimo ir rizikuo­ damas tapti status quo ideologu (nėra nieko žalingesnio ir labiau neprideramo filosofui už vulgarų rėmimąsi tariamai prieštaraujančiu idėjai patyrimu; Kantas 1982; 277), kita vertus, realų asmenį – be jį saugančių ir jam atstovaujančių kultūros sąsajų – perduoda „mirtingojo dievo“ (Hobbes 1999; 183), žinojimo, jėgos ir galios supirkėjo Leviatano, žinion. Palaima ir baimė yra tinkamos dorybės: einant prieš einantį (tekančio vidurio palai­ moje) ir einant po einančio (lenkiant pakraščio baimėje) liberalizmo ženklai darosi vis tikresni. Tik kadangi jie neturi saistančios socialinio au­ toriteto galios, – tai tik erdvėje judančių kūnų juslinio gyvenimo „ornamentai“ (Strauss 1963; 115), – tuščios moralės sąvokos sutvirtinamos stipriomis teisės-įstatymo diskursų, verčių ir vertinimų, kurie kyla iš kautynėms nusiteikusių kūnų trinties, grandinėmis. Jei dorybės priklauso patiems judantiems kūnams, tai vertybės – teisės-įstatymo diskursui 154 Metodologija Sociologija. Y dalies tezės: gindamas rinką nuo sąmonės ko nors intencio­ nalumo (politinės bendruomenės, vertybių, socialinių sąsajų), kitaip tariant, ne neutralaus, o šališko žmogaus proto „ribotumų“, liberalizmas pasirenka „vidurkių tobulėjančioje begalybėje“ dėsnį, evoliucinį „išlieka tik geriausieji“ protą, vadinamą „nematomoji FELICITY ranka“. Reikia aiškiai skirti: tai nėra būtinybės ranka, o tik subjektyvistinė liberalizmo teorija: a) sponta­ niškumo, arba laisvumo, b) priežastinio proto kaip „žodžių grandinės“ (Hobbes 1999; 51) ir 155 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 priešinant, išskleidžiami geroje matricoje, kuri remiasi „teisingu kalbos vartojimu“ (ten pat; 90). Kalbant elementariai, daugybę tuštumoje judančių teisių į laisves, arba individualių norų, galima sudėti ir atimti (išsprendžiant lygtį: kiek laisvių vidutinis žmogus leistų kitam viduti­ niam žmogui spontaniškoje bandymų ir klaidų erdvėje), – galiojant liberaliai atomizuotos vi­ suomenės, kuriai natūraliai būdingas statistinis dėsningumas, sąlygoms. „Bandyk, bet klysk apdairiai.“ Liberalių „tiks­ lų“ rinkos tvarkoje pliuralumas – lygu „teisių apėjimo priemonių“ laisvumas. Rinkos laisvė turi būtiną norą netrukdomai tekėti: grįsti savo laisvumą ne galimai šališkais ekonominiais pa­ skaičiavimais, o savo jėga (veržlumu) ir vidurio taisyklės protu (inercija, prisitaikymu, apskri­ tomis apskrito stalo diskusijomis, visuomenės nuomonės apklausomis). Viskas yra pateisina­ ma, jei tai būtina įgyvendinant nepriklausomy­ bę, arba rinkos tėkmę, kurios priešakyje – du flagmanai (įsidėmėtina eilės tvarka): a) jėga ir b) protas (Hobbes 1999; 218). Vėlyvojo liberalizmo laikais, kaip ir „Nojaus laikais“ (Hobbes 1999; 436), „laisvių“ netrūks­ ta. Priežastis paprasta: nors valstybė, visuomenė, bendruomenė, socialinė grupė, politinė partija, klasė, šeima ir bet kokia kita saistanti prigimtis ir galia tebevadinamos minėtais vardais, būtent saistančios prigimties ir galios jos jau nebeturi: „Viskas yra apibrėžta savo paskirties ir galios, taigi, kas jų netenka, nebevadinamas tuo pa­ čiu, o tik turinčiu tą patį vardą“ (Aristotelis 1997; 67 [1253a 28–30]). Laisvė sutampa su laisvumu: bandymų ir klaidų spontaniškumu. Liber-izmas, ne laisvės, o teisinio egalitarizmo filosofija, pasiekia savo aukščiausiąją išsivystymo stadiją: padarinys eina po priežasties kaip dešinė koja po kairės, – juo dažiau ir sparčiau žingsniai kartojasi, tuo „tikresnis yra ženklas“ (Hobbes 1999; 48). Pirma, kairė koja išspiria valstybės gebėjimo integruoti socialinį gyvenimą ramstį. Antra, dešinė, policijos koja (stingant politikos lieka policija), išspiria laisvę gręždamasi į rinkos laisvumą, tiksliau, jos gebėjimą veiksmingai pa­ naudoti teisių ir laisvių, tariant Carlo Schmitto žodžiais, Technik und Politik, jungtuką, arba laisvių diskursą (o tai yra viena ir tas pat). Laisvė padalijama į teisių apėjimo priemonių aibes ir tampa įkainojama preke. Kainų mecha­ nizmas, nuvertėjant laisvės, bet kylant laisvumo ir teisių akcijoms, padeda pasiūlą pritaikyti prie paklausos. Y dalies tezės: Vertybės laisvai keičiamos į pasirin­ kimo laisvę rinkoje. O tai ir reikėjo įrodyti: kad liberalizmas prekiauja pliuralizmu daug geriau, nei demokratija. Viskuo: tikslais, užda­ viniais, gyvenimo planais, paskirtimis, galiomis, valdžiomis, autoritetas, laisvėmis, teisėmis, lygybėmis, nelygybėmis, įgalinimais, įgalioji­ mais, pavedimais, pervedimais... Kai viskas yra pateisinama, politika ne marginalizuojama, kaip mus bando guosti melancholiški liberalai (labai reta individualistų rūšis), o tiesiog išnyksta. Apėjimo laisvės materializuojamos at­ sitiktinėse progose ir galimybėse. „Įprastai individams netrūksta progų ir išteklių tenkinti pagrindines savo reikmes“, – kaip pasakytų bet kuris sąžiningas, tikintis Tėkmės teisingumu, liberalas. Liberalizmas išlaisvina net laisvę nuo jos pačios. Viskas, kas tik juda, yra pateisinama: individas privalo turėti tiek laisvės kitų žmonių atžvilgiu, kiek jis leistų kitiems žmonėms turėti jo Viskas (!) yra pateisinama: teisės gina laisves, o laisvės ieško priemonių, kaip apeiti teises: 156 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija perteikia kraują stingdančia (ne tokių laisvų pažiūrų žmogui) skirtimi tarp mirties (negyvų daiktų) ir gyvenimo (judančių kūnų). Tradicijos prilyginimas mirčiai (subtili liberalizmo ironija arba atvirai ciniškas sarkazmas, – nelygu liberaus žmogus) – tai bene vienintelės aštriu jausminiu patyrimu grindžiamos liberalizmo ištakos. Liberalizmas – lygu dirbtinė teorija, sugrįžtanti į gyvenimo-pasaulį faktiškai: faktiniai mokslai, kaip rašo Husserlis pirmuose Europos mokslų krizės puslapiuose, formuoja faktinius žmones. O faktiniai žmonės sutampa su savižiniais: tik absurdo filosofija, – ironizuoja rūstusis Hobbe­ sas, – gali tvirtinti, kad „žinojimas yra didesnis už žmogaus žinojimą“ (ten pat). atžvilgiu (ten pat; 142). Tai ne tik graži ir dai­ ninga melodija, įprastai atliekama liberalizmo dainų šventėse, bet ir svarbi žinia individui. Gręžkis nuo savo prigimties  – „žmogus iš prigimties yra pilietinis gyvūnas“ (Aristotelis 1997; 66 [1253a 45]) – į savo judėjimo išorėn teisę, – pliuralistinės ir heterogeniškos laisvių sudėjimo ir atėmimo operacijos yra pirmesnės už homogenišką politinių ir socialinių sąsajų prigimtį. Valstybės prigimtis, paskirtis ir galia atgręžiama prieš ją pačią. Klaidinga manyti, kad liberalus požiūris į pliuralizmą yra įvairovės vienovėje koncep­ cija. Priešingai, tai karingo pliuralizmo, kurio bambagyslė niekad neatrišama nuo kraštutinai dirbtinių ir karinių „moralės mokslo teoremų“ (Hobbes 1999; 360) įsčių: „Kad pilietinės visuomenės pradžia kyla iš abipusės baimės“ (Hobbes 1983; 41). Politinio intencionalumo – vidinio sąmonės ko nors horizonto – samprata liberalizmui nuo pačios pradžios yra svetima: graikai sukuria žmogaus kaip politinio gyvūno koncepciją, – stebisi Hobbesas, – tarsi taikos iš­ saugojimui ir žmogiškos rūšies valdžiai daugiau nieko ir nereikėtų, o tik pritarimo dėl bendrų sutarčių bei sąlygų, kurios pavadinamos įstaty­ mais (ten pat; 42). Y dalies tezės: Stigdamas vidinio žmogaus sąmonės horizonto analizės, Hobbesas „lengvai“ išardo esminį žmogaus prigimties, vietos ir rele­ vantiškumo (tinkamumo) sąryšį (Hobbes 1999; 37–8), kritikuodamas „absurdiškas filosofines mokyklas“, kurios, postuluodamos kalbamąjį sąryšį, neva ir negyviems daiktams priskiria troš­ kimą ir žinojimą (ten pat; 38). Ar tai reiškia, kad žinojimas – lygu žmogaus žinojimas? Atskyręs žmogų nuo natūralios aplinkos, laiko, erdvės, socialinio kultūros pa- saulio, Hobbesas mums neatsako. Užbaigti lygtį tenka patiems. Kai žmogus samprotauja, jis nedaro nieko kito, o vien suvokia visą sumą (ten pat; 60). O tai reiškia, kad Hobbesas randasi aukštojoje socialaritmetinėje matricoje (plg. su identiškais šiuolaikiniais projektais): erdvė natū­ ralioje laikysenoje, taigi ir fenomenologinėje, yra išgyvenama erdvė, o laikas – išgyvenamas laikas. Kas kita – liberalizmo laikysena: čia erdvė yra pergyventa erdvė, o laikas – pergyventas laikas. Dirbtinės geometrinės Hobbeso teorijos įsčios nuo pradžios yra meta-pliuralistinės (lygu – neoliberalios). Meta-pliuralizmui svetima rele­ vantiškumo samprata. Rūstų hobsizmo žvilgsnį meta-liberalizmas nukreipia į kultūros priešduotis gyvenimo pasaulyje, siekdamas jas išardyti, atimti iš jų laisvę, politinio intencionalumo sąsajas. Viskas, kas tik juda tam, kad ardytų įprotį ir politiškai socialinius sąryšius, yra legitimu. Vietos ir judėjimo, tradicijos ir modernybės, politikos filosofijos ir politikos mokslo, nelibe­ ralizmo ir liberalizmo priešpriešą Hobbesas 157 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Liberalus požiūris nuo pradžios yra skaidus, analitinis, aritmetinis, statiškai ir statistiškai „buvojantis pats savyje“: „<...> ten, kur negalima nei sudėti, nei atimti, samprotavimui nėra kas veikti“ (Hobbes 1999; 60). Tik šioje epistemo­ loginėje melioruojančioje erdvės aritmetikoje gali rastis „vienetai“, jų „sudėtys“, „atimtys“, dirbtinės „prigimties būklės“ ir „sutarties būklės“ konstrukcijos. Tik čia „atskirtumas“ gali pasiekti tokį stulbinantį lygmenį, kad racionalistinė fantazija lengvai virsta tikrove. Jei natūraliąją ir fenomenologinę laikysenas do­ mina žmogaus gyvenimo-planas, tai liberaliąją Hobbeso – dirbtinio asmens gyvenimo-planas. Hobbeso metodas, kaip pažymi Leo Straussas, imituoja Galileo, – kuris pirmasis iškelia fiziką į mokslo rangą, – metodą (Strauss 1963; 2). tinė. Liberali pilietinė visuomenė kaip iš anksto teisinga politinė tvarka randasi ne tik tam, kad saugotų sutartį ir kad „sutarties iš baimės“ sargai nesnaustų, o tam, kad galutinai išlaisvintų mus nuo bet kokių tradicinių sąryšių. Liberalus požiūris – ne šališkas, bet šališkas (indiferentiškas tikrovei už savęs, bet artimas tikrovei savyje): man relevantiški dėmenys sude­ dami ir atimami, o man nerelevantiški – pralei­ džiami ir anuliuojami, todėl sudėties rezultatas visada lieka tas pats, kad ir kokia tvarka šiuos pastovius dėmenis (racionalius, skirtinus nuo neracionalių, neliberalių) aš sudėčiau ir atimčiau. Y dalies tezės: Sudėti ir atimti galima viską, kas tiesiogiai dedukuojama iš epistemologinės erdvės aritme­ tikos teorijos. (Sudėti ir atimti negalima to, ko trūksta šioje liberalioje biurokratinėje erdvėje: partnerių, porininkų, amžininkų, pirmtakų, įpėdinių intencionalių sąsajų bendroje erdvėje, bendrame laike ir bendroje ap-linkoje.) Naujųjų laikų socialgeometrinė fantazija, Geometrija (Geometry), idealus visuomenės vaizdas vaizduotėje, akivaizdžiai, anot Hobbeso, pranoksta „stačiokišką Antikos primityvumą“ („the rude simplenesse of Antiquity“; Hobbes 1983; 25). Hobbesui turėtų žemai lenkti galvą šiuo-laikinis žmogus, kurio veiksmo laikysenoje įkūnyta moderni psichologinė priešprieša tarp vadinamųjų „vinerių“ ir „lūzerių“: hobsiška „Naujųjų kentaurų“ (ten pat; 31) karta savaime suprantamai pranašesnė už „<...> hermafrodi­ tines moralinių filosofų nuomones, iš dalies teisingas ir gražias, iš dalies brutalias ir laukines, visų kovų ir kraujo praliejimų priežastis“ (ten pat). Dvinarė priešprieša, žinoma bet kuriam šiuo-laikiniam žmogui: liberalizmas visad pra­ našesnis už neliberalizmą (rytdiena – už vakar­ dieną). Jei sutarties (pilietinė, modernioji) būklė yra racionali, politinė, tai prigimties (tradicijos, socialumo) būklė yra ne politinė, netgi antipoli­ Individus: liberali politikos tradicija visų pirma sudeda, arba gina į priekį, ir tik į priekį, išlaisvintus individus, gaudama tikimybiškai pastovias laisvųjų individų sumas. Individų laisves: sudėdama judėjimo be pasipriešinimo (nelygu vakarietiškų visuomenių laisvių tan­ kumas) ir atimdama judėjimo su pasipriešinimu (nelygu nevakarietiškų visuomenių laisvių retu­ mas ir nelaisvių tankumas) išeikvotas energijas, nustatydama laisvės skirtingose visuomenėje koeficientus (kad būtų aišku, kam ką vytis ir kam ką ginti: į priekį, ir tik į priekį). Individų laisves ir baimes: simetriškai erdvėje sudėdami baimes gauname baimės padarinius, laisvumo grandinę, dirbtinę visuomenę, racionalią sutartį, o iš jos atimdami iš baimės tirtančius žmones gauname laisvas diskusijas ir jas atspin­ 158 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija jama daugybė nepriklausomų libervalių, laisvo oro balionų: ir ne tik virš Vingio parko, o virš Žemės, virš vietos, ap-linkos, virš žmogaus tėvynės. dinčias spaudos laisves. Individų teises: sudėdami individų nuomones apie bendrąjį turtą ir privatų jo suvartojimą, gauname viešąjį gėrį ir teises turėti apie tai savo nuomones. Ne tik privačias nuomones (kultūra liberalioje visuomenėje yra „nuomonės apie kultūrą turėjimas“), bet ir bendrą dirbtinę, nominalistinę nuomonę: jungdamas dalių vardus, suvoktų visumos vardą arba iš visumos ir vienos dalies vardų – kitos dalies vardą (ten pat; 60). Y dalies tezės: Leo Straussas nurodo penkias būdingas Hobbeso konstrukcionizmui negatyviosios laisvės – naujo dirbtinio kūno gręžimosi nuo pasenusios savo sąmonės –„nuogrąžas“ (šio proceso metu natūralios relevancijos tampa nerelevantiškomis gręžiantis į dirbtines relevan­ cijas): 1) gręžiantis nuo monarchijos kaip natū­ raliausios valstybės formos idėjos, gręžiamasi į monarchiją kaip tobuliausią dirbtinę valstybę; 2) gręžiantis nuo prigimtinio įpareigojimo kaip moralės, įstatymo ir valstybės pripažini­ mo, gręžiamasi į moralės, įstatymo ir valstybės dedukavimą iš „prigimties teisės“ („the right of nature“), t.y. natūralistinės pretenzijos, kuri vienintelė visiškai pateisina moralę, įstatymą, valstybę; 3) gręžiantis nuo viršžmogiško auto­ riteto pripažinimo, gręžiamasi į tik žmogaus pateisinamo valstybės autoriteto pripažinimą; 4) gręžiantis nuo praeities (ir dabarties) valstybių pripažinimo, gręžiamasi į laisvą ateities valstybės konstrukciją; 5) gręžiantis nuo garbės principo, gręžiamasi į smurtinės mirties baimės principą. Tai naujojo politikos mokslo kaip galutinio šių procesų – „priežasties“ ir „padarinio“ – susieji­ mo programa (ten pat). Hobbesas išranda dirbtinę individo episte­ mologiją, tapusią universalia (!) naujojo individo valios ir visų valių pliuralizmo ideologija. Hob­ beso dirbtinis asmuo atgyja visuotinio pliuralizmo dvasioje, begalinėje tolerancijoje, kuri ne leidžia, o verčia: „Pasirink skirtingai, tiksliau, atsiskirk (nesvarbu: nuo vyro ar nuo žmonos, ar nuo jų abiejų, ar nuo paties savęs...)!“ Šiandien esame pasmerkti liberalizmui: globaliai policinei-proce­ dūrinei technologijai. Dirbtinumo virsmo natūralizmu momentas, ilgainiui tampantis universalumu, stulbina. Nepriklausomo individo valia, naujojo tikėji­ mo – be abejonės šešėlio – dirbtinė konstrukcija, tampa tradicinės sielos pakaitu. Hobbesas išran­ da ne bet ką, o dirbtinę akivaizdą: nepriklauso­ mo individo valią, priekin tvarkingai judančių kūnų sielą, naujojo gnosticizmo kryžių, kuris lengvai – dėl abejonės negalimumo tikėjime – nugali tradicinę politikos filosofiją. Artėjančiam masinių ideologijų amžiui Hobbesas svarbus dar ir dėl savo įžūlumo tradicinės politinės filosofijos atžvilgiu. Tiksliau, netikėtumo veiksnio: absurdo filosofiją atmetęs agnostikas, savo nepriklauso­ mą valią iš suviskam mirusios vietos ištraukia už plaukų! Virš konkrečiosios ontologinės kultūros pasaulio struktūros aukštai iškyla nesuskaičiuo­ Šiandieną penkianarė Hobbeso tezė jau įrodyta. Tai ir reikėjo įrodyti: kad demokratija (tik įprotis) negali gyvuoti be liberalizmo (net proto). Ir atvirkščiai: kad liberalizmas (politinės visuomenės gėris be politinės bendruomenės gėrio) gali gyvuoti ir be demokratijos (substancinės doktrinos). Priežasčiai (įpročiui, demokratijai) sunykus lieka padarinys – negatyvioji laisvė kaip 159 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija savaime suprantamos leviataniškos, biurokrati­ nės poistorijos priežastis. Štai kodėl nėra būtino ryšio, kaip vienu balsu (unisonu) tvirtina liberalai, tarp demokratinės valdžios ir individualios laisvės. Dar daugiau: šį ryšį nutraukdami, „mes – visi liberalai“ išsilaisviname, išlaisvėjame. Y dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija tai valia – valiojimas – priežasties-padarinio grandinėje gali būti tik vertybiškai neutrali bet kokios sąmonės kilmės judėjimui. „Kad tik į priekį...“: neutralumo triukšme viskas valiojama (позволено всё). „Nestruktūroje“ nerasime natūraliajai lai­ kysenai būdingo tikėjimo ir pasitikėjimo tautos, arba politinio intencionalumo (o tai yra viena ir tas pat), papročiais ir jos žmonių įpročiais. Čia, nestruktūroje, viešpatauja dirbtinis hobsizmas, pliuralizmas: a) panieka bendrojo tautos laiko prasmei ir laisvei, kuri pavojingai suplakama su lėkštumu (betarpiškų įspūdžių keliamų malonumų aibėmis); b) dirbtinių grandinių – oligarchinio elitizmo uni-formų – savivalė. Išlais­ vėjusių individų pliuralizmą Hobbesas statė ant baimės – laisvai medžioti su varovais, bet įpročio gūžtis vos tik pamačius šeimininką – pamatų. Šiuolaikinis pliuralizmas, laisvės ieškoti einantis irgi su varovais, žiniaskaida, yra, psichologiniu požiūriu, rafinuotas: prisileidžia laisvosios šalies piliečius gerokai arčiau, į rinkimų „Teisės lais­ vėms, laisvės teisėms“ kabinas, jausdamas tą patį pasidygėjimą, tačiau gebėdamas jį nuslėpti. Pa­ sidygėjimas yra psichologinis paniekos giminaitis. Pasidygėjimas – psichologinė reakcija priešduoto socialinio ir politinio intencionalumo atžvilgiu, o panieka – institucionalizuota dabarties laikyse­ na. Abi šios negatyviosios laisvės psichologinės ištakos c) hierarchizuoja policines struktūras atskirdamos jas nuo gyvenimo-pasaulio ir d) išlaisvina – iki anarchiškos būklės – kasdienio gyvenimo pasaulį, kuris veržiasi dėkingai kle­ gėdamas seklia ir lėkšta būtinybės upe. Pasitikėjimo ir tikėjimo aplinka, – papro­ čiais, įpročiais, kurių iš esmės trūksta sparčioje ir triukšmingoje plunkančios minios klegesio tėkmėje, – stoką kompensuoja viskas valiojama „stabilumas“. Tobulą išganymo dabartyje regi­ mybę suplūkia šeimininkų varovai, žiniaskaida. Keturmečių ir „visad sėkmingų“ rinkimų, visuo­ menės apklausos nuomonių „apie ką nors“, „už ką nors“ ir „prieš ką nors“ kaupimų, gražuolių, sveikuolių ir puikuolių reitingų įsukimų, pa­ sirinkimų tarp vienodų pasirinkimų klegesyje pereinama nuo pasirinkimo galimybių (laisvės) prie pasirinkimo prievartos (klegančios nelaisvės, valiojimo triukšmo). Kasdienybės pasaulis reliaty­ vizuojamas radikaliai: etiniai žmonių įsitikinimai virsta estetiniais skoniais (pupyčių ir pupuliukų). p p p p Trūkstant tęstinio, laiko pilnumos požiūriu, atsakingo politinio (iš pareigos politinio inten­ cionalumo laikysenai) ir moralinio (iš pagarbos socialinio intencionalumo laikysenai) veiksmo, laisvė virsta laisvumu, ginčytina valia, savivale, „netrukdymu žmogui daryti tai, ką nori“ (Hobbesas 1999; 218); apatiška vidurio taisy­ klės su policiniais švyturėliais inercija. Veiksmo kokybę pakeičia veiksmo kiekybė: ne kaip, o kiek. O drauge išardomas tikrovės ir galimybės santykis. Laisvės – lygybės norų ir vidinio šių norų apribojimo – priežiūros ištakos. Kai gali­ mybė „sutampa“ su tikrove, lieka objektyvios prasmės, o išnyksta subjektyvios laikiškumo dimensijos. Dabar esame išlaisvinti: išvaduoti net nuo mirties ... Y dalies tezės: Dabar esame pasmerkti vien negatyviosios laisvės būti­ nybei, kuriai įpročio gręžtis į vertybes prievarta jau nebegresia. Esame pasmerkti pliuralizmui: faktų aibėms, faktinių žmonių aibėms, biurokratinių potvarkių aibėms, teisių aibėms, laisvių aibėms, savi-valės (valingo kūnų judėjimo be socialinio pasipriešinimo) aibėms... rasčiau pusiausvyros ir savo vertės kitų žmonių akyse, – o „laisvės“, „reto ir netvaraus dalyko“ (Tocqueville 1996; 70), man netgi nereikia, nes ji yra pernelyg trapi objektyvaus laiko, priežas­ ties-padarinio, aplinkoje. Štai kodėl liberalus „demokratijos“ vaizdi­ nys – nestruktūrizuotas, nesusidedantis iš čia gyvenančių žmonių prasminių prieš-atrankų, prieš-interpretacijų. O susidedantis tik iš rankų sukirtimų „tam kartui“ („konsensų“). Būtent šioje „nestruktūroje“ įgyvendinami racionalūs ir nešališki procedūriniai liberalizmo eksperi­ mentai. Iš esmės konservatyvus, fenomeno-loginis tradicinės politinės filosofijos klausimas: „Kas ir kaip valdo, kas ir kaip įgyvendina?“ – libera­ lizmui nerūpi. Valdžia yra dirbtinė. Individas yra dirbtinis. Procedūros yra dirbtinės. Svarbu ne kas ir kaip valdo, o kad valdo. Liberalizmo „nestruktūra“ – tai baltas popieriaus lapas, kurio skiriamasis bruožas – numanomos dirbtinės linijos, abejingo požiūrio į kitą žmogų šaltinis. Politinis veiksmas suaižomas į izoliuotų da­ bartiškumo faktų aibes fiksuojant tik tam tikrus statistinio pobūdžio su-tapimus suerdvintame laike. Prarandama galimybė suprasti socialinės politinės veiklos tęstinumo, arba politinės ben­ druomenės biografiškumo, reikšmes ir prasmes. Liberalioje geometrinėje matricoje galioja tik priežastinio adekvatumo, arba mokslinio aiškini­ mo, kriterijai, atskirti nuo vertybinių sprendinių gyvenimo-pasaulio struktūroje. Čia nerasime bet kokiai lanksčiai socialinei metodologijai būdingos skirties tarp pirmojo lygmens konstrukcijų (priešinterpretacijų kasdie­ niame pasaulyje) ir antrojo lygmens konstrukcijų, grindžiamų pirmosiomis (Schutz 1962; 6–10). Čia rasime tik mokslines konstrukcijas, grin­ džiamas abejingomis, todėl idealiai tiesiomis „jei..., tai...“ (priežasties-padarinio) linijomis, kurių centre – vertybiškai neutrali neoklasikinės ekonomikos figūra: kraštutinai anonimizuota, kraštutinai racionali marionetė, homo econo­ micus. Šioje „nestruktūroje“ laisvumui visiškai saugu: apdairiai prarandama galimybė atrasti relevantiškus daugelio ko nors sąmonės sąryšius tęstinėje a) laikiškumo, b) erdviškumo, c) inten­ cionalumo, d) daiktiškumo patyrimo vienovėje. Tai reiškia, kad liberalizmas ir socialinis gamtos mokslas (mokslas negali būti ne socia­ linis) sutampa su „grynojo sekuliarizmo religija“ (susisaistymo tarp žmonių priešybe). Grynasis susvetimėjimas – tai brangi kaina už pigią ne-laisvę. Pirma. Kad kitą pirmąkart galėtum save atrasti subjektyviai laikiškoje porininkų, amžininkų, pirmtakų ir įpėdinių ap-linkoje, privalai save prarasti natūralizmo / liberalizmo primesties aplinkoje, – mechaniško priežasties (nepriklausomų individų) ir padarinio (dirb­ tinės visuomenės) santykio matricoje. Antra. Panašių vienas į kitą ir lygių žmonių minioje aš laikausi įsitvėręs tik „lygybės“, – kad nepra­ 160 Sociologija. Y dalies tezės: Laisvės pavertimas laisvumu, – nepaliauja­ mai steigiant naujas „neskaidrumo priežiūros“ ir „neskaidrumo priežiūros priežiūrų“ insti­ tucijas, – nėra tik „šiaip sau“ atsitiktinumas. „Nestruktūroje“ randasi šiuolaikiškumo esmė: nepriklausomo individo valia, norėjimas, valioji­ mas. Jei siela – są-monė – politikos filosofijoje yra savigrąži bendro gyvenimo polinkiui ir polinkyje, 161 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Demokratija kaip kintantis santykis tarp laisvės ir lygybės yra iš esmės neužbaigta, tęstinė politinio intencionalumo, arba pa- tyrimo, forma. Kitaip tariant, ji reikalauja ne fragmentiško, dalinio, faktografinio patyrimo, o pa-tyrimo, tęstinio-tyrimo, o ypač, vartojant tinkamą Williamo Jameso terminą, pa-kraščių pa-tyrimo. Demokratija kaip fenomenologinis daiktas būtinai reikalauja eksplikuoti jos erdvi­ nius-laikinius, išorinius-vidinius horizontus, ją sudarančių daiktų ta-patumo kylius. Tiksliau, deontologinius demokratijos kylius, priešduotas esmes be esencializmo: be liberalizmo teorijos ir praktikos. O ypač be „nepriklausomo individo“ esencializmo. antra, puoselėjant „išvirkščias“ praeities atžvilgiu vertybes: naujus a) negatyviosios laisvės idealus, nerūpestingumą, nedėmesingumą, abejingumą Kitam, suporuojant su b) pozityviosios laisvės idealais, – rūpestingumu, dėmesingumu, aistrin­ gumu dabarčiai-savyje? Liberalizmas įgyvendina „negalimybės projektą“, kuris teoriškai, o paskui praktiškai naikina sąsajas, intencionalumą, konstituacijas. Socialinis politinis intencionalumas susiaurina­ mas iki minimalistinės racionalaus pasirinkimo karikatūros: individo intencijos, tikslo ir šio individo pasirenkamų efektyvių priemonių. Atitinkamai išrandamos dvi racionalistinio individualizmo vertybės – negatyviosios laisvės ir politinio liberalizmo. Demokratijos kylis, nugarkaulis – sociali­ niame politiniame intencionalume, tapatybių jungtyse, tęstinume: kiekvienas demokratijos „dabar“ modifikuoja save į „ką tik“; „kitas dabar“, atliepdamas ankstesnį „dabar“, tampa „ką tik“, atliesdamas ankstesnį „ką tik“, ir t.t. (Schutz 1966; 10–1). Dabartis esti laikiškumo kylio rodmenyje. Atidžiau įsižiūrėję į šias dvi dirbtines ver­ tybes, kurios sukuriamos tam, kad abipusiškai stiprintų viena kitą, matome, kad, pirma, nega­ tyvioji laisvė yra a) „ko nors racionaliai norinčių kūnų judėjimas be pasipriešinimo“ (vulgariame empiristų liberalizme), b) „alternatyvų pasirin­ kimas“ (rafinuotesniame liberalizme). Antra, politinis liberalizmas yra pliuralizmas (šiandienos tikrovė kaip tikrovė). Šiomis dienomis liberaliz­ mas praktiškai laikomas užbaigtu, savaime su­ prantamu: „Visi mes – liberalai.“ Sudėję pirmą ir antrą dalis gauname: liberalizmas yra individo negatyvioji laisvė, o negatyvioji laisvė yra alterna­ tyvų pasirinkimas. O alternatyvų pasirinkimas yra negalimybė, – iš esmės trūkstant alternatyvų, kokybiškai nepalyginamų vertybių ir dorybių eros pabaigoje, arba šiuolaikinėje tikrovėje, kurioje „visi mes – liberalai“. Liberalizmas įgyvendina negalimybės projektą. O tai ir reikėjo įrodyti. Liberalizmas, tiksliau, racionalistinis indi­ vidualizmas, imasi užduoties, kuri iš esmės, t.y. ne tik epistemologiškai, o ir ontologiškai, yra neįgyvendinama: pagrįsti negalimybės – grynosios dabarties – sąlygas. Dėl prievartos, daromos laikui, liberalizmas praranda tikslą, likdamas vien su priemonėmis. Y dalies tezės: Priemonių-priemonių lygmuo – tai pirmtakų, porininkų (ne tik verslo partnerių) ir įpėdinių atžvilgiais visiškai susveti­ mėjusi dabarties pasmerktis. Kad rastųsi tikrojo individualizmo negalimybė, pri-mesties (praei­ ties), sąlygos turi būti išardomos. Juk kaip galima išrasti dirbtinę individo suvereniteto iliuziją, jei ne, pirma, nukarpant visus ryšius su praeitimi ir, Liberalizmo skirtis tarp negatyviosios lais­ vės ir pozityviosios laisvės sampratų tarnauja, 162 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija pirma, kitų vertybių negalimybės daiginimo ir, antra, liberalių vertybių galimybės aktualizavi­ mo tikslams. Viena vertus, pozityvioji laisvė ir negatyvioji laisvė viena nuo kitos atskiriamos analitiškai (šitaip dirbtinai išrandant skiria­ muosius liberalizmo bruožus). Kita vertus, šioji skirtis sustiprina kraštutinai minimalistinės „nepriklausomo individo“ prielaidos vertę. Nuo šiol nebereikia aiškintis, kokių reikia sąlygų, kad rastųsi esminė darna tarp esamų ir galimų pozityviosios laisvės ir negatyviosios laisvės įgy­ vendinimų (intencionalumų), nes negatyvioji laisvė pagal apibrėžimą neigia pozityviąją laisvę. Liberalizmas yra kokybiškai nepalyginama – su kitomis vertybių koncepcijomis – metodologija. Liberalios subjektyvizmo žirklės atkerpa praeities laiką – pirmtakų, ankstesnių kartų, tradicinės šeimos, draugų ir iš principo bet kokių kitų soci­ alinių saitų – nuo dabarties laiko, toleruodamos tik naują laisvės kaip individo teisių koncepciją. teisė save atskirti nuo ko nors, ir vėl nuo ko nors, ir t.t. nuo pradžios yra šizofreniška. Begalinio atsiskyrimo – gyvenant visuome­ nėje, o ne iki visuomenės arba už visuomenės – negalimybė (nuo kūno atskirta ąmonė) verčia kūną skubėti sudarant vis naujas „rankų sukir­ timo“ operacijas ir vėl jas nutraukiant. Skubėji­ mas – vienintelis išvirkščiai pastovus liberalizmo bruožas, dėl kurio randasi staigūs trūkčiojimai, mėšlungio sąlygojami „konsensai“. Išvirkštinis stabilumas „individualioje sąmonėje“ virsta tiesioginiu nestabilumu „individualiame kūne“. O nuolatinis nestabilumo patyrimas kūne kelia pirmykštę baimę, kurios racionalūs padariniai – saugos ir paklusimo lūkesčiai: „Nuo Hobbeso iki Carlo Schmitto, taip pat iki šiuolaikiškiausių elektroninės Amerikos imperijos (kartais priar­ tėjančios prie fašizmo) teoretikų, santykis tarp saugos ir paklusimo lieka vieninteliu valdžios paaiškinimu“ (Joxe 2002; 122). „Nauja“ pozityvioji laisvė, atsiribojusi nuo tradicinių vertybių ir dorybių, tampa stebėtinai panaši į negatyviąją. Dirbtinai išrasta „individo sąmonė“ yra grynoji negalimybė, todėl negali būti pozityvi. Individo sąmonė – be savo kūno patyrimo – visad negatyvi. Tik sveiką nuovoką ir savitvardą praradęs liberalas gali teigti, kad ji ar jis neturi kūno, – žinant ką jiems abiem reiškia kūno judėjimas be pasipriešinimo (malonumas be skausmo)! Y dalies tezės: Esminė liberalizmo problema ir yra ta, kad net liberalo kūnas pareina tik iš praei­ ties: „Ką daryti?!“ Ką daryti, kai kūnas pareina iš motinos ir tėvo, iš praeities, iš kultūros, iš laikiškumo erdviškume tėvynės, o visos šios tarsi-regimybės naktimis tebesivaidena? Libera­ lus individualizmas siekia atsigriebti dienomis. Tačiau dirbtinai išrasta „individualios sąmonės“ Pirma, negatyvioji laisvė išlieka substanciškai hobsiška: „<...> be kitų asmenų įsikišimo asme­ niui leidžiama daryti tai, ką jis sugeba daryti, arba būti tuo, kuo jis sugeba“ (Berlin 1995; 275). Antra, pozityvioji laisvė apibrėžiama kaip tik negatyviosios konkrečioji variacija: „<...> kas – asmuo ar daiktas – trukdo...“ (ten pat). Pozityvioji laisvė sukonkretinama į formalistines „laisvių nuo ko nors“ aibes, kurios liberalizmo biurokratų kruopščiai registruojamos, rūšiuo­ jamos ir paverčiamos žmogaus teisių ir laisvių kiekybinio pobūdžio skaičiuotėmis. T. Hobbeso koncepcija idealiai atitinka du „liberaliosios demokratijos“ principus: 1) formaliosios / nega­ tyviosios laisvės, 2) saugumo pasiūlos ir paklusimo už išorinę globą kainos, – žmogaus vertė smunka iki „tinkamumo“ arba „pritaikytumo“ (Hobbes 163 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 1999; 111) „tokiai didelei jėgai ir galiai“, kuri garantuoja karinės ekonominės visuomenės pobūdžio tikslus – „taiką ir mūsų apgynimą“ (ten pat; 183). ne-buvojimo, atsitolinimo, nerimo, mirties, kaltės sąvokos“ (Schrag 1983; xi), – buhalterinę revi­ ziją. Johno Rawlso abstraktusis racionalizmas – aukščiausioji dabartiškumo negalimybės legiti­ macijos fazė – demokratinės minties revizija, akcentuodama išorinį vertybinio neutralumo požiūrį, atskiria asmens tapatumą ir asmens laisvę (laisvės patyrimo aktą): „Šioji revizija rodo, kad laisvei kaip laisvei nesuteikiama jokio priorite­ to, – tarsi įgyvendinimas kažko, kas vadinama „laisve“ („liberty“), turėtų iškilią vertę, ir būtų pagrindinis, jei ne vienintelis, politinio ir soci­ alinio teisingumo tikslas“ (ten pat; 44). Potencialus karinio saugumo garantas Le­ viatanas už formaliąją laisvę ir saugumą politiką priverčia tarnauti laisvajai rinkai, mirtingojo dievo bendra-turtui (common-wealth), o pilietį modifikuoja į vartotoją, kuriam suteikia „sau­ gumo“ garantijas ir „teisių“ sąrašus. Saugumo garantijos šiandieną neabejotinai tampa dar reikalingesnėmis: randantis teroristiniams „mir­ ties kultams“ ir priešingiems antiteroristinams „keršto kultams“, formuojantis nihilistinės „aristokratijos“ (vadinamojo „elito“) policinei politikai, kurios interesai yra aiškiai antidemo­ kratiniai, – išardyti sąsajas tarp suvereniteto ir tautos, užtikrinant neatskaitingos aristokratijos viešpatavimą, grindžiamą agresyviu neoliberaliz­ mo tikėjimu (Curtis 2004; 142). g p Nelygu laisvės prasmė: jei šį žodį įsprausime į liberalią „laisvumo“ (liberty) formą, jai iš tikro neverta suteikti pirmumo teisės. Tačiau laisvė (freedom) yra ne mokslinė, ne teisinė ir net ne filosofinė, o fenomenologinė ir egzistencinė patyri­ mo kategorija. Y dalies tezės: Patyrimas yra originalo suvokimas; kai patiriame kitą žmogų, tai sakome, kad Kitas stovi prieš mus kūniškai (Husserl 2005; 134). Atskirdamas tapatumą, tik pasyvų vardą, nuo laisvės, patyrimo išgyvenant, Rawlsas konstruoja racionalią marionetę, dovanodamas jai iš išorės „specifines teises ir laisves“. Subjektyvistinėmis liberalaus sodininko žirklėmis visų pirma nukerpama demokratinė politinės filosofijos tradicija, akcentuojant kraš­ tutinai formalius ir tariamai neutralius pasirink­ tų teorinių prielaidų interesus: „demokratinės minties istorijos akiratyje“ yra „specifinės teisės ir laisvės“ (Rawls 2001; 45). Istorinis demokrati­ jos apibrėžimas dabartiškai susiaurinamas vado­ vaujantis ne laisvos filosofijos, o priklausomybės nuo abstrakčios teisės principais. Šioji marionetė turi išorinį vardą: tai-patu­ mą. Ne savasties vardą, savigrąžą, o iš išorės duo­ damą pravardę: „Aš esti vertybinis neutralumas (a lithuanian:)“; „Aš esu Vakarų vertybių, taigi liberalizmo atspindys.“ Kai iš žmogaus atimamas vidinio laiko ir esmingo laisvės trapumo paty­ rimas, jai ar jam netekti laisvės – pigiau grybo. Tereikia – jaučiant dėkingumą šeimininkui – gręžtis į dovanotų „Teisių laisvėms, laisvių tei­ sėms“ sąrašus. Juose aš niekad neprarasiu laisvės, o atgausiu trokštamą laisvumą. Tereikia paleisti trapią laisvę iš rankų, – ir gauni dovanų ne-laisvę Politikos filosofija už-miršta. Tai liberalizmui įprasta nedėmesingumo Kitam (kitoms politinės minties tradicijoms) nerelevancija. Rawlsas, tariant jo paties žodžiais, tiesiog „įgyvendina reviziją“. Įgyvendindamas ne bet ką, o laisvės ir žmogaus baigtinumo filosofijos, – čia pagrindinės yra „baigtinumo laisvės, laikiškumo, istoriškumo, 164 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija sąsajų laikas sutraukomas ir suerdvinamas. Intencionalumas paverčiamas racionaliu pasi­ rinkimu. Politiniai įvykiai, kurių ištakose – vi­ sada ginčytinas konkretaus veiksmo teisingumas arba neteisingumas, redukuojami į procedūrines operacijas, dalijami į elementus statistiškai ap­ dorojant „individualių sąmonių“ laivo judėjimo padarinius. O paskui – viską sudėjus ir atėmus – sugrįžtama prie „viešosios valios“ (būtina skirti nuo bendrosios valios): naujų paskaičiavimų, po rinkimų. Radikali bendrosios tautos valios fragmentacija, kaip galbūt patikslintų Charles’as Montesquieu, „susimaišo“ taip, kad liaudis tampa „būtinai laisva“ (ji gali daryti viską, ką nori), – čia laisvės nebeatskirsi nuo valdžios: „Kadangi demokratinės valstybės liaudis, at­ rodo, gali daryti viską, ką nori, tai laisvė buvo priskirta šiai santvarkai, tokiu būdu sumaišant liaudies valdžią su liaudies laisve“ (Montesquieu 2004; 159). (į laisvę iš pažiūros panašų vardą), a) Leviatano saugumo tikimybės garantą ir b) lygybės laisvėms regimybių sąrašą: „O politinės laisvės netekti labai lengva: tik reikia ją paleisti iš rankų, ir jos nebelieka“ (Tocqueville 1996; 556). Kai nelieka vidinės laiko sąmonės, nelieka subjektyvaus laiko ir intersubjektyvaus tapa­ tumo sąsajų. Y dalies tezės: Randasi, kaip įprasta istorijoje, apimančioje begalinį skaičių jau palaidotų galimybių, istorizmas, šįkart vadinamas kitu pirmu, – „utopinio realizmo“, arba „niekuo netikinčiųjų pliuralizmo“, – vardu. Dirbtinio vardo vaikai – irgi dirbtiniai, arba pasyvūs (o tai yra viena ir tas pat). O tai ir norėta įrodyti pačių liberalų (nuo Hobbeso iki Rawlso): kad natūralus asmuo yra dirbtinis asmuo ir kad libe­ ralizmo vardas, Pliuralizmas, turi be galo daug suprantamų ir nesuprantamų pravardžių, tarp pastarųjų – „a lithuanian“. Išskaidžius aktyvią, konstituacinę, tęstinai gimstančią-mirštančią-gimstančią... socialinio viseto sandarą į paskiras pasyvias dalis (štai kodėl pozityvistų ir liberalų šitaip gerbiama analitika), ir jau po to, kai pliuralizmas, heterogeniškumas, reliatyvizmas padaro savo darbą (vieniems juodą, kitiems – baltą), randasi vadinamoji liaudiška pasirinkimo laisvė – tarp „Persil arba Ariel, VW arba Saab“ (Berger, Luckmann 1995; 45) – ir karinės arba kol kas dar tik karingos visuomenės standartų imperializmas, laisvių anarchizmas, teisių komunizmas (teisinėmis nesvarumo sąly­ gomis skraidančių tuščių oro balionų NVO ir sprogstančių per siūles savivaldžios savivaldos teisių). Štai kodėl liberalai trokšta tik minimalios po­ litinės moralės. Jeigu (priežastis) liaudies valdžia gerai sumaišoma su liaudies laisve (laisvumu daryti ką tik nori), tai padariniai – amžinas libe­ ralių atomų judėjimas laisvumo rojuje, kuriame laisvių tiek daug, kiek ir teismų. Visi juda kiek tik nori ir kaip tik nori. Ir svarbiausia: vieni į kitų reikalus nekiša nosies, nes bendroji vertybinio neutralumo būklė yra visų, o vertybės – tik libe­ ralų, neutralumas – irgi tik jų. Rankų sukirtimas po skaičiavimų (konsensas) – visų, o nepriklauso­ mybė – liberalų, kaip ir laisvumas – tik jų. Z dalies tezės: a) juo blogiau visuomenėje įsisąmoninami ir, atitinkamai, juo neaiškiau skiriami du nelygūs, visada skirtingi – tas pats ir tas „Individo sąmonė“ (romantizmo konsti­ tucijoje: individuali valia) tampa pamatiniu šiuolaikinės visuomenės elementu. Prasmingas a) juo blogiau visuomenėje įsisąmoninami ir, atitinkamai, juo neaiškiau skiriami du nelygūs, visada skirtingi – tas pats ir tas 165 Metodologija Sociologija. Z dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija apeina net patys save. Kaip sako Tereza – kie­ kvieno mūsų išgyventu, bet kažkodėl dažniau nei rečiau primirštamu transcendentalinio subjekto patyrimu – Eliaso Canetti romano Apakimas skyriuje „Mirtis“: „Nė neapsižiūrėsi, kaip tau sukaks keturiasdešimt ir pats gražiausias laikas bus praėjęs“ (Canetti 2007; 84). m) juo labiau aiškėja, kad laisvės prigim­ tis yra pirmesnė už nepriklausomybės prigimtį, tuo akivaizdžiau skaidrėja teleologija, ateities-muzika, prasmingo bendrabuvojimo laikiškume prasmė, – kad laisvė yra gręžimasis į valstybės prigimtį, buvojimą mūsump, kuris yra pirmesnis už kiekvieną paskirą politinio inten­ cionalumo dalį, nes esti visetas, esmė, – vertybių kaip idealiųjų žinojimo objektų tarpusavio sąsajos. Grožis, jei tik jis grožis, kaip ir gėris, jei tik jis gėris, ne sunyksta, o esti tęstinume. Liberaliu požiūriu, tai, kas „jau nejuda“, „praeitis“, yra ne laisvės, o nelaisvės, prietarų sankaupos, libera­ laus kūno judėjimo priekin trukdžiai. Fenome­ nologiniu požiūriu, tai, kas nejuda, ir gali būti, ir dažniau nei rečiau yra integrali laikiškumo ir erdviškumo prasminių sąsajų viseto dalis. Jei šios sąsajos labai baugina net ir aštuoniolikos metų sulaukusį liberalą, tai fenomenologą – ne. Fenomenologija gręžiasi į jau pasirinktas galimybes ir jau paaukotas kitas galimas veiksmo kryptis. Gręžiasi į tai, kad „nejuda“. Kad valios ir būtinybės susidūrimo aki-vaizdoje kitą pirmą­ kart patikrintų žmogiškos būklės ir ypač laisvės egzistavimo sąlygas. Laisvė yra sudėtinga filosofinė ir, dar svar­ biau, egzistencinė samprata. Laikiškoje trimatėje laikysenoje, teleologijoje, laisvė, – kitaip nei vien­ matiškoje istoristinėje liberalizmo utopijoje, – nuolat gręžiasi į priešduotį, į primestį, ieškodama atramos dabartin ir ateitin gyvoje praeities galimybių struktūroje. Laisvė intuityviai nušvinta akivaizdoje, niekad neleisdama jos „analizuoti“, „padalinti“, „apskaičiuoti“, „sudėti“, „atimti“... Kitaip sakant, malonūs, nors ne visai veltui gaunami lygybės sudėties be atimties žaidimai („o ir aš šito noriu“, „o ir man dar mažai“, „o man kaip tik šito ir trūksta“) laisvės nedomina. Laisvė – egzistencinė sąvoka, buvojanti gyveni­ mo-pasaulio brūkšnelyje, gyvenimo atogrąžoje pasaulin. Liberalizmo teorijose, nuo pradžios nepa­ kančiose praeičiai, arba kultūrai, egzistenci­ nėms žmogaus gyvenimo ir pasaulio santykio sąlygoms, laisvė visada tapatinama su laisvumu, arba nepriklausomybe: ką noriu daryti savo jėga ir inercija. Skirties tarp laisvės (egzistencinės aki-vaizdos) ir būtinybės (lemties) nepaisymas siaurina akiratį: verčia gręžtis nuo galimybių srities į vien aktualybės, arba priemonių dėl pačių priemonių, sritį. Liberalizme – ir teorijoje ir praktikoje – sutampa būtinybė ir laisvumas. Z dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 liekama likimo valiai, – ir virsta trečiojo asmens nuosavybe; aktualiosios galimybės įkūnijimu, – laisve ko nors norėti ir ką nors turėti atsiskiriant nuo ko nors ir be paliovos sudarant vis naujas „dirbtinių sutarčių“ kombinacijas; pats – daiktai, tuo joje mažiau laisvės; ir atvirkščiai, kuo visuomenėje daugiau žmonių, gebančių skirti du – tas pats ir tas pats – daiktus, tuo visuomenė laisvesnė, tuo geriau ji jaučia ir supranta išlaisvėjimo ribas; b) b) juo labiau lygybė paverčiama vien paliepimo sakinių vartojimo – „laisvai krentančių žodžių ir ženklų srautų“, „statistinių faktų“, „visuomenės nuomo­ nės apklausų“, „vidurkių“, „vidutiniško žmogaus gyvenimo visuomenėje dizai­ nerių (skonio, mados, politikos, ekonomi­ kos, bankininkystės, šventinių ir karinių paradų ekspertų...)“ – rūšimi, tuo labiau sunyksta gyvenimo-pasauliui prasmin­ gos tvirtinimo sakinių ir klausimo sakinių rūšys ir juo mažiau lieka galimybių galėti ir gebėti laisvai ne pasirinkti šioje paliepimo sakinių  – „lygia greta iš laisvų šalių laisvai krentančių žodžių ir ženklų srautų“ – prie-vartoje, tvoskiančioje pilkais kaip šleikšti-ne-laisvė pelenais; b) juo labiau lygybė paverčiama vien paliepimo sakinių vartojimo – „laisvai krentančių žodžių ir ženklų srautų“, „statistinių faktų“, „visuomenės nuomo­ nės apklausų“, „vidurkių“, „vidutiniško žmogaus gyvenimo visuomenėje dizai­ nerių (skonio, mados, politikos, ekonomi­ kos, bankininkystės, šventinių ir karinių paradų ekspertų...)“ – rūšimi, tuo labiau sunyksta gyvenimo-pasauliui prasmin­ gos tvirtinimo sakinių ir klausimo sakinių rūšys ir juo mažiau lieka galimybių galėti ir gebėti laisvai ne pasirinkti šioje paliepimo sakinių  – „lygia greta iš laisvų šalių laisvai krentančių žodžių ir ženklų srautų“ – prie-vartoje, tvoskiančioje pilkais kaip šleikšti-ne-laisvė pelenais; c) juo, kiekybiniu požiūriu, daugiau pa­ liepimo sakinių ir juo mažiau tvirtinimo sakinių ir klausimo sakinių, tuo labiau lygybė praranda kokybės dimensiją ir virs­ ta lygumu, arba suvienodinimu, nelygių iš gimties ir laisvų iš galėjimo ir gebėjimo žmonių išlyginimu; d) juo natūraliau veržiamasi į išlyginimą g) juo natūraliau veržiamasi į laisvę, tuo labiau laisvė tampa pirmojo asmens nuo- savybe; grynosios galimybės įkūnijimu; teleologine savigrąža bendrosios laiko prasmės laikysenoje; g) h) juo natūraliau veržiamasi į lygybę, tuo labiau aiškėja, kad lygybė yra grynoji negalimybė; i) juo natūraliau veržiamasi į laisvę, tuo labiau aiškėja, kad nelygybė yra aktu­ aliai nelygi prigimtis priešduotyje, o laisvė – grynoji galimybė priešduotyje, arba teleologija, kuri yra ankstesnė už bet kokį aš; j) juo arčiau lygybės negalimybės refleksijos, tuo aiškesnė – laisvės prasmė; ir atvirkš­ čiai, juo arčiau lygybės regimybės natūra­ lizmo, tuo toliau supratimo, kad daiktai yra daugiau ar mažiau lygūs tuomet, kai jie yra relevantiški ko nors atžvilgiu; c) juo, kiekybiniu požiūriu, daugiau pa­ liepimo sakinių ir juo mažiau tvirtinimo sakinių ir klausimo sakinių, tuo labiau lygybė praranda kokybės dimensiją ir virs­ ta lygumu, arba suvienodinimu, nelygių iš gimties ir laisvų iš galėjimo ir gebėjimo žmonių išlyginimu; k) juo arčiau relevantiško laisvės ir lygybės tarpusavio santykio supratimo, tuo labiau aiškėja, kad laisvės prigimtis yra pirmesnė už nepriklausomybės prigimtį; d) juo natūraliau veržiamasi į išlyginimą, tuo labiau sutampa laisvumas ir būtinybė; l) relevantiškumas yra santykiškas dydis: juo arčiau mažųjų tautų, tuo relevan­ tiškesnis yra laisvė stiprina lygybę, o lygybė – laisvę santykis, o kuo arčiau didžiųjų tautų ir imperijų, tuo relevan­ tiškesnis – lygybė stiprina laisvę, o laisvė lygybę santykis; e) juo natūraliau veržiamasi į išlyginimą, tuo labiau laisvė virsta nepermatomo vidurio natūralizmu; f) juo labiau laisvė virsta nepermatomo vidurio natūralizmu, tuo greičiau ji pa­ 166 Sociologija. Z dalies tezės: Intencionalus demokratinės tautos veržimasis į laisvę (bendroji valia, politinis intencionalu­ mas) – „<...> demokratinėms tautoms būdingas Laisvės patyrimas, kitaip nei lygybės patyri­ mas, kuris įprastai užsibaigia realiu ar tariamu „lygybės tam kartui“ įgijimu, yra akivaizdos išgy­ venimas. Kitaip nei lygybės patyrimas, kuris daž­ niau nei rečiau yra malonus, laisvės išgyvenimas dažniau nei rečiau yra ne malonus, o, priešingai, skausmingai aštrus, akinančiai akivaizdus, netgi galintis apakinti. Kai tiktai tampa laisvi, žmonės neretai apanka, susvetimėja savo pačių atžvilgiu, klydinėdami – maloniais lygumo klystkeliais – 167 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 natūralus veržimasis į laisvę“ (Tocqueville 1996; 558; išskirta mano – A.V.) – virsta nepermato­ mu natūralizmu. Bendroji valia, arba veržimasis į laisvę, paliekama likimo valiai. aistringas potroškis turėti – pačiais įvairiausiais atžvilgiais – ne mažiau, nei kiti. Laisvė – neabe­ jinga savo porininkės, lygybės, laisvės patyrimo sąlygoms. O lygybė – abejinga pačiai laisvės kryp­ čiai savigrąžon, todėl laisvę laiko ne porininke, o tik laikinų sutarčių partnere. Lygybė savo ruožtu neabejinga nuolatiniam gręžimuisi stokon. Drauge su tik laikinais partneriais, įskaitant laisvę, ji išlygina žmonių tikslus pasitelkdama teisines priemones. Tai vidurio natūralizmas. Čia niekada nesu­ siduria savęsp ir mūsump: subjektyvi asmens valia ir lemtis, subjektyvumas ir inter-subjektyvumas. Palikti likimo valiai žmonės turi dvi pagrindines galimybes: arba 1) gręžiasi į laisvę („siekia laisvės, brangina ją ir sielvartauja jos netekusios“; ten pat), arba 2) gręžiasi nuo laisvės gręždamiesi į lygybę („O lygybė yra karšta, nepasotinama, amžina, nenugalima jų aistra“; ten pat). Visi žmonės, net ir liberalai, kartodami indiferen­ tišką pliuralizmo „vertybinio neutralumo“ maldą, – kad liberalizmo politinės struktūros turi laikytis atokiai nuo skirtingų gero gyvenimo koncepcijų, – įsitraukia į atkaklią vidinę kovą tarp esminių – laisvės ir lygybės – vertybių, ir ne tik minimalios politinės moralės lygmenyje. Laisvės niekaip negalima paversti „-izmu“: nei ideologija, nei filosofija. Juo labiau laisvės filosofija teorinė, tuo labiau ji klaidina. O lygybė pati nori tapti egalitar-izmu (teorija), reikalaudama, kad kai kurie turi gauti arba daugiau, arba mažiau, kadangi kiti gauna daugiau arba mažiau. Darkart: laisvė – aštrus prarajos politinio intencionalumo laikysenoje patyrimas, akivaizdos nušvitimas ir štai-šio pa­ tyrimo išgyvenant laisvę sąlygų nuskaidrinimas, o lygybė – geometrinės matricos hierarchinės sandaros vidinis pertvarkymas, po kurio prasi­ deda sistemingas kitų lygybės stokos atžvilgių – skonių, malonumų, pomėgių, agresyvumo ir minkštakūniškumo instinktų, pavydo, geltos, tulžingumo, pagiežos  – išlyginimas: „Nebepa­ kanka pripažinti ir gerbti kiekvieno žmogaus žmogiškumo. Mes turime matyti kitą kaip tokį pat“ (Manent 2008; 9). Z dalies tezės: Gyvenimo-pasaulyje grynosios lygybės nie­ kur ir niekada nerasime: grynoji lygybė – tik abstrakčioje geometrinėje Leviatano matricoje. Kaip tik šis abstraktus lygmuo, kursai iš tikro neegzistuoja, labiausiai traukia negalimybės filosofiją, liberalizmą. Reikalas tas, kad tiek ma­ trica, tiek lygybė turi mažai moralinės reikšmės. O tai labai tinka vertybinio neutralumo ir (jei tik išlaisvintiems žmonėms prireikia dirbtinės moralės) moralinio minimalizmo prielaidoms. Laisvės patyrimas yra originalo suvokimas. Laisvose laisvės akyse du daiktai – visada skir­ tingi: tas pats ir tas pats. O jeigu du daiktai yra daugiau ar mažiau lygūs, tai tik ko nors atžvilgiu, kaip teigia fenomenologinis relevantiškumo principas. Ir priešingai, liberalizmo teorijose lygybė, paversta geometriniu lygumo principu, tampa lygybės saviverte. Ne lygybės įgyvendini­ Laisvė yra neįkainojama (moralės dėsnis nepriklauso nuo rinkos diktuojamų pasiūlos- paklausos kreivių dinamikos) pirmojo asmens nuo-savybė. Drąsi savigrąža į absurdo brūkšnelį, jungiantį tikrovę kaip prie-vartos būtinybę ir tikrovę kaip kitas tos pačios tikrovės galimybes. Lygybė yra įkainojama trečiojo asmens nuosavybė: 168 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija motyvų ir interesų, iš kurių jau per tarpines – gręžimosi nuo baimės į savo išlaisvintą kūną arba gręžimosi į rūsčias, nes priešduotas laisvės savastyje prielaidas – priemones meistraujama lygybės sąvoka. Liberalios teorijos motyvai ir inte­ resai nesikerta ir netgi dažnu atveju akivaizdžiai sutampa su ekonomikos teorijos paskirtimi ir uždaviniais, o dar prieš tai – jų abiejų bendrais interesais ir motyvais: juo toliau nuo baimės, tuo toliau nuo mirties; juo toliau nuo savęs, tuo arčiau dabartiškumo tikrovės, juo arčiau šeimi­ ninko, „utopinio realizmo“, durų: „Šuo mano, kad jo šeimininkas stovi prie durų“ (ten pat). mu relevantiškų gyvenimo-pasaulio struktūrų aplinkose, o aukščiausiosios vertės suteikimu formaliam principui: jei kiti gauna daugiau (o man įprastai nerelevantiški tie, kurie gauna ma­ žiau), tai ir aš turiu gauti ne mažiau už juos, bet, tikėtina, daugiau. Su nekintama išlyga – bent jau ne mažiau. Darkart: laisvose laisvės akyse du daiktai – visada nelygūs, visada skirtingi: tas pats ir tas pats. Todėl galima sakyti, kad lygybės sąvoka gyvenimo-pasaulio vertybių sistemoje yra „tretinė“: po laisvės ir teisingumo. Tai reiškia: jei aš žinau tvarką man priešduotoje vertybių sistemoje ir jei aš laikausi jos, tai galiu įžvelgti ir bandyti atitolinti „blogos lemties“ pirštą, kuris prikišamai liepia man pradėti lygybe, paskui teisingumu (dažniau nei rečiau liberaliu: reika­ laudamas teisių laisvėms tulžingumo išlyginimo vardu), ir tik paskui laisve. Z dalies tezės: Štai kodėl tikrųjų individualistų, arba uto­ pinių realistų, tikrovėje į-vertinama palaima, sutampanti tik su empiriniais dabarties lūkesčių instinktais, ypač piktumo (veržlumo durų link ir eilės taisyklių normų nesilaikymo), įžeidumo (nesėkmės virsmo dar siautulingesne paska­ ta), juslinių potyrių pertekliaus (saldumynų džiaugsmų), ir tik retsykiais išgąsčio (arešto galimybės). Ir priešingai, nu-vertinama laimė, socialinio politinio intencionalumo laikysena, viltis. (Plg.: „Galima įsivaizduoti piktą, bailų, liūdną, linksmą, išgąsdintą žvėrį. O kupiną vilties? Kodėl gi ne?“; Wittgenstein 1995; 330.) Egzistencija, taigi ir laisvė, – aš pats savigrą­ žoje įgyvendinu savo gyvenimo laisvės sąlygas, įsižiūriu į jas ir pri-žiūriu (nebus per daug pakartoti: net aštuoniolikos sulaukęs liberalas baiminasi būtent prie-žiūros: savasties priežiū­ ros, kuri trukdo netrukdomai išlaisvinti kūną, trukdo nevaržomai ir be jokių ribų judėti), – yra prieš-duotyje, taigi dar prieš teisingumo ir lygybės duotis. Plg. 1) neįsisąmonintą liberalią baimę ir 2) įsisąmonintą fenomenologinę baimę: 1) „Žinok: aš bijau.“ „Žinok: mane apima šiurpas.“; 2) „Tą galima pasakyti ir juokaujamu tonu.“ (Wittgenstein 1995; 330) Liberali „lygiųjų“ visuomenė utopijoje vaiz­ duojama kaip vertybiškai neutrali bet kokių gėrio koncepcijų atžvilgiu, nors ir ne abejinga tam tikrų dorų priemonių, akylai saugančių bendruosius individualistų motyvus ir interesus, atžvilgiu. Kaip kad laisvė ir teisingumas fenomenologi­ nėje veiksmo laikysenoje reikalauja lygybės, taip lygybė (visiems vienodų pliuralizmo sąlygų įtvirtinimas visose visuomenės gyvenimo srityse ir trečiojo asmens kūne bei sąmonėje) liberalioje veiksmo laikysenoje reikalauja teisingumo, ir nie­ Taigi tvarkingai: visų pirma laisvės ir teisingu­ mo veiksmo laikysenos (relevancijos) apibrėžia ir reikalauja vienokios ar kitokios lygybės koncepci­ jos. Kitaip sakant, dar prieš nuskaidrinant vieną ar kitą lygybės figūrą, reikia ieškoti pirminių 169 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ko daugiau. Štai kodėl abstrakčioje, dirbtinėje liberalizmo matricoje – viskas teisinga, gražu ir švaru. Teisingumas vaizduojamas kaip sekuliarus pliuralistinio teisingumo kryžius, kurį 1) neša, teisingai pasiskirstę naštą pagal nešulio svorį, tenkantį konkretaus nešančiojo – išlyginto net pagal lytį – kūno masei, visi iki vieno, vartojant Hobbeso terminą, „mirtingojo dievo“ nariai, 2) gaudami iš to „mažiau“, „daugiau“, „daug“ ir „labai daug“ naudos (pagal individualios autono­ mijos svorį ir už tai atitinkamai gaunamas teises). įprotis: „Žiūrėk, aš visąlaik darau tą patį: aš ...“ (Wittgenstein; 221 [223]). Šis visąlaik įprotis virsta ego-perviršiu. Aš matau tik griežta seka judančių kūnų tikrovę, bet neturiu galimybės pažvelgti į šio judėjimo pradžias, pasitikrinti ir įsitikinti, ar teisingai paaukojau – dėl šios „nea­ bejotinos tikrovės“ – kitas ir kokybiškai kitokias judėjimo galimybes. Ego-perviršis pasiglemžia net ir pirmojo asmens laisvės patyrimo gali­ mybę. Z dalies tezės: Pagrįskime subjektyvumo – „Bet ar bent jau tas pats nėra: tas pats?“ – paradoksą, iš kurio randasi ontologinė intersubjektyvumo figūra, savo išgyvenamu patyrimu. Kai egalitarinio liberaliz­ mo paradigmos „2 2 2 2 ...“ keliai jau išvaikščioti skersai ir išilgai pagal vieną ir tą pačią seką, aš kitą pirmąkart klausiu: „Tačiau ar tai viskas?“ (ten pat; 218 [209]). Neturėdamas pasirinkimo grynojoje pasirinkimo negalimybėje, aš radika­ liai gręžiuosi savęsp suskliausdamas – tai veiksmo pakraštys, paribys, transcendentalinė savimo­ nė – kitus, kuriems vis dėlto lieku įsipareigojęs. Gręždamasis į tą pačią demokratijos paradigmą, aš atrandu priešduotyje, demokratijos-man- primestyje, esmingai absurdišką laisvės prigimtį: „Visi žingsniai iš tikrųjų jau žengti“ reiškia: aš nebeturiu pasirinkimo“ (ten pat). „Tai“, arba „2 2 2 2 ...“ seka, nėra, kaip aiškina mano šeiminin­ kas kažkur už durų, „tik“ skaičiuotė, naudinga parankiniams policinės politikos tikslams. Aš pats matau intencionalių sąsajų visetą: štai kaip jis man atrodo (ten pat; 220 [219]). Kadangi giminingumo brūkšnelis – ma­ tomas, tačiau nepastebimas, įprotis sutampa su įpročiu, sutarimas tampa taisykle, o laisvė – tuš­ čia procedūrine dabarties išlygų laukimo poza: „Šuo mano, kad jo šeimininkas stovi prie durų“ (ten pat; 330). Dabar jau esu pasirengęs liberalių, arba lais­ vųjų, diskusijų ginčui su filosofijos profesoriais, kurie domisi teisingumo klausimais ir praktiniu racionalumu, vienas su kitu nesutardami stipriai ir, rodos, nesutaikomai (MacIntyre 1988; 4). Esu pasirengęs ir ginčui su dešimtkart libera­ lesniais, išskyrus nebent visiškai „kairį žodyną“, sociologijos profesoriais: „Sociologai įprastai rašo apie nelygybę, o ne apie lygybę“ (Turner 1986; 15). „Aš neturiu laiko galimybės turėti laiko galimybę, išskyrus dabarties, erdvės be laiko, išlygą“, – tai liberalo, išdidžiai nukirtusio savo socialinę bambagyslę ir žvelgiančio žmogui tik į nugarą poza. Aš įsižiūriu į laisvę, mįslingiausią politinio intencionalumo slėpinį, aiškiai skirdamas tapa­ tumo (tas pats ir tas pats) formas ir suprasdamas, kad laisvė – ne duotis, o priešduoties refleksija mano relevantiškoje arba nerelevantiškoje – jos atžvilgiu – veiksmo laikysenoje. Bendra-erdviš­ kumo – su porininkais, partneriais, amžinin­ kais – draugėje, pačiame bendros kalbos var­ tojime jau kaip veiksmo laikysenoje aš įžvelgiu kažkur kažkada matytas laikiškumo dimensijas: „Ne be pagrindo sakoma, kad ankstyvoje vai­ kystėje turėjome mokytis matyti daiktus ir kad šios rūšies patyrimai turėjo būti genetiškai Kokybiškai kitokią, socialinio politinio in­ tencionalumo, laikyseną, sudarančią tinkamas sąlygas laisvei, perteikia vienas trumpiausių Wittgensteino fragmentų: „Bet ar bent jau tas pats nėra: tas pats?“ (ten pat; 219 [215]; išskirta autoriaus). Z dalies tezės: Aš nematau savo socialinės ap-linkos, ir negaliu intencionaliai, draugiškai arba taktiškai, linktelėti pasauliui, kursai davė man gyvenimą (tai bent duotis!). Gręždamasis savęsp gręžiuosi nuo savo kilmės, darydamasis nekilnus. Teisingumas pliuralistinėje visuomenėje ku­ pinas ne vilties ir, žinoma, ne kaltės, o judančių skaičių sekos. Tas, kursai miršta, atimamas, o tas, kursai eina, sudedamas. Štai kodėl „Seka turi mums vieną išvaizdą!“ (Wittgenstein 1995; 221 [228]). Seka, išėjusi iš vaizdo, yra iš-vaizda. Ne bendrasis gėris, o visų gėris. Kai tik priartėju prie savęs, gręžiuosi nuo – dėl mirties-gimties ir gimties-mirties brūkšnelių akivaizdos baimės – są-monės ir atsigręžiu į sąmonės, arba kūno (šįkart tai yra viena ir tas pat), įprotį. Tampu tik savo vidinio psichinio patyrimo „pluoštu“, arba „egoistu“: „Egoiz­ mas – tai karšta, besaikė savimeilė, skatinanti žmogų pirmiausia galvoti tik apie save ir tik sau teikti pirmenybę“ (Tocqueville 1996; 559). Galbūt ir suvokiu, kad esu „greičiau sąmonin­ gas“, negu „nesąmoningas“, bet neturiu su kuo šiuo savo suvokimu pasidalinti. Turiu tikrovę, bet neturiu galimybės. Neturiu galimybės transcenduoti savo sąmonės srautų „pluošto“, „egoizmo“, „tik subjektyvių įspūdžių“ aibių. Neturiu galimybės nuskaidrinti intenciona­ lią – sąmonės ko nors už jos pačios – kryptį. Negaliu pajausti ir apglėbti patyrimo viseto. Negaliu pratęsti, nes negaliu sugriebti gimto polinkio buvoti subjektu-pasauliui ir drauge buvoti objektu-pasaulyje. Iš-vaizdi seka (žr. „Stiliaus“ žurnalą, natū­ ralizmo konstituciją): judantys, sudedami ir atimami, kūnai tam tikroje teritorijoje, einantys nežinia kuria ir nežinia kokia kryptimi. Po­ licinė seka: kūnų priežiūra, kontroliavimas, mokesčių surinkimas, įkalinimas, išlaisvinimas, įforminimas, išforminimas, įregistravimas, išregistravimas... Aš, kaip trečiasis asmuo, nešu sekuliarų – judantį su malonumu – kryžių ne todėl, kad klausčiau „Kodėl?“ ir patvirtinčiau esmines laikiškas įsisąmonintos lemties „Taip nešu“ sąlygas, o tiesiog kad nešu: kaip „prieš einantis įvykis po einančio, ir, priešingai, po einantis prieš einantį“ (Hobbes 1999; 48). Prie­ šingai nei teigia „utopiniai pliuralistai“, utopijos čia – nė kvapo. Tik prie-vartos suerdvinamas sąmonės srautas, be laikiškumo refleksijos. Čia įprastu „mano“ metodu tampa procedū­ rinis – „Žiūrėk žmogui ne į akis, o į nugarą!“ – Neturiu galimybės turėti galimybę. Grynoji negalimybė tampa mano įpročiu, metodu. Už- 170 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija gimsta intencionali figūra: laiko prasmės tėkmė, viltis ir atsakinga laikysena. miršu. Pra-randu są-monės įprotį, o randu – ir t.t. įprotį. Už-mirštu: kad į-protis ir įprotis yra vienas kitam giminingi. Plg.: žodis „sutarimas“ ir žodis „taisyklė“ yra vienas kitam giminingi (Wittgenstein 1995; [225]). Z dalies tezės: Jei egalitarinio liberalizmo seka „2 2 2 2 ...“ yra tik einantis prieš einantį ir einantis po einančio, be jokio intencionalumo, taigi ir be vilties, tai Wittgensteino fragmentas į bet kurią „2 2 2 2 ...“ seką įsižiūri kaip į bet kokią „2 2 2 2 ...“ seką. Štai-šioje sekoje randasi vidinio laiko – drauge su išoriniu laiku – judėjimas, vadinasi, 171 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija pirmesni už bet kokius kitus daiktų suvokimo būdus“ (Husserl 2005; 98–9). Aš praturtinu savo domesio (inter-esse) aplinką tipizacijomis, figūracijomis, asociacijomis, kurios susieja mane su pirmtakais ir, – vaizduotės dėka, – netgi su įpėdiniais. ne klasifikacinė trečiojo asmens teisių ir laisvių sąrašų, o pirmojo asmens patyrimo ir žinojimo ryšių eksplikavimo problema. Pasaulis yra duo­ tas-kaip-jis-pasirodo-man: ir kaip man savaime suprantamas, ir kaip mano tematizuojamas. Mūsų taikomojo pobūdžio tezė yra ta, kad bet koks eidetinis socialinio veiksmo ir politinio veiksmo (veikiančio asmens, socialinės grupės, sąjūdžio, minios, štai-šios jėgos, valdžios, valsty­ bės, bendruomenės ir pan.) tyrimas, reikalauja (o ypač viešpataujant biurokratinio liberaliz­ mo, – menkiausią procedūrinį savo nemenkos apimties kūno judesį siejančio su „politikos“ vardu, – sąlygoms) nuskaidrinti 1) esmines pirmojo asmens politinio intencionalumo patyrimo sąlygas; 2) esmines pirmojo asmens ir bendruomenės socialinio ir politinio patyrimo sąlygas, būdingas abipusiškai veiksmo laikysenai. Kitaip sakant, aš įsižiūriu į laisvės figūracijas amžininkų visuomenėje: nuo betarpiškų sociali­ nių (pasirinkimo galėjimo, čiabūtiškumo laisvės namų) iki anonimiškų socialinių santykių, kurie struktūrizuojami tam tikrose lygybės ko nors at­ žvilgiu tikrovėse ir regimybėse. Aš įsižiūriu į tai, kas paslėpta supratimo tankmėje (Wittgenstein 1995; 172 [102]). Pirmoji aptarta laikysena – egalitarinio libe­ ralizmo „2 2 2 2 ...“  – leidžia aiškiai pamatyti „nepriekaištingą tapatumo paradigmą“, natū­ ralius „gamtos faktus“ ir suvokti „priežastinius ryšius“ (ten pat; 168 [89]), o antroji – įsižiūrėti į kažką, kas slypi po paviršiumi: „“Esmė yra pa­ slėpta nuo mūsų“: štai kokią formą dabar įgyja mūsų problema“ (ten pat; 169 [92]). Skirtini trys įprasti konvenciniai būdai, nagrinėjantys dviejų tarp savęs glaudžiai susiju­ sių – politinio ir socialinio – fenomenų sąsajas, – a) politologinis, b) sociologinis ir c) mišrus. Pirmojo tyrimų būdo akiratyje yra politinio instituto sudedamųjų dalių sandaros ir politinės inžinerijos mechanizmo veikimo būdų tyrimai. Antroji laikysena yra socialiai ir politiškai esminga: prasmė randasi iš a) asmens ir b) visuo­ menės c) sąveikos (atitinkamai: są-monės, tikrovės, laisvės). Z dalies tezės: O pirmoji laikysena yra išvestinė: prasmė randasi vadovaujantis taisyklėmis aklai: ne kitą pirmą kartą (kaip veiksmo laikysenos refleksijoje atveju), o pirmą, antrą, trečią... kartus (pagal procedūrinę sekos liniją). Kai politika – lygu kitas pirmas rinkimų- vajus, o paskui – viskas-yra-politika, tai ir po­ litikos tyrimai yra visko – nuo administravimo iki psichologinio portretavimo – tyrimai, išskyrus nerelevantiškus politologijai nelaisvės, neteisingumo, nelygybės sąlygų bei aplinkybių ir struktūrinių laisvės, teisingumo ir lygybės sąlygų tyrimus. Štai kodėl, – nes lygybė yra nuolat pigiai per­ vertinama laisvės sąskaita, – svarbu nuolat skai­ drinti relevantiškus politinio intencionalumo problemos aspektus. Nebus per daug pakartoti: „trumpa“, relevantiškumo pilnumos požiūriu, čia išskleidžiama X, Y, Z išnaša yra tik bandymas parodyti, kad relevantiškumo problema – tai Antrojo politikos tyrimų būdo akiratyje yra einančių po einančių ir einančių prieš einančius, arba „2 2 2 2 ...“ visuomenės“, vidurkių, arba vadinamosios nuomonės, tyrimai ir kokybinės 172 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija tikrovės („tik lankstinuko“), ir susiejama su oligarchiniais kompleksiško politekonominio klano (balos, valkos, telkšos) interesais. (bet nebūtinai visada kokybiškos) subjektyvių nuomonių interpretacijos. Trečiojo būdo paran­ kiniai interesai – daugiau ar mažiau eklektiškas pirmųjų dviejų būdų derinimas (vadovaujantis savaime suprantama samprata, kad viskas yra politika, o politika yra viskas). Tai savitas (tik empiriniu požiūriu) komu­ nalliberalizmas, pakeitęs komunalkomunizmą. Neskaidri pirminė, antrinė, tretinė... privačios nuosavybės savisklaida, kurią pakeičia dualis­ tinis aukštesnės vertės policinių, teisinių ir eko­ nominių struktūrų ir žemesnės vertės kultūros, atiduotos pliuralistų savivalėn, valdymo mode­ lis. Spontaniškai agresyvus komunalliberalizmas, cikliškai gaminantis ir atgaminantis „prestiži­ nes“ padėtis, statusus, vaidmenis. „Laisvumo vanago“ akimi skirstantis, pasiskirstantis ir perskirstantis išteklius. Savivaldaus „viršaus“ tikrovė. Liberalizmo demokratija turi dvi puses. „Racionalioji“ liberalizmo pusė traukia mus visus tik į grynąją procedūrinę dabartį (nuo rinkimų iki kitų pirmų rinkimų). „Romantiš­ koji“ rodo į grynąją utopinę ateitį: išlaisvinto nepriklausomo individo, gyvenančio „tuščiuo­ se“ tarpuose tarp socialinių politinių sąsajų. Būtina pažymėti (nes kalbame ne apie proce­ dūrinę ir ne utopinę vertę), kad esminė laisvės ir lygybės santykio problema – ne politologinė: dėl šios disciplinos polinkio šlietis prie jau duotų analitinių, natūralistinių prielaidų. Nors ir čia, žinoma, yra ką veikti aiškinantis procesus, kurie įgyja pagreitį aktualiame gyvenime (kai aukštyn kojomis apverčiamas idealusis Tocqueville’io abipusiškumo principas): kai lygybė stiprina ne laisvę, o pačią save, o dvigubai pamaloninta lygybė – atima laisvę. Štai kodėl politinio relevantiškumo proble­ ma – netgi ne fenomenologinė, nors ir čia yra ką veikti skaidrinant subjektyvumo, racionalu­ mo, relevantiškumo sąsajas ikipredikatiniame ir predikatiniame lygmenyse. Z dalies tezės: Problema yra filosofinė antropologinė (darkart: tik žmonės iš visų gyvūnų išsiskiria tuo, kad jie vieninteliai suvokia, kas yra gera ir bloga, teisinga ir netei­ singa; Aristotelis 1997; 1253a 19–20), sietina su konkrečiąja gyvenimo-pasaulio ontologija (inter­ subjektyvumu ir dviem pagrindiniais – socialumo ir politiškumo – sandais). Lygybė, geometrinė bendruomenės abstrakci­ ja, kai kurioms visuomenės grupėms dovanoja vaidmenų ir statusų išskirtinumo – atitinkamai suranguotose scenos pakylose – teisę, o drau­ ge – ir gerą ūpą: „Laisvumo pakyloje stoviu štai čia prieš jus, o ne su jumis.“ Tautos „socialinė“ erdvė pripildoma tuščių „laisvės“, „lygybės“, „teisingumo“ parametrų. Erdvės abstrakcija, viena vertus, tampa parankiniu „tautos vaiz­ diniu“ (lankstinuku, lankstomu kaip tik nori ir kieno tik nori), kita vertus, egalitarizuojama tam tikruose scenos pakylų lygmenyse ir atker­ tama nuo gyvosios dabarties, pirminės politinės Tai reiškia, kad sociologinę ir politologinę nacionalinės valstybės ir globalizacijos santykio problemą dera „išversti“ į konkrečiosios gyve­ nimo-pasaulio ontologijos kalbą. Dera grįžti į pačią pradžią: nuskaidrinti politinio ir socialinio intencionalumo (abi šios rūšys konstituojamos drauge, o ne viena iš jų „prieš“, o kita – „po“), erdviškumo, o ypač laikiškumo radimosi iki­ predikatiniame patyrime problemas, ir klausti, 173 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija kaip konstituojami veiksmo tipai ir tipizacijos, socialinės ir politinės erdviškumo ir laikiškumo figūros, ir asmeniškame, ir bendruomeniniame patyrime? [1253a 23–26]). Esminę skirtį tarp, viena vertus, pirminio politiškumo pasaulio, ir, kita vertus, antrinio (deleguoto) socialinio institucinio bei politikos pasaulio kažkodėl noriai griauna pati politologija (pagal paskirtį mokslas ne dėl politikos, o apie politiką), kuri, užuot prižiūrėjusi pirminės politinės tikrovės išteklius, o ypač jos galimybes, rūpinasi tariamai tikslių ir realiai fabrikuojamų reitingų grynaisiais aprašymais, apklausų duomenimis (prieš reitingus ir po reitingų), politinėmis šventėmis ir ritualais, arba politinių agitbrigadų ir jų lyderių stilistinių profilių portretais bei šaržais (visada kas nors laimi, ir kas nors pralaimi). Juk iš prigimties politinė pirminės socialinės tikrovės struktūra grindžia visus kitus struktūri­ nius – socializacijos, komunalizacijos, hierarchi­ zacijos, valdumo ir pavaldumo, subordinacijos sąmonės, arba paklusimo laikysenos radimosi, koordinacijos sąmonės, arba autoriteto regionų formavimosi – santykius. Šis pirminis – jau struktūriškai tipizuotas dar iki liberalizmo konstitucijos – tautos konstitu­ cijos principas lengvai paminamas absoliutaus vertybinio reliatyvizmo (žr. Jokubaitis 2012), arba privačiai suprantamo liberalizmo (o tai yra viena ir tas pat). Biurokratijos ir technokratijai kylant kaip ant mielių, visai neatsitiktinai gausė­ ja tarsi-viešų-diskusijų ir racionalių „iracionalaus laisvumo“ progresavimo indeksų, kurių paskir­ tis – prižiūrėti, valdyti ir prikaustyti žmonių dėmesį. Z dalies tezės: Tokia laisve, žinoma, mėgaujasi visi liberalesni už kitus subjektai, dirbtinėje grandinėje (visuo­ menėje) vienbalsiai priimdami romų „landų“ įstatymą: „Didžiausia pavaldinių laisvė priklauso nuo įstatymo nutylėjimo“; „The greatest liberty of subjects, dependeth on the silence of the law“ (ten pat; 226; 146). Šios istorijos pradžioje dirbtinių grandinių „vienas galas“ pritvirtinamas prie grandininės motinėlės „lūpų“ (ten pat), arba realistinės politikos, kuri turi aukščiausią valdžią. „Kitas galas“ pritvirtinamas prie motinėlės „ausų“ („in­ ter-tele-žurnalistikos“: kad visi viską girdėtų ir įsidėtų į galvą): „<...> vieną galą pritvirtino prie lūpų..., o kitą galą – prie savo ausų“ (ten pat). g „Lūpos“ išmoko žemąjį – „kitą galą“ – liberalaus multikultūralizmo (reliatyvizmo dorovės ir atitinkamos politinės etikos). Net neklausdamos, ar derėtų, neišnagrinėjus eide­ tinių pirminio pasaulio ontologijos galimybių klausimų, įsileisti į gyvenimo-pasaulį strateginės gyvenimo fragmentacijos ir taktinės privatizacijos (skaldyk ir valdyk) reiškinių, galimai naudingų, bet galimai žalingų, galbūt griaunančių pačias bendrabuvojimo sąlygas. Darkart: kai visos prielaidos laikomos „savaime suprantamomis“, viešpatauja konsensas, arba demokratijai AMEN. „Landus laisvumo principas“ skelbia: ten, kur suverenas nėra nustatęs jokių taisyklių, liberalus subjektas laisvas veikti ar neveikti savo nuožiūra (Hobbes 1998; 146); tik ne pavaldinys (Hobbes 1999; 226), kuriam privalu paklusti. Šis į lygiųjų klanus (įgyvendinant komunalizaciją) organizuojantis principas paverčiamas „švininio svambalo“ (vertybinio neutralumo) principu. p j j Dominuojantis požiūris (administracinių po-tvarkių etika) – ne liberaliosios-demokratijos, o konsenso-demokratijos. Pirmoji – eidetinė, idea­ lių bendrabuvojimo galimybių, o antroji – fakti­ nė, buvojimo valdžioje aktualybių. Išsilaisvinusi iš galimybių srities, laisvė nuo bet ko išsilaisvina ir iš bet kokios opozicijos: „LIBERTY, or FREEDOM, signifieth (properly) the absence of oposition <...>“ (Hobbes 1998; 139). Štai kodėl dirbtinėse grandinėse šiandieną aktyviai blaškosi kitas galas „Dar nuo ko nors laisvės!“ Kito galo, arba natūralizmo / materializmo (tai yra viena ir tas pat) laisvė dirbtinėse grandinėse anksčiau ar vėliau tampa tik „nešvankių juok­ darių“ (Hobbes 1999; 221) laisve. Tai reiškia: jei suverenas nėra nustatęs jokių taisyklių vertinimo (gėrio ir blogio, grožio ir bjaurumo, teisingumo ir neteisingumo kri­ terijų) srityje, išskyrus spontaniškai privatų vertinimų laisvumą, vadovaukis vertybinio neutralumo, švininio svambalo (jėgos kaip pro­ to), principu. Vertybinio neutralumo principas, pirma, politinį bendrabuvojimą suveržia „dirb­ tinėmis grandinėmis“ / „artificial chains“ (ten pat; 219; 141), antra, klasifikuoja, apskaičiuoja vidurkius, organizuoja, ranguoja ir ringuoja (mažorine „Mūsų trys milijonai“ derme). Tai graži ir pamokanti liberalaus įkvėpimo pabaiga – dirbtinis iškvėpimas. Tautos socialinė organizacija (galėjimo, galimybių, galių sferos) parklupdoma prieš paskirą dalį (savo motyvus ir interesus be paliovos komunalizuojančią jėgą). Z dalies tezės: Dvi pagrindinės – politinės sąmonės (socializacijos, intersubjektyvumo) ir politinio kūno (komunalizacijos, privačios nuosavybės savivaldos) – dalys išsiskiria, ir eina kas sau, kel­ damos daug gardaus juoko (liberaliosios ironijos). „Šizofrenija“, rodos, užkoduota septynioliktojo amžiaus mokslo ir filosofijos Relevancijoje Nr. 1: subjektas atskiriamas nuo savo tikslų ir gyvenimo- planų ir „iškeliamas“ dar prieš tikslus (Sandel 1998; 175). Palikta be priežiūros tikslinė indi­ vido laisvė lengvai virsta laisvumu be moralinės intencijos (Seidman 1992). Šiuo atveju net ir Hobbesas, ko gero, pri­ pažintų, kad „štai šis ilgai tyrinėjamas daiktas“ neturi potencialumo, galėjimo, galios intencio­ naliai judėti, todėl yra prikaustytas prie ligos patalo: „Tačiau, kai judėjimo kliūtis yra paties daikto struktūroje, paprastai nesakome, kad jis neturi laisvės, o sakome, kad jis neturi galios judėti, pavyzdžiui, kai akmuo stūkso vietoje arba žmogus yra prikaustytas prie ligos patalo“ (Hobbes 1999; 217). Antra vertus, Hobbesas pasiūlo individualis­ tinę išlygą, tikėdamasis išjudinti, progresuojant liberalizmui, net akmenį: „Te niekas netrukdo man daryti tai, ką noriu.“ Kūniškojo liberalizmo tėvas, viena vertus, laisvę apibrėžia kaip nevaržo­ mą kūno judėjimą (šiandieną griežtai vartotina tik daugiskaita: kūnų judėjimą), priklausomą nuo tikslingo racionalumo (jėgos-proto): „<...> laisvas žmogus yra tas, kuriam nekas netrukdo daryti tai, ką nori, nes jis savo jėga ir protu gali tai padaryti“ (Hobbes 1999; 217–8). Nebus per daug pakartoti: „Taigi valstybė pagal prigimtį yra pirmesnė už ūkį ar kie­ kvieną iš mūsų, nes visuma neišvengiamai yra pirmesnė už dalį <...>“ (Aristotelis 1997; 67; Pamaloninęs net „nelaisvą akmenį“ Hobbe­ sas laisvą kūną įbruka į tokias „dirbtines grandi­ 174 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija nes“, kad vargu ar nuo šiol turime teisę kalbėti apie subjektų laisvę („Of the Liberty of Subjects“ 1998; 139). Veikiau apie pavaldinių laisvumą (plg. tą patį XXI-ojo sk. lietuvišką pavadinimą: „Apie pavaldinių laisvę“; Hobbes 1999; 217). Tokia laisve, žinoma, mėgaujasi visi liberalesni už kitus subjektai, dirbtinėje grandinėje (visuo­ menėje) vienbalsiai priimdami romų „landų“ įstatymą: „Didžiausia pavaldinių laisvė priklauso nuo įstatymo nutylėjimo“; „The greatest liberty of subjects, dependeth on the silence of the law“ (ten pat; 226; 146). nes“, kad vargu ar nuo šiol turime teisę kalbėti apie subjektų laisvę („Of the Liberty of Subjects“ 1998; 139). Veikiau apie pavaldinių laisvumą (plg. tą patį XXI-ojo sk. lietuvišką pavadinimą: „Apie pavaldinių laisvę“; Hobbes 1999; 217). Z dalies tezės: Dalykas tas, kad pirminė politinė tikrovė drauge yra pirminė socialinė tikrovė. Prie jos 175 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija arčiausiai gali prieiti – atitinkamai – politikos filosofija ir fenomenologiškai grindžiama meto­ dologija. Politinis intencionalumas ir socialinis intencionaluamas – abipusiškai relevantiški. Štai kodėl keičiantis politinei sandarai keičiasi ir socialinė epistemologija. Reliatyvizmas kaip „multikultūralizmas“ randasi po slegiančios Wittgensteino tylos: apie ką negalima kalbė­ ti, apie tai reikia tylėti. Gyvenimo-pasaulyje (pirminėje socialumo ir politiškumo tikrovėje) suveši gausūs paskirų empiriniai faktų kas­ dienybėje, bet ne gyvenimo-pasaulio sąlygų ir prielaidų tyrimai. Dekartiškas filosofinis abejojimo principas įgauna sofistinio natūraliz­ mo – „abejojimo bet kokiu abejojimu“ – formą. Plg. su „private men“ nugvelbtu ir privatizuotu visad abejojančio filosofo fragmentu, itin dažnai kartojamu anglų liberalizmo knygose: „Kam žmogus galvoja? Kokia iš to nauda?“ (Wittgens­ tein 1995; 281 [466]). metodą, orientuojantį į dvi paminėtas sąlygų, arba galimybių analizės, sritis. Fenomenologinis aprašymas ir analizė turi įtraukti relevantiškus – tiek Aristotelio, tiek Platono bei kitų „ikimodernių“ tyrėjų (žr., pavyzdžiui, Schutz 1964; 226–76), tiek šiuo­ laikinių tyrėjų – analizės pavyzdžius. Pirma, atsižvelgti į šiuolaikinius struktūrinius santykio tarp nuomonės ir žinojimo (doxa ir episteme) pokyčius. Antra, dėl šiuo-laikinių pokyčių ne­ aukoti esminės gyvenimo-pasaulio relevancijos ir drauge svarbiausio veiksmo bruožo – vertybių ir dorybių teorijos. Šios teorijos akiratyje, radika­ laus fenomenologinio metodo požiūriu, privalo išlikti intencionalūs jausmų objektai (intentionale Gegenstände des Fühlens). Šie „daiktai“ (laikantis Husserlio nuorodų) tinkamai eksplikuoti Maxo Schelerio, Alfredo Schutzo, taip pat Erico Voegelino bei kitų me­ todologų, tačiau politikos logikos ir socialinių mokslų metodų išstumti į pačias paraštes, tipiš­ kai klausant: „Ar reikia ir kam reikia nagrinėti politinį intencionalumą, intersubjektyvumą, ir apskritai ar verta (čia slypi šiuo-laikinio žmo­ gaus esmės modifikacija: vertė susiaurinama į tik ekonominę vertę) aukoti brangų sėkmės dabartyje laiką? Doxa ir episteme skirtis vėlyvo­ sios sėkmės pasaulyje sunyksta: esminę ne tik žinojimo, bet ir politinės ontologijos skirtį išlygina standartizuojanti ideologija, kurios veiksniau­ sias mokslinis instrumentas yra visuomeninės nuomonės apklausos (lygu – ideologiniai stan­ dartizacijos lūkesčių šaukiniai) ir nekokybiškos (subjektyvistinės) kokybinių tyrimų surenkamos nuomonės. Tai, kas, fenomenologiniu požiūriu, mi­ nėtais tyrimų tik į dabartį orientacijos atvejais išleidžiama iš akių, yra politinio patyrimo reflek­ sija, kuri galima tik aiškiai skiriant 1) esmines pirmojo asmens politinio intencionalumo patyrimo sąlygas; 2) esmines pirmojo asmens ir bendruomenės socialinio ir politinio patyrimo sąlygas, būdingas abipusiškai veiksmo laikysenai. Z dalies tezės: Fenomenologinė žiūra privalo (reikalauda­ ma taikyti radikalų fenomenologinės redukcijos metodą) aiškiai skirti savaime suprantamą po­ litiką (policinės veiklos – priežiūros, kontrolės, administravimo – inžinerijas, viešosios nuomo­ nės apklausas ir kokybiniams tyrimams būdingą atsitiktinį psichologizmą, atspindintį, dažniau, nei rečiau, vienodinančias sutankintos būklės inter-tinkluose ideologemas) ir fenomenologinį Trys būdingos fenomenologijos metodo sudedamosios dalys – 1) konstituacinė, 2) su­ 176 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija prasmės provincijos, bruožus. Svarbu radikaliai suskliausti vyraujantį pozityvistinį, šiandieną vadinamą švelnesniais vardais, socialinių mokslų modelį, kuris etiką ir konkrečiąją ontologiją – esą tarsi-problemas – beveik be liekanų ištirpdo natūralistiniuose tikslingai racionalaus veiksmo eksperimentuose (žr. ypač Schutzo ir Voegelino tarpusavio susirašinėjimą vertybių teorijos klausi­ mais; Wagner, Weiss 2011) ir „formuluojamų, tikrinamų ir kodifikuojamų nesuinteresuotais būdais“ empirinių tyrimų išskleistinėse. pratimo / hermeneutinė, 3) egzistencinė – skatina neapsiriboti savaime suprantamomis duotimis ir vien tik „hobsizmo“ – „sudėjimo“ ir „atė­ mimo“ – metodais. Politinės fenomenologijos pagrindinis objektas yra prie-varta, kuri – vos tik paleidus laisvę iš rankų – bematant virsta prievarta: „Prievartą (violence) tirti nelengva, – kaip pažymi Hannah Arendt, – nors Sorelis jau kadais rašė, kad „Prievartos problemos tebėra nesuskaidrintos“, tačiau lygiai ta pati padėtis – ir dabar“ (Arendt 1969; 35). Vertybes ir dorybes reikia nagrinėti ne kaip tyrėjų iš anksto parengtų klasifikacinių schemų projektus, išraižytus iš anksto duotų pliuraliz­ mo vertybinių skalių, – apdairiai užglaistomų „multikultūralizmo“ kyšiais nuovokiam pro­ tui, – punktyrais, o kaip intencionalius jausmų objektus: „Husserlis atskleidžia, kad esti idealūs žinojimo objektai, kurie yra nepriklausomi nuo pažįstančiojo subjekto lygiai taip pat, kaip ir realūs objektai“ (Schutz 1966; 163). Vargu ar yra svarbesnis šiuo-laikinis idealaus žinojimo objektas, nei laisvės ir lygybės santykis, ir iš šio santykio išsišakojantys kiti relevantiški santy­ kiai, – ypač moralinio veiksmo ir politinio veiksmo tarpusavio sąsajų. Ar kas nors pasikeitė šiandieną, praėjus daugiau nei pusei šimto metų? Liberalus atsakymas yra tas, kad pasaulis keičiasi tik į gerą, vadinasi pasaulis pasikeitė gerokai: tam ir tarnauja optimistiškos „pasiūlų-paklausų kreivės“, „sumavimo-minusavimo lentelės“ ir objektyvūs „pažangaus lenktyniavimo rodi­ kliai“. Fenomenologinis atsakymas yra kitas: „Danielis Bellas atsargiai pasitiki, kad mokslinis ir techninis darbas priklauso nuo „teorinio pažinimo, kuris yra formuluojamas, tikrinamas ir kodifikuojamas nesuinteresuotais būdais“. Galbūt šis optimizmas gali būti pateisinamas iki tol, kol mokslininkai ir technologai išlieka nesuinteresuoti valdžia ir nieko daugiau nesie­ kia, išskyrus prestižą, t.y. kol jie dar nei valdo, nei dominuoja“ (ten pat; 100). Kaip ir nagrinėtu subjektyvumo paradokso atveju, svarbu neišleisti iš akių pirminio visu­ mos ir dalies tarpusavio santykio. Z dalies tezės: Politiškumo paradoksą Aristotelis apibrėžia kaip politinio intencionalumo (viseto lūkesčių) ir socialinio in­ tencionalumo (oikoi, arba aplinkų, kurias sudaro parankiniai įrankiai ir kultūriniai įgūdžiai, gy­ vuojantys gero gyvenimo labui) tarpusavio san­ tykį. Geras žmogaus veiksmo laikysenos sąlygas, arba labas (tikslines) sąlygas, lemia relevantiška politikos prigimties ir žmogaus prigimties dermė Ir galima, ir būtina tirti prie-vartą ir prievartą, vengiant patogių parankinių „prie­ vartos duotybių“ – psichologinių nuomonių ir vidurkių – sampratų. Vengiant kaupiamosios ideologijos (visuomenės nuomonės apklausų vidurkių ir subjektyvistinių kokybinių duo­ menų „pluoštų“), reikia įsižiūrėti ir analizuoti struktūrinius santykiškai natūralių laikysenų gyvenimo-pasaulyje, kurį sudaro skirtingos 177 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija (atitikimas, atliepimas, susietumas reikšmingais viseto ir dalies santykių atžvilgiais). 1975; 13), – žmogus, svarbu aiškiai skirti dviejų galimos analizės elementariųjų pradžių – feno­ menologinės asociacijos ir liberaliosios disociaci­ jos – sampratas. Ne ūkis ir netgi ne socialumas (dalys) yra ankstesni už visetą. Ankstesnė yra esmė, eidos, prigimtis, valstybė. Ankstesnis – politinis in­ tencionalumas: „Taigi valstybė pagal prigimtį yra pirmesnė už ūkį ar kiekvieną iš mūsų, nes visuma neišvengiamai yra pirmesnė už dalį <...>“ (Aristotelis 1997; 67; [1253a 23–26]). Žmogaus prigimties esmė yra politinis inten­ cionalumas drauge su relevantiškais šios esmės, politinio bendrumo, išsišakojimais tiesos, gėrio, grožio, teisingumo, tinkamumo, supratingumo labui: „<...> o žmonės iš visų gyvūnų išsiskiria tuo, kad jie vieninteliai suvokia, kas yra gera ir bloga, teisinga ir neteisinga ir t.t., ir šių dalykų bendrumo pagrindu kuriamas ūkis bei valstybė“ (ten pat; 66; [1253a 19–22]). Šiuo-laikinis žmogus aukštyn kojomis ap­ verčia esmės ir dalies santykį, todėl aristoteliškas, arba tokviliškas, santykis laikytinas relevantišku, o antrasis, hobsiškas, – nerelevantišku. Relevantiš­ kumas yra santykiškas dydis: juo arčiau mažųjų tautų, tuo relevantiškesnis yra laisvė stiprina lygybę, o lygybė – laisvę santykis, o kuo arčiau didžiųjų tautų ir imperijų, tuo relevantiškesnis – lygybė stiprina laisvę, o laisvė lygybę santykis. Me­ džiaginei, o ne tik formaliai, – tariant Edmundo Husserlio žodžiais, – fenomenologinei geometrijai svarbu priešduotys: daiktai / objektai ir nori, ir gali būti suprasti intencionaliai, kaip asociacinės „panašumo į“ dar pasyvioje genezėje figūros. O liberalizmui, pozityvizmui, analitikai, natūraliz­ mui ir kitoms panašioms grynosios-formaliosios geometrijos metodologijoms svarbu jau-duotys, su kuriomis – savaime suprantama – galima įgyvendinti racionalias operacijas. Vadinasi, mes, kaip racionalūs (lygu – kal­ bantys), gyvūnai ir galime, ir privalome eks­ plikuoti politinio socialinio laisvės – skirtumo tarp tas pats ir tas pats – figūras. Z dalies tezės: Valstybė – ne ūkinė ir netgi ne socialinė organizacija, – Aris­ totelis iš arčiau įsižiūri į Lietuvos esmę (ne tik pavadinimą) ir galimas jos modifikacijas, – „<...> suardžius visą [kūną], nebebus nei rankos, nei kojos, nebent išliks tik pavadinimas <...>“ (ten pat; [1253a 26–27]). Taikant čia aptariamą skirtį Tocqueville’io apibrėžtam santykiui tarp laisvės ir lygybės iš­ ryškėja du priešingi – saistantis-fenomenologinis ir taškinis-liberalus (pertraukiantis laisvės ir lygy­ bės tarpusavio santykį) – modeliai. Kai santykis nurodomas netinkamai, „abipusiškumo nebus“ (Aristotelis 1990a; 39 [7a 29–30]). Antrasis, taškinis-liberalus, modelis randasi analitiškai skaidant ir atsiribojant nuo to, ką Alfredas Schutzas vadina abipusiškumo perspektyvų (šįkart tarp laisvės ir lygybės) principu: numanomai supo­ nuojamų idealizacijų, kuriomis remiantis veidu į veidą santykyje steigiami abipusiškai suprantamos aplinkos bruožai (Schutz 1962; 315–29). Intencionali politinė galia / galėjimas randasi ne iš aktualiai administruojančios jėgos ir netgi ne iš galimų teisinių konstitucijos (dalies, o ne viseto) interpretacijų, o iš bendrabuvojimo – po­ litinio veiksmo intencionalumo – laikysenos: bendro gyvenimo gero gyvenimo labui. Dėl šios pirminės, teleologinės, priežasties, kurią arogan­ tiškai atmeta šiuo-laikinis, – Hobbesą, kaip ir Epikūrą, jaudina originalumas (Oakeshott 178 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Taškinis-liberalusis modelis, pabrėžtinai ignoruojantis politinio ir socialinio intencionalu­ mo ir tipizavimo būdus, steigiamus gyvenimo- pasaulyje dar iki teorinių liberalizmo predikatų, lengvai virsta sisteminiu-funkciniu. O tai yra naujojo geometrinio mokslo esmė: išrasdamas ir išpūsdamas vis naujus liberalizmo – ne tik politinio, o ir analitinio – idealus (net iki globa­ laus „jurisdikcinio gėrio“), sisteminis-funkcinis modelis dirbtinę „visuomenės“ abstrakciją su­ skaido į „ekonomikos“, „politikos“, „kultūros“, „socialinę“ posistemes, kurios savo ruožtu tar­ nauja, – samprotavimas yra nieko kito nedarymas, o vien sudėjimas ir atėmimas, – parankinėms liberalizmo reikmėms. nebuvo jokios naudos. <...> Už tai, ką mes turime iš geometrijos, kuri yra visų gamtos mokslų motina, neprivalome būti dėkingi šioms mokykloms.“ (Hobbes 1999; 629–30) Nauda, gaunama iš geometrų mokyklos, yra ta: visas sąvokas, net ir laisvės (!), galima apibrėžti remiantis vien tik išorinėmis veiksmo – kūnų ir jų judėjimo – sąlygomis, kai laisvumas, „tiksliai sakant, yra pasipriešinimo nebuvimas“ (ten pat; 217). Hobbesas yra „modernus“, t.y. iš tikrųjų nutraukia ryšius su politikos filosofija, ku­ riai rūpi politinio intencionalumo sąlygos. (Visi šiuolaikiniai bandymai „pridėti“ prie Hobbeso teorijos dar ir „psichologiją“, yra kilnūs, bet ne­ relevantiški, „modernaus“ Hobbeso motyvo ir „modernių“ instrumentinių interesų atžvilgiais). Šiuolaikiniai liberalai-idealistai visai be reikalo kratosi ankstyvojo liberalaus – vulga­ raus-minimalistinio – T. Hobbeso modelio. Z dalies tezės: Pirminė Hobbeso intuicija – visiškos laisvės nuo prie-tarų (darkart pažymėtina: fenomenologija savitai įsipareigoja prie-tarams, reikalaudama po fenomenologinės redukcijos nepakeisti jų prasmės) – yra liberalizmo esmė: kad socialumas kaip esminės bendruomeninio intencionalumo istorijos atsargos yra tik dirbtinės socialinės konstrukcijos. Hobbesas politinei filosofijai būdingą viseto ir dalies santykį apverčia aukštyn kojomis. Natūralistinėje, abstrakčioje, geometri­ nėje Leviatano formoje tauta drauge su politinio socialinio intencionalumo lūkesčiais, išskaidoma į pavienius taškus. Būtent Hobbeso gamtos mokslų metodas leidžia vis skaidriau apibrėžti liberalizmo hori­ zontą, kuriame Hobbeso intuityviai nepalikta vietos socialumo įstatymams, papročiams. Dėl šios priežasties reikia aiškiai skirti dvi pagal skambesį „tas pačias“ laisvės sąlygas: priežiūrą ir priežiū­ rą. Pirmoji, prie-žiūra, yra politinio socialinio intencionalumo atitikmuo. Antroji, priežiūra, yra kontrolės, viešojo administravimo, atitikmuo. Hobbesas – iš tikrųjų modernus. Žmogus iš principo negalįs pats prižiūrėti savo prigimties, nes sudedamieji savasties elementai yra lengvi it pienės pūkai arba Leukipo ir Demokrito atomai: „<...> nes nieko nėra lengvesnio žmogui, kaip sulaužyti savo žodį“ (ten pat; 144). Štai kodėl teisingumas natūralioje laikysenoje (tradicijoje) esąs labai lengvai pažeidžiamas: niekas čia nesilaiko žodžio ir kaip pašėlę kariauja vienas su kitu. Ir tik „sudarius racionalią sutartį“ – perdavus pirmojo asmens kūno ir sąmonės nuo-savybės teisę – įgaliotiniams (ten pat; 145), Dalis, arba formali sutartis iškeliama į pirmą vietą, o visetas pajungdamas dirbtinei daliai, kurią išranda subjektyvios teoretiko, tam tikru laikotarpiu išgyvenančio pilietinio karo sumaištį, pretenzijos. Sutrikusio žmogaus, bet nesutrikusio teoretiko: „Iš graikų mokyklų 179 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 randasi „tvarka“, „dirbtinės grandinės“, „baimė“ (dirbtinis ne natūralios, o racionalios „baimės“ principas pradeda galioti tik racionalistinėje socialinėje struktūroje: į tai neatsižvelgia nu­ rodantieji, kad baimė Hobbeso teorijoje yra „vidinė“ laisvės sąlyga). Są-monės, arba gebėjimo būti savęs ap­ mąstymo objektu ir drauge sprendinio subjektu, galia perduodama anonimiškam įgaliotojui. O pastarasis – kad rastųsi statiška geometrinio teatro konstitucija  – maskuotę, antveidį, kaukę, išorinę išvaizdą (savęs pateikimą auditorijai) nuo aukštosios scenos „<...> perkelia kiekvienam kalbos ir veiksmų atstovui ir tribunoluose, ir teatruose“ (ten pat; 171). Dramaturginis li­ beralizmas steigia lygybę tarp politikos ir teatro, tiksliau, išranda transformacijos lygtis, formules, kurios įrodo vaidybos politinį tikrumą ne tik vyriausybinėse ataskaitose, bet ypač tiesioginėse apeigose: „Daugelyje krikščioniškų religijų ti­ kintysis dalyvauja apeigose taip, kaip filmo arba TV stebėtojas dalyvauja pateikiamame spekta­ klyje“ (Wolin 2008; 2). Transformacinių lygybių kūrimo ir platinimo – stebėjimo dalyvaujant ir dalyvavimo stebint (kokybinių metodų) – srityse liberalizmo pasiekimai yra išties nepranokstami. Z dalies tezės: Liberaliojo natūralizmo logikoje sangrąži­ nė – įsipareigojimų – dalelytė ilgainiui tampa neveiksminga: įsipareigojimų politiką pakeičia įpareigojimų procedūros. Įsipareigojimus pakeičia įpareigojimai (lygu – baimės aplinkos). Egoistinė, be polinkių pasauliui žmogaus prigimtis rūsčiai prižiūrima Įstatymo, Leviatano: pažeidusi raci­ onalius įpareigojimus, lengva žmogaus prigimtis privalo sulaukti sunkaus atsako. Atsitiktinumą – santykį tarp asmens valios ir šanso – pakeičia būtinumas: tuščia „nepriklausomų individų“, judančių pagal kūnų judėjimo, jų „sudėjimo“ ir „atėmimo“ koordinates, laisvė. „Nepriklausomas individas“ keistame Hobbeso liberalizme neturi jokių „individua­ lių“ refleksijos gebėjimų (neįstengia spręsti ir teigti, kas yra gera, o kas bloga, kas teisinga, o kas neteisinga): „Teisingumas ir neteisingumas nėra nei kūno, nei proto sugebėjimas“, nes <...> tai žmonių, gyvenančių ne vienatvėje, o visuomenėje savybės“ (Hobbes 1999; 140). Klystų tie, kurie manytų, kad Hobbesas gręžiasi į konkrečiosios ontologijos – bendrabūvio ir intencionalumo – sferą. Kadangi jam „visuome­ nė“ – tik dirbtinis kūnas, dirbtinės grandinės, formalioji procedūrinė teisė, tai ir „žmogus, gy­ venantis visuomenėje“, yra ne bendruomeniškai savigrąžus asmuo, turintis nuo-savus „kūno ir proto gebėjimus jausti ir spręsti“, o tik trečiasis asmuo, „netikras asmuo“, perduodantis nuo- savybę „procedūriniam teisingumui“. Išgalvoto, netikro asmens, liberalo, nepriklau­ somo individo tikrovėje nerasime. Visus juos, – atstovą, įgaliotąjį, įgaliojimus (ten pat), – rasime įgaliotinių atstovybėse ir žodžiuose, skirtuose abstrakčiai praeičiai, dabarčiai ir ateičiai (ten pat; 147). Jei politikos filosofijoje politinė žmogaus prigimtis susiejama su teisingumu kaip valstybės pradu (Aristotelis 1997; 66 [1253a]), tai natūralistinių potraukių teorijoje – su bega­ liniu norų dauginimu galiojant netrukdymo, „pasipriešinimo nebuvimo, arba laisvės (sic)“ (Hobbes 1999; 217–8) principui. Drauge su negatyviosios laisvės samprata ran­ dasi negatyviosios moralės samprata. Šios sampra­ tos ne tik kad paremia viena kitą, bet ir sudaro vienatį, arba vienatinį: „liberality“ (Hobbes 1998; 58). Šioji vienatis yra „išvirkščia“ (kaip ir 180 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija 76). Žmogaus racionalumas yra tiesiog teisingų žodžių vartojimas (use), o teisingai sudedamos sumos, kurios tikrinamos platesnės apimties panašių operacijų grandinėse, sudaro teisingumą (Hobbes 1999; 62) negatyvioji laisvė yra „išvirkščia“), – apgaulingai priešinga tam, ką nurodo šis daugiareikšmis – nuo tolerantiškumo iki dosnumo – žodis. Libe­ rality moralė kaip amžina šiuolaikinio žmogaus „moralė“ natūraliai įgyvendina du pagrindinius dalykus. Moralė „liberality“ – ne stebėtinai, o jau na­ tūraliai – yra šiuolaikinio racionalumo sampratos ir atitinkamos elgsenos institucija, susidedanti iš dviejų pagrindinių dalių: 1) natūralių apetitų skatinimo racionalaus pasirinkimo – priemonių- priemonių – veikose; 2) nuomonių apie galingą valdžią kaupimo (sudedamosios socialinio proto, arba teisingumo, sampratos). Z dalies tezės: Darkart: suside­ danti iš „apetitų“ ir „socialinio proto“, arba „visuomenės nuomonės apklausų“ (nuomonių apie galingą valdžią bei kitų laisvųjų „apskritų­ jų stalų“ diskusijų). Tai amžinoji šiuolaikinio žmogaus liberality: „Hobbeso politinė filosofija grindžiama ne amoralios moralės iliuzija, bet naująja morale, arba, turint galvoje Hobbeso ketinimą, naujais vienos amžinos moralės pa­ grindais“ (Strauss 1963; 15). Pirma, postuluoja, veikiau pripažįsta, kad savi-sauga yra pagrindinis judėjimo – nusigrę­ žiant nuo mirties (blogio) ir atsigręžiant į gyvybę (gėrį) – šaltinis, jusliškai susijęs su nekalto prigim­ tinio ydingumo paskatomis, tarp jų – ypač trimis apetitais (appetite): pride (arogancija), ambition (savimana), vanity (tuštybe). Antra, nors liberality moralės atomas, judantis žmogaus kūnas, iš pri­ gimties (ne natūralioje laikysenoje, bet liberality teorijoje) yra blogas, šią „tiesą“ gali ilgainiui gali sušvelninti arba netgi visai paslėpti natūralius blogio instinktus ir skatinantis, ir varžantis „visų arogancijos vaikų karalius“ („king of all the children of pride“), patsai „arogancijos karalius“ („King of the Proud“; Hobbes 1998; 212). Leo Straussas atkreipia mūsų dėmesį į es­ minį Leviatano kaip naujosios amžinos moralės tarpininko kilmės aspektą: ne galinga valdžia pati savaime, bet „galinga valdžia, kuri slopina aroganciją“ (Strauss 1963; 13). Kitaip sakant, ne galinga valdžia ir ne racionalistinės kilmės baimė yra pagrindiniai sudedamieji Hobbeso natūraliųjų potraukių teorijos komponentai, o šiuo-laikiškos moralės koncepcija, kurios pagrindinis argumentas yra tas, kad žmogaus nekalto prigimtinio ydingumo paskatas ilgainiui gali apmalšinti tik galinga valdžia. Žmogaus protas ir vaizduotė šioje natūralizmo „moralėje“ redukuojami į aritmetines operacijas: prigimties įstatymai yra tik tam tikros išvados, kurias daro protas sudėdamas ir atimdamas (Hobbes 1983; „Pagarba – tai vien nuomonė apie galią“ [„for honour consisteth only in the opinion of power“] (Hobbes 1999; 108; Hobbes 1998; 62). Verta pratęsti šį neįstabų apibrėžimą, labiau nesvetimą nei tolimą šioms dienoms: „Todėl ir senovės pagonys („the ancient heathen“; tiksliau būtų „pirmykščiai stabmeldžiai“; Hobbes 1998; 62) manė, kad jie neniekina, o, priešingai, kad didžiai šlovina dievus, vaizduodami juos savo poemose prievartaujančius, vagiančius ir atliekančius kitus didžius, bet neteisingus ar nešvarius veiksmus <...>“ (Hobbes 1999; 108). Natūralios paskatos, norai, troškimai gali būti arba tinkami, geri, leistini, arba netinkami, blogi, neleistini. Hobbesas, kaip ir bet kuris 181 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija šiuolaikinis liberalas, šiai „tradicinei“ skirčiai abejingas, indiferentiškas, vertybiškai neutralus. Svarbu, Hobbeso požiūriu, tik nuomonių apie galią kaupimas (svarbu visuomenės nuomonės apklausos, grindžiamos abejingumo, arba ver­ tybinio neutralumo, principu): pagarbumo nekeičia tai, ar tas veiksmas kilnus, teisingas, neteisingas, nes pagarba – tai vien nuomonė apie galią... (ten pat). Z dalies tezės: „Nuomonių apie galią aritmetinis kaupimas“ yra vienintelė teisinga partinė („visi mes – liberalai“) linija: „Linija man įteigia, kaip turiu eiti (Wittgenstein 1995; 221 [222]). moralinius, tuo labiau išryškėtų atžvilgių nely­ gumas. O juo labiau ryškėtų atžvilgių nelygu­ mas, tuo labiau aiškėtų, kad trūksta pamatinio politinio gyvenimo santykio: tarp, viena vertus, subjektyvumo, racionalumo ir relevantiškumo, kita vertus, laisvės. Tuomet išaiškėtų esminė libe­ ralizmo, postuluojančio pirmykštį stabmeldišką ryšį tarp baimės ir laisvės, yda. Liberalizmas iš principo negali apibrėžti ryšio tarp ko nors ir laisvės, nes, pirma, visiškai nuskurdina raciona­ lumo sampratą, atimdamas iš jos jai artimiausias subjektyvumo ir relevantiškumo dimensijas, antra, nuskurdina asmens socialinio politinio veiksmo laikysenos sampratą, atimdamas iš jos intencionalumo ir laikiškumo dimensijas. Liberalizmas, yra vienintelė politikos filosofija, kuri geba normatyvumą, esminį po­ litikos teorijos dėmenį, prarasti atgaudama tik normatyvumo regimybę grynajame natūralizme. Vulgariojo liberalizmo (bet kokių liberalizmo rū­ šių esmės) žavesys ir skurdas yra tas, kad žmogaus instinktus, poreikius ir potroškius jis paverčia „idealais“: 1) laimė yra ne kitame, o šiame gy­ venime (Hobbes 1999; 81), 2) gyvenimas – tai tik judėjimas tarp norų ir baimės (ten pat), t.y. malonumų vaikymasis ir skausmo vengimas. Laisvė negalima be trinario reflektyvaus – racionalaus, subjektyvaus ir intersubjektyvaus (relevantiško) – savigrąžos akto, o tai, ką su­ griebiame reflektyviu aktu, niekada nėra mūsų minčių srauto dabartis, kaip nėra ir tikėtina dabartis; tai visada yra praeitis“ (Schutz 1962; 172–3). Liberalizmas negali priartėti prie savo savasties akto („praeities“), o eina tik prie išorinio nepriklausomybės nuo kito akto (kaip negatyvaus santykio su kitu dabartyje įgyvendinimo). Li­ beralioje visuomenėje laisvė apibrėžiama vien tik remiantis individo nepriklausomybės nuo kitų, – ar tai būtų vyriausybės, korporacijos, ar privatūs asmenys, – galimo trukdymo (interfe­ rence) terminais (Taylor 1979; 175). Geometrinė logika lygybės ir laisvės idealus, o ką jau kalbėti apie konkretų jų tarpusavio san­ tykį, paverčia statistiniais kintamaisiais dydžiais. Tai paprasta, nes gyvenimas kaip judėjimas susi­ deda iš a) norų, b) baimės ir 3) pojūčių, akstinų (ten pat). „Viską sudėjus“ (Hobbes 1999; 135), žmonės, kūno ir proto gebėjimų atžvilgiais, yra „labiau lygūs, negu nelygūs“ (ten pat; 136). Šioje statistiškai patikimų sąlygų lygybėje galioja tie­ siai proporcingas ryšys tarp baimės ir laisvės (žr. sk. „Baimė ir laisvė yra suderinamos“; ten pat; 218). Juo daugiau į šią primityvią skaičiuotę Liberalizmas renkasi ne laisvę (nepainioti su liberalizmo pavadinimu), – moralinis indi­ ferentiškumas (gręžimasis nuo tradicijos) gali rastis tik abstrahuojantis nuo gyvenimo-pasaulio tikrovės. Z dalies tezės: Laikydamasis ne socialinio politinio veiksmo, o abstrakcijos, indiferentiškumo, ver­ tybinio neutralumo nuostatos, liberalizmas Juo daugiau į šią primityvią skaičiuotę įtrauktume santykių ir atžvilgių, įskaitant ypač 182 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija savo taisykles platesnei, veikiau saistančiai, racionalumo, subjektyvumo ir relevantiškumo sampratai, apimančiai racionalumo formų var­ tojimo įvairovę natūralioje laikysenoje, kurioje sveika nuovoka, supratingumas ir racionalumas nėra aiškiai skiriami dalykai (Schutz 1962; 27). gręžiasi į lygybę (geometrinių abstrakcijų šaltinį), reikalaujančią turėti lygų, vienodai atvirą ir nie­ kieno nepažeidžiamą šansą ko nors norėti arba šansą ką nors daryti, galėti norėti arba galėti daryti, arba galėti nedaryti, – tik, žinoma, ne negalėti nedaryti (tai principinis liberalių teisių pažeidimas, primetant įsipareigojimo instituciją, įsitikinimų etiką). Analitinėse liberalizmo 2 2 2 2... schemose moralė yra šanso – ko nors norėti arba ko nors nenorėti, ką nors daryti arba galėti nedaryti... – privilegija. Vienas dalykas yra teoretiko įsitikinimas – „Įsitikinimas (belief) yra teorinio racionalumo pagrindas“ (Audi 2004; 17) – racionalumo apibrėžimu ir universaliu šio švento arba seku­ liaraus dalyko pritaikomumu (kai įsitikinimas paverčiamas įtikinimu). O kitas dalykas yra teo­ retiko įsitikinimo ištakos: problema, nuo kurios nusigręžia liberalus teoretikas. Kad racionalumo konstituacijoje aš dalyvauju ne vienas (kaip nepriklausomas protas), – o dalyvauju drauge su savo biografine aplinka. Liberali racionalumo samprata – „Netrukdyk savaime suprantamam mano veiksmo raciona­ lumui, mano šansui!“ – yra ir prieštaringa, ir infliacinė. Skirdamas „socialinę kooperaciją“ ir „tik socialiai koordinuojamą veiklą“, ku­ rią koordinuoja centrinės valdžios įsakai, J. Rawlsas nurodo pirmą bendrąjį racionalumo kriterijų: 1) socialinė kooperacija galima dėl viešai pripažintų taisyklių ir procedūrų, kurios pačių kooperuojančiųjų laikomos tinkamomis reguliuoti jų elgseną. Antrasis racionalumo bruožas – 2) nešališko sąžiningumo kooperaci­ joje idėja (the idea of fair terms of cooperation): kad kiekvienas dalyvis supratingai (reasonable) laikytųsi apibusiškumo taisyklės. Trečiasis raci­ onalumo bruožas – 3) racionalaus naudingumo (rational advantage), arba gerovės (good), idėja: „Racionalaus naudingumo idėja sukonkretina tai, ko siekia dalyvaujantieji kooperacijoje jų pačių gerovės atžvilgiu“ (Rawls 2001; 6). Teorinio racionalumo, vaizduojančio pa- saulį, įskaitant vidinį mūsų patyrimo pasaulį, pagrindas, yra mūsų įsitikinimų sistema. Įsitiki­ nimai, tinkamai pagrįsti, konstituoja žinojimą, kuris tautologiškai laikomas (įprastai sulaikant bet kokią abejonę natūraliu savo žinojimu, nes abejonė reikalautų radikaliai suskliausti natūralų žinojimą) teorinio proto siekiamybe. Maždaug: mokslo tikslas yra sukurti gerą teoriją. Problema, kurios Rawlsas nepastebi arba nenori pastebėti, yra teoretiko laisvai sau priskiriama privilegija ne tik apibrėžti, bet ir taikyti racionalumo api­ brėžimą. Z dalies tezės: Kad ir kaip, loginiu požiūriu, tobulai būtų apibrėžta racionalumo samprata, ji apima tik siauresnį, nei daugybė kitų įvairių raciona­ lumo gyvenimo-pasaulyje sampratų, – politikos teorijos lygmenį. Darant aiškią arba numanomą prielaidą, būdingą racionalizmo teorijoms (kad mokslas yra racionalus pasiekimas par excellence; Parsons 1949; 58), viešai pripažintos taisyklės Racionalumas, supratingumas, naudingu­ mas, tinkamumas, sveika nuovoka randa savo vietą vieningoje teorinėje racionalumo sistemoje. Tačiau racionalumas yra gerokai platesnė ir įvairesnė samprata už tik teorinį racionalumą. Siauresnė racionalumo samprata čia diktuoja 183 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ir procedūros (o visų pirma politikos ir teisės sferų racionalumas) sutapatinamos su „visos visuomenės protu“. atsisieja nuo afektinių arba tradicinių priemonių (Schutz 1962; 28). Darydami neįtikėtiną prielaidą, – kad galbūt Johno Rawlso racionalumo samprata, tobulai jungdama racionalų naudingumą, supratingu­ mą, sveiką nuovoką, apibusiškumo taisykles, viešąją pagarbą (kurią savo ruožtu reikia skirti nuo šių sferų darbuotojams tradiciškai nesveti­ mos „viešosios arogancijos“) daugiau ar mažau atitinka politikos ir teisės sferų, nuo kurių ir pradeda savo kelią liberalizmo teorija, praktinę laikyseną, turėtume remtis dar labiau neįtikėtina prielaida, kad poistorijoje, civilizacijoje, kurioje tobulai viešpatauja pliuralizmas, mes visi liauja­ mės būti pagarbiais, arogantiškais ar dar „kokiais nors“ dėl paprasčiausios priežasties: grynasis pliuralizmas yra grynasis liberalizmas, kuris ne­ pažįsta ir nepripažįsta ne tik jokių „tradicinių“ socialinių politinių intencionalumo sąsajų, bet ir „tradicinių“ socialinių psichologinių fenomenų. Pliuralizmas yra indiferentiškas, o liberalizmas yra vertybiškai neutralus. O tai yra viena ir tas pat. Ir vienas, ir kitas – moraliai indiferentiški ir racionaliai abstraktūs kaip biurokratinės taisyklės (o tai yra viena ir tas pat). Racionalumo tyrimai visų pirma reikalauja užklausti, ką mes turime galvoje, kai kalbame apie skirtingus socialinio pasaulio stebėjimo lygmenis: tik daugiau ar mažiau išsiaiškinę, ką racionalumas reiškia konkrečių prasminių kas­ dienybės provincijų gyventojams, mes galime klausti, ar mūsų postuluojamos kategorijos – viešai pripažintos taisyklės ir procedūros – su-tam­ pa (Rawlso vartojamas terminas: overlapping) su tomis racionalumo formuluotėmis, kurias įgyvendina socialinio pasaulio gyventojai. Gy­ venimo-pasaulio racionalumo struktūros, – pasitelkiant siauresnį biurokratinį politikos ir teisės sferų racionalumo supratimą, liberaliu paliepimu su-tampa su socialiniu protu, viešai pripažintomis taisyklėmis ir procedūromis. Formaliai konstruojama ir apibusiškumo taisyklė, – visiškai nepaisant teorinio racionalumo ir praktinio racionalumo tarpusavio santykio problemos: kad asmens žinojimo atsargos (racio­ nalumo, kylančio iš įsitikinimų) būtinai skiriasi nuo stebėtojo žinojimo atsargų (susisteminto racionalumo, kylančio iš įsitikinimų). Rawlsas nepaaiškina savo minėtų pirmojo, antrojo ir trečiojo lygmenų tarpusavio sąsajų. Z dalies tezės: Trečiojo lygmens, racionalaus naudingumo idėja, teikia nuorodą į racionalų veiksmą, kuris visada turi aiškų ir ryškų tikslų, priemonių ir numanomų rezultatų supratimą. Kalbant griežtai, „racio­ nalus veiksmas“ ir kiti „labiau tradiciški“ (kad ir abipusiškai supratingi) veiksmai tarp savęs sunkiai suderinami arba visiškai nesuderinami: aiškus ir ryškus tikslų, priemonių ir numanomų rezultatų, alternatyvių priemonių konstravimas Nebus per daug pakartoti, kad liberaliz­ mas nustato indiferentišką ryšį (abstrakcijos santykį) ne tarp ko nors (baimės, malonumo, šiurpo, laimės...) ir laisvės, o tarp laisvumo ir nepriklausomumo nuo bet ko nors. Iš laisvės, kaip ir iš racionalumo iš karto atimamos ją konstituojančios subjektyvumo ir relevantiškumo dimensijos. Veiksmo racionalumas (priklauso­ mumas laisvumui ir nepriklausomumas nuo ko nors) suporuojamas su poelgio verte (vartojant: sudedant ir atimant apetitus ir norus, atimant aversijas ir nenorus). Pasirenkant arba nepasi­ 184 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija nia-skaidos – „spėliojimų apie įvykių tėkmę“ (Wittgenstein 2005; 130 [23]) – priemonėmis natūralizuodamos praeities baimę, – „kad kitaip bus tik blogiau“. Tai konstitucinė, apibrėžta natūraliai susiklosčiusių spontaniškų kūnų ju­ dėjimo sąlygų ir aplinkybių, liberalizmo tvarka. Ji būtinai reikalauja, kad visi jos piliečiai, var­ tojant Pirmtako terminus, vienodai džiaugtųsi FELICITY (Hobbes 1998), – galiojant proce­ dūrinėmis apdairumo konkrečiose situacijose teisėms, teikiančioms galimybę tamsoje vorele judantiems kūnams nepakliūti į bėdą, teisingumo (teismų) akiratį. renkant tam tikrą (relevantišką vienu atžvilgiu, bet nerelevantišką kitu atžvilgiu) santykį su išoriniais daiktais ir procedūriškai apskaičiuo­ jant tikėtiną konkrečios relevancijos vertę. Jei savigrąžos vertybė ir dorybė skaidrėja sąmonės vidinio horizonto lauke, tai į išorę nukreipto veiksmo  – pačiame veiksmo įgyvendinime. Racionalumui sutampant su verte (skirti nuo vertybių). „Filosofinių radikalų“ (Schutz 1964; 254) racionalumo, subjektyvumo, relevantišku­ mo, laisvės sampratos konstituojamos ne pagal santykį su pilna žmogaus asmenybe, bet pagal santykį su „gerove“ ir „malonumu“ (ten pat). Jei politikos filosofijoje politinio intenciona­ lumo pradas yra žmogaus ir valstybės pri-gimčių derinimas (teisingumas), – „<...> nes teismas, kuris sprendžia, kas teisinga, yra piliečių ben­ drijos nustatyta tvarka“ (Aristotelis 1997; 67; [47–49], – tai natūralių potroškių teorijoje teisingumas yra procedūrinis neteisingumo padarinių procedūrinis apdorojimas plečiantis ne­ teisingumo mastams visuomenėje ir perskirstant vaidmenis, padėtis, funkcijas pagal augančias profesinio darbo apimtis. Jei pirmuoju atveju analizės akiratyje yra ap-linka, tai antruoju – ap-imtis. Pirmuoju atveju gręžiamasi link ko nors, antruoju atveju gręžiamasi nuo ko nors. Pirmuoju atveju gręžiamasi žmonių aplinkos link, o antruoju – natūraliosios aplinkos (gam­ tinės ir socialinės geometrinės aplinkos vienetų visumos) link. Baimė neįkliūti lygiųjų apeigose yra didelė. Z dalies tezės: Todėl visiems lygiai prieinamos transformacijos lygtys užtikrina liberaliosios lygybės sąlygas: visiems vienodų įstatymų apeigų. Nepaklusti apeigų įstatymams – lygu neapdairiai įkliūti esant lygioms priėjimo prie neribotų apeigų išteklių sąlygoms; t.y. atsitiktinai suklysti lygia greta artėjant prie visiems atvirų įstatymų landų. Apeigoms paklūstantiems – nerašyta teisingumo išlyga: „Šįkart nemenkai nusidėjai, brolau mano, – triskart sukalbėk „Nuolatinė sėkmė mūsų“. Gerokai didesnė bausmė skiriama egzistuojantiems nuodėmėje (be jokių laisvumo ir lygybės idealų): jiems teisingumas – lygu nuoširdi politiko šypsena, kylantis ekonomikos eksperto nykštys ir viltis senatvėje. Tinkamo bausmių paskirstymo visuome­ nėje – tinkamas rezultatas: pirma, tikintieji tampa apdairesni; antra, netikintieji praranda laisvės egzistencinės vertės (jos autonomiškumo savigrąžoje) skonį ir tampa tik nepasitikinčiais savo jėgomis: „Nuolatinė neviltis yra vadinama NEPASITIKĖJIMU savo jėgomis“; Hobbes 1999; 73). O tai ir reikėjo įrodyti: netikintys Gero apetito sąlygos liberalioje visuomenėje progresuoja ne pertraukiamai, o tolygiai. Draus­ damos varžyti kylančią ekonomikos eksperto nykščio kreivę. Drausdamos nevartoti: netenkinti savo alkio ir malšinti troškulio dabar, rytoj net daugiau nei vakar, bet mažiau nei poryt... Ži­ 185 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija liberalizmo negatyvioji esmė neišnyktų kaip dūmas jam praradus būdingus vidinius prieš­ taravimus? Tai, ką Leo Straussas tiksliu api­ brėžimu sugriebia Hobbeso teorijoje – ir norą samprotauti griežtai moksliškai, ir konceptualių prieštaravimų apėjimą (tikėtino galvos skausmo vengimą) – taikytina visai liberalizmo tradicijai: viena vertus, aiškumas, griežtumas ir mąstymo apibrėžtumas, kita vertus, aibė prieštaravimų (Strauss 1963; x). Jei tarsime, kad Hobbeso ir liberalų įsitikinimai, kurių pagrindas yra negatyvusis „anti-bendruomeninis anti-inten­ cionalumas“, sutampa, matysime, kad tradicinės politikos filosofijos pastangas tipiškas liberalas tipiškose aplinkybėse laiko „klaida“. Įstatymo, arba bendrojo gėrio (dorybės), koncepciją Hobbesas drastiškai nuvertina iki tik „žodžių ir popieriaus“ ir pavadina Aristotelio Politikos „klaida“ (Hobbes 1999; 643), pridurdamas, kad „Vakarų šalys“ (sic) per brangiai sumoka už senovės graikų ir lotynų kalbų mokymąsi“ (Hobbes 1999; 223). vis labiau nepasitiki savo jėgomis, todėl šlie­ jasi ne prie nesaugios laisvės, o prie saugios lygybės. Šliejasi ne prie laisvę konstituojančio gyvenimo, o prie oficialiosios politikos ir teisės a priori, konstitucinio egalitarinio liberalizmo mito: „<...> centrinio mito, kuriuo remiantis pagrindžiamas socialinės grupės savęs pačios supratimas“ (Schutz 1964; 262). Visų karas su visais baigiasi, o Žemėje, ypač centrinėje, geranoriškai – „norėdama gero kitam“ (Hobbes 1999; 73) – įsivyrauja amžina taika, liberalizmo teisinė procedūrinė politolo­ gijos paradigma: dominuoja „jurisprudentinė paradigma, bent jau JAV“ (Gray 1993; 238). Tai dėsninga progresuojančio žmogaus savi-saugos noro pabaiga. Z dalies tezės: Samprotavimo tikslas, kaip teigia­ ma septintame Leviatano skyriuje, yra galutinis sprendimas, o jį pasiekti galima pirmojo asmens įgaliojimus perdavus kam nors kitam. Pirmojo asmens įgaliojimus perdavus kam nors kitam, liberalizmo negatyvioji laisvė tam­ pa dar negatyvesne. Liberalizmas priverstas imtis, – liberalioje Žemėje viešpataujant ne liberalizmui, o kam nors kitam, savirefleksijos, kuri būtų bergždžia, jei ne kitų, pozityviųjų, politinės filosofijos tradicijų intencionalios sąvokos ir idėjos. Pozityvių politinės filosofijos tradicijų priemonėmis liberalizmas ima glaistyti savo vidinius kraštutinai negatyvios koncepcijos prieštaravimus: „Mano argumentas yra tas: kadangi liberalizmas turi polinkį į nestabilumą ir disociaciją, jis reikalauja nuolatinių bendruo­ meninio intencionalumo (communitarian) korekcijų“ (žr.: Walzer 1990). Tai nemenka dalimi randasi iš paviršinės Hobbeso filosofinės antropologijos: būdingo empirizmui natūralizmo („dirbtinis asmuo“, „dirbtinė visuomenė“ – tik racionalistinės konstrukcijos, grindžiamos „sudėjimu ir atė­ mimu“, arba natūralistiniu empiriniu tyrimu) ir būdingo sveikam protavimui materializmo. Natūralūs asmenys, savo žodžių ir savo veiksmų nuo-savybės šeimininkai, vienišame Hobbeso sakinyje („Jei žmogaus žodžiai ar veiksmai laikomi jo paties, tada jis vadinamas natūraliu asmeniu“; Hobbes 1999; 170), pasirodo tik kaip racionaliai samprotaujančio teoretiko priemo­ nės, kurios tuoj pat užmirštamos išradus vieną ar kitą parankinę sąvoką. Tačiau ar liberalizmas, pirma, nori nesiprie­ šinti (sic: juk tai liberalizmo esmė!) tradicinės politinės filosofijos korekcijoms ir, antra, ar 186 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Hobbeso natūralizme randasi netgi lygybės ženklas tarp dviejų autonomiškų savasčių: „Kai kurių netikrų asmenų žodžiai ir veiksmai pri­ pažįstami kaip savi tų, kuriems jie atstovauja“ (ten pat; 171; išskirta autoriaus). Kitaip sakant, įgaliotiniai pasisavina, panaudodami „savą“ lygybės ženklą („Argi šie asmenys nėra mūsų analogijos, mūsų nuosavybės?“), pavaldinių savastis, net nekeldami klausimo apie galimus iš prigimties nelygių savasčių skirtumus (plg. su panašiais J. Rawlso arba T. Parsonso racionalis­ tiniais „lygybių“ ženklais). mūsų pačių nuomonė (visuomenės nuomonės apklausų kaupiama baimė) apie beribę jų galią. Žmogus, praradęs save kaip sąmonę, atranda save kaip kūną dirbtinėje, vis ilgėjančioje gran­ dinėje: žmogus visų pirma nori vis didesnės valdžios (ten pat; 113). Žmogus, gręždamasis į išorėn-relevanciją (iš vieno noro – į kitą), nuo pradžios susvetimėja ir neišvengiamai užbaigia savo kelią iš savi-saugos į ap-saugą: „Laimė yra nuolatinis noro judėji­ mas nuo vieno objekto prie kito; ir pirmesnio objekto pasiekimas yra tik žingsnis prie antrojo“ (ten pat; 112). Šių dienų progreso, hobsizmo, su­ pratimas ir jo įgyvendinimas – vienas ir tas pat: judėjimas-priemonių-priemonių-ne-kryptimi. Progresas – lygu natūrmaterializmas: nuolatinė sėkmė įgyjant tai, ko žmogus vis ir vis (from time to time) nori. Z dalies tezės: Grandininėje Hobbeso teorijoje išlaisvinto žmogaus norai pašėlsta iki tokio aukšto lygmens (liberalizmas ribų neturi: VISI TAI ŽINO!), kad galiausiai žmogus panorsta norėti nenorėti savo norų. Nors širdies gilumoje jis tebenori savo norų, kuriuos iš jo racionaliai, tinkamai paakintas filosofo, nugvelbia jis pats (ko laukti, kai niekas nebesipriešina!), žmogų vis tiek kaus­ to baimė. Juk atsigręžęs į varganą savo aplinką, kiaurai perpūstą iracionalių „visų karo su visais“ vėjų, – tarp likusių kelių epušių, drebulių ir kitų liaunų medelių jis gali pamatyti tik silpną (dėl mažų norų, o didelių baimių) vaikiščią, prara­ dusį apetitą ir tirtantį iš siaubo, – įrėmusį akis į savo Įgaliotinio, kaip du vandens lašai panašaus į jį patį, akis. Svarbūs tam tikri žodžiai, akcentai, Hobbeso žymimi didžiosiomis raidėmis. Ne paprastas malonumas, o tęstinė PA-LAIMA, FELICI­ TY (Hobbes 1998; 41; lietuviškame vertime didžiosios raidės sumažėja: felicity; Hobbes 1999; 81), – tokia yra racionalistinė Hobbeso išvada. Nors tai panašu į šių dienų įgaliotinių hedonizmą, tačiau nereikia apsigauti. Toli gražu ne hedonizmas rūpi rūsčiajam liberalui, kaip interpretuoja Michaelas Oakeshottas ir Johnas Gray’us, o „perėjimo tobulėjimas“ (a transitory perfection), neturintis pabaigos (Oakeshott 1975; 73; Gray 1993; 5). Štai kodėl žmogus, paakintas filosofo, nu­ taria perduoti nuo-savybės galias, savo politinį socialinį intencionalumą, savo įgaliotiniams. Savi-sauga virsta ap-sauga. Nors savi įgaliotieji turi menkučių ydų, didesnių už didelius norų, bet užtat kaip klasė neturi jokios baimės: „Nuo­ latinėje sėkmėje įgydami tai, ko žmogus vis nori <...>“ (ten pat; 81). Tai ir reikėjo įrodyti. Mūsų rodoma jiems pagarba – klasinės kilmės: „Viskas“ turi pradžią ir pabaigą, ir liberaliz­ mas turi pradžią ir pabaigą – mirusią poistoriją be ideologijų ir be skirtingų gero bendro gyvenimo koncepcijų. Nusigręždamas nuo „tuščių“ senųjų filosofų, kurie norį tik idėjų, ir pašiepdamas jų mokymus kaip tik „patenkintos sielos ramybės“ 187 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija (Hobbes 1999; 112) paieškas, Hobbesas Aris­ totelio teleologiją traktuoja kaip statišką, t.y. tik aktualią (be galimybės modusų išsklaidos!) doktriną, – o pats tuo pačiu metu nesnaudžia konstruodamas dirbtinę ir statišką erdvę, kurioje „<...> žmonės valingais veiksmais ir polinkiais siekia ne tik [ką nors] įgyti, bet ir užsitikrinti patenkinamą bei sotų gyvenimą <...>“ (ten pat; 113). prilyginama formalių perdavimo teisių sričiai. Pirmojo asmens galėjimas suvaržomas iki bran­ dos (sic) sulaukusių asmenų balsavimo teisių veikimo balsavimo apylinkėse aktų ir perduoda­ mas atstovams-vykdytojams: „išgalvotiems, arba netikriems asmenims“, kalbant grėsmingiau, „jurisdikcinei paradigmai“. Z dalies tezės: Pirmojo asmens nuo-savybės galia (pradžioje „perduodama“, o paskui „atimama“ ir „sumuojama“) tampa tiesiogine įgaliotųjų atstovų, visad su privalo­ mais „sceniniais antveidžiais“, nuosavybe. O kai nuo-savybė (są-monės galėjimas ir gebėjimas gręžtis į pačią save) priklauso ne sau pačiai, ji virsta nuosavybe kam nors kitam. Hobbeso individualizmas yra pernelyg stiprus, kad leistų bent trumpam pasirodyti bendrajai valiai“ (Oakeshott 1975; 66), – rašo Michaelas Oakeshottas. Manytume, kad šią tezę reikėtų skaidyti į dvi dalis. Nors Hobbesas yra tipiškas individualistas, tačiau Hobbeso dirbtinėje orbitoje pasirodo ne ben­ droji Rousseau valia (volonté générale), kuri dar turi palaukti, kol išmuš jos laikas, o „dirbtinės grandinės“. Šis „visuomenės“ surogatas, kitaip nei teigia Oakeshottas (ten pat), ne išsaugo, o sugriauna manyje mano kaip asmens individu­ alumą, – perduoda mano nuo-savybę įgaliotinių nuosavybei ( „savų“ įgaliotinų, – nors „nežinia kodėl?“). Ko nors sąmonės virsmą kam nors sąmone verta (jei vertė mums yra daugiau, nei tik eko­ nominė apetito arba administracinių potvarkių vertė) palyginti su Aristotelio brėžiama skirtimi tarp laisvo žmogaus ir savo galėjimą sterilizavusio vergo: „<...> kas, būdamas žmogus, priklauso ne sau pačiam, bet kitam, tas iš prigimties yra vergas <...>“ (Aristotelis 1997; 69 [1254a 16–17]). Sau pačiam nepriklausančiam žmogui būtinai reikia dirbtinių grandinių (įgaliotųjų visuomenės). Štai kodėl įgaliotinių filosofijoje dalies (grandinių) dominavimas viseto atžvilgiu yra būtinas, o ne tikimybinis. Labai trumpai pakartotina. Pirma. Žmo­ gaus motyvai, interesai, norai susiaurinami iki noro perduoti savo sprendinius (aš-teiginius!) ĮGALIOTINIAMS (policinėms instituci­ joms, tarp kurių neabejotinai draugiškiausia, arčiausia kasdienio gyvenimo, policija). Antra. Laisvai, be jokių išorinių kliūčių, kurios gali trukdyti žmogui visomis jėgomis daryti tai, ko jis norėtų (Hobbes 1999; 141), pertvarkoma transformacijos – iš politinės bendruomenės į policinę visuomenę (dirbtines grandines) – lygtis. Filosofija virsta jurisprudencija. Asmens galių (politinio ir socialinio intencionalumo) sritis Paverskime (tik akimirką, niekam nematant, tik mums girdint) Ludwigą Wittgensteiną šiuo- laikiniu lietuviu: „Ar mes neturime kiekviename žingsnyje sakyti „Aš tvirtai (mit Bestimmtheit) tuo tikiu“?“ (Wittgenstein 2009; 68 [242]) Jei tik ir kai tik du trys žmonės įgaliotųjų grandinė­ je „sukerta rankomis (kol dar kiti nespėjo)“, tuoj pat pasigirsta neįgalus – visad pritariantis mano- kaip-įgaliotinio – „Taip, taip taip...!“ murmesys. Neįgalus, taigi beprasmiškas. Kad jį kur galas! 188 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Ne atrasti, o išrasti vis naują skirtį: tam, kad tęstųsi atominių taškų – liberalios pažangos begalybėje – linija-iliuzija. Atsiskyrimas nuo konkrečios vietos yra ypatingas brandaus libera­ lizmo atsiskyrimas. Z dalies tezės: Pirmojo asmens savastis, kuri, visuotinai sukirtus rankomis, laikoma tik „socia­ line konstrukcija“, tik „iliuzija“, atplėšiama nuo šaknų, nuo žemės tiesiogine šio žodžio prasme. Subjektyvi vieta, o ir erdvė, yra glaudžiai susi­ jusios su socialiniu ir politiniu intencionalumu, egzistencine žmogaus ir bendruomenės istorija. Atsiskyrusi nuo konkrečios vietos, erdvė sulaiki­ nama, jei ne sulaukinama (savasčiai trūkstant že­ mės, šaknų), ir virsta objektyvaus laiko fabriku: formaliu procedūriniu racionalizmu, istorizmu, techninių simuliacijų scenarijumi, projektu; sau patiems nepriklausančių žmonių laisvo judėjimo projektų dirbtinėmis grandinėmis. Tai mums išmokama kompensacija už esminį atsiplėšimą nuo savo šaknų: „Nepūsk prieš vėją, nesišiaušk prieš multikultūralizmą!“ Neracionalu, neprotinga, netgi iracionalu prie­ šintis policinio pliuralizmo „Taip, taip, taip...“ Reikia aiškiai pasakyti: liberalų tolerancija kultūrų skirtumams (taigi ir prisirišimui prie skirtingų šaknų), grąžinanti kultūroms tik po­ litinio intencionalumo regimybę, prieštarauja, viena vertus, jų iš esmės atomistiniam galvojimo būdui, iš principo skaidančiam viską į dalis, kita vertus, jų vertybiniam reliatyvizmui, – empiris­ tiniam pliuralizmui be esmių ir jų modifikacijų apibrėžčių. Liber-izmo modeliai sudaryti iš begalinio skaičiaus idealiųjų taškų: privataus- sukirpimo-žmonių („private men“; Hobbes 1998; 142), – visų antra reikalaujančių laisvės dar nuo ko nors, o visų pirma – visuotinai ly­ gaus, aklo paklusimo liberalios pasaulio tvarkos „parametrams“ ir racionalioms procedūrinėms liberalizmo „vertybėms“: Žmogus pagaliau – pasitelkiant anonimi­ zacijos padargus – išlaisvinimas iš čiabūtiškumo prietarų: objektyviame globalizacijos laike libe­ ralūs multikultūriniai „mainai“ ne tik visuotinai išjudinami, bet ir tampa „<...> tokiu žaidimu, kurį žaisdami vaikai išmoksta savo gimtąją kalbą“ (Wittgenstein 1995; 122 [7]). Pagal ins­ trumentinę „skaitvardžių“ tvarką išaugęs visiškai naujas žmogus pagaliau (tai vadinama poistorija) išsivaduoja iš pirmtakų žaistų žaidimų: relevan­ tiškai gręždamasis į skaitvardžių kalbos žaidimus, ji ir jis gręžiasi nuo nerelevantiškų bendruomenės, visuomenės, tautos vietų, sudarančių visetą, žmogui susiformuoti būtinų kalbos, kultūros, įpročių ir papročių – įrankių, įgūdžių, vaizduo­ tės – lopšį, mores (Montesquieu 2004). Liberalizmas yra laisvumas. Ir nepastovu­ mas. VISI TAI ŽINO. (Jei dar tuo abejoji, bet kuris aukštasis ar žemasis konstitucinės teisės ekspertas išaiškins tau šiuos momentus kaip FELICITY momentus.) Laisvė nuo santykio iki kito laikino santykio, ir t.t. Kadangi santykis yra būdingas bet kokios santvarkos socialinio politinio gyvenimo bruožas, visada galima – siekiant išlaikyti liberalizmo tęstinumą begaly­ bėje – atsiskirti nuo ko nors įprasto. O drauge išrasti (liberalizmui privalu atsinaujinti kas dieną, o liberalui – kas valandą) neįprastą proto arba elgsenos formą kaip laisvę dar nuo ko nors: draugo, priešo, kaimyno, katino, šeimos, lyties, bendruomenės, visuomenės, papročio, bendrojo gėrio, konkrečios vietos. Z dalies tezės: Mores – tai socialinio ir politinio intencio­ nalumo lopšys, kurį metodiškai – žingsnis po žingsnio – ardo ne naujas, bet dėl ypatingo 189 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija ir tas pat), viešoji diskusija. Skambant senajam „Nepriklausomo individo“ himnui ir sukantis nepriklausomiems, jaudulio kupiniems kūnų judėjimų uždaruose apskritimuose šokiams: „Ei, berniukai, į ratelį!“ (Plg. su kitais panašiais kūnų judėjimo uždarame rate žaidimais: Reigenspielen, ring-a-ring-a-roses; Wittgenstein 1995; 122 [7].) Tebesaugodamas procedūrinę skaistybę, liberalizmas kas ketveri metai grįžta į jį maiti­ nančią demokratijos žemę, kurioje „vėl ir vėl“ atgyja viltys, – užverda arši, bet teisinga visų kovos su visais („kiek balsų, prieš ką ir už ką“) politika. Tereikia „sudėti“ ir „atimti“, sutam­ pant atsitiktiniams interesams parankinėje politikoje: juk kai tikslas ranka pasiekiamas, rankas ir kojas gi reikia pramankštinti. Į žemę trumpam grįžta viltis ir kai kam žadanti laimė: po rinkimų visada kai kas laimi. Trumpam sugrįžta ir įgaliotinių – visi juda, skuba, bėga, neša, praneša ... – teisingumas: „Tai yra visa ap­ imantis fonas, kuriuo remdamasis aš skiriu tai, kas teisinga, ir tai, kas klaidinga“ (Wittgenstein 2009; 46 [94]). Ir ne be reikalo skiriu: kad man vėl nusišypsotų laimė. banalumo („aš“ esu viseto sąlyga) vis patrau­ klesnis atomistinio pliuralizmo projektas. Projekto tikslą („nuolatinę sėkmę“, „laimę šiame gyvenime“) pateisina priemonės (tikslūs procedūriniai administraciniai judesiai). Tikslai gręžiasi į priemones: valdžios gaminimą, o pas­ kui – atgaminimą (pačius tikslus). Į gaminius (tariant Aristotelio žodžiais). Į technologiją (vartojant Martino Heideggerio, Walterio Benjamino, Carlo Schmitto terminus). Į triko­ jus projektus, „savo kojomis“ atkeliaujančius į lygių mainų tarp priemonių ir tikslų susirinki­ mus, kuriuos savo ruožtu tiksliai prognozuoja tiksliųjų socialinių gamtos mokslų funkcijos. Kadangi objektyvus Laikas iš prigimties yra nepolitiškas, – juk politinį intencionalumą gali turėti tik pirmasis asmuo, politika (reikia skirti nuo policijos) vietose jau nepasirodo. Pasirodo politikos regimybė: „Viskas yra politika.“ Nors tai nereiškia, kad lieka tik policija. Lieka kons­ titucinė (reikia skirti nuo konstituacinės) teisė balsuoti ir laisvė dalyvauti rinkimuose (sulaukęs liberalaus brandumo amžiaus gali eiti kur nori, o gali neiti, jei nenori, – net į rinkimus). ir tas pat), viešoji diskusija. Skambant senajam „Nepriklausomo individo“ himnui ir sukantis nepriklausomiems, jaudulio kupiniems kūnų judėjimų uždaruose apskritimuose šokiams: „Ei, berniukai, į ratelį!“ (Plg. Z dalies tezės: su kitais panašiais kūnų judėjimo uždarame rate žaidimais: Reigenspielen, ring-a-ring-a-roses; Wittgenstein 1995; 122 [7].) Tebesaugodamas procedūrinę skaistybę, liberalizmas kas ketveri metai grįžta į jį maiti­ nančią demokratijos žemę, kurioje „vėl ir vėl“ atgyja viltys, – užverda arši, bet teisinga visų kovos su visais („kiek balsų, prieš ką ir už ką“) politika. Tereikia „sudėti“ ir „atimti“, sutam­ pant atsitiktiniams interesams parankinėje politikoje: juk kai tikslas ranka pasiekiamas, rankas ir kojas gi reikia pramankštinti. Į žemę trumpam grįžta viltis ir kai kam žadanti laimė: po rinkimų visada kai kas laimi. Trumpam sugrįžta ir įgaliotinių – visi juda, skuba, bėga, neša, praneša ... – teisingumas: „Tai yra visa ap­ imantis fonas, kuriuo remdamasis aš skiriu tai, kas teisinga, ir tai, kas klaidinga“ (Wittgenstein 2009; 46 [94]). Ir ne be reikalo skiriu: kad man vėl nusišypsotų laimė. Auditorijai, kuri savo likimu sumoka už įėjimą į biurokratinio iliuzionizmo teatrą, leidžia­ ma dvidešimt keturias valandas per parą stebėti dalyvaujant TV projektuose valdžią dėl pačios valdžios, o kas keturi metai dalyvauti renkant butaforus, išraiškingų valdžios vaidmenų atlikė­ jus. Šį nuostabų draminį stebėjimo dalyvaujant ir dalyvavimo stebint vodevilį tiksliai aprašo Carlas Schmittas: pliuralizmas-viešumas-diskusija-repre­ zentacija. Ten, kur politinio ir metodologinio pilnumo samprata matuojama tik minėto „brandumo“ kriterijumi, užsimezga ne kokia nors kita, o tik liberali, arba brandi (tai yra viena Brandžios laimės – vertybinės liberalizmo policijos-ir-politikos (žodžių-skaitvardžių inflia­ cijos) – modelis, bent jau Lietuvoje, viešpatauja visose, be išimties, visuomenės gyvenimo srityse. Be jokio pasipriešinimo (be realios opozicijos, be strateginio požiūrio į pamatinį laisvės ir lygybės santykį, be atitinkamų taktinių kriterijų). Tai totalus viseto pajungimas policinei priemonių- priemonių daliai. Viseto pajungimas išmaniajam metodui „Skaldyk ir valdyk!“, kuris net oficialią politiką paverčia atsitiktinių įvykių apytakos (drauge su laisviausio žmonių gyvenimo ele­ mento, PINIGŲ, apykaita) įkaite. 190 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija Liberalizmas itin gerai išmoksta vulgaraus marksizmo pamoką banaliojo veiksmo ly­ gmenyje. Drauge su visais nuskriaustaisiais ir pažemintaisiais, – dabar jau prakutusiais ir gerai vartojančiais viduriniosios klasės proletarais,  – liberalizmas viena savo ranka smaginasi daužy­ damas it bokso kriaušę tikslingai konstruojamą abstraktų priešą, DISKURSĄ (ir augindamas ideologinius raumenis, ir autoritetinius ište­ klius). O kita ranka sistemingai naikina žmogui relevantiškas politinio intencionalumo, socialinio intencionalumo ir kultūrinio intencionalumo sampratas, – tarp jų ypač padorumo ir santūrumo (Berger 1984; 149), kurios priskiriamos jau „ne šio pasaulio“ dorybėms. (sic, net ir liberalizmas turi pastarąją – viešosios nuomonės apklausų – dalį). Z dalies tezės: Jei nenorime ir ki­ tiems, ir sau kelti nerimo, geriau apskritai viešai neprabilti, – kad, be šios realaus dominavimo tikrovių įvairovės, liberalizmo ir netgi liberaliz­ mo filosofijos žodžiai dar ką nors reiškia. Jei šio vardo filosofija, kaip ji pati teigia, visų pirma gina politikos koncepciją (ir tik prie siauresnio diskusijų stalo apdairiai prisimena gero gyvenimo sampratą), tai pačioje politikoje (praktikoje) pakėlę „nežinojimo uždangėlę“ (liberalizmo prielaidas), mes nieko daugiau ir nesužinosime. Išskyrus tai, kad „dirbtinės grandinės“ („visuomenė“) privalo būti „neutrali“ bendrojo gėrio koncepcijų atžvilgiu, – kad nepertraukia­ mai tęstųsi nepriklausomo individo sėkmė šiame vienatiniame (liberaliame) pasaulyje. Kadangi liberalizmas yra legitimi (neutrali, išskyrus pačią save) visų valdžia, tam prireikia trečiojo kilnaus (be „individualizmo“ ir „pliuralizmo“) žodžio – „multikultūralizmo“. O kai tik šie trys žodžiai įeina į pačią praktiką, vėl – ne pirmą ir ne paskutinį kartą – išryškėja liberalizmo vaikystės negalia, „šizofrenija“. Tiek motyvacijų ir interesų ratų – socialinės politinės visumos atžvilgiu – siaurėjimas, randantis itin agresyviai „skaldančio solidarumo“ formai, pakeitusiai ro­ mantinį Sąjūdžio solidarumą. „Kūnas“ (partinių, administracinių, teisinių procedūrinių, karinių, t.y. aktualių policinių, interesų politika) atskiria­ mas nuo „galvos“ (tautos kaip suvereno steigtos galėjimo, o ne tik aktualios jėgos), konstitucijos. O kadangi visada yra pavojus, jog kas nors kada nors gali bandyti įsiskverbti į pačius tikė­ jimo liberalizmu pagrindus – „Pagrįsto tikėjimo pagrinduose glūdi nepagrįstas tikėjimas“ (Witt­ genstein 2009; 69 [253]) – liberalų partijos gerai prižiūri ant sienų gausiai kabančius laikrodžius, kurie labai tiksliai muša objektyvias priemo­ nių-priemonių valandas, minutes ir sekundes: vertybes, normas ir taisykles. Liberalizmas yra tikslus, nes gerai prižiūrimas, valdžios dėl val­ džios policijos, teismų policijos, vertybių policijos (pusiausvyros jausenos ir laikysenos užtikrinimo masių sąmonėje) laikrodis. Liberalizmo konstrukcijų ir dekonstrukcijų cikliškumas – tai banaliausia tipiškų gaminimo ir tipiškų vartojimo pavyzdžių diegimo karto­ tinė praktika – padėčių rangavimo, statistinio reitingavimo ir inter-tele-žurnalistikos ringavi­ mo – abstrakčiuose visuomenės lygmenyse: po­ litikos (viešosios administracijos), ekonomikos (ekonominės statistikos), kultūros (projektų šaukimo ir jų atšaukimo), socialinio solidarumo Abi šias dalis sujungia dirbtinė visuome­ nės grandinė: „viešųjų“ scenų vietose užverda pliuralistinių „diskusijų“ aktualiomis dienos, valandos ir minutės temomis procesai; įvairiose simuliacijų, numanomų scenarijų ir žaidimų 191 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija teorijose smagiai aptariami „atsitiktiniai“ savai­ tės, dienos ir minutės įvykiai. Z dalies tezės: O „atsitiktiniai“ įvykiai niekur neišnyksta, nes pasiekiamas skai­ drus liberalizmo politikos lygmuo: atomizmas, individualizmas, egoizmas, geras apetitas ir bendruomenės sukaustymas į dirbtines veikė­ jų, patarėjų, suflerių ir – būtinai – juokdarių grandines, prigaminančias daug gardaus juoko nugalėtoje nugalėtojų istorijoje: liberaliosios ironijos, arba reliatyvizmo (o tai yra viena ir tas pat). Nerelevantiškumo ir relevantiškumo ben­ drajam gero gyvenimo pasauliui pusės šiandieną išsiskiria, ir eina kas sau. intersubjektyvumo – problemą, nuo pačios pra­ džios prislegia rūsti ilgaamžė visuomeninės sutar­ ties tradicija, keistai derinanti valstybės prie-vartą ir racionalią piliečių sutartį be socialinių sąsajų, be kultūros tarpininkų. Be sąsajų poistorija išauga iš tvirtai sukerojusio liberalaus statisti­ nio lygybės augalo: vidurkių konstrukcionizmo. „Viską galima padaryti ir išdaryti“, – skelbia iš pažiūros linksmas socialkonstrukcionistų partijos, sėkmingai užsiimančios dirbtinėmis operacijomis – nuo krūtų didinimo iki smegenų mažinimo – šūkis. Šio – tik iš pažiūros smulkaus – verslo sklaidos rezultatas yra paradoksalus: juo daugiau operacijų, tuo daugiau poreikių (įskaitant nau­ jus technologinius jau operuotųjų norus). Juo daugiau poreikių ir norų, tuo daugiau lygybės. Visi balsai – rinkimų visuomenės po aštuoniolikos metų (dirbtinių grandinių) balsadėžėse – vienodi, vadinasi, tolygūs, vadinasi, vienodi, vadinasi, lygūs... Lygybė – geometrinė, bet bekūnė. Iš čia – geometrinė bekūnė liberaliosios demokratijos darna. Teiginys l yra teisingas liberalui, o teiginys d – demokratui, tačiau teiginys d yra klaidingas liberalui, o teiginys l – demokratui, tačiau (ta­ čiau – iki begalybės – ir t.t.) pačiame „tačiau“ rato viduryje l ir d puikiai sutaria „liberaliosios demokratijos“ regimybėje, šukuojamoje pagal viešosios nuomonės plauką. L ir d viena kitai jaučia bekūnės kilmės aistrą: panašumo „ir t.t.“ Tariant tikslinančiais Pierre’o Manent žodžiais, „būtino suvienodėjimo“ (Manent 2008; 9). „Jei tik ir kai tik galiu, man viskas leistina (jau po diskusijos!)“ Postmodernūs sociologai, įskaitant Z. Baumaną, iš dalies yra teisūs, teig­ dami, kad Rytų Europoje, taigi ir Lietuvoje, trūksta subtilios postmoderniosios ironijos (gy­ venimo formos, būdingos socialiniam ir politi­ niam intencionalumui). Nuo subtilios ironijos at-graso (tai viena pagrindinių matomos, bet nepastebimos prie-vartos formų) sarkastiška, išskirtinai grubi ir tiesmuka laikysena Kito atžvil­ giu: „ciniška šaipa“, ateinanti iš pačių laisvumo pasaulio gelmių „Cool“. At-grasa, kurią dėl „aukštųjų“ reitingų prie savęs glaudžia liberalūs iner-tele-žurnalistikos portalai, – ir matomai, ir pastebimai grasindami (drauge su savo bičiuliais ir bičiulėmis: „sarkazmu“, „karikatūra žemiau juosmens“) „pa-nieka“... Nebus per daug pakartoti. „Naujojo politi­ kos mokslo“ (Strauss 1963; 129) pradininkas T. Z dalies tezės: Hobbesas sistemingai ardo viską, kas tik gali būti susiję su politinio intencionalumo (valstybės, viseto ir dalies santykio, socialumo, sąmonės nu­ kreiptumo, teleologijos) principais. Mįslingiausią filosofijos ir socialinių mokslų – socialumo kaip Nors tebevartojami tradicinių socialumo sąvokų pavadinimai, tačiau jų turiniai – visiš­ kai skirtingi (žr. dešimtą Hobbeso Leviatano skyrių): „Godumas didelio turto, didelis pasi­ žymėjimo ir garbės ženklų troškimas yra tam 192 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija tikras garbingumas, nes tai galios juos įgyti ženklai“ (ten pat; 108). Šiuolaikiniai liberalai praktiškai tęsia tą pačią liniją: visos vertybės įtraukiamos į egalitarinės politikos prekių apy­ vartos sąrašus ir, reikalui esant, perkainojamos teisių ir teismų diskursuose, vadovaujantis, jei tik ir kai tik kyla teisiniai ginčai, pamatiniu ir neprilygstamu liberalizmo kriterijumi, – kad iš tradicijos būtinai pareina suvaržymai, galimai pažeidžiantys negatyviąją laisvę. Didžiuosiuose liberalios prekybos tinkluose, kurie ir iš mažųjų reikalauja ginti vien liberalų teisingumą, verty­ bių atranka yra liberalių vertybių atranka. Štai kodėl „<...> teisingumas kaip nešališkumas iš tikrųjų atmeta politinės bendruomenės idealą“ (Rawls 2001; 198–9). konsensu, yra jų racionalistinė konstitucinė pri-mestis bendruomeniškam (saistančiam) gyvenimo-pasauliui. Natūralią politinę pri-mestį pakeičia dirb­ tinė konstitucinė pri-mestis. Neutralumas ir instrumentinis procedūrinis požiūris į politiką iš esmės pakeičia politinio intencionalumo prigimtį ir politikos apimtį: stingant politinės atramos natūralioje laikysenoje, gręžiamasi į dirbtinę dirbtinės teritorijos metafiziką, „dirb­ tinių individų“ teises, kurios, negalėdamos sukurti realaus politinio etoso, bendrojo gėrio sampratą pakeičia konstitucinėmis vertybėmis. Vertybės virsta vertėmis: „Žmogaus VERTĖ (worth), arba vertingumas, kaip ir kitų daiktų, yra jo kaina, t.y. kiek būdų duodama už naudo­ jimąsi jo galia, todėl ji nėra absoliutus dalykas, o priklauso nuo kito poreikių ir sprendimų“ (Hobbes 1999; 103). Laisvė kaip asmens subjek­ tyvumo, racionalumo ir relevantiškumo sąlygų prie-žiūra (tiek vidinė, tiek išorinė), glaudžiai susijusi su žmogaus kaip vertinančios, morali­ nės, tikslingos būtybės samprata, traktuojama tik kaip parduodami-perkami „atvejo faktai“, šiomis dienomis nuvertėjantys dėl viešpatau­ jančio metodologinio subjektyvizmo. Kitaip tariant, transformacinės lygties – iš vertybių į vertes – formulė yra šiuolaikiška, arba liberali (o tai yra viena ir tas pat). Politinės bendruomenės vertybės – ne modernios. O nemodernios vertybės yra – pagal liberalų api­ brėžimą – antiliberalios. Naujoji liberalizmo metafizika – nuo Hobbeso iki šių dienų – visų teisingų žmonių vardu kovodama su tradicine metafizika perša save modernėjančiai auditorijai kaip naujasis mokslas. „Dirbtinės visuomenės“ mokslas. Ir „dirbtinio individo“ mokslas. „In­ divido“, kadais buvusio natūraliu (laisvu) asme­ niu, kursai savo vaizdiniuose neatpažintų nei ti­ krovės, nei pats savęs. Z dalies tezės: Formuojantis liberalizmo metafizikai drauge randasi „faktiniai žmonės“, „statistiniai vidurkiai“, „visuomenės nuomonės apklausos“, visiškai abejingos fenomenologi­ niam adekvatumo kriterijui. Faktiniai mokslai, kaip pažymi Husserlis pirmuose Europos mokslų krizės puslapiuose, formuoja faktinius žmones. Tai, ką faktiniai liberalai vadina su-tampančiu Liberalizmas – lygu negatyviosios laisvės pro­ gresas, susidedantis iš dviejų pagrindinių dalių: 1) pliuralizmo, kuris apdairiai suardo trinarę ir realiai vienintelę politinių vertybių – konserva­ tizmo, radikalizmo ir liberalizmo – struktūrą, šiandieną siūlydamas vien tik liberaliųjų verty­ bių sąrašus (tik pasak liberalų, šiandieną mes visi esame liberalai), 2) progresyviai prisitaikančio individo, gebančio eiti į priekį be pasipriešini­ mo ardant tradicines aplinkas, tipo. Progresas, gręždamasis atgalios, negali žiūrėti į socialinį 193 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija politinį savo veidą: visos vertybinio neutralu­ mo ir nešališkumo idėjos gali išnykti tradicijos veidrodyje pamačius „jau kažkur matytą“ veidą. Taigi liberalizmas vis dėlto turi tradiciją: įgimtą kompleksą, tamsaus kampo baimės. Kad natūralus liberalizmo racionalumas nėra „natūralus“, taigi galbūt nėra racionalus. Būdamas racionalus (nebaikštus), aš „atmetu“ tradiciją, mano paties socialinį veidą, neracionalų argumentą. Liberalus asmuo visuomet „skilęs“: nepriklausomas nuo visiškai atomizuotos dabarties, bet priklausomas nuo socialinės praeities. džiantys liberaliems kūnams judėti be socialinio pasipriešinimo. Visus vienodinančioje, kraštuti­ nai egalitarinėje liberalizmo erdvėje nėra vietos nei visuomenei (įsipareigojantiems socialiniams saitams, o ne laikiniems kooperaciniams), nei politinei bendruomenei. Liberaliame – tik pliuralizmo faktų ir nuo jų švariai atskirtų vertybių – pasaulyje, kurį konstruoja racionalistas teoretikas, politika yra negalima. O gyvenimo-pasaulyje, saugančiame savo vidinių ribų, kurias figūruoja saviti faktų ir vertybių susietumo deriniai, politika yra įgimta: gyvenimo-pasaulis yra vienovė įvairovėje, sudaryta iš skirtingų prasminių provincijų, – visiškai ne­ panaši į viena-visuomenės vaizdinį, kuris, atka­ kliai diegiant vieną „vertybę be substancijos“, „pliuralizmą“, virsta homogeniška procedūrine nelaisvės praktika ir žiniaskaida. Susvetimėjimas savo paties atžvilgiu, skilimas į nepriklausomą individą ir socialinę savastį, vi­ dinio horizonto ir išorinio horizonto atskyrimas tampa veiksmo įpročiu. Tai istoriškai sąlygota susvetimėjimo, arba istorizmo, forma: kai liberalizmas virsta savaime suprantama institu­ cija. O istorizmas – lygu racionalistinė utopija, kurios sąvokos neturi nieko bendro su socialine tikrove. Liberalizmo baimė ir pagrindinis nega­ tyvusis argumentas, – kad „pliuralizmas“, arba „amžinoji taika“, gali sugrūti, jei sugrįš „politinė visuomenė, kurią vienija viena (dalinė ir pilna) esminė religinė filosofinė arba moralinė doktri­ na“ (Rawls 2001; 198–9), egzistuoja arba libe­ ralizmo politinės filosofijos vaizduotėje, arba tik kai kuriuose realiuose, bet kraštutiniuose karinio homogeniškumo (kad ir fašizmo) pavyzdžiuose. Z dalies tezės: Racionalistinis vertybių klasifikavimas (ten pat; 102–5), išskleidžiamas Talcotto Parsonso „socialinės sistemos“, „sisteminių tarpusavio sąsajų“ (Parsons 1951; 101) arba Johno Rawlso „socialinės vienovės“ (social unity) lygmenyje (Rawls 2001; 199), yra dirbtinis, „ir vėl, ir vėl“ pateisinamas visuomenės nuomonės apklausų ir liberalaus mokslo kartotinių vertybių „patvir­ tinimų“ (empirinių tyrimų laisvųjų visuomenių lyginamosiose analizėse). Abiem šiais atvejais tradicija griežtai atskiriama nuo šiuolaikiškumo; neracionalumas – nuo racionalumo; neliberali so­ ciologija ir politika – nuo liberalios sociologijos ir politikos. Įtikrinimo ideologijos projektas grę­ žiasi į mus, o mes – į jį sutampančiame konsense, „realistiškai utopinėje politikos filosofijoje“ (ten pat; 4). Ir jis, ir mes gręžiamės vienas į kitą: politinį teisingumą kaip nešališkumą. Keičiantis teritoriniams geopolitikos akcentams, drauge Viena-visuomenės vaizdas piešiamas taip, kaip reikalauja teorinė liberalizmo, nepri­ pažįstančio kultūros struktūrų įvairovės prieš­ duotiškumo, schema. Viena-visuomenės vaiz­ das – egalitarinis pliuralizmas – yra paskesnių dedukcinių žingsnių padarinys, liberalizmas! Iki kraštutinumo standartizuotas: be vertybių, be socialinių tarpininkų. Tik pliuralūs faktai, lei­ 194 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija nesuvaržytų laisvųjų šnekalų... Juo daugiau..., tuo mažiau pirminės socialinės politinės ti­ krovės – visuomenės gyvųjų gyvenimo formų, skirtingų gyvenimo būdų ir stilių, moralinių ir estetinių veiksmo-laikysenų, gyvenimo-planų įvairovės – supratimo. Juo daugiau..., tuo labiau laikoma savaime suprantamu dalyku, „jau įro­ dytu pozityvistų“, kad ypač politinio veiksmo srityje etikos ir metafizikos problemos yra tik pseudo-problemos. su Hegeliu „ir vėl, ir vėl“ gręžiamės į praktinio racionalumo virsta grynuoju racionalumu: „Kai žvelgiame į pasaulį racionaliai, ir pasaulis racionaliai žvelgia į mus“ (ten pat; 2). Laisvė, kaip taria globalizacijos teorijos, o tiksliau, prasmės simetrizacijos objektyviame laike, racionalizme, „simetriškame išsidėstyme (Rawls 2001; 82),k pradininkas Hobbesas, gręždamasis į Lietuvą, yra tai, ką dabar ir turite. Tai laisvas kūnų judėjimas, jau nebesipriešinant vietai; negatyviųjų laisvių aibės, pretenduojančios tapti pozityviosiomis teisėmis, apibrėžtomis pagal išankstinius liberalių vertybinių gairių sąrašus, vadinamus aktualiomis ir potencialiomis teisių gynimo teismuose darbotvarkėmis. Tai netiesa. Vertybių figūracijų gyvenimo- pasaulyje, relevantiškų tipizacijų eksplikavimas ir supratimas ne trukdo, o priešingai, padeda skaidrinti žmogaus esminio priklausomumo savo-gyvenimo-aplinkai bruožus. Plg. su nuo pradžios politiškai intencionaliu Montesquieu laisvės kaip visų pirma galimybių refleksijos srities apibrėžimu ir skirtimi tarp laisvės ir nepriklauso­ mybės: „Valstybėje, tai yra visuomenėje, kurioje yra įstatymai, laisvė gali būti suprantama tik kaip galimybė daryti tai, ko reikia norėti, ir nebūti verčiamam daryti tai, ko nereikia no­ rėti. Būtina išsiaiškinti, kas yra laisvė ir kas yra nepriklausomybė. Laisvė yra teisė daryti viską, kas leidžiama įstatymais. Z dalies tezės: Jei pilietis galėtų da­ ryti tai, kas įstatymais draudžiama, jis neturėtų laisvės, nes tą patį galėtų daryti ir kiti piliečiai“ (Montesquieu 2004; 159). Nepriklausomybė, kaip taria socialinio pilnumo metodologijos pirmtakas gręždamasis į Lietuvą, – nėra tas pat, kas yra laisvė. Tai automatinės, natūralistinės valdžios policijos <objektyvaus> laiko darbotvarkės. Yra darbotvarkės laikysenos, pakeitusios turėtų būti laikysenas, kurios visuomet grindžiamos verty­ bėmis: „Kitaip nei gamtos moksluose, dėsniai socialiniuose ir humanistiniuose moksluose yra prasmės dėsniai. Šitaip esti todėl, kad jie įgyven­ dinami sąmonėje, o ne <objektyviame> laike“ (Schutz 1996; 79). Kitaip nei automatinės policijos objektyvios laiko prasmės, žmogiškos subjektyvios prasmės problema yra išgyvenamo laiko-patyrimo, vidinės laiko-prasmės (Phänomen der Erlebniszeit; des inneren Zeitsinne) problema (Schutz 1932; iii). Juo daugiau išlaisvinto – nepriklausomo (kai dalis iškeliamas priešpriešiais visetą)– judė­ jimo, juo daugiau nepriklausomų (nešališkų, vertybiškai neutralių) teisių gynimo teismuose darbotvarkių. Juo daugiau kruopščių procedū­ rinių darbotvarkių, tuo daugiau ataskaitų. Juo daugiau ataskaitų, tuo daugiau žiniaskaidų... Juo daugiau žiniaskaidų, tuo daugiau tikrai Laisvė – ne nepaliaujamas socialinių kons­ trukcijų gaminimas, ne vartojimo formų per­ tekliaus atgaminimas visuomenėje ( „Perskaitau kurį nors Paryžiaus laikraštį iš ryto, ir kaskart randu vis naują dėsnį...“, – su šypsena lūpose rašo Tocqueville’is). Laisvė randasi iš savigrąžos 195 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Minimalios socialinės politinės moralės, arba laisvumo, sąlygomis verda, nes savaimingai ataušti nebegali, ženklų pliuralizmas, dažniau neįsisąmoninamas nei įsisąmoninamas auto­ matizmas. Liberalizmas nuo mažens yra laimės vaikas: „Tokių rūšių yra nesuskaičiuojama aibė: nesuskaičiuojama įvairovė vartojimo būdų to, ką vadiname „ženklais“, „žodžiais“, „sakiniais“ (Wittgenstein 1995; 128–9; [21, 23]). Tai tikra palaima liberalizmui, nuo mažens jaučiančiam aversiją socialinams-politiniams saitams. „Neša­ liškos vertės politinėms laisvėms garantija“ („the garantee of the fair value of the political liberties“), kaip pripažįsta patys liberalizmo filosofai, turi prigimtinį polinkį skaidyti-ir-tik-skaidyti: tik stumtelėk ją lygybės (plačių garantijų politinėms laisvėms) kryptimi, ir ji bematant virsta iracio­ nalia, paviršutiniška, socialiai skaidančia (Rawls 2001; 150–1). į esminį pasirinkimą ir iš įsipareigojimo, o ne iš jusles dirginančio išpardavimų reliatyvizmo, kurio pabaiga – apriorinis, t.y. be pasirinki­ mo multikultūralizmas ir į liberalią mišrainę sumestos moralinės ir ne-moralinės (estetinės, gyvenimo būdų, laiko praleidimo būdų, stilių kopijavimo būdų, svetimų tekstų kopijavimo būdų, apskritų stalų ir pailgų menių diskusi­ jų...) vertybės. Tsai vertybės, kurios, be perstojo daugin­ damos išorinius norus, išlaisvina iš savigrąžos. Jos rūšiuojamos pagal liberalią vertybių skalę, remiantis aiškiai ir ryškiai skaidančiais vulga­ raus, arba holivudinio, liberalizmo – „Nr. 1 / nr. 2“, „Nr. 2 / nr. Z dalies tezės: 11“, „viršesnis / žemesnis“, „greitesnis / lėtesnis“, „pirmesnis / antresnis“, „vertingesnis / menkesnis“, „jaunesnis / senes­ nis“, „brangesnis / pigesnis“... – principais. Vienintelis svertas, kuris šiek tiek išlygina šiuos kraštinumus – kūnas be pusiausvyros pargriū­ tų – yra visuomenės nuomonės apklausos. Apklausos išaiškina demokratijos pusiausvyros – „daugumos“ ir „mažumų“ – tendencijas (net procentų tikslumu!), didesnius ar mažesnius „nuomonių apie ką nors“ nukrypimus nuo bendrųjų liaudies demokratijos tendencijų, ir skelbia naują „ir vėl, ir vėl“ vertinimų visuome­ nėje – lyginimų, išlyginamų, vertinimų, nuver­ tinamų, pervertinamų... – bangą. Šias vertybes, puikiai nujausdamas jų sėkmės priežastis sveiko protavimo aplinkose, pradėjo klasifikuoti jau Hobbesas. Tiksliau, klasifikuoti nesaistančius ženklus, nerodančius į daiktus, – kurių apstu liberalioje žinia-skaidos „tikrovėje“: „Tai yra lyg ženklai, pažymintys, kad koks nors žodis nėra daikto, apie kurį kalbama, vardas <...>“ (Hobbes 1999; 57). Juo daugiau minimalios politinės moralės principų, tuo dažniau jie panaudojami priemo­ nių-priemonių, arba visų karo su visais, atvirose ir uždarose kovose. Skambant liberalizmo himnui FELICITY: užtikrinti kiekvienam piliečiui neša­ liškas ir maždaug lygias viešųjų gebėjimų vartoji­ mo („the use of a public facility“) galimybes, kurių politinė paskirtis ir yra pats viešųjų gebėjimų vartojimas sukonkretinant konstitucines taisy­ kles ir procedūras, garantuojančias nešališką, kiek tai įmanoma paklūstantį įstatymų tvarkai, politinės valdžios padėčių paskirstymą (ten pat). Minimaliosios politinės moralės, kaip ir tobulo pliuralizmo, koncepcija yra tik teorinis principas. Kaip daiktas pats savaime pliuralizmas yra nie­ kas. Kaip ir sąmonė pati savaime (reikia skirti nuo są-monės kaip transcendentalinio savigrąžos fenomeno) yra niekas: „Sąmonė pati savaime yra 196 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija kuriame eiliškai, nors nebūtinai tvarkingai, sukasi liberalios vertės – valdžia, pinigai, padė­ tys, vaidmenys, statusai, pareigos, jėga, prievarta, įtaka, įtaiga, orūs vardai, įtikrinami reitingų ir visuomenės nuomonės apklausų priemonėmis. niekas; ji visada yra ko nors sąmonė“ (Berger, Luckmann 1995; 10). „Minimaliosios politinės moralės pliuralizmas“ – tai tik racionalistinė liberalizmo prielaida, kaip su kirviu atkertanti savo idealiuosius tipus nuo praktinio raciona­ lumo tipizacijų intersubjektyviame gyvenimo- pasaulyje. Natūralizmo, arba globalizacijos įtikrinimo (o tai yra viena ir tas pat), požiūriu, tai, kas juda, yra laisvumas, nepriklausomumas, jėga, inercija, šiuo-laikiškumo vertė. Ir priešingai, visa tai, kas stūkso vietose – praeitis, kultūra, čiabūtiškumas, čia-buvis, savigrąža, aš-intencionalumas (skirti­ nas nuo judančio tavęsp-intencionalumo), įprotis ir į-protis (absurdo, arba būtinybės ir laisvės santykio aki-vaizda) – yra be vertės. Tai esą tik „nykstančios“ – vieniems dar gražios, kitiems apgailėtinos – vertybės. Z dalies tezės: Pliuralizmo ženklų prie-vartinio, ne nuosai­ kaus, o infliacinio vartojimo (nuo policijos poli­ tikos iki žinia-skaidos), kurį Durkheimas kadais tikėjosi taikiai suderinti su organiškuoju – kad stiprėjant diferenciacijai stiprės profesinio pobū­ džio tarpusavio sąsajos – solidarumu visuomenė­ je, rezultatai visiškai tenkina tik pačius liberalus, okupavusius gyvenimo-pasaulį: „Augantis socialinis ir vertybinis pliuralizmas taip pat ardo politikos teoriją ir praktiką. Šiuolaikinės valstybės darosi vis labiau heterogeniškos, o jų piliečiai įgyja vis daugiau skirtingų ir dažnai tarp savęs nesuderinamų tapatumų, idealų ir interesų. Viešasis, ne mažiau negu privatus, gyvenimas susiduria su problemiškais, o dažnai ir tragiškais, pasirinkimais“ (Bellamy 1999; 1). Natūralus kūnų judėjimas, laisvumas, norų skiemenavimas globaliame Hobbeso projekte kyla, kaip paviršinis garavimo reiškinys, dėl priežasties, o ši – dėl kitos priežasties nenutrūksta­ moje priežasčių grandinėje (ten pat; 218). Laisvė ir būtinybė srūva savo vaga, savaime suprantama, inertiška tikrove, be galimybės atsigręžti į šią tėkmę sąlygų, be galimybės prarasti ir atgauti prasmę, atrasti simbolines reikšmes, atgauti gy­ venimo sąsajų relevantiškumą. Plg.: „Taisyklė, kartą pažymėta apibrėžta reikšme, per visą erdvę nubrėžia linijas, kurių turi būti laikomasi. <..> Ne; mano aprašymas tik tuomet turėjo pras­ mę, jeigu jis turėjo būti suprastas simboliškai“ (Wittgenstein 1995; 220 [219]). Trūkstant politinio ir vertybinio intencio­ nalumo, įsigalioja minimalistinė liberalizmo moralė, vadinamasis „apribojančio pliuralizmo faktas“: turintieji daugiau priemonių-priemonių išstumia ne tik turinčiuosius mažiau priemonių, bet ir visus tuos, kurie turi tikslus. Turintys dau­ giau priemonių, arba priemonių-priemonių, gali „drauge kombinuoti“ (angl. „combine together“) ir išstumti turinčius mažiau priemonių (Rawls 2001; 150). Tai nuolatinis nusiteikimas karui, – primena mums Klasikas, – nes žmogus negali pasitenkinti nuosaikia valdžia „neįgydamas jos dar daugiau“ (Hobbes 1999; 113). Nusiteiki­ mas karui – lygu liberalus valdžios vartojimas, Taikant šią Wittgensteino skirtį – nepri­ klausomos linijos erdvėje ir simbolinio šios linijos supratimo – politinei tikrovei, galima ir reikia aiškiai skirti, viena vertus, nepriklausomybę, ir, kita vertus, laisvę. Pirmoji yra visad santykiška: žmogus yra įmestas į savo aplinką, bet visada 197 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija gali santykiškai modifikuoti įmesties būklę kaip savo ap-linką, savo pa-saulį, savo pri-mestį. Kitaip sakant, žmogus gali įgyvendinti nepriklausomybę kaip intencionalų ap-linkos atžvilgiu aktą. Savi­ grąžoje aplinkon žmogus įgyja są-monę, arba, ne-priklausomybę, tačiau drauge – natūralų polinkį laikyti šią ap-linką aplinka (be brūkš­ nelio). Aplinka be brūkšnelio, natūralioji laiky­ sena, suteikia įpročių visetą, laisvumo inerciją, santykišką nepriklausomybę gamtinės aplinkos prigimties, įskaitant instinktų ir impulsų pri­ gimtį, atžvilgiu. inter-esminių sąsajų ir, kita vertus, taisyklingo gyvenimo su-vartojimo. Ir t.t. Z dalies tezės: geležinkelio linija yra tik vaizdas. Jei tik ir kai tik įsisąmoniname, kad šis-vaizdas „kažkodėl“ slegia, trukdydamas judėti ne laisvame išoriniame, o nelaisvame vidi­ niame horizonte, trukdydamas ap-linkoje, savo namuose, jaustis prasmingai, randasi simboli­ niai atsakingo pasirinkimo klausimai, – kodėl ir kaip turėtų būti: „„Linija man įteigia, kaip turiu eiti.“ – Tačiau tai, aišku, yra tik vaizdas. Ir jei nuspręsčiau, kad ji įteigė man tą ar kitą dalyką neatsakingai, tuomet nesakyčiau, kad laikausi jos kaip taisyklės.“ (Ten pat; 221 [222].) Laisvės prigimtis yra pirmesnė už nepri­ klausomybės prigimtį: tai atskleidžia teleologija, ateities-muzika, kaip ją vadina Husserlis, verty­ bių kaip idealiųjų žinojimo objektų tarpusavio sąsajos. Laisvė yra gręžimasis į valstybės (vertybių viseto ir atitinkamų relevancijų tarpusavio sąsajų) prigimtį, buvojimą mūsump, kuris yra pirmesnis už kiekvieną paskirą politinio intencionalumo dalį, – drauge su kodėl ir kaip turėtų būti (atsa­ komybės ir atskaitomybės) modusais. Vartojant Kanto terminus, laisvė perteikia tam tikrą būti­ numo ir ryšio su pagrindais santykį, privalėjimą, kurio gamtoje nerastume ir kurį atranda ne empiriškesnis intelektas, o apriorinio pažinimo principus, pastovias sąlygas – turėtų būti – teikian­ tis protas (Kantas 1982; 69, 396, 399). Kitaip sakant, tik relevancijose, – atogrąžų ir savęsp, ir mūsump linkmėse, – pasirodo laisvės vaizdo priešduotys drauge su klausimais-links­ niais: kas išlaisvinamas, o kas iš-laisvinamas (liberalia hobsiška išorinio kūnų judėjimo pra­ sme), kam laisvė, o kam laisvumas ko nors norėti kad turėti be trukdžių...? Ir priešingai, be rele­ vancijų nepriklausomybė (pradžios laisvumas) įteigia linijos vaizdą: atskaitingą (loginį), bet ne­ būtinai atsakingą (teleologinį). Būtent inercijo­ je – nepriklausomybėje ir t.t. – kyla mintis kaip tik iš atsakingiausių žodžių (laisvės, tiesos, gėrio, grožio, teisingumo, lygybės) daryti vardus, nors ir akivaizdu, jog tai nėra jokie vardai: lysvė nėra laisvė (žr. 39-ąjį Wittgensteino fragmentą apie „Nothung“ kaip tikrinį vardą; ten pat; 139–40). Laisvė yra gyvenimo, gero bendro gyvenimo, teleologija, o lysvė – nelaisvės trūkumo ir t.t. bėgių linija, randantis intercijai, kuri subjekty­ vumo, racionalumo, laisvės paradoksus, būdingus šiuo-laikinei tikrovei, redukuoja į lygybės lygčių aibes liberalizmo teisinėje paradigmoje. Nepriklausomybė konstituoja pradžios vertes, tačiau be relevantiškų viseto sąsajų ne­ priklausomybė gali ir įprastai tampa inercija, loginio sąlygojimo ir t.t. bėgiais: „<...> o be galo ilgi bėgiai atitinka neribotą taisyklės taikymą“ (Wittgenstein 1995; 220 [218]). Laisvę kons­ tituoja simbolinis supratimas, kurio paskirtis – atskleisti skirtumą tarp priežastinio ir loginio sąlygojimo (ten pat; [220]), tarp, viena vertus, Lysvėje (santykiškoje nepriklausomybėje, atplėštoje nuo gyvenimo-pasaulio) tikroji ti­ 198 Sociologija. Z dalies tezės: Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija krovė konstruojama gręžiantis nuo netikrosios. Gaudant nepagaunamus savo norus kažkurlink judančių kūnų praktikoje. Šiame judėjime tobulai skleidžiasi liberalizmo ne tiek dviveidiš­ kumas, kiek prigimtinis dualizmas. Liberalizmas jaučiasi it žuvis vandenyje todėl, kad vertybių teleologiją, kuri yra pirmesnė už paskirą žmogų, institutą, kokią nors politikos filosofiją, padaro priklausomą nuo empirinių kasdienio liberalizmo reikmių (norų laisvai judėti tarp kitų kūnų be pasipriešinimo). Nors liberali politikos filosofija yra tik viseto dalis, tačiau veržlia jėga-ir-protu (versdama laikytis linijos taisyklės: tai užtikrina liberalūs teisių ir teismų diskursai) nepriklauso­ mą vertybių teleologiją padaro priklausomą nuo dalies: politiniai institutai sutampa su liberaliais institutais. visada sako tą patį, o mes darome tai, ką ji mums sako.“ (Ten pat; 221 [223]) Liberalizmas – jau kaip multikultūralizmas (dar gražesnis už ankstesnįjį gražų) – gręžiasi nuo prasminių moralinio patyrimo ir moralinio vertinimo regionų. Gręžiasi nuo bendrabuvoji­ mo (inter-esse) sąsajų, gręždamasis į negatyviosios laisvės priežasčių grandinę, FELICITY. Laisvė, kurią reikia skirti nuo nepriklausomybės, galima tik kaip savigrąža į subjektyvaus buvojimo- pasauliui ir drauge objektyvaus buvojimo-pa­ saulyje struktūras, bendrosios kultūros sąsajas. Multikultūralizmo projektas sudarytas iš dviejų geometrinių Hobbeso predikatų: 1) „dirbtinių asmenų pliuralizmo“ ir 2) „dirbtinės visuome­ nės grandinės indiferentiškumo“. Šie predikatai šiandieną įgyvendinami Projekto šaukinių ir jų šauktinių grandinėje. Griežtai laikykis dviejų procedūrinių taisyklių: 1) naivaus individua­ lizmo (gręžimosi nuo ko nors) ir 2) moralinio solipsizmo (gręžimosi tik savęsp) verčių. Šitaip randasi naujas filosofinis liberalizmo vardas, multikultūralizmas, kuris nėra joks vardas, nes neatitinka paprastos tikrovės, natū­ raliosios laikysenos. Multikultūralizmo vardas tarsi „šnibžda“, kad, viena vertus, kultūros turi sąsajas, kad skirtingos kultūros „ko-egzis­ tuoja“, kita vertus, būtent sąsajas, relevancijas, intersubjektyvumą (priešduotas moralines gy­ venimo struktūras) liberalizmo filosofija labai sistemingai ardo. O kai kultūros neparemia indiferentiška filosofija, kuriai iš esmės trūksta subjektyvumo, racionalumo ir relevantiškumo sąsajų, liberalizmas tiesiog sutampa su paprasta tikrove, o liberalizmo filosofija – su liaudies-val­ džia, rinkimų-turais. Liaudies-valdžia nelaukia liberalizmo filosofijos „pašnibždėjimo“, nors ir daro tai, ką ji „šnibžda“. Plg.: „Nejaučiame, lyg turėtume visuomet laukti taisyklės linktelėjimo (pašnibždėjimo). Atvirkščiai. Nelaukiame įsi­ tempę, ką dabar ji mums pasakys, nes ji mums Liberalizmas yra Projekto šauktinių, arba šiuolaikinių automatizmas, kurio pridėtinė ver­ tė – indiferentiškumas. Reikia aiškiai skirti aske­ tiškai rūsčią liberalizmo filosofiją, susidedančią iš dviejų dalių, a) dirbtinių asmenų laisvumo ir b) dirbtinių grandinių įtemptumo, ir pridėtinę šios filosofijos vertę. „Vertybinio neutralumo“ nuos­ tata, kitaip nei įprastai teigiama, yra ne rūsčiosios filosofijos, o jos pridėtinės vertės, kurios dalis yra pozityvus mokslas, komponentas. Z dalies tezės: Tik „vertybinis neutralumas“, arba „moralinis minimalizmas“, gali suteikti „universalistinę“ priedangą filoso­ fijai kaip teisiniam norų nevaržymo diskursui: „Politiniai institutai – lygu liberalūs institutai.“ Liberalizmas yra jau užbaigta laisvės teorija (turinti griežtą sandarą ir tik liepiamąją nuo­ 199 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 saką: jau nebe ženklų prie-vartą, o institutų prievartą), kuri pridėtinę vertę gauna iš kito pagrindinio demokratinės santvarkos principo, lygybės. Plg. su esminiu politinės filosofijos, fe­ nomenologinio ir egzistencinio intencionalumo bendrabuvojime principu: „Taigi pagrindinis demokratinės santvarkos principas yra laisvė“ (Aristotelis 1997; 264 [1317b 1–2]). Laisvė reikalauja aukščiausios moralinės laikysenos gręžiantis į transcendentalinio-aš ir empirinio- aš, laisvės ir būtinybės, asmens ir bendruomenės susidūrimo paradoksą, egzistencinį absurdą. O lygybė tenkinasi minimalia moraline laikysena, egalitarizmo potroškiu; verte, kuri naudinga, moraliniu požiūriu, minimalistinėms liberalizmo teorijoms dėl savo šiuo-laikiškumo, lygybės aistros (Hobbesą skaitant jau be vaizduotės, o kaip paliepimo taisyklę: kai pinigai metami daugumai, tereikia gebėti juos sugauti). Liberalizmas (ne filosofija, o pridėtinė jos vertė) yra stilistinė priemonė, vartojant tikslų Wittgensteino terminą, „išsigimusi hiperbolė“ (ten pat; 126 [19]). Jei Hobbeso filosofijoje „sudedama“ ir „atimama“, tai liberalizmo hiper­ bolėje – „perdedama“ siekiant didesnio įspūdžio ir įspūdžio valdymo (įtaigos). Liberalizmas yra eliptinis stilius, esminis trūkumas, esmė be esmės, kuri randasi iš negatyviosios laisvės darant pozityvų vardą, kuris nėra joks vardas. Jautri ir tolerantiška skirtumams „liberalioji sąžinė“ (Bauman 1996; 80) visiškai indiferen­ tiška sąsajoms. Kultūros „struktūros“, arba „mul­ tikultūralizmas“, liberalui svarbu ne todėl, kad turi savaiminę moralinę prigimtį, o todėl, kad turtinga ir saugi „kultūros struktūra“ suteikia žmogui galimybę tinkamai pasirinkti (Kymlicka 1989). Nors daugelį daugelį šimtmečių kultūra buvoja kaip kultūra, kaip esminės laikiškumo prasmės sąsajos, tačiau liberalizmo privačioje rekonstrukcjoje ji staiga virsta „struktūra“. Liberalizmas yra egalitarinė pagunda prieš­ tarauti (arba aversiškai arba paprasčiausiai: „ir aš noriu...“). Ypač tam kas vadinama laikiškumo, istoriškumo, priešduoties, tradicijos, kultūros, sąsajų – pirmtakų, amžininkų ir įpėdinių – vardu. Kodėl kyla tokia pagunda, kuri niekaip ir niekad nepatenkinama?: „Kaip tik todėl“ (Wittgenstein 1995; 139 [39]). Kodėl vieniems vaikams brendimo laikotarpiu, o kitiems iki žilo plauko norisi nerasti atsakymo „taip“, o rasti tik atsakymą „ne“? „Kaip tik todėl“ Kad tik: „Bu-bu-bu“ (ten pat). Liberalizmas (ne libera­ lizmo filosofija) yra brandaus amžiaus kalbinis žaidimas: „Sulaukei aštuoniolikos?“ – Tuomet: „Bu-bu-bu.“ Lengva skiemenuoti, nes trys skie­ menys: 1) laisvas individas, 2) laisva visuomenė, 3) pridėtinė laisvumo vertė. Galima tik numanyti, kaip „struktūra“ atsi­ skiria nuo kultūros: vienu ypu trukteli save pačią už plaukų ir ištempia iš ankančios tradicijos ba­ los. Z dalies tezės: Jei dabar pravertume liberalizmo prielaidų skrynelę, „nežinojimo uždangą“, ten rastume tik nieką, pliuralizmą, indiferentiškumą. Štai kodėl liberalizmas ir jo vedinys, pozityvizmas (atitinkamai: grynasis pliuralizmas ir grynasis indiferentiškumas) reikalauja skirti, o ne jungti du nelygius dalykus: tradiciją ir modernybę, pozityviąją laisvę ir negatyviąją laisvę. Skirti dvi vertes, kad rastųsi viena: aiški, ryški, aki­ vaizdi. Štai kodėl visi be išimties šiuo-laikiniai vadovėliai pabrėžia: modernybė randasi „pati iš savęs“. O drauge – jau ištempus save už plaukų iš ankančios tradicijos balos – negatyvioji laisvė 200 Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 Metodologija tampa antrąja pozityviąja (pirmoji pozityvioji išnyksta). įprastų vietų, gyventojais. Tam neprieštarauja ir „šiandieną mes visi – liberalai“ (Gray 1996; 18). Mes galime tapti stokais, kaip siūlo Johnas Gray’us (iš esmės taip visad buvo, o ir ne kitaip visad bus): „Mes visi priklausome daugeliui ben­ druomenių, ir susikertančių, ir nesiderinančių, tarp kurių išlieka tam tikros erdvės (some space) autonomiškam pasirinkimui“ (ten pat). Pasirinkimas, – išskyrus tikrai savigrąžų, egzistencinį pasirinkimą, kuris visada gali­ mas, – įprastoje kasdienybėje išnyksta. Arba / arba pakeičia ir / ir. Randasi reliatyvistinė (pomoderni) „skirtis“. Viskas – ir teisinga, ir neteisinga: nelygu kontekstas. Tai reiškia, kad liberalizmas virsta egalitarizmu, užmirš­ tančiu nelygybės formos išvaizdą, – o ką jau kalbėti apie jos spalvą, kvapą, skonį. Nelygu kontekstas, o ne nelygus kontekstas. Hobbeso „ir aš noriu, kad būtų nelygu“ virsta „ir aš noriu, kad būtų lygu“. Aš tik noriu, kad visur ir visada būtų vienoda, ir man būtų vienodai. Noriu tolerancijos sunkiai beišrandamam nevienodumui išlygintame vienodume. Kul­ tūros „struktūros“ susideda iš pavienių nelygu individų. Kultūra – niekad lygu. p p Mes galime tapti – dar kilniau – egzisten­ cialistais. Egzistencinė laisvė ragina skaidrinti gyvenimo-planą kiekvieną gyvenimo-valandą. Juk egzistencija, taigi ir laisvė, įsisąmoninina­ ma – visų pirma! – kaip savo paties tamsių kampų baimė, kuri sugriebiama kokiu nors savigrąžiu, bet ne savigražiu, būdu. Laisvei nereikia gražaus blizgesio. O nelaisvei ir lygybei – reikia. Laisvė gali išlikti esant bet kokioms sąlygoms, – ir netgi gyvenimo be spalvų. Juk ir nespalvoto baltuko ar naktinio pelėdgalvio laisvės esmė yra ypatingas jautrumas konstituacinėms savigrąžos „Jei..., tai...“, „Kaip..., taip...“, „Nes jei..., tai ir paskui...“ sąlygoms. Lygybės noras, arba reliatyvizmo noras (ne­ lygu kontekstas) yra vienintelis (nes absoliučiai indiferentiškas) liberalizmo noras. Vienintelis noras, dėl įvairovės vadinamas skirtingais vardais: multikultūralizmo, indiferentiškumo, negatyviosios laisvės, pliuralizmo, tolerancijos, nelygu gerėjimo... Z dalies tezės: Negatyvioji laisvė, individo grąžos nuo bendruomenės laisvė, išardžiusi tai, ką jos priemonėmis galima išardyti, atsigręžia prieš pačią save: gręžiantis nuo prasminių sąsajų gręžiamasi į konfliktą, kurio padariniai – skaida (ir patyrimo sąsajų dispersija, ir šio išsisklaidymo įtikrinimas, žinia-skaida), nedarna, išsklidimas, išsibarstymas, išdrikimas, iškrikimas, nerimas, nepasitikėjimas... 3 + 1 tezė (nebus per daug pakartoti, – ir dar kai ką pridurti) Laisvė yra grąžos fenomenas: ir savi-grąžos, ir bendra-grąžos. Laisvė yra prasminių tikrovės ir galimybės horizontų durų atverties drąsa. Pirmojo asmens šypsena nušviečiant – paliekant, bet nieko nepakeičiant – savo vaikišką baimės kampą. Laisvė – ne tik pirmojo asmens mįslė, bet ir priešduotos egzistencijos dovana: ne tik tikrovė, bet ir galimybė. Jei aš, gliaudydamas mįslę, drąsiai sulaikysiu įprastą, natūralų, kasdienį, malonų, kartotinį – „Žiūrėk, aš visąlaik darau tą patį: aš ...“ – abejonės šios sekos tikslingumu sulaikymą, kurį aš iki šiol vadinau protu (į-pro­ Absoliučiai indiferentiškame „struktūrų“ pasaulyje mes galime – vis dėlto galime (laisvė yra dar prieš lygybę, priešduotyje) – tapti vietų, 201 Metodologija Sociologija. Mintis ir veiksmas 2015/1(36), ISSN 1392-3358 tis – lygu protas), man gali nušvisti ne laisvė, o ne-laisvė. Gali atsiverti bedugnė, kuri yra ir už mano abejojimo, ir už neabejojimo ribų. Tik objektyvios sekos – 1 1 1 1 ... arba 2 2 2 2 (skirtumo tarp šių alternatyvų, matyt, niekas nežino ir turbūt nesužinos) – laikas. tis – lygu protas), man gali nušvisti ne laisvė, o ne-laisvė. Gali atsiverti bedugnė, kuri yra ir už mano abejojimo, ir už neabejojimo ribų. Tik objektyvios sekos – 1 1 1 1 ... arba 2 2 2 2 (skirtumo tarp šių alternatyvų, matyt, niekas nežino ir turbūt nesužinos) – laikas. Kilmės ištakas, arba kilnumo sąlygas, kurios nuskaidrins vaikiškos baimės – laisvės negalimu­ mo sekoje – kampą, išnarstys į-protį jo nesugriau­ damos. Iškels į dienos šviesą aki-vaizdą mano aki-ratyje. Kad laisvė galima tik egzistenciniame įgyvendinime: autentiškame pasirinkime įsipar­ eigojant proto ir vaizduotės, tikrovės ir galimybės, tradicijos ir tęstinumo santuokoms. Ir jei aš sau pasakysiu, kad nėra tokios sekos, kurios aš negalėčiau įveikti savo panieka arba savo griausmingu juoku, aš vėlgi nesužinosiu tiesos, – ar tik ne toji pati seka priveda mane prie paniekos ar juoko? Laisvė yra grąžos fenomenas: ir savi-grąžos, ir bendra-grąžos. Galima pasakyti ir kitaip: ir savigrąžos, ir bendragrąžos. O tai reiškia: Lais­ vę nuo ne-laisvės skiria vos mažas brūkšnelis. Nelaisvę nuo laisvės neskiria niekas. Niekas yra matomas, bet nepastebimas savaime supranta­ mumas. Vadinasi, laisvė yra štai-čia: įprastume, paprastume, kasdieniškume... Štai-čia tikrovėje: savi-grąžos ir bendra-grąžos galimybėje... Tačiau kai, – supratęs, kad susidūriau su pas­ kesnio proto (savo įpročio) spąstais ankstesnėje laisvės mįslėje, – gręžiuosi savęsp dar stipriau, artėju – kitaip ir negali būti! – prie proto kilmės. Artėju – jausdamas, o ne žinodamas: jei aptiksiu proto kilmę, tai galbūt surasiu kilnumo ištakas? 202
https://openalex.org/W2930056136
https://europepmc.org/articles/pmc6825946?pdf=render
English
null
Fat‐specific protein 27α inhibits autophagy‐dependent lipid droplet breakdown in white adipocytes
Journal of diabetes investigation
2,019
cc-by
8,898
Keywords Aims/Introduction: Fat-specific protein 27 (FSP27) a is the major isoform of FSP27 in white adipose tissue (WAT), and is essential for large unilocular lipid droplet (LD) formation in white adipocytes. In contrast, FSP27b is abundantly expressed in brown adipose tissue (BAT), and plays an important role in small multilocular LD formation. In FSP27 KO mice in which FSP27a and b are both depleted, WAT is characterized by multilocular LD forma- tion, and by increased mitochondrial abundance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs and reduced energy expenditure. *Correspondence Yoshikazu Tamori Tel.: +81-78-382-5861 Fax: +81-78-382-2080 E-mail address: tamori@med.kobe-u.ac.jp J Diabetes Investig 2019; 10: 1419–1429 doi: 10.1111/jdi.13050 Materials and Methods: We investigated the effects of autophagy in WAT and BAT of wild type (WT) and FSP27 knockout (KO) mice. In addition, we examined the effects of FSP27a and FSP27b to the induction of autophagy in COS cells. Results: Food deprivation induced autophagy in BAT of WT mice, as well as in WAT of FSP27 KO mice, suggesting that enhanced autophagy is characteristic of adipocytes with small multilocular LDs. Pharmacological inhibition of autophagy attenuated the fasting- induced loss of LD area in adipocytes with small multilocular LDs (BAT of WT mice and WAT of FSP27 KO mice), without affecting that in adipocytes with large unilocular or oligolocular LDs (WAT of WT mice or in BAT of FSP27 KO mice). Overexpression of FSP27a inhibited autophagy induction by serum deprivation in COS cells, whereas that of FSP27b had no such effect. Conclusions: The present results thus showed that FSP27a inhibits autophagy and might thereby contribute to the energy-storage function of WAT. ª 2019 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd J Diabetes Investig Vol. 10 No. 6 November 2019 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. Fat-specific protein 27a inhibits autophagy- dependent lipid droplet breakdown in white adipocytes Nishimoto1, Sanshiro Tateya1,2, Yasuyuki Iwahashi1, Yuko Okamatsu-Ogura3, Masayuki Saito3, k T i14* 1Department of Internal Medicine, Division of Diabetes and Endocrinology, Kobe University Graduate School of Medicine, Kobe, 2Department of Internal Medicine, Division of Diabetes, Kakogawa Central City Hospital, Kakogawa, 3Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, and 4Department of Social/Community Medicine and Health Science, Division of Creative Health Promotion, Kobe University Graduate School of Medicine, Kobe, Japan ORIGINAL ARTICLE Received 10 December 2018; revised 26 February 2019; accepted 19 March 2019 INTRODUCTION The a isoform of fat-specific protein 27 (FSP27a), which belongs to the cell death-inducing deoxyribonucleic acid (DNA) fragmentation factor A-like effector (Cide) family of proteins, has been shown to be indispensable for large unilocular LD formation in WAT3–6. We also recently found that FSP27b, a novel isoform of FSP27 that is abundant in BAT, plays a key role in small multi- locular LD formation in this tissue by inhibiting the homodimer- ization of CideA7. FSP27a and FSP27b are the splice variants driven by distinct promoters from the same gene8. Thus, FSP27a and FSP27b are both deficient in the FSP27 knockout (KO) mice we produced3. In FSP27 KO mice, WAT is characterized by mul- tilocular LD formation, and by increased mitochondrial abun- dance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs, and reduced mitochondrial Mammals possess two main types of adipose tissue with dis- tinct functions. White adipose tissue (WAT) stores energy in the form of triglyceride (TG) for expenditure during periods of food deprivation, whereas brown adipose tissue (BAT) con- sumes stored energy for heat production in a cold environ- ment1. These two types of adipose tissue also differ in the morphology of intracellular lipid droplets (LDs), which are large and unilocular in WAT, and small and multilocular in BAT2, with this difference likely reflecting the metabolic charac- teristics of the adipocytes. Received 10 December 2018; revised 26 February 2019; accepted 19 March 2019 1419 O R I G I N A L A R T I C L E Nakajima et al. O R I G I N A L A R T I C L E http://wileyonlinelibrary.com/journal/jdi Nakajima et al. RNeasy kit (Qiagen, Hilden, Germany), and the resulting com- plementary DNA was subjected to real-time polymerase chain reaction (PCR) analysis with specific primers (Table 1) and Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) in a Sequence Detector (model 7500; PE Applied Biosystems). The abundance of target messenger RNAs (mRNAs) was normalized by that of 36B4 mRNA as the invariant control. abundance and energy expenditure3. Unilocular LD formation might contribute to efficient lipid storage in WAT, because lipoly- sis from the LD surface is restricted by the minimal surface area, with the free fatty acids (FFAs) and glycerol generated by TG hydrolysis then entering the circulation for transport to other tis- sues. Cell culture White adipocyte (HW) and brown adipocyte (HB2) cells were prepared from epididymal WAT (eWAT) and interscapular BAT, respectively, of homozygous p53 KO mice, as described previously16. The cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supple- mented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), streptomycin (50 lg/mL) and penicillin (50 U/mL). For induction of adipocyte differentiation, complete medium sup- plemented with 0.5 mmol/L 3-isobutyl-1-methylxanthine and 1 lmol/L dexamethasone was added to the cells at confluence. After 2 days, the medium was changed to Dulbecco’s modified Eagle’s medium supplemented with 50 nmol/L triiodothyronine and insulin (10 lg/mL), and was refreshed every 2 days. The cells were subjected to experiments at 6–8 days after the onset of differentiation induction. COS cells were maintained in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. types of adipose tissue . Hydrolysis of TG in LDs is generally mediated by cytosolic lipases9. However, LDs also serve as a substrate for macroau- tophagy. Autophagy is a lysosomal degradative pathway for the removal and breakdown of cellular components, such as orga- nelles and proteins, that are especially important during periods of food deprivation. Lipolysis and autophagy share certain simi- larities, with both processes being important for adaptation to nutrient deprivation. In addition, autophagy has been shown to play an important role in lipid metabolism and storage10. For example, inhibition of autophagy was found to increase lipid accumulation in the liver of mice11, whereas activation of autop- hagy reduced hepatocellular lipid accumulation and lipotoxic- ity12. Autophagy contributes to the regulation of adipocyte differentiation13,14 and the beige-to-white fat transition15, but it has remained unclear whether it also plays a role in lipid meta- bolism in mature adipocytes. We have now investigated the pos- sible contribution of autophagy to lipid degradation in WAT, which supplies FFAs to other tissues during food deprivation, as well as to that in BAT, which metabolizes FFAs during exposure to cold environments. Immunoblot analysis h Tissue homogenates were prepared with the use of a Teflon homogenizer in a lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 250 mmol/L NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/L ethylene- diaminetetraacetic acid, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride and 1% protease inhibitors (Sigma-Aldrich). Lysates of HW, HB2 or COS cells were INTRODUCTION In contrast, small multilocular LD formation might promote efficient intracellular lipolysis from the LD surface and subse- quent transport of FFAs to adjacent mitochondria for b-oxidation in BAT. Intracellular LD morphology and lipolysis are thus clo- sely related to the functions and characteristics of the different types of adipose tissue2. RNeasy kit (Qiagen, Hilden, Germany), and the resulting com- plementary DNA was subjected to real-time polymerase chain reaction (PCR) analysis with specific primers (Table 1) and Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) in a Sequence Detector (model 7500; PE Applied Biosystems). The abundance of target messenger RNAs (mRNAs) was normalized by that of 36B4 mRNA as the invariant control. Overexpression of FSP27 and immunofluorescence analysis in COS cells We next examined the expression of autophagy-related pro- teins by immunoblot analysis. The amounts of Beclin1 (Atg6), FOXO1, the lipidated form of LC3 (LC3-II), and both total and phosphorylated (p) forms of AMPKa were increased in eWAT and sWAT, but decreased in BAT of FSP27 KO mice compared with WT mice (Figure 1d). Conversely, the abun- dance of p62 was decreased in eWAT and sWAT, but increased in BAT of FSP27 KO mice (Figure 1d). Overall, these results were thus consistent with the notion that autophagy is enhanced in WAT and downregulated in BAT of FSP27 KO mice. Full-length complementary DNAs for mouse FSP27a or FSP27b were subcloned into the expression plasmid pIRES2-DsRed2 (Clontech, Mountain View, CA, USA) 7, and the resulting vectors were introduced into COS cells with the use of the X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science, Penzberg, Upper Bavaria, Germany). The cells were subsequently fixed with phosphate-buffered saline (PBS) containing 4% paraformalde- hyde at room temperature, washed with PBS and exposed to PBS containing 5% bovine serum albumin. For immunostaining of LC3, the cells were permeabilized with 0.2% Triton X-100 for 5 min, exposed to PBS containing 5% bovine serum albumin, and then incubated consecutively with rabbit antibodies to LC3 (Medical & Biological Laboratories) and Dylight405-conjugated goat antibodies to rabbit immunoglobulin G (ThermoScientific, Inhibition of autophagy in mice FSP27 KO mice were generated as described previously3. The mice used in the present study are all male. Mice at 12 weeks of age were injected intraperitoneally with the lysosomal pro- tease inhibitor, leupeptin (40 mg/kg), at 0, 4, 8 and 12 h after the onset of food deprivation, and were analyzed at 14 h. All mouse experiments were approved by the Animal Ethics Com- mittee of Kobe University Graduate School of Medicine (Regis- tration No: A170314). Autophagy is increased in WAT, but attenuated in BAT of FSP27 KO mice We first examined the expression of autophagy-related genes in eWAT, sWAT and BAT of food-deprived FSP27 KO and WT mice by quantitative RT–PCR analysis. The amounts of mRNAs for autophagy-related protein 7 (Atg7)17, the autop- hagy regulator Gabarapl118, the autophagy mediator FOXO119 and SIRT1, which mediates histone deacetylation at autop- hagy-related genes20, were all significantly increased in eWAT and sWAT of FSP27 KO mice compared with WT mice (Figure 1a,b). The expression of genes for p62, an adaptor protein that functions coordinately with the autophagy medi- ator LC321; for the autophagy initiator Unc51-like kinase 1 (Ulk1)22; and for AMPKa1, which promotes autophagy by activating Ulk1 through direct phosphorylation23, was also increased in eWAT of FSP27 KO mice (Figure 1a). In BAT, however, the abundance of Gabarapl1, p62, Ulk1, Sirt1 and AMPKa1 mRNAs was decreased in FSP27 KO mice com- pared with WT mice (Figure 1c). These results suggested that the extent of autophagy is increased in WAT, but attenuated in BAT of FSP27 KO mice. With regard to lipases, the amount of mRNA for adipose triacylglycerol lipase (ATGL), a promoter of autophagy24, was increased in eWAT and sWAT of FSP27 KO mice, whereas that of lysosomal acid lipase mRNA was increased in eWAT, and that of hormone-sensitive lipase mRNA was unaffected in adipose tissues of FSP27 KO mice (Figure 1a,b). Quantitative reverse transcription polymerase chain reaction analysis 6 November 2019 O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Autophagy in adipocytes http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocytes Waltham, MA, USA). The cells were finally examined with a confocal laser-scanning microscope (BZ-X710; Keyence). Waltham, MA, USA). The cells were finally examined with a confocal laser-scanning microscope (BZ-X710; Keyence). Waltham, MA, USA). The cells were finally examined with a confocal laser-scanning microscope (BZ-X710; Keyence). prepared with a lysis buffer containing 25 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Nonidet P40, 1 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride and 1% protease inhibitors. Pri- mary antibodies for immunoblot analysis included rabbit poly- clonal antibodies to FSP27 generated as described previously3; rabbit antibodies to p62 and to Beclin1, as well as mouse anti- bodies to LC3 from Medical & Biological Laboratories (Aichi, Japan); rabbit antibodies to forkhead box protein O1 (FOXO1) and to total or phosphorylated adenosine monophosphate-acti- vated protein kinase-a (AMPKa) from Cell Signaling Technol- ogy (Danvers, MA, USA); and mouse antibodies to a-tubulin and to b-actin from Sigma-Aldrich. Immune complexes were detected with horseradish peroxidase-conjugated secondary anti- bodies and enhanced chemiluminescence reagents (GE Health- care, Chicago, IL, USA). Statistical analysis Quantitative data are presented as the mean + standard error of the mean, and were compared between two groups with the use of the two-tailed Student’s t-test. A P-value of <0.05 was considered statistically significant. Histological analysis Epididymal WAT, subcutaneous WAT (sWAT) and interscapu- lar BAT of mice were fixed with 10% formalin, embedded in paraffin, sectioned at a thickness of 6 lm and mounted on glass slides according to standard procedures. The sections were stained with hematoxylin–eosin and were examined with a BZ- X710 microscope (Keyence, Osaka, Japan) for determination of LD area. Quantitative reverse transcription polymerase chain reaction analysis Quantitative reverse transcription polymerase chain reaction analysis Total ribonucleic acid (RNA) extracted from adipose tissues was subjected to reverse transcription (RT) with the use of an Table 1 | Primer sequences for quantitative reverse transcription polymerase chain reaction analysis mRNA Sense (50?30) Antisense (50?30) Atg7 TCCGTTGAAGTCCTCTGCTT CCACTGAGGTTCACCATCCT p62 TGTGGAACATGGAGGGAAGAG TGTGCCTGTGCTGGAACTTTC Ulk1 AGATTGCTGACTTTGGATTC AGCCATGTACATAGGAGAAC Gabarapl1 TCGTGGAGAAGGCTCCTAAA ATACAGCTGGCCCATGGTAG AMPKa1 GCTCACCCAACTATGCTGCAC TATCTACCTCTGGGCCTGCATACAA Sirt1 GACGGTATCTATGCTCGCCT ATTCCTGCAACCTGCTCCAAG FOXO1 ACATTTCGTCCTCGAACCAGCTCA ATTTCAGACAGACTGGGCAGCGTA ATGL GGAGACCAAGTGGAACATCTCA AATAATGTTGGCACCTGCTTCA HSL TGTGGCACAGACCTCTAAAT GGCATATCCGCTCTC LAL GACCACTCCCGATGCAACTC GACCACTCCTTGTGAGCCAG AMPKa1, adenosine monophosphate-activated protein kinase-a; Atg7, autophagy-related protein 7; ATGL, adipose triacylglycerol lipase; FOXO1, fork- head box protein O1; Gabarapl1, GABA type A receptor-associated protein like 1; HSL, hormone-sensitive lipase; LAL, lysosomal acid lipase; mRNA, messenger ribonucleic acid; Sirt1, silent mating type information regulation 2 homolog 1; Ulk1, Unc51-like kinase 1. Table 1 | Primer sequences for quantitative reverse transcription polymerase chain reaction analysis Table 1 | Primer sequences for quantitative reverse transcription polymerase chain reaction analysis Table 1 | Primer sequences for quantitative reverse transcription polymerase chain reaction analysis mRNA Sense (50?30) Antisense (50?30) Atg7 TCCGTTGAAGTCCTCTGCTT CCACTGAGGTTCACCATCCT p62 TGTGGAACATGGAGGGAAGAG TGTGCCTGTGCTGGAACTTTC Ulk1 AGATTGCTGACTTTGGATTC AGCCATGTACATAGGAGAAC Gabarapl1 TCGTGGAGAAGGCTCCTAAA ATACAGCTGGCCCATGGTAG AMPKa1 GCTCACCCAACTATGCTGCAC TATCTACCTCTGGGCCTGCATACAA Sirt1 GACGGTATCTATGCTCGCCT ATTCCTGCAACCTGCTCCAAG FOXO1 ACATTTCGTCCTCGAACCAGCTCA ATTTCAGACAGACTGGGCAGCGTA ATGL GGAGACCAAGTGGAACATCTCA AATAATGTTGGCACCTGCTTCA HSL TGTGGCACAGACCTCTAAAT GGCATATCCGCTCTC LAL GACCACTCCCGATGCAACTC GACCACTCCTTGTGAGCCAG AMPKa1, adenosine monophosphate-activated protein kinase-a; Atg7, autophagy-related protein 7; ATGL, adipose triacylglycerol lipase; FOXO1, fork- head box protein O1; Gabarapl1, GABA type A receptor-associated protein like 1; HSL, hormone-sensitive lipase; LAL, lysosomal acid lipase; mRNA, messenger ribonucleic acid; Sirt1, silent mating type information regulation 2 homolog 1; Ulk1, Unc51-like kinase 1. AMPKa1, adenosine monophosphate-activated protein kinase-a; Atg7, autophagy-related protein 7; ATGL, adipose triacylglycerol lipase; FOXO1, fork- head box protein O1; Gabarapl1, GABA type A receptor-associated protein like 1; HSL, hormone-sensitive lipase; LAL, lysosomal acid lipase; mRNA, messenger ribonucleic acid; Sirt1, silent mating type information regulation 2 homolog 1; Ulk1, Unc51-like kinase 1. AMPKa1, adenosine monophosphate-activated protein kinase-a; Atg7, autophagy-related protein 7; ATGL, adipose triacylglycerol lipase; FOXO1, fork- head box protein O1; Gabarapl1, GABA type A receptor-associated protein like 1; HSL, hormone-sensitive lipase; LAL, lysosomal acid lipase; mRNA, messenger ribonucleic acid; Sirt1, silent mating type information regulation 2 homolog 1; Ulk1, Unc51-like kinase 1. ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd 1420 J Diabetes Investig Vol. 10 No. ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd Autophagy is enhanced in a cultured brown adipocyte cell line The present results also suggested that autophagy is increased in BAT compared with WAT in WT mice (Figure 1d). To test J Diabetes Investig Vol. 10 No. 6 November 2019 1421 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd J Diabetes Investig Vol. 10 No. 6 November 2019 1421 O R I G I N A L A R T I C L E Nakajima et al. O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Nakajima et al. http://wileyonlinelibrary.com/journal/jdi http://wileyonlinelibrary.com/journal/jdi 6 (a) (d) 5 Relative mRNA amount 4 3 eWAT WT KO KO eWAT sWAT BAT WT Beclin1 p62 FOXO1 AMPKα KO WT KO WT Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 ** ** ** ** ** ** ** 2 1 0 * * 6 (a) (d) (e) (b) (c) 5 Relative mRNA amount 4 3 eWAT WT KO KO eWAT sWAT BAT WT Beclin1 p62 FOXO1 AMPKα pAMPKα α-tubulin LC3 I II KO WT KO WT WT KO WT HW HB2 β-actin LC3 I II β α FSP27 KO BAT sWAT Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 * * * * * * ** ** ** ** ** ** ** ** ** ** ** 2 1 0 5 Relative mRNA amount Relative mRNA amount 4 3 2 1 0 2.0 1.5 1.0 0.5 0 * * 1422 J Diabetes Investig Vol. 10 No. 6 November 2019 ª 2019 The Authors. Autophagy is enhanced in a cultured brown adipocyte cell line Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, (d) (e) (b) pAMPKα α-tubulin LC3 I II WT KO HW HB2 sWAT Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 * * * ** ** 5 Relative mRNA amount 4 3 2 1 0 (e) (c) WT HW HB2 β-actin LC3 I II β α FSP27 KO BAT Gab Am Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 * * * ** ** Relative mRNA amount 2.0 1.5 1.0 0.5 0 (c) WT KO BAT Atg7 Lal Sirt1 Foxo1 UlK1 p62 Gabarapl1 Hsl Atgl Ampkα1 * * * ** ** Relative mRNA amount 2.0 1.5 1.0 0.5 0 1422 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd J Diabetes Investig Vol. 10 No. 6 November 2019 O R I G I N A L A R T I C L E http://wileyonlinelibrary.com/journal/jdi http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocytes Figure 1 | Enhancement of autophagy in white adipose tissue (WAT) of fat-specific protein 27 (FSP27) knockout (KO) mice and brown adipose tissue (BAT) of wild-type (WT) mice. (a–c) Quantitative reverse transcription polymerase chain reaction analysis of messenger ribonucleic acids (mRNAs) for autophagy-related proteins in (a) epididymal WAT (eWAT), (b) subcutaneous WAT (sWAT) and (c) BAT of WT or FSP27 KO mice at 12 weeks of age that had been deprived of food for 14 h. Data were normalized by the amount of 36B4 mRNA, are expressed relative to the corresponding value for WT mice and are the mean + standard error of the mean (n = 6 mice per genotype). *P < 0.05, **P < 0.01 versus the corresponding value for WT mice. (d) Immunoblot analysis of autophagy-related proteins and a-tubulin (loading control) in eWAT, sWAT and BAT of 12-week-old WT or FSP27 KO mice that had been deprived of food for 14 h. (e) Immunoblot analysis of LC3, FSP27 and b-actin (loading control) in white adipocyte (HW) and brown adipocyte (HB2) cells cultured in serum-free medium for 40 h. AMPK, adenosine monophosphate- activated protein kinase-a; FOXO1, forkhead box protein O1. We next investigated LDs in the treated mice. Autophagy is enhanced in a cultured brown adipocyte cell line LD area was greater in eWAT and sWAT of FSP27 KO mice treated with leupeptin than in those injected with saline (Figure 3b,c,d), whereas inhibition of autophagy by leupeptin did not affect LD morphology in WAT of WT mice (Figure 3a,c,d). Conversely, the LD area was greater in BAT of WT mice treated with leu- peptin than in those injected with saline (Figure 3a,e). The pre- sent results showing that autophagy is augmented in WAT of FSP27 KO mice and in BAT of WT mice thus suggested that increased autophagy might contribute to the fasting-induced decrease in LD area in cells with multilocular LDs. To exclude the possibility that the effects of leupeptin on LD area in WAT of FSP27 KO mice and in BAT of WT mice were attributable to changes in the expression of cytosolic lipases, we measured the abundance of ATGL and hormone-sensitive lipase mRNAs. Quantitative RT–PCR analysis showed no significant effects of leupeptin on the amounts of these mRNAs in WAT or BAT of FSP27 KO and WT mice, respectively (Figure 3f). this possibility further, we examined autophagy in HW and HB2 cell lines that were established from p53 KO mice. With regard to expression of FSP27, HW and HB2 adipocytes pre- dominantly expressed FSP27a and FSP27b, respectively (Fig- ure 1e), which are the major isoforms in WAT and BAT, respectively7 – indicating that these cell lines retain the charac- teristics of the parental adipocytes. Immunoblot analysis of serum-deprived cells with antibodies to LC3 showed that autophagy was increased in HB2 cells compared with HW cells (Figure 1e), consistent with the conclusion that autophagy is enhanced in brown adipocytes. this possibility further, we examined autophagy in HW and HB2 cell lines that were established from p53 KO mice. With regard to expression of FSP27, HW and HB2 adipocytes pre- dominantly expressed FSP27a and FSP27b, respectively (Fig- ure 1e), which are the major isoforms in WAT and BAT, respectively7 – indicating that these cell lines retain the charac- teristics of the parental adipocytes. Immunoblot analysis of serum-deprived cells with antibodies to LC3 showed that autophagy was increased in HB2 cells compared with HW cells (Figure 1e), consistent with the conclusion that autophagy is enhanced in brown adipocytes. Pharmacological inhibition of autophagy attenuates fasting- induced changes in eWAT weight as well as LD area in white adipocytes of FSP27 KO mice We previously showed that WAT of FSP27 KO mice is charac- terized by multilocular LD formation, whereas BAT of these mice conversely manifests large oligolocular LDs3.To investigate the effects of autophagy on lipid storage in FSP27 KO mice, we injected food-deprived WT and mutant mice with saline (con- trol) or with the protease inhibitor, leupeptin, to inhibit autop- hagy in WAT and BAT (Figure 2a). Consistent with the results shown in Figure 1d, immunoblot analysis with antibodies to LC3 showed that autophagy was enhanced in WAT and atten- uated in BAT of saline-treated FSP27 KO mice compared with WT mice, as well as enhanced in BAT compared with WAT of WT mice (Figure 2b). Injection with leupeptin increased the amount of LC3-II in each adipose tissue of FSP27 KO mice, as well as in BAT of WT mice (Figure 2b), suggesting that leu- peptin inhibited autophagy before breakdown of the contents of autolysosomes. There were no significant changes in body- weight after 14 h fasting between saline injection and leupeptin injection in WT mice and in FSP27 KO mice (Figure 2c). However, the weight of eWAT at 14 h after the onset of food deprivation was greater in FSP27 KO mice treated with leu- peptin than in those injected with saline (Figure 2d). There were no significant differences in the weight of sWAT and BAT between saline injection and leupeptin injection in WT mice and FSP27 KO mice (Figure 2d). In addition, no signifi- cant differences were detected in the weight of liver, kidney, heart and gastrocnemius after 14 h fasting between saline injec- tion and leupeptin injection in WT mice and FSP27 KO mice (Figure S1). Overexpression of FSP27a inhibits autophagy induction by serum deprivation in COS cells p The present results showed that autophagy is restrained in cells with large unilocular LDs, suggesting the possibility that FSP27a, the white adipocyte-specific isoform of FSP27, might inhibit autophagy. We therefore investigated whether FSP27a can indeed inhibit autophagy in COS cells. We first confirmed that serum deprivation for 18 h markedly increased LC3 immunostaining in the presence of the autophagy inhibitor, chloroquine (Figure 4a), suggesting that serum deprivation induces autophagy in COS cells. Immunoblot analysis with antibodies to LC3 also showed that serum deprivation increased autophagic flow in COS cells (Figure 4b). We then transfected COS cells with an expression vector for both FSP27a and the fluorescent protein, DsRed2, and examined the possible effect of FSP27a overexpression on autophagy induced by serum deprivation. Immunofluorescence analysis showed that LC3 staining was attenuated in serum-deprived cells overexpressing FSP27a compared with that apparent in cells negative for DsRed2 fluorescence (Figure 4c,d), suggesting that FSP27a inhibited autophagy. In contrast, overexpression of FSP27b had no apparent effect on autophagy (Figure 4c,d). These results might thus account for our observation that autophagy is enhanced in BAT, in which FSP27b is predominantly 1423 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd J Diabetes Investig Vol. 10 No. 6 November 2019 O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E N k ji l O R I G I N A L A R T I C L E Nakajima et al. http://wileyonlinelibrary.com/journal/jdi Nakajima et al. 14 h fasting (a) (b) (c) (d) 4 h 4 h 4 h 2 h Analysis Saline or leupeptin i.p. Overexpression of FSP27a inhibits autophagy induction by serum deprivation in COS cells FSP27KO mice WT mice eWAT WT Leupeptin: LC3 I II 30 20 10 Bodyweight at base line (g) Bodyweight after 14 h fasting (g) 0 30 20 10 Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin KO WT 160 40 30 20 10 0 60 50 30 40 20 10 0 60 60 80 50 30 40 40 20 20 10 0 0 140 120 100 80 60 40 20 0 120 Weight of eWAT (mg) Weight of sWAT (mg) Weight of BAT (mg) 80 40 0 KO ** WT KO WT KO WT 0 KO – + – + – + – + – + – + WT KO WT KO sWAT BAT KO WT Figure 2 | Effects of pharmacological inhibition of autophagy on fasting-induced changes in body and adipose tissue weight in fat-specific protein 27 (FSP27) knockout (KO) and wild-type (WT) mice. (a) Experimental schedule for intraperitoneal (i.p.) injection of leupeptin or saline in 12- week-old mice deprived of food. (b) Immunoblot analysis of LC3 in epididymal white adipose tissue (eWAT), subcutaneous WAT (sWAT) and brown adipose tissue (BAT) of WT or FSP27 KO mice at the end of the experimental period. (c) Bodyweight at the baseline and the end of the experimental period and (d) adipose tissue weight of mice at the end of the experimental period. Data are the mean + standard error of the mean (n = 5 mice per group). **P < 0.01 versus the corresponding value for saline. 14 h fasting (a) (b) 4 h 4 h 4 h 2 h Analysis Saline or leupeptin i.p. Overexpression of FSP27a inhibits autophagy induction by serum deprivation in COS cells 6 November 2019 1424 O R I G I N A L A R T I C L E http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocytes WT (a) (c) (f) (d) (e) (b) 1,000 800 LD area in sWAT (µm2) LD area in BAT (µm2) LD area in eWAT (µm2) Relative mRNA amount Relative mRNA amount Relative mRNA amount 600 400 200 0 16 12 8 4 0 WT KO + – + – Leupeptin: WT sWAT of KO BAT of WT eWAT of KO KO + – + – Leupeptin: WT KO + – + – Leupeptin: ATGL HSL + – + – Leupeptin: ATGL HSL + – + – Leupeptin: ATGL HSL + – + – Leupeptin: * * * 16 400 300 200 100 0 300 200 150 250 100 50 0 30 20 15 25 10 5 0 3.0 2.0 1.5 2.5 1.0 0.5 0 2.0 1.5 1.0 0.5 0 2.0 1.5 1.0 0.5 0 12 8 4 0 KO Saline Leupeptin Leupeptin Saline eWAT sWAT BAT WT (a) (b) KO Saline Leupeptin Leupeptin Saline eWAT sWAT BAT WT (a) Leupeptin Saline eWAT sWAT BAT (a) (b) KO b) KO Saline Leupeptin (d) (e) LD area in sWAT (µm2) LD area in BAT (µm2) 16 12 8 4 0 WT KO + – + – WT KO + – + – Leupeptin: WT KO + – + – Leupeptin: * * * 16 400 300 200 100 0 300 200 150 250 100 50 0 30 20 15 25 10 5 0 12 8 4 0 (c) 1,000 800 LD area in eWAT (µm2) 600 400 200 0 16 12 8 4 0 WT KO + – + – Leupeptin: * (d) LD area in sWAT (µm2) WT KO + – + – Leupeptin: * 16 400 300 200 100 0 12 8 4 0 (e) LD area in BAT (µm2) WT KO + – + – Leupeptin: * 300 200 150 250 100 50 0 30 20 15 25 10 5 0 (e) (d) (c) Relative mRNA amount BAT of WT ATGL HSL + – + – Leupeptin: 2.0 1.5 1.0 0.5 0 (f) Relative mRNA amount eWAT of KO ATGL HSL + – + – Leupeptin: 3.0 2.0 1.5 2.5 1.0 0.5 0 Relative mRNA amount sWAT of KO ATGL HSL + – + – Leupeptin: 2.0 1.5 1.0 0.5 0 (f) ª 2019 The Authors. Overexpression of FSP27a inhibits autophagy induction by serum deprivation in COS cells FSP27KO mice WT mice eWAT WT Leupeptin: LC3 I II KO – + – + – + – + – + – + WT KO WT KO sWAT BAT (a) (b) Leupeptin: LC3 I II – + – + Bodyweight after 14 h fasting (g) 30 20 10 Saline Leupeptin Saline Leupeptin 0 KO WT (c) 30 20 10 Bodyweight at base line (g) 0 Saline Leupeptin Saline Leupeptin KO WT (d) Saline Leupeptin Saline Leupeptin 160 40 30 20 10 0 120 Weight of eWAT (mg) 80 40 0 KO ** WT ) Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin 160 40 30 20 10 0 60 50 30 40 20 10 0 60 60 80 50 30 40 40 20 20 10 0 0 140 120 100 80 60 40 20 0 120 g ( g) Weight of sWAT (mg) Weight of BAT (mg) 80 40 0 KO ** WT KO WT KO WT Saline Leupeptin Saline Leupeptin Saline Leupeptin Saline Leupeptin 60 50 30 40 20 10 0 60 60 80 50 30 40 40 20 20 10 0 0 140 120 100 80 60 40 20 0 Weight of sWAT (mg) Weight of BAT (mg) KO WT KO WT n Saline Leupeptin Saline Leupeptin 60 60 80 50 30 40 40 20 20 10 0 0 Weight of BAT (mg) KO WT Figure 2 | Effects of pharmacological inhibition of autophagy on fasting-induced changes in body and adipose tissue weight in fat-specific protein 27 (FSP27) knockout (KO) and wild-type (WT) mice. (a) Experimental schedule for intraperitoneal (i.p.) injection of leupeptin or saline in 12- week-old mice deprived of food. (b) Immunoblot analysis of LC3 in epididymal white adipose tissue (eWAT), subcutaneous WAT (sWAT) and brown adipose tissue (BAT) of WT or FSP27 KO mice at the end of the experimental period. (c) Bodyweight at the baseline and the end of the experimental period and (d) adipose tissue weight of mice at the end of the experimental period. Data are the mean + standard error of the mean (n = 5 mice per group). **P < 0.01 versus the corresponding value for saline. ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd J Diabetes Investig Vol. 10 No. DISCUSSION p g p p y Adipocytes in visceral fat of Atg4b KO mice, which manifest limited autophagic activity, were recently found to undergo a larger increase in size in response to feeding with a high-calorie diet compared with those in control mice27. In addition, phar- macological stimulation of autophagy with spermidine resulted in an increase in the level of autophagy and a decrease in cell size in visceral fat of WT mice27. These data suggest that fat content is tightly associated with autophagic activity in fat cells. In contrast, several studies have shown the importance of autophagy in adipocyte differentiation and the beige-to-white fat transition13–15. For example, inhibition of autophagy in 3T3- L1 preadipocytes, either pharmacologically or by knockdown of Atg5 or Atg7, blocked their differentiation into mature adipo- cytes and, hence, the increase in their lipid-storing capacity13,14. An adipogenic role for autophagy was further suggested by analysis of adipocyte-specific Atg7 KO mice, which manifest reduced WAT mass and a lean, insulin-sensitive phenotype14. Loss of WAT mass in these mice was accompanied by increased BAT-like features in white adipose depots13. Given that these various studies were carried out with undifferentiated adipocytes or with mice generated by germline genetic manipu- lation, it was difficult to evaluate the effects of autophagy alone on the physiology of mature adipocytes independent of the effect on adipocyte differentiation. However, a recent study showed that cold exposure activated autophagy in BAT of con- trol mice, and that prevention of autophagy by genetic ablation of Atg7 specifically in BAT of adult mice reduced LD turnover and blocked lipophagy28. Conversely, activation of autophagy induced breakdown of LDs in BAT28. In the present study, we We have shown that autophagy is enhanced in WAT, especially in eWAT, of FSP27 KO mice and in BAT of WT mice. In addition, overexpression of FSP27a, the major isoform of FSP27 in WAT, inhibited the induction of autophagy by serum derivation in COS cells, whereas overexpression of FSP27b, the major isoform in BAT, had no such effect. Given that WAT in FSP27 KO mice and BAT in WT mice both show multilocular LD formation and contain abundant mitochondria, the enhanced autophagy apparent in these adipocytes might be related to increased metabolic capacity. y Autophagy is an evolutionarily conserved process that is essential for cell survival during periods of nutrient deprivation. O R I G I N A L A R T I C L E Nakajima et al. http://wileyonlinelibrary.com/journal/jdi Figure 3 | Effects of pharmacological inhibition of autophagy on fasting-induced changes in lipid droplet (LD) area in white adipose tissue (WAT) and brown adipose tissue (BAT) of fat-specific protein 27 (FSP27) knockout (KO) and wild-type (WT) mice. Hematoxylin–eosin staining of sections of epididymal WAT (eWAT), subcutaneous WAT (sWAT) and BAT of (a) WT or (b) FSP27 KO mice treated as in Figure 2a. Scale bars, 100 μm. Mean LD area of (c) eWAT, (d) sWAT and (e) BAT was quantitated from images similar to those in (a) and (b). Data are the mean + standard error of the mean (n = 5 mice per group). *P < 0.05 versus the corresponding value for saline. (f) Quantitative reverse transcription polymerase chain reaction analysis of adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) messenger ribonucleic acids (mRNAs) in eWAT and sWAT of FSP27 KO mice or in BAT of WT mice as in (a) and (b). Data were normalized by the amount of 36B4 mRNA, are expressed relative to the corresponding value for saline-treated control mice and are the mean + standard error of the mean (n = 5 mice per group). expressed, compared with that in WAT, in which FSP27a is the major FSP27 isoform. lipolysis resulting from decreased delivery of lipid substrates to lysosomes rather than to increased de novo lipogenesis11. Lipo- phagy might therefore play a key role in lipid homeostasis and protect cells against hepatolipotoxicity. DISCUSSION Dysregulation of autophagy is associated with various patholo- gies, including cancer, as well as cardiovascular, neurodegenera- tive, muscular and metabolic diseases25. In addition, the role of autophagy in lipid metabolism is important because of its potential implications for obesity and metabolic syndrome26. LDs can be selectively sequestered in autophagosomes and delivered to lysosomes for degradation by lysosomal acid lipases – a process known as lipophagy, which is distinct from classical lipolysis whereby lipids in LDs are directly hydrolyzed by cytosolic lipases without sequestration by autophagosomes11. Indeed, targeted inhibition of autophagy in the liver increased hepatic lipid accumulation in mice after starvation or feeding with a high-fat diet11. Furthermore, inhibition of autophagy in hepatocytes also increased cellular TG content during exposure to oleate. The increased lipid storage was characterized by an increase in both the size and number of LDs in the autophagy- deficient hepatocytes, and it was due to reduced lysosomal Autophagy is an evolutionarily conserved process that is essential for cell survival during periods of nutrient deprivation. Dysregulation of autophagy is associated with various patholo- gies, including cancer, as well as cardiovascular, neurodegenera- tive, muscular and metabolic diseases25. In addition, the role of autophagy in lipid metabolism is important because of its potential implications for obesity and metabolic syndrome26. LDs can be selectively sequestered in autophagosomes and delivered to lysosomes for degradation by lysosomal acid lipases Figure 4 | Overexpression of fat-specific protein 27 (FSP27)a, but not FSP27b, inhibits autophagy induced by serum deprivation in COS cells. (a) COS cells incubated in Dulbecco’s modified Eagle’s medium with or without 10% fetal bovine serum and 50 μmol/L chloroquine for 18 h were subjected to immunofluorescence staining with antibodies to LC3 and observed with a confocal laser-scanning microscope. The boxed regions of the upper panels are shown at higher magnification in the lower panels. Scale bars, 50 μm. (b) Immunoblot analysis of LC3 in cells treated as in (a). (c) COS cells were transfected with pIRES2-DsRed2 encoding (or not, top panels) FSP27a or FSP27b for 2 days, incubated with serum-free Dulbecco’s modified Eagle’s medium containing 50 μmol/L chloroquine for 18 h and then subjected to immunofluorescence staining with antibodies to LC3. DsRed2 fluorescence was also observed directly with a confocal laser-scanning microscope. Scale bars, 50 μm. (d) Quantitation of LC3 staining was carried out by calculating the ratios of LC3-positive cells in DSRed-positive cells. Figure 4 | Overexpression of fat-specific protein 27 (FSP27)a, but not FSP27b, inhibits autophagy induced by serum deprivation in COS cells. (a) COS cells incubated in Dulbecco’s modified Eagle’s medium with or without 10% fetal bovine serum and 50 μmol/L chloroquine for 18 h were subjected to immunofluorescence staining with antibodies to LC3 and observed with a confocal laser-scanning microscope. The boxed regions of the upper panels are shown at higher magnification in the lower panels. Scale bars, 50 μm. (b) Immunoblot analysis of LC3 in cells treated as in (a). (c) COS cells were transfected with pIRES2-DsRed2 encoding (or not, top panels) FSP27a or FSP27b for 2 days, incubated with serum-free Dulbecco’s modified Eagle’s medium containing 50 μmol/L chloroquine for 18 h and then subjected to immunofluorescence staining with antibodies to LC3. DsRed2 fluorescence was also observed directly with a confocal laser-scanning microscope. Scale bars, 50 μm. (d) Quantitation of LC3 staining was carried out by calculating the ratios of LC3-positive cells in DSRed-positive cells. Data are the mean + standard error of the mean (n = 5). **P < 0.01 versus the corresponding value for DSRed and FSP27b. Overexpression of FSP27a inhibits autophagy induction by serum deprivation in COS cells Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd 1425 J Diabetes Investig Vol. 10 No. 6 November 2019 O R I G I N A L A R T I C L E Nakajima et al. REFERENCES 1. Cohen P, Spiegelman BM. Cell biology of fat storage. Mol Biol Cell 2016; 27: 2523–2527. 1. Cohen P, Spiegelman BM. Cell biology of fat storage. Mol Biol Cell 2016; 27: 2523–2527. 2. Nishimoto Y, Tamori Y. CIDE family-mediated unique lipid droplet morphology in white adipose tissue and brown adipose tissue determines the adipocyte energy metabolism. J Atheroscler Thromb 2017; 24: 989–998. 2. Nishimoto Y, Tamori Y. CIDE family-mediated unique lipid droplet morphology in white adipose tissue and brown adipose tissue determines the adipocyte energy metabolism. J Atheroscler Thromb 2017; 24: 989–998. 3. Nishino N, Tamori Y, Tateya S, et al. FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J Clin Invest 2008; 118: 2808–2821. 3. Nishino N, Tamori Y, Tateya S, et al. FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J Clin Invest 2008; 118: 2808–2821. 4. Puri V, Ranjit S, Konda S, et al. Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc Natl Acad Sci USA 2008; 105: 7833–7838. 4. Puri V, Ranjit S, Konda S, et al. Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc Natl Acad Sci USA 2008; 105: 7833–7838. 5. Toh SY, Gong J, Du G, et al. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS ONE 2008; 3: e2890. 5. Toh SY, Gong J, Du G, et al. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS ONE 2008; 3: e2890. 6. Sun Z, Gong J, Wu H, et al. Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes. Nat Commun 2013; 4: 1594. The enhancement of fasting-induced autophagy in WAT of FSP27 KO mice and BAT of WT mice suggests that autophagy might be associated with the presence of multilocular LDs and abundant mitochondria, both of which are characteristics of metabolically active, energy-consuming adipocytes. FFAs gener- ated in energy-consuming BAT are taken up by mitochondria for beta-oxidation. In contrast, FFAs generated in energy-storing WAT are delivered to other tissues through the circulation for utilization as an energy substrate. DISCUSSION Data are the mean + standard error of the mean (n = 5). **P < 0.01 versus the corresponding value for DSRed and FSP27b. 1426 J Diabetes Investig Vol. 10 No. 6 November 2019 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd O R I G I N A L A R T I C L E Autophagy inhibitor (–) (a) (b) (c) (d) Serum deprivation (–) Serum deprivation: Autophagy inhibitor: β-actin LC3 I II – – – + + 100 80 60 Percentage of LC3-positive cells 40 20 0 DsRed DsRed FSP27α FSP27β LC3 Merge LC3 Merge LC3 Merge FSP27α FSP27β ** – + + Serum deprivation (+) Serum deprivation (–) Serum deprivation (+) Autophagy inhibitor (+) O R I G I N A L A R T I C L http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocyte http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocytes Autophagy inhibitor (–) Autophagy inhibitor (+) Autophagy inhibitor (–) (a) Serum deprivation (–) Serum deprivation (+) (a) Serum deprivation (–) Serum deprivation (+) (b) Serum deprivation: Autophagy inhibitor: (b) Serum deprivation: Autophagy inhibitor: (b) β-actin LC3 I II (c) (d) 100 80 60 Percentage of LC3-positive cells 40 20 0 DsRed DsRed FSP27α FSP27β LC3 Merge LC3 Merge LC3 Merge FSP27α FSP27β ** (c) DsRed LC3 (d) (c) LC3 FSP27α LC3 LC3 LC3 FSP27β LC3 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd 1427 J Diabetes Investig Vol. 10 No. 6 November 2019 O R I G I N A L A R T I C L E Nakajima et al. O R I G I N A L A R T I C L E http://wileyonlinelibrary.com/journal/jd http://wileyonlinelibrary.com/journal/jdi underlying mechanism of the different energy metabolism between white adipocytes and brown adipocytes. inhibited autophagy pharmacologically in adult mice in order to exclude the effect of such inhibition on BAT differentiation. Our finding that inhibition of autophagy attenuated the fasting- induced disappearance of LDs in BAT is consistent with the previous observation that inhibition of autophagy in BAT reduced the cold-induced degradation of LDs28. ACKNOWLEDGMENTS FSP27a forms a homodimer and induces unilocular large LD formation in white adipocytes6,32,33, whereas FSP27b promotes multilocular small LD formation by inhibiting the homodimerization of CideA in brown adipocytes7. Therefore, 10 amino acids in the amino-terminal domain of FSP27b are supposed to play a critical role in LD formation through the interaction with other molecules. Given the possibility that FSP27a might inhibit autophagy by interacting with other molecules, as mentioned above, FSP27b might not be able to show the inhibitory effects because of the alteration of the steric structure induced by the 10 amino acids in its amino-terminal domain. DISCLOSURE The authors declare no conflict of interest. The authors declare no conflict of interest. ACKNOWLEDGMENTS This work was partially supported by JSPS KAKENHI (grant number 18K16201). We thank S Shigeta for technical assistance. This work was partially supported by JSPS KAKENHI (grant number 18K16201). We thank S Shigeta for technical assistance. Although the precise mechanism by which FSP27a inhibits autophagy remains to be elucidated, several mechanisms are assumed. AMPK is a major activator of autophagy that is mediated through the mammalian target of rapamycin and Ulk123,29. FSP27a decreases the expression of AMPK through the ubiquitin/proteasome pathway in cultured cells30. This reduction of AMPK is one of the possible mechanisms that might underlie the inhibitory effect of FSP27a on autophagy. In addition, ATGL promotes autophagy through SIRT124. Given that FSP27a interacts with ATGL and inhibits its activity through perilipin in human adipocytes31, FSP27a might inhibit autophagy through the interaction of ATGL. FSP27b is a molecule to which 10 amino acids were added to the amino-terminal domain of FSP27a8. However, these two molecules show the opposite effects to the LD forma- tion. FSP27a forms a homodimer and induces unilocular large LD formation in white adipocytes6,32,33, whereas FSP27b promotes multilocular small LD formation by inhibiting the homodimerization of CideA in brown adipocytes7. Therefore, 10 amino acids in the amino-terminal domain of FSP27b are supposed to play a critical role in LD formation through the interaction with other molecules. Given the possibility that FSP27a might inhibit autophagy by interacting with other molecules, as mentioned above, FSP27b might not be able to show the inhibitory effects because of the alteration of the steric structure induced by the 10 amino acids in its amino-terminal domain. Although the precise mechanism by which FSP27a inhibits autophagy remains to be elucidated, several mechanisms are assumed. AMPK is a major activator of autophagy that is mediated through the mammalian target of rapamycin and Ulk123,29. FSP27a decreases the expression of AMPK through the ubiquitin/proteasome pathway in cultured cells30. This reduction of AMPK is one of the possible mechanisms that might underlie the inhibitory effect of FSP27a on autophagy. In addition, ATGL promotes autophagy through SIRT124. Given that FSP27a interacts with ATGL and inhibits its activity through perilipin in human adipocytes31, FSP27a might inhibit autophagy through the interaction of ATGL. FSP27b is a molecule to which 10 amino acids were added to the amino-terminal domain of FSP27a8. However, these two molecules show the opposite effects to the LD forma- tion. REFERENCES Lipolysis through autophagy might promote the effective flux of FFAs into mitochondria in cells with multilocular LDs and thereby contribute to energy expenditure in metabolically active cells. Indeed, BAT from cold- exposed mice in which autophagy was inhibited by Atg7 ablation manifested an ~50% reduction in the rate of oxygen consump- tion28. In addition, oxygen consumption was previously shown to be reduced in BAT of FSP27 KO mice3, in which we have now shown that autophagy is attenuated. FSP27a might thus con- tribute to the metabolic characteristics of energy-storing white adipocytes by inhibiting lipophagy, as well as by promoting large unilocular LD formation. These findings might elucidate the 7. Nishimoto Y, Nakajima S, Tateya S, et al. Cell death- inducing DNA fragmentation factor A-like effector A and fat-specific protein 27b coordinately control lipid droplet size in brown adipocytes. J Biol Chem 2017; 292: 10824– 10834. 8. Xu X, Park JG, So JS, et al. Transcriptional activation of Fsp27 by the liver-enriched transcription factor CREBH promotes lipid droplet growth and hepatic steatosis. Hepatology 2015; 61: 857–869. 9. Gibbons GF, Islam K, Pease RJ. Mobilisation of triacylglycerol stores. Biochim Biophys Acta 2000; 1483: 37–57. 10. Jaishy B, Abel ED. Lipids, lysosomes, and autophagy. J Lipid Res 2016; 57: 1619–1635. 10. Jaishy B, Abel ED. Lipids, lysosomes, and autophagy. J Lipid Res 2016; 57: 1619–1635. 11. Singh R, Kaushik S, Wang Y, et al. Autophagy regulates lipid metabolism. Nature 2009; 458: 1131–1135. 11. Singh R, Kaushik S, Wang Y, et al. Autophagy regulates lipid metabolism. Nature 2009; 458: 1131–1135. 12. Nissar AU, Sharma L, Mudasir MA, et al. Chemical chaperone 4-phenyl butyric acid (4-PBA) reduces hepatocellular lipid accumulation and lipotoxicity through induction of autophagy. J Lipid Res 2017; 58: 1855–1868. J Diabetes Investig Vol. 10 No. 6 November 2019 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd 1428 O R I G I N A L A R T I C L E http://wileyonlinelibrary.com/journal/jdi Autophagy in adipocytes 13. Zhang Y, Goldman S, Baerga R, et al. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc Natl Acad Sci USA 2009; 106: 19860–19865. 24. Sathyanarayan A, Mashek MT, Mashek DG. ATGL promotes autophagy/lipophagy via SIRT1 to control hepatic lipid droplet catabolism. Cell Rep 2017; 19: 1–9. 25. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. REFERENCES Cell 2008; 132: 27–42. 14. Singh R, Xiang Y, Wang Y, et al. Autophagy regulates adipose mass and differentiation in mice. J Clin Invest 2009; 119: 3329–3339. 26. Czaja MJ. Autophagy in health and disease. 2. Regulation of lipid metabolism and storage by autophagy: pathophysiological implications. Am J Physiol Cell Physiol 2010; 298: C973–C978. 15. Altshuler-Keylin S, Shinoda K, Hasegawa Y, et al. Beige adipocyte maintenance is regulated by autophagy- induced mitochondrial clearance. Cell Metab 2016; 24: 402–419. 27. Fernandez AF, Barcena C, Martınez-Garcıa GG, et al. Autophagy couteracts weight gain, lipotoxicity and pancreatic b-cell death upon hypercaloric pro-diabetic regimens. Cell Death Dis 2017; 8: e2970. 16. Irie Y, Asano A, Canas X, et al. Immortal brown adipocytes from p53-knockout mice: differentiation and expression of uncoupling proteins. Biochem Biophys Res Commun 1999; 255: 221–225. 28. Martinez-Lopez N, Garcia-Macia M, Sahu S, et al. Autophagy in the CNS and periphery coordinate lipophagy and lipolysis in the brown adipose tissue and liver. Cell Metab 2016; 23: 113–127. 17. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011; 27: 107–132. 29. Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 2011; 331: 456–461. 18. Chakrama FZ, Seguin-Py S, Le Grand JN, et al. GABARAPL1 (GEC1) associates with autophagic vesicles. Autophagy 2010; 6: 495–505. 30. Xu Y, Gu Y, Liu G, et al. Cidec promotes the differentiation of human adipocytes by degradation of AMPKa through ubiquitin-proteasome pathway. Biochim Biophys Acta 2015; 1850: 2552–2562. 19. Zhao Y, Yang J, Liao W, et al. Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nat Cell Biol 2010; 12: 665–675. 20. Lee IH, Cao L, Mostoslavsky R, et al. A role for the NAD- dependent deacetylase Sirt1 in the regulation of autophagy. Proc Natl Acad Sci USA 2008; 105: 3374–3379. 31. Grahn TH, Kaur R, Yin J, et al. Fat-specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) to regulate lipolysis and insulin sensitivity in human adipocytes. J Biol Chem 2014; 289: 12029–12039. 21. Bjørkøy G, Lamark T, Brech A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005; 171: 603–614. 32. Gong J, Sun Z, Wu L, et al. REFERENCES Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. J Cell Biol 2011; 195: 953–963. 22. Chan EY, Kir S, Tooze SA. siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy. J Biol Chem 2007; 282: 25464–25474. 33. Jambunathan S, Yin J, Khan W, et al. FSP27 promotes lipid droplet clustering and then fusion to regulate triglyceride accumulation. PLoS ONE 2011; 6: e28614. 23. Kim J, Kundu M, Viollet B, et al. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 2011; 13: 132–141. J Diabetes Investig Vol. 10 No. 6 November 2019 ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article. ditional supporting information may be found online in the Supporting Information section at the en Figure S1 | Effects of pharmacological inhibition of autophagy on fasting-induced changes in liver, kidney, heart, and gastrocne- mius weight in WT and FSP27 KO mice. ª 2019 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd 1429 J Diabetes Investig Vol. 10 No. 6 November 2019
https://openalex.org/W4322767271
https://www.nature.com/articles/s41598-023-30702-z.pdf
English
null
Size distribution and relationship of airborne SARS-CoV-2 RNA to indoor aerosol in hospital ward environments
Scientific reports
2,023
cc-by
10,691
Size distribution and relationship of airborne SARS‑CoV‑2 RNA to indoor aerosol in hospital ward environments V. Groma 1, Sz. Kugler 1, Á. Farkas 1, P. Füri 1, B. Madas 1, A. Nagy 2, T. Erdélyi 3, A. Horváth 3,5, V. Müller 3, R. Szántó‑Egész 4, A. Micsinai 4, G. Gálffy 5 & J. Osán 1* OPEN Aerosol particles proved to play a key role in airborne transmission of SARS-CoV-2 viruses. Therefore, their size-fractionated collection and analysis is invaluable. However, aerosol sampling in COVID departments is not straightforward, especially in the sub-500-nm size range. In this study, particle number concentrations were measured with high temporal resolution using an optical particle counter, and several 8 h daytime sample sets were collected simultaneously on gelatin filters with cascade impactors in two different hospital wards during both alpha and delta variants of concern periods. Due to the large number (152) of size-fractionated samples, SARS-CoV-2 RNA copies could be statistically analyzed over a wide range of aerosol particle diameters (70–10 µm). Our results revealed that SARS-CoV-2 RNA is most likely to exist in particles with 0.5–4 µm aerodynamic diameter, but also in ultrafine particles. Correlation analysis of particulate matter (PM) and RNA copies highlighted the importance of indoor medical activity. It was found that the daily maximum increment of PM mass concentration correlated the most with the number concentration of SARS-CoV-2 RNA in the corresponding size fractions. Our results suggest that particle resuspension from surrounding surfaces is an important source of SARS-CoV-2 RNA present in the air of hospital rooms. Since the COVID-19 pandemic started, a lot of attention has been drawn towards the emission, transport, infec- tion properties, collection, detection, and particle sizing of the SARS-CoV-2 virus, but still, a lot of challenges and obstacles ­exist1–7. For instance, aerosol sampling, in the environment of patients treated in a hospital must be done in a way that is not disturbing the work of the hospital staff nor the patient’s calm and recovery.hi y gf y The first studies highlighted that the SARS-CoV-2 spread through direct human-to-human droplet trans- mission, indirect means such as contact with contaminated objects or surfaces (fomites) and by airborne ­transmission8–10. At the beginning of 2021, the scientific community already emphasized the predominance of airborne mode of ­transmission11–13, which means that floating aerosol particles play an essential role.i l According to Birgand et al.14, during the first 11 months of the pandemic, 23 studies were carried out studying air contamination by SARS-CoV-2 RNA at hospital sites. The studies focused on the measurement of total aerosol concentration without size fractionation or studied only a few particle size ranges. The measurements yielded surprisingly few positive samples, only 82 out of 471 air samples (17.4%) were positive for SARS-CoV-2 RNA. www.nature.com/scientificreports www.nature.com/scientificreports Methods Study design, participants and setting. Measurements were performed in two different hospital wards. At the Pulmonology Hospital of Törökbálint (hereafter Hospital A) only one, whilst at the Department of Pul- monology of Semmelweis University (hereafter Hospital B) three patients were treated at the same time in the studied wards. While in Hospital A a normal pulmonary patient room, in Hospital B a high-intensity non-inva- sive ventilation unit (HDU) was sampled. Patients treated in a non-invasive ventilation unit require continuous observation and advanced level management, however invasive mechanical ventilation with closed breathing circuit is not used but non-invasive mechanical ventilation can be applied which is able to generate additional airflow around the ­patient30. The aerosol sampling and monitoring instruments were installed as close as pos- sible on the nightstand beside the bed, at the height of the lying patients’ heads at both hospitals. To avoid the disturbing of the patients, the noise generating part of the equipment was placed outside the ward. g g g In the case of Hospital A, the sampling and monitoring devices were operated at a distance of 3 m from the bed of the patient in a room of 5.0 × 4.8 × 2.5 ­m3 during the entire measurement period (see Figure S1a in Sup- plementary Material). The patient was hospitalized on the ­4th day after the positive test, and he got respiratory support by using a nasal cannula during the measurement period. His medical condition allowed him to move around in the room, and the patient logged his main activities in a diary. Medical treatment, daily cleaning and room ventilation activities were logged by the staff. f In Hospital B HDU the room size was 6.7 × 4.3 × 2.5 ­m3 and the patients were undergoing treatment in all three beds of the ward for varying lengths of time, with varying degrees of ventilator support, including non-invasive ventilation, ­O2 treatment via high flow nasal cannula or reservoir mask. As the patients changed within a short time, there were only 3–4 consecutive days when the same patients were in the ward, but the type of non-invasive respiratory support might have changed according to the improvement or worsening of the clinical condition of the patients (for details see Table S1). Thus, we could define 6 groups of measurement days, which are numbered from B1 to B6 (see Tables 1 and 2). Table 1.   Basic information on PM measurements and sampling in hospital wards. *No measurement was performed in 11 24 2021. **One patient was replaced on 11 24 2021. www.nature.com/scientificreports/ size distribution associated the virus can be utilized to inform disease transmission modeling, as well as better understand the inhalability/respiratory deposition pattern of aerosols in exposed individuals. The size of the respiratory droplets/droplet nuclei has an important role in both disease transmission and in airway deposition distribution. Many authors presented numerical models, that described the effect of gravitational settling and evaporation of the droplets in the ­air24–28. Depending on the solute content, small droplets can remain airborne as droplet nuclei for a long time as they can almost completely evaporate during their descent to the ground. Viruses are likely to remain infectious in aerosols for hours, resulting in an increase in the infectious viral air load. According to the review article of Ribaric et al.29, several (11) studies were performed to study the size distribution of aerosol particles bearing virus copies in the size range of 0.25–10 µm collected at the patient and non-patient areas of the hospitals. However, none of them was performed in patient areas including size ranges below 250 nm. Overall, it was found that SARS-CoV-2 RNA could be detected in all size ranges, from which the positivity rate is higher in the larger size ranges (2.5–10 µm). The positivity rate of SARS-CoV-2 RNA in the air samples was found to be similar to the overall detection rate of non-size-fractionated airborne particulate matter (PM) samples (approx. 16%).h The primary objective of the present study was to quantify the SARS-CoV-2 RNA prevalence in a hospital ward, and in a specific high-dependency unit (HDU) for a wide size range in numerous fractions from the ultrafine mode (particulate matter of nanoscale size) up to coarse mode (aerodynamic diameter ranging from 2.5 to 10 µm), for many different patient groups allowing statistical evaluation. Based on this, our aim was to determine the characteristic size range of airborne SARS-CoV-2 RNA and its possible change over time due to the disease progresses. By determining the distribution of the particle number concentration with high time resolution, our second aim was to study the effect of particle mass fluctuation due to indoor human activity and the relationship between the number of SARS-CoV-2 RNA copies and the aerosol particle number/mass concentration in hospital wards. Size distribution and relationship of airborne SARS‑CoV‑2 RNA to indoor aerosol in hospital ward environments V. Groma 1, Sz. Kugler 1, Á. Farkas 1, P. Füri 1, B. Madas 1, A. Nagy 2, T. Erdélyi 3, A. Horváth 3,5, V. Müller 3, R. Szántó‑Egész 4, A. Micsinai 4, G. Gálffy 5 & J. Osán 1* OPEN In intensive care unit settings, the positivity rate was 25.2% (27 of 107 samples), which is significantly higher than the value of 10.7% (39 of 364) characterizing the non-intensive care units. It was also found that the distance between the patients and the sampling point did not play an important ­role14, except for higher distances (more than 2 m)15. The mentioned studies found the airborne SARS-CoV-2 RNA concentration between 1 to 113 cop- ies/m316–21, except the work of Lednicky et al.22, who detected three orders of magnitude higher concentration with a maximum of 94,000 copies/m3. p As a result of the developments in aerosol measurement technology and the increase of the sensitivity of PCR methods, and also due to the expansion of opportunities in sampling conditions, more and more aerosol samples were found to be evaluable for SARS-CoV-2 ­RNA23. This provided an opportunity for the scientific community to conduct research oriented towards size fractional aerosol sampling and analysis. Information on the particle 1Environmental Physics Department, Centre for Energy Research, Budapest  1121, Hungary. 2Department of Applied and Nonlinear Optics, Wigner Research Centre for Physics, Budapest  1121, Hungary. 3Department of Pulmonology, Semmelweis University, Budapest  1085, Hungary. 4BIOMI Ltd., Gödöllő  2100, Hungary. 5Pest County Pulmonology Hospital, Törökbálint 2045, Hungary. *email: osan.janos@ek-cer.hu Scientific Reports | (2023) 13:3566 | https://doi.org/10.1038/s41598-023-30702-z www.nature.com/scientificreports/ Methods Patient group Measurement date Positivity rate (number of samples with N2 above detection limit/ number of all samples) Quantifiability rate (number of samples ≥ 10 copies for N2 positive/number of all samples) A1 4 26 2021 6/7 2/7 4 27 2021 3/7 3/7 4 28 2021 2/7 2/7 4 29 2021 3/7 3/7 4 30 2021 5/7 5/7 5 1 2021 6/7 4/7 B1 3 1 2021 4/5 3/5 3 2 2021 4/5 4/5 3 3 2021 3/5 3/5 3 4 2021 3/5 3/5 B2 11 3 2021 1/5 0/5 11 4 2021 0/5 0/5 11 5 2021 0/5 0/5 B3 11 9 2021 2/5 1/5 11 10 2021 5/5 0/5 11 11 2021 4/5 1/5 11 12 2021 5/5 3/5 B4 11 16 2021 3/5 3/5 11 17 2021 2/5 2/5 11 18 2021 4/5 4/5 11 19 2021 4/5 4/5 B5 11 22 2021 1/5 0/5 11 23 2021 1/5 0/5 11 25 2021 3/5 0/5 11 26 2021 4/5 1/5 B6 12 6 2021 4/5 0/5 12 7 2021 3/5 0/5 12 8 2021 5/5 4/5 Table 2. Rate of positivity and quantifiability for each set of size-fractionated PM samples. Table 2. Rate of positivity and quantifiability for each set of size-fractionated PM samples. department is cleaned regularly according to local protocol. All cleaning procedures were performed prior to start of the measurement each day (6:00–6:20 AM). department is cleaned regularly according to local protocol. All cleaning procedures were performed prior to start of the measurement each day (6:00–6:20 AM). y During the sampling period, the variant of concern (VOC) of SARS-CoV-2 was alpha VOC for Hospital A and included periods of alpha and delta VOC for Hospital B. It is important to note that during delta VOC, most patients have already received their baseline vaccinations. This could affect the shedding of patients as reported in Refs.31,32. In Hospital A only one patient, whilst in Hospital B nineteen patients were studied. This allowed us to study both the time evolution of virus concentration for a single patient and differences of virus concentration resulting from multiple patients with different degrees of disease severity. Sampling and data collection. An in-house built May-type cascade ­impactor33 was used to sample size- fractionated aerosol particles on presterilized gelatin disc filters (Sartorius, Göttingen, Germany) as collecting substrate. The easily dissolvable gelatin filers have suitable performance for collecting viruses for PCR ­analysis34. Methods The time of infection and the technique used for respiratory support are summarized in Table S1 (Supplementary Material). In the room at Hospital B, the measurement instruments were placed on a nightstand between the window and the bed on the side opposite the door. The measurement point was 90, 270 and 450 cm far from the three patients (see Figure S1b in Supplementary Material). HDU Table 1. Basic information on PM measurements and sampling in hospital wards. *No measurement was performed in 11 24 2021. **One patient was replaced on 11 24 2021. Location Patient group notation Measurement period (year 2021) # of days # of patients VOC # of impactor stages Hospital A A1 04 26–05 01 6 1 alpha 7 Hospital B B1 03 01–03 04 4 3 alpha 5 Hospital B B2 11 03–11 05 3 3 delta 5 Hospital B B3 11 09–11 12 4 3 delta 5 Hospital B B4 11 16–11 19 4 3 delta 5 Hospital B B5 11 22–11 26* 4 4** delta 5 Hospital B B6 12 06–12 08 3 3 delta 5 Table 1. Basic information on PM measurements and sampling in hospital wards. *No measurement was performed in 11 24 2021. **One patient was replaced on 11 24 2021. https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 | www.nature.com/scientificreports/ Table 2. Rate of positivity and quantifiability for each set of size-fractionated PM samples. Methods Two versions of the impactor were used, a 7 stage basic version, which has aerodynamic cut-off diameters of 16, 8, 4, 2, 1, 0.5 and 0.25 μm for stages 1 to 7, respectively at 20 L/min sampling flowrate, whilst the 9 stage in-house developed extended version is suitable for collecting further size fractions with cutting diameters of 0.18 and 0.07 μm (stages 8 and 9 respectively)35. Cut-off diameters below 1 µm were verified through a comparison meas- urement using an aerosol ­spectrometer36. Total particle mass agreed well with reference filter sampling and size distributions were found to be similar to those obtained using a commercial Dekati ­impactor37. The sampling time was 8 h in both cases, during daytime including the main time for medical interventions. No sampling was performed on stages 1 and 2 since particles larger than 10 μm aerodynamic diameter are beyond the range of ­PM10. Sampling periods are summarized in Table 1. In Hospital B, 22 sample sets were collected (5 stages each, altogether 110 samples), while in Hospital A, only 6 sample sets (7 stages each, altogether 42 samples). g p p y p g g p Simultaneously with the 8-h impactor samplings, an optical particle counter (GRIMM PAS 1.109) was used to measure the indoor particle mass concentration of ­PM1, ­PM2.5 and ­PM10 (particle matter with an aerodynamic diameter less than 1, 2.5 and 10 µm, respectively) with a time resolution of 1 min. Sampling by the impactor provides information on the concentration and size fractions of virus laden particles without any information on the concentration and size distribution of all the aerosol particles from the ward. Sampling by the OPC provides Scientific Reports | (2023) 13:3566 | https://doi.org/10.1038/s41598-023-30702-z www.nature.com/scientificreports/ useful information on the number and size of all particles. Simultaneous sampling by the impactor and OPC allowed the analysis of correlation between the size distribution of the aerosol and SARS-CoV-2 RNA concentra- tion in aerosol particles for each patient group. Since the OPC data are obtained at a high time resolution, and the values are influenced also by the outdoor air quality, first a preprocessing of the PM mass concentration data measured by OPC was applied for each 8-h sampling period. Detection and quantification of SARS‑CoV‑2. Size-fractionated PM samples were collected onto pres- terilized gelatin filters (Sartorius) at stages 3 to 7/9 of the impactor. Results and discussion Several studies investigating the aerosol concentration and elemental composition in hospital ­wards43–46 high- lighted the importance of the outdoor aerosol concentration influencing the indoor concentration of the par- ticulate matter. It has long been known that outdoor particles are the main contributors to indoor particles in hospital ­environments47, and a linear relationship exists between indoor and outdoor PM ­concentrations48,49. As direct outdoor PM measurement were not performed, correlation between the measured indoor and outdoor PM concentrations available from the closest urban reference station was studied. Therefore, first, the connection between indoor and outdoor aerosol load was investigated in the present study for both measurement locations. The highest correlation was found between the minimum ­PM2.5 concentrations measured indoors and those reported at the closest urban environmental reference station in Budapest (see Figure S2 in Supplementary Mate- rial). Taking into account the 8-h sampling period each day, the Pearson correlation coefficients were r = 0.94 and r = 0.87 for Hospitals A and B, respectively. Thus, it can be assumed that the minimum concentration measured indoors can be considered as a lower estimate, practically a baseline that is dependent on the outdoor aerosol concentration. The concentration increment above the baseline is characteristic of the indoor events and sources of PM. Therefore, the outdoor environmental effect was minimized by considering the PM concentration incre- ments for each 8-h sampling period, by subtracting the respective minimum PM concentrations from all 1-min PM values, as preprocessing of the OPC data. In the next step, we examined the characteristics of the high time- resolution size-fractionated PM parameters measured by the optical counter (trend, maximum, mean, fluctuation of concentration increment) for each day. In the final step, a Pearson correlation analysis of aerosol parameters and SARS-CoV-2 RNA concentration in each size range was performed for each patient group. Particle number size distribution trends. There are significant differences between the two hospital sites in terms of environmental and internal conditions. Hospital A is located in a suburban area, while Hospital B is in downtown Budapest. The impact of the different sites is clearly reflected in the PM concentrations and their daily trends (see Fig S2). During the whole measurement campaign, the daily outdoor minimum ­PM2.5 concentration was between 3.8 and 6 µg/m3 at Hospital A, whereas generally higher values were detected at Hospital B for the same variable (5.4–29.6 µg/m3). Methods The gelatin filters were removed and placed in a clean and sterile holder and were forwarded within 12–72 h for virus testing, which is an acceptable time- frame for specimen transportation and ­storage38. The gelatin filters were dissolved in 600 µl RAV1 buffer of the NucleoSpin RNA Virus kit (Macharey-Nagel, Düren, Germany), and the isolation of viral RNA was carried out following the kit’s protocol. This included incubation at 70 °C for 5 min, adding 600 μL ethanol and loading it on the MN NucleoSpin Viral RNA isolation spin-columns. A centrifugation step of 1 min at 8000 g followed, and the washing steps included three washing steps (1st wash: 500 μL RAW buffer 1 min at 8000 g, 2nd wash 600 μL RAV3 buffer 1 min at 8000 g, 3rd wash 200 μL RAV3 buffer 5 min at 11,000 g). The final elution step included adding 60 μL prewarmed (70 °C) TE buffer, incubating it on the column for 1–2 min, and carrying out a centrifugation at 11,000 g for 1 ­min39.h g g The standard curve was derived from the ATCC Heat Inactivated 2019 Novel Coronavirus VR-1986HK (ATCC, Manassas, USA) by isolating RNA with the above-described method and preparing ten-fold dilu- tions. The samples were analyzed according to the methods by Refs.40,41, see details in Supplementary Material (Table S2). Sensitivity, specificity and validity tests of the method are discussed in detail in Ref.41. Only samples with at least 10 copies of the N2 sequence for a given aerosol size gelatin were used for statistical analysis. Sampling with the May-type cascade impactor was performed at nearly similar daytime periods of 8 h to reach the appropriate detection limit of the PCR test. According to Ribaric et al.29, among sampling methods used at different hospital sites, impactors were found to yield one of the lowest mean concentrations of detected SARS-CoV-2 RNA. The detection limit of the above-detailed methodology is similar to that reported in other studies (around 2 copies/sample16,42), while the quantification limit was 10 copies/sample. The results for airborne concentrations of the SARS-CoV-2 N2 gene are expressed in copies/m3, taking into account the 9.6 ­m3 sampled air volume (operating flow rate of 20 L/min during 8 h). Thus, in the present study, the quantification limit for airborne concentrations is 1.04 copies/m3 for each size fraction. Ethical approval. Methods The measurements of the multicenter observational, non-interventional case-only study were completed based on a protocol approved by the Semmelweis University Regional and Institutional Com- mittee of Science and Research Ethics (SE RKEB) (approval no 219/2020). All methods were performed in accordance with the relevant guidelines and regulations. All voluntarily participating patients signed written consent after being informed both orally and in written form. www.nature.com/scientificreports/ the emission rates of this equipment are highly dependent on the respiratory activities and the settings. Depend- ing on the health status of the patients, we can assume that the direct contribution of the respiratory support units to aerosol concentration is not significant (~ 1.5–5 particles/cm3)50,51. However, in the case of HDU, due to the frequent activity of medical staff and the often displacement of the respiratory mask, which results in high airflow inside the ward, air turbulence-related dispersion of airborne particles can play a very important ­role52. Therefore, besides the passive room ventilation (through the adjacent control room for Hospital B), the resus- pension generating processes basically determined the quantity and typical size distribution of aerosol concen- tration in the ward. In general, the detected size distributions showed two maxima observed in size ranges below 1 μm and around 10 μm, which is typical for ambient aerosols in urban environments. In addition, especially at Hospital B, an additional small maximum at around 4 μm was observed occasionally, presumably related to indoor activity. y Based on an itemized event log available in the case of Hospital A, the daily time trends of PM concentrations could be studied in detail in relation to indoor human activity. It is important to note that short events, such as coughing, were not registered. It was found that for all activities, an increment in ­PM10 concentration can be assigned to each movement, while for smaller size ranges ­(PM2.5 and ­PM1) an increase can only be observed during certain events (room ventilation, cleaning). O’Neil et al.53 found typically just a few µg/m3 total mass con- centration increase due patient’s activity (like eating, bathing, tidying), while Nagy et al.54, highlighted that each (even similar) activities could cause very different particle number increments in the different size ranges. Health care professional (HCP) activities related to physician and/or nurse visits for medical diagnostic examinations and nursing care are among the most important events causing the increase in the number of submicron particles. By the same token, activities related to full patient care, but especially bedding, have contributed significantly to the rise of larger particles’ concentration in the air. Moreover, increases in supermicron particle concentration were associated with the number of HCPs and the duration of the activity, while submicron particles increased with all ­activities54. All these demonstrates that particle size distribution is a function of the activity type. Results and discussion In addition, only one patient was treated in Hospital A during the sampling period, whose medical condition allowed more active forms of free movement (getting out of bed, telephony, and so on). By contrast, in the downtown ward (Hospital B), three patients were treated in the respira- tory HDU setting using different aerosol-generating therapeutic devices and the patients were not able to leave their beds. Although high-flow nasal oxygen (HFNO) respiratory supports are aerosol-generating procedures, https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ establishing the fact of positivity. In the present study, several 8-h samples collected using a 20 L/min flow rate are discussed for 5/7 size fractions.h The number of SARS-CoV-2 virus copies was found to be highly variable in the studied size fractions. In the case of Hospital A, within the 6 sample sets, 25 out of the 42 samples (59.5%) were determined to be positive of which 19 (45.2%) samples were quantifiable for the SARS-CoV-2 virus (see details in Table 2). p qi Size variation of the number of copies shows a bimodal distribution in all cases except for one day (04 28 2021), however the locations of maxima vary in size from day to day. The average of SARS-CoV-2 N2 gene num- ber concentration ranged between 18 and 184 copies/m3 for the measurement period (see Fig. 2) between 0.07 and 8 µm. A significantly higher value was detected on 04 30 2021, when an extremely high number of SARS- CoV-2 RNA was found in the 230 nm and 800 nm mode size ranges. Based on the event log, it can be concluded that no extraordinary event occurred during that day. The patient was more and more active, however his PCR test was still positive, which might have cause the significant virus release detected. p g gi In the case of Hospital B, 65 out of the 110 samples (59.1%) was found to be positive of which 35 (32.8%) was quantifiable, besides a narrower size range (250 nm–8 μm see details in Tables 1 and 2). For this size interval the total number of SARS-CoV-2 varied in the range of 0.9–59.9 copies/m3 on an 8-h daily average covering the most active 8 h of patient care at the HDU. As the patients changed within a short time we could define 6 groups of measurement days, however we could obtain quantitative information for only two groups (marked with green (B1) and orange (B4), see Fig. 2), where the number of SARS-CoV-2 RNA copies could be determined for all days and the entire size range. Based on these measurement results, it was found that an unimodal size distribution is typical in all cases, of which the maximum varies within the 1–4 μm diameter range. www.nature.com/scientificreports/ Our results show that the magnitude of increments linked to different events was diverse. While in the case of room cleaning, the increase of ­PM10 concentration was up to 60 µg/m3, the effect of bathing led only to an increment of 8–13 µg/m3. Generally, 7–9 significant events could be registered in one day (see Fig. 1.). µg y gi g y g In the case of Hospital B, although three patients were treated in parallel, the patients were not active, and room ventilation was indirect, so much fewer—typically 1–4—events were detected each day indirectly based on the OPC data. However, the effect of these events was found to be much larger at this site. The increase in ­PM10 concentration was found to be up to 180–315 µg/m3. As this is a HDU, this is in line with the high-intensity activity of HCPs. Size distribution of SARS‑CoV‑2 RNA copies. Based on the results of published studies so far, it can be generally concluded that the positivity rate of SARS-CoV-2 RNA surface and indoor air samples collected from the patient’s environment is ­similar23,29. In addition to direct contact, deposition of airborne particles also con- tributes to surface contamination. At the same time, from the aspect of airborne transmission, it is important to know the abundance of each particle size range that the virus is likely to be attached to. To date, only a few studies have been performed investigating the size distribution of infectious aerosol. In the work of Liu et al.16, a size distribution of 6 size ranges was obtained as a result of a long 16-h sampling. Stern et al.42,55 performed successful measurements for three size ranges with a sampling time of 48 h, while Moharir et al.56 presented measurements with a much larger sampling flow rate (100 L/min) and a much shorter sampling time (10 min) suitable only for Figure 1. Event log and particle mass concentration trends at Hospital A, 04 29 2021. Figure 1. Event log and particle mass concentration trends at Hospital A, 04 29 2021. Scientific Reports | (2023) 13:3566 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Coefficients of linear regression for size fractionated SARS-CoV-2 and PM mass concentration variables for three group of patients. Patient group Coefficients of linear regression R2 Slope of linear fit PM1 PM2.5 PM10 PM1 PM2.5 PM10 A1 (7 stage data) 0.67 0.79 0.21 15.71 26.57 1.52 A1 (5 stage data) 0.50 0.74 0.53 4.90 8.63 1.03 B1 0.97 0.30 0.21 0.07 0.26 0.31 B4 0.77 0.98 0.64 0.35 0.16 0.01 Patient group Coefficients of linear regression R2 Slope of linear fit PM1 PM2.5 PM10 PM1 PM2.5 PM10 A1 (7 stage data) 0.67 0.79 0.21 15.71 26.57 1.52 A1 (5 stage data) 0.50 0.74 0.53 4.90 8.63 1.03 B1 0.97 0.30 0.21 0.07 0.26 0.31 B4 0.77 0.98 0.64 0.35 0.16 0.01 Table 3. Coefficients of linear regression for size fractionated SARS-CoV-2 and PM mass concentration variables for three group of patients. Table 3. Coefficients of linear regression for size fractionated SARS-CoV-2 and PM mass concentration variables for three group of patients. typically varies in the size range of 0.5–4 µm. In the work of Stern et al.55 it was also reported that SARS-CoV-2 RNA positive samples could be found in a wide size range, however it was most frequent in the 2.5–10 µm size range. It should be noted that the differing results of the A1 group could be the consequence of high infectious SARS-CoV-2 alpha VOC as vaccination was low at that time. Tan et al.31 demonstrates a decrease in the probabil- ity of transmission in previously exposed/vaccinated individuals, which would suggest decreased viral shedding. The difference could also be due to a range of other possible factors, such as the wide interpersonal variability in shedding reported in COVID-19 ­patients32. Among the many factors that determine the exact location of the maximum in the size distribution (atmospheric conditions, indoor air quality, patients’ virulence, indoor human activity, etc.), our aim was to examine the effect of particle load and fluctuations. It should be mentioned the fact that some of the patients may have not released detectable amounts of virus under the conditions tested, thus they were no longer infectious during hospitalization (see Table S1), which suggests that the patients’ virus emission rate has a very high personal variety, in accordance with the results reported in Ref.31,57. www.nature.com/scientificreports/ Considering this, and the fact that three patients could have released the virus in parallel, the discussion on the number of days since symptom onset in relation with airborne virus load is not straightforward. However, in case of Hospital B, where 18 patients were examined in total, it was found that the virus release was much lower for those patients who had spent more than 20 days in the hospital since the onset of symptoms (63% of days studied).i y y y To investigate the significance of the aerosol load status of the wards, a correlation analysis was performed between the PM mass concentration increment and the number of SARS-CoV-2 RNA N2 copies in each size range. Those periods were evaluated for which at least the 80 percent of samples were quantifiable by PCR (i.e., A1 and B1 and B4 groups). Among the statistical values of daily PM mass concentration trends (min, max, mean, increment etc.) medical staff and patient activity related highest concentration increment was found to show the strongest correlation for the size ranges studied ­(PM10, ­PM2.5 and ­PM1). SARS-CoV-2 copies were summed for all impactor stages within the size range studied (i.e. stages (9,8,)7,6 for ­PM1; all stages for ­PM10). Correlation coefficients of linear regression for the three periods (patient groups) and the studied size ranges are summarized in Table 3, while the corresponding scatter plots are shown in Fig. 3.hf The most important difference between the two sites in terms of sampling is that the extended version of May impactor was used at Hospital A, which allowed us the collection of particles below 250 nm. As the number of SARS-CoV-2 in this size range was remarkably high on certain days it should be emphasized that the investigation of this size range is also recommended. This is also confirmed by the correlation analysis results, since a more accurate conformity is found (rows 1 and 2 of Table 3) if the smallest size ranges (stages 8 and 9) are taken into account. To the best of our knowledge our study is the first to provide data for this important particle size range.f i According to different investigators, the median/mean equivalent spherical diameter of SARS-CoV-2 (without the spikes) was between 60 and 140 ­nm16,58–62. The size depended on the sample collection (e.g. dissection) and preparation (freezing, fixation etc.) method and also on the measurement technique (e.g. www.nature.com/scientificreports/ In case of these 3 sample sets all samples were found to be SARS-CoV-2 RNA positive in the 2–4 μm size range, however the number of SARS-CoV-2 RNA copies was found to be below the quantification limit in the size range of 250–500 nm. Also, no clear time trend in size distribution was observed during any of the 4-day periods. g y y p For the remaining four groups (B2, B3, B5 and B6), the number of SARS-CoV-2 RNA copies were below the detection limit in a major proportion of the collected samples. Mostly samples corresponding to the size fraction of 0.5–1 μm showed values above the detection limit, suggesting that if highly virus-laden particles are present, they are more likely to exist on particles in this size range. According to Ribaric et al.29 summarizing the few available measurement results, it is not possible to establish a clear size distribution of the SARS-CoV-2 RNA copy number, as the summarized results of the collected studies show that the positivity rate in hospital rooms and waiting rooms is similar (~ 30%) for both 1–4 µm and < 1 µm size ranges (Table 2). In the current study, based on larger number of samples with a higher size resolution, we can confirm that the maximum of the size distribution Figure 2. Size distribution of the number of SARS-CoV-2 RNA copies for A1 group (purple) and B1 (green) and B4 (orange) two patient groups. The relative standard deviation of PCR is estimated as 31%, indicated on the bars. Transparent bars refer to positivity but below the quantification limit. Figure 2. Size distribution of the number of SARS-CoV-2 RNA copies for A1 group (purple) and B1 (green) and B4 (orange) two patient groups. The relative standard deviation of PCR is estimated as 31%, indicated on the bars. Transparent bars refer to positivity but below the quantification limit. https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 | www.nature.com/scientificreports/ Table 3. Coefficients of linear regression for size fractionated SARS-CoV-2 and PM mass concentration variables for three group of patients. Patient group Coefficients of linear regression R2 Slope of linear fit PM1 PM2.5 PM10 PM1 PM2.5 PM10 A1 (7 stage data) 0.67 0.79 0.21 15.71 26.57 1.52 A1 (5 stage data) 0.50 0.74 0.53 4.90 8.63 1.03 B1 0.97 0.30 0.21 0.07 0.26 0.31 B4 0.77 0.98 0.64 0.35 0.16 0.01 Table 3. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Exhaled droplets range in diameter from 0.01 and 1000 µm depending on the physical activity, health status, generation mechanism and the site of origin, among ­others63. The smallest particles are formed in the smallest airways due to the mechanism of elasto-capillary instability related to airway reopening. Though the median mass is represented by particles in the size range of 0.7 to 1 µm64, exhaled droplets of 100–200 µm are also common and these particles represent a significant particle fraction of the total number ­concentration65. After exhalation, the size of these droplets can further decrease by the evaporation of volatile content, thus at least theoretically it is possible that the RNA copies detected on the lowest stages (especially the second lowest stage) of the impactor originate from entire virions. Obviously, it cannot be excluded that the particles impacting here contained or were only virion fragments containing the N2 sequence. y g g q A strong linear relationship was found for the smaller ­(PM1 and ­PM2.5) size ranges, while it was moderate for the greater (total, under 10 µm) size range. In general, the aerosol sources in hospital wards can basically be (i) the mixing of outdoor particles due to room ­ventilation47–49, (ii) the emission of people by breathing and speaking, etc.63–65, (iii) resuspension as a result of their or staff ­activities54, and (iv) the direct and indirect particle emission of machines (respirator apparatus) operating in the ­room30,50,51. As the effect of outdoor sources was eliminated, and the quantity of particles emitted during breathing by the hospital staff wearing masks and by patients even with respiratory ­support50,51 was found to be orders of magnitude smaller than the concentration fluctuations due to human activities, it can be stated that the resuspension effect of inside movements are determinant for indoor PM concentration increase. Although patients are the primary source of airborne SARS-CoV-2 RNA, these virus laden particles are transmitted to the surfaces by direct contact or by sedimentation. Direct contact might be responsible for SARS-CoV-2 RNA identified on the patients’ bed, nightstand and patients’ phone as we detected it in surface swabs in Hospital B. As resuspension of all particles are significant it can be concluded that the medical stuff activities (such as movement and treatment) could result in an excess of virus-laden aerosols in the indoor atmosphere. Conclusion h In the present study, high numbers of size-fractionated aerosol samples collected in hospital wards were analyzed to investigate the potential viral load of aerosol particles over a wide range of particle sizes. As a result, we could also obtain information about the ultrafine size range that, to the best of our knowledge, had not been studied so far. The particle size distribution associated the virus is important, since the lung deposition probability has a minimum at around 400 nm, alveolar deposition increases with decreasing particle diameter 67. As fine ­(PM1) particles can reach the alveolar surface, they might cause direct alveolar infection with highly contagious pathogens.hi p g The detected SARS-CoV-2 RNA positivity rate of nearly 60% and the quantification rate of air samples of nearly 50% indicate that sampling and analysis were successful in the 70 nm–8 µm size range. In addition, the length of the sampling period allowed for statistical evaluation of the collected data. The detected size distribu- tions were typically unimodal in the 0.25–10 µm range, and significant quantities of SARS-CoV-2 RNA were detected even in the sub-300 nm size range. This suggests that the enveloped SARS-CoV-2 virus might even be able to spread in the air without being attached to a carrier aerosol particle. The location of the maximum in the size distribution was different for different patient groups, and there was no temporal trend, suggesting that the viral load of the hospital room is not primarily determined by the duration of the patient’s positivity. y y y Statistical evaluation of size-fractionated particle number concentrations and SARS-CoV-2 RNA number concentrations indicated that the number of virus-containing particles increased with the increased aerosol particle number due to human activity. This is likely to be the consequence of the observed large proportion of infected surfaces, as discussed in numerous ­studies29. Moreover, the results suggest that the virus RNA number concentrations in the air are determined by indoor aerosol sources and do not appear to be dependent on total indoor PM concentrations. Surface disinfection, therefore, plays a key role not only in preventing infection through contact but also in reducing the airborne spread of infectious particles.hi The significant fraction of positive samples in the < 250 nm size range can be important also from the per- spective of face masks. www.nature.com/scientificreports/ It is worth noting that the virus detection method used in this work could not distinguish between viable and non-viable viruses/virions.h The slope of the linear regression lines (Fig. 3) was generally much higher for Hospital A than for Hospital B, while in case of ­PM2.5 it was in the same magnitude at Hospital A for different patient groups. This suggests that under identical site layout and measurement setup, the viral content of the indoor air is proportional to the increase in particle number concentration caused by indoor activities, taking into account that the characteristics of viral emission of each individual person is highly ­variable32,66. www.nature.com/scientificreports/ atomic force micros- copy, transmission electron microscopy). There was also an inherent inter-individual variability of the virion size, but the diameter of the investigated individual virions regularly fell into the 50–160 nm size range. As in our study on the last two stages of the extended impactor the sampled particles were 180–250 nm and 70–180 nm large, it is a plausible question whether the detected RNA fragments originated from virions contained in very small, eventually dried out droplets, unattached virions or merely virus fragments. Figure 3. PM vs SARS-CoV-2 number concentrations for three measurement periods (A1, B1 and B4 patient groups) for the (a) below 1 µm, (b) below 2.5 µm and (c) below 10 µm size ranges and the fitted linear regression lines. Figure 3. PM vs SARS-CoV-2 number concentrations for three measurement periods (A1, B1 and B4 patient groups) for the (a) below 1 µm, (b) below 2.5 µm and (c) below 10 µm size ranges and the fitted linear regression lines. Scientific Reports | (2023) 13:3566 | https://doi.org/10.1038/s41598-023-30702-z Limitations of the study One of the weaknesses of our study is that sampling was carried out over an 8-h period to ensure the collec- tion of a critical amount of samples without the disruption of the ward’s activity. As the RNA copy results were integrated to this 8-h period, the relative impact of individual activities in the wards could not be assessed. In addition, we did not have sufficient and evaluable samples with high N2 copies to evaluate the infectivity, as the virus could be deactivated during sampling with the cascade impactor and PCR is not giving information on viability of the virus.fi An additional limitation was the fact that sampling spanned over two different VOC periods with significant changes in the vaccination status of patients, associated with shorter recovery, implying different degrees of virus ­emissions69,70, however, this supposition needs further investigations. Conclusion h It is well known that the filtering efficiency of the masks is related to their capacity to filter out particles as large as or larger than 300 nm. Virtually, these masks could not be effective in the case of viruses found in the < 250 nm size fraction. However, it has been demonstrated that impaction and interception characterizing the > 300 nm particles are not the only filtering mechanisms and smaller particles are also well filtered due to diffusional and electrostatic attraction ­mechanisms68. if During the COVID pandemic, the use of standardized ventilator devices was not allowed in the sampled wards, so these data might be different in non-pandemic times. Nevertheless, it is likely, that the current findings are valid for a number of other respiratory pathogens. Therefore, present results may serve as valuable inputs when designing hospital wards, but also when planning the accommodation and treatment of infected patients. Scientific Reports | (2023) 13:3566 | https://doi.org/10.1038/s41598-023-30702-z www.nature.com/scientificreports/ Data availabilityh y The data related to the present study can be obtained from the corresponding author J. Osán (osan.janos@ek-cer u) upon personal request. Received: 25 November 2022; Accepted: 28 February 2023 Received: 25 November 2022; Accepted: 28 February 2023 References h l f l l l f d h h l d of educational hospital. Environ. Sci. Pollut. Res. 29(10), 14143–14157. https://​doi.​org/​10.​1007/​s11356-​021-​16733-x (2022). 8. Chan, J. F. W. et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-perso p ( ), p g ( ) 8. Chan, J. F. W. et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster. Lancet 395(10223), 514–523. https://​doi.​org/​10.​1016/​S0140-​6736(20)​30154-9 (2020). p p g 8. Chan, J. F. W. et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster Lancet 395(10223) 514–523 https://doi org/10 1016/S0140-6736(20)30154-9 (2020) J p g p p transmission: A study of a family cluster. Lancet 395(10223), 514–523. https://​doi.​org/​10.​1016/​S0140-​6736(20)​30154-9 (202 transmission: A study of a family cluster. Lancet 395(10223), 514 523. https://​doi.​org/​10.​1016/​S0140 ​6736(20)​30154 9 (2020). 9. Ryu, B. H. et al. Environmental contamination of SARS-CoV-2 during the COVID-19 outbreak in South Korea. Am. J. Infect Control 48(8), 875–879. https://​doi.​org/​10.​1016/j.​ajic.​2020.​05.​027 (2020).i ( ) p g j j ( ) 0. Lotfi, M., Hamblin, M. R. & Rezaei, N. COVID-19: Transmission, prevention, and potential therapeutic opportunities. Clin. Chim Acta 508, 254–266. https://​doi.​org/​10.​1016/j.​cca.​2020.​05.​044 (2020).ih , p g j ( ) 11. Greenhalgh, T. et al. Ten scientific reasons in support of airborne transmission of SARS-CoV-2. The Lancet 397(10285), 1603–1605. https://​doi.​org/​10.​1016/​S0140-​6736(21)​00869-2 (2021). , p g j ( ) 11. Greenhalgh, T. et al. Ten scientific reasons in support of airborne transmission of SARS-CoV-2. The Lancet 397(10285), 1603–1605. https://​doi.​org/​10.​1016/​S0140-​6736(21)​00869-2 (2021). p g 12. Wang, C. C. et al. Airborne transmission of respiratory viruse 13. Lewis, D. Mounting evidence suggests coronavirus is airborne - But health advice has not caught up. Nature 583(7817), 510–513. https://​doi.​org/​10.​1038/​d41586-​020-​02058-1 (2020). 3. Lewis, D. Mounting evidence suggests coronavirus is airborne - But health advice has not caught up. Nature 583(7817), 510–513 https://​doi.​org/​10.​1038/​d41586-​020-​02058-1 (2020). p g 4. Birgand, G. et al. Assessment of air contamination by SARS-CoV-2 in hospital settings. JAMA Netw. Open 3(12), 1–14. https:// doi.​org/​10.​1001/​jaman​etwor​kopen.​2020.​33232 (2020).hf g j p ( ) 5. Thuresson, S. et al. Airborne severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospitals: Effects of aerosol- generating procedures, HEPA-filtration units, patient viral load, and physical distance. Clin. Infect. Dis. 75(1), e89–e96. https:// doi.​org/​10.​1093/​cid/​ciac1​61 (2022). g 16. Liu, Y. et al. Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals. Nature 582(7813), 557–560. References https://​doi.​org/​10.​1038/​ s41586-​020-​2271-3 (2020). 7. Guo, Z. D. et al. Aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards, Wuhan China, 2020. Emerg. Infect. Dis. 26(7), 1586–1591. https://​doi.​org/​10.​3201/​eid26​07.​200885 (2020). g f p g 8. Razzini, K. et al. SARS-CoV-2 RNA detection in the air and on surfaces in the COVID-19 ward of a hospital in Milan, Italy. Sci Total Environ 742 140540 https //doi org/10 1016/j scitoten 2020 140540 (2020) g f p g 8. Razzini, K. et al. SARS-CoV-2 RNA detection in the air and on surfaces in the COVID-19 ward of a hospital in Milan, Italy. Sci Total Environ. 742, 140540. https://​doi.​org/​10.​1016/j.​scito​tenv.​2020.​140540 (2020). p g j 19. Chia, P. Y. et al. Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients. Nat. Commun. 11(1), 1–7. https://​doi.​org/​10.​1038/​s41467-​020-​16670-2 (2020). g 20. Zhou, J. et al. Investigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface and air contamination in an acute healthcare setting during the peak of the coronavirus disease 2019 (COVID-19) pandemic in London. Clin. Infect. Dis. 73(7), e1870–e1877. https://​doi.​org/​10.​1093/​cid/​ciaa9​05 (2021).h p g 21. Santarpia, J. L. et al. The size and culturability of patient-generated SARS-CoV-2 aerosol. J. Expo. Sci. Environ. Epidemiol. https://​ doi.​org/​10.​1038/​s41370-​021-​00376-8 (2021). g 2. Lednicky, J. A. et al. Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients. Int. J. Infect. Dis. 100, 476–482 https://​doi.​org/​10.​1016/j.​ijid.​2020.​09.​025 (2020). 23. Dinoi, A. et al. A review on measurements of SARS-CoV-2 genetic material in air in outdoor and indoor environments: Implica- tion for airborne transmission. Sci. Total Environ. 809, 151137. https://​doi.​org/​10.​1016/j.​scito​tenv.​2021.​151137 (2022).f p g j 24. Rezaei, M. & Netz, R. R. Airborne virus transmission via respiratory droplets: Effects of droplet e Curr. Opin. Colloid Interface Sci. 55, 101471. https://​doi.​org/​10.​1016/j.​cocis.​2021.​101471 (2021). p g j ( ) 4. Rezaei, M. & Netz, R. R. Airborne virus transmission via respiratory droplets: Effects of droplet evaporation and sedimentation C O i C ll id I f S i 1014 1 h //d i /10 1016/j i 2021 1014 1 (2021) 24. Rezaei, M. & Netz, R. R. Airborne virus transmission via respiratory droplets: Effects of droplet evaporation and sedimentation. Curr. Opin. Colloid Interface Sci. 55, 101471. https://​doi.​org/​10.​1016/j.​cocis.​2021.​101471 (2021). 24. Rezaei, M. & Netz, R. R. Airborne virus transmission via respiratory droplets: Effects of droplet evaporation and sedimentation. Curr. Opin. Colloid Interface Sci. 55, 101471. https://​doi.​org/​10.​1016/j.​cocis.​2021.​101471 (2021). p f p g j 5. References References 1. Pan, M., Lednicky, J. A. & Wu, C. Y. Collection, particle sizing and detection of airborne viruses. J. Appl. Microbiol. 127(6), 1596–1611. https://​doi.​org/​10.​1111/​jam.​14278 (2019). 1. Pan, M., Lednicky, J. A. & Wu, C. Y. Collection, particle sizing and detection of airborne viruses. J. Appl. Microbiol. 127(6), 1596–1611. https://​doi.​org/​10.​1111/​jam.​14278 (2019). 1. Pan, M., Lednicky, J. A. & Wu, C. Y. Collection, particle sizing and detection of airborne viruses. J. Appl. Microbiol. 127(6), 1596–1611. https://​doi.​org/​10.​1111/​jam.​14278 (2019). p g j ( ) 2. Borges, J. T., Nakada, L. Y. K., Maniero, M. G. & Guimarães, J. R. SARS-CoV-2: A systematic review of indoor air sampling for virus detection. Environ. Sci. Pollut. Res. 28(30), 40460–40473. https://​doi.​org/​10.​1007/​s11356-​021-​13001-w (2021). p g j 2. Borges, J. T., Nakada, L. Y. K., Maniero, M. G. & Guimarães, J. R. SARS-CoV-2: A systematic review of indoor air sampling for virus detection. Environ. Sci. Pollut. Res. 28(30), 40460–40473. https://​doi.​org/​10.​1007/​s11356-​021-​13001-w (2021). p g 3. Duchaine, C. & Roy, C. J. Bioaerosols and airborne transmission: Integrating biological complexity into our perspective. Sci. Tota Environ. 825, 1–2. https://​doi.​org/​10.​1016/j.​scito​tenv.​2022.​154117 (2022). p g j 4. Duval, D. et al. Long distance airborne transmission of SARS-CoV-2: Rapid systematic review. BMJ 377, 1–14. https://​doi.​org/​10.​ 1136/​bmj-​2021-​068743 (2022). p g j 4. Duval, D. et al. Long distance airborne transmission of SARS-CoV-2: Rapid systematic review. BMJ 377, 1–14. https://​doi.​org/​10 1136/​bmj-​2021-​068743 (2022). j 5. Li, X. et al. A spatiotemporally resolved infection risk model for airborne transmission of COVID-19 variants in indoor spaces. Sci. Total Environ. 812, 152592. https://​doi.​org/​10.​1016/j.​scito​tenv.​2021.​152592 (2022). 5. Li, X. et al. A spatiotemporally resolved infection risk model for airborne transmission of COVID-19 variants in indoor spaces. Sci. Total Environ. 812, 152592. https://​doi.​org/​10.​1016/j.​scito​tenv.​2021.​152592 (2022). p g j 6. Auvinen, M., Kuula, J., Grönholm, T., Sühring, M. & Hellsten, A. High-resolution large-eddy simulation of indoor turbulence and its effect on airborne transmission of respiratory pathogens - Model validation and infection probability analysis. Phys. Fluids 34(1), 1–23. https://​doi.​org/​10.​1063/5.​00764​95 (2022).f 6. Auvinen, M., Kuula, J., Grönholm, T., Sühring, M. & Hellsten, A. High-resolution large-eddy simulation of indoor turbulence and its effect on airborne transmission of respiratory pathogens - Model validation and infection probability analysis. Phys. Fluids 34(1), 1–23. https://​doi.​org/​10.​1063/5.​00764​95 (2022).f p g 7. Yousefzadeh, A. et al. Evaluation of bio-aerosols type, density, and modeling of dispersion in inside and outside of different ward of educational hospital. Environ. Sci. Pollut. Res. 29(10), 14143–14157. https://​doi.​org/​10.​1007/​s11356-​021-​16733-x (2022). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 29. Ribaric, N. L., Vincent, C., Jonitz, G., Hellinger, A. & Ribaric, G. Hidden hazards of SARS-CoV-2 transmission in hospitals: A systematic review. Indoor Air 32(1), 1–34. https://​doi.​org/​10.​1111/​ina.​12968 (2022).lf y g 0. Hui, D. S. et al. Exhaled air dispersion during high-flow nasal cannula therapy versus CPAP via different masks. Eur. Respir. J https://​doi.​org/​10.​1183/​13993​003.​02339-​2018 (2019). p g ( ) 1. Tan, S. T. et al. Infectiousness of SARS-CoV-2 breakthrough infections and reinfections during the Omicron wave. Nat. Med https://​doi.​org/​10.​1038/​s41591-​022-​02138-x (2023). g 2. Yang, Q. et al. Just 2% of SARS-CoV-2−positive individuals carry 90% of the virus circulating in communities. Proc. Natl. Acad Sci. U. S. A. 118(21), 1–6. https://​doi.​org/​10.​1073/​pnas.​21045​47118 (2021). g 3. May, K. R. An ‘ultimate’ cascade impactor for aerosol assessment. J. Aerosol Sci. 6(6), 413–419. https://​doi.​org/​10.​1016/​0021- 8502(75)​90057-9 (1975). ( ) ( ) 34. Rahmani, A. R., Leili, M., Azarian, G. & Poormohammadi, A. Sampling and detection of corona viruses in air: A mini review. Sci. Total Environ. 740, 140207. https://​doi.​org/​10.​1016/j.​scito​tenv.​2020.​140207 (2020).illi 35. Osán, J. et al. Experimental evaluation of the in-the-field capabilities of total-reflection X-ray fluorescence analysis to trace fine and ultrafine aerosol particles in populated areas. Spectrochim. Acta - Part B At. Spectrosc. 167, 105852. https://​doi.​org/​10.​1016/j.​ sab.​2020.​105852 (2020).ii 36. Kugler, S. et al. “Characterization of the ultrafine and fine particles formed during laser cladding with the Inconel 718 metal powder by means of X-ray spectroscopic techniques. Spectrochim Acta - Part B At. Spectrosc. 177, 106110. https://​doi.​org/​10.​1016/j.​sab.​ 2021.​106110 (2021).i 7. Seeger, S. et al. Quantification of element mass concentrations in ambient aerosols by combination of cascade impactor sampling and mobile total reflection x-ray fluorescence spectroscopy. Atmosphere (Basel) https://​doi.​org/​10.​3390/​atmos​12030​309 (2021). l yl y g 38. Agaoglu, N. B. et al. COVID-19 PCR test performance on samples stored at ambient temperature. J. Virol. Methods https://​doi.​ org/​10.​1016/j.​jviro​met.​2021.​114404 (2022). g j j 9. Kolarević, S. et al. Detection of SARS-CoV-2 RNA in the Danube River in Serbia associated with the discharge of untreated wastewaters. Sci. Total Environ. 783, 146967. https://​doi.​org/​10.​1016/j.​scito​tenv.​2021.​146967 (2021). p g j 0. “Real-time RT-PCR Primers and Probes for COVID-19 | CDC.” https://​www.​cdc.​gov/​coron​avirus/​2019-​ncov/​lab/​rt-​pcr-​panel- primer-​probes.​html (accessed Jun. 24, 2022). p p 1. Corman, V. M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance 25(3), 2000045 https://​doi.​org/​10.​2807/​1560-​7917.​ES.​2020.​25.3.​20000​45 (2020). 42. Stern, R. A. et al. Characterization of hospital airborne SARS-CoV-2. Respir. Res. 22(1), 73. https://​doi.​org/​10.​1186/​s12931-​021-​ 01637-8 (2021). 43. Loupa, G., Zarogianni, A. M., Karali, D., Kosmadakis, I. www.nature.com/scientificreports/ & Rapsomanikis, S. Indoor/outdoor PM2.5 elemental composition and organic fraction medications, in a Greek hospital. Sci. Total Environ. 550, 727–735. https://​doi.​org/​10.​1016/j.​scito​tenv.​2016.​01.​ 070 (2016). 44. Lomboy, M. F. T. C. et al. Characterization of particulate matter 2.5 in an urban tertiary care hospital in the Philippines. B Environ. 92, 432–439. https://​doi.​org/​10.​1016/j.​build​env.​2015.​05.​018 (2015). 45. Li, R. et al. Physiochemical characteristics of aerosol particles in the typical microenvironmen Total Environ. 580, 651–659. https://​doi.​org/​10.​1016/j.​scito​tenv.​2016.​12.​011 (2017). 45. Li, R. et al. Physiochemical characteristics of aerosol particles in the typical microenvironment of hospital in Shanghai, China. Sci. Total Environ. 580, 651–659. https://​doi.​org/​10.​1016/j.​scito​tenv.​2016.​12.​011 (2017). 46. Slezakova, K., Morais, S., Do, M. & Pereira, C. Trace metals in size-fractionated particulate matter in a Portuguese hospital: Exposure risks assessment and comparisons with other countries. Env. Sci. Pollut. Res. 21, 3604–3620. https://​doi.​org/​10.​1007/​ s11356-​013-​2316-3 (2014). 47. Morawska, L. Particulate matter in the hospital environment. Indoor Air 8(4), 285–294. https://​doi.​org/​10.​1111/j.​1600-​0668.​1998.​ 00009.x (1998). 8. Wang, X., Bi, X., Sheng, G. & Fu, J. Hospital indoor PM10/PM2.5 and associated trace elements in Guangzhou, China. Sci. Tota Environ. 366(1), 124–135. https://​doi.​org/​10.​1016/j.​scito​tenv.​2005.​09.​004 (2006). p g j 9. Nimra, A., Ali, Z., Nasir, Z. A., Tyrrel, S. & Sidra, S. characterization of indoor air quality in relation to ventilation practices in h i l f L h P ki S i M l 50(6) 1609 1620 h //d i /10 17576/j 2021 5006 09 (2021) p g j 49. Nimra, A., Ali, Z., Nasir, Z. A., Tyrrel, S. & Sidra, S. characterization of indoor air quality in relation to ventilation practices in hospitals of Lahore, Pakistan. Sains Malays. 50(6), 1609–1620. https://​doi.​org/​10.​17576/​jsm-​2021-​5006-​09 (2021). 9. N a, ., , ., Nas , . ., y e , S. & S d a, S. c a acte at o o doo a qua ty e at o to ve t at o p act ces hospitals of Lahore, Pakistan. Sains Malays. 50(6), 1609–1620. https://​doi.​org/​10.​17576/​jsm-​2021-​5006-​09 (2021).hf p y p g j 50. Wilson, N. M. et al. The effect of respiratory activity, non-invasive respiratory support and facemasks on aerosol generatio its relevance to COVID-19. Anaesthesia 76(11), 1465–1474. https://​doi.​org/​10.​1111/​anae.​15475 (2021). 51. Hamilton, F. W. et al. Aerosol emission from the respiratory tract: An analysis of aerosol generation from oxygen delivery syst Thorax 77(3), 276–282. https://​doi.​org/​10.​1136/​thora​xjnl-​2021-​217577 (2022). h p g j 52. Heinzerling, A. et al. www.nature.com/scientificreports/ Transmission of COVID-19 to health care personnel during exposures to a hospitalized patient — Solano County, California, February 2020. MMWR. Morb. Mortal. Wkly. Rep. 69(15), 472–476. https://​doi.​org/​10.​15585/​mmwr.​mm691​ 5e5 (2020). 3. O’Neil, C. A. et al. Characterization of aerosols generated during patient care activities. Clin. Infect. Dis. 65(8), 1342–1348. https:// doi.​org/​10.​1093/​cid/​cix535 (2017). g 4. Nagy, A., Horváth, A., Farkas, Á., Füri, P. & Erdélyi, T. Modeling of nursing care - associated airborne transmission of SARS - CoV - 2 in a real - world hospital setting. GeroScience 0123456789, 1–11 (2022). p g 5. Stern, R. A. et al. Concordance of SARS-CoV-2 RNA in aerosols from a nurses station and in nurses and patients during a hospita ward outbreak JAMA Netw Open 5(6) E2216176 https //doi org/10 1001/jamanetworkopen 2022 16176 (2022) g 55. Stern, R. A. et al. Concordance of SARS-CoV-2 RNA in aerosols from a nurses station and in nurses and patients during a hospital ward outbreak. JAMA Netw. Open 5(6), E2216176. https://​doi.​org/​10.​1001/​jaman​etwor​kopen.​2022.​16176 (2022). 56. Moharir, S. C. et al. Detection of SARS-CoV-2 in the air from hospitals and closed rooms occupied by COVID-19 patients. Med https://​doi.​org/​10.​1101/​2020.​12.​30.​20248​890 (2021). p g 57. Riediker, M. et al. Higher viral load and infectivity increase risk of aerosol transmission for Delta and Omicron variants of SA CoV-2. Swiss Med. Wkly. 152(w30133), 1–5. https://​doi.​org/​10.​4414/​smw.​2022.​w30133 (2022). 58. Kiss, B., Kis, Z., Pályi, B. & Kellermayer, M. S. Z. Topography, spike dynamics, and nanomechanics of individual native SARS-CoV-2 virions. Nano Lett. 21(6), 2675–2680. https://​doi.​org/​10.​1021/​acs.​nanol​ett.​0c044​65 (2021). g 59. Yao, H. et al. Molecular architecture of the SARS-CoV-2 virus. Cell 183(3), 730-738.e13. https://​doi.​org/​10.​1016/j.​cell.​2020.​09 (2020). 0. Zhu, N. et al. A Novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 382(8), 727–733. https://​doi org/​10.​1056/​nejmo​a2001​017 (2020). g j 1. Laue, M. et al. Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures Sci. Rep. 11(1), 3515. https://​doi.​org/​10.​1038/​s41598-​021-​82852-7 (2021). 2. Tai, L. et al. Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein. Proc. Natl. Acad. Sci. U. S. A 118(48), e2112703118. https://​doi.​org/​10.​1073/​pnas.​21127​03118 (2021). ( ) p g p ( ) 3. Morawska, L. et al. Size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities. J. Aerosol Sci. 40(3), 256–269. https://​doi.​org/​10.​1016/j.​jaero​sci.​2008.​11.​002 (2009). p g j j 4. Bake, B., Larsson, P., Ljungkvist, G., Ljungström, E. & Olin, A. C. Exhaled particles and small airways. References Chen, W., Zhang, N., Wei, J., Yen, H. L. & Li, Y. Short-range airborne route dominates exposure of respiratory infection during close contact. Build. Environ. 176, 1–33. https://​doi.​org/​10.​1016/j.​build​env.​2020.​106859 (2020). p f p g j 25. Chen, W., Zhang, N., Wei, J., Yen, H. L. & Li, Y. Short-range airborne route dominates exposure close contact. Build. Environ. 176, 1–33. https://​doi.​org/​10.​1016/j.​build​env.​2020.​106859 (2020).f 5. Chen, W., Zhang, N., Wei, J., Yen, H. L. & Li, Y. Short-range airborne route dominates exposure of respiratory infection during close contact. Build. Environ. 176, 1–33. https://​doi.​org/​10.​1016/j.​build​env.​2020.​106859 (2020).f p g j 6. Nicas, M., Nazaroff, W. W. & Hubbard, A. Toward understanding the risk of secondary airborne infection: Emission of respirable pathogens. J. Occup. Environ. Hyg. 2(3), 143–154. https://​doi.​org/​10.​1080/​15459​62059​09184​66 (2005).hl p g p yg p g 27. Mittal, R., Ni, R. & Seo, J. H. The flow physics of COVID-19. J. Fluid Mech. 894, F2. https://​doi.​org/​10.​1017/​jfm.​2020.​330 (2020). 28. Netz, R. R. Mechanisms of airborne infection via evaporating and sedimenting droplets produced by speaking. J. Phys. Chem. B 124(33), 7093–7101. https://​doi.​org/​10.​1021/​acs.​jpcb.​0c052​29 (2020). Jhl p y J p g j ( ) 28. Netz, R. R. Mechanisms of airborne infection via evaporating and sedimenting droplets produced by speaking. J. Phys. Chem. B 124(33), 7093–7101. https://​doi.​org/​10.​1021/​acs.​jpcb.​0c052​29 (2020). https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 | Acknowledgementsh g This research was supported by the Eötvös Loránd Research Network (Grant number: SA-45/2021) and by European Structural and Investment Funds jointly financed by the European Commission and the Hungarian Government under grant no. VEKOP-2.3.2-16-2016-00011. g This research was supported by the Eötvös Loránd Research Network (Grant number: SA-45/2021) and by European Structural and Investment Funds jointly financed by the European Commission and the Hungarian Government under grant no. VEKOP-2.3.2-16-2016-00011. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Groma, V. et al. Sources and health effects of fine and ultrafine aerosol particles in an urban environment. Atmos. Pollut. Res. 13(2) 101302. https://​doi.​org/​10.​1016/j.​apr.​2021.​101302 (2022).ifi p g j p 8. Konda, A. et al. Aerosol filtration efficiency of common fabrics used in respiratory cloth masks. ACS Nano 14(5), 6339–6347 https://​doi.​org/​10.​1021/​acsna​no.​0c032​52 (2020). p g ( ) 69. Puhach, O. et al. Infectious viral load in unvaccinated and vaccinated individuals infected with ancestral, Delta or Omicron SARS- CoV-2. Nat. Med. 28(7), 1491–1500. https://​doi.​org/​10.​1038/​s41591-​022-​01816-0 (2022). p g 0. King, K. L. et al. SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva. PLoS ONE https://​doi.​org/ 10.​1101/​2022.​02.​10.​22270​797 (2022). 70. King, K. L. et al. SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva. PLoS ONE https://​doi.​org/​ 10.​1101/​2022.​02.​10.​22270​797 (2022). Author contributions Conceptualization, B.M., Á.F., V.M., A.N. and J.O.; methodology, V.G., R.S.-E., A.M. and J.O.; formal analysis, V.G. and S.K.; investigation, A.H., G.G., T.E., R.S.-E., A.M.; data curation, V.G., P.F. and S.K.; writing—original draft preparation, V.G., S.K. and J.O; writing—review and editing, A.N., Á.F., V.M., B.M., A.H., R.S.-E., T.E. and A.M.; visualization, V.G. and S.K.; supervision, A.N., Á.F., B.M. and V.M.; funding acquisition, B.M. All authors have read and agreed to the published version of the manuscript. www.nature.com/scientificreports/ Respir. Res. 20(1), 8. https:// doi.​org/​10.​1186/​s12931-​019-​0970-9 (2019). g 65. Holmgren, H., Ljungström, E., Almstrand, A. C., Bake, B. & Olin, A. C. Size distribution of exhaled particles in the range from 0.01 to 2.0μm. J. Aerosol Sci. 41(5), 439–446. https://​doi.​org/​10.​1016/j.​jaero​sci.​2010.​02.​011 (2010).l μ ( ) p g j j ( ) 6. Scheuch, G. Breathing is enough: For the spread of influenza virus and SARS-CoV-2 by breathing only. J. Aerosol Med. Pulm. Drug Deliv. 33(4), 230–234. https://​doi.​org/​10.​1089/​jamp.​2020.​1616 (2020). https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 | Funding Funding Open access funding provided by Centre for Energy Research. Competing interests  The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​30702-z. Correspondence and requests for materials should be addressed to J.O. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2023 Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​30702-z. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​30702-z. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​30702-z. Correspondence and requests for materials should be addressed to J.O. Correspondence and requests for materials should be addressed to J.O. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2023 https://doi.org/10.1038/s41598-023-30702-z Scientific Reports | (2023) 13:3566 |
https://openalex.org/W3002462287
https://scindeks-clanci.ceon.rs/data/pdf/2217-8139/2017/2217-81391702049B.pdf
English
null
Determination of the in situ coefficient of friction and imperfection of prestressing cables
Građevinski materijali i konstrukcije
2,017
cc-by-sa
5,782
Dragan Bojović, MSc, research assistant, IMS Institute, Bul. vojvode Mišića 43, Belgrade, dragan.bojovic@institutims.rs Bojan Aranđelović, PhD student, IMS Institute, Bul. vojvode Mišića 43, Belgrade, bojan.arandjelovic@institutims.rs Ksenija Janković, PhD, senior research fellow, IMS Institute, Bul. vojvode Mišića 43, Belgrade, ksenija.jankovic@institutims.rs Aleksandar Senić, assistant, Faculty of Civil Engineering, Bul. kralja Aleksandra 73, Belgrade Marko Stojanović, MSc, research assistant, IMS Institute, Bul. vojvode Mišića 43, Belgrade, marko.stojanovic@institutims.rs ODREĐIVANJE IN-SITU KOEFICIJENTA TRENJA I IMPERFEKCIJE KABLOVA ZA PREDNAPREZANJE DETERMINATION OF THE IN SITU COEFFICIENT OF FRICTION AND IMPERFECTION OF PRESTRESSING CABLES Dragan BOJOVIĆ Bojan ARANĐELOVIĆ Ksenija JANKOVIĆ Aleksandar SENIĆ Marko STOJANOVIĆ STRUČNI RAD PROFESSIONAL PAPER UDK: 624.042.7 doi:10.5937/grmk1702049B Mr Dragan Bojović, istraživač saradnik, IMS Institut, Bul. vojvode Mišića 43, Beograd, dragan.bojovic@institutims.rs Bojan Aranđelović, IMS Institut, Bul. vojovde Mišića 43, Beograd, bojan.arandjelovic@institutims.rs Dr Ksenija Janković, viši naučni saradnik, IMS Institut, Bul. vojvode Mišića 43, Beograd, ksenija.jankovic@institutims.rs Aleksandar Senić, asistent, Građevinski fakultet Beograd, Bul. kralja Aleksandra 73, Beograd Marko Stojanović, MSc, istraživač saradnik, IMS Institut, Bul. vojovde Mišića 43, Beograd, marko.stojanovic@institutims.rs 1 UVOD Modern reinforced concrete structure of significant spans cannot be imagined without its prestressing. Nowadays, the design established at the very beginning of such systems, with small changes introduced in the recent past, is in use worldwide. One of the basic premises is that the force in the cable at the active end during tensioning must not exceed the value given in the following formula (1): Savremena armiranobetonska konstrukcija značajnih raspona ne može se zamisliti bez njenog prednapre- zanja. Projektovanje ustanovljeno na samim počecima ovakvih sistema, s malim izmenama nedavno uvedenim, u upotrebi je i danas širom sveta. Jedna od osnovnih premisa jeste to da sila u kablu na aktivnom kraju za vreme zatezanja ne sme preći vrednost datu obrascem (1): (1) Pmax = A p·σp,max where Ap represents the cross-sectional area of the cable and σp,max, the maximum stress in the cable. Value σp,max is determined by the lower value of k1·fpk and k2·fp0,1k. The values of k1 and k2 are defined by national standards. The most commonly used values are k1=0.8 and k2=0.9 [1,2]. Exceeding the highest tension force is allowed in special cases when highly accurate equipment for prestressing is used. Then the maximum tensioning force is determined by using the formula k3·fp0,1k⋅Ap where the coefficient k3 adopts the value of 0.95. where Ap represents the cross-sectional area of the cable and σp,max, the maximum stress in the cable. Value σp,max is determined by the lower value of k1·fpk and k2·fp0,1k. The values of k1 and k2 are defined by national standards. The most commonly used values are k1=0.8 and k2=0.9 [1,2]. Exceeding the highest tension force is allowed in special cases when highly accurate equipment for prestressing is used. Then the maximum tensioning force is determined by using the formula k3·fp0,1k⋅Ap where the coefficient k3 adopts the value of 0.95. gde A p predstavlja površinu poprečnog preseka kabla i σp,max maksimalni napon u kablu. Vrednost σp,max određena je manjom vrednošću od k 1·fpk i k 2·fp0,1k. Vrednosti k 1 i k 2 definisane su nacionalnim standardima. Najčešće se koriste vrednosti k 1=0,8 i k 2=0.9 [1,2]. Prekoračenje najveće sile zatezanja dopušta se u posebnim slučajevima kada se koristi veoma precizna oprema za prednaprezanje. Tada se najveća sila prednaprezanja određuje pomoću obrasca k 3·fp0,1k⋅Ap, gde se za koeficijent k3 usvaja vrednost 0,95. 1 UVOD GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 49 For good design of prestressed structures, the coefficients of friction and imperfection, or angular deviation are introduced in the calculation. Za dobro projektovanje prednapregnutih konstrukcija u proračun se uvode i koeficijenti trenja i nepravilnosti, odnosno ugaonog odstupanja. g g p j U proizvodnji čelika i plastičnih materijala, svakodnevno se uvode novine koje omogućavaju izradu bezbednijih konstrukcija. Tako, primenom savremenih čelika za prednaprezanje, relaksacija kablova svedena je na minimum. U građevinskoj praksi veoma često, u slučaju prednapregnutih konstrukcija, za trasiranje kablova upotrebljavaju se plastične orebljene cevi od novih vrsta plastike. Uticaj primene novih vrsta materijala na vrednosti koeficijenta trenja često se ostavlja na procenu projektantu. Najčešće se na nivou projekta uzimaju vrednosti koeficijenata iz važećih pravilnika i propisa koji možda i nisu uzeli u obzir primenu novih materijala u prednaprezanju. Dosadašnja iskustva na polju određivanja koeficijenata trenja i nepravilnosti izvođenja u slučaju prednaprezanja uglavnom su u vezi s teorijskim i laboratorijskim ispitivanjima. Kao što je već napomenuto, koeficijent trenja umnogome zavisi od primenjenih materijala, dok koeficijent nepravilnosti zavisi ponajviše od stepena obučenosti i savesnosti izvođača da izvede radove na prednaprezanju [3,4]. Parametri se usvajaju iskustveno, u granicama koje propisuju nacionalni standardi i propisi zemlje u kojoj se konstrukcija izvodi. Stvarno određivanje navedenih parametara jeste veoma komplikovano i zahteva dodatna sredstva od izvođača, kao i angažovanje specifične opreme. Samim tim, projektovani parametri najčešće se ne proveravaju. In the production of steel and plastic materials new things which enable the production of safer structures are introduced every day. Thus, with the application of modern steel for prestressing the relaxation of cables is reduced to a minimum. In construction practice in the case of prestressed structures for tracing cables plastic corugated pipes made of new types of plastics are used very often. The effect of the application of new types of materials on the values of the coefficient of friction is often left for the designer to estimate. Most often at the design level coefficient values are taken from applicable policies and regulations that may not take into account the application of new materials in prestressing. 1 UVOD Previous experience in the field of determining the coefficients of friction and imperfection of the execution of prestressing are mainly related to the theoretical and laboratory testing. As it is already mentioned, the coefficient of friction depends largely on the applied materials, while the coefficient of imperfection depends largely on the level of training and conscientiousness of the contractor to perform work on the prestressing [3.4]. The parameters are adopted empirically within the limits prescribed by national standards and regulations of the country where the structure is performed. Actual determination of the aforementioned parameters is a very complicated process which requires additional resources from the contractor, as well as the invol- vement of specific equipment. Therefore the designed parameters are usually not checked. Pri naknadnom utezanju kablova, sila prethodnog naprezanja i odgovarajuće izduženje kabla moraju da se provere merenjima, a moraju da se kontrolišu i stvarni gubici usled trenja. Pored ovih parametara, za projektante i izvođače na konstrukciji veoma bi korisno bilo da se analiziraju parametri u vezi s koeficijentom trenja i koeficijentom nepravilnosti izvođenja radova na prethodnom naprezanju [5]. In post-tensioning of the cables the prestressing force and the corresponding elongation of the cable must be checked by measurements, and actual losses since the friction have to be controlled. In addition to these parameters, it would be of great use for designers and contractors of the structure to analyze the parameters related to the coefficient of friction and the coefficient of imperfection of execution of work on prestressing [5]. 2 THEORETICAL PREMISES − losses due to the short-term effects of relaxation - ∆Pr , − losses due to the short-term effects of relaxation - ∆Pr , Srednja vrednost sile prethodnog naprezanja Pm,t(x) u vremenu t > t 0 određuje se u zavisnosti od metode utezanja. Pored navedenih trenutnih gubitaka, treba da se imaju u vidu i gubici prednaprezanja, koji zavise od vremena ∆Pc+s+r (x), a rezultat su tečenja i skupljanja betona i dugotrajne relaksacije čelika za prethodno naprezanje, tako da je − losses due to friction - ∆Pµ(x). − losses due to friction - ∆Pµ(x). Mean value of prestressing force Pm,t(x) at time t > t 0 is determined depending on the method of tensioning. In addition to these current losses, the losses of prestressing which depend on the time ∆Pc+s+r (x), which are the result of creeping and shrinkage of concrete and long-lasting relaxation of steel for prestressing should be taken into account, so that: Pm,t(x) = Pm0(x) − ∆Pc+s+r(x). Pm,t(x) = Pm0(x) − ∆Pc+s+r(x). Početna sila u kablu (Ppo) manja je od početne sile na presi (Pp), usled uvlačenja klinova u toku prenošenja sile s prese na kotvu. Prilikom uvlačenja klinova istovremeno, u približno istoj veličini, uvlače se i užad, što dovodi do pada sile u kablu usled izgubljenog izduženja. Uvlačenje klinova javlja se na strani aktivne i pasivne kotve. Uvlačenje klinova kod pasivne kotve ne mora uticati na smanjenje početne sile ukoliko se ukupno izduženje kod prese poveća za ovu vrednost. Uvlačenje klinova na strani prese, aktivne kotve, dovodi do smanjenja početne sile u kablu za veličinu izgubljenog izduženja, što može dati znatnu vrednost kod kratkih kablova. Uvlačenje klinova kod aktivne i pasivne kotve približno je isto i uzima se na nivou od 3 mm do 5 mm. Postoje načini da se ovaj gubitak delimično ili potpuno eliminiše, uz primenu posebnih procedura. Initial force in cable (Ppo) is less than the initial force on the press (Pp) due to the insertion of wedges during the transmission of force to the anchor. During the insertion of wedges simultaneously at approximately the same size, cables are also drawn, leading to a drop in force in the cable due to the lost elongation. Insertion of wedges occurs on the side of the active and passive anchor. 2 THEORETICAL PREMISES GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 50 Sila prednaprezanja najčešće nije konstantna duž kabla usled trenja kabla o zidove kanala kroz koji prolazi i trenja usled promene pravca kabla. Pored toga, sila prednaprezanja se tokom vremena i smanjuje usled relaksacije čelika, skupljanja i tečenja betona. The prestressing force is usually not constant along the cable due to the friction of the cable on the walls of the channels through which it passes and friction due to the changes in the direction of the cable. In addition, the tensioning force is reduced over time due to relaxation of steel, shrinkage and creeping of concrete. Prema tome, u jednom preseku kabla imamo početnu silu Pm0 u trenutku prednaprezanja, silu Pm,t u nekom vremenu „t” i trajnu silu za t ∞ → , Pm∞. Za trajnu silu uzima se u obzir i promena sile usled dejstva stalnog i pokretnog opterećenja nakon prednaprezanja. Gubici koji se proračunavaju dati su u standardu SRPS EN:1992-1-1:2004 [6]. Accordingly, in a cross-section of the cable there is the initial force Pm0 in the moment of prestressing, force Pm,t in some time „t” and permanent force for t ∞ → , Pm∞. For the permanent force the changes of the force due to the effect of permanent and moving load after prestressing should also be taken into account. Losses that are computed are presented in standard SRPS EN:1992-1-1:2004 [6]. Kada se određuju trenutni t=t 0 gubici ∆Pi(x), razmatraju se sledeći trenutni uticaji usled kojih nastaju gubici pri utezanju, u zavisnosti koji od tih uticaja je relevantan: When determining the current t=t 0 losses ∆Pi(x) the following current impacts which lead to losses in tensioning are considered, depending on which of these impacts is relevant: − gubici pri ankerovanju - usled uvlačenja klina - ∆Psl; − losses at anchorage - due to insertion of the wedge - ∆Psl, − gubici usled elastičnih deformacija betona - ∆Pel; − gubici usled kratkotrajnog dejstva relaksacije - − gubici usled kratkotrajnog dejstva relaksacije - ∆Pr; − losses due to elastic deformation of concrete - ∆Pel, − gubici usled trenja - ∆Pµ(x). − gubici usled trenja - ∆Pµ(x). 2 THEORETICAL PREMISES Mean value of prestressing force Pm,t(x) at a given time t, at a distance x from the active end of the cable is equal to the maximum force Pmax which tension the cable on the active end, reduced for current losses and losses that depend on the time. All losses are considered in absolute values. Srednja vrednost sile prethodnog naprezanja Pm,t(x) u datom trenutku vremena t, na rastojanju x od aktivnog kraja kabla, jednaka je najvećoj sili Pmax kojom se kabl zateže na aktivnom kraju, umanjenoj za trenutne gubitke i gubitke koji zavise od vremena. Svi gubici razmatraju se u apsolutnim vrednostima. Vrednost početne sile prednaprezanja Pm0(x) za t=t 0, kojoj je beton izložen neposredno posle utezanja i ankerovanja kablova, ili posle prenošenja prethodnog naprezanja na beton, dobija se kada se sila prednaprezanja Pmax umanji za trenutne gubitke ∆P i (x), i ne treba da prekorači vrednost dobijenu obrascem (2): The value of the initial prestressing force Pm0(x) for t=t 0, where the concrete is exposed immediately after the tensioning and anchoring of the cables, or after the transmission of the prestressing to concrete, is obtained when the prestressing force Pmax is reduced by the current loss ∆Pi (x), and should not exceed the value obtained by the equation (2): x) = Ap σpm0(x) (2) (2) Pm0(x) = Ap σpm0(x) Pm0(x) = Ap σpm0(x) gde je σpm0(x) napon u kablu neposredno posle utezanja ili prenošenja sile kao manja vrednost od k 7·fpk i k 8·fp0,1. Preporučene vrednosti za koeficijente jesu k 7=0,75 i k 8=0.85. gde je σpm0(x) napon u kablu neposredno posle utezanja ili prenošenja sile kao manja vrednost od k 7·fpk i k 8·fp0,1. Preporučene vrednosti za koeficijente jesu k 7=0,75 i k 8=0.85. where σpm0(x) is the stress in the cable immediately after the tensioning or transmitting the force as a smaller value of k 7·fpk and k 8·fp0.1. The recommended values for the coefficients are k 7=0.75 and k 8=0.85. where σpm0(x) is the stress in the cable immediately after the tensioning or transmitting the force as a smaller value of k 7·fpk and k 8·fp0.1. The recommended values for the coefficients are k 7=0.75 and k 8=0.85. 2 THEORETICAL PREMISES The values of coefficients that define the losses of force is often specified while the preparation of the project by selecting different types and shapes of cable route is being done. As the effect of the curvature is predetermined, angular deviation is the result of accidental or unavoidable deviation, as there is no possibility for the trace of protective pipes to be ideally instaled [7]. Treba imati u vidu da će maksimalna vrednost gubitka sile usled trenja biti na drugom kraju elementa, ako se zateže s jednog kraja. Stoga, gubitak usled trenja se menja linearno duž raspona elementa i može se interpolirati za posebne položaje ako je potrebna veća tačnost. y [ ] It should be kept into account that the maximum value of the loss of force due to friction will be on the other end of the element, if it is tension from one end. Therefore, loss due to friction varies linearly along the element span and can be interpolated for specific positions if there is a need for greater accuracy. Gubici usled trenja ∆Pµ(x), pri utezanju kablova, mogu da se procene prema obrascu (3): Losses due to friction ∆Pµ(x) when tensioning cables can be assessed according to the formula (3): −µ (θ + kx)) (3) ∆Pµ(x)=Pmax(1–е−µ (θ + kx)) (3) U prethodnom obrascu θ predstavlja zbir skretnih uglova na rastojanju x izražen u rad; µ je koeficijent trenja između kabla i cevi u koju je postavljen izražen u rad-1; k određuje nenamerno ugaono skretanje unutraš- njih kablova izraženo u rad/m; i x predstavlja rastojanje izraženo u metrima duž kabla od tačke u kojoj je sila u kablu jednaka Pmax to jest sila na aktivnom kraju kabla za vreme utezanja. In previous formula θ represents the sum of the diverting angles at a distance x expressed in rad; µ is the coefficient of friction between the cable and pipes in which it is placed, expressed in rad-1; k determines accidental diverting angle of internal cables expressed in rad/m; and x is the distance expressed in m` along the cable from the point where the force in the cable is equal to Pmax i.e. the force on the active end of the cable during tension time. Za koeficijent trenja µ treba naglasiti da veoma zavisi od površinskih karakteristika kablova i cevi, a naročito od stepena korozije dodirnih površina. 2 THEORETICAL PREMISES Insertion of wedges in the passive anchor need not to affect the reduction of the initial force if total elongation at press is increased by this amount. Insertion of wedges on the side of the press, active anchor, leads to a reduction in the initial force in the cable for the size of the lost elongation, which can have significant value for short cables. Insertion of wedges of active and passive anchor is approximately the same and is taken at the level of 3-5mm. There are ways to partially or completely eliminate this loss with the use of special procedures. Elastično skraćenje konstrukcije dovodi do gubitka ukupne početne sile konstrukcije ukoliko se uteže više od jednog kabla. Gubitak je znatan u slučaju jako napregnutih elemenata malog preseka (npr. vešaljke, stubovi). Tada treba voditi računa o gubitku sile u kablu usled deformacije betona, kao i o zavisnosti od redosleda zatezanja kablova (prema SRPS EN:1992-1- 1:2004). Elastic deformation of structures, leads to a total loss of the initial force of the structure if more than one cable is being tensioned. The loss is significant with highly prestressed elements with small cross-section: hangers, piles and the like. Then the loss of force in the cable due to deformation of concrete, as well as dependence from the order of tensioning cables (according to EN: 1992-1- 1: 2004) have to be taken into account. Gubitak sile prednaprezanja javlja se u slučaju naknadno prednapregnutih elemenata usled trenja između kablova i zaštitnih cevi u betonu. Veličina ovog gubitka jeste funkcija oblika trase kabla - efekat zakrivljenosti i odstupanja prilikom montaže trase kablova - ugaono odstupanje. Vrednosti koeficijenata koji definišu gubitke sile često se preciziraju dok se Loss of tensioning force occurs in post-prestressed elements due to friction between the cables and protective pipes in concrete. The value of this loss is a function of the form of the route of the cable-effect of GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 51 obavlja priprema projekta izborom različitih tipova i oblika trase kablova. Pošto je efekat zakrivljenosti unapred određen, ugaono odstupanje jeste rezultat slučajnog ili neizbežnog odstupanja, pošto ne postoji mogućnost da se trasa zaštitnih cevi idealno montira [7]. curvature and deviation during the installation of cable route-angular deviation. 2 THEORETICAL PREMISES Veličina trenja zavisi i od vrste cevi koja se koristi i od tipa kabla. Postoje dva mehanizma koja proizvode trenje; prvi je zakrivljenost kabla, a drugi - slučajna razlika između težišta kablova i cevi. For the friction coefficient µ it should be emphasized that it is highly dependent on surface characteristics of cables and pipes, in particular on the degree of corrosion of contact surfaces. Amout of friction also depends on the type of pipes used and the type of cable used. There are two mechanisms that produce friction. The first is the curvature of the cable, and the other is a random difference between the center of gravity of the cables and the pipe. 3 EXPERIMENTAL WORK IN SITU Kompletan eksperiment urađen je na gradilištu, prilikom izgradnje mosta Borča-Zemun. Izvođač radova bio je CRBC (China Road and Bridge Corporation). Konstrukcija mosta van vode imala je dva sistema. Jedan od sistema uključivao je izradu prednapregnutih nosača statičke dužine 26 i 36 metara. U eksperimentu je korišćen nosač dužine 26 metara. Projektom je bilo predviđeno utezanje nosača sa četiri kabla od po sedam užadi prečnika 15,2 mm, dok je za trasu kablova korišćena plastična rebrasta cev. Karakteristični poprečni preseci i podužni presek nosača s trasama dva ispitivana kabla prikazani su na slici 1. The complete experiment was performed on construction site during the construction of the bridge Zemun-Borca. The contractor was the CRBC (China Road and Bridge Corporation). The construction of the bridge outside the water had two systems. One of the system included the making of prestressed girders with static length 26 and 36m. The experiment used the girder with the length of 26 meters. In the design tensioning of the girder with 4 cables of 7 ropes each with the diameter of 15,2mm was planned, while for the trace of cables corrugated plastic pipe was used. A typical cross-sections and longitudinal-section of the girder with traces of two tested cables are shown in Figure 1. Slika 1. Karakteristični poprečni i podužni presek AB nosača Figure 1. Typical cross section and longitudinal section of RC girders Slika 1. Karakteristični poprečni i podužni presek AB nosača Figure 1. Typical cross section and longitudinal section of RC girders Slika 1. Karakteristični poprečni i podužni presek AB nosača Figure 1. Typical cross section and longitudinal section of RC girders Slika 1. Karakteristični poprečni i podužni presek AB nosača Figure 1. Typical cross section and longitudinal section of RC girders GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 52 Slika 2. Aktivni kraj Figure 2. Active end Slika 3. Pasivni kraj Figure 3. Passive end Slika 3. Pasivni kraj Figure 3. Passive end Slika 2. Aktivni kraj Figure 2. Active end Slika 3. Pasivni kraj Figure 3. Passive end Slika 2. Aktivni kraj Figure 2. Active end Slika 2. Aktivni kraj Figure 2. Active end The experiment included the determination of the coefficient of friction and the coefficient of imperfection, except that the effects of wedging and elastic deforma- tion of concrete were not taken into account. 3 EXPERIMENTAL WORK IN SITU The effects of wedging were not taken into account because the procedures and modifications of the IMS system of presstresing minimized the impact. Press for tensioning has been modified so that wedge done before releasing the cables. By respecting procedures of using press wedging effect can be ignored. Design is anticipated tensioning of the complete girder with 4 cables, while the experiment tensioning with one cable up to ~90% designed force is planned. Effect of elastic deformation of concrete at such girders in adopted disposition tests can be ignored becouse it performs tensioning with only one cable which is released before tensioning second cable. Eksperiment je obuhvatio određivanje koeficijenta trenja i koeficijenta imperfekcije, s tim što nisu uzimani u obzir efekti zaklinjavanja i elastične deformacije betona. Efekti zaklinjavanja nisu se imali u vidu, jer su procedurama i modifikacijama IMS sistema prednapre- zanja uticaji svedeni na minimum. Presa za utezanje modifikovana je tako da se zaklinjavanje vrši pre otpuštanja kablova. Poštujući proceduru upravljanja presom, efekat zaklinjavanja se može zanemariti. Projektom je predviđeno utezanje kompletnog nosača sa četiri kabla, a u eksperimentu je planirano utezanje s jednim kablom do ~90% projektovane sile. Efekat elastične deformacije betona na ovom nosaču i pri usvojenoj dispoziciji ispitivanja može se zanemariti, jer se vrši utezanje samo s jednim kablom koji se otpušta pre utezanja sledećeg kabla. It was planned for cable N1 to be tensioned first and up to ~90% of the designed force in the cables. Then wedging was carried out and the force was monitored for the next 24 hours. While tensioning the cable measure- ments were conducted for every 20% of the maximum designed force. Practically force measurements were conducted at 20, 40, 60 and 85-95% of the maximum designed force in the cables. Measurements were also carried out after 24 hours. Then the released of tensioned cable N1 was performed and tensioning of cable N2 was started. All measurements were performed the same as for cable N1. After reaching the planned force wedging was performed and the force was followed for the next 24 hours. At the end released of cable N2 was performed. The results of the tensioning force measurements on the active and passive end are given in table 1. Planirano je da se prvo uteže kabl N1 do 95% projektovane sile u kablovima. Potom, vršeno je zaklinjavanje i praćenje sile naredna 24 sata. 3 EXPERIMENTAL WORK IN SITU Pri utezanju kabla, merenja su sprovođena na svakih 20% maksimalno projektovane sile. Praktično, sila se merila na 20%, 40%, 60% i 80% maksimalne projektovane sile u kablovima. Merenja su natavljena i nakon 24 sata. Potom, izvršeno je rasklinjavanje utegnutog kabla N1 i pristupilo se utezanju kabla N2. Merilo se sve, kao i u slučaju kabla N1. Nakon postizanja predviđene sile, vršeno je zaklinjavanje i praćenje sile naredna 24 sata. Naposletku, izvršeno je rasklinjavanje kabla N2. Rezultati merenja sile utezanja na aktivnom i pasivnom kraju dati su u tabeli 1. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 53 Tabela 1. Rezultati merenja sile utezanja u kablovima N1 i N2 Table 1. The results of tensioning force measurements in cables N1 and N2 Tabela 1. Rezultati merenja sile utezanja u kablovima N1 i N2 Table 1. The results of tensioning force measurements in cables N1 and N Tabela 1. Rezultati merenja sile utezanja u kablovima N1 i N2 Table 1. The results of tensioning force measurements in cables N1 and N2 Kabal N1 / Cable N1 Kabal N2 / Cable N2 Aktivni kraj Active end Pasivni kraj / Passive end Aktivni kraj Active end Pasivni kraj / Passive end Sila / Force t→0 t→0 t→24h t→0 t→0 t→24h 20% 347kN 314kN - 348kN 312kN - 40% 694kN 649kN - 694kN 675kN - 60% 1041kN 982kN - 1041kN 1026kN - ~90% 1379kN 1304kN 1302kN 1276kN 1180kN 1179kN 4 DISCUSION AND CONCLUSIONS Rezultati ispitivanja obrađeni su na dva načina. Izračunati su koeficijenti trenja i koeficijenti imperfekcije na osnovu sila utezanja na dva kabla, kao i procena poređenja koeficijenta trenja za različite nivoe utezanja kablova. kabal Test results processing was done in two ways. Coefficients of friction and coefficients of imperfection on the basis of tension forces in two cables as well as an evaluation of the comparison of the coefficient of friction for different levels of tension cables. Proračun koeficijenta trenja i koeficijenta imperfekcije svodi se na rešavanje sistema jednačina s dve nepoznate. Jednačine su prikazane obrascima (4) i (5). Calculation of the coefficient of friction and the coefficient of imperfection is reduced to solving a system of equations ((4) and (5)) with two unknowns. μ*(θ1+kx) (4) μ*(θ2+kx) (5) (4) ln(P1,x / P1,max)=-μ*(θ1+kx) ln(P2,x / P2,max)=-μ*(θ2+kx) (4) (5) (5) Ukupni skretni ugao θ1 u kablu N1 iznosio je 15.346°, a θ2 u kablu N2 iznosio - 12.136°. Dužina na kojoj je merena vrednost smanjenja sile jeste kraj kabla x=26,097 m. Na osnovu ulaznih podataka i rešavanja sistema jednačina s dve nepoznate, dobijene su vrednosti za koeficijent trenja kablova μ=0,22 i koeficijent imperfekcije k=0,004. Total deviation angle θ1 in the cable N1 was 15.346°, and θ2 in the cable N2 amounted to 12.136°. Length at which the measured value of the reduction in force is the end of the cable x=26.097m. Based on the input data and solving the system of equations with two unknowns, the values for the coefficient of friction of cables μ=0.22 and imperfection coefficient k=0.004 were obtained. After solving the system of equations a calculation of the coefficient of friction of cables was made separately for different levels of cables tensioning. The calculation was done separately for each cable and imperfection coefficient ranging from 0.001 to 0.01 with step 0.001. The obtained results are shown in Figures 4 and 5 Nakon rešavanja sistema jednačina, izvršen je i proračun koeficijenta trenja pojedinačno za kablove za različite nivoe utezanja kablova. Proračun je vršen posebno za svaki kabl i raspon koeficijenta imperfekcije od 0,001 do 0,01 s korakom 0,001. Dobijeni rezultati prikazani su na slikama 4 i 5. g g p The obtained results are shown in Figures 4 and 5. Slika 4. Koeficijent trenja kabla N1 u zavisnosti od koeficijenta imperfekcije Figure 4. The coefficient of friction of the cable N1 depending on the coefficient of imperfection Slika 4. 4 DISCUSION AND CONCLUSIONS Koeficijent trenja kabla N1 u zavisnosti od koeficijenta imperfekcije Slika 4. Koeficijent trenja kabla N1 u zavisnosti od koeficijenta imperfekcije . The coefficient of friction of the cable N1 depending on the coefficient of imperfection Slika 4. Koeficijent trenja kabla N1 u zavisnosti od koeficijenta imperfekcije Figure 4. The coefficient of friction of the cable N1 depending on the coefficient of imperfection j j j p j Figure 4. The coefficient of friction of the cable N1 depending on the coefficient of imperfection GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 54 Slika 5. Koeficijent trenja kabla N2 u zavisnosti od koeficijenta imperfekcije Figure 5. The coefficient of friction of the cable N2 depending on the coefficient of imperfection Slika 5. Koeficijent trenja kabla N2 u zavisnosti od koeficijenta imperfekcije Figure 5. The coefficient of friction of the cable N2 depending on the coefficient of imperfection Zajedničkom obradom rezultata ispitivanja dva kabla, dobijeni su rezultati koji se mogu uporediti sa stan- dardom i preporukama definisanim vrednostima iz tabele 2. Kao što se može videti, dobijena vrednost za plastične rebraste cevi, koeficijenta trenja μ=0,22 nešto je veća, dok je koeficijent imperfekcije k=0,004 u predloženim granicama. Koeficijent imperfekcije zavisi umnogome od izvođača radova i od njegove sposobnosti da što bolje postavi kabl. Time se imperfekcije znatno smanjuju i uticaj se može svesti na minimum. Koeficijent trenja jeste nešto na šta se ne može uticati i njegova vrednost će zavisiti najviše od materijala koji se koriste za vođenje kabla. By joint processing of the test results for two cables the obtained results are comparable with the standard and recommendations defined by the values from Table 2. As it can be seen, the value obtained for plastic corrugated pipes, the friction coefficient μ=0.22 is slightly higher, while the coefficient of imperfection k=0.004 is in the suggested limits. The coefficient of imperfections depends greatly on the contractor and its ability to instal the cord as good as possible. This significantly reduces the imperfections and the impact can be brought down to a minimum. The coefficient of friction cannot be influenced and its value depends the most on the materials used for tracing the cable. Tabela 2. Preporučene vrednosti proizvođača za koeficijente trenja i imperfekcije Table 2. 4 DISCUSION AND CONCLUSIONS Manufacturers recommended values for the coefficients of friction and imperfection µ (rad-1) k (rad/m) Tip cevi Type of pipe Opseg vrednosti Value range Preporuka za proračun Recommendation for design Opseg vrednosti Value range Preporuka za proračun Recommendation for design Rebrasta metalna Corugated metal 0.18 ÷ 0.26 0.22 (1-10)·10-3 3·10-3 Rebrasta plastična Corugated plastic 0.10 ÷ 0.14 0.12 (1-10)·10-3 4·10-3 Plastična sa mašću Plastic with grease 0.05 ÷ 0.08 0.06 (2-6)·10-3 4·10-3 Beton / Concrete 0.34 ÷ 0.62 0.48 (1-10)·10-3 5·10-3 Tabela 2. Preporučene vrednosti proizvođača za koeficijente trenja i imperfekcije Table 2. Manufacturers recommended values for the coefficients of friction and imperfection Considering the results according to the levels of force application in the cables, it is clear that for lower tension forces coefficient of friction is far greater. When setting up the cable and lower forces in the cables, the cables are first being installed and losses from the active to the passive end are considerable. Later, when the cable is installed in the final position the friction decreased and the values that can be assumed depending on the material of the pipes for cables are obtained. Razmatrajući rezultate po nivoima unošenja sile u kablove, jasno je da je na manjim silama utezanja koeficijent trenja daleko veći. Prilikom postavljanja kabla i manjih sila u kablovima, prvo dolazi do nameštanja kablova i gubici od aktivnog do pasivnog kraja su znatni. Kasnije, kada se kabl namesti u konačnu poziciju, trenje se smanjuje i dolazi na vrednosti koje se mogu pretpostaviti, u zavisnosti od materijala cevi za vođenje kablova. GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 55 5 LITERATURA REFERENCES [1] ETAG 013 (Guideline for European Technical Approval of post-tensioning kits for prestressing of structures), EOTA, 2002. [6] SRPS EN:1992-1-1, European Standard, 2004. [7] Robert Benaim, The design of prestressed concrete bridges; concepts and principles, Taylor and francis Group, London and New York, 2008. ) [2] Sistem za prednaprezanje SPB SUPER, Institut IMS, Beograd, 2009. [8] Jordan Milev, Problems and Their Solutions in Practical Application of Eurocode in Seismic design of RC Structures, Building Materials and Constructiion, ISSN 2217-8139, 2016. [3] Nigel R. Hewson Prestressed concrete bridges: design and construction, Thomas Telford LTD, III Edition, 2003. [9] Miloš Čokić, Petronijević P., Todorović M., Pecić N., Analiza Rezerve Energije i Emisije CO2 za izvođenje mostovske konstrukcije sa stanovišta održivosti, Building Materials and Construction, ISSN 2217-8139, 2015. [4] Edward G. Nawy, Prestressed concrete: a fundamental approach, Prentice Hall, V edition, 2009. [5] M.K. Hurst, Prestressed concrete design, CRC Press, II Edition, 2003. DETERMINATION OF THE IN SITU COEFFICIENT OF FRICTION AND IMPERFECTION OF PRESTRESSING CABLES Dragan BOJOVIĆ Bojan ARANĐELOVIĆ Ksenija JANKOVIĆ Aleksandar SENIĆ Marko STOJANOVIĆ Dragan BOJOVIĆ Bojan ARANĐELOVIĆ Ksenija JANKOVIĆ Aleksandar SENIĆ Marko STOJANOVIĆ Dragan BOJOVIC Bojan ARANDJELOVIC Ksenija JANKOVIC Aleksandar SENIC Marko STOJANOVIC Prednaprezanjem konstruktivnih elemenata omogućava se povećavanje raspona armiranobetonskih konstrukcija. Pri projektovanju i dimenzionisanju prednapregnutih armiranobetonskih konstrukcija, neophodno je poznavanje, odnosno usvajanje većeg broja parametara koji su empirijski određeni. Za prednapregnute konstrukcije od osnovnog je značaja da se odredi nivo sile naprezanja kablova. Nivo sile prednaprezanja, pored drugih parametara, umnogome zavisi od koeficijenta trenja kablova i koeficijenta imperfekcije izvođenja. Ovi koeficijenti definisani su standardima za projektovanje ili su ih definisali proizvođači sistema prednaprezanja. Koeficijenti su definisani u širokim opsezima, dok je za projektovanje neophodno poznavanje što tačnijih vrednosti, kako bi se dobio dobar projekat i jeftinija konstrukcija. Putem eksperimentalnog rada in-situ, određen je opseg vrednosti koeficijenta trenja kablova i upoređen je sa standardom definisanim opsezima. Prestressing structural elements allows an increase in the spans of reinforced concrete structures. In the design and dimensioning of prestressed reinforced concrete structures it is necessary to know, that is to adopt a number of parameters which are empirically determined. Determining the level of the tension force of the cables is of primary importance for prestressed structures. The level of tensioning force, in addition to other parameters, greatly depends on the friction coefficient of cables and the imperfection coefficient of execution. These coefficients are defined by standards for the design or by the manufacturer of the prestressing system. The coefficients are defined in wide ranges, while for the design, the knowledge of the most accurate values is necessary in order to obtain a good design and cheaper structures. Through experimental work in-situ the range of the values of the coefficient of friction of cables is determined and compared with ranges defined by the standard. Key words: prestressing, coefficient of friction, coefficient of imperfection Ključne reči: prednaprezanje, koeficijent trenja, koeficijent imperfekcije GRAĐEVINSKI MATERIJALI I KONSTRUKCIJE 60 (2017) 2 (49-56) BUILDING MATERIALS AND STRUCTURES 60 (2017) 2 (49-56) 56
https://openalex.org/W4244641608
https://www.researchsquare.com/article/rs-51185/latest.pdf
English
null
A GPU Based Multidimensional Amplitude Analysis to Search for Tetraquark Candidates
Research Square (Research Square)
2,020
cc-by
11,807
A GPU based multidimensional amplitude analysis to search for tetraquark candidates Nairit Sur  (  surnairit.pointer@gmail.com ) Tata Institute of Fundamental Research https://orcid.org/0000-0001-5233-553X Leonardo Cristella  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Adriano Di Florio  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Vincenzo Mastrapasqua  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Methodology Keywords: High Energy Physics, Flavour Physics, search, Exotic Hadron Spectroscopy, GPU, CUDA, GooFit, Unbinned Maximum Likelihood, Fitting Posted Date: January 7th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-51185/v4 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 on January 7th, 2021. See the published version at https://doi.org/10.1186/s40537-020-00408-4. A GPU based multidimensional amplitude analysis to search for tetraquark candidates Nairit Sur  (  surnairit.pointer@gmail.com ) Tata Institute of Fundamental Research https://orcid.org/0000-0001-5233-553X Leonardo Cristella  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Adriano Di Florio  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Vincenzo Mastrapasqua  Universita degli Studi di Bari and I.N.F.N. - Sezione di Bari Methodology Keywords: High Energy Physics, Flavour Physics, search, Exotic Hadron Spectroscopy, GPU, CUDA, GooFit, Unbinned Maximum Likelihood, Fitting Posted Date: January 7th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-51185/v4 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 on January 7th, 2021. See the published version at https://doi.org/10.1186/s40537-020-00408-4. A GPU based multidimensional amplitude analysis to search for tetraquark candidates Nairit Sur1*†, Leonardo Cristella2†, Adriano Di Florio2† and Vincenzo Mastrapasqua2† *Correspondence: nairit.sur@cern.ch 1Tata Institute of Fundamental Research, Mumbai, India Full list of author information is available at the end of the article †Equal contributor Methodology Version of Record: A version of this preprint was published on January 7th, 2021. See the published version at https://doi.org/10.1186/s40537-020-00408-4. Sur et al. RESEARCH Abstract The demand for computational resources is steadily increasing in experimental high energy physics as the current collider experiments continue to accumulate huge amounts of data and physicists indulge in more complex and ambitious analysis strategies. This is especially true in the fields of hadron spectroscopy and flavour physics where the analyses often depend on complex multidimensional unbinned maximum-likelihood fits, with several dozens of free parameters, with an aim to study the internal structure of hadrons. Graphics processing units (GPUs) represent one of the most sophisticated and versatile parallel computing architectures that are becoming popular toolkits for high energy physicists to meet their computational demands. GooFit is an upcoming open-source tool interfacing ROOT/RooFit to the CUDA platform on NVIDIA GPUs that acts as a bridge between the MINUIT minimization algorithm and a parallel processor, allowing probability density functions to be estimated on multiple cores simultaneously. In this article, a full-fledged amplitude analysis framework developed using GooFit is tested for its speed and reliability. The four-dimensional fitter framework, one of the firsts of its kind to be built on GooFit, is geared towards the search for exotic tetraquark states in the B0 →J/ψKπ decays and can also be seamlessly adapted for other similar analyses. The GooFit fitter, running on GPUs, shows a remarkable improvement in the computing speed compared to a ROOT/RooFit implementation of the same analysis running on multi-core CPU clusters. Furthermore, it shows sensitivity to components with small contributions to the overall fit. It has the potential to be a powerful tool for sensitive and computationally intensive physics analyses. Keywords: High Energy Physics; Flavour Physics; search; Exotic Hadron Spectroscopy; GPU; CUDA; GooFit; Unbinned Maximum Likelihood; Fitting *Correspondence: nairit.sur@cern.ch 1Tata Institute of Fundamental Research, Mumbai, India Full list of author information is available at the end of the article †Equal contributor Introduction Big data usually includes data sets with sizes beyond the ability of commonly used software tools to capture, curate, manage, and process data within a tolerable elapsed time, thus calling for parallel computing tools to analyze them [1, 2]. Modern particle accelerators such as the LHC (Large Hadron Collider) [3] at CERN (Eu- ropean Organization for Nuclear Research) produce data at a phenomenal rate [4]. CERN operates the largest particle physics laboratory in the world providing req- uisite infrastructures for research in high-energy physics, viz. powerful computing facilities primarily used to store and analyse data from experiments, as well as to simulate events. The LHC is a superconducting accelerator and collider of protons and heavy ions at teraelectronvolt (TeV) energy scales. An electronvolt is a unit of Page 2 of 20 Sur et al. energy defined as the amount of kinetic energy gained or lost by a single electron accelerating from rest through an electric potential difference of one volt in vacuum. The LHC consists of a 27-kilometre circular underground tunnel of superconducting magnets with a number of accelerating structures to boost the energy of the parti- cles along the way. The accelerated protons flow in opposite directions through two parallel beam pipes of the LHC tunnel and collide with each other at four points where the beam pipes cross each other. Massive and intricate detectors, such as ATLAS [5], CMS [6], and LHCb [7], are built around these collision points to detect the huge number of particles created due to the 600 million collisions taking place per second. Such particle accelerators and the associated detectors are collectively referred to as “collider experiments”. The LHC experiments represent about 150 million sensors delivering data at the rate of 40 MHz. The raw data flow from the LHC detectors exceeds 500 exabytes per day which is almost 200 times more than all the other sources combined in the world. Even after preserving only a fraction of that data stream for physics analysis, hundreds of petabytes of complex data are stored and processed [8, 9]. One of the key challenges in analysing and interpreting these data is to accurately model the distributions of observable quantities in terms of the physics parameters of interest.The result of repeating an experiment (like tossing a coin or rolling dice) many times does not lead to the same result but produce a distribution of answers. Introduction The form of the distribution depends on the nature of the experiment and can be represented by mathematical models [10]. In addition, a large number of other parameters may be needed to accurately describe the resolution and efficiency of complex detectors. These mathematical models are constructed in terms of probabil- ity density functions (PDFs) [11] normalized over the allowed range of observables with respect to the parameters. Due to the large amount of data, as well as the ever-increasing complexity of physics models, the running time of estimation of the parameters (“fitting” [10]) has become a major bottleneck. The PDFs become particularly complex while probing different aspects of quan- tum chromodynamics (QCD), the quantum field theoretical description of strong interaction between the quarks and gluons [12]. Quarks and gluons are elementary constituents of matter. They combine together to form composite particles called hadrons. The most common hadrons, namely protons and neutrons, form atomic nuclei and are thus responsible for most of the mass of the visible matter in the universe. Hadrons are generally of two types - baryons (bound state of three quarks) and mesons (bound states of a quark and an antiquark). The study of masses and decays of hadrons is called hadron spectroscopy which is a key to understand QCD. Due to the complex nature of this nonabelian gauge theory including peculiar fea- tures like “colour confinement” and “asymptotic freedom” [13–15], it is very hard to study the nature of this interaction analytically, especially at low energy regimes. In the last 15 years, experimental evidence has been mounting [16] for a large number of multiquark bound states that are allowed in principle by QCD but do not fit the expectations for the conventional quark model (i.e., the baryons or the mesons) and relative spectra. These new particles are often called “exotic” states. The exact nature of many of these states still remains a puzzle; even though some of them are confirmed by multiple experiments, not all the quantum numbers of these states Page 3 of 20 Sur et al. have yet been determined. Spectroscopic studies of such heavy-flavor states can provide a deeper understanding of the underlying dynamics of quarks and gluons at the hadron mass scales as well as a valuable insight into various QCD inspired phenomenological models [16, 17]. Introduction The charged charmonium-like Z states, which are strong candidates for tetraquark states with a possible quark content of |c¯cd¯u⟩, can be studied in ongoing collider experiments, ATLAS, Belle II [18], BESIII [19], CMS, and LHCb. To ascertain, with a high degree of statistical significance, the presence of such intermediate states in three-body decays B0 →ψ(nS)K+π−, complex multidimensional un- binned maximum-likelihood (UML) [11] fits on tens of thousands of data points, with several dozens of free parameters, must be performed, thus requiring a con- siderable amount of computational resources. The traditional high-energy physics (HEP) analysis tools such as ROOT [20] and RooFit [21], which are designed to run on CPUs, require excessively long processing times amounting to days even when they are run on servers comprising several multi-core CPUs. In this article, we explore the scope of an advanced GPU-accelerated computing framework to reduce the processing times of such complex multidimensional fits fre- quently occurring in the field of HEP. We expand the usability of existing software keeping in mind the particular needs of a typical HEP analysis. This article starts with a comprehensive overview of existing literature in the emerging field of GPU- assisted HEP analysis, followed by a detailed methodology of a four-dimensional amplitude analysis. The findings are discussed in the “Results” section and con- cluding remarks are elaborated in the “Discussions” and “Conclusion” sections. Our framework is based on the novel GPU based GooFit [22, 23] package. GooFit is an open-source analysis tool, presently under development, which can be used in the HEP applications for parameters estimation, and which interfaces ROOT to the CUDA parallel computing platform on NVIDIA GPUs [24]. GPU-accelerated com- puting enhances application performances by offloading a sequence of elementary but computationally intensive operations to the GPU to be processed in parallel, while the remaining code still runs on the CPUs. MINUIT [25] is a numerical min- imization program that searches for a minimum in a user-defined function with respect to one or more parameters using several different methods as specified by the user. MINUIT cannot be distributed as an executable binary to be run by a relatively unskilled user. The user must write and compile a subroutine defining the function to be optimized, and oversee the optimization process. GooFit acts as an interface between MINUIT and the GPU, which allows any PDF to be evalu- ated in parallel over a huge amount of data. Introduction Fit parameters are estimated at each negative-log-likelihood (NLL) minimization step on the host side (CPU) while the PDF/NLL is evaluated on the device side (GPU). GooFit is still a limited open- source tool, being mainly developed by the users themselves for their specific needs. A very few applications in HEP analysis have been designed using GooFit. Signifi- cant sections needed for our fit implementation have been either newly encoded or adapted starting from the existing classes and methods. State-of-the-Art Literature The need for GPU-based analysis frameworks to meet the demands of current and future HEP experiments has been acknowledged within the community for quite Page 4 of 20 Sur et al. some time [26]. To that end, the GPU-based GooFit package was developed to mimic the functionalities and flexibilities of the widely popular RooFit one. GooFit is designed to minimize the amount of CUDA coding required by a general user while exploiting the full potential of GPU parallelization. GooFit objects, viz. PDFs, can be created and combined in standard C++ if the PDFs are already encoded in existing classes. However, the available classes are limited in number and many other functionalities widely used for HEP analyses are not yet developed within the framework. The general algorithm to develop new PDF models within GooFit and test their functionalities involves coding with the help of CUDA while keeping in mind the complex data organization in GooFit that facilitates an efficient transfer of bytes between the host and device. During the fitting process the PDF must be normalized accurately. As it is not feasible to find an analytic expression for complicated functions in general, the normalization is computed numerically which requires evaluation of the function at several million phase space points. One of the first performance comparison studies of GooFit vs RooFit was con- ducted in Ref. [27]. Here, a high-statistics toy Monte Carlo technique was imple- mented for a simple 2D PDF model with a few parameters and the fit performances were compared for binned maximum likelihood fits. A further extension can be found in Ref. [28] where pseudo-experiments are coupled with a complex clustering technique in order to include the Look-Elsewhere-Effect when assessing the statis- tical significance of a new physics signal. Models of higher complexity viz. time-dependent Dalitz plot analysis and model- independent partial wave analysis have been gradually added to the GooFit package as demonstrated in Refs. [29–31]. All these are extensions of the standard UML fit of the Dalitz plot [32], in which the matrix element describing the decay process is represented by a coherent sum of quantum mechanical amplitudes. The models developed so far within GooFit could not perform a full-fledged am- plitude analysis fit for complex processes such as a pseudoscalar meson decaying into at least one vector state along with another zero- or a higher-spin particle, with an eventual four-particle final state. State-of-the-Art Literature The complexity arises due to an addi- tional angle-dependent part of the PDF needed to describe the more complicated decay dynamics. This functionality has now been introduced for the first time and is described in detail in this article. An amplitude analysis of the three body decay B0 →J/ψKπ An amplitude analysis of the three body decay B0 →J/ψKπ / An amplitude analysis of the three body decay B →J/ψKπ The rare exotic Z states can appear as J/ψπ resonances in the quasi two-body decay B0 →Z−K+ →J/ψπ−K+, where the J/ψ decays into a µ+µ−pair (inclusion of the charge conjugate mode ¯B0 →Z+K−→J/ψπ+K−is always implied). However, the decay process is dominated by the intermediate K∗(→Kπ) resonances in the quasi two-body decay B0 →J/ψK∗[33]. These ten kinematically allowed kaonic resonances can interfere with one another as well as with the Z states. Three-body decays with intermediate resonant states, such as P →D1 + Dres, Dres →D2 + D3, are generally analysed using a technique pioneered by Dalitz [32]. Here, P is the parent particle, D1 is one of its daughters, Dres is the other daughter which, being an intermediate resonance, decays into D2 and D3. A two-dimensional Page 5 of 20 Sur et al. scatter plot of m2 D1D2 vs. m2 D2D3 (invariant mass squared of any two daughters), known as the Dalitz plot, shows a nonuniform distribution due to the interfering intermediate resonances, thus to the decay dynamics. If at least one of the three daughters in the decay is a vector state instead of being a pseudoscalar, the tra- ditional Dalitz plot approach becomes insufficient as the angular variables are im- plicitly integrated over, leading to a loss of information about angular correlations among the decay products. The K∗-only model The kinematics of the process B0 →J/ψKπ, J/ψ →µ+µ−can be completely described by a four-dimensional variable space (1) Φ ≡ mKπ, mJ/ψπ, θJ/ψ, ϕ  . (1) Φ ≡ mKπ, mJ/ψπ, θJ/ψ, ϕ  . The two angles, θJ/ψ and ϕ are illustrated in Fig. 1. The number of dimensions required to describe any decay process is given by the difference between the degrees of freedom of the system and the total number of constraints. A three-body decay in general has twelve degrees of freedom due to the four-momenta of each particle. As one of the particles in the B0 →J/ψKπ decay is a vector state (spin 1), it has two extra degrees of freedom. The corresponding constraints are the conservation of four-momenta, the three masses, and the three euler angles. Thus the number of dimensions required becomes (12 + 2 −4 −3 −3) = 4. The two angles, θJ/ψ and ϕ are illustrated in Fig. 1. An amplitude analysis of the three body decay B0 →J/ψKπ R is given by [34]: R is given by [34]: ARm2 R  = F (LB) B  pB MB LB F (LR) R  pR mR LR M 2 R −m2 R −iMRΓ(mR) , (2) (2) where the mass-dependent width of R is: where the mass-dependent width of R is: Γ(mR) = Γ0  pR pR0 2LR+1 MR mR  F 2 R (3) (3) and • mR is the running invariant mass of the two daughters of R (e.g., mR = mKπ for a K∗); • mR is the running invariant mass of the two daughters of R (e.g., mR = mKπ for a K∗); ) • MB is the B0 meson mass; • MB is the B0 meson mass; • MR is the nominal mass of R; • MR is the nominal mass of R; • LB (LR) is the orbital angular momentum in the B0 (R) decay; • pB is the B0 daughter momentum (i.e., R momentum) in the B0 rest frame; (LB) d (LR) h Bl W k f f f [ ] f B0 d R d pB g ( , ) ; • F (LB) B and F (LR) R are the Blatt-Weisskopf form factors [35] for B0 and R decay, respectively, with the superscript denoting the orbital angular momentum of the (sub-)decay; • Γ0 is the nominal width of R; • pR and pR0 are the momenta of R daughters in the former’s rest frame, cal- l t d f th i d l f R ti l • pR and pR0 are the momenta of R daughters in the former’s rest frame, cal- culated from the running and pole mass of R, respectively. culated from the running and pole mass of R, respectively. For K∗resonances with spin (J) of one or more units, LB can take several values (S, P, and D-waves for J = 1; P, D, and F-waves for J = 2; and D, F, and G-waves for J = 3). The lowest LB is taken as the default value while the other possibilities are considered as part of the uncertainty in measurements due to their small contributions. For K∗resonances with spin (J) of one or more units, LB can take several values (S, P, and D-waves for J = 1; P, D, and F-waves for J = 2; and D, F, and G-waves for J = 3). An amplitude analysis of the three body decay B0 →J/ψKπ The number of dimensions required to describe any decay process is given by the difference between the degrees of freedom of the system and the total number of constraints. A three-body decay in general has twelve degrees of freedom due to the four-momenta of each particle. As one of the particles in the B0 →J/ψKπ decay is a vector state (spin 1), it has two extra degrees of freedom. The corresponding constraints are the conservation of four-momenta, the three masses, and the three euler angles. Thus the number of dimensions required becomes (12 + 2 −4 −3 −3) = 4. Figure 1 A sketch illustrating the definition of two independent angular variables, θJ/ψ and ϕ, for the amplitude analysis of B0 →J/ψKπ decays. Figure 1 A sketch illustrating the definition of two independent angular variables, θJ/ψ and ϕ, for the amplitude analysis of B0 →J/ψKπ decays. The relativistic Breit–Wigner (BW) function is a continuous probability distribu- tion used to model resonances (unstable particles). The total decay amplitude of B0 →J/ψKπ is represented by a coherent sum of the BW contributions associated with all the kinematically allowed intermediate resonant states. Simple field theory assumes all particles to be point like. In real life, however, the finite size of bound states of hadrons is modeled by form factors that are used to modify the original BW shape. The angle-independent part of the decay amplitude for each resonance Page 6 of 20 Sur et al. An amplitude analysis of the three body decay B0 →J/ψKπ The lowest LB is taken as the default value while the other possibilities are considered as part of the uncertainty in measurements due to their small contributions. A sequential decay of the B0 meson via an intermediate resonance into a four- body final state involves multiple decay planes requiring the application of Lorentz boosts and rotations to go from one rest frame to another, as can be seen in Fig. 1. As the helicity remains invariant under both Lorentz boost and rotation, the angle- dependent part of the amplitude is obtained using the helicity formalism [36]. For each K∗resonance, it is given by AK∗ λξ (Φ) = HK∗ λ AK∗m2 Kπ  dJ(K∗) λ0 (θK∗)eiλϕd1 λξ(θJ/ψ) (4) (4) where, AK∗m2 Kπ  , defined in Eq. (2), is explicitly written for R ≡K∗and where, AK∗m2 Kπ  , defined in Eq. (2), is explicitly written for R ≡K∗and • J(K∗) is the spin of the considered K∗resonance; • λ is the helicity of the J/ψ (the quantisation axis being parallel to the K∗ momentum in the J/ψ rest frame). In general, λ can take the values −1, 0 and 1. For K∗s with zero spin, only λ = 0 is allowed; • ξ is the helicity of the µ+µ−system; • HK∗ λ is the complex helicity amplitude for the decay via the intermediate K∗; J(K∗) λ p y p y ; • dJ(K∗) λ0 (θK∗) and d1 λξ(θJ/ψ) are the Wigner small-d functions that represent rotations; Page 7 of 20 Sur et al. • θK∗is the K∗helicity angle, i.e. the angle between K momentum in the K∗ rest frame and the K∗momentum in the B0 rest frame (Fig. 1); • θJ/ψ is the J/ψ helicity angle, i.e. the angle between µ+ momentum in the • θJ/ψ is the J/ψ helicity angle, i.e. An amplitude analysis of the three body decay B0 →J/ψKπ the angle between µ+ momentum in the J/ψ rest frame and the J/ψ momentum in the B0 rest frame; and J/ψ rest frame and the J/ψ momentum in the B0 rest frame; and J/ψ rest frame and the J/ψ momentum in the B0 rest frame; and / / • ϕ is the angle between the J/ψ →µ+µ−and K∗→Kπ decay • ϕ is the angle between the J/ψ →µ+µ−and K∗→Kπ decay plan The signal density function, to be used in the UML fit, is obtained after appro- priately summing over the helicity states and is given by S(Φ) = X ξ=1,−1 X K∗ X λ=−1,0,1 AK∗ λξ 2 (5) (5) The sum over K∗includes all kinematically allowed resonance states up to mKπ = 2.183 GeV, namely K∗ 0(800), K∗(892), K∗(1410), K∗ 0(1430), K∗ 2(1430), K∗(1680), K∗ 3(1780), K∗ 0(1950), K∗ 2(1980), and K∗ 4(2045). As the expression in Eq. (5) is sensitive only to the relative phases and amplitudes, we have the freedom to fix one overall phase and amplitude in the fit. The helicity amplitude of the K∗(892), the dominant resonance, is chosen to be fixed, for λ = 0: The sum over K∗includes all kinematically allowed resonance states up to mKπ = 2.183 GeV, namely K∗ 0(800), K∗(892), K∗(1410), K∗ 0(1430), K∗ 2(1430), K∗(1680), K∗ 3(1780), K∗ 0(1950), K∗ 2(1980), and K∗ 4(2045). As the expression in Eq. (5) is sensitive only to the relative phases and amplitudes, we have the freedom to fix one overall phase and amplitude in the fit. The helicity amplitude of the K∗(892), the dominant resonance, is chosen to be fixed, for λ = 0: HK∗(892) 0 = 1 , arg  HK∗(892) 0  = 0 (6) (6) The masses and widths of all the resonances are fixed to their world-average val- ues [37]. The masses and widths of all the resonances are fixed to their world-average val- ues [37]. The LASS parametrization (9) (9) Model including exotic Z resonances Model including exotic Z resonances For the decay B0 →KZ (→J/ψπ), J/ψ →µ+µ−where the Z can either be a Z(4200), and/or a Z(4430), or any other exotic (charmonium-like) state, the angle- dependent amplitude is given as AZ λ′ξ(Φ) = HZ λ′AZ m2 J/ψπ+  dJ(Z) 0λ′ (θZ)eiλ′ ˜ϕd1 λ′ξ(˜θJ/ψ)eiξα (10) (10) where, , • J(Z) is the spin of the Z resonance, we consider only 1+ spin-parity of the Zs as per Belle’s result [33]; • J(Z) is the spin of the Z resonance, we consider only 1+ spin-parity of the Zs as per Belle’s result [33]; • λ′ is the helicity of the J/ψ (quantisation axis parallel to the π momentum in the J/ψ rest frame); • ξ is the helicity of the µ+µ−system; • ξ is the helicity of the µ+µ−system; • HZ λ′ is the complex helicity amplitude for the decay via the intermediate Z; J(Z) • dJ(Z) 0λ′ (θZ) and d1 λ′ξ(˜θJ/ψ) are the Wigner small-d functions; • θZ is the Z helicity angle, i.e. the angle between K and π momenta in the Z rest frame; • ˜θJ/ψ is the J/ψ helicity angle, i.e. the angle between µ and π momenta in the J/ψ rest frame; • ˜ϕ is the angle between the (µ+, µ−) and (K, π) planes in the J/ψ rest frame; • ˜ϕ is the angle between the (µ+, µ−) and (K, π) planes in the J/ψ rest frame; • α is the angle between the (µ+, π) and (µ+, Kπ) planes in the J/ψ rest frame. The amplitudes for different λ′ values are related by parity conservation: • α is the angle between the (µ+, π) and (µ+, Kπ) planes in the J/ψ rest frame. The amplitudes for different λ′ values are related by parity conservation: • α is the angle between the (µ+, π) and (µ+, Kπ) planes in the J/ψ rest frame. The amplitudes for different λ′ values are related by parity conservation: HZ λ′ = −P(Z)(−1)J(Z)HZ −λ′ (11) (11) After inclusion of the Z component, the signal density function of Eq. (5) becomes After inclusion of the Z component, the signal density function of Eq. The LASS parametrization Generally, P- and D-wave states are considered to be well described by narrow reso- nance approximations. For the Kπ system, the low mass S-wave K∗ 0(800) appears as a broad peak calling for a more careful treatment. The LASS experiment at SLAC used an effective range expansion to model the low-energy behaviour of such Kπ S-wave [38]. We use a similar parametrization where the angle-independent part of the amplitude is a nonresonant contribution interfering with the scalar K∗ 0(1430) BW amplitude: ALASS = mKπ qKπ sin θBeiθB + 2e2iθB  m2 K∗ 0 (1430)/qK∗ 0 (1430)  ΓK∗ 0 (1430) M 2 K∗ 0 (1430) −m2 Kπ −iMK∗ 0 (1430)Γ(mKπ), (7) with cot θB = 1 a qKπ + 1 2 b qKπ and, a = 1.95 GeV−1, b = 1.76 GeV−1, (8) where ALASS = mKπ qKπ sin θBeiθB + 2e2iθB  m2 K∗ 0 (1430)/qK∗ 0 (1430)  ΓK∗ 0 (1430) M 2 K∗ 0 (1430) −m2 Kπ −iMK∗ 0 (1430)Γ(mKπ), (7) ALASS = mKπ qKπ sin θBeiθB + 2e2iθB  m2 K∗ 0 (1430)/qK∗ 0 (1430)  ΓK∗ 0 (1430) M 2 K∗ 0 (1430) −m2 Kπ −iMK∗ 0 (1430)Γ(mKπ), (7) with cot θB = 1 a qKπ + 1 2 b qKπ and, a = 1.95 GeV−1, b = 1.76 GeV−1, (8) h with cot θB = 1 a qKπ + 1 2 b qKπ and, a = 1.95 GeV−1, b = 1.76 GeV−1, (8) where • mKπ is the running mass of the Kπ system; • mKπ is the running mass of the Kπ system; • qKπ is the momentum of one of the K∗daughters in the K∗rest frame; • qKπ is the momentum of one of the K daughters in the K rest frame; • Γ (mKπ) is the running resonance width. • Γ (mKπ) is the running resonance width. Sur et al. Page 8 of 20 Therefore, the signal density with the LASS parametrization for the low-mass Kπ S-wave becomes Therefore, the signal density with the LASS parametrization for the low-mass Kπ S-wave becomes S-wave becomes S(Φ) = X ξ=1,−1 HLASS 0 ALASS 0ξ + X K∗′ X λ=−1,0,1 AK∗′ λξ 2 . Timing comparison The computing capabilities of GPUs versus CPUs are tested by generating and fitting three sets, each comprising 10,000 Monte Carlo (MC) events (pseudo- experiments) of increasing complexity (number of K∗s) of the fit model previously described. The fitter implemented in ROOT/RooFit is run on an Intel Xeon cluster with 24 CPUs whereas the GooFit version is run on NVIDIA Tesla K40 GPU with 2880 CUDA cores. As the timing test models are for the demonstration purpose only, they are much less complex than the full model required for the analysis. Also, they process a smaller number of events than that expected from a collider experiment. As shown in Fig. 2, it becomes almost impossible to run the fitter on CPUs within any reasonable timescale when the number of fit parameters is increased. The GPU-based GooFit application provides a striking speed-up in per- formance compared to the CPU-based RooFit application. The latter gets so slow that it can become unreliable once the full number of parameters is adopted in the fit model. Figure 2 Comparison of time required by RooFit (CPU-based) and GooFit (GPU+CPU based) fitter frameworks to fit three data sets of 10,000 pseudo-experiments, each generated and fitted according to models of increasing complexity in terms of the number of K∗components. Figure 2 Comparison of time required by RooFit (CPU-based) and GooFit (GPU+CPU based) fitter frameworks to fit three data sets of 10,000 pseudo-experiments, each generated and fitted according to models of increasing complexity in terms of the number of K∗components. The LASS parametrization (5) becomes S(Φ) = X ξ=1,−1 X K∗ X λ=−1,0,1 AK∗ λξ + X Z X λ′=−1,0,1 AZ λ′ξ 2 (12) (12) The signal density function of the charge conjugate decay, identified through the charge of the K (or π) differs only in the sign of ϕ. The implementation of this model takes into account this switching of sign and also allows for a possible flavour mis- tagging (typically a few %). For the full fit model with ten K∗s and two Zs as well as considering the floating masses and widths for some of the resonances, the total number of free parameters in the 4D probability density function can exceed 60. The large number of free parameters coupled with a complex PDF, which requires many internal mathematical operations to be executed at each step of the UML fit, poses a real computational challenge. Page 9 of 20 Sur et al. Validation with the K∗-only model A pseudo-data sample of one million events is generated with the ten K∗s mentioned in Section 2 with their masses and widths fixed to the nominal values. The helicity amplitude parameters for each of these resonances are fixed to the values obtained by Belle [33]. Figure 3 Projection of mKπ spectrum of the 4D dataset (black points with error bars) generated according to an ideal signal model. The fit result (red points with error bars) is superimposed along with the individual signal components corresponding to the different K∗s. The post-fit values of the helicity amplitude parameters and fit fractions for each component are also displayed. Figure 3 Projection of mKπ spectrum of the 4D dataset (black points with error bars) generated according to an ideal signal model. The fit result (red points with error bars) is superimposed along with the individual signal components corresponding to the different K∗s. The post-fit values of the helicity amplitude parameters and fit fractions for each component are also displayed. As the PDF is four-dimensional, the fit results are presented as projections in each of the dimensions. The mKπ projection of the fit to the generated dataset is shown in Fig. 3 and the other three projections, mJ/ψπ, cos θJ/ψ, and ϕ, are presented in Fig. 4. The fit results are found to be in excellent agreement with the generated pseudo-data in each of the four dimensions signifying a good fit overall. The consistency of the post-fit values of the free parameters is checked by comparing the pull distributions (normalised residuals) with their generated values as shown in Figs. 5 and 6. As the PDF is four-dimensional, the fit results are presented as projections in each of the dimensions. The mKπ projection of the fit to the generated dataset is shown in Fig. 3 and the other three projections, mJ/ψπ, cos θJ/ψ, and ϕ, are presented in Fig. 4. The fit results are found to be in excellent agreement with the generated pseudo-data in each of the four dimensions signifying a good fit overall. The consistency of the post-fit values of the free parameters is checked by comparing the pull distributions (normalised residuals) with their generated values as shown in Figs. 5 and 6. As the exact contribution of each resonance to the total signal cannot be precisely evaluated due to interference effects, an approximate measure is provided through the fit fractions. Fit validation To validate the framework, a distribution according to the fit model is generated through MC techniques. These generated events mimic real data that are recorded Page 10 of 20 Sur et al. by the collider experiments. A fit to that distribution is performed to check whether the best estimates of parameters returned by the fit are consistent with their input values. Validation with the K∗-only model The fit fraction of the j-th resonance Rj is given by FFj = R Ω|ARj(Φ)|2d⃗x R Ω|S(Φ)|2d⃗x (13) (13) where Ωis the four-dimensional domain for the set of variables Φ [Eq. (1)] and S(Φ) is the signal function defined in Eq. (12). The numerator of Eq. (13) is obtained by Page 11 of 20 Sur et al. Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ, cos θJ/ψ, and ϕ of the 4D dataset generated according to an ideal signal model (black points with error bars). The fit result (red points with error bars) is superimposed along with the individual fit components corresponding to different K∗s. Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ, cos θJ/ψ, and ϕ of the 4D dataset generated according to an ideal signal model (black points with error bars). The fit result (red points with error bars) is superimposed along with the individual fit components corresponding to different K∗s. Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ, cos θJ/ψ, and ϕ of the 4D dataset generated according to an ideal signal model (black points with error bars). The fit result (red points with error bars) is superimposed along with the individual fit components corresponding to different K∗s. setting to zero all the other helicity amplitudes at the post-fit level. The sum of all the fit fractions is not constrained to 100% as a consequence of the nonunitarity of the model which stems from the constructive and destructive effects of interference between the resonances. Page 12 of 20 Sur et al. a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 10 2 10 1 100 101 102 103 Amplitudes Amplitude Values Gen values Post-fit values a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Amplitude Pull Values Figure 5 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with all the ten K∗resonances. The green lines define a ±3σ band. Validation with the K∗-only model Amplitude Pull Values Amplitude Pull Values Figure 5 Comparison of generated and post-fit values of the amplitude parameters (above) and he corresponding pull distribution (below) obtained from a fit to events generated with all the en K∗resonances. The green lines define a ±3σ band. Sensitivity of the fitter to Z contributions Fit validation exercises are performed for a) the K∗-only model but with the LASS lineshape used for the S-wave, and b) model with all ten K∗s together with Z(4200) and Z(4430) resonances. The mass, width, and helicity amplitudes of the Z reso- nances are fixed to the values obtained by Belle [33]. It is found that the post-fit values of parameters are consistent with the ones used for generation in both cases. The fit fractions of the Z-components are found to be small (about a few percent) as expected from the Belle results. This confirms that the fitter is capable of correctly detecting Z contributions even if they are relatively small. Since the Z contributions are expected to be small, we need to ensure that the fitter does not artificially generate Z peaks due to statistical fluctuations or al- ternative parametrizations of K∗signals such as the LASS lineshape. Pseudo-data is generated with only ten K∗s and fitted with a [ten K∗s + Z(4200) + Z(4430)] model. The fit fraction for both Z(4200) and Z(4430) are found to be 0.01%. From Figs. 7 and 8, it can be seen that the post-fit helicity amplitude values for the K∗s are close to their generated values indicating that the contribution of the Zs are indeed consistent with zero. Similarly, another set of pseudo-data was generated with all K∗s (with LASS for the S-wave) and fitted with an “all K∗s (with LASS) + Z(4200) + Z(4430)” model. The fit fractions for Z(4200) and Z(4430) are found to be 0.002% and 0.003%, respectively. Similar to the previous test, the post-fit helicity amplitude Page 13 of 20 Sur et al. Validation with the K∗-only model b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 3 2 1 0 1 2 3 Phases [rad] Phase Values Gen values Post-fit values b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Phase Pull Values Figure 6 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with all the ten K∗resonances. The green lines define a ±3σ band. Phase Pull Values Figure 6 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with all the ten K∗resonances. The green lines define a ±3σ band. Figure 6 Comparison of generated and post-fit values of the phase parameters (above) and the orresponding pull distribution (below) obtained from a fit to events generated with all the ten K∗resonances. The green lines define a ±3σ band. values for the K∗s are found to be close to their generated values signifying that the contribution of the Zs are again consistent with zero. Applicability to real-life use cases Amplitude Pull Values Figure 7 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. Figure 7 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. model, whereas the background is added with a fixed coefficient (equal to [1−signal purity] which in this study is assumed to be 15%). Therefore, the 4D PDF f(Φ) on which the UML fit is to be performed takes the form f(Φ) = p · ǫ(Φ) · S(Φ) + (1 −p) · b(Φ) (14) (14) f(Φ) = p · ǫ(Φ) · S(Φ) + (1 −p) · b(Φ) where, where, • p is the signal purity; • p is the signal purity; • ǫ(Φ) is the 4D relative signal efficiency; • ǫ(Φ) is the 4D relative signal efficiency; • S(Φ) is the signal density function defined in Eq. (12) and • b(Φ) is the 4D background PDF model. From Figs. 11 and 12, it can be seen that the post-fit helicity amplitude values for the K∗s and the Zs are close to their generated values. The fit fractions of Z(4200) (3.49%) and Z(4200) (1.17%) are found to be a few percent as expected from the Belle result [33]. The almost identical post-fit values of the parameters from both the ideal-world case and the real-life case indicate that the fitter can produce reliable results while taking into account the detection efficiency and background contributions. From Figs. 11 and 12, it can be seen that the post-fit helicity amplitude values for the K∗s and the Zs are close to their generated values. The fit fractions of Z(4200) (3.49%) and Z(4200) (1.17%) are found to be a few percent as expected from the Belle result [33]. Applicability to real-life use cases An accurate representation of real data from collider experiments would require the inclusion of detection efficiency and background contamination. Keeping that in mind, the fit framework is developed in such a way that the efficiency and back- ground models of suitable dimensions can be easily included in the form of analytical functions or binned templates. Generic shapes for efficiency (Fig. 9) and background (Fig. 10) in the form of 2D Bernstein polynomials are adopted to test the effective- ness of the fitter with efficiency and background included. Each of the 4D efficiency and background shapes is passed into the fitter as 2D (mass variables) × 2D (an- gular variables) histograms since the masses and angles are expected to be fully (or largely) uncorrelated. Typically the background levels found in dedicated flavour-physics experiments (e.g. Belle and LHCb) are of the order of a few percent [33]. For this test, the fraction is set to a higher value keeping in mind general purpose detectors like CMS and ATLAS that may record signals with less purity due to the absence of dedicated hadron identification systems. One million simulated decays are generated and fitted with a model including all ten K∗s, two Zs as well as the relative efficiency and background parametrizations. The relative efficiencies are used to weight the signal Page 14 of 20 Sur et al. a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 a_Z_4200_0 a_Z_4200_p1 a_Z_4430_0 a_Z_4430_p1 10 1 100 101 102 103 Amplitudes Amplitude Values Gen values Post-fit values a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 a_Z_4200_0 a_Z_4200_p1 a_Z_4430_0 a_Z_4430_p1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Amplitude Pull Values Figure 7 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. Applicability to real-life use cases The almost identical post-fit values of the parameters from both the ideal-world case and the real-life case indicate that the fitter can produce reliable results while taking into account the detection efficiency and background contributions. Page 15 of 20 Sur et al. b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 b_Z_4200_0 b_Z_4200_p1 b_Z_4430_0 b_Z_4430_p1 3 2 1 0 1 2 3 Phases [rad] Phase Values Gen values Post-fit values b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 b_Z_4200_0 b_Z_4200_p1 b_Z_4430_0 b_Z_4430_p1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Phase Pull Values Figure 8 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. Phase Pull Values Figure 8 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. Figure 8 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K∗s is fitted with the [ten K∗s + Z(4200) + Z(4430)] model. The green lines define a ±3σ band. The pulls for the Z components are not defined because the Zs are not present in the generation model. Applicability to real-life use cases 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 ) [GeV] π m(K 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 ) [GeV] π ψ m(J/ 0.9 1.0 1.1 1 − 0.5 − 0 0.5 1) ψ J/ θ cos( 3 − 2 − 1 − 0 1 2 3 φ 0.9 1.0 1.1 Figure 9 Simulated template of the relative reconstruction efficiency for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary reflects the decay kinematics. Figure 9 Simulated template of the relative reconstruction efficiency for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary reflects the decay kinematics. Page 16 of 20 Sur et al. Figure 10 Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary reflects the decay kinematics. The z-axis values are arbitrary. Figure 10 Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary reflects the decay kinematics. The z-axis values are arbitrary. Figure 10 Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary reflects the decay kinematics. The z-axis values are arbitrary. a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 a_Z_4200_0 a_Z_4200_p1 a_Z_4430_0 a_Z_4430_p1 10 1 100 101 102 103 Amplitudes Amplitude Values Gen values Post-fit values a_K_892_0 a_K_892_p1 a_K_892_m1 a_K_800_0 a_K_1410_0 a_K_1410_p1 a_K_1410_m1 a_K_1430_0 a_K_1430_2_0 a_K_1430_2_p1 a_K_1430_2_m1 a_K_1680_0 a_K_1680_p1 a_K_1680_m1 a_K_1780_3_0 a_K_1780_3_p1 a_K_1780_3_m1 a_K_1950_0_0 a_K_1980_2_0 a_K_1980_2_p1 a_K_1980_2_m1 a_K_2045_4_0 a_K_2045_4_p1 a_K_2045_4_m1 a_Z_4200_0 a_Z_4200_p1 a_Z_4430_0 a_Z_4430_p1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Amplitude Pull Values Figure 11 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with ten K∗s + Z(4200) + Z(4430) model including efficiency and 15% background contribution. The green lines define a ±3σ band. Applicability to real-life use cases Amplitude Pull Values Figure 11 Comparison of generated and post-fit values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with ten K∗s + Z(4200) + Z(4430) model including efficiency and 15% background contribution. The green lines define a ±3σ band. Page 17 of 20 Sur et al. b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 b_Z_4200_0 b_Z_4200_p1 b_Z_4430_0 b_Z_4430_p1 3 2 1 0 1 2 3 Phases [rad] Phase Values Gen values Post-fit values b_K_892_0 b_K_892_p1 b_K_892_m1 b_K_800_0 b_K_1410_0 b_K_1410_p1 b_K_1410_m1 b_K_1430_0 b_K_1430_2_0 b_K_1430_2_p1 b_K_1430_2_m1 b_K_1680_0 b_K_1680_p1 b_K_1680_m1 b_K_1780_3_0 b_K_1780_3_p1 b_K_1780_3_m1 b_K_1950_0_0 b_K_1980_2_0 b_K_1980_2_p1 b_K_1980_2_m1 b_K_2045_4_0 b_K_2045_4_p1 b_K_2045_4_m1 b_Z_4200_0 b_Z_4200_p1 b_Z_4430_0 b_Z_4430_p1 7.5 5.0 2.5 0.0 2.5 5.0 7.5 = postfit gen error Phase Pull Values Figure 12 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with ten K∗s + Z(4200) + Z(4430) model including relative efficiency and 15% background contribution. The green lines define a ±3σ band. Phase Pull Values Figure 12 Comparison of generated and post-fit values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a fit to events generated with ten K∗s + Z(4200) + Z(4430) model including relative efficiency and 15% background contribution. The green lines define a ±3σ band. Page 18 of 20 Sur et al. Discussion Searches for exotic multiquark states in collider experiments require complex mul- tidimensional analyses involving several (or even hundreds of) thousands of events that demand considerable computational resources. Conventional CPU-based tech- niques may fall short to meet these ever increasing demands. In this study, by using the helicity formalism, a four-dimensional amplitude analysis framework for an un- binned maximum-likelihood fit has been implemented. The fitting framework has been developed using the novel GPU based GooFit, an open-source tool under de- velopment which is used in HEP applications for parameters’ estimation, interfacing ROOT to the CUDA parallel computing platform on NVIDIA GPUs. It has been shown that the choice to use GooFit and the accelerated performance provided by GPUs is crucial to carry out these extreme fits. The fit model has been validated by a “closure test”, i.e., by a multi-step proce- dure in which pseudo-experiments under different conditions and assumptions were generated and fitted. The starting model is assumed to be composed of the known set of K∗resonances. Since the low mass S-wave K∗ 0(800) is not yet satisfacto- rily described by a Breit-Wigner amplitude, the alternative LASS parametrization has been implemented on GooFit and thoroughly tested. The fitter has been addi- tionally equipped with the capability of handling relative detection efficiency and background contamination. The possible contribution of the exotic Z states has been calculated and incorporated within the fitter framework with reasonable ro- bustness to allow for testing any combination of their spin-parity values as well as without any constraints. Lastly, the fitter, though designed for a 4D amplitude analysis of a pseudoscalar decaying into a vector and two pseudoscalars, can be easily adapted to other types of decays with higher or lower dimensions, occurring in flavour physics studies. Conclusion The ability of the fitter to efficiently handle higher dimensionality of fit models with great accuracy, its inbuilt functions to calculate complex operations like vector algebra while evaluating PDFs on the GPU-side, its systematized application of Gaussian constraints on fit parameters if required, and its sensitivity to very small contributions of different varieties of hitherto unknown signals make it a formidable toolkit built into an already powerful framework. It is hoped that this kind of fitter implemented within the GooFit framework, along with the flexibility to be easily adapted for even more complex PDFs, will considerably augment the capabilities of collider experiments in searches and measurements in the field of exotic hadron spectroscopy and beyond. List of abbreviations GPU: graphics processing unit; QCD: quantum chromodynamics; UML: unbinned maximum likelihood; HEP: high-energy physics; BW: Breit-Wigner; PDF: probability density function; NLL: negative log likelihood; MC: Monte Carlo. List of abbreviations GPU: graphics processing unit; QCD: quantum chromodynamics; UML: unbinned maximum likelihood; HEP: high-energy physics; BW: Breit-Wigner; PDF: probability density function; NLL: negative log likelihood; MC: Monte Carlo. Author details 1Tata Institute of Fundamental Research, Mumbai, India. 2Universit´a degli Studi di Bari and I.N.F.N. - Sezione di Bari, Italy. References 1. Snijders, C., Matzat, U., Reips, U.-D.: “Big Data” : Big gaps of knowledge in the field of internet science. International Journal of Internet Science 7, 1–5 (2012) 2. Fox, C.: Data science for transport: A self-study guide with computer exercises. Springer (2018) 2. Fox, C.: Data science for transport: A self-study guide with computer exerc 3. Evans, L., Bryant, P.: LHC Machine. JINST 3, 08001 (2008) ( ) 4. Mascetti, L., et al.: Cern disk storage services: Report from last data taking, evolution and future outlook 4. Mascetti, L., et al.: Cern disk storage services: Report from last data taking, evolution and future outlook d b l EPJ W b C f 245 ( ) 4. Mascetti, L., et al.: Cern disk storage services: Report from last data taking, evolution and future outlook towards exabyte-scale storage EPJ Web Conf 245 04038 (2020) 4. Mascetti, L., et al.: Cern disk storage services: Report from last data taking, evolution a towards exabyte-scale storage. EPJ Web Conf. 245, 04038 (2020) towards exabyte-scale storage. EPJ Web Conf. 245, 04038 (202 Aad, G., et al.: The ATLAS Experiment at the CERN Large Hadron Collider. JINST 3, 08003 (2008) 5. Aad, G., et al.: The ATLAS Experiment at the CERN Large Hadron Collider. JINST 3, 08003 (2008) Aad, G., et al.: The ATLAS Experiment at the CERN Large Had 6. Chatrchyan, S., et al.: The CMS Experiment at the CERN LHC. JINST 3, 08004 (2008) 7. Alves, J. A.Augusto, et al.: The LHCb Detector at the LHC. JINST 3, 08005 (2008) lves, J. A.Augusto, et al.: The LHCb Detector at the LHC. JINS 8. Lefevre, C.: LHC brochure (English version). LHC brochure (version anglaise) (2010) 9. Brumfiel, G.: High-energy physics: Down the petabyte highway. Nature 469(7330), 282–283 (2011) 10. Lyons, L.: Statistics for Nuclear and Particle Physicists. Cambridge University Press, ??? (1986 0. Lyons, L.: Statistics for Nuclear and Particle Physicists. Camb 11. Barlow, R.J.: Statistics: a Guide to the Use of Statistical Methods in the Physical Sciences. Manchester physics series. Wiley, Chichester (1989) 12. Gross, D.J., Wilczek, F.: Ultraviolet Behavior of Nonabelian Gauge Theories. Phys. Rev. Lett. 30, 1343–1346 (1973) Politzer, H.D.: Reliable Perturbative Results for Strong Interactions? Phys. Rev. Lett. 30, 1346–1349 (1973) Gross D J Wilczek F : Asymptotically Free Gauge Theories - I Phys Rev D8 3633–3652 (1973) 13. Politzer, H.D.: Reliable Perturbative Results for Strong Interactions? Phys. Rev. Lett. Acknowledgements We acknowledge Prof. Alexis Pompili (Universit´a degli Studi di Bari and I.N.F.N. - Sezione di Bari) and Prof. Gagan Mohanty (Tata Institute of Fundamental Research, Mumbai) for their guidance and constant support as well as helpful comments on this article. We are indebted to Prof. John Yelton (University of Florida) for his valuable comments and suggestions on the content as well as the language and presentation of this article. The computational work has been executed on the IT resources of the ReCaS-Bari data centre, which have been made available by two projects financed by the MIUR (Italian Ministry for Education, University and Research) in the “PON Ricerca e Competitivit`a 2007-2013” Program: ReCaS (Azione I - Interventi di rafforzamento strutturale, PONa3 00052, Avviso 254/Ric) and PRISMA (Asse II - Sostegno all’innovazione, PON04a2 A). Author’s contributions All authors have equal contribution. They have read and approved the manuscript. Funding Open access funding is partially provided by Tata Institute of Fundamental Research, Mumbai, India and Universit´a degli Studi di Bari and I.N.F.N. - Sezione di Bari, Italy. The research received no external funding. Declarations Consent for publication Not applicable Page 19 of 20 Sur et al. Availability of data and materials The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. References 30, 1346–1349 (1973) 13. Politzer, H.D.: Reliable Perturbative Results for Strong Interactions? Phys. Rev. Lett. 30, 1346–1349 (1973) 14. Gross, D.J., Wilczek, F.: Asymptotically Free Gauge Theories - I. Phys. Rev. D8, 3633–3652 (1973) , g y , ( ) 14. Gross, D.J., Wilczek, F.: Asymptotically Free Gauge Theories - I. Phys. Rev. D8, 3633–3652 (1973) ., Wilczek, F.: Asymptotically Free Gauge Theories - I. Phys. Re 15. Politzer, H.D.: Asymptotic Freedom: An Approach to Strong Interactions. Phys. Rept. 14, 129–180 (1974) 16. Ali, A., Maiani, L., Polosa, A.D.: Multiquark Hadrons. Cambridge University Press, Cambridge (2019) 17. Olsen, S.L., Skwarnicki, T., Zieminska, D.: Nonstandard heavy mesons and baryons: Experimental evidence. Rev. Mod. Phys. 90, 015003 (2018) 18. Abe, T., et al.: Belle II Technical Design Report (2010). 1011.0352 19. Ablikim, M., et al.: Design and Construction of the BESIII Detector. Nucl. Instrum. Meth. A 614, 345–399 (2010) 20. Brun, R., Rademakers, F.: ROOT: An object oriented data analysis framework. Nucl. Instrum. Meth. A 389, 81–86 (1997) ( ) 21. Verkerke, W., Kirkby, D.P.: The RooFit toolkit for data modeling. eConf C0303241, 007 (2003) 22. R. Andreassen, B. T. Meadows, M. de Silva, M. D. Sokoloffand K. Tomko: Goofit: A library for massively parallelising maximum-likelihood fits. Journal of Physics: Conference Series 513(5), 052003 (2014) 22. R. Andreassen, B. T. Meadows, M. de Silva, M. D. Sokoloffand K. Tomko: Goofit: A li parallelising maximum-likelihood fits. Journal of Physics: Conference Series 513(5), 0520 er, H., et al.: GooFit 2.0. J. Phys. Conf. Ser. 1085(4), 042014 (2 ( ) ( ) 24. Nickolls, J., Buck, I., Garland, M., Skadron, K.: Scalable parallel programming with cuda. ACM Queue 6 (2008) 24. Nickolls, J., Buck, I., Garland, M., Skadron, K.: Scalable parallel programming with cuda. ACM Queue 6 (2008) 25 James F Roos M : Minuit – a system for function minimization and analysis of the parameter errors and 24. Nickolls, J., Buck, I., Garland, M., Skadron, K.: Scalable parallel programming w 24. Nickolls, J., Buck, I., Garland, M., Skadron, K.: Scalable parallel programming with cuda. ACM Queue 6 (2008) 25. James, F., Roos, M.: Minuit – a system for function minimization and analysis of the parameter errors and correlations. Comput. Phys. Commun. 10, 343 (1975) correlations. Comput. Phys. Commun. 10, 343 (1975) 26. Albrecht, J., et al.: A Roadmap for HEP Software and Computing R&D for the 2020s. References Comput Softw Big Sci 3(7) (2019) et al.: GPUs for statistical data analysis in HEP: a performance 27. Pompili, A., et al.: GPUs for statistical data analysis in HEP: a performance stu vs.RooFit on CPUs. Journal of Physics: Conference Series 762, 012044 (2016) vs.RooFit on CPUs. Journal of Physics: Conference Series 762, 012044 (2016) 28. Di Florio, A.: Estimation of global statistical significance of a new signal within the GooFit framework on GPUs. PoS Confinement2018, 229 (2019) 29. Lees, J.P., et al.: Measurement of the neutral D meson mixing parameters in a time-dependent amplitude analysis of the D0 →π+π−π0 decay. Phys. Rev. D 93(11), 112014 (2016) Page 20 of 20 Sur et al. 30. Sun, L., et al.: Model-independent partial wave analysis using a massively-parallel fitting framework. Journal of Physics: Conference Series 898, 072025 (2017) 31. Hasse, C., et al.: Amplitude analysis of four-body decays using a massively-parallel fitting framework. J. Phys. Conf. Ser. 898(7), 072016 (2017) 32. Dalitz, R.H.: On the analysis of τ-meson data and the nature of the τ-meson. Phil. Mag. Ser.7, 1068–1080 (1953) 0 33. Chilikin, K., et al.: Observation of a new charged charmoniumlike state in B 0 →J/ψK−π+ decays. Phys. Rev. D 90, 112009 (2014) 34. Chilikin, K., et al.: Experimental constraints on the spin and parity of the Z(4430)+. Phys. Rev. D 88(7), 074026 (2013) pf, V.F.: Theoretical Nuclear Physics. Springer, New York, NY (1 36. Richman, J.D.: An Experimenter’s Guide to the Helicity Formalism. CALT-68-1148 (1984) 37. Tanabashi, M., et al.: Review of particle physics. Phys. Rev. D 98, 030001 (2018) 38. Aston, D., et al.: A Study of K−π+ Scattering in the Reaction K−p →K−π+n at 11 GeV/c. Nucl. Phys. B 296, 493 (1988) Figures Figures Figure 1 A sketch illustrating the de¦nition of two independent angular variables, θJ/ψ and φ, for the amplitude analysis of B0 ฀ J/ψKπ decays. Figure 1 A sketch illustrating the de¦nition of two independent angular variables, θJ/ψ and φ, for the amplitude analysis of B0 ฀ J/ψKπ decays. Figure 2 Figure 2 Comparison of time required by RooFit (CPU-based) and GooFit (GPU+CPU based) ¦tter frameworks to ¦t three data sets of 10,000 pseudo-experiments, each generated and ¦tted according to models of increasing complexity in terms of the number of K* components. Comparison of time required by RooFit (CPU-based) and GooFit (GPU+CPU based) ¦tter frameworks to ¦t three data sets of 10,000 pseudo-experiments, each generated and ¦tted according to models of increasing complexity in terms of the number of K* components. Figure 3 Projection of mKπ spectrum of the 4D dataset (black points with error bars) generated according to an ideal signal model. The ¦ t result (red points with error bars) is superimposed along with the individual signal components corresponding to the different K*s. The post-¦t values of the helicity amplitude parameters and ¦t fractions for each component are also displayed. Figure 3 Projection of mKπ spectrum of the 4D dataset (black points with error bars) generated according to an ideal signal model. The ¦ t result (red points with error bars) is superimposed along with the individual signal components corresponding to the different K*s. The post-¦t values of the helicity amplitude parameters and ¦t fractions for each component are also displayed. Projection of mKπ spectrum of the 4D dataset (black points with error bars) generated according to an ideal signal model. The ¦ t result (red points with error bars) is superimposed along with the individual signal components corresponding to the different K*s. The post-¦t values of the helicity amplitude parameters and ¦t fractions for each component are also displayed. Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ', cos θJ/ψ, generated according to an ideal signal model (black points with error bars). The error bars) is superimposed along with the individual ¦t components correspond Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ', cos θJ/ψ, and φ of the 4D dataset generated according to an ideal signal model (black points with error bars). The ¦t result (red points with error bars) is superimposed along with the individual ¦t components corresponding to different K*s. Figure 4 Projections of the other three variables: (from top to bottom) mJ/ψπ', cos θJ/ψ, and φ of the 4D dataset generated according to an ideal signal model (black points with error bars). The ¦t result (red points with error bars) is superimposed along with the individual ¦t components corresponding to different K*s. Figure 5 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 5 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 5 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 3 Figure 6 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 6 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 6 Figure 6 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with all the ten K* resonances. The green lines de¦ne a ±3σ band. Figure 7 Comparison of generated and post- ¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K*s is ¦tted with the [ten K*s + Z(4200) + Z(4430)] model. The green lines de¦ne a ±3σ band. The pulls for the Z components are not de ¦ned because the Zs are not present in the generation model. Figure 9 Simulated template of the relative reconstruction e¨ciency for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary re§ects the decay kinematics. Figure 10 Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary re§ects the decay kinematics. The z-axis values are arbitrary. Figure 7 Figure 7 Comparison of generated and post- ¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K*s is ¦tted with the [ten K*s + Z(4200) + Z(4430)] model. The green lines de¦ne a ±3σ band. The pulls for the Z components are not de ¦ned because the Zs are not present in the generation model. Comparison of generated and post- ¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K*s is ¦tted with the [ten K*s + Z(4200) + Z(4430)] model. The green lines de¦ne a ±3σ band. The pulls for the Z components are not de ¦ned because the Zs are not present in the generation model. Figure 8 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K*s is ¦tted with the [ten K*s + Z(4200) + Z(4430)] model. The green lines de¦ne a ±3σ band. The pulls for the Z components are not de¦ne because the Zs are not present in the generation model. Figure 8 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained when a dataset generated with ten K*s is ¦tted with the [ten K*s + Z(4200) + Z(4430)] model. The green lines de¦ne a ±3σ band. The pulls for the Z components are not de¦ne because the Zs are not present in the generation model. Figure 9 Simulated template of the relative reconstruction e¨ciency for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary re§ects the decay kinematics. Figure 10 Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary re§ects the decay kinematics. The z-axis values are arbitrary. Simulated background template for the scatter plots of the two mass variables (left) and the two angular variables (right) of the decay. In the former, the 2D kinematic boundary re§ects the decay kinematics. The z-axis values are arbitrary. Figure 11 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Figure 11 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Figure 11 Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Comparison of generated and post-¦t values of the amplitude parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Figure 12 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including relative e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Figure 12 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pul distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including relative e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Figure 12 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pul Figure 12 Figure 12 Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including relative e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including relative e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band. Comparison of generated and post-¦t values of the phase parameters (above) and the corresponding pull distribution (below) obtained from a ¦t to events generated with ten K*s + Z(4200) + Z(4430) model including relative e¨ciency and 15% background contribution. The green lines de¦ne a ±3σ band.
https://openalex.org/W3045594978
http://doc.rero.ch/record/329401/files/wei_chc.pdf
English
null
Contribution of Hydrogen Cyanide to the Antagonistic Activity of Pseudomonas Strains Against Phytophthora infestans
Microorganisms
2,020
cc-by
7,729
Received: 11 July 2020; Accepted: 27 July 2020; Published: 28 July 2020 Abstract: Plants face many biotic and abiotic challenges in nature; one of them is attack by disease-causing microbes. Phytophthora infestans, the causal agent of late blight is one of the most prominent pathogens of the potato responsible for multi-billion-dollar losses every year. We have previously reported that potato-associated Pseudomonas strains inhibited P. infestans at various developmental stages. A comparative genomics approach identified several factors putatively involved in this anti-oomycete activity, among which was the production of hydrogen cyanide (HCN). Here, we report the relative contribution of HCN emission to the overall anti-Phytophthora activity of two cyanogenic Pseudomonas strains, P. putida R32 and P. chlororaphis R47. To quantify this contribution, we generated HCN-negative mutants (∆hcn) and compared their activities to those of their respective wild types in different experiments assessing P. infestans mycelial growth, zoospore germination, and infection of potato leaf disks. Using in vitro experiments allowing only volatile-mediated interactions, we observed that HCN accounted for most of the mycelial growth inhibition (57% in R47 and 80% in R32). However, when allowing both volatile and diffusible compound-mediated interactions, HCN only accounted for 1% (R47) and 18% (R32) of mycelial growth inhibition. Likewise, both mutants inhibited zoospore germination in a similar way as their respective wild types. More importantly, leaf disk experiments showed that both wild-type and ∆hcn strains of R47 and R32 were able to limit P. infestans infection to a similar extent. Our results suggest that while HCN is a major contributor to the in vitro volatile-mediated restriction of P. infestans mycelial growth, it does not play a major role in the inhibition of other disease-related features such as zoospore germination or infection of plant tissues. Keywords: potato late blight; pseudomonads; hydrogen cyanide; biocontrol Abhishek Anand , Delphine Chinchilla, Christopher Tan, Laurent Mène-Saffrané, Floriane L’Haridon and Laure Weisskopf * Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland; abhishek.anand@unifr.ch (A.A.); delphine.chinchilla@unibas.ch (D.C.); christopher.tan@unifr.ch (C.T.); laurent.mene-saffrane@unifr.ch (L.M.-S.); floriane.lharidon@unifr.ch (F.L.) * Correspondence: laure.weisskopf@unifr.ch Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland; abhishek.anand@unifr.ch (A.A.); delphine.chinchilla@unibas.ch (D.C.); christopher.tan@unifr.ch (C.T.); laurent.mene-saffrane@unifr.ch (L.M.-S.); floriane.lharidon@unifr.ch (F.L.) Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland; abhishek.anand@unifr.ch (A.A.); delphine.chinchilla@unibas.ch (D.C.); christopher.tan@unifr.ch (C.T.); laurent.mene-saffrane@unifr.ch (L.M.-S.); floriane.lharidon@unifr.ch (F.L.) Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland; abhishek.anand@unifr.ch (A.A.); delphine.chinchilla@unibas.ch (D.C.); christopher.tan@unifr.ch (C.T.); laurent.mene-saffrane@unifr.ch (L.M.-S.); floriane.lharidon@unifr.ch (F.L.) * Correspondence: laure.weisskopf@unifr.ch R i d 11 J l 2020 A d 27 J l 2020 P bli h d 28 J l 2020    delphine.chinchilla@unibas.ch (D.C.); christopher.tan@unifr.ch (C.T.); laurent.mene-saffrane@unifr.ch (L.M.-S.); floriane.lharidon@unifr.ch (F.L.) * Correspondence: laure.weisskopf@unifr.ch * Correspondence: laure.weisskopf@unifr.ch Received: 11 July 2020; Accepted: 27 July 2020; Published: 28 July 2020 microorganisms microorganisms Microorganisms 2020, 8, 1144; doi:10.3390/microorganisms8081144 Contribution of Hydrogen Cyanide to the Antagonistic Activity of Pseudomonas Strains Against Phytophthora infestans Abhishek Anand , Delphine Chinchilla, Christopher Tan, Laurent Mène-Saffrané, Floriane L’Haridon and Laure Weisskopf * www.mdpi.com/journal/microorganisms 1. Introduction In nature, plants face a variety of biotic and abiotic challenges such as climate change and infection by disease-causing microorganisms. Late blight caused by Phytophthora infestans (a hemibiotrophic pathogen) is one of the most devastating diseases in potato [1]. Worldwide, P. infestans is associated with a cost of several billion dollars per year, including the control efforts and production losses [2]. The current control methods mostly rely on the use of synthetic fungicides or copper-based products, both of which represent environmental hazards [3,4]. As an alternative tool to control plant diseases, researchers have focused their interest on identifying biocontrol agents [5], many of which are isolated from the host plants or soil [6]. Biocontrol agents (BCAs) are a group of microorganisms which show potential to restrict plant diseases either by inducing plant defense responses and/or by directly Microorganisms 2020, 8, 1144; doi:10.3390/microorganisms8081144 www.mdpi.com/journal/microorganisms 2 of 10 Microorganisms 2020, 8, 1144 inhibiting the pathogen by producing antimicrobial agents [7]. Among the antimicrobial agents, hydrogen cyanide (HCN) has been reported by many studies to be a key biocontrol trait and was shown to be produced by multiple Pseudomonas species [8–13]. It is generally considered as a secondary metabolite produced at the end of the exponential phase and the start of the stationary phase. It is synthesized by hcnABC, which oxidizes glycine to produce HCN and CO2 [14]. HCN is a strong inhibitor of cytochrome c oxidase and other metalloenzymes and hence is a toxic molecule for all aerobic organisms. Nevertheless, cyanogenesis has been reported in many plants, fungi, myriapods, beetles, butterflies, and bacteria [15,16]. Many organisms have developed different strategies to avoid cyanide intoxication such as chemical conversion of HCN to thiocyanate by the rhodanese enzyme or the use of cyanide-insensitive oxidases [17–19]. In a recent comparative genomics study investigating the putative genetic determinants underlying the anti-Phytophthora activity of potato-associated Pseudomonas strains, we observed that the strains showing strong in vitro inhibition of P. infestans mycelial growth all harbored the HCN locus in their genome [20]. We therefore asked the question of the relative contribution of this particular biocontrol trait to the overall protective effect of cyanogenic Pseudomonas known for their strong biocontrol activity against different pathogens like Phytophthora infestans, Helminthosporium solani, Rhizoctonia solani, Fusarium oxysporum, and Dickeya dianthicola [6,21]. We selected P. chlororaphis (R47) and P. 1. Introduction putida (R32), which were shown to inhibit mycelium growth and zoospore release in vitro but also to restrict the infection by P. infestans in leaf disk experiments [22]. The main aim of this study was therefore to quantify the relative contribution of HCN in the overall anti-oomycete activity of two efficient biocontrol Pseudomonas strains by generating HCN-negative mutants and assessing their effect on the different life stages and leaf infection ability of Phytophthora infestans. Understanding the mechanisms underlying the efficient inhibition of different developmental stages of P. infestans by biocontrol agents could help us in designing better crop protection strategies against the devastating late blight disease. 2.1. Strains and Culture Media Pseudomonas strains R47 and R32 [6] were routinely grown on Luria-Bertani (LB) agar medium made with 20 g/L of Difco LB broth (Lennox, Fisher Bioreagents, Geel, Belgium) and 15 g/L of agar, (Agar-agar, Kobe I, Carl Roth, Karlsruhe, Germany). Phytophthora infestans (Rec01) originally obtained from Agroscope Reckenholz was grown on unclarified V8 medium for dual assays and routine culture. To prepare unclarified V8 agar medium, 100 mL/L V8 juice (V8 Vegetable Juice, Campbell Soup Company, Camden, NJ, USA), 1 g/L calcium carbonate (Fluka Chemika, Buchs, Switzerland), and 15 g/L agar were dissolved in 1 L of distilled water and autoclaved. To prepare clarified V8 medium, the V8 juice was centrifuged twice at 5000 rpm for 15 min, and then the supernatant was used as above. 2.2. Generation of ∆hcn Strains For the generation of HCN-negative mutant strains (∆hcn), an allelic replacement technique using the I-SceI system was used [10,23]. A 2.8kb deletion was created within the HCN locus (hcnABC locus–3 kb). A suicide plasmid pEMG with a kanamycin resistance cassette and restriction sites for I-SceI, EcoRI, and BamHI was used. Two 500bp fragments were PCR-amplified from both ends of the HCN locus retaining the start codon of hcnA and the stop codon of the hcnC gene. Briefly, fragments 1 & 2 were generated by PCR amplification from the genomic DNA (gDNA) of R47 using the primers hcn47-1, hcn47-2 and hcn47-3, hcn47-4 containing the EcoRI-KpnI restriction sites for fragment 1 and the KpnI-BamHI restriction sites for fragment 2 (Supplementary Table S1). Fragments and pEMG were digested with their respective restriction enzymes, and the digested products were then triple-ligated to generate pEMG::∆hcn_R47 plasmid (Supplementary Table S2). Similarly, pEMG::∆hcn_R32 plasmid was generated using the primers hcn32-1, hcn32-2 and hcn32-3, hcn32-4. The final pEMG::∆hcn_R47 and pEMG::∆hcn_R32 plasmids were then transformed into 3 of 10 Microorganisms 2020, 8, 1144 R47 wild-type and R32 wild-type strains, respectively, to allow the plasmid to integrate into the chromosome using homologous recombination (HR). Following the HR event, kanamycin-resistant colonies were selected and screened for the presence of the inserted plasmid in gDNA. Positive clones were then transformed with pSW-2 plasmid, which contained the cassette for gentamycin resistance and the gene for the inducible production of I-SceI enzyme. Then, gentamycin-resistant clones were induced with m-toluate for the production of I-SceI enzyme, which facilitated the second crossover event to excise the suicide plasmid pEMG and HCN locus from the chromosome. Clones were then screened for their sensitivity to kanamycin. Kanamycin-sensitive clones were screened for the presence of mutated HCN fragments by PCR amplification using primers hcn47-1 and hcn47-4 for R47 and hcn32-1 and hcn32-4 for R32. The putative mutant strains were confirmed by a cyanide detection assay (refer to Section 2.4) (Supplementary Figure S1). 2.3. Growth Curves Two to three bacterial colonies from a 1-day-old plate were inoculated in LB broth for 16 h and then centrifuged at 5000 rpm for 3 min. The pellet was washed twice with 0.9% NaCl and then adjusted to OD600 = 1. Clarified V8 liquid medium (95 µL) was inoculated with 5 µL of bacterial suspension and grown for 20 h at 30 ◦C with shaking in a 24-well plate (Costar, Corning, NY, USA). A Cytation5 plate reader (BioTek, Winooski, VT, USA) was used to read absorbance every 30 min at 600 nm. The experiment was replicated twice with 3 technical replicates each. Uninoculated clarified V8 liquid medium was used as blank. 2.4. Cyanide Detection Assay Filter papers were soaked in a premixed solution of 10 mL chloroform (Fisher Scientific, Leicestershire, UK), 50 mg copper(II) ethylacetoacetate (Sigma-Aldrich, St. Louis, MO, USA), and 50 mg 4,4-methylenebis(N,N-dimethylaniline) (Sigma Aldrich, St. Louis, MO, USA) and allowed to dry overnight at room temperature [24]. These filter papers were then placed in one empty compartment of split-plate Petri dishes, and the other compartment was filled with LB agar. The LB side of the Petri dish was then inoculated with 3 drops (10 µL each) of bacterial suspension, which was pre-adjusted to OD600 = 1. The images were taken after 24 h, and the experiment was repeated twice with 3 technical replicates each. Uninoculated LB medium was used as negative control. 2.5. Dual Assays For volatile compound-mediated dual assays, split Petri dishes were used and both sides were filled with V8 agar medium. For diffusible compound-mediated assays, full plates with V8 agar medium were used. For both types of experiment, Pseudomonas strains and P. infestans were inoculated on the same day. Three drops of 10 µL bacterial suspension (OD600 = 1) were inoculated, and a plug from 2-week-old plates of P. infestans was used. Plates were incubated at 20 ◦C in the dark (upside down) for 2 weeks for full plate assays and for 1 week for split plate assays. Images were taken after the experiment and used for mycelial growth analysis using the ImageJ software. The experiment was repeated thrice with 3–4 technical replicates each. Mycelial growth in control plates (with no bacterial inoculation) was considered as 100%, and relative growth percentages were calculated for the mycelial growth in inoculated samples. Inhibition percentage was calculated by subtracting mycelial growth percentage from 100. 2.7. Leaf Disk Experiment Leaf disks were obtained from 3–4 independent potato plants (aged ~4 weeks, Bintje cultivar) and placed on 0.8% water agar medium in Petri dishes. Zoospores were harvested as mentioned in the zoospore germination experiment. The bacterial suspensions in 0.9% NaCl adjusted to OD600 = 1 were mixed with zoospores in a 1:1 ratio, and 10 µL of the mix was spotted on the abaxial surface of each leaf disk. For this experiment, plates were incubated in a humid chamber (polystyrene box with wet papers on the bottom) for 6 days at 18 ◦C in the dark. Pictures were taken after 6 days of inoculation with zoospores. Later, leaf disks were harvested and stored in 0.05% BHT in MeOH at −80 ◦C until fatty acid methyl esters (FAMEs) analysis. The experiment was repeated twice with 12–16 leaf disks from 3–4 plants in each experiment. 2.6. Zoospore Germination Experiment Zoospores were harvested from sporangia present in a 2-week-old P. infestans plate after treatment with ice-cold sterile water for 2 h at 4 ◦C followed by 20 min at room temperature. Zoospores were harvested by pipetting from the water surface and counted using Jessen cell counting chamber. The number of zoospores was adjusted to 150,000 zoospores per mL. Zoospores were mixed in 1:1 ratio with bacterial suspension (in 0.9% NaCl adjusted to OD600 = 1) and incubated for 3 h in the dark at room temperature. 4 of 10 Microorganisms 2020, 8, 1144 After incubation, 30 µL of the samples were pipetted into the wells of a 24-well plate (Costar, Corning, NY, USA), and 4 images were taken randomly at 4× magnification using a Cytation5 plate reader. The percentage of germination was calculated by counting the total number of zoospores and the number of germinated zoospores on the images. Inhibition percentage was calculated by subtracting zoospore germination percentage from 100. The experiment was repeated thrice with 3 technical replicates each. After incubation, 30 µL of the samples were pipetted into the wells of a 24-well plate (Costar, Corning, NY, USA), and 4 images were taken randomly at 4× magnification using a Cytation5 plate reader. The percentage of germination was calculated by counting the total number of zoospores and the number of germinated zoospores on the images. Inhibition percentage was calculated by subtracting zoospore germination percentage from 100. The experiment was repeated thrice with 3 technical replicates each. 2.8. Fatty Acid Methyl Esters Analysis (FAMEs) For this assay, leaf disks were harvested and processed using acid-catalysed transesterification to obtain FAMEs. Three leaf disks were pooled and treated with 1 mL of 5% H2SO4 in MeOH (v/v) and 50 µL of 0.05% butylated hydroxytoluene (w/v) in MeOH. Ten micrograms of glyceryl triheptadecanoate (Sigma-Aldrich, St. Louis, MO, USA) were used as an internal standard. Samples were placed in 7 mL glass tubes and were incubated at 85 ◦C for 45 min followed by cooling of tubes at room temperature and addition of 1.5 mL of 0.9% NaCl (w/v) and 2 mL of n-hexane. Samples were mixed by shaking for 5 min and centrifuged at 240 g for 5 min to separate the organic phase. The organic phase was then transferred into a new 7 mL glass tube, and the extraction was repeated two additional times with 2 mL of n-hexane. The pooled organic phases containing FAMEs were evaporated under a stream of nitrogen gas, and the FAMEs were resuspended in 200 µL heptane. FAMEs (2 µL) were injected into a gas chromatograph coupled to a flame ionization dectector (GC-FID) in split mode (50:1) for separation and quantification. The capillary column used was DB-23 by Agilent technologies (30 m × 250 µm × 0.25 µm). Supelco 37 component FAME mix from Sigma was used as a standard. The identification of Eicosapentaenoic acid was performed as described [25]. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth Cyanide-negative mutants were generated in Pseudomonas putida R32 and in Pseudomonas chlororaphis R47 strains. A qualitative detection assay revealed the expected absence of HCN emission in both mutants (Supplementary Figure S1). We performed two types of dual assays to quantify the impact of HCN on the mycelium growth of P. infestans. First, a volatile compound-mediated dual assay was performed to quantify the relative contribution of HCN on the overall volatile-mediated activity of R47 and R32. Second, a diffusible compound-mediated dual assay was performed to include the effects of non-volatile compounds (e.g., siderophores, phenazine) in the overall activity of R47 and R32 against P. infestans mycelial growth. In volatile compound-mediated assays, R47 wt reduced mycelium growth by 90% while R47 ∆hcn strain only reduced it by 33% (Figure 1a). Similarly, when exposed to the volatiles of R32 wt, mycelium growth was reduced by 84% but only by 4% when exposed to the volatiles from R32 ∆hcn (Figure 1b). Diffusible compound-mediated assay showed that the lack of HCN emission did not significantly impact the inhibition potential of R47 against P. infestans mycelial growth. It showed that R47 wt restricted the mycelial growth by 99%, while R47 ∆hcn restricted it by 98% (Figure 1c). Similarly, R32 wt was able to inhibit the mycelial growth by 98%, while an inhibition of 80% was observed 5 of 10 5 f Microorganisms 2020, 8, 1144 in co-cultivation with R32 ∆hcn strain (Figure 1d). We also observed that both volatiles and diffusible compounds produced by R32 ∆hcn altered the mycelium phenotype as visualised by thin peripheral mycelium density in the volatile compound-mediated assay and the grainy mycelium phenotype in diffusible compound-mediated assay (Figure 1b,d). (Figure 1d). We also observed that both volatiles and diffusible compounds produced by R32 ∆h altered the mycelium phenotype as visualised by thin peripheral mycelium density in the volati compound-mediated assay and the grainy mycelium phenotype in diffusible compound-mediate assay (Figure 1b,d). Figure 1. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on Phytophthora infestans mycelium growth. (a,b) Volatile compound-mediated dual assay; (c,d) diffusible compound-mediated dua assay. Left panels: quantification of mycelium growth of P. infestans represented as inhibition percentages. The bars show averages (n = 3 independent assays with 3–4 technical replicates) with error bars indicating standard error of the mean. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s honestly significant difference (HSD) (threshold p < 0 05) Ri ht l t ti i f d l di t th h th l ft Figure 1. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on Phytophthora infestans mycelium growth. (a,b) Volatile compound-mediated dual assay; (c,d) diffusible compound-mediated dual assay. Left panels: quantification of mycelium growth of P. infestans represented as inhibition percentages. The bars show averages (n = 3 independent assays with 3–4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s honestly significant difference (HSD) (threshold p < 0.05); Right panels: representative images of dual assays corresponding to the graphs on the left. Figure 1. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on Phytophthora infestans myceliu growth. (a,b) Volatile compound-mediated dual assay; (c,d) diffusible compound-mediated du assay. Left panels: quantification of mycelium growth of P. infestans represented as inhibitio percentages. The bars show averages (n = 3 independent assays with 3–4 technical replicates) wi error bars indicating standard error of the mean. Letters indicate significant differences betwee treatments as per ANOVA followed by Tukey’s honestly significant difference (HSD) (threshold p Figure 1. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on Phytophthora infestans mycelium growth. (a,b) Volatile compound-mediated dual assay; (c,d) diffusible compound-mediated dual assay. Left panels: quantification of mycelium growth of P. infestans represented as inhibition percentages. The bars show averages (n = 3 independent assays with 3–4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s honestly significant difference (HSD) (threshold p < 0.05); Right panels: representative images of dual assays corresponding to the graphs on the left. 0.05); Right panels: representative images of dual assays corresponding to 3.2. Contribution of HCN to the Inhibition of P. infestans Zoospore Germination 0.05); Right panels: representative images of dual assays corresponding to 3.2. Contribution of HCN to the Inhibition of P. infestans Zoospore Germination 3.2. Contribution of HCN to the Inhibition of P. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth (a) Graph representing the inhibition percentages of germinated zoospores when exposed to different strains and control (unexposed to bacterial strains). The bars show averages (n = 3 independent assays with 3-4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). (b) Representative images of zoospores after 3 h incubation with or without bacterial strains at room temperature. Scale bar = 100 μm. Figure 2. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on zoospore germination. (a) Graph representing the inhibition percentages of germinated zoospores when exposed to different strains and control (unexposed to bacterial strains). The bars show averages (n = 3 independent assays with 3–4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). (b) Representative images of zoospores after 3 h incubation with or without bacterial strains at room temperature. Scale bar = 100 µm. 3 3 Contribution of HCN to the Inhibition of Infection by P infestans on Potato Leaf Disks 3.3. Contribution of HCN to the Inhibition of Infection by P. infestans on Potato Leaf Disks f f f y f Following the in vitro experiments which showed that HCN only accounts for a small portion of the anti-oomycete activity of Pseudomonas strains in direct contact-mediated assays (diffusible compound-mediated assay and zoospore germination experiment), we performed leaf disk experiments to quantify its relative contribution in the strains’ anti-Phytophthora activity in planta. Visual inspection of the leaf disks revealed that both R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains led to comparable levels of P. infestans infection on leaf disks, which were significantly lower than in the non-inoculated controls (Figure 3a). To quantify the infection level of P. infestans in plant tissues, we performed fatty acid methyl esters analysis of a P. infestans-specific fatty acid, the eicosapentaenoic acid (EPA; C20:5) [25]. Control leaf disks contained an average of 72 μg of EPA per sample, while leaf disks inoculated with either R47 wt, R47 ∆hcn, R32 wt, or R32 ∆hcn showed an average of 15, 18, 24, and 10 μg of EPA per sample, respectively (Figure 3b). No phytotoxic effect was observed when the bacterial strains were inoculated alone (without P. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth infestans Zoospore Germination Exposing harvested zoospores to cyanogenic wild types and their HCN-negative mutan revealed that both mutants retained the ability to significantly inhibit the germination of zoospor as compared to the control. The inhibition percentage upon exposure to R47 decreased from 88% f wt to 71% for the mutant, while the ∆hcn mutant strain of R32 caused even stronger inhibition Exposing harvested zoospores to cyanogenic wild types and their HCN-negative mutants revealed that both mutants retained the ability to significantly inhibit the germination of zoospores as compared to the control. The inhibition percentage upon exposure to R47 decreased from 88% for wt to 71% for the mutant, while the ∆hcn mutant strain of R32 caused even stronger inhibition of zoospore germination compared to its wt counterpart. Exposure to R32 wt led to 70% inhibition of zoospores 6 of 10 Microorganisms 2020, 8, 1144 germination while exposure to R32 ∆hcn increased the inhibition percentage to 90% (Figure 2a). Representative images for the zoospores germination experiment are shown in Figure 2b. Microorganisms 2020, 8, x FOR PEER REVIEW 6 of 10 Figure 2. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on zoospore germination. (a) Graph representing the inhibition percentages of germinated zoospores when exposed to different strains and control (unexposed to bacterial strains). The bars show averages (n = 3 independent assays with 3-4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). (b) Representative images of zoospores after 3 h incubation with or without bacterial strains at room temperature. Scale bar = 100 μm. Figure 2. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on zoospore germination. (a) Graph representing the inhibition percentages of germinated zoospores when exposed to different strains and control (unexposed to bacterial strains). The bars show averages (n = 3 independent assays with 3–4 technical replicates) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). (b) Representative images of zoospores after 3 h incubation with or without bacterial strains at room temperature. Scale bar = 100 µm. Figure 2. Effect of R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains on zoospore germination. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth infestans) on the leaf disks l Following the in vitro experiments which showed that HCN only accounts for a small portion of the anti-oomycete activity of Pseudomonas strains in direct contact-mediated assays (diffusible compound-mediated assay and zoospore germination experiment), we performed leaf disk experiments to quantify its relative contribution in the strains’ anti-Phytophthora activity in planta. Visual inspection of the leaf disks revealed that both R47 (wt and ∆hcn) and R32 (wt and ∆hcn) strains led to comparable levels of P. infestans infection on leaf disks, which were significantly lower than in the non-inoculated controls (Figure 3a). To quantify the infection level of P. infestans in plant tissues, we performed fatty acid methyl esters analysis of a P. infestans-specific fatty acid, the eicosapentaenoic acid (EPA; C20:5) [25]. Control leaf disks contained an average of 72 µg of EPA per sample, while leaf disks inoculated with either R47 wt, R47 ∆hcn, R32 wt, or R32 ∆hcn showed an average of 15, 18, 24, and 10 µg of EPA per sample, respectively (Figure 3b). No phytotoxic effect was observed when the bacterial strains were inoculated alone (without P. infestans) on the leaf disks (Supplementary Figure S2). 7 of 10 Microorganisms 2020, 8, 1144 Figure 3. Pseudomonas strains (R32 and R47) restrict the infection by P. infestans on leaf disks. (a) Left panel: representative images of leaf disks co-inoculated with Pseudomonas strains and P. infestans zoospores. Pictures were taken 6 days post-inoculation. Right panel: zoomed images of leaf disks showing P. infestans infection (white hyphal mat). (b) Quantification of infection by measuring P. infestans-specific fatty acid (EPA; C20:5) content in leaf tissues. The bars show averages (n = 3) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). Figure 3. Pseudomonas strains (R32 and R47) restrict the infection by P. infestans on leaf disks. (a) Left panel: representative images of leaf disks co-inoculated with Pseudomonas strains and P. infestans zoospores. Pictures were taken 6 days post-inoculation. Right panel: zoomed images of leaf disks showing P. infestans infection (white hyphal mat). (b) Quantification of infection by measuring P. infestans-specific fatty acid (EPA; C20:5) content in leaf tissues. The bars show averages (n = 3) with error bars indicating standard error of the mean. ea e 4. Discussion 4. Discussion Even after more than 170 years since the most infamous plant epidemic in Europe, the Irish potato famine, its causative agent P. infestans continues to challenge us with severe outbreaks overcoming our attempts to control the disease [26,27]. Pseudomonas bacteria, which are known for their widespread distribution across habitats, their catabolic versatility, and their abilities to efficiently colonize roots as well as to produce a variety of anti-microbial compounds, present promising possibilities as biocontrol agents [28]. Morrison and colleagues showed in 2017 that P. fluorescens LBUM636 could restrict P. infestans infection of potato tubers [29]. Similarly, promising results were obtained in our previous studies exploring the potential of Pseudomonas strains isolated from the potato microbiome for late blight control [6,21,22,30,31]. We have recently identified some candidate genes putatively involved in this anti-Phytophthora activity, among which those encoding production of the respiratory toxin HCN [20]. Here, we verified the involvement of this known particular biocontrol trait and quantified its relative contribution to the overall anti-oomycete activity of two cyanogenic Pseudomonas using in vitro and leaf disk experiments. In our volatile-mediated dual assays, R32 wild type restricted the mycelial growth by 84% and R47 wild type by 90% compared to the unexposed P. infestans control. In contrast, R32 ∆hcn showed only 4% growth inhibition while R47 ∆hcn restricted the P. infestans mycelial growth by 33% (Figure 1a,b). This suggests that HCN might be the sole anti-oomycete volatile emitted by R32 on V8 medium, or that other active compounds might be produced in too low concentrations on this medium to lead to observable inhibition. In the case of R47, our result suggests that HCN is not the only volatile compound active against P. infestans but that it plays a major role. Although we did not investigate the entire volatilome of R47 grown on V8, we might speculate that the remaining inhibition activity could be due to volatile Even after more than 170 years since the most infamous plant epidemic in Europe, the Irish potato famine, its causative agent P. infestans continues to challenge us with severe outbreaks overcoming our attempts to control the disease [26,27]. Pseudomonas bacteria, which are known for their widespread distribution across habitats, their catabolic versatility, and their abilities to efficiently colonize roots as well as to produce a variety of anti-microbial compounds, present promising possibilities as biocontrol agents [28]. Morrison and colleagues showed in 2017 that P. 3.1. Contribution of HCN to the Inhibition of P. infestans Mycelium Growth Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). Figure 3. Pseudomonas strains (R32 and R47) restrict the infection by P. infestans on leaf disks. (a) Left panel: representative images of leaf disks co-inoculated with Pseudomonas strains and P. infestans zoospores. Pictures were taken 6 days post-inoculation. Right panel: zoomed images of leaf disks showing P. infestans infection (white hyphal mat). (b) Quantification of infection by measuring P. infestans-specific fatty acid (EPA; C20:5) content in leaf tissues. The bars show averages (n = 3) with error bars indicating standard error of the mean. Letters indicate significant differences between t t t ANOVA f ll d b T k ’ HSD (th h ld < 0 05) Figure 3. Pseudomonas strains (R32 and R47) restrict the infection by P. infestans on leaf disks. (a) Left panel: representative images of leaf disks co-inoculated with Pseudomonas strains and P. infestans zoospores. Pictures were taken 6 days post-inoculation. Right panel: zoomed images of leaf disks showing P. infestans infection (white hyphal mat). (b) Quantification of infection by measuring P. infestans-specific fatty acid (EPA; C20:5) content in leaf tissues. The bars show averages (n = 3) with error bars indicating standard error of the mean. Letters indicate significant differences between treatments as per ANOVA followed by Tukey’s HSD (threshold p < 0.05). ea e 4. Discussion infestans infection on potato tubers [29], and the siderophore pyoverdine could also play an important role, as competition for iron is a well-known mechanism by which bacteria inhibit the growth of fungal pathogens [32]. S-methyl methanethiosulfonate, acetophenone, or 2-hexanone, which were previously shown to be produced by R47 and to have anti-Phytophthora activities [6,25,30]. When diffusible compounds were also allowed to participate in the P. infestans–Pseudomonas warfare, experiments showed no loss of activity of R47 ∆hcn compared to its wild ype (98 vs. 99% inhibition respectively, Figure 1c), and only moderate loss of activity (from 98 to 80%) for the mutant of R32 (Figure 1d). This finding reveals the presence of other determinants of mycelial growth inhibition in both strains, and of very strong one(s) in the case of R47. Based on the strains’ genome content [20], possible candidate molecules explaining the remaining inhibitory activity of the cyanide deficient mutants are e.g., pyoverdine, which is encoded in the genome of both strains, and phenazine, which is encoded only in R47 [20]. Phenazines have been previously shown to inhibit P. infestans infection on potato tubers [29], and the siderophore pyoverdine could also play an important role, as competition for iron is a well-known mechanism by which bacteria inhibit the growth of fungal pathogens [32]. Beyond mycelia, zoospores are an important life form of P. infestans that is of high relevance for infection. Our results indicated that more efficient compounds than HCN were likely involved in this process. Interestingly, the cyanide-deficient mutant inhibited zoospore germination more efficiently than the wild type (Figure 2a). This could have been caused by better growth due to the absence of the fitness cost generated by HCN biosynthesis, but the growth curves generated indicated that the deletion of hcnABC genes did not provide the mutant strains with growth advantages on V8 medium (Figure S3). Our current hypothesis is that independently of growth rate, strains lacking HCN might have compensated for the loss of this important weapon by increasing the production of other compounds, leading to slightly but significantly higher activity than the wild type in the P. putida R32 strain. Such a phenomenon could also explain the altered mycelium phenotype of P. infestans exposed to the R32 cyanide deficient mutant observed in both our dual assays (Figure 1b,d). This putative effect, which we are currently investigating, was however not observed in P. ea e 4. Discussion fluorescens LBUM636 could restrict P. infestans infection of potato tubers [29]. Similarly, promising results were obtained in our previous studies exploring the potential of Pseudomonas strains isolated from the potato microbiome for late blight control [6,21,22,30,31]. We have recently identified some candidate genes putatively involved in this anti-Phytophthora activity, among which those encoding production of the respiratory toxin HCN [20]. Here, we verified the involvement of this known particular biocontrol trait and quantified its relative contribution to the overall anti-oomycete activity of two cyanogenic Pseudomonas using in vitro and leaf disk experiments. In our volatile-mediated dual assays, R32 wild type restricted the mycelial growth by 84% and R47 wild type by 90% compared to the unexposed P. infestans control. In contrast, R32 ∆hcn showed only 4% growth inhibition while R47 ∆hcn restricted the P. infestans mycelial growth by 33% (Figure 1a,b). This suggests that HCN might be the sole anti-oomycete volatile emitted by R32 on V8 medium, or that other active compounds might be produced in too low concentrations on this medium to lead to observable inhibition. In the case of R47, our result suggests that HCN is not the only volatile compound active against P. infestans but that it plays a major role. Although we did not investigate the entire volatilome of R47 grown on V8, we might speculate that the remaining inhibition activity could be due to volatile compounds such as 1-undecene, dimethyl disulfide, dimethyl trisulfide, 8 of 10 Microorganisms 2020, 8, 1144 S-methyl methanethiosulfonate, acetophenone, or 2-hexanone, which were previously shown to be produced by R47 and to have anti-Phytophthora activities [6,25,30]. When diffusible compounds were also allowed to participate in the P. infestans–Pseudomonas warfare, experiments showed no loss of activity of R47 ∆hcn compared to its wild ype (98 vs. 99% inhibition respectively, Figure 1c), and only moderate loss of activity (from 98 to 80%) for the mutant of R32 (Figure 1d). This finding reveals the presence of other determinants of mycelial growth inhibition in both strains, and of very strong one(s) in the case of R47. Based on the strains’ genome content [20], possible candidate molecules explaining the remaining inhibitory activity of the cyanide deficient mutants are e.g., pyoverdine, which is encoded in the genome of both strains, and phenazine, which is encoded only in R47 [20]. Phenazines have been previously shown to inhibit P. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/8/1144/s1, Figure S1: Cyanide detection assay, Figure S2: Representative images from potato leaf disc experiments where only Pseudomonas strains R47 (wt and ∆hcn) and R32 (wt and ∆hcn) were inoculated. Figure S3: Growth curves for R47 (wt and ∆hcn) and R32 (wt and ∆hcn). Table S1: Primers used in this study, Table S2: Bacterial strains and plasmids used in this study. ea e 4. Discussion chlororaphis R47, where the cyanide-deficient mutant exhibited slightly but significantly lower inhibiting activity than the wild type, suggesting minor contribution of HCN to the inhibition of zoospore germination (Figure 2a). Leaf disk infection assays showed that both ∆hcn strains reduced P. infestans infection on plant tissues to a similar extent as their corresponding wild types (Figure 3). A similar trend to higher efficacy of the mutant of P. putida R32 compared to the wild type could be observed, although this was not significant (Figure 3b). Since zoospores were used to infect leaf tissues, it might indicate that the mode of action of these bacterial strains lies in direct inhibition of the pathogen rather than in inducing defense-related genes in the plant host. In conclusion, our experiments show that HCN is the major (in R47) or only (in R32) contributor to the inhibition of P. infestans mycelial growth in volatile mediated experiments but that it is not involved in the inhibition of P. infestans by the biocontrol strains when they are in direct contact with the pathogen. However, this does not mean that the ability to emit HCN is not an important biocontrol trait in other experimental setups or in other types of plant–pathogen interactions. More importantly, our results highlight the occurrence of other, yet-unknown efficient compounds of great interest in the fight against P. infestans in the relatively small genome of P. putida R32 (ca 5.6 MB), which we aim to identify in the near future using a mutant bank approach. Moreover, the indication of higher activity or altered phenotype observed in the cyanide deficient mutant of this strain suggests that the presence of cyanogenic strains e.g., in synthetic communities designed as biocontrol consortia, might alter the metabolic potential of such consortia in a previously unsuspected way, an aspect that also deserves further investigation. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/8/1144/s1, Figure S1: Cyanide detection assay, Figure S2: Representative images from potato leaf disc experiments where only Pseudomonas strains R47 (wt and ∆hcn) and R32 (wt and ∆hcn) were inoculated. Figure S3: Growth curves for R47 (wt and ∆hcn) and R32 (wt and ∆hcn). Table S1: Primers used in this study, Table S2: Bacterial strains and plasmids used in this study. ea e 4. Discussion 9 of 10 Microorganisms 2020, 8, 1144 Author Contributions: Conceptualization, L.W.; formal analysis, A.A., C.T., and L.M.-S.; investigation, A.A., D.C., C.T., and L.M.-S.; resources, F.L.; writing—original draft preparation, A.A. and L.W.; writing—review and editing, A.A., D.C., C.T., L.M.-S., F.L., and L.W.; visualization, A.A.; supervision, L.W., D.C., and F.L.; project administration, L.W.; funding acquisition, L.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Swiss National Science Foundation, grant numbers 31003A_179310 and 316030_177093. ding: This research was funded by the Swiss National Science Foundation, grant numbers 31003A_179310 316030_177093. Acknowledgments: We would like to sincerely thank Christoph Keel and Maria Péchy-Tarr for sharing their expertise and providing tools and methods for generating the mutants. We are thankful to Fiona Invernizzi for technical help and to Mout De Vrieze for sharing expertise on P. infestans biology. 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 y, y p f p ( g ) y , , [ ] 2. Haverkort, A.J.; Boonekamp, P.M.; Hutten, R.; Jacobsen, E.; Lotz, L.A.P.; Kessel, G.J.T.; Visser, R.G.F.; Van Der Vossen, E.A.G. Societal costs of late blight in potato and prospects of durable resistance through cisgenic modification. Potato Res. 2008, 51, 47–57. [CrossRef] 3. Cooke, L.R.; Schepers, H.T.A.M.; Hermansen, A.; Bain, R.A.; Bradshaw, N.J.; Ritchie, F.; Shaw, D.S.; Evenhuis, A.; Kessel, G.J.T.; Wander, J.G.N.; et al. Epidemiology and Integrated Control of Potato Late Blight in Europe. Potato Res. 2011, 54, 183–222. [CrossRef] 4. Axel, C.; Zannini, E.; Coffey, A.; Guo, J.; Waters, D.M.; Arendt, E.K. Ecofriendly control of potato late blight causative agent and the potential role of lactic acid bacteria: A review. Appl. Microbiol. Biotechnol. 2012, 96, 37–48. [CrossRef] 5. Velivelli, S.L.S.; De Vos, P.; Kromann, P.; Declerck, S.; Prestwich, B.D. Biological control agents: From field to market, problems, and challenges. Trends Biotechnol. 2014, 32, 493–496. [CrossRef] [PubMed] 6. Hunziker, L.; Bönisch, D.; Groenhagen, U.; Bailly, A.; Schulz, S.; Weisskopf, L. Pseudomonas strains naturally associated with potato plants produce volatiles with high potential for inhibition of Phytophthora infestans. Appl. Environ. Microbiol. 2015, 81, 821–830. [CrossRef] [PubMed] 7. Haas, D.; Défago, G. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat. Rev. Microbiol. 2005, 3, 307–319. [CrossRef] 8. Siddiqui, I.A.; Shaukat, S.S.; Sheikh, I.H.; Khan, A. Role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of root-knot nematode, Meloidogyne javanica in tomato. World J. Microbiol. Biotechnol. 2006, 22, 641–650. [CrossRef] 9. Kang, B.R.; Anderson, A.J.; Kim, Y.C. Hydrogen cyanide produced by Pseudomonas chlororaphis O6 is a key aphicidal metabolite. Can. J. Microbiol. 2019. [CrossRef] 10. Flury, P.; Vesga, P.; Péchy-Tarr, M.; Aellen, N.; Dennert, F.; Hofer, N.; Kupferschmied, K.P.; Kupferschmied, P.; Metla, Z.; Ma, Z.; et al. Antimicrobial and insecticidal: Cyclic lipopeptides and hydrogen cyanide produced by plant-beneficial Pseudomonas strains CHA0, CMR12a and PCL1391 contribute to insect killing. Front. Microbiol. 2017, 8, 100. [CrossRef] 11. Kang, B.R.; Anderson, A.J.; Kim, Y.C. Hydrogen cyanide produced by Pseudomonas chlororaphis O6 exhibits nematicidal activity against meloidogyne hapla. Plant. Pathol. J. 2018. [CrossRef] 12. Stutz, E.W. Naturally Occurring Fluorescent Pseudomonads Involved in Suppression of Black Root Rot of Tobacco. Phytopathology 1986. [CrossRef] 3. Ahl, P.; Voisard, C.; Défago, G. Iron Bound-Siderophores, Cyanic Acid, and Antibiotics Involved Suppression of Thielaviopsis basicola by a Pseudomonas fluorescens Strain. J. Phytopathol. 1986. References Martínez-García, E.; de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: Analysis of the multi resistant antibiotic profile of Pseudomonas putida KT2440 Environ Microbiol 2011 , ; , ; , ; p , g Strains for Improved Biocontrol of Phytophthora infestans. Front. Microbiol. 2018, 9, 1–13. [CrossRef] [PubMed] 23. Martínez-García, E.; de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: Analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 2011. [CrossRef] [PubMed] 23. Martínez-García, E.; de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: Analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 2011. [CrossRef] [PubMed] 24. Cernava, T.; Aschenbrenner, I.A.; Grube, M.; Liebminger, S.; Berg, G. A novel assay for the detection of bioactive volatiles evaluated by screening of lichen-associated bacteria. Front. Microbiol. 2015, 6, 1–9. [CrossRef] [PubMed] 25. Chinchilla, D.; Bruisson, S.; Meyer, S.; Zühlke, D.; Hirschfeld, C.; Joller, C.; L’Haridon, F.; Mène-Saffrané, L.; Riedel, K.; Weisskopf, L. A sulfur-containing volatile emitted by potato-associated bacteria confers protection against late blight through direct anti-oomycete activity. Sci. Rep. 2019, 9, 1–15. [CrossRef] [PubMed] 26. Fry, W.E. Phytophthora infestans: New Tools (and Old Ones) Lead to New Understanding and Precision Management. Annu. Rev. Phytopathol. 2016, 54, 529–547. [CrossRef] 27. Grünwald, N.J.; Flier, W.G. The Biology of Phytophthora infestans at Its Center of Origin. Annu. Rev. Phytopathol. 2005, 43, 171–190. [CrossRef] Walsh, U.F.; Morrissey, J.P.; O’Gara, F. Pseudomonas for biocontrol of phytopathogens: From functiona genomics to commercial exploitation. Curr. Opin. Biotechnol. 2001, 12, 289–295. [CrossRef] 29. Morrison, C.K.; Arseneault, T.; Novinscak, A.; Filion, M. Phenazine-1-carboxylic acid production by Pseudomonas fluorescens LBUM636 alters Phytophthora infestans’ growth and late blight development. Phytopathology 2016, 107, 273–279. [CrossRef] 30. De Vrieze, M.; Pandey, P.; Bucheli, T.D.; Varadarajan, A.R.; Ahrens, C.H.; Weisskopf, L.; Bailly, A. Volatile organic compounds from native potato-associated Pseudomonas as potential anti-oomycete agents. Front. Microbiol. 2015, 6, 1295. [CrossRef] 31. De Vrieze, M.; Gloor, R.; Massana Codina, J.; Torriani, S.; Gindro, K.; L’Haridon, F.; Bailly, A.; Weisskopf, L. Biocontrol activity of three Pseudomonas on a newly assembled collection of Phytophthora infestans isolates. Phytopathology 2019, 109, 1555–1565. [CrossRef] [PubMed] 32. Kloepper, J.W.; Leongt, J.; Teintzet, M.; Schroth, M.N. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 1980, 286, 885–886. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. References [CrossRe Suppression of Thielaviopsis basicola by a Pseudomonas fluorescens Strain. J. Phytopathol. 1986. [CrossRef] 14. Laville, J.; Blumer, C.; Von Schroetter, C.; Gaia, V.; Défago, G.; Keel, C.; Haas, D. Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0. J. Bacteriol. 1998, 180, 3187–3196. [CrossRef] [PubMed] 15. Zdor, R.E. Bacterial cyanogenesis: Impact on biotic interactions. J. Appl. Microbiol. 2015, 118, 267–274. [CrossRef] 14. Laville, J.; Blumer, C.; Von Schroetter, C.; Gaia, V.; Défago, G.; Keel, C.; Haas, D. Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0. J. Bacteriol. 1998, 180, 3187–3196. [CrossRef] [PubMed] g 15. Zdor, R.E. Bacterial cyanogenesis: Impact on biotic interactions. J. Appl. Microbiol. 2015, 118, 267–274. [C 16. Bröckner, A.; Raspotnig, G.; Wehner, K.; Meusinger, R.; Norton, R.A.; Heethoff, M. Storage and release of hydrogen cyanide in a chelicerate (Oribatula tibialis). Proc. Natl. Acad. Sci. USA 2017. [CrossRef] 10 of 10 Microorganisms 2020, 8, 1144 17. Frangipani, E.; Pérez-Martínez, I.; Williams, H.D.; Cherbuin, G.; Haas, D. A novel cyanide-inducible gene cluster helps protect Pseudomonas aeruginosa from cyanide. Environ. Microbiol. Rep. 2014. [CrossRef] 18. Cunningham, L.; Pitt, M.; Williams, H.D. The cioAB genes from Pseudomonas aeruginosa code for a novel cyanide-insensitive terminal oxidase related to the cytochrome bd quinol oxidases. Mol. Microbiol. 1997. [CrossRef] 19. Cipollone, R.; Frangipani, E.; Tiburzi, F.; Imperi, F.; Ascenzi, P.; Visca, P. Involvement of Pseudomonas aeruginosa rhodanese in protection from cyanide toxicity. Appl. Environ. Microbiol. 2007. [CrossRef] 20. De Vrieze, M.; Varadarajan, A.R.; Schneeberger, K.; Bailly, A.; Ahrens, C.H.; Weisskopf, L. Linking comparative genomics of nine potato-associated Pseudomonas isolates with their differing biocontrol potential against late blight. Front. Microbiol. 2020, 11, 857. [CrossRef] 21. Guyer, A.; De Vrieze, M.; Bönisch, D.; Gloor, R.; Musa, T.; Bodenhausen, N.; Bailly, A.; Weisskopf, L. The anti-phytophthora effect of selected potato-associated Pseudomonas strains: From the laboratory to the field. Front. Microbiol. 2015, 6, 1–13. [CrossRef] [PubMed] 22. De Vrieze, M.; Germanier, F.; Vuille, N.; Weisskopf, L. Combining Different Potato-Associated Pseudomonas Strains for Improved Biocontrol of Phytophthora infestans. Front. Microbiol. 2018, 9, 1–13. [CrossRef] [PubMed] 22. De Vrieze, M.; Germanier, F.; Vuille, N.; Weisskopf, L. Combining Different Potato-Associated Pseudomonas Strains for Improved Biocontrol of Phytophthora infestans. Front. Microbiol. 2018, 9, 1–13. [CrossRef] [PubMed] 23. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4214950780
https://bmcinfectdis.biomedcentral.com/track/pdf/10.1186/s12879-020-05712-1
English
null
Trend and treatment outcomes of latent tuberculosis infection among migrant persons in Japan: analysis of Japan Tuberculosis Surveillance data
Research Square (Research Square)
2,020
cc-by
7,377
Kawatsu et al. BMC Infectious Diseases (2021) 21:42 https://doi.org/10.1186/s12879-020-05712-1 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 https://doi.org/10.1186/s12879-020-05712-1 Open Access Trend and treatment outcomes of latent tuberculosis infection among migrant persons in Japan: retrospective analysis of Japan tuberculosis surveillance data Lisa Kawatsu* , Kazuhiro Uchimura and Akihiro Ohkado * Correspondence: kawatsu@jata.or.jp Department of Epidemiology and Clinical Research, The Research Institute of Tuberculosis, 3-1-24, Matsuyama Kiyose, Tokyo, Japan Abstract Keywords: Latent tuberculosis infection, Migrants, Trend, Treatment outcome * Correspondence: kawatsu@jata.or.jp Department of Epidemiology and Clinical Research, The Research Institute of Tuberculosis, 3-1-24, Matsuyama Kiyose, Tokyo, Japan * Correspondence: kawatsu@jata.or.jp Department of Epidemiology and Clinical Research, The Research Institute of Tuberculosis, 3-1-24, Matsuyama Kiyose, Tokyo, Japan Abstract Background: Screening for latent tuberculosis infection (LTBI) among migrant population has become a critical issue for many low tuberculosis (TB) burden countries. Evidence regarding effectiveness of LTBI programs are limited, however, partly because of paucity of national data on treatment outcomes for LTBI. In Japan, notification of LTBI is mandatory, and its treatment outcome is reported as part of Japan’s national TB surveillance system. We thus conducted a detailed analysis of LTBI among foreign-born persons, to update the epidemiological trend of newly notified LTBI between 2007 and 2018, and to examine the treatment regimen and outcome of those notified in 2016 and 2017, focusing specifically on the potential risk factors for lost to follow-up. Methods: We extracted and analyzed the data of newly notified LTBI patients from the Japan Tuberculosis Surveillance System to examine the overall trend of notification and by age groups and modes of detection between 2007 and 2018, and the cohort data for treatment regimen and outcomes of foreign-born persons notified with LTBI in 2016 and 2017. Trends and proportions were summarized descriptively, and logistic regression analysis was conducted to identify potential risk factors for lost to follow-up. Comparisons were made with the Japan-born patients where appropriate, using chi-squared tests. Results: Both the number and proportion of LTBI among foreign-born persons have been constantly increasing, reaching 963 cases in 2018. Cohort analysis of the surveillance data indicated that the proportion of those on shorter regimen was higher among the foreign- than Japan-born patients (5.5% vs. 1.8%, p < 0.001). The proportion of those who have been lost to follow-up and transferred outside of Japan combined was higher among the foreign- than Japan-born patients (12.0% vs, 8.2%, p < 0.001). Risk factors for lost to follow-up were being employed on a temporal basis, and job status unknown (adjusted odds ratios 3.11 and 4.09, 95% confidence intervals 1.34– 7.26 and 1.60–10.48, respectively). Conclusions: Migrant population face greater risk of interrupting LTBI treatment, and interventions to improve adherence are a critical component of programmatic management of LTBI. Further studies are needed to explore the cultural and socioeconomic situation in which foreign-born persons undergo LTBI treatment in Japan. © 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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 2 of 11 Page 2 of 11 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Kawatsu et al. BMC Infectious Diseases Background possible for all types of TB, including extra-pulmonary TB and LTBI, for notifications from 2017 and onward. We thus conducted a detailed analysis of LTBI among foreign-born persons, with two main objectives; firstly to provide a detailed description of the epidemiological trend of newly notified LTBI between 2007 and 2018, and secondly, to examine the treatment regimen and outcome of those notified in 2016 and 2017, focusing specifically on the potential risk factors for lost to follow-up among foreign-born LTBI patients, with the ultimate goal of providing epidemiological evidence which can be used to guide decision-making on screen- ing and testing policies for LTBI among foreign-born persons in Japan. g Foreign-born populations contribute considerably to both the number and proportion of all tuberculosis (TB) cases, especially in countries with low TB incidence [1– 3]. In Japan as well, which is a TB middle-burden coun- try with a notification rate of 12.3 per 100,000 in 2018, although the proportion of foreign-born persons out of all TB cases was 9.5%, that among those aged between 15 and 24 years old has reached 70.8% in the same year [4]. It has been pointed out that the majority of these TB cases occurs due to the reactivation of latent tuber- culosis infection (LTBI) acquired in their country of ori- gin. As a large proportion of migrants is born in high- TB burden countries, where it has been estimated that approximately 26 to 46% of the population are latently infected with TB [5, 6], screening for not only active TB but also LTBI among the migrants, has become a critical issue for many low TB burden countries (< 10 cases/100, 000 population). In Japan as well, 58.2% of foreign-born TB patients come from 5 countries in Southeast Asia, with a TB incidence of more than 100 per 100,000 popu- lation [4]. Study population and data source In order to examine the overall trend of notification and by age groups and modes of detection, we extracted the data of LTBI patients newly notified between 2007 and 2018, from the “newly notified dataset”. In order to analyze the treatment regimen and treatment outcomes, we extracted the cohort data of foreign-born LTBI patient who had been notified between 2016 and 2017 from the “cohort dataset”. y In Japan, however, when a physician diagnoses LTBI and determines that treatment is necessary, he or she is required to notify the local public health center, just as is the case with active TB, under the Infectious Diseases Control Law. Once notified, treatment outcome is evalu- ated and reported as part of Japan’s national TB surveil- lance system (JTBS) [4]. There is no national guideline that indicates clear priorities for targeted LTBI screening, however, approximately 60% of notified LTBI patients are detected via contact investigation [4]. Treatment is strongly recommended for selected populations consid- ered to be at a higher risk of developing TB, including people living with HIV/AIDS, silicosis, people on treat- ment with immunosuppressants, and those who have been recently infected [8]. Methods Japan introduced its first electronic surveillance system in 1987, which since then has undergone several major system revisions. Details of the system can be found elsewhere [4]. Briefly, at the end of each year, it pro- duces four sets of data; 1) a list of all active TB and LTBI cases newly notified in that year (“newly notified data- set”), 2) a list of all active TB and LTBI cases registered at the end of the year (“end of year dataset”), 3) a list of active TB and LTBI cases notified in the previous year, with treatment outcomes (“cohort dataset”), and 4) a list of all cases who were de-registered in that year (“de-reg- istered dataset”). Yet in its recently revised guideline, the World Health Organization (WHO) has only conditionally recom- mended LTBI screening among migrants living in low TB burden countries [7]. The guideline points to the low-quality evidence for the effectiveness and cost- effectiveness of LTBI programs in these settings, includ- ing challenges in achieving treatment adherence and completion rate. Studies reporting national data on treatment outcomes for LTBI are however limited, due mainly to LTBI not requiring mandatory notification in many countries. Trend in LTBI notification Figure 1 shows the annual trend of the number of over- all LTBI notifications by country of birth, and the pro- portion of foreign-born persons among the total cases. The number of total LTBI notifications has reached a peak in 2011, and since then it has declined and in the past five years or so, has stabilized around 7000 a year. However, both the number and the proportion of foreign-born LTBI patients have increased steadily, reaching 963 cases in 2018 (13.0%, 963/7414). Looking at the trend in the notification by age groups, while among the Japan-born patients, the number of notifica- tions has increased only among those aged 55 years old and above, among the foreign-born patients, the number has increased rapidly among all age groups except those aged between 0 and 14 years old (Fig. 2). Regarding the modes of detection, for both Japan and foreign-born pa- tients, the majority were detected via contact investiga- tion over the study period (foreign-born; 80.3%, and Japan-born; 72.3%). However, while among the Japan- born, only the number of those detected at hospital has increased, among the foreign-born, the numbers of those detected via contact investigation, routine screening, and at hospital all similarly expressed steady increase (Fig. 3). Treatment outcomes of those who have started treat- ment, i.e. excluding those whose treatment regimen was recorded as “no treatment”, are summarized in Table 2. The overall treatment success was slightly higher among the Japan-born than foreign-born patients (85.8% vs. 82.2%, p < 0.001). While the proportion of lost to follow- up (8.2% vs. 8.4%, p = 0.78) were similar among the Japan-and foreign-born patients, the proportion of transferred-out was much higher among the foreign- than Japan-born patients (8.3% vs. 1.5%, p < 0.001). How- ever, the “transfer-out” refers to a move outside the jur- isdiction of the public health center to where the patient was initially notified, including both a move to another jurisdiction within Japan, and a move to another coun- try. A closer analysis has revealed that of patients who have transferred-out, while among the Japan-born, 94.8% (183/193) were transfer-out within Japan, among the foreign-born, 43.0% (50/116) had transferred out of Japan. Trend in LTBI notification Considering that treatment outcome of those who have transferred outside of Japan is practically non- traceable, these were re-categorized as lost to follow-up – this had the effect of making lost to follow-up consid- erably higher among the foreign- than Japan-born pa- tients (12.0% vs, 8.2%, p < 0.001). Data analysis T d d Trends and proportions were summarized descriptively, and comparisons were made with the Japan-born pa- tients where appropriate, using chi-squared analysis. Treatment outcomes were analyzed for those who have started treatment, as according to the outcome variables of the JTBS i.e. “treatment success”, “died”, “treatment failed”, “lost to follow-up”, “transferred-out”, “still on treatment” and “unknown”. From the variables that are collected via the surveillance, potential reasons for lost to follow-up were further explored, and logistic regres- sion analysis was conducted to identify risk factors. The crude odds ratio (OR) and adjusted odds ratio (AOR) were reported, with their corresponding 95% confidence interval and p-values. The definitions for the main The authors have previously estimated the treatment outcomes of LTBI notified between 2007 and 2014 – the outcomes were however determined using proxy vari- ables because the JTBS then only evaluated treatment outcome for pulmonary TB [9]. In 2017, the JTBS underwent a system revision, making cohort analysis Page 3 of 11 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Kawatsu et al. BMC Infectious Diseases variables can be found in the Annex (S1 Appendix). R version 3.6.3 (R Development Core Team, Vienna, Austria) was used for all statistical analyses. variables can be found in the Annex (S1 Appendix). R version 3.6.3 (R Development Core Team, Vienna, Austria) was used for all statistical analyses. INH monotherapy, respectively. Higher proportion of foreign-born than Japan-born patients had started their treatment with RFP monotherapy (5.5% vs. 1.8%, p < 0.001) (Table 1). Risk of lost to follow-up within foreign-born patients From the “cohort dataset”, treatment regimen and out- come were available for 12,817 Japan-born and 1418 foreign-born LTBI patients. Of the 12,817 Japan-born and 1418 foreign-born LTBI patients, 96.0% (n = 12,298) and 92.3% (n = 1309) had started their treatment with p g p Proportions of lost to follow-up and those who have transferred out of Japan (n = 167), among various socio- demographic characteristics within foreign-born persons are summarized in Table 3. Fig. 1 Annual trend of LTBI notification by country of birth, and proportion of foreign-born patients, 2007–2018 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 4 of 11 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 4 of 11 Fig. 2 Trend of LTBI notification by country of birth and age groups, 2007–2018 The results of logistic regression analysis, whereby all of the above characteristics were entered as explanatory variables, are summarized in Table 4. the Japan-born patients, the rise was mainly seen among the older persons, which can be attributable largely due to the abolition of age limit for LTBI treatment in the national guideline in 2010 [11] and an increasing num- ber of elderly persons being tested for and diagnosed with LTBI before being treated for other medical condi- tions such as rheumatoid arthritis (i.e. hence LTBI being detected “at hospital”). On the other hand, among the foreign-born persons, the LTBI notifications increased in all age groups except those aged between 0 and 14 years old, and among all modes of detection. Among the Japan-born persons, “routine” screening for LTBI has only really been conducted at selected workplaces, such as among high-risk healthcare workers – however, the constant increase in the notification of LTBI among foreign-born persons via “routine screening” may indi- cate that increasingly, schools and workplaces are mak- ing individual decisions to introduce LTBI screening as part of the routine health-check for foreign-born per- sons. Indeed, there have been sporadic reports of LTBI screening being conducted as part of medical Variable which remained statistically significant at P- value of < 0.05 was being employed on a temporal basis, and job status unknown. Discussion Table 2 Treatment outcomes of LTBI patients (2016–2017), by county of birth Table 2 Treatment outcomes of LTBI patients (2016–2017), by county of birth Japan-born Foreign-born n % n % Treatment success 10,798 85.8 1142 82.2 Died 310 2.5 1 0.1 Failed 41 0.3 5 0.4 Lost to follow-up 1026 8.2 117 8.4 Transferred-out 193 1.5 116 8.3 within Japan 183 – 63 – outside Japan 8 – 50 – unknown 2 – 3 – Still on treatment 132 1.0 8 0.6 Unknown 82 0.7 2 0.1 TOTAL 12,582 100.0 1391 100.0 Lost to follow-up, recategorizeda 1034 8.2 167 12.0 aSum of Lost to follow-up and Transferred outside of Japan Table 1 Treatment regimen of LTBI patients upon registration (2016–2017) Japan-born Foreign-born n % n % INH monotherapy 12,298 96.0 1309 92.3 RFP monotherapy 225 1.8 78 5.5 No treatment 235 1.8 27 1.9 Othersa 59 0.5 4 0.3 TOTAL 12,817 100.0 1418 100.0 INH isoniazid, RFP rifampicin a“Others” consist of all other treatment categories, excluding “no treatment” – please see Annex (S1 Appendix) Table 1 Treatment regimen of LTBI patients upon registration (2016–2017) INH isoniazid, RFP rifampicin a“Others” consist of all other treatment categories, excluding “no treatment” – please see Annex (S1 Appendix) Kawatsu et al. Discussion Our results indicated that LTBI notifications among the foreign-born persons have continued to increase, al- though this is not surprising considering that the number of foreign-born persons from high TB-burden countries entering Japan have continued to increase [9]. It is certainly possible that foreign-born LTBI patients acquired their infection after immigrating to Japan, how- ever, a molecular-epidemiological study which examined the transmission dynamics of TB among foreign- and Japan-born patients in a large urban area in Japan has suggested otherwise, indicating that most of the foreign- born TB cases were attributable to reactivation of LTBI that were acquired in their home country [10]. Among Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 5 of 11 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Kawatsu et al. BMC Infectious Diseases Page 5 of 11 Fig. 3 Trend of LTBI notification by mode of detection, 2007–2018 examination upon admissions to universities [12, 13]. In the absence of a clear national guideline on priorities for LTBI among foreign-born persons in Japan, rigorous studies on effectiveness and cost-effectiveness are ur- gently needed to determine the most appropriate inter- vention for case finding of LTBI targeting foreign-born persons in Japan. Discussion Considering that LTBI treatment is publicly subsidized with minimum out-of- pocket payment, and is unlikely to place a significant fi- nancial burden for the patients, being employed on a daily or temporal basis may represent not necessarily poor economic condition but more an unstable lifestyle and greater mobility which are putting patients at risk of becoming lost to follow-up. The majority of the patients had started LTBI treat- ment with INH monotherapy. This is understandable considering that, although the WHO guideline recom- mends both INH monotherapy for 6 months for adults and children in countries of both low and high TB inci- dence, and RFP monotherapy for 3 to 4 months for both adults and children in countries with low TB incidence [7], the Japanese national guideline recommends INH monotherapy above RFP monotherapy [8]. As to why a higher proportion of foreign-born patients were started with RFP monotherapy – we were unable to find an ex- planation from our results. We may however speculate that physicians in Japan are more inclined to RFP mono- therapy because of their knowledge and awareness re- garding higher prevalence of INH resistance among foreign-born TB patients [4]. As for the issue of treatment adherence, to our know- ledge, this is the first detailed analysis of LTBI treatment outcome among foreign-born persons at a national level in Japan. Outside of Japan, several systematic reviews on adherence for and outcomes of LTBI treatment have been conducted – for example, a review that was con- ducted for the WHO 2015 LTBI Guideline has con- cluded that treatment completion rates varied across different risk groups, ranging from 6 to 94%, with lower completion rates for prisoners and immigrant [14]. An- other study which looked at studies from US and Canada has reported completion rates to be ranging be- tween 22 and 90% [15], and a more recent review re- ported rates between 7 to 86%, both for foreign-born person [16]. A meta-analysis that was published in 2016 has estimated the pooled treatment completion rates for migrants to be 14.3% [17]. The studies which were in- cluded in these systematic reviews do, however, vary considerably in terms of sample size, study design, types of immigrant and treatment regimen, and hence caution is required when interpreting the results. Discussion BMC Infectious Diseases (2021) 21:42 Page 7 of 11 Table 3 Proportions of “lost to follow-up” among various characteristics, foreign-born LTBI patients (n = 1391) (Continued) Total LTFU Transferred out of Japan LTFU + transferred out of Japan % of LTFU + transferred out of Japan Time between entry to Japan and diagnosis within 2 years 381 22 15 37 9.7 more than 2 years, within 5 years 222 18 15 33 14.9 more than 5 years, within 10 years 74 10 1 11 14.9 10 years or more 199 18 1 19 9.5 Unknown 515 49 18 67 13.0 INH isoniazid, RFP rifampicin, LTFU lost to follow-up to follow-up” among various characteristics, foreign-born LTBI patients (n = 1391) (Continued) Many studies have also examined the predictors for adherence to LTBI treatment, and in general, have tended to conclude that demographic factors such as age, sex, place of birth and race do not seem to influence completion rate [15]. In our study too, neither sex nor age were conclusive risk factors for lost to follow-up among foreign-born LTBI patients. However, as for age, although not statistically significant, there was a clear tendency for the risk to increase with age. This is quite understandable, as younger children are usually overseen by their guardians and are therefore less likely to be- come lost to follow-up, as well as for the fact that anti- tuberculosis drugs are unusually better tolerated at younger age. Other socio-economic factors, such as un- employment and lack of health insurance, have previ- ously been associated with failure to complete treatment [18, 19] – in our study, none of the foreign-born LTBI patients who had started treatment were reported as “non-insured”. Receiving social welfare assistance and having “others and unknown” for insurance status, which could include short-term visitors with private health insurance, were associated with elevated risk but were not statistically significant. As for job status, being employed on a daily or temporal basis, and having “others and unknown” for job status, were both associ- ated with being lost to follow-up. Discussion BMC Infectious Diseases (2021) 21:42 Page 6 of 11 Table 3 Proportions of “lost to follow-up” among various characteristics, foreign-born LTBI patients (n = 1391) Total LTFU Transferred out of Japan LTFU + transferred out of Japan % of LTFU + transferred out of Japan Sex Male 665 58 19 77 11.6 Female 726 59 31 90 12.4 Age groups (years) 0–14 49 3 2 5 10.2 15–19 61 4 1 5 8.2 20–29 630 47 21 68 10.8 30–39 302 29 17 46 15.2 40–49 195 21 6 27 13.8 50 plus 154 13 3 16 10.4 Job status Healthcare workers (HCW) 110 10 0 10 9.1 Full-time work, other than HCW 495 28 24 52 10.5 Self-employed 19 1 1 2 10.5 Employed on daily/temporal basis 114 19 5 24 21.1 Houseworkers 16 2 0 2 12.5 Students, high school and above 432 38 9 47 10.9 Secondary, primary school students, other children and infants 54 4 2 6 11.1 Others and unknown 67 8 8 16 23.9 Unemployed 84 7 1 8 9.5 Country of Birth the Philippines 320 33 11 44 13.8 China 268 14 18 32 11.9 Vietnam 196 19 8 27 13.8 Nepal 194 17 0 17 8.8 Indonesia 66 3 4 7 10.6 Myanmar 56 3 2 5 8.9 Others 291 28 7 35 12.0 Health insurance National health insurance 1313 107 48 155 11.8 Social Welfare 18 3 0 3 16.7 Others and unknown 60 7 2 9 15.0 Regimen INH monotherapy 1309 113 48 161 12.3 RFP monotherapy 78 3 2 5 6.4 Other 4 1 0 1 25.0 Modes of detection Routine screening 95 10 1 11 11.6 Contact investigation 1101 89 46 135 12.3 At hospital 120 13 3 16 13.3 Other mass screening 29 1 0 1 3.4 Others 46 4 0 4 8 7 ost to follow-up” among various characteristics, foreign-born LTBI patients (n = 1391) Table 3 Proportions of “lost to follow-up” among various characteristics, foreign-born LTBI patients (n = 1 Kawatsu et al. Discussion There could also be various cofounding factors, and a separate survey may be necessary before conclusions can be drawn. It must also be noted that the information regarding treatment regimen is only of that upon notification – in reality, regimen can and do change during the course of the treatment [9], however, neither the change nor the new regimen after change is captured in the JTBS. It has been reported that one of the major challenges in ensuring adherence especially for LTBI treatment is to overcome the psychological resistance held by pa- tients to take drugs for a non-contagious and non- symptomatic infection that may never develop into ac- tive disease, but which could cause potential adverse ef- fects, and convince them of the potential benefits of prevention [15]. This could be challenging especially when targeting patients who come from culture that is unfamiliar with the concept of screening and prevention [20]. For example, a prospective study that has examined the predictors for non-completion of LTBI treatment has concluded that a perceived risk of progression to ac- tive TB is strongly associated with better adherence [21]. However, discussions regarding which factors may influ- ence the construction of such perceived risk and benefit are still inconclusive. One possible factor is the status of being a contact of an active TB patient, and having a close experience with the disease. Indeed, some studies have indicated that contacts tended to have higher com- pletion rates than other population groups [22–24]. In our study, whether or not the patient was a contact of a TB case could be identified from the variable “mode of detection” – those who were detected by contact investi- gation are obviously case contacts, however, our results indicated that being a contact was not associated with better treatment adherence. This may suggest that being a contact of a TB case alone is a weak motivating factor to adhere to and complete LTBI treatment, at least among foreign-born persons in Japan. Finally, we considered the time between entry to Japan and diagnosis of LTBI as a potential risk factor for lost to follow-up. Discussion Our study re- sults indicate the treatment completion rate for foreign- born persons in Japan is relatively high, compared to other studies included in the abovementioned reviews, albeit not reaching the national target of 85%. As for the length of treatment regimen, while the WHO 2015 Guideline has concluded that longer treat- ment duration was detrimental to treatment completion, a systematic review that was published a year later has concluded that study results were inconclusive, with some showing better treatment outcomes for shorter regimens using rifampicin, pyrazinamide, rifabutin and/ or INH, than the standard regimen (6 or 9 months of Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 8 of 11 Table 4 Risk factors for lost to follow-up and transfer-out combined among foreign-born LTBI patients Crude OR 95% CI Adjusted OR 95% CI P Sex Male Reference Female 1.08 0.78–1.49 1.16 0.81–1.66 0.41 Age groups (age groups) 0–14 Reference 15–19 0.79 0.21–2.88 2.02 0.20–20.39 0.55 20–29 1.06 0.41–2.78 3.06 0.24–38.79 0.39 30–39 1.58 0.60–4.20 5.50 0.42–71.34 0.19 40–49 1.41 0.51–3.88 5.01 0.37–67.1 0.22 50 plus 1.02 0.35–2.94 3.58 0.26–49.37 0.34 Job status Healthcare workers (HCW) Reference Full-time work, other than HCW 1.17 0.58–2.39 1.29 0.60–2.76 0.51 Self-employed 1.18 0.24–5.84 1.21 0.23–6.39 0.82 Employed on daily/temporal basis 2.67 1.21–5.88 3.11 1.34–7.26 < 0.05 Houseworkers 1.43 0.28–7.20 1.12 0.21–5.89 0.89 Students, high school and above 1.22 0.60–2.50 1.98 0.85–4.64 0.11 Secondary, primary school students, other children and infants 1.25 0.43–3.64 4.97 0.42–58.75 0.20 Unemployed 1.05 0.40–2.79 1.07 0.35–3.26 0.90 Others and unknown 3.14 1.33–7.41 4.09 1.60–10.48 < 0.05 Country of Birth Others Reference the Philippines 1.17 0.72–1.88 1.05 0.63–1.77 0.84 China 0.99 0.59–1.65 0.92 0.54–1.57 0.75 Vietnam 1.17 0.68–2.00 1.38 0.76–2.49 0.29 Nepal 0.70 0.38–1.29 0.64 0.33–1.24 0.18 Indonesia 0.87 0.37–2.05 1.19 0.48–2.97 0.71 Regimen RFP monotherapy Reference INH monotherapy 2.05 0.82–5.14 2.14 0.82–5.54 0.12 Other 4.87 0.43–55.7 4.57 0.29–71.53 0.28 Health insurance National health insurance Reference Social welfare 1.49 0.43–5.22 1.85 0.45–7.61 0.39 Others and unknown 1.32 0.64–2.73 1.13 0.53–2.43 0.76 Modes of detection Contact investigation Reference Routine screening 0.94 0.49–1.80 1.09 0.54–2.21 0.80 At hospital 1.10 0.63–1.92 1.17 0.63–2.17 0.63 Other mass screening 0.26 0.03–1.89 0.19 0.02–1.47 0.11 Others 0.68 0.24–1.93 0.64 0.22–1.89 0.42 Kawatsu et al. Discussion BMC Infectious Diseases (2021) 21:42 Page 9 of 11 Table 4 Risk factors for lost to follow-up and transfer-out combined among foreign-born LTBI patients (Continued) Crude OR 95% CI Adjusted OR 95% CI P Time between entry to Japan and diagnosis within 2 years Reference more than 2 years, within 5 years 1.62 0.98–2.68 1.69 1.00–2.87 0.05 more than 5 years, within 10 years 1.62 0.79–3.35 1.50 0.69–3.26 0.31 10 years or more 0.98 0.55–1.76 0.85 0.43–1.68 0.65 INH isoniazid, RFP rifampicin, CI confidence interval, OR odds ratio NH isoniazid, RFP rifampicin, CI confidence interval, OR odds ratio Another potential factor is patient education and counselling – for example, studies from prison have in- dicated that those who have received education sessions about LTBI prior to being released had higher treatment completion rates post-release, than those who did not [25]. Several studies have shown that well-designed edu- cational intervention using culturally and socially sensi- tive languages can assist patients make better-informed decisions about the potential risks and benefits of LTBI treatment [26, 27]. We were unable to assess the pos- sible impact of education as such is not collected in the JTBS. However, numerous studies on adherence support to foreign-born active TB patients in Japan have pointed to the challenge of language [28, 29]. Even today, Japan continues to be a largely mono-cultural society and it is quite common that local healthcare workers only speak Japanese. There is an increasing awareness of the need to improve the language capacity of health services, against the rising number of foreign-born patients in Japan – however, the resources are still very much lim- ited. Under such situation, it is not difficult to imagine that foreign-born patients in Japan are not receiving the appropriate and adequate information that they need to make informed decisions about LTBI and treatment. Studies are needed to explore the most effective infor- mation, education, and communication for foreign-born patients regarding LTBI. INH) while others showing similar completion rates, and also that the studies themselves were too heterogeneous to conduct pooled analysis [16]. In our study, the proportion of lost to follow-up was bigger among those who have started treatment with INH than those with RFP mono- therapy (12.3% vs. 6.4%), however, the difference was shown not to be significant in the logistic regression analysis (p = 0.12). Authors’ contributions LK and UK initiated and designed the study. LK and UK analyzed the data, LK was responsible for writing up, and all (LK, UK, AO) were involved in the discussion of the results. All authors have read and approved the final manuscript. 2. Centers for Disease Control and Prevention (CDC). Reported Tuberculosis in the United States, 2017. Atlanta, GA: US Department of health and human services, CDC; 2018. Abbreviations d INH: Isoniazid; JTBS: Japan Tuberculosis Surveillance; LTBI: Latent tuberculosis infection; RFP: Rifampicin; TB: Tuberculosis INH: Isoniazid; JTBS: Japan Tuberculosis Surveillance; LTBI: Latent tuberculosis infection; RFP: Rifampicin; TB: Tuberculosis Acknowledgements The authors would like to acknowledge all those who contributed to notifying and registering information on TB patients in Japan, including physicians, public health nurses, microbiologists and administrative staff. Funding The study was supported by JSPS Grants-in-Aids for Scientific Research (C), grant number: 20 K10491. The funders had no role in study design, data col- lection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Conclusions Our study results have added to the growing evidence that migrant population face greater risk of interrupting LTBI treatment. While the WHO updated guideline for LTBI management states that “concerns about adher- ence should not be a barrier to use of preventive treat- ment” [7], the authors nevertheless believe that clinical benefit to individual patient and the success of control programs are subject to completion of LTBI treatment, and hence interventions to improve adherence are a crit- ical component of programmatic management of LTBI. Supplementary Information usually 1 year, 3 years or 5 years. In other words, those who are transferring out of Japan may simply be doing so, because their permit to stay is expiring. This brings up another important issue surrounding the continuum of care for LTBI patients across na- tional borders, but the discussions are beyond the scope of this study. The online version contains supplementary material available at https://doi. org/10.1186/s12879-020-05712-1. The online version contains supplementary material available at https://doi. org/10.1186/s12879-020-05712-1. Additional file 1. Definitions of selected variable from the Japan Tuberculosis Surveillance system. Additional file 2. Annual trend of LTBI notification by country of birth and age groups, 2007–2018. Excel file (.xls) showing the number of LTBI notifications by country of birth and age groups. Additional file 2. Annual trend of LTBI notification by country of birth and age groups, 2007–2018. Excel file (.xls) showing the number of LTBI notifications by country of birth and age groups. Our study is not without limitations. Firstly, as our sole source of data was the JTBS, we were unable to examine the effects of variables which were not collected in the JTBS, such as frequency of side-effect [30, 31], and alcohol and/or drug dependence [28, 32], which have previously been identified as being potentially detri- mental to treatment adherence. Secondly, our study did not take into account those who have become lost to follow-up at various stages in the cascade of care in diagnosis and treatment of LTBI, including those who, despite being eligible, were not tested for LTBI and those, after being diagnosed as LTBI, did not initiate treatment. For example, the previously mentioned meta- analysis has concluded that screening completion rates was the lowest among the migrants (43%), compared with other populations such as medical personnel (86.1%), marginalized persons (83.3%) and contacts of active case (79.3%) [17]. A separate study is probably re- quired to examine the entire cascade of care for LTBI among foreign-born persons in Japan. Additional file 3. Trend of LTBI notification by mode of detection, 2007–2018. Excel file (.xls) showing the number of LTBI notifications by mode of detection, 2007–2018. Additional file 3. Trend of LTBI notification by mode of detection, 2007–2018. Excel file (.xls) showing the number of LTBI notifications by mode of detection, 2007–2018. Discussion Compared with those who had been diag- nosed within 2 years of arriving to Japan, those who had been in Japan for more than 2 years but less than 5 years had a slightly raised risk of becoming lost to follow-up (adjusted odds ratio 1.69, 95% confidence interval 1.00– 2.87). The risk probably reflects the risk of transferring- out of Japan, rather than becoming lost to follow-up in Japan, as the proportion of international transfer-out was the highest among those who had been in Japan for more than 2 years but less than 5 years (n = 15/222, 6.7%). The reasons are not clear, though this may be related to the duration of permit to stay, which majority of foreign-born students and workers are requested to apply when staying in Japan. The duration of stay de- pends on the type of permit, however, and it is Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Page 10 of 11 Ethics approval and consent to participate Ethical clearance obtained from the Institution al Review Board of the Research Institute of Tuberculosis (reference number RIT/IRB 2020–08). Informed consent as deemed unnecessary as the JTBS data does not include case identifiers, as according to the Ethical Guidelines for Epidemiological Research established by Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour and Welfare of Japan. Competing interests As previous studies have already shown, efforts to es- tablish a “one-size fits-all” approach to improve adher- ence to LTBI treatment are probably not likely to succeed. Our results have indicated risk factors for lost to follow-up, some of which were potentially unique to Japan. On the other hand, we have also pointed to the need for a further, in-depth study to explore the socio- economic and cultural situation in which foreign-born persons initiate LTBI treatment in Japan. Whatever the target population, sharing the experience of trying to understand the population and identifying potential bar- riers, and of innovative approaches to improving adher- ence can be beneficial to the global effort in tackling LTBI. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 16 July 2020 Accepted: 15 December 2020 Received: 16 July 2020 Accepted: 15 December 2020 References License: CC BY-NC-SA 3.0 IGO. 28. Shigeto E. Nihon no kekkakuchiryou niokeru shomondai (issues in treatment of tuberculosis in Japan). Kekkaku. 2018;93:563–8 In Japanese. 28. Shigeto E. Nihon no kekkakuchiryou niokeru shomondai (issues in treatment of tuberculosis in Japan). Kekkaku. 2018;93:563–8 In Japanese. of tuberculosis in Japan). Kekkaku. 2018;93:563–8 In Japanese. 8. The Prevention Committee and the Treatment Committee of the Japanese Society for Tuberculosis. Treatment Guideline on LTBI. Kekkaku. 2013;88:497– 512. 29. Nagata Y, Takayanagi K, Yano R, Sawada T, Morita N, Murakami K, Takasaki J. Utilization of medical interpreters for tuberculosis patients in Japan: a questionnaire survey. Kekkaku. 2020;95:41–6. 29. Nagata Y, Takayanagi K, Yano R, Sawada T, Morita N, Murakami K, Takasaki J. Utilization of medical interpreters for tuberculosis patients in Japan: a questionnaire survey. Kekkaku. 2020;95:41–6. 9. Ministry of Justice. Annual Report on Statistics on Legal Migrants, 2019. http://www.moj.go.jp/housei/toukei/toukei_ichiran_nyukan.html. 30. Ailinger RL, Dear MR. Adherence to tuberculosis preventive therapy among Latino immigrants. Public Health Nurs. 1998;15:19–24. 30. Ailinger RL, Dear MR. Adherence to tuberculosis preventive therapy among Latino immigrants. Public Health Nurs. 1998;15:19–24. 10. Murase Y, Ohkado A, Watanabe Y, Kaguraoka S, Ishihara K, Homareda C, Uchimura K, Maeda S, Takii T, Ishikawa N. Transmission dynamics of mycobacterium tuberculosis between foreign-nationals and Japanese tuberculosis patients living in Shinjuku-City, Tokyo. Japan Kekkaku. 2017;92: 431–9. 31. LoBue A, Moser S. Use of isoniazid for latent tuberculosis infection in a public health clinic. Am J Respir Crit Care Med. 2003;168:443–7. 32. Lobato N, Reves R, Jasmar M, Grabau C, Bock N, Shang N, et al. Adverse events and treatment completion for latent tuberculosis in jail inmates and homeless persons. Chest. 2005;127:1296–303. 11. Kawatsu L, Uchimura K, Ohkado A. Trend and treatment status of latent tuberculosis infection patients in Japan - analysis of Japan TB surveillance data. PLoS One. 2017;12(11):e0186588. https://doi.org/10.1371/journal.pone. 0186588. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 12. Tanaka Y, Suzuki M. Gaikokujin ryugakusei no senzaisei kekkaku kansenshou ni taisuru IGRA to kyoubu X sen niyoru kenko kanri (Medical examination via IGRA and chest X-ray for foreign-born students). Campus Health. 2018; 55(1):462–4 In Japanese. 13. Nishimura T, Mori M. Gaikokujin ryugakusei no kekkaku kansen chousa (screening for TB infection among foreign-born students). Bull Keio University Health Center. 2019;37(1):35–9 In Japanese. 14. Getahun H, Matteelli A, Abubaker I, Abdel Aziz M, Baddeley A, Barreira D, et al. Management of latent mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries. European Respir J. 2015; 46(6):1563–76. https://doi.org/10.1183/13993003.01245-2015. 15. Hirsch-Moverman Y, Daftary A, Franks J, Colson P. Adherence to treatment for latent tuberculosis infection: systematic review of studies in the US and Canada. Int J Tuberc Lung Dis. 2008;12(11):1235–54. 16. Sandgren A, Noordegraaf-Shouten MV, van Kessel F, Stuuman A, Oordt- Speets A and van der Werf MS. Initiation and completion rates for latent tuberculosis infection treatment: a systematic review. BMC Infect Dis. 2016; 16:204. DOI: https://doi.org/10.1186/s12879-016-1550y. 17. Alsdurf H, Hill PC, Metteelli A, Getahun H, Menzies D. The cascade of care in diagnosis and treatment of latent tuberculosis infection: a systematic review and meta-analysis. Lancet Infect Dis. 2016;16(11):1269–78. https://doi.org/10. 1016/S1473-3099(16)30216-X. 17. Alsdurf H, Hill PC, Metteelli A, Getahun H, Menzies D. The cascade of care in diagnosis and treatment of latent tuberculosis infection: a systematic review and meta-analysis. Lancet Infect Dis. 2016;16(11):1269–78. https://doi.org/10. 1016/S1473-3099(16)30216-X. 18. Nyamathi AM, Christiani A, Nahid P, Gregerson P, Leake B. A randomized controlled trial of two treatment program for homeless adults with latent tuberculosis infection. Int J Tuberc Lung Dis. 2006;10:775–82. 18. Nyamathi AM, Christiani A, Nahid P, Gregerson P, Leake B. A randomized controlled trial of two treatment program for homeless adults with latent tuberculosis infection. Int J Tuberc Lung Dis. 2006;10:775–82. 19. Lardizabal A, Passannante M, Kojakali F, Hayden C, Reichman LB. Enhancement of treatment completion for latent tuberculosis infection with 4 months of rifampin. Chest. 2006;130:1715–7. 19. Lardizabal A, Passannante M, Kojakali F, Hayden C, Reichman LB. Enhancement of treatment completion for latent tuberculosis infection with 4 months of rifampin. Chest. 2006;130:1715–7. 20. References 1. European Centre for Disease Prevention and Control (ECDC)/World Health Organization Regional Office for Europe. Tuberculosis surveillance and monitoring in Europe 2018–2016 data. Stockholm: ECDC; 2018. Available from: https://ecdc.europa.eu/sites/portal/files/documents/ecdc-tuberculosis- surveillance-monitoring-Europe-2018-19mar2018.pdf WHO. Accessed 3 Apr 2020. 1. European Centre for Disease Prevention and Control (ECDC)/World Health Organization Regional Office for Europe. Tuberculosis surveillance and monitoring in Europe 2018–2016 data. Stockholm: ECDC; 2018. Available from: https://ecdc.europa.eu/sites/portal/files/documents/ecdc-tuberculosis- surveillance-monitoring-Europe-2018-19mar2018.pdf WHO. Accessed 3 Apr 2020. 3. Toms C, Stapledon R, Coulter C, Douglas P. The National Tuberculosis Advisory Committee. Tuberculosis notifications in Australia, 2014. Commun Dis Intell. 2017;41:3. Page 11 of 11 Page 11 of 11 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 Kawatsu et al. BMC Infectious Diseases (2021) 21:42 (2021) 21:42 4. Tuberculosis Surveillance Center. Tuberculosis in Japan – annual report 2019. Tokyo: Department of Epidemiology and Clinical Research, the Research Institute of Tuberculosis; 2019. 4. Tuberculosis Surveillance Center. Tuberculosis in Japan – annual report 2019. Tokyo: Department of Epidemiology and Clinical Research, the Research Institute of Tuberculosis; 2019. 24. Reichler MR, Reves R, Bur S, Ford J, Thompson V, Mangura B. Treatment of latent tuberculosis infection in contacts in new tuberculosis cases in the United States. South Med J. 2002;95:414–20. 5. Pareek M, Baussano I, Abubakar I, Dye C, Lalvani A. Evaluation of immigrant tuberculosis screening in industrialized countries. Emerg Infect Dis. 2012; 18(9):1422–9. https://doi.org/10.3201/eid1809.120128. 25. White MC, Tulsky JP, Menedez E, Goldenson J, Kawamura LM. Incidence of TB in inmates with latent TB infection: 5 year follow-up. Am J Prev Med. 2005;29:295–301. 5. Pareek M, Baussano I, Abubakar I, Dye C, Lalvani A. Evaluation of immigrant tuberculosis screening in industrialized countries. Emerg Infect Dis. 2012; 18(9):1422–9. https://doi.org/10.3201/eid1809.120128. 26. Kim DY, Rizdon R, Giles B, Mireles T, Garrity K, Hatchcock AL, Crowder D, Jackson R, Taylor Z. A no-name tuberculosis tracking system. Am J Public Health. 2003;93:1637–9. 6. Campbell JR, Chen W, Johnston J, Cook V, Elwood K, Krot J, Marra F. Latent tuberculosis infection screening in immigrants to low-incidence countries: a meta-analysis. Mol Diagn Ther. 2015;19(2):107–17. https://doi.org/10.1007/ s40291-015-0135-6. 27. Goldberg SV, Wallace J, Jackson JC, Cahulk CP, Nolan CM. Cultural case management of latent tuberculosis infection. Int J Tuberc Lung Dis. 2004;8: 76–82. 7. Latent tuberculosis infection. updated and consolidated guidelines for programmatic management. Geneva: World Health Organization; 2018. License: CC BY-NC-SA 3.0 IGO. 7. Latent tuberculosis infection. updated and consolidated guidelines for programmatic management. Geneva: World Health Organization; 2018. Publisher’s Note Spruijt I, Haile DT, Erkens C, van den Hof S, Goosen S, ten Kate A, Tshome H, Karels M, Koenders M, Suurmond J. Strategies to reach and motivate migrant communities at high risk for TB to participate in a latent tuberculosis infection screening program: a community-engaged, mixed methods study among Eritreans. BMC Public Health. 2020;20:315. https://doi. org/10.1186/s12889-020-8390-9. 20. Spruijt I, Haile DT, Erkens C, van den Hof S, Goosen S, ten Kate A, Tshome H, Karels M, Koenders M, Suurmond J. Strategies to reach and motivate migrant communities at high risk for TB to participate in a latent tuberculosis infection screening program: a community-engaged, mixed methods study among Eritreans. BMC Public Health. 2020;20:315. https://doi. org/10.1186/s12889-020-8390-9. 21. Shieh FK, Snyder G, Horsburgh CR, Bernado J, Murphy C, Saukkonen JJ. Predicting non-completion of treatment for latent Tuberculous infection. A prospective survey. Am J Respir Crit Care Med. 2006;174:717–21. 22. Center for Disease Control and Prevention. Transmission of mycobacterium tuberculosis in a high-school and school-based supervision of an isoniazid- rifapentine regimen for preventing tuberculosis - Colorado, 2011-2012. MMWR Morb Mortal Wkly Rep. 2013;62:805–9. 22. Center for Disease Control and Prevention. Transmission of mycobacterium tuberculosis in a high-school and school-based supervision of an isoniazid- rifapentine regimen for preventing tuberculosis - Colorado, 2011-2012. MMWR Morb Mortal Wkly Rep. 2013;62:805–9. 23. Sloot R, van der Loeff MF S, Kouw PM, Borgdorff MW. Yield of tuberculosis contact investigation in Amsterdam: opportunities for improvement. Eur Respir J. 2014;44:714–24. 23. Sloot R, van der Loeff MF S, Kouw PM, Borgdorff MW. Yield of tuberculosis contact investigation in Amsterdam: opportunities for improvement. Eur Respir J. 2014;44:714–24.
https://openalex.org/W2894950287
https://www.nature.com/articles/s41598-018-33487-8.pdf
English
null
Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission
Scientific reports
2,018
cc-by
10,490
Attenuation of replication by a 29 nucleotide deletion in SARS- coronavirus acquired during the early stages of human-to-human transmission 018 mber 2018 xx OPEN Received: 16 July 2018 Accepted: 27 September 2018 Published: xx xx xxxx Doreen Muth1,2,3, Victor Max Corman   1,2,3, Hanna Roth3, Tabea Binger3, Ronald Dijkman   4,5, Lina Theresa Gottula1,2,3, Florian Gloza-Rausch6, Andrea Balboni   7, Mara Battilani7, Danijela Rihtarič8, Ivan Toplak8, Ramón Seage Ameneiros9,10, Alexander Pfeifer11, Volker Thiel4,5, Jan Felix Drexler1,2,3, Marcel Alexander Müller   1,2,3 & Christian Drosten1,2,3 A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (−29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23- fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS. Emerging zoonotic viruses such as Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) are a consider- able concern for public health1. Bats of the genus Rhinolophus are the bona-fide animal reservoir for SARS- and SARS-related CoV (SARSr-CoV)2. Epidemics may emerge from wild animal reservoirs in which a plethora of viral variants exists. After initial cross-host infection, the occurrence of positive selection with adaptive changes 1Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Charitéplatz 1, 10117, Berlin, Germany. 2German Center for Infection Research (DZIF), Berlin, Germany. 3Institute of Virology, University of Bonn Medical Centre, Sigmund- Freud-Str. 25, 53127, Bonn, Germany. 4Federal Department of Home Affairs, Institute of Virology and Immunology IVI, Bern and Mittelhäusern, Sensemattstrasse 293, 3147, Mittelhäusern, Switzerland. 5Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, 3012, Bern, Switzerland. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Attenuation of replication by a 29 nucleotide deletion in SARS- coronavirus acquired during the early stages of human-to-human transmission 018 mber 2018 xx OPEN We have previously found that a European SARS-related CoV carried by rhinolophid bats in Bulgaria did not contain any ORF8 or similar gene13 while virtually all SARS-related CoV from Asia possess a single, continuous ORF88,14,15.h g y p g , The 29 nt deletion in ORF8 was the most obvious genetic change during human-to-human transmission of SARS, causing the expression of truncated gene products termed ORF8a and ORF8b. It has been widely hypoth- esized that the truncated products led to a modulation of pathogenicity or replication that favored adaptation of SARS-CoV to humans9,11,12. For instance, it was found that replication of SARS-CoV is increased in cells that overexpress the protein encoded by ORF8a16. In contrast, the same protein led to an attenuation of replication when engineered into recombinant infectious bronchitis virus (IBV)17. The 8b protein, when overexpressed, was found to induce apoptosis and to be involved in cellular degradation of the viral envelope protein during SARS-CoV infection16,18,19. When engineered into an IBV infectious clone, protein 8b inhibited the induction of interferon (IFN) as triggered by poly(I:C)-stimulation and thus enhanced IBV replication. This effect was found to involve binding of ubiquitin and interference with induction of the IFN type 1 promoter via IRF317. Another study found that the full 122 amino acid protein encoded by ORF8 induces ATF6-dependent transcription, which triggers the expression of chaperones and leads to a general attenuation of the protein translation level, thus mod- ifying the unfolded protein response20. Loss of ORF8’s original cellular localization in the endoplasmatic retic- ulum would have ablated this function in human viruses of the middle and late epidemic phases20,21. However, the effect that this change had on SARS-CoV replication level remains unclear. Studies of SARS-CoV replication in experimental mice have not provided indications as to essential functions of ORF8a or 8b22. However, these studies involved high inoculated virus doses and utilized a mouse model that may not reflect relative changes of replication in transition from bats to humans. In sum, the available data leave it unclear whether the changes that occurred in ORF8 led to a modification of viral replication when comparing bat versus human hosts, and specifically, whether the deletion of 29 nt involved an increase of replication level in human cells as often impli- cated. Attenuation of replication by a 29 nucleotide deletion in SARS- coronavirus acquired during the early stages of human-to-human transmission 018 mber 2018 xx OPEN 6Noctalis, Centre for Bat Protection and Information, Oberbergstraße 27, 23795, Bad Segeberg, Germany. 7Dipartimento di Scienze Mediche Veterinarie, Facoltà di Medicina Veterinaria, Alma Mater Studiorum-Università di Bologna, Via Tolara di Sopra 50, 40064, Ozzano Emilia, (BO), Italy. 8Virology Unit, Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000, Ljubljana, Slovenia. 9Institute of Evolutionary Ecology and Conservation Genomics, University of Ulm, Albert-Einstein Allee 11, 89069, Ulm, Germany. 10Group Morcegos de Galicia, Drosera Society, Pdo. Magdalena, G-2, 2° esq, 15320, As Pontes, Spain. 11Institute for Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany. Correspondence and requests for materials should be addressed to C.D. (email: christian.drosten@charite.de) SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 1 www.nature.com/scientificreports/ is thought to be essential for viral emergence3,4. The confirmation of phenotypic changes acquired in the process of adaptation requires virus isolation, which is not always possible. In the case of SARS-CoV, isolation of virus strains from the bat reservoir has widely failed5, except in singular instances involving bat-borne viruses whose spike protein showed an exceptionally high amino acid identity with the epidemic strain6–8. Additional reasons for low isolation success include the lack of appropriate cell culture systems as well as low virus concentrations and often inappropriate storage conditions for samples from field investigations. Studies of phenotypic properties of reservoir-borne viruses can be facilitated by the reconstruction of genetic traits through reverse genetics.f y g g g In the present study we reconstructed variant SARS-CoVs carrying different forms of open reading frame (ORF) 8, an accessory gene in the SARS-CoV genome that is among the most variable genes in bat-associated SARSr-CoV. ORF8 is also one of the most relevant genes when studying potential viral adaptation to humans, as the ORF8 coding sequence has undergone gradual deletion during the human epidemic. Early epidemic SARS-CoVs contained a full ORF8 that is also present in the genomes of almost all bat- and carnivore-associated precursor viruses9–12. A 29 nucleotide (nt) deletion within ORF8 occurred in all strains involved in the middle and late phase of the human epidemic9,10. Subtotal or total deletions of up to 415 nts occurred within and around the ORF8 region in SARS-CoV strains circulating in the very late phase of the epidemic. Attenuation of replication by a 29 nucleotide deletion in SARS- coronavirus acquired during the early stages of human-to-human transmission 018 mber 2018 xx OPEN Genome deletions have also been observed in MERS-CoV, and also here it was speculated that deletion may reflect adaptation to the human host or the release of selective pressure exerted exclusively in the zoonotic reservoir23,24.ii In the present study on SARS-CoV, we first followed up on our initial finding of the absence of ORF8 in genomes of SARSr-CoV from European bats, and sequenced rhinolophid bat-associated SARSr-CoV from four countries across Europe (Spain, Bulgaria, Italy, Slovenia). We then studied the influence of ORF8 on replication in the context of a full replicating SARS-CoV genome, based on a host transition scenario represented by cell cul- tures. We constructed recombinant viruses with full ORF8, truncated ORF8 (29 nt deletion), as well as completely deleted ORF8. Replication was compared in primate cell lines (VeroFM, MA104), a novel cell line generated from the lung of a rhinolophid bat, three additional non-chiropteran cell lines, as well as human airway epithelial cul- tures. We find that the 29 nt deletion conferred an attenuation of replication level irrespective of the host system studied. Results B t i E Generation and evaluation of ORF8 variant recombinant SARS-CoV. Variants of the open reading frame 8 (ORF8) were designed in accordance to their appearance in nature. (a) rSCV8full represents a single ORF8 as found in reservoir bats and amplification hosts in China, as well as in the early phase of the SARS epidemic. The middle phase of the epidemic was dominated by a virus variant carrying a 29 nt deletion resulting in the disruption of ORF8 in 2 reading frames, 8a and 8b, as seen in rSCVepi. The absence of ORF8 is a genomic feature of reservoir bats in Europe and the epidemic virus in the late phase. Two deletion variants were constructed. Variant 1, rSCVdel8–1, perfectly misses ORF8. In variant 2, rSCVdel8–2, ORF8 is replaced by the short substitutional sequence AATAA in accordance to the upstream region of the nucleocapsid gene of the European SARS-related bat-CoV BtCoV/BM48–31/Rhi bla/Bulgaria/2008 (NC_014470). (b) Plaque morphology of rSCV8full and rSCVepi were very similar, while rSCVdel8–1 produced only diffuse and rSCVdel8–2 reduced plaques. Western Blot analysis revealed that only rSCV8full, rSCVepi, and rSCVdel8–2 infected cells expressed the nucleocapsid protein, while none could be detected in cells infected with rSCVdel8–1. Detection of β actin Table 1. Bat specimens included in this study and confirmation of absence of ORF8. Absence of ORF8 was determined by RT-PCR using reverse primers F29260R (TTTGTATGCGTCAATGTGCTTG) for reverse transcription and F28182R (GGGTCCACCAAATGTAATGCGG) and forward primer F27626F (GAGAAAGACAGAATGAATGAGC) for PCR. Figure 1. Generation and evaluation of ORF8 variant recombinant SARS-CoV. Variants of the open reading frame 8 (ORF8) were designed in accordance to their appearance in nature. (a) rSCV8full represents a single ORF8 as found in reservoir bats and amplification hosts in China, as well as in the early phase of the SARS epidemic. The middle phase of the epidemic was dominated by a virus variant carrying a 29 nt deletion resulting in the disruption of ORF8 in 2 reading frames, 8a and 8b, as seen in rSCVepi. The absence of ORF8 is a genomic feature of reservoir bats in Europe and the epidemic virus in the late phase. Two deletion variants were constructed. Variant 1, rSCVdel8–1, perfectly misses ORF8. In variant 2, rSCVdel8–2, ORF8 is replaced by the short substitutional sequence AATAA in accordance to the upstream region of the nucleocapsid gene of the European SARS-related bat-CoV BtCoV/BM48–31/Rhi bla/Bulgaria/2008 (NC_014470). Results B t i E (b) Plaque morphology of rSCV8full and rSCVepi were very similar, while rSCVdel8–1 produced only diffuse and rSCVdel8–2 reduced plaques. Western Blot analysis revealed that only rSCV8full, rSCVepi, and rSCVdel8–2 infected cells expressed the nucleocapsid protein, while none could be detected in cells infected with rSCVdel8–1. Detection of β actin served as loading control. Virus replication of the three ORF8 variants, (rSCVdel8 = rSCVdel8–2) was monitored by plaque titration after infection of VeroFM cells at two different multiplicities of infection, 1 (c) and 0.001 (d). Virus growth was determined in at least 3 independent experiments in triplicates. Shown is one representative experiment. Error bars represent standard deviation of the mean. Figure 1. Generation and evaluation of ORF8 variant recombinant SARS-CoV. Variants of the open reading frame 8 (ORF8) were designed in accordance to their appearance in nature. (a) rSCV8full represents a single ORF8 as found in reservoir bats and amplification hosts in China, as well as in the early phase of the SARS epidemic. The middle phase of the epidemic was dominated by a virus variant carrying a 29 nt deletion resulting in the disruption of ORF8 in 2 reading frames, 8a and 8b, as seen in rSCVepi. The absence of ORF8 is a genomic feature of reservoir bats in Europe and the epidemic virus in the late phase. Two deletion variants were constructed. Variant 1, rSCVdel8–1, perfectly misses ORF8. In variant 2, rSCVdel8–2, ORF8 is replaced by the short substitutional sequence AATAA in accordance to the upstream region of the nucleocapsid gene of the European SARS-related bat-CoV BtCoV/BM48–31/Rhi bla/Bulgaria/2008 (NC_014470). (b) Plaque morphology of rSCV8full and rSCVepi were very similar, while rSCVdel8–1 produced only diffuse and rSCVdel8–2 reduced plaques. Western Blot analysis revealed that only rSCV8full, rSCVepi, and rSCVdel8–2 infected cells expressed the nucleocapsid protein, while none could be detected in cells infected with rSCVdel8–1. Detection of β actin served as loading control. Virus replication of the three ORF8 variants, (rSCVdel8 = rSCVdel8–2) was monitored by plaque titration after infection of VeroFM cells at two different multiplicities of infection, 1 (c) and 0.001 (d). Virus growth was determined in at least 3 independent experiments in triplicates. Shown is one representative experiment. Error bars represent standard deviation of the mean. Infectious viruses were rescued from cDNA as described25. Results B t i E Bats in Europe carry SARS-related CoV that lack ORF8. We have previously described the detection of SARS-related CoV in Rhinolophus in Bulgaria13. The full-length genome of the virus sequenced in that study con- tained no ORF8. To further investigate the presence of ORF8 in SARS-related CoV in Europe, we analyzed fecal samples from rhinolophid bats in Spain, Bulgaria, Italy, as well as Slovenia. All five Rhinolophus species commonly encountered in Europe were tested positive for coronavirus RNA (Table 1). However, none of 92 geographically and phylogenetically representative viruses provided evidence for the presence of an ORF8 gene. 19 of these viruses were fully sequenced, confirming absence of ORF8 at orthotopic or heterotopic genome positions. Generation of recombinant SARS-CoV encoding ORF8 variants. To understand the consequences of the absence of ORF8, recombinant SARS-CoV (rSCV) were generated as shown in Fig. 1a. The variant termed rSCV8full contained a complete ORF8 as encountered in bats, civet cats, as well as in early human cases of SARS, thus resembling the pre-epidemic and starting phase of the SARS outbreak. The variant termed rSCVepi corre- sponds to isolates from the main phase (also referred to as middle and late phases) of the epidemic containing a 29 nt deletion that introduces a ribosomal frameshift separating ORF8 into ORF8a and ORF8b. Two alternative virus variants without ORF8 were constructed. Variant 1, rSCVdel8–1, had ORF8 removed without any replace- ment. Variant 2, rSCVdel8–2, had ORF8 replaced by a 5 nt spacer sequence (AATAA) which occurs instead of ORF8 between ORF7b and the nucleocapsid gene in a natural SARS-related CoV variant from European rhinolophid bats13. SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 2 www.nature.com/scientificreports/ Country N individuals tested Rhinolophid species and number of individuals (n) yielding positive RT-PCR Number of RT-PCR results indicating absence of ORF8 Bulgaria 506 R. euryale (n = 44) R. blasii (n = 11) R. ferrumequinum (n = 1) R. mehelyi (n = 4) 44 11 1 4 Italy 45 R. ferrumequinum (n = 6) 6 Slovenia53 36 R. hipposideros (n = 5) 5 Spain 285 R. hipposideros (n = 21) 21 Total 872 All 5 species known in Europe 92 (100%) Table 1. Bat specimens included in this study and confirmation of absence of ORF8. Absence of ORF8 was determined by RT-PCR using reverse primers F29260R (TTTGTATGCGTCAATGTGCTTG) for reverse transcription and F28182R (GGGTCCACCAAATGTAATGCGG) and forward primer F27626F (GAGAAAGACAGAATGAATGAGC) for PCR. Figure 1. Results B t i E 24 h after co-electroporation of in-vitro transcribed RNA and nucleocapsid subgenomic RNA, parts of the cell culture supernatant were transferred to VeroFM cells and virus replication was monitored by real-time RT-PCR. 72 h post infection (hpi), rSCV8full, rSCVepi and rSCVdel8–2 gave evidence of replication by real-time RT-PCR, while rSCVdel8–1 RNA increased more slowly, reach- ing a plateau level only by 96 hpi. SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 3 www.nature.com/scientificreports/ Figure 2. Replication of ORF8 variants in primate cell cultures with and without IFN pre-treatment. (a) VeroFM and MA104 cells were infected with three ORF8 variant viruses at MOI = 0.001. Supernatants were harvested at 24 hpi and virus titers determined by plaque titration. The experiment was done in triplicates. Significance of replication differences between virus variants was determined by t-Test (***p < 0.001, *p < 0.05). (b) VeroFM cells were incubated with universal type I IFN at indicated concentrations 16 h prior to infection with rSCV8full and rSCVdel8 at MOI = 0.001. At 24 hpi supernatants were plaque titered. Experiments were done at least twice in triplicates. Shown is one representative experiment each. Error bars represent standard deviation of the mean. Figure 2. Replication of ORF8 variants in primate cell cultures with and without IFN pre-treatment. (a) Figure 2. Replication of ORF8 variants in primate cell cultures with and without IFN pre-treatment. (a) VeroFM and MA104 cells were infected with three ORF8 variant viruses at MOI = 0.001. Supernatants were harvested at 24 hpi and virus titers determined by plaque titration. The experiment was done in triplicates. Significance of replication differences between virus variants was determined by t-Test (***p < 0.001, *p < 0.05). (b) VeroFM cells were incubated with universal type I IFN at indicated concentrations 16 h prior to infection with rSCV8full and rSCVdel8 at MOI = 0.001. At 24 hpi supernatants were plaque titered. Experiments were done at least twice in triplicates. Shown is one representative experiment each. Error bars represent standard deviation of the mean. All variants were plaque-quantified. During this process, differences in plaque morphology were noted. While rSCV8full and rSCVepi yielded plaques of similar sizes, rSCVdel8–2 showed smaller plaque size and rSCVdel8–1 gener- ated no distinct plaques (Fig. 1b).h All variants were plaque-quantified. During this process, differences in plaque morphology were noted. Results B t i E While rSCV8full and rSCVepi yielded plaques of similar sizes, rSCVdel8–2 showed smaller plaque size and rSCVdel8–1 gener- ated no distinct plaques (Fig. 1b).h p q g The absence of plaques in rSCVdel8–1 could point to a replication defect caused by the loss of essential gene func- tions, including the nucleocapsid gene whose expression is indispensable for virus replication26. Modifications of ORF8 affect the upstream context of the transcription regulatory sequence (TRS) of the nucleocapsid gene27. Western blot analyses showed that rSCV8full, rSCVepi, as well as rSCVdel8–2 expressed similar amounts of the nucle- ocapsid protein, while no expression was seen in cells infected with rSCVdel8–1 (Fig. 1b). Subsequent experiments were therefore carried out using variant 2, which is hereafter referred to as rSCVdel8. Integrity of ORF8 facilitates the replication of recombinant SARS-CoV. Replication of rSCV8full, rSCVepi, and rSCVdel8 were tested in VeroFM cells at high and low multiplicity of infection (MOI) (1 and 0.001 plaque forming unit (PFU)/cell). Supernatants were harvested at 8, 24, 48, and 72 hpi and titered in VeroFM cells (Fig. 1c, d). At MOI = 1, 30–40% of the cells were already lysed by 24 hpi, precluding meaningful comparisons of viruses (Fig. 1c). At MOI = 0.001, replication was in the exponential phase by 24 hpi (Fig. 1d). rSCV8full grew significantly more efficiently than both other variants at 24 hpi (p < 0.05 and p < 0.001 comparing to rSCVepi and rSCVdel8, respectively, using t-Test). The fully deleted variant rSCVdel8 replicated significantly less efficiently than rSCVepi at 24 hpi (p = 0.002, t-Test). By 48 hpi, replication of all variants reached plateau levels. The replication facilitating effect of ORF8 is IFN independent. Due to the essential role of the type I IFN system in the restriction of virus infection, it was tested whether the ORF8-dependent replication phenotype might be linked to the type I IFN response. MA104 monkey kidney cells were used because they are competent for IFN induction and signaling28. VeroFM cells would only reflect differences in IFN signaling, due to their defect in IFN beta gene expression29. According to previous results, virus infections with all three variants were carried out at MOI = 0.001 and virus progeny was measured at 24 hpi by plaque assay.fif g y y q y All viruses generally replicated more efficiently in VeroFM cells than MA104 cells. Results B t i E However, relative differ- ences between strains were very similar in both cell lines, suggesting that IFN beta gene induction that is only functional in MA104 cells may not play a significant role for the observed phenotypes (Fig. 2a).lt To test the potential influence of the ORF8 gene product on IFN signaling, virus growth after external addition of IFN was compared. To this end, VeroFM cells were incubated with indicated concentrations of universal type I IFN alpha prior to infection with rSCV8full and rSCV8del. At 24 h p.i., rSCV8full grew to titers 30 times higher than rSCVdel8 (Fig. 2b). If cells were treated with IFN prior to infection the overall replication of both viruses decreased in a dose-dependent manner. However, the replication difference between both viruses remained constant. This suggests that ORF8 has a SARS-CoV-specific replication-enhancing effect that is independent of IFN antagonism. ORF8 improves replication of recombinant SARS-CoV in a reservoir bat cell line. In previous studies, it has been suggested that the function of ORF8 may be more relevant for replication in cells from the actual animal reservoir, than for replication in human or primate cell cultures30. Because bats of the genus Rhinolophus are the bona-fide animal reservoir for SARS-related CoV2, an epithelial cell line was generated from the lung of Rhinolophus alcyone (Fig. 3a). One pregnant female individual was euthanized and embryonal tissues were prepared using techniques described previously (Fig. 3b)31–33. The resulting immortalized Rhinolophus lung cell SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 4 www.nature.com/scientificreports/ Figure 3. Generation of an hACE2-transgenic bat cell line for infection experiments with ORF8 variant rSCV. (a) Picture of an African Rhinolophus alcyone bat (copyright Victor Max Corman). Primary cell culture and immortalization of R. alcyone embryonic lung cells (b) was done as described in the Material and Methods section. To determine the RhiLu cell type the epithelial protein marker cytokeratin (c) and the fibroblast marker S-100A4 (d) were detected by immunofluorescence assay using mouse-anti cytokeratin or rabbit-anti S-100A4 Ig. Secondary detection was performed by incubating with goat-anti-mouse cyanin 2- or goat-anti-rabbit cyanin 3-labeled Igs. The bars represent 20 µm. (e) Integration of hACE2 into the genome of RhiLu cells was verified by PCR. The vector containing the hACE2-puromycin resistance gene construct was used as a PCR positive control. (f) Expression of hACE2 was confirmed by Western blot analysis using mouse anti-hACE2 Ig (1:1,000). Results B t i E In addition, β actin protein was detected using rabbit anti-β-actin Ig (1:2,000) to ensure that similar protein amounts were applied. MA104 cells, expressing hACE2 naturally, served as positive control. (g) Virus replication of rSCV8full, rSCVepi and rSCVdel8 was observed by titration of supernatants sampled at 24, 48, and 72 hpi. Cells were infected in triplicates at an MOI of 0.001. Error bars represent standard deviation of the mean. Figure 3. Generation of an hACE2-transgenic bat cell line for infection experiments with ORF8 variant rSCV. (a) Picture of an African Rhinolophus alcyone bat (copyright Victor Max Corman). Primary cell culture and immortalization of R. alcyone embryonic lung cells (b) was done as described in the Material and Methods section. To determine the RhiLu cell type the epithelial protein marker cytokeratin (c) and the fibroblast marker S-100A4 (d) were detected by immunofluorescence assay using mouse-anti cytokeratin or rabbit-anti S-100A4 Ig. Secondary detection was performed by incubating with goat-anti-mouse cyanin 2- or goat-anti-rabbit cyanin 3-labeled Igs. The bars represent 20 µm. (e) Integration of hACE2 into the genome of RhiLu cells was verified by PCR. The vector containing the hACE2-puromycin resistance gene construct was used as a PCR positive control. (f) Expression of hACE2 was confirmed by Western blot analysis using mouse anti-hACE2 Ig (1:1,000). In addition, β actin protein was detected using rabbit anti-β-actin Ig (1:2,000) to ensure that similar protein amounts were applied. MA104 cells, expressing hACE2 naturally, served as positive control. (g) Virus replication of rSCV8full, rSCVepi and rSCVdel8 was observed by titration of supernatants sampled at 24, 48, and 72 hpi. Cells were infected in triplicates at an MOI of 0.001. Error bars represent standard deviation of the mean. culture, termed RhiLu, clearly expressed cytokeratin as a marker for epithelium (Fig. 3c), and hardly showed any expression of the fibroblast marker S-100A4 (Fig. 3d), suggesting that the cell culture mainly contains epithelial cells. Initial infection experiments in RhiLu cells identified these cells to be non-permissive for SARS-CoV infec- tion. By use of VSV-G protein-pseudotyped lentiviral particles, RhiLu cells were therefore transduced with the gene for the SARS-CoV entry receptor, human angiotensin-converting enzyme 2 (hACE2), and selected to stably express hACE2 upon co-expression of a puromycin resistance gene from the same lentiviral vector. Chromosomal culture, termed RhiLu, clearly expressed cytokeratin as a marker for epithelium (Fig. 3c), and hardly showed any expression of the fibroblast marker S-100A4 (Fig. ARS-CoV ORF8 determines replication in non-host cell lines and in human airway epithelia lf SARS-CoV ORF8 determines replication in non-host cell lines and in human airway epithelial cultures. To determine whether the effect was more general and independent of host taxa, a low-passage air- way epithelial cell line from Sigmodon hispidus (cotton rat)34 and primary lung cell lines from Capra hircus (goat) and Ovis aries (sheep) were transduced using vesicular stromatitis virus G protein pseudotyped lentiviruses to transiently express hACE2 as described above (Fig. 4a), and subsequently infected with the ORF8 virus vari- ants at MOI = 0.001. Again the same hierarchy of viral replication levels, namely rSCV8full > rSCVepi > rSCVdel8, was observed at 24 hpi (Fig. 4b). Virus rSCV8full replicated significantly less efficiently than rSCVepi in all three “non-host” cell lines (t-Test: p = 0.001, p < 0.05, p < 0.01 for cotton rat, goat and sheep, respectively). Replication differences were levelled off by 48 hpi, when all viruses reached the plateau of replication. Of note, the utilized cell cultures in total represent hosts from four different orders of Euarchontoglires and Laurasiatheria, the two main groups of mammals. g p As the virus variants constructed in this study corresponded to viruses from the three major phases of the human SARS epidemic, we were interested to see whether the deletions have an effect on human respiratory tract infection as represented by in-vitro differentiated human airway epithelial cultures (HAE). Infection was done at MOI = 0.01, and virus production was measured by plaque titration after 48 and 72 hpi in supernatants. At both time points rSCV8full replicated to significantly higher titers than rSCVepi or rSCVdel8 in HAE (Fig. 4c). Results B t i E 3d), suggesting that the cell culture mainly contains epithelial cells. Initial infection experiments in RhiLu cells identified these cells to be non-permissive for SARS-CoV infec- tion. By use of VSV-G protein-pseudotyped lentiviral particles, RhiLu cells were therefore transduced with the gene for the SARS-CoV entry receptor, human angiotensin-converting enzyme 2 (hACE2), and selected to stably express hACE2 upon co-expression of a puromycin resistance gene from the same lentiviral vector. Chromosomal SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 5 www.nature.com/scientificreports/ Figure 4. Replication of ORF8 variants in different “non-SARS-CoV-host” cell lines and differentiated human airway epithelial cells. (a) Cotton rat, goat, and sheep cells were transduced with lentiviruses to transiently express the SARS-CoV receptor hACE2 for at least 72 h. Expression of hACE2 was verified by Western Blot analysis, detection of β actin served as a loading control. To improve clarity blots were cropped. Full length blots are presented in Supplementary Fig. S1. (b) Cells were infected in triplicates with different ORF8 variants at MOI 0.001 24 h after lentiviral transduction, 24 hpi supernatants were sampled and virus replication determined by plaque titration. (c) Differentiated human airway epithelial cells were infected in triplicates at MOI = 0.1, supernatants were sampled at 48 and 72 hpi for plaque titration. The experiment was done twice. Shown is one representative experiment. Error bars represent standard deviation of the mean. Significance of replication differences between virus variants was determined by t-Test (***p ≤ 0.001, **p ≤ 0.001, *p ≤ 0.05, n.s., not significant p > 0.05). Figure 4. Replication of ORF8 variants in different “non-SARS-CoV-host” cell lines and differentiated hum Figure 4. Replication of ORF8 variants in different “non-SARS-CoV-host” cell lines and differentiated human airway epithelial cells. (a) Cotton rat, goat, and sheep cells were transduced with lentiviruses to transiently express the SARS-CoV receptor hACE2 for at least 72 h. Expression of hACE2 was verified by Western Blot analysis, detection of β actin served as a loading control. To improve clarity blots were cropped. Full length blots are presented in Supplementary Fig. S1. (b) Cells were infected in triplicates with different ORF8 variants at MOI 0.001 24 h after lentiviral transduction, 24 hpi supernatants were sampled and virus replication determined by plaque titration. (c) Differentiated human airway epithelial cells were infected in triplicates at MOI = 0.1, supernatants were sampled at 48 and 72 hpi for plaque titration. The experiment was done twice. Results B t i E Shown is one representative experiment. Error bars represent standard deviation of the mean. Significance of replication differences between virus variants was determined by t-Test (***p ≤ 0.001, **p ≤ 0.001, *p ≤ 0.05, n.s., not significant p > 0.05). integration of the hACE2 gene and expression of the hACE2 protein were verified by RT-PCR and Western blot analysis (Fig. 3e, f). The resulting hACE2-transgenic cells termed RhiLu-hACE2 were highly permissive for SARS-CoV infection. RhiLu-ACE2 cells were infected with all three ORF8 virus variants at MOI = 0.001. Supernatants were har- vested 24, 48 and 72 hpi and titered. As shown in Fig. 3g, the same hierarchy of viral replication levels as in VeroFM cells was observed (rSCV8full > rSCVepi > rSCVdel8). These results did not point to a specific effect of the gene product of ORF8 on replication in bat cells but rather suggested a general promoting effect on virus replica- tion conferred by ORF8. RhiLu-ACE2 cells were infected with all three ORF8 virus variants at MOI = 0.001. Supernatants were har- vested 24, 48 and 72 hpi and titered. As shown in Fig. 3g, the same hierarchy of viral replication levels as in VeroFM cells was observed (rSCV8full > rSCVepi > rSCVdel8). These results did not point to a specific effect of the gene product of ORF8 on replication in bat cells but rather suggested a general promoting effect on virus replica- tion conferred by ORF8. Discussion Here we have shown by viral reverse genetics and advanced cell culture models that SARS-CoV ORF8 facilitates viral replication irrespective of the host cell system. These data suggest the occurrence of an attenuating mutation in the initial phase of the human SARS epidemic. p p SARSr-CoV is a paradigmatic pathogen for the study of viral reservoirs and the processes involved in epi- demic emergence8,14,15,35. The acquisition of certain spike proteins by recombination may have formed the viral lineage that emerged from the reservoir and established itself in humans during the SARS epidemic6–8. SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 6 www.nature.com/scientificreports/ The gradual deletion of ORF8 constituted the most obvious change in SARS-CoV after emergence. Because few changes occurred in other parts of the genome, we have focused our study on ORF8 and kept the rest of the genome constant representing the sequence of SARS-CoV strain Frankfurt-1. This prototype virus was isolated from the late phase of the SARS-CoV epidemic when transmission chains already occurred in countries outside China36. Even in this late epidemic strain, a reconstitution of the full ORF8 reading frame led to an increased replicative capability, suggesting that the viral genome was still compatible with its primordial ORF8 element in spite of possible onward evolution in humans9.f p p Compared to earlier observations with ORF8-deleted SARS-CoV, our results show a clear phenotypic differ- ence in replication in relevant models of human respiratory tract infection. The phenotypic difference depends on the presence of full ORF8, with enhanced replication as opposed to the deletion variant. Earlier experiments in which no clear differences in replication have been noted were aimed at the identification of strong attenuation markers as necessary for live vaccine development, and hence worked at very high MOIs at or above 122. We have also included such high MOIs in our experiments for comparison, and our results were highly concordant with those earlier studies. For instance, like in the study by Yount et al.22, differences between viruses with full ORF8 and 29nt deletions were only about three-fold at MOI = 1. Natural infection, however, does not involve high virus doses37. When a virus is passed from human to human, the stochastic nature of infection success implicates that inocula just below or above one unit of human-infectious virus are transmitted. For instance, during influenza A transmission in ferrets as few as 2 virus units were transmitted between animals38. Discussion Under conditions of inter-host transmission, the observed phenotypic differences as observed in our study may have significant effects on viral fitness37.i i In endemic viral infections, loss of fitness should cause viral lineage extinction in competition with more reproductively capable lineages that co-circulate within the host population39. After a single-time zoonotic intro- duction, however, competing viral lineages are unlikely to exist. Variants with slightly deleterious mutations, ran- domly selected through transmission bottlenecks, can continue to reproduce in spite of reduced fitness – an effect known as founder effect. Interestingly, it has been shown by in-vitro studies that virus populations with reduced initial fitness suffer less from slightly deleterious mutations than populations that replicate on peak fitness level40. Reduced initial fitness is a condition that can be expected in early-stage zoonotic epidemics when the virus is not yet adapted to the new host environment41. y p Considering the present results, we therefore suggest that the 29 nt deletion in SARS-CoV is the result of a founder effect that has permitted survival in spite of reduction of fitness. This interpretation contrasts with the earlier notion that the 29 nt deletion reflects adaptation to humans. The conclusion of adaptation is partly based on results from expression of ORF8 or its truncation products in overexpression systems or expression in heterologous virus genomes, suggesting various influences on virus-cell interaction16–21. For instance, one study provided evidence for IFN evasion mediated by ORF8b17. This is not confirmed by our experiments studying ORF8 in full virus context. Other authors have proposed that the 29 nt deletion may have been neutral for fit- ness after viral host transition to humans, assuming that ORF8 may elicit a function that is only relevant in the bat host9,22. However, we have not observed any bat-specific effects in cell culture, and rather show that ORF8 optimizes fitness irrespective of the host cell system, including hosts that are irrelevant for the SARS-CoV chain of emergence11. Our experiments suggest that only the deletion of further portions after initial fragmentation of ORF8 may have been neutral to fitness. Such deletions were seen during very late phases of the SARS epidemic in Hong Kong9. g g Deletions in accessory reading frames were also oberserved in MERS-CoV23,24,42,43. Transmission of deleted variants was confirmed in an outbreak in Jordan, involving deletions in ORF4a, ORF3 and potentially other parts of the genome23,24. Discussion The available studies leave it open whether these deletions involved changes of repli- cation level or virulence. However, it is known that ORF4a acts as an effective antagonist of MDA5-dependent induction of type I IFN44, and that MERS-CoV is highly sensitive against type I IFN in human airway epithelial cultures45. Based on these known mechanisms, attenuation rather than human adaptation should be considered. Our results are also relevant in the context of a series of experimental studies recently conducted to understand potential adaptive changes in the 2014 Ebola virus Makona outbreak in West Africa. Whereas initial studies suggested human adaptation with increase of replication level during the outbreak46–50, later studies found that a late-outbreak strain rather caused reduced virulence and prolonged survival of experimental animals51. These results provide another reminder of the fact that outbreak-associated mutations do not have to increase replica- tion or virulence. It is interesting to consider the consequences for viral propagation conferred by a reduction of viral replication level. As pointed out in Marzi et al. for Ebola virus Makona, a prolonged survival time as seen with late epidemic strains could have increased the duration of infectious virus shedding in humans and may thus have increased the long-term fitness of those viruses51. We cannot, at present, exclude whether similar effects may have pro- vided a fitness advantage to SARS-CoV on host population level, such as by keeping infected individuals socially interactive for prolonged times while infected with a slightly attenuated virus. On an individual level, however, a reduction of replication level is likely to attenuate the pathogenicity of infection and reduce the health burden caused by a given outbreak. y g It may be seen as a weakness in our present study that no experimental animals were infected as in Marzi et al. However, mouse models of SARS-CoV do not reflect human disease as accurate as macaques do for Ebola virus, and the HAE culture system used in the present study already provides an appropriate model for the authentic site of replication of SARS-CoV in the human body (Ebola virus infection cannot be modeled by organ-specific cultures). Moreover, the infection phenotypes as seen in our study for ORF8full and ORF8epi have already been demonstrated in mice, and the corresponding effects in cell culture based on those variants have been reproduced in our study22. Materials and Methods To determine the RhiLu cell type the epithelial protein marker cytokeratin and the fibroblast marker S-100A4 (calcium binding protein A4 or fibroblast specific protein 1) were stained by immunofluorescence assay using mouse anti-cytokeratin (ab7753) or rabbit anti-S-100A4 immu- noglobulins (Ig, ab27957; both supplied by abcam, Cambridge, UK). Secondary detection was performed by incubation with goat anti-mouse cyanin 2- or goat anti-rabbit cyanin 3-labeled Igs. Generation of a SARS-CoV susceptible bat cell line by lentiviral transduction. Since RhiLu cells were not susceptible to SARS-CoV the receptor hACE2 was stably transfected by lentiviral transduction58. Genomic integration of hACE was verified by PCR on genomic DNA. PCR was performed using a hACE2 gene specific for- ward primer (GAATGTAAGGCCACTGCTCAACTA) and a puromycin resistance gene specific reverse primer (TCAGGCACCGGGCTTGC) yielding a 1.9 kb amplicon. Expression of hACE2 was confirmed by Western blot analysis as described earlier58,59. Protein lysates were separated on a 10% SDS-PAGE gel, and blotted onto a 0.45 µm polyvinylidene fluoride membrane. Primary detection of hACE2 was done using a mouse anti-hACE2 Ig (1:1,000; R&D Systems, Wiesbaden-Nordenstadt, Germany), for secondary detection a goat anti-mouse horse- radish peroxidase (HRP)-conjugated Ig (1:20,000) and SuperSignal® West Femto Chemiluminescence Substrate (Fisher Scientific, Schwerte, Germany) were used60. As a loading control samples were analyzed for β actin expres- sion with a rabbit anti-βactin Ig (1:2,000; Sigma-Aldrich, Munich, Germany) and a goat anti-rabbit horseradish peroxidase-labeled Ig (1:20,000)61. Generation of recombinant SARS-CoV. Recombinant SCV was generated as previously described25. The single full-length ORF8 was generated by inserting 29 nts into ORF8a/b. In an overlap extension PCR two templates, generated by using primers F26020F (CGGCTCTTCAGGAGTTGCTA) in combination with 29nt-rev (TCCATTCAGGTTGGTAACCAGTAGGACAAGGATCTTCAAGCACATGA) and 29nt-fwd (CTGGTTA CCAACCTGAATGGAATATAAGGTACAACACTAGGGGTAATACT) with pB-fwd (GCCCTTAAACGCCT GGTTGCTAC), were fused. Underlined nts indicate overlapping regions leading to the introduction of 29 nts. The PCR product was inserted into subclone pEF via restriction sites BamHI and NotI. ORF8 was deleted from subclone pDEF by Phusion® Site-Directed Mutagenesis (Fisher Scientific) using 5′-phosphorylated primers delO8-1-fwd (AATGTCTGATAATGGACCCCAATCAAACCAACGTAGTGC) and delO8-1-rev (GTTCGTTTAGACTTTGGTACAAGGTTCTTCTAGATCC) or delO8-2-fwd (TAAAATGTCTGATAATGGA CCCCAATCAAACCAACG) and delO8-2-rev (TTGTTCGTTTAGACTTTGGTACAAGGTTCTTCTAGATCC). Underlined nts are the substitutional sequence for ORF8 compared to delO8-1. Assembly of full-length SARS-CoV genome plasmids and rescue of recombinant viruses were done as described before25. Briefly, full-length SARS-CoV plasmid was linearized by NotI and in-vitro transcribed (mMESSAGE mMACHINE® Kit, Applied Biosystems, Darmstadt, Germany). Materials and Methods Sample collection and processing. In total, 827 bats were sampled in four countries (Bulgaria, Italy, Slovenia, Spain). All animals were handled according to national and European legislation for the protection of animals (EU council directive 86/609/EEC). Licenses for sampling of bats using mist nets, hand nets or harp traps were obtained from the respective countries and authorities: Bulgarian Ministry of Environment and Water, permit No. 192/26.03.200913, Italien Ministry of the Environment, permit No. 192/26.03.200952, Slovenian Environment Agency, permit No. 35701-80/200453, Service for the Biodiversity Conservation of the Rural Counseling of the Xunta de Galicia, Spain, permit No. 52/2010 n.s. 13697. No animals were sacrificed during this study. All animal handling and sampling was done by trained personnel, with animal safety and comfort as the first priority during minimally invasive sampling (collection of faeces). Bat species were identified on site and, if necessary, mitochondrial DNA in representative fecal samples was amplified and sequenced for species confirma- tion as described previously54. Captured bats were freed from nets immediately and put into cotton bags for 2 to 15 min to allow them to calm down before examination. While being kept in bags, bats produced fecal pellets that were transferred to 500 µl RNAlater RNA stabilization solution (Qiagen, Hilden, Germany) for sample processing. After homogenization, 50 µl of the suspension was resuspended in 560 µl of buffer AVL from the Qiagen viral RNA minikit and processed according to manufacturers instructions. The elution volume was 50 µl. General cell culture procedures. Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) as described earlier31. For titration of rSCV Vero E6 cells (ATCC CRL-1586) were used. For infection studies African green monkey kidney cells, VeroFM (kindly provided by Jindrich Cinatl, University of Frankfurt) and MA104 (a gift from Friedemann Weber, University of Marburg), Rhinolophus alcyone (R.alcyone) embryonic lung cells (RhiLu, prepared in-house as described below), as well as bronchial epithelial cells from Sigmodon hispidus (cot- ton rat) and lung cells from Capra hircus (domestic goat) and Ovis aries (domestic sheep) were used55,56. HAE cultures were generated and cultured as described elsewhere57. Generation and characterization of R. alcyone lung cell cultures. Bats were caught in Ghana under research permit no. CHRPE49/09; A04957 Wildlife Division, Forestry Commission, Accra, Ghana. Primary bat cell culture and immortalization of RhiLu cells by lentiviral transduction of the simian virus 40 large T antigen and genotyping were done as previously described31,33. Discussion We have therefore avoided additional animal experimentation and focused on human epithelial models with low inoculation doses such as seen in natural infections. SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 7 www.nature.com/scientificreports/ Our data suggest that SARS-CoV has suffered an attenuating mutation by the 29 nt deletion that constitutes a landmark genetic change. The SARS epidemic in 2003 may have taken a more severe course if not involving this mutation. Further work, including work in experimental animals, will be required to understand and confirm whether the absence of ORF8 in European bat-associated SARSr-CoV correctly predicts lesser epidemic risks as compared to Asian strains. Materials and Methods Capped mRNA was electroporated into baby hamster kidney cells and supernatant was subsequently transferred to susceptible VeroFM cell culture 24 h post electropora- tion. Recombinant virus was harvested three days post infection. The ORF8 mutations were verified by PCR SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 8 www.nature.com/scientificreports/ using a specific reverse primer F29260R (TTTGTATGCGTCAATGTGCTTG) for reverse transcription and ORF8 covering forward primer F27626F (GAGAAAGACAGAATGAATGAGC) and reverse primer F28182R (GGGTCCACCAAATGTAATGCGG) for conventional PCR. Sequencing of the PCR product ensured the integrity of the introduced mutations. Expression of the nucleocpasid was confirmed by Western blot analysis as described above. Primary detection was done using a rabbit anti-nucleocapsid Ig (1: 500; abcam). For secondary detection a goat anti-rabbit HRP-conjugated Ig (1:20,000) and and SuperSignal® West Femto Chemiluminescence Substrate (Fisher Scientific) was used. Generation of transiently hACE2 expressing cell lines. Cells were seeded according to their size and growth rate to yield 80% confluence in 24-well plates. After attachment overnight, cells were infected with lenti- viruses to yield 50 ng reverse transcriptase activity per well in a reduced cultivation volume of 200 µL DMEM per well for at least 24 h. Expression levels of hACE2 were determined by Western blot analysis as described above in a time course experiment. Protein expression levels were constant for three days after transduction. Therefore, cells were infected with rSCV 24 h post transduction as described below. Virus infection. Cells (4 × 105 cells/mL) were seeded in 24-well plates and if necessary pre-incubated with 200 µl recombinant universal type I IFN alpha (PBL InterferonSource, Piscataway, USA) for 16 h prior to infec- tion. Infections with rSCV8full, rSCVepi and rSCVdel8 were done at an MOI ranging from 0.001 to 1. Virus was diluted in OptiPROTM serum-free medium or in HBSS buffer in case of HAE cultures. Cells were inoculated for 1 to 2 h at 37 °C, washed twice with PBS or HBSS, and supplied with fresh DMEM or HAE cell culture medium57. Supernatants were taken at designated time points usually 8, 24, 48, and 72 h post infection and stored at −70 °C for titration or real-time RT-PCR analysis. All virus containing samples were mixed with equal volumes of 0.5% gelatin in OptiPROTM (stock solution 5% gelatin in water) for stabilization of infectious particles. All infec- tion experiments were done under biosafetly level 3 conditions with enhanced respiratory personal protection equipment. Plaque titration. Materials and Methods Titration of rSCV was done as previously described25,62. Vero E6 cells (3.5 × 105 cells/mL) were infected with a serial dilution (in OptiPROTM) of virus infected cell culture supernatants for 1 h at 37 °C. After removing the inoculum cells were overlaid with 2.4% Avicel (FMC BioPolymers, Brussels, Belgium) 1:2 diluted in 2 × DMEM supplemented with 2% Penicillin/Streptomycin, 2% L-glutamine, 2% non-essential amino acids, 2% sodium pyruvate and 20% fetal bovine serum. Three days after infection the overlay was discarded, cells were fixed in 6% formaldehyde and stained with a 0.2% crystal violet, 2% ethanol and 10% formaldehyde containing solution.h g The datasets generated during and/or analysed during the current study are available from the corresponding uthor on reasonable request. References Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and l ifi ti f i b d ti l RNA d d t RNA l J Vi l 84 11336 11349 (2010) 3. Drexler, J. F. et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J Virol 84, 11336–11349 (2010) 4. Lau, S. K. et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronaviru 13. Drexler, J. F. et al. Genomic characterization of severe acute respiratory syndrome related coronavirus in European bats classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J Virol 84, 11336–11349 (20 14. Lau, S. K. et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Corona from Greater Horseshoe Bats through Recombination. Journal of virology 89, 10532–10547 (2015). i p p p y g q 14. Lau, S. K. et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination. Journal of virology 89, 10532–10547 (2015). 4. Lau, S. K. et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS Related Coronaviru from Greater Horseshoe Bats through Recombination. Journal of virology 89, 10532–10547 (2015). g f gy 15. Wu, Z. et al. ORF8-Related Genetic Evidence for Chinese Horseshoe Bats as the Source of Syndrome Coronavirus. The Journal of infectious diseases 213, 579–583 (2016). g f gy 5. Wu, Z. et al. ORF8-Related Genetic Evidence for Chinese Horseshoe Bats as the Source of Human Severe Acute Respiratory Syndrome Coronavirus. The Journal of infectious diseases 213, 579–583 (2016). yh f f 16. Chen, C. Y. et al. Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes replication but also induces apoptosis. The Journal of infectious diseases 196, 405–415 (2007). p p ph f f 17. Wong, H. H. et al. Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon sign h 17. Wong, H. H. et al. Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3. Virology 515, 165–175 (2018).h 18. Keng, C. T. et al. References 1. Jones, K. E. et al. Global trends in emerging infectious diseases. Nature 451, 990–993 (2008).i 2. Drexler, J. F., Corman, V. M. & Drosten, C. Ecology, evolution and classification of bat coronaviruses in the aftermath of S Antiviral research 101, 45–56 (2014). ( ) 3. Wolfe, N. D., Dunavan, C. P. & Diamond, J. Origins of major human infectious diseases. Nature 447, 279–283 (2007).h 4. Holmes, E. C., Dudas, G., Rambaut, A. & Andersen, K. G. The evolution of Ebola virus: Insights from the 2013-2016 epidemic Nature 538, 193–200 (2016).t 5. Graham, R. L., Donaldson, E. F. & Baric, R. S. A decade after SARS: strategies for controlling emerging coronaviruses. Nature reviews. Microbiology 11, 836–848 (2013). gy 6. Ge, X. Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538 (2013). 7. Yang, X. L. et al. Isolation and Characterization of a Novel Bat C 7. Yang, X. L. et al. Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus. Journal of virology 90, 3253–3256 (2015). 8. Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS pathogens 13, e1006698 (2017). y g p coronavirus. PLoS pathogens 13, e1006698 (2017). p g ( ) 9. Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 303, 1666–1669 (2004 g p 10. Chiu, R. W. et al. Tracing SARS-coronavirus variant with large genomic deletion. Emerging infectious diseases 11, 168–170 (20 11. Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276–278 (2003). 12. Lau, S. K. et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proceedings of the National Academy of Sciences of the United States of America 102, 14040–14045 (2005). y f f f 13. Drexler, J. F. et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J Virol 84 y f f f 3. Drexler, J. F. et al. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 21. Oostra, M., de Haan, C. A. & Rottier, P. J. The 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8. Journal of virology 81, 13876–13888 (2007). l d fi d f d l f 2. Yount, B. et al. Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential function for replication in cell cultures and mice. Journal of virology 79, 14909–14922 (2005). p f gy 3. Payne, D. C. et al. Multihospital Outbreak of a Middle East Respiratory Syndrome Coronavirus Deletion Variant, Jordan: A Molecular, Serologic, and Epidemiologic Investigation. Open Forum Infect Di 5 (2018). g p g g p f 4. Lamers, M. M. et al. Deletion Variants of Middle East Respiratory Syndrome Coronavirus from Humans, Jordan, 2015. Emerging infectious diseases 22, 716–719 (2016).f f 25. Pfefferle, S. et al. Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. Virology journal 6, 131 (2009).h g g p gy j , ( ) 26. Schelle, B., Karl, N., Ludewig, B., Siddell, S. G. & Thiel, V. Selective replication of coronavirus genomes that express nucleocapsid protein. Journal of virology 79, 6620–6630 (2005).l p f gy 7. Sola, I., Moreno, J. L., Zuniga, S., Alonso, S. & Enjuanes, L. Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. Journal of virology 79, 2506–2516 (2005). 8. McKimm-Breschkin, J. L. & Holmes, I. H. Conditions required for induction of interferon by rotaviruses and for their sensitivity to its action. Infect Immun 36, 857–863 (1982). 9. Emeny, J. M. & Morgan, M. J. Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. The Journal of general virology 43, 247–252 (1979). ph f g gy ( ) 0. Perlman, S. & Netland, J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7, 439–450 (2009). 30. Perlman, S. & Netland, J. Coronaviruses post SARS: update on replication and pathogenesis. Nat Rev Microbiol 7, 439 450 (2009). 31. Biesold, S. E. et al. Type I interferon reaction to viral infection in interferon-competent, immortalized cell lines from the African fruit bat Eidolon helvum. PloS one 6, e28131 (2011).h 2. Kuhl, A. et al. www.nature.com/scientificreports/ Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells. The Journal of infectiou diseases 204(Suppl 3), S840–849 (2011).h ( pp ) ( ) 33. Hofmann, A. et al. Epigenetic regulation of lentiviral transgene vectors in a large animal model. Mol Ther 13, 59–66 (2006). 33. Hofmann, A. et al. Epigenetic regulation of lentiviral transgene 4. Ehlen, L. et al. Epithelial cell lines of the cotton rat (Sigmodon hispidus) are highly susceptible in vitro models to zoonotic Bunya- Rhabdo-, and Flaviviruses. Virology journal 13, 74 (2016).f Rhabdo-, and Flaviviruses. Virology journal 13, 74 (2016). 35. Lau, S. K. et al. Ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related Rhinolophus bat coronavirus in China reveal bats as a reservoir for acute, self-limiting infection that allows recombination events. Journal of virology 84, 2808–2819 (2010).ih f gy ( ) 36. Drosten, C. et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. The New England journal of medicine 348, 1967–1976 (2003).h 37. Zwart, M. P. & Elena, S. F. Matters of Size: Genetic Bottlenecks in Virus Infection and Their Potential Impact on Evolution. Annu Rev Virol 2, 161–179 (2015).li 8. Varble, A. et al. Influenza A virus transmission bottlenecks are defined by infection route and recipient host. Cell Host Microbe 16 691–700 (2014). 39. Bergstrom, C. T., McElhany, P. & Real, L. A. Transmission bottlenecks as determinants of virulence in rapidly evolving pathogens. Proceedings of the National Academy of Sciences of the United States of America 96, 5095–5100 (1999).i 40. Novella, I. S., Elena, S. F., Moya, A., Domingo, E. & Holland, J. J. Size of genetic bottlenecks leading to virus fitness loss is determ by mean initial population fitness. Journal of virology 69, 2869–2872 (1995). y p pi f gy 41. McCrone, J. T. & Lauring, A. S. Genetic bottlenecks in intraspecies virus transmission. Curr Opin Virol 28, 20–25 (2018). 42. Xie, Q. et al. Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characte symptoms. Archives of virology 162, 2445–2449 (2017). 3. Lu, X. et al. Spike gene deletion quasispecies in serum of patient with acute MERS-CoV infection. Journal of medical virology 89 542–545 (2017). 44. Niemeyer, D. et al. Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist. J Virol 87, 12489–12495 (2013).fi 5. Kindler, E. et al. References The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells. Virology 354, 132–142 (2006). 19. Law, P. Y. et al. Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome- associated coronavirus. FEBS letters 580, 3643–3648 (2006). 18. Keng, C. T. et al. The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counte in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells. Virology 354, 132–142 (2006 19. Law, P. Y. et al. Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndr in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells. Virology 354, 132–142 (2006). 19. Law, P. Y. et al. Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome- associated coronavirus. FEBS letters 580, 3643–3648 (2006).h p associated coronavirus. FEBS letters 580, 3643–3648 (2006).h , ( ) 20. Sung, S. C., Chao, C. Y., Jeng, K. S., Yang, J. Y. & Lai, M. M. The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6. Virology 387, 402–413 (2009). SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 9 www.nature.com/scientificreports/ Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights It Zoonotic Potential. mBio 4 (2013). 46. Diehl, W. E. et al. Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic. Cell 167, 1088– e1086 (2016). ( ) 47. Urbanowicz, R. A. et al. Human Adaptation of Ebola Virus during the West African Outbreak. Cell 167, 1079–1087 e1075 (20 47. Urbanowicz, R. A. et al. Human Adaptation of Ebola Virus during the West African Outbreak. Cell 167, 1079–1087 e1075 (2016). 48. Dietzel, E., Schudt, G., Krahling, V., Matrosovich, M. & Becker, S. Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak. Journal of virology 91 (2017).fi p g 48. Dietzel, E., Schudt, G., Krahling, V., Matrosovich, M. & Becker, S. Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak. Journal of virology 91 (2017).fi f gy 49. Ueda, M. T. et al. Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency. Genes Cells 22, 148–159 (2017). 0. Wang, M. K., Lim, S. Y., Lee, S. M. & Cunningham, J. M. Biochemical Basis for Increased Activity of Ebola Glycoprotein in the 2013–16 Epidemic. Cell Host Microbe 21, 367–375 (2017).i 51. Marzi, A. et al. Recently Identified Mutations in the Ebola Virus-Makona Genome Do Not Alter Pathogenicity in Animal Models. Cell Rep 23, 1806–1816 (2018). p 2. Balboni, A., Palladini, A., Bogliani, G. & Battilani, M. Detection of a virus related to betacoronaviruses in Italian greater horseshoe bats. Epidemiol Infect 139, 216–219 (2011).i p f 3. Rihtaric, D., Hostnik, P., Steyer, A., Grom, J. & Toplak, I. Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophu hipposideros) in Slovenia. Archives of virology 155, 507–514 (2010).f pp ) f gy ( ) 54. Vallo, P., Guillen-Servent, A., Benda, P., Pires, D. B. & Koubek, P. Variation of mitochondrial DNA in the Hipposideros caffer complex (Chiroptera: Hipposideridae) and its taxonomic implications. Acta Chiropterol 10, 193–206 (2008). p 55. Eckerle, I. et al. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines. Emerging infectious diseases 20, 27 56. Eckerle, I. et al. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses. PloS one 9, e84679 (2014). p 55. Eckerle, I. et al. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines. Acknowledgements g We would like to thank Artem Siemens, Stephan Kallies,Tobias Bleicker, Sebastian Brünink, and Monika Eschbach-Bludau for excellent technical assistance. We are grateful to Matthias Lenk (Friedrich-Loeffler-Institut, Greifswald-Insel Riems) for providing goat and sheep cell lines. This study was supported by the Deutsche Forschungsgemeinschaft (DFG DR 772/12-1) and EU-funded project COMPARE GA no. 643476. Furthermore, we acknowledge support from the Deutsche Forschungsgemeinschaft (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin. www.nature.com/scientificreports/ Emerging infectious diseases 20, 2 k l l h l l ll l l f h d f l ( ) 55. Eckerle, I. et al. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines. Emerging infectious diseases 20, 276–279 (2014) 56. Eckerle, I. et al. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses. PloS one 9, e84679 (2014). p p y g g f 56. Eckerle, I. et al. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses. PloS one 9, e84679 (2014). 56. Eckerle, I. et al. Bat airway epithelial cells: a novel tool for the st 57. Dijkman, R. et al. Isolation and characterization of current human coronavirus strains in primary human epithelia cultures re differences in target cell tropism. Journal of virology (2013). differences in target cell tropism. Journal of virology (2013). f 58. Muller, M. A. et al. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines. MBio 3 (2012). 59. Muller, M. A. et al. Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein. Virology journal 7, 6 (2010). p gy j 60. Hofmann, H. et al. Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiote converting enzyme 2 and infection can be blocked by soluble receptor. Biochem Biophys Res Commun 319, 1216–1221 (2004). l h d d l h d ( ) k f f h 61. Morris, D. W. et al. An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis. Hum Mol Genet 23, 3316–3326 (2014). 62. Matrosovich, M., Matrosovich, T., Garten, W. & Klenk, H. D. New low-viscosity overlay medium for viral plaque assays. Virology journal 3, 63 (2006). SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 10 www.nature.com/scientificreports/ Author Contributions C.D., M.A.M., D.M. conceived and designed the experiments. V.M.C., F.G.R., A.B., M.B., D.R., I.T., R.S.A. and J.F.D. sampled and processed bat specimens. T.B., H.R. and L.T.G. established and characterized transgenic bat cell line. R.D., V.T. and A.P. provided essential materials. D.M. and L.T.G. performed cloning and infection experiments with recombinant viruses. D.M., C.D. and M.A.M. interpreted the data and wrote the manuscript with input from all authors. © The Author(s) 2018 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-33487-8 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. 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 Cre- ative 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 per- mitted 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/. SCIentIFIC RePorts | (2018) 8:15177 | DOI:10.1038/s41598-018-33487-8 11
https://openalex.org/W2473569893
https://europepmc.org/articles/pmc4944215?pdf=render
English
null
Structures and electrical properties of single nanoparticle junctions assembled using LaC2-encapsulating carbon nanocapsules
Scientific reports
2,016
cc-by
5,361
www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Structures and electrical properties of single nanoparticle junctions assembled using LaC2- encapsulating carbon nanocapsules received: 16 February 2016 accepted: 21 June 2016 Published: 14 July 2016 Manabu Tezura & Tokushi Kizuka As the miniaturization of integrated circuits advances, electronics using single molecules and nanosize particles are being studied increasingly. Single nanoparticle junctions (SNPJs) consist of two electrodes sandwiching a single nanoparticle. Nanocarbons with nanospaces in their center, such as fullerenes, carbon nanotubes, and carbon nanocapsules (CNCs), are expected to be elements of advanced SNPJs. In this study, SNPJs were assembled using lanthanum dicarbide (LaC2)-encapsulating CNCs and two gold (Au) electrodes by a nanotip operation inside a high-resolution transmission electron microscope. The atomic configuration and electrical resistance of the SNPJs were investigated in situ. The results implied that the electrical resistance of the SNPJ depended on the interface structures of the contacts between the CNC and Au electrodes, i.e., the contact electrical resistance, and the greatest portion of the current through the SNPJ flowed along the outermost carbon layer of the CNC. Thus, the resistance of the SNPJs using the CNCs was demonstrated and the electrical conduction mechanism of one of the CNC was discussed in this study. Carbon nanocapsules (CNCs) are hollow carbon-layered shells that are occasionally referred to as giant fullerene molecules1–4. Various pure metals and carbides are encapsulated in the nanospaces5–13. The first studies of CNCs encapsulating nanoparticles addressed the encapsulation of lanthanum dicarbide (LaC2)5,6. Such hollow and nanoparticle-encapsulating CNCs are expected to be major elements of single nanoparticle junctions (SNPJs)14–28. The electrical properties of SNPJs are affected by the encapsulated particles and the structures of the nanoparticle and contact interfaces between the nanoparticles and electrodes. However, the structures of each component and interface in SNPJs have not been observed; therefore, the relationships between the structures and electrical properties are unclear. In particular, although the resistance and its origin of one isolated nanoparticle, including CNCs, are essential to designing SNPJs, they remain an open question because the structure and resistance of the contact interfaces between the nanoparticles and electrodes have not been estimated. In situ transmission elec- tron microscopy (TEM) combined with nanotip operation techniques allows such simultaneous observations of structures and electrical properties29. In this study, we investigated the relationships between the structures and electrical properties of SNPJs assembled using LaC2-encapsulating CNCs. received: 16 February 2016 accepted: 21 June 2016 Published: 14 July 2016 Division of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305- 8573, Japan. Correspondence and requests for materials should be addressed to T.K. (email: kizuka@ims.tsukuba. ac jp) Results A /L C Au/LaC2-encapsulating CNC/Au. Figure 1(a) shows a high-resolution TEM image of an SNPJ assembled using a LaC2-encapsulating CNC and two gold (Au) electrodes (Supplementary Movie 1). The lower left and right dark regions are two Au electrodes biased positively and negatively, respectively. The regions around the CNC and Au electrodes correspond to vacuum. The diameters along the major and minor axes of the CNC are 18 and 12 nm, respectively. The CNC consists of 12 carbon layers. Figure 1(b) shows an enlarged image of the particle encapsulated in the center of the CNC shown in Fig. 1(a). The spacings of the lattice fringes observed in Fig. 1(b) are 0.20 and 0.18 nm, which correspond to the (112) and (200) planes of LaC2 with a tetragonal structure, respectively. Figure 2 shows the current–voltage characteristics of the SNPJ shown in Fig. 1(a), with fixed contact Division of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305- 8573, Japan. Correspondence and requests for materials should be addressed to T.K. (email: kizuka@ims.tsukuba. ac.jp) Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. (a) High-resolution TEM image of a Au/LaC2-encapsulating CNC/Au SNPJ; (b) enlarged image of the LaC2 particle encapsulated in the CNC center. The two Au electrodes in the lower left and right regions shown in (a) are biased positively and negatively, respectively. Figure 1. (a) High-resolution TEM image of a Au/LaC2-encapsulating CNC/Au SNPJ; (b) enlarged image of the LaC2 particle encapsulated in the CNC center. The two Au electrodes in the lower left and right region shown in (a) are biased positively and negatively, respectively. Figure 1. (a) High-resolution TEM image of a Au/LaC2-encapsulating CNC/Au SNPJ; (b) enlarged image of the LaC2 particle encapsulated in the CNC center. The two Au electrodes in the lower left and right regions shown in (a) are biased positively and negatively, respectively. Figure 2. Current–voltage characteristics of the SNPJ presented in Fig. 1(a). The resistance near 0 V is 4.2 kΩ. Figure 2. Current–voltage characteristics of the SNPJ presented in Fig. 1(a). The resistance near 0 V is 4.2 kΩ. interfaces between the CNC and Au electrodes. The current–voltage curve of the SNPJ is linear at bias voltages from −​50 to +​50 mV. The resistance near 0 V is 4.2 kΩ. Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 Results A /L C h We also measured the current of other SNPJs, in which the size of the CNCs and the contact areas between the CNC and the positive and negative electrodes were similar to those shown in Fig. 1, at bias voltages up to 500 mV. The current–voltage curve was linear at this voltage range with an almost constant differential conductance; the result was similar to that shown in Fig. 2. When higher bias voltage was applied, the encapsulated LaC2 nanopar- ticle moved outside the carbon shells. Therefore, the voltage was found to be critical for structural stability and electrical resistance measurements of the SNPJs. Figure 3 shows a time-sequence series of high-resolution images during the contact, retraction, compression, and separation process between the LaC2-encapsulating CNC and the negative Au electrode at the SNPJ shown in Fig. 1(a). In this process, the contact region between the CNC and the positive Au electrode was fixed. The applied bias voltage was fixed at 47 mV. The size of the contact region between the CNC and the negative Au electrode was controlled by manipulating the positive Au electrode. The electrical conductance (resistance) of the SNPJ and the contact areas during the process shown in Fig. 3 are illustrated in Fig. 4(a). To calculate the contact area, we assumed the shape of the contact area to be circular; the area was calculated from the diameter measured by the observed interface width. The times in Fig. 4(a–f) correspond to the observed times in Fig. 3(a–f), respectively. Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Time-sequence series of high-resolution TEM images of the contact region at the SNPJ shown in Fig. 1(a) during the contact, retraction, compression, and separation process between the CNC and the negative Au electrode: approaching the Au electrode (a); contact and retraction (b,c); compression (c–e); and separation (f). Both edges of the contact region between the carbon layers and Au electrode are indicated by two arrowheads, i.e., the contact region is located between the two arrowheads, as shown in (b–e). Figure 3. Time-sequence series of high-resolution TEM images of the contact region at the SNPJ shown in Fig. 1(a) during the contact, retraction, compression, and separation process between the CNC and the negative Au electrode: approaching the Au electrode (a); contact and retraction (b,c); compression (c–e); and separation (f). Results A /L C Both edges of the contact region between the carbon layers and Au electrode are indicated by two arrowheads, i.e., the contact region is located between the two arrowheads, as shown in (b–e). Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 3 www.nature.com/scientificreports/ Figure 4. (a) Variations in electrical conductance (resistance) and contact areas during the process presented in Fig. 3 as a function of time. (b) Variations in electrical resistance as a function of the inverse of the contact areas observed in Fig. 3(b–e). The applied bias voltage was 47 mV. The times indicated by a–f in (a) correspond to the recording times of the images in Fig. 3(a–f), respectively. The cross in (a) indicates fracture. The dashed line in (b) is a linear approximation. Figure 4. (a) Variations in electrical conductance (resistance) and contact areas during the process presented in Fig. 3 as a function of time. (b) Variations in electrical resistance as a function of the inverse of the contact areas observed in Fig. 3(b–e). The applied bias voltage was 47 mV. The times indicated by a–f in (a) correspond to the recording times of the images in Fig. 3(a–f), respectively. The cross in (a) indicates fracture. The dashed line in (b) is a linear approximation. First, the CNC was allowed to approach the Au electrode (Fig. 4(a), time a), followed by contact and retraction (Fig. 3(b–c)) and compression (Fig. 3(c–e)). When the contact area became 54 nm2 (Fig. 3(b)), the electrical conductance increased to 0.5 mS (Fig. 4(a), time b). Then, the electrical conductance decreased as the CNC was pulled back from the Au electrode. When the contact area decreased to 36 nm2 (Fig. 3(c)), the electrical conduct- ance decreased to 0.3 mS (Fig. 4(a), time c). Subsequently, the electrical conductance increased (Fig. 4, time c–e) as the contact area increased again (Fig. 3(c–e)). Finally, when the contact area increased to 91 nm2 (Fig. 3(e)), the electrical conductance increased to 1.2 mS (Fig. 4(a), time e). The electrical conductance (resistance) of the SNPJ shown in Fig. 1(a) was changed by the contact area of the interface between the CNC and the Au electrode. Since the electrical resistance depends on the inverse of the contact area, we plot the electrical resistance against the inverse of the areas in Fig. 4(b). A linear relationship was found between both parameters. Electrical resistance of capsule part. Results A /L C Figure 5 shows a high-resolution TEM image of an SNPJ assembled using a LaC2-encapsulating CNC and two Au electrodes, similar to the case in Fig. 1(a). Figure 6 shows enlarged images of the interface between the carbon layers and the negative Au electrode in the SNPJ shown in Fig. 5. The sizes of the contact regions shown in Fig. 6(a,b) were controlled by manipulating the negative Au electrode with a fixed contact region between the CNC and the positive Au electrode (Supplementary Movie 2). The spacings of the lattice fringes observed in Fig. 6(a) are 0.34 and 0.24 nm, which correspond to (0002)carbon layers and (111)Au, respectively. The widths of the interfaces between the carbon layers and the negative Au electrode shown in Fig. 6(a,b) are 3.4 nm and 5.0 nm, respectively. The total electrical resistance of the SNPJ (Rt Ω) is estimated using the following formula: α α = + + = + + R R R R A R A / / (1) t Au c Au 1 1 c 2 2 1 2 (1) where RAu1 Ω and RAu2 Ω, Rc Ω, α1 Ω nm2 and α2 Ω nm2, and A1 nm2 and A2 nm2 are the contact electrical resist- ance between the CNC and the Au electrodes 1 and 2, the electrical resistance of the capsule part in the SNPJ, the contact electrical resistivity between the CNC and the Au electrodes 1 and 2, and the contact area between the CNC and the Au electrodes 1 and 2, respectively. The index number 1 or 2 is given by a case between the CNC and the positive or negative Au electrode. Here, we assumed that α​1 is approximately equal to α​2; thus, Equation (2) is given as follows. Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. High-resolution TEM image of a Au/LaC2-encapsulating CNC/Au SNPJ. Au(+) and Au(−) show the positive and negative Au electrodes, respectively. Figure 5. High-resolution TEM image of a Au/LaC2-encapsulating CNC/Au SNPJ. Au(+) and Au(−) show the positive and negative Au electrodes, respectively. Figure 6. Enlarged images of the interface between the carbon layers and the negative Au electrode in the SNPJ shown in Fig. 5. The two large arrowheads indicate the edges of the contact region, i.e., the contact region is located between the two arrowheads. Figure 6. Results A /L C Enlarged images of the interface between the carbon layers and the negative Au electrode in the SNPJ shown in Fig. 5. The two large arrowheads indicate the edges of the contact region, i.e., the contact region is located between the two arrowheads. α = + + R R A A /( ) (2) t c 1 1 2 (2) α = + + R R A A /( ) t c 1 1 2 Rt and A1, A2 were 9.7 kΩ and 80.5 nm2, 9.1 nm2 (Fig. 6(a)) and 6.6 kΩ and 80.5 nm2, 19.4 nm2 (Fig. 6(b)), respec- tively. By substituting these values in Equation (2), Rc was obtained as 3.2 kΩ, and Rc, RAu1, and RAu2 were 33%, 7%, and 60% of Rt, respectively (Fig. 6(a)).h Rt and A1, A2 were 9.7 kΩ and 80.5 nm2, 9.1 nm2 (Fig. 6(a)) and 6.6 kΩ and 80.5 nm2, 19.4 nm2 (Fig. 6(b)), respec- tively. By substituting these values in Equation (2), Rc was obtained as 3.2 kΩ, and Rc, RAu1, and RAu2 were 33%, 7%, and 60% of Rt, respectively (Fig. 6(a)).h t p y g In this study, contact electrical resistivity is expressed using a unit of Ω∙nm2 30,31. This is because we quoted the values of the contact electrical resistivity of graphene, which was expressed using Ω∙nm2 from previous stud- ies32,33, as described in discussion, and developed the expression for the contact electrical resistance between the CNC and the Au electrode (Equation (1)). Discussion h l was measured from the observed length between the positive and negative Au electrodes along the outermost carbon layer of the CNC. w is the perimeter of the contact area between the CNC and the negative Au electrode, and was calculated from the observed interface widths. The contact area between the CNC and the negative Au electrode was less than that of the positive electrode, as shown in Fig. 5, and the resistance of the graphene layers was dominated by the smaller contact area. Thus, we focused on the contact area between the CNC and the negative Au electrode using w in the contact region of the negative Au electrode to cal- culate Rg. When ρ​s (1.8 Ω per square38), l, and w were substituted into Equation (3), Rg corresponding to the outer- most layer of the CNC shown in Fig. 5 was approximately 3.0 kΩ. This value is similar to that of Rc in Equation (2). In addition, the relationships between Rc and Rg in the other three SNPJs assembled using LaC2-encapsulating CNCs shown in Fig. 7 were investigated. The results are shown in Table 1. We found that the Rc and Rg for each CNC show similar values.h There are wrinkles and kinks in the carbon layers of the CNC and it is considered that these contribute to current flows between the layers. Since it is difficult to evaluate the characteristics and quantity of these defects quantitatively, we used a simple model, in which the outermost layer of the CNC corresponded to graphene, and calculated the resistance of the CNC. As a result, we found that the measured resistance of the CNC agreed well with the calculated resistance using this simple model. This result suggests that although a certain amount of the winkles and kinks contributed to the current through between the layers, the contribution was substantially low in comparison with that of the current along the outermost layer. p g y In this study, we operated the electron microscope with an acceleration voltage of 200 kV and thus we also need to consider the introduction of radiation damages to the carbon layers and the effect on the conductance39. To reduce radiation damages as possible, we decreased the electron beam current to less than 4 A/cm2 and the total observation time to shorter than 1.5 min. Discussion h As shown in Figs 4(b) and 6, Rt decreased as A2 increased. According to Equation (2), RAu1 and RAu2 decreased as A1 and A2 increased. The contact area between the CNC and the Au electrode of the SNPJ increased; thus, the contact electrical resistance decreased, resulting in decreased total electrical resistance of the SNPJ. In this study, the sum of RAu1 and RAu2 reached 67% of Rt. Since the projected densities of states (PDOSs) of the π​-orbitals of the graphene and the d-orbitals of the Au at near-Fermi energy overlap by a small amount, the electrical coupling of the metal with graphene becomes weak and the number of conduction channels decreases34. In addition, the transmission function near the Fermi energy mirrors the PDOS behaviors; therefore, the contact electrical resist- ance of the graphene and Au junctions becomes greater than that of the graphene and other metal junctions34. Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 5 www.nature.com/scientificreports/ SNPJ encapsulating CNC width (nm) minor the number of carbon layers resistance (kΩ) particles major Rc Rg Fig. 5 LaC2 26 21 17 3.2 ±​ 0.4 3.0 ±​ 0.9 Fig. 7(a) // 27 17 14 1.8 ±​ 0.4 2.4 ±​ 0.9 Fig. 7(b) // 25 21 10 2.3 ±​ 0.4 2.4 ±​ 0.9 Fig. 7(c) // 28 27 16 2.8 ±​ 0.4 2.4 ±​ 0.9 Table 1. Resistance of Au/LaC2-encapsulating CNC/Au SNPJs shown in Figs 5(a) and 7(a–c). Table 1. Resistance of Au/LaC2-encapsulating CNC/Au SNPJs shown in Figs 5(a) and 7(a–c). The I–V characteristics of the SNPJ presented in Fig. 1(a) show a linear relationship and no current region near 0 V, as shown in Fig. 2. These features are the same as those of graphene and the metal junctions35,36. Since the electrical conductivity of graphite along the in-plane direction is 104 times greater than that along the c-axis direction37, we presume that the greatest portion of the current through the SNPJ flowed along the outermost carbon layer of the CNC. Here, we consider the electrical resistance of the graphene layers (Rg) corresponding to the outermost layer of the CNC. Rg is expressed as follows36: = ρ R l w ( / ), (3) g s = ρ R l w ( / ), g s (3) where ρ​s Ω per square, l nm, and w nm are the sheet resistance of graphene layers, the channel length, and the channel width, respectively. Discussion h During the image observation and the conductance measurement, no variation in the interlayer spacing of the carbon layers and no additional formation of radiation defects were observed although a certain amount of the radiation defects were inferred to be introduced. In this study, the major variation in electrical conductance of the SNPJ was observed when the contact area between the CNC and Au electrode was changed. Thus, although the introduction of a certain amount of the radiation damages also contributed to the variation in conductance, the contribution was minor and the total conductance was governed by the control of the contact areas.h y The contact areas of the interfaces observed in this study were 30−​90 nm2, which were near the lower limit for applying the Maxwell resistance approximation. As shown in the Fig. 4(b), since the inverse of the areas and the resistance have a linear relationship, it is considered that the approximation can be applied to the observed contact interfaces. In summary, we investigated the structures and electrical properties of SNPJs assembled using LaC2-encapsulating CNCs. The electrical resistance of the CNC was estimated to be similar to that of the graphene layers corresponding to the outermost layer of the CNC, implying that the greatest portion of the current through the SNPJ flowed along the outermost carbon layer of the CNC. The contact electrical resistance decreased as the contact area between the CNC and the Au electrodes of the SNPJ increased; therefore, the total electrical resistance decreased. Based on this observation, the resistance and its origin of one isolated nanoparticle were evaluated. The contact electrical resistance was 67% of the total electrical resistance. This suggests that the electrical resistance of the SNPJ was significantly affected by the contact resistance. In this study, an electrical conduction mechanism of the SNPJ using the CNC was revealed. These results are expected to be applied to the design of SNPJs. References H., Guinea, F., Peres, N. M. R., Novoselov, K. S. & Geim, A. K. The electronic properties of graphene. Rev. Mod. Phys. 81, 109–162 (2009).hfi ( ) 36. Peng, S.-A. et al. The sheet resistance of graphene under contact and its effect on the derived specific contact resistivity. Carbon 82, 500–505 (2015). ( ) 37. Primak, W. & Fuchs, L. H. Electrical conductivities of natural graphite crystals. Phys. Rev. 95, 22–30 (1954). 37. Primak, W. & Fuchs, L. H. Electrical conductivities of natural graphite crystals. Phys. Rev. 95, 22 30 (1954). 38. Wang, X., Zhi, L. & Müllen, K. Transparent, conductive graphene electrodes for dye-sensitized solar cells. Nano Lett. 8, 323–327 (2008).f ( ) 39. Smith, B. W. & Luzzi, D. E. Electron irradiation effects in single wall carbon nanotubes. J. Appl. Phys. 90, 3509–3515 (2001). Methodsh Thus, to improve accuracy of the production and the control of SNPJs, we selected LaC2 as encapsulated nanoparticles. References Acknowledgementsh g The author (M.T.) thanks to Dr. Tomo-o Terasawa of University of Tsukuba for useful discussion. The authors (M.T. and T.K.) thank the members of our laboratory for help with a part of experiment. This study was partly supported by Cross-ministerial Strategic Innovation Promotion Program–Unit D66–Innovative measurement and analysis for structural materials, and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos 26630032 and 16H03839). References & Venkataraman, L. Single molecule junctions beyond electronic transport. Nat. Nanotechnol. 8, 399 410 (2013). 27 Evangeli, C et al Engineering the thermopower of C60 molecular junctions Nano Lett 13 (2013) y g j y p ( 27. Evangeli, C. et al. Engineering the thermopower of C60 molecular junctions. Nano Lett. 13 (2013). 27. Evangeli, C. et al. Engineering the thermopower of C60 molecular junctions. Nano Lett. 13 (2013). g g g j 8. Matsuura, D. & Kizuka, T. Electrical conductivity of single molecular junctions assembled from Co- and Co3C-encapsulating carbon nanocapsules. J. Nanosci. Nanotechnol. 14, 2441–2445 (2014).i 28. Matsuura, D. & Kizuka, T. Electrical conductivity of single molecular junctions assembled fro nanocapsules. J. Nanosci. Nanotechnol. 14, 2441–2445 (2014).i 28. Matsuura, D. & Kizuka, T. Electrical conductivity of single mole nanocapsules. J. Nanosci. Nanotechnol. 14, 2441–2445 (20i p 29. Kizuka, T. Atomic configuration and mechanical and electrical properties of stable gold wires of single-atom width. Phys. Rev. B 77, 155401 (2008). 30. Yu, A. Y. C. Electron tunneling and contact resistance of metal-silicon contact barriers. Solid·State Electron. 13, 239–247 (1970 31 Lin M E et al Low resistance ohmic contacts on wide band gap GaN Appl Phys Lett 64 1003 1005 (1994) 30. Yu, A. Y. C. Electron tunneling and contact resistance of metal-silicon contact barriers. Solid·State Electron. 13, 2 30. Yu, A. Y. C. Electron tunneling and contact resistance of metal-silicon contact barriers. Solid·State Electron. 13, 239–247 (1970). 31. Lin, M. E. et al. Low resistance ohmic contacts on wide band-gap GaN. Appl. Phys. Lett. 64, 1003–1005 (1994). 30. Yu, A. Y. C. Electron tunneling and contact resistance of metal-silicon contact barriers. Solid·State Electron. 13, 239–247 (1970). 31. Lin, M. E. et al. Low resistance ohmic contacts on wide band-gap GaN. Appl. Phys. Lett. 64, 1003–1005 (1994). g 31. Lin, M. E. et al. Low resistance ohmic contacts on wide band-gap GaN. Appl. Phys. Lett. 64, 1003–1005 (1994). g pp y 32. Venugopal, A., Colombo, L. & Vogel, E. M. Contact resistance in few and multilayer graphene devices. Appl. Phys. Lett. 96, 01 (2010). 33. Moon, J. S. et al. Ultra-low resistance ohmic contacts in graphene field effect transistors. Appl. Phys. Lett. 100, 203512 (2012). if 34. Matsuda, Y., Deng, W.-Q. & Goddard, W. A. Contact resistance properties between nanotubes and various metals from quan mechanics. J. Phys. Chem. C 111, 11113–11116 (2007).h 35. Castro Neto, A. References 1. Heidenreich, R. D., Hess, W. M. & Ban, L. L. A test object and criteria for high resolution electron microscopy. J. Appl. Crystallogr. 1 1–19 (1968). ( ) 2. Iijima, S. Direct observation of the tetrahedral bonding in graphitized carbon black by high resolution electron microscopy. J. C Growth 50, 675–683 (1980). , ( ) 3. Smith, P. P. & Buseck, P. R. Graphitic carbon in the allende meteorite: a microstructural study. Science 212, 322 (1981). 3. Smith, P. P. & Buseck, P. R. Graphitic carbon in the allende meteorite: a microstructural study. Science 212, 322 (1981) 4. Ugarte, D. Curling and closure of graphitic networks under electron-beam irradiation. Nature 359, 707–709 (1992). 4. Ugarte, D. Curling and closure of graphitic networks under electron-beam irradiation. Nature 359, 707–709 (199 5. Tomita, M., Saito, Y. & Hayashi, T. LaC2 encapsulated in graphite nano-particle. Jpn. J. Appl. Phys. 32, L280 (1993). 6. Rodney, S. R., Donald, C. L., Bryan, C., Ripudaman, M. & Shekhar, S. Single crystal metals encapsulated in carbon nanoparticles Science 259, 346 (1993). 7. Ugarte, D. How to fill or empty a graphitic onion. Chem. Phys. Lett. 209, 99–103 (1993). 7. Ugarte, D. How to fill or empty a graphitic onion. Chem. Phys. L gi p y g p y 8. Majetich, S. A., Artman, J. O., McHenry, M. E., Nuhfer, N. T. & Staley, S. W. Preparation and properties of carbon-coated magnetic nanocrystallites. Phys. Rev. B 48, 16845–16848 (1993). i 8. Majetich, S. A., Artman, J. O., McHenry, M. E., Nuhfer, N. T. & Staley, S. W. Preparation and nanocrystallites. Phys. Rev. B 48, 16845–16848 (1993). 9. Brunsman, E. M. et al. Magnetic properties of carbon-coated, ferromagnetic nanoparticles produced by a carbon-arc method. J Appl. Phys. 75, 5882–5884 (1994). pp y 10. Hihara, T. et al. Magnetic properties of Iron in nanocapsules. Jpn. J. Appl. Phys. 33, L24 (1994).h 11. El-Gendy, A. A. et al. The synthesis of carbon coated Fe, Co and Ni nanoparticles and an examination of their magnetic properties. Carbon 47, 2821–2828 (2009).i 2. Saito, Y. Nanoparticles and filled nanocapsules. Carbon 33, 979–988 (1995).i 12. Saito, Y. Nanoparticles and filled nanocapsules. Carbon 33, 979–988 (1995).i i 13. Saito, Y. & Matsumoto, T. Hollow and filled rectangular parallelopiped carbon nanocapsules catalyzed by calcium and strontiu Cryst. Growth 187, 402–409 (1998). y 14. Reed, M. A., Zhou, C., Muller, C. J., Burgin, T. P. Methodsh The experimental method in this study was developed based on in situ TEM combined with piezomanipula- tion of nanotips and electric conductance measurements29. LaC2-encapsulating CNCs were synthesized via a gas evaporation method using dilanthanum trioxide (La2O3) and carbon5,6 evaporation sources. The synthesized 6 Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. High-resolution TEM images of three Au/L h h i i d i A l d i Figure 7. High-resolution TEM images of three Au/ show the positive and negative Au electrodes respectiv Figure 7. High-resolution TEM images of three Au/LaC2-encapsulating CNC/Au SNPJs. Au(+​ ) and Au(−) show the positive and negative Au electrodes, respectively. Figure 7. High-resolution TEM images of three Au/LaC2-encapsulating CNC/Au SNPJs. Au(+​ ) and Au(−) show the positive and negative Au electrodes, respectively. LaC2-encapsulating CNCs were dispersed on the surfaces of a Au nanotip (the positive electrode). The nanotip, on which the CNCs were dispersed and a bare Au nanotip (the negative electrode) were mounted on the first and second sample holders, respectively and were inserted into a transmission electron microscope equipped with two goniometer stages and a sample piezodriving system. Inside the microscope, the negative Au electrode was manipulated using the piezodriving system at the picometer scale to pick up one CNC on the positive Au Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 7 www.nature.com/scientificreports/ electrode, followed by sandwiching the CNC between the two electrodes. Bias voltage was then applied between the Au electrodes at room temperature at a vacuum of 1 ×​ 10−5 Pa. electrode, followed by sandwiching the CNC between the two electrodes. Bias voltage was then applied between the Au electrodes at room temperature at a vacuum of 1 ×​ 10−5 Pa. electrode, followed by sandwiching the CNC between the two electrodes. Bias voltage was then applied between the Au electrodes at room temperature at a vacuum of 1 ×​ 10−5 Pa. p It is essential for this study to assemble controllable SNPJs. For this purpose, highly reproducible stable CNCs were required. The encapsulation of LaC2 into CNCs satisfies this requirement. Thus, to improve accuracy of the production and the control of SNPJs, we selected LaC2 as encapsulated nanoparticles. p It is essential for this study to assemble controllable SNPJs. For this purpose, highly reproducible stable CNCs were required. The encapsulation of LaC2 into CNCs satisfies this requirement. References & Tour, J. M. Conductance of a molecular junction. Science 278, 252–254 (19 15 P k H t l N h i l ill ti i i l C t i t N t 407 57 60 (2000) 14. Reed, M. A., Zhou, C., Muller, C. J., Burgin, T. P. & Tour, J. M. Conductance of a molecular junction. Science 278, 252–254 (1997). 15. Park, H. et al. Nanomechanical oscillations in a single-C60 transistor. Nature 407, 57–60 (2000). g j 15. Park, H. et al. Nanomechanical oscillations in a single-C60 transistor. Nature 407, 57–60 (2000). 16. Taylor, J., Guo, H. & Wang, J. Ab initio modeling of quantum transport properties of molecular electronic devices. Phys. Rev. 245407 (2001). 17. LeRoy, B. J., Lemay, S. G., Kong, J. & Dekker, C. Electrical generation and absorption of phonons in carbon nanotubes. Nature 432, 371–374 (2004). 18. Yaish, Y. et al. Electrical nanoprobing of semiconducting carbon nanotubes using an atomic force microscope. Phys. Rev. Let 046401 (2004). ( ) 19. Dadosh, T. et al. Measurement of the conductance of single conjugated molecules. Nature 436, 677–680 (2005).h g j g 20. LeRoy, B. J., Kong, J., Pahilwani, V. K., Dekker, C. & Lemay, S. G. Three-terminal scanning tunneling spectroscopy of suspended carbon nanotubes. Phys. Rev. B 72, 075413 (2005). y 21. Tao, N. J. Electron transport in molecular junctions. Nat. Nanotechnol. 1, 173–181 (2006). 22. Asaka, K., Kato, R., Yoshizaki, R., Miyazawa, K. & Kizuka, T. Conductance of carbon nanocapsule junctions. Phys. Rev. B 76, 11 (2007).hh 23. Sun, C. Q. Thermo-mechanical behavior of low-dimensional systems: The local bond average approach. Prog. Mater. Sc 179–307 (2009). ( ) 24. Huang, T. et al. A molecular switch based on current-driven rotation of an encapsulated cluster within a fullerene cage. Nano Lett. 11, 5327–5332 (2011). 25. Néel, N., Kröger, J. & Berndt, R. Two-level conductance fluctuations of a single-molecule junction. Nano Lett. 11, 3593–3596 (2011). 26. Aradhya, S. V. & Venkataraman, L. Single-molecule junctions beyond electronic transport. Nat. Nanotechnol. 8, 399–410 (2013). 27. Evangeli, C. et al. Engineering the thermopower of C60 molecular junctions. Nano Lett. 13 (2013). gl g j 6. Aradhya, S. V. & Venkataraman, L. Single-molecule junctions beyond electronic transport. Nat. Nanotechnol. 8, 399–410 (2013). l l h h f l l ( ) l 26. Aradhya, S. V. & Venkataraman, L. Single-molecule junctions beyond electronic transport. Nat. Nanotec 26. Aradhya, S. V. Author Contributions T.K. developed the experimental setup of the in situ TEM. M.T. and T.K. designed this experiment. M.T. performed the experiment and data analysis under the direction of T.K. M.T. and T.K wrote the manuscript. 8 Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Tezura, M. and Kizuka, T. Structures and electrical properties of single nanoparticle junctions assembled using LaC2-encapsulating carbon nanocapsules. Sci. Rep. 6, 29708; doi: 10.1038/srep29708 (2016). 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/ Scientific Reports | 6:29708 | DOI: 10.1038/srep29708 9
https://openalex.org/W2146068366
https://scielo.conicyt.cl/pdf/jtaer/v8n3/art06.pdf
English
null
Incentives to Apply Green Cloud Computing
Journal of theoretical and applied electronic commerce research
2,013
cc-by
12,742
Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Incentives to Apply Green Cloud Computing Tommi Makela1 and Sakari Luukkainen2 Aalto University, Department of Computer Science and Engineering, Espoo, Finland, 1 tommi.makela@aalto.fi, 2 sakari.luukkainen@aalto.fi Received 29 March 2013; received in revised form 6 August 2013; accepted 22 August 2013 Received 29 March 2013; received in revised form 6 August 2013; accepted 22 August 2013 Keywords: Private cloud, Hosting service, Derived demand, Efficiency metric, Green service leve agreement 1 Introduction The information and communication technology (ICT) industry utilizes a variety of equipment and infrastructures to offer services. Large-scale infrastructures that host equipment are referred to as data centers. Companies can either own a data center or they can rent floor space from data centers maintained by commercial actors. Even though the ICT industry can be beneficial for environment, such as by reducing the need for travelling, it also causes plenty of emissions when the equipment used by the ICT industry is designed, manufactured, operated, and disposed. The research group Gartner estimates that the ICT industry produced up to 2% of the worldwide carbon dioxide (CO2) emissions in 2007. The figure included most of the equipment and infrastructures used by enterprises and governments, but also computers and mobile phones purchased by consumers. Gartner forecasts that financial and legislative issues would eventually force the ICT industry to become greener in future. Furthermore, Gartner predicts that customers will start to demand more environmentally friendly ICT services. (Site 1) In addition to environmental awareness, cloud computing is an another trend which has had a major impact on the ICT industry. Companies no longer need to possess IT equipment or plan resource provision to provide services, because they can purchase all of the required resources from cloud providers, which in theory have an unlimited amount of resources. Proper resource provision has typically been problematic, because under-provision causes revenue losses while over-provision wastes capital and electricity. Cloud computing allows companies to eliminate their up-front costs and shift them to operational costs. Furthermore, companies only need to pay for resources that they actually use, which improves the companies' ability to adapt to market changes and avoid potential losses when demand suddenly decreases. [2] However, these problems become cloud providers’ problems and it is up to them to optimize computation and minimize latencies in order to gain some profits [8]. Cloud computing should strive for more green computation, because cloud resources are presumably highly utilized, when compared to the traditional client-server model [9]. However, what are the most green cloud configurations in the market? Furthermore, what are the incentives for companies to apply more green computation, especially when cloud services have characteristic threads, including data compromises, insecure interfaces, and vulnerable shared infrastructures [4]? 2 Literature Review The main literature findings related to green cloud computing are presented in this section. 1 Introduction To study these questions, this paper is divided into three main sections: a literature review with the evaluation of green computing, case studies of novel green hosting services for private clouds, and a discussion about the incentives of green cloud computing. Abstract In recent years, there have been two major trends in the ICT industry: green computing and cloud computing. Green computing implies that the ICT industry has become a significant energy consumer and consequently, a major source of CO2 emissions. Cloud computing makes it possible to purchase IT resources as a service without upfront costs. In this paper, the combination of these two trends, green cloud computing, will first be evaluated based on existing research findings, which indicate that private clouds are the most green option to offer services. Hosting of private clouds can be outsourced, which allows companies to focus on their core competences. Furthermore, three case studies of state-of-the-art companies offering green hosting services are presented and incentives affecting their energy-efficiency development are analyzed. The results reveal that currently there is no demand in the market for green hosting services, because the only incentive for companies is low costs. Service providers should illustrate their greenness with transparent efficiency metrics, draw up green service level agreements and compete with greenness. Then it is up to end users to require more green web services and create derived demand for green cloud services and green hosting services. Keywords: Private cloud, Hosting service, Derived demand, Efficiency metric, Green service level agreement 74 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Incentives to Apply Green Cloud Computing This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile 2.1 Green Computation Microsoft has studied if its cloud services are more green than its on-premise applications for business customers. Three business applications, Exchange, Dynamics CRM, and SharePoint, were selected for comparison. The environmental impacts of these applications were defined by using small, medium, and large user groups. The comparison indicates that cloud services, which provided applications for a large group of 10 000 users, had 30% smaller carbon footprint than on-premise solutions. When a small group of 1 000 users utilized these products, an even larger gap existed, because cloud services released 90% less CO2 emissions than on-premise solutions. [1] Microsoft has also listed the key factors of cloud services that enable low electricity consumption per end user. One of the factors is server utilization, which is high when compared to similar on-premise solutions. The power draw of servers only slightly increases when utilization rate rises. For example, a four times higher utilization rate causes two times higher electricity consumption. Better use of resources reduces cloud service providers’ need to purchase new servers in the short term. High server utilization is achieved with multi-tenancy, which centralizes computation and offers uniform infrastructure to multiple companies and their web services. Because of multi-tenancy, demand peaks of individual companies can be combined to a more predictable and steady demand rate which then helps to allocate resources. Furthermore, the gap between peak and average demand in cloud services is small in comparison to conventional servers and as a result cloud services require less extra resources during rush hours. [1], [9] Cloud services can be scaled based on workloads [2]. For example, during demand peaks web services scale out to acquire more instances that serve extra end users. On the other hand, when web services do not need all their instances, they shut down their spare resources and reduce operational expenses. If several hosted services scale in their resources at the same time, cloud providers should put their underutilized servers into sleep in order to centralize workloads to remaining servers and to reduce electricity consumption [17]. This feature is also known as dynamic provisioning, and it is based on advanced IT equipment which monitor network traffic and predict upcoming demand. It strives to prevent the over-provision of servers and to reduce capital and operational expenses. [1] Some measurements, however, indicate that cloud services still have underutilized servers, even if they supposedly have advanced mechanisms to centralize computation. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 same time or instances are mostly idle, and as a consequence the average utilization rate in cloud services can be below 10%. [12] Furthermore, there are also some performance issues related to provisioning and booting instances, because latencies vary from one to ten minutes. If instances are activated too slowly, web service providers will permanently lose some of their end users that are too impatient to revisit on their services. Notable latencies imply that cloud software stacks still need to be improved so that cloud service providers could use sleep modes on their servers without compromising the quality of their services. [9], [16] Scheduling algorithms that enable sleep modes on servers and instances need to perform so efficiently that the end user will not notice their existence. [15] Jayant Baliga and his associates have also examined the greenness of cloud services. The study revealed that energy savings can be gained by using cloud services, when compared to thick clients. Nevertheless, the increased amount of network traffic seems to have a negative impact on the overall power draw and, therefore, the most green of cloud services is not unambiguous. Conducted measurements and simulations indicate that private cloud as a deployment model is the most energy efficient option. The main reason for this result is short distances between data centers and end users. Especially if private clouds are placed in the same premises where the majority of the staff is working, only a few network devices are required to transfer network traffic between end points. Because of the large amount of network traffic, frequently needed data should be stored locally, while rarely used data, such as backup files, could be stored in cloud services. Furthermore, large files, including high-quality videos, should be stored close to the end users, even if files were fetched rarely. [5] To maximize the greenness of cloud services, highly utilized servers need to be hosted within highly energy-efficient data centers. The energy efficiency of a data center infrastructure can be measured with power usage effectiveness (PUE) metric, which is calculated by dividing total electricity consumption with IT electricity consumption [1], [20]. 2.2 Towards Green Private Clouds In addition to environmental aspects, the overall expenses of different cloud solutions need to be carefully evaluated before adopting them. Conventional facilities and especially green data centers tend to have substantial investment costs, whereas public clouds have no upfront costs. However, public clouds could be more expensive than acquiring own infrastructure and servers in the long term. First of all, cloud service providers need to have a marginal profit in order to renew their hardware from time to time and to develop their processes. As a consequence, instances are not as affordable as they could be. Secondly, transferring data can be a greater item of expenditure than instances for some customers. [21] It seems that data transfer costs can be substantial for those companies which have many end users and often need to shift a significant amount of data to a cloud service. In the long term, a private cloud would be more affordable as well as more environmentally friendly when it is hosted within a green data center. Perhaps the most significant drawbacks of cloud computing are related to data privacy. First of all, customers need to trust that cloud providers are able to protect the confidentiality of data on shared platforms [4]. Furthermore, they also need to trust that cloud providers will not exploit their sensitive data for commercial purposes. Especially major actors could have a great interest to sell customers' data for advertisers. In this sense, private clouds in companies' own premises are safer options to store sensitive data than public clouds. Even though companies trusted public cloud providers, existing legislation could be a major problem. Some data protection laws might require that certain information must be kept inside specific regions, such as the European Union [23]. Many of these restrictions are justified, because some other countries might have loose laws to protect privacy and some governments possibly have excessive rights to explore stored data on cloud services in their regions. As a result, private clouds could be the only option for those companies who want to apply cloud services and store sensitive data. The literature implies that private clouds are currently the most environmentally friendly solutions in the market [5]. Furthermore, private clouds overcome many of the cloud-related issues, including the previously listed privacy issues. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile The average PUE value is around 2.0, which indicates that half of the electricity is used by the infrastructure, including cooling systems, power transformers, and lighting [1], [19]. Due to the magnitude of cloud services, cooling costs in data centers can be substantial. For example, in 2010, data centers used globally 130 billion kilowatt-hours to cool servers [6]. Traditionally data centers are cooled using Computer Room Air Conditioning (CRAC) units, which blow chilled air though raised floor in front of the server racks and suck hot air near the ceiling. Units utilize cool liquid to exchange heat with air and the liquid is cooled using chillers, which are significant energy consumers. [6], [17] According to Microsoft, state-of-the-art data centers for cloud services strive for extremely low 1.1-1.2 PUE values, which can be achieved by adopting new energy-efficient methods to cool servers, including modular design, outside air and water evaporation based cooling [1]. Furthermore, novel cooling systems should be dynamic in order to adapt different workloads. For example, a cooling system should notice when servers are powered down and modify its air flow patterns. It would require numerous sensors to collect information about servers’ cooling needs. [15] There are also more energy-efficient methods, including liquid cooling, to shift heat in data centers than CRAC units. Liquid as a medium is more capable to obtain heat energy than air, and therefore liquid should be brought as close as possible to server racks. [19] Another strategy to gain energy savings is more hot data centers, which require less cooling. Recommend operating temperatures for servers are 20-25°C [8], [17]. 2.1 Green Computation It seems that servers rarely have multiple instances at the 75 75 Tommi Makela Sakari Luukkainen centives to Apply Green Cloud Computing Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing 3 Case Analysis The selected research methodology and applied framework for this study are introduced in this section. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Figure 1: Different types of private clouds Hosted and operated by a third party Self-hosted, operated by a third party Hosted by a third party, self-operated Figure 1: Different types of private clouds Hosted and operated by a third party Self-hosted, operated by a third party Hosted by a third party, self-operated Figure 1: Different types of private clouds Based on the literature, it could be argued that cloud computing is greener than the traditional client-server model [1], [5], [21]. However, are there any incentives to apply green cloud computing in practice? The next section introduces three companies with novel facilities and cloud services. Their services resemble the first and the third private cloud types (figure 1), where the third parties host and operate private clouds or only host private clouds. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile their servers should consider using public clouds or virtualizing their current servers to gain energy savings. Then it is up to small companies themselves to determine which of the public clouds would be the most desirable for them; for example, the most affordable instances near the end users. Large companies should, however, consider adopting private clouds, mainly because data transmission costs and distances to cloud data centers are so substantial. For those companies that decide to put instances into operation, there are at least three sub-deployment models of private clouds from which to choose (figure 1). A private cloud is commonly referred as a cloud which is operated and hosted by the company itself. IBM specifies that there are also private clouds that are both operated and hosted by a third party. [18] This option resembles a public cloud deployment model, because IT equipment and data center infrastructure are fully controlled by a service provider. This option, however, separates customers more firmly than public clouds, because each company has its own resources that are not shared with others. As a consequence, this option provides better privacy than public clouds. Then there are private clouds that are operated by a third party and hosted by the company itself [18]. In this option, the IT maintenance is outsourced to service providers with more IT expertise, such as Hewlett-Packard, Dell, or IBM. Furthermore, there are also private clouds that are operated by the company and hosted by a third party [18]. This option allows companies to focus on their core competences, because they do not need to take care of the data center infrastructure. Because the infrastructure is fully outsourced, it is up to the companies to select a service provider which would host servers within a novel facility with minimal electricity consumption and CO2 emissions. As a matter of fact, the combination of green hosting services and self- operated private cloud services could be the most energy efficient option in the market. 2.2 Towards Green Private Clouds Because of the small number of end users, individual households and small businesses cannot benefit from private clouds, which would lack of economies of scale. Furthermore, centralized resources in a small scale would not offer significant energy savings compared to servers and workstations [1]. Companies with less than a thousand users on 76 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile As a result, those who want to perform surveys should train themselves to become skilled interviewers. Furthermore, performing interviews is time-consuming, because suitable interviewees first need to be located and then interviews need to be arranged separately. Transcribing informal material after interviews also requires plenty of time, because there are no ready-made models that would simplify the process. Moreover, results typically suffer from reliability issues, because people could behave abnormally in social interactions; for example, an interviewee wants to please an interviewer and therefore he provides answers that are expected by the interviewer. [11] In this study, some of the proposed drawbacks were recognized. Definitely more experience about conducting inter- views would have been helpful, but all the interviewees were so competent and cooperative that they offered plenty of useful information, which was not directly related to the questions. In other words, it is very important to locate the correct people for interviews, who are able to answer questions and provide additional information. In this study it is unlikely that interviewees would have provided any false information, which would have impaired the reliability of the interview study, because some of the data was already public. The interviews, however, offered numerous technical details and provided a better overview of facilities. Furthermore, the fact that the companies remain anonymous in this study increases the reliability of their answers. However, if the questions were unintentionally related to their trade secrets, they declined to answer, which is completely understandable. It is rather difficult to determine if some of the questions are too sensitive; for instance, information related to the exact locations of facilities. The goal was to locate domestic companies, who have promoted their services or data centers as green, and then agree appointments. Up to ten companies were asked to participate in the interviews. In the end, three companies agreed to participate, but luckily those companies had the most promising green data centers in the market. The participating companies had rather different business models, so the main common factor of the companies was brand-new facilities and different types of cloud services. All the participating companies could be considered as major actors in their field in Northern Europe. The main goal was to discuss new facilities and offered services with personnel, who have great knowledge of the topic. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 between the energy efficiency development and related incentives. Case studies also facilitate concentration on the new factors that arose during the research and the reasons for the differences in the case companies’ behavior. [24] The information for this study was collected by interviews. According to the literature, interview studies as a research method have several benefits. They allow interviewees to express themselves as freely as possible. Secondly, if the field of research is uncharted or unknown, it is difficult to expect certain answers beforehand in which case interview studies are a very flexible method to apply. Moreover, they are also suitable when the collected data needs to be used in a wide context. In some cases it is predictable that the topic of research will result in complex answers, which require clarification. Furthermore, supplementary questions can be asked during the interviews, if the original questions were imperfect or the answers were so insufficient that they needed clarification. [11] In this study, all the listed benefits were identified. It is useful that an interviewer can ask for clarification if he needs more justification for some of the responses. Moreover, it seems that questions for interviews do not need to be as accurate and explicit as questions for questionnaires, because an interviewer can ask follow-up questions, which are especially useful, if interviewees offer unexpected new information. For example, in this study additional questions clarified what kind of cloud services participating companies offered and how their business customers evaluated environmental aspects. Moreover, the research topic was somewhat uncharted, because the green data centers were either under construction or just put into operation. Furthermore, the main goal was to utilize the interviews in a holistic view to green cloud computing incentives and as a result the interview case study was selected as a method to collect empirical evidence to support the conclusions of this study. Naturally, interview studies also have some drawbacks when compared to other methods to collect information. To maximize the amount of useful data collected from social interactions, an interviewer needs to have experience. 3.1 Methodology The literature review forms a basis for a framework (table 1), which will used be in the empirical section of this paper. It helps to collect relevant information about green cloud computing and limit the scope of the research. Based on the literature, there are four factors, which define green cloud computing. The first category focuses on the methods to centralize computation in order to increase server utilization. The second category examines cooling systems which would use significantly less energy than traditional CRAC units to cool data centers. The third category concentrates on the possible issues between energy efficiency and other requirements of IT services. Finally the fourth category emphasizes the importance of efficiency metrics to illustrate the greenness of services to customers and end users. Table 1: Research framework Dimension Meaning Server utilization [1], [5], [21] Approaches to centralize computation in companies Cooling systems [6], [19]-[20] Solutions to chill facilities more energy efficiently Quality of Service [15], [17], [22] The relationship between greenness and quality Efficiency metrics [3], [7], [15], [20] Methods to illustrate the greenness of services Table 1: Research framework Dimension Meaning Server utilization [1], [5], [21] Approaches to centralize computation in companies Cooling systems [6], [19]-[20] Solutions to chill facilities more energy efficiently Quality of Service [15], [17], [22] The relationship between greenness and quality Efficiency metrics [3], [7], [15], [20] Methods to illustrate the greenness of services Table 1: Research framework The goal of this empirical section is to identify features of state-of-the-art facilities that would provide new knowledge about the incentives of green cloud computing and green hosting. This study was conducted by using the multiple- case-study research methodology, where each case should be treated as a single experiment. If similar results are obtained in several cases, a replication has taken place. By using the case study method, it is possible to observe the phenomena specific to the cloud computing industry in a detailed level. While the secondary sources mainly describe what has been done, the explanatory case studies provide more detailed insight into the interrelationship 77 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing 77 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Incentives to Apply Green Cloud Computing 3.2 Company A This sounds a little odd, as company A could be considered as a small scale energy company, because the first data center generates so much heat that up to 500 detached houses can be maintained warm all year round. Moreover, when the second data center is full of server equipment, the amount of released waste heat equals the heating requirements of 4 500 households with 80 m² of floor space. These figures indicate that company A provides notable quantities of heat for the energy company and therefore it would be reasonable that data centers would at least have low prices for district cooling and electricity. To achieve substantially high energy efficiency and to maximize the benefits of district cooling, the company utilizes liquid-cooled racks to chill hosted equipment. There is one slight difference between company A’s two data centers. Due to the limited space in the first facility, all the IT equipment, including servers, network switches, and storage devices are cooled by liquid cooling. The second facility, however, uses liquid cooled racks only for IT equipment with high heat density and the rest of the devices are cooled with CRAC units. Based on the interview, liquid cooling for all IT equipment does not guarantee energy savings while it has rather high upfront costs. Company A considered applying economizers to replace CRAC units during wintertime, but there have been permission related issues. The energy efficiency of hosted servers is customers' concern, meaning that the customers decide what kind of server models they want to purchase. For some reason, many customers, including telecommunication companies and major enterprises, want to host rack servers instead of blade servers. The majority of company A’s business is related to hosting equipment. However, it also offers virtual machines, which operate in fault-tolerant clusters. Customers can upgrade purchased virtual machines afterwards, if they need more resources. Due to some missing cloud characteristics, company A wisely does not promote its virtual machines as instances. At the time of the interview, it was difficult to determine how quickly the capacity of the data centers runs out. For example, if several large-scale companies decide to outsource the hosting of their private clouds, the data center capacity would then rapidly be exhausted. 3.2 Company A Nowadays, company A offers numerous IT services, including equipment hosting, communication solutions, support, and voice-related solutions for business customers. Company A has its own data center facilities, core networks, and system experts. The first listed service, equipment hosting, allows companies to rent an infrastructure for their IT equipment, particularly for rack servers. Company A also offers some management services for hosted servers, but customers typically purchase and configure their servers by themselves. Equipment hosting has similar benefits as public cloud services, because companies can focus on their main businesses, thus reducing capital expenses. All the servers are hosted in brand-new data centers in Helsinki. The first facility was officially introduced in the fall of 2010 and only one year later the second facility was in operation. The first one is compact, because it is built in an old bomb shelter with 500 m² of floor space. It could be considered as a proof-of-concept data center, because the second data center has as much as 5 000 m² of floor space. Nowadays, company A claims that their facilities are one of the most energy efficient in the market, because they have 1.25 PUE and they reuse plenty of waste heat. To achieve substantially low PUE, the cooling system of a data center needs to be extremely energy efficient. Both of company A’s data centers are quite unique, because they use district cooling to chill hosted servers, and the same pipe system is used to transfer waste heat to the city's district heating system. A local energy company mainly uses sea water to chill circulating refrigerant within the district cooling system. During the summer, however, the energy company utilizes waste heat generated by its power plant to create pressure to the refrigerant, which evaporates, releases its own heat, and finally cools down. Typically, the circulating refrigerant has too low a temperature when it returns from the data centers, and therefore, heat pumps are required to provide additional heat before waste heat can be shifted from the district cooling system to the district heating system. At the time of the interview, company A did not benefit from the waste heat, which was shifted to the city's district heating system. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Luckily all of them who were initially asked for the interviews were able to participate, which then assured high quality answers. Suitable personnel were located from the companies' web sites, which often contained news sections, where the new data centers were announced and their project managers were listed. The personnel were mainly contacted via email. All the interviews were conducted in the premises of the companies. Two of the interviewees were IT managers and one of the interviewees was the CEO of a company, which then co-owns the actual company with new facilities. The questions for the interviews were posed based on previous experience from data center infrastructures and basic knowledge of cloud computing. The questions involved three different themes: infrastructures, cloud services, and transparent efficiency metrics. Two of the interviews took an hour to perform, which was the estimated time for all interviews, but one of interviews took over one and half hours, because the company had so much say about their products and services. Nevertheless, the resulting wide range of answers was just a positive outcome for this study. Moreover, two of the companies had one extra person with technical knowledge to offer more details of the facilities. To ensure that all the available data could be fully exploited, all the interviews were taped with a voice recorder. Furthermore, notes, which were written during the interviews, were also utilized. The summary of the interviews is presented in the following sections and its content is approved by the companies. 78 78 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Tommi Makela Sakari Luukkainen Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing 3.3 Company B Company B provides IT support and computational resources for companies, research institutes, and universities. It is administrated by the Finnish Ministry of Education, which naturally means that the company does not try to make any profit, but tries to maximize the value of taxpayers' money. In Finland, company B is one of the actors with the expertise, equipment, and all the necessary software tools to help scientists conduct their research. Company B has two known data centers and a few secret facilities. To provide plenty of computational resources for scientists, company B has four supercomputers for performing computationally heavy tasks. When compared to conventional servers, supercomputers have substantially more processor units and main memory modules. The conducted interview indicates that the company has a challenging task to maintain its supercomputers on the top 500 most powerful machines listing, but at the same time they should be on the 500 most energy-efficient machines listing as well. Therefore, company B will acquire a new supercomputer for its new data center. In the early stage of the project, the general confusion in the public was that company B was going to build a data center in the premises of an old paper mill in Kajaani. Actually, company B is a customer of a data center park, which is being developed mainly by a local energy company, a telecommunication company, and a paper manufacturer. In the first phase, 1 000 m² floor space will be allocated for a new supercomputer and other IT equipment, which will be shifted from existing data centers. In total, around 4 000 m² of floor space is reserved for the company. Because the premises are still under construction, it is difficult to determine the final PUE value for the data center. However, the company expects that the PUE would be around 1.15, which means that the infrastructure would drain significantly less electricity than hosted servers, which are rather power intensive due to their heavy tasks. When discussing the potential risks of the building project, the only major issue for company B would be unrealized energy efficiencies for the infrastructure. Therefore, it is important that the local energy company and the telecommunication company measure the electricity consumption of individual customers as accurately as possible. Low PUE values require an especially energy-efficient cooling system. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile 3.3 Company B The company has decided to focus on airside economizers, because outside air is chilly the whole year around in Kajaani. As a matter of fact, the region is so cold during the winter that airside economizers will be modified to handle outside air below -45°C, before air is shifted directly indoors. Because the data center will be built in the paper mill, there are no residential areas nearby, which would make it difficult to utilize waste heat. The offices and warehouses inside the data center park, however, can be warmed by using hot exhaust air from the servers. Obviously, it does not make sense to build a separate district heating system if the distances are too long. To gain more energy savings, the company will follow the American Society of Heating, Refrigerating, and Air-conditioning Engineers’ (ASHRAE) latest recommendations, which state that the maximum temperature for supply air is 27°C. Because certain computer components are regularly upgraded and replaced, higher operational temperatures should not significantly affect supercomputers’ reliability. Company B has several blade systems providing an infrastructure for cloud services, which are available for partner companies and universities. The company uses open source software stacks to manage cloud resources. The cloud is small in comparison with commercial products, which means that resources will not scale endlessly. This private cloud is an alternative for scientists who need to process sensitive data. In addition to a lack of support for software tools, commercial high-performance cloud clusters become notably expensive in the long term. To be green as possible, company B will buy hydro-electric power for its IT equipment. As a matter of fact, there are hydro-electric power plants providing clean electricity near the data center park. These power plants, however, are rather small and therefore they are better suited for backup power. Due to the magnitude of the power draw, hydro- electric power will be purchased from Nordic electricity markets. There are up to five major transmission lines supplying electricity from the main grid and it is very unlikely that all of them would break down at the same time. As a result, the company will not install any uninterruptible power supplies (UPS) into its data center. 3.2 Company A To minimize the costs of a partially empty facility, the second data center is built by using modular design, meaning that new sections will be put into operation when demand rises. When the facility was introduced in 2011, it already had a major customer, which offers numerous IT services and outsourcing solutions, including a cloud infrastructure for companies. It is interesting that the customer promotes its new product as green cloud computing. In other words, the combination of the customer’s highly utilized cloud resources and the company A’s highly efficient infrastructure can be considered as green cloud. From the business point of view, this is exactly what companies should do when they host their IT equipment in a green data center. Company A has other unique features as well. For instance, the electricity consumption of every hosted server is separately measured, and the billing is based on these measurements instead of theoretical maximum electricity consumptions and fixed prices. This arrangement encourages companies to purchase energy-efficient server models. The company also offers water and wind power for companies which want to provide even greener services. The conducted interview revealed that none of the customers had purchased renewable energy. The price tag of supplied electricity was more important than the amount of released CO2 emissions. 79 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 company C tends to host high-density IT equipment. Therefore, the sufficiency of local power distribution alone is a major reason to favor energy-efficient solutions. Based on early measurements and future estimates, the PUE value of the data center will vary between 1.2-1.3. PUE value is the only metric which is constantly measured in company C's facilities. The interview indicates that accurate measurements are problematic. A data center infrastructure needs to possess equipment that measure every rack full of servers and storage devices as well as the components of cooling systems and backup systems separately. Furthermore, company C has additional challenges, because in the same data centers it hosts both customers' servers and equipment that are dedicated only for its own internal processes. Measurements are even more difficult to conduct if the customers and the company itself utilize the same IT resources, such as a shared cloud platform. In other words, there are many complex equipment-level measurement issues. For instance, how to collect relevant information from individual servers, instances, and software and then compose useful analyses without interfering with hosted services? company C tends to host high-density IT equipment. Therefore, the sufficiency of local power distribution alone is a major reason to favor energy-efficient solutions. Based on early measurements and future estimates, the PUE value of the data center will vary between 1.2-1.3. PUE value is the only metric which is constantly measured in company C's facilities. The interview indicates that accurate measurements are problematic. A data center infrastructure needs to possess equipment that measure every rack full of servers and storage devices as well as the components of cooling systems and backup systems separately. Furthermore, company C has additional challenges, because in the same data centers it hosts both customers' servers and equipment that are dedicated only for its own internal processes. Measurements are even more difficult to conduct if the customers and the company itself utilize the same IT resources, such as a shared cloud platform. In other words, there are many complex equipment-level measurement issues. For instance, how to collect relevant information from individual servers, instances, and software and then compose useful analyses without interfering with hosted services? Similar to company A in Helsinki, company C also utilizes district cooling to chill its devices. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile In practice, a local energy company uses heat pumps to cool down the refrigerant that returns to the data center and to warm up the refrigerant that comes from the data center before waste heat is shifted to the city's district heating system. In Espoo, the district cooling does not involve any free cooling. Even though only 1 000 m² of floor space is currently equipped with data center infrastructure, as much as 1 500 detached houses can be warmed the whole year around. When the full capacity of the new data center is in operation, up to 90 000 households can be heated merely by using waste heat and heat pumps. It has been estimated that the data center makes it possible to reduce CO2 emissions by roughly 10 000 tons, when the data center replaces other heat production that utilizes coal or oil in its processes. When the interview was carried out, company C’s negotiations with the local energy company about the contracts were still underway. Nevertheless, it was clear that district cooling would be more affordable, because company C provides plenty of waste heat to the local energy company in return. Company C uses liquid-cooled racks to chill equipment energy efficiently. There are products that isolate servers and keep the air loops short inside the cabinets. Company C has tested different types of liquid cooling solutions and it has determined that fully closed and tailored solutions are not necessarily more green or energy efficient. According to company C, inexpensive alternatives, which isolate commodity racks with extra doors and walls, are capable enough to prevent cold and hot air mixing with each other, and it is not completely necessary that the liquid cooling units are attached to the racks themselves. These kinds of solutions provide flexibility, because they can be applied in existing data centers. As previously stated, liquid cooling has major upfront costs compared to CRAC units. Due to the very competitive markets, keeping investment costs as low as possible is important for the company, which is why it favors flexible and modular solutions with both low initial and operational costs. Company C offers two types of cloud services: individual private clouds for business customers as well as a hybrid cloud. For major customers, it also provides additional security services for private clouds. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile A customer can select if its data is stored in genuine private storage or in shared storage. Those customers who want to outsource even more can purchase software as a service with shared resources on the equipment level. The hybrid cloud is an alternative for customers who do not need massive amounts of instances and for whom privacy is not an issue. The billing of these cloud services is rather fixed compared to commercial public cloud services. 3.5 Discussion The results of the multiple-case-study are summarized in the research framework 3.5.1 Server Utilization In order to minimize the power draw of the hosted servers and to reduce the need for future server investments, all three companies use server virtualization to centralize their computation. Two of the companies exploit commercial products, including Citrix XenServer and VMware vSphere, to offer virtual machines to business customers. These kinds of products guarantee a certain level of quality of service and provide extensive IT support, which are often required by business customers. Company B, however, uses open source software stacks, such as OpenStack, to offer cloud services to selected partners. As a consequence, company B has a great role to ensure the sufficient functionality and reliability of the services. Both commercial products and open source solutions allow the companies to monitor their servers and to determine when extra resources need to be put into operation. When the servers are fully reserved for numerous instances, then it is up to customers to maximize the utilization of their instances. In addition to better use of resources, cloud software stacks improve the quality of hosted services, because running instances can be moved to other devices when server malfunction occurs. Reliable software allows service providers to use unreliable commodity hardware to reduce their capital expenses and to offer affordable services to end users [8]. All three companies, however, utilize conventional blade and rack servers together with software stacks and therefore their services should be fault-tolerant enough to fulfill the high demands of business customers. 3.4 Company C Company C offers several IT services and product development services for business customers in Northern Europe. Typically, all the services are tailored for each customer, which means that the content of services must be negotiated separately. Company C wants to provide extra value for its customers and as a result it will always have some control over hosted IT equipment and infrastructure. In this sense, company C does not offer similar hosting services as company A, where only the infrastructure is rent for rack servers. Company C typically purchases or allocates existing IT equipment for its customers, who then pay monthly fees. In 2011, company C put its novel data center into operation. This particular facility is located at an industrial area in Espoo, which is one of the major cities in Finland. Furthermore, the facility has up to 6 000 m² of floor space to host customers' services. It is quite large in comparison with company C’s five other facilities located in Finland and Sweden, because they have around 7 600 m² of floor space in total. In the first phase, only 1 000 m² of the new facility is fully equipped with a cooling system, redundant power distribution, and backup energy. The conducted interview indicates that it is more likely that electricity would run out before available floor space, especially because 80 Tommi Makela Sakari Luukkainen ncentives to Apply Green Cloud Computing 80 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile 3.5.3 Quality of Service y The interviews indicate that even the most green companies in the market are not willing to sacrifice the quality of their services. Both hardware and software solutions need to be highly reliable. Naturally the supply of electricity to data centers is very important for all service providers. For example, company A uses a large diesel generator to produce electricity for its facility, if nearby major transmission lines are down. The company also owns UPS devices to handle short-term power outages. Both of these measures of precaution reduce the greenness of the facility, due to the occasional CO2 emissions of the diesel generator and the power losses of the UPS devices. Companies A and C state that these kinds of measures are mandatory for cloud and hosting service providers, because business customers require high availability and reliability in all circumstances. In order to avoid confusion between different actors, the quality of services need to be determined in service level agreements (SLA). The expectations towards quality are the reason why the companies have not applied any experimental products which would provide energy savings, but would possibly have a negative impact on the reliability of the services. For example, there are some network solutions, including ElasticTree, which utilize algorithms and alternative network topologies to minimize the power draw of network equipment, but all three companies have decided to use traditional network switches and topologies [10]. Furthermore, they have not experimented network virtualization, even though it could have similar benefits as server virtualization. For instance, OpenFlow allows centralizing network traffic flows and turning off network switches with no traffic [13]. Based on the interviews, cloud and hosting service providers need to carefully evaluate their software and hardware investments and try to find the balance between different requirements, because extremely green services are useless, if end users are unable to use them. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 minimize the risks related to major facility investments. Furthermore, modular design guarantees sufficient workloads for cooling systems, which then operate more energy efficiently. Two of the data centers use district cooling systems to chill hosted devices and one of the facilities utilizes a fully independent cooling system, which is based on airside economizers that exploit the cool of outside air. Energy-efficient liquid cooling seems to be the perfect combination with district cooling, especially when the waste heat is collected and then utilized. Furthermore, heat pumps are an essential part of district cooling and heating, because they offer both additional cool and heat. The ability to collect heat seems to be one of the features which modern data centers should support nowadays. The energy efficiency of the facilities is around 1.2, which is the same as the estimated 1.1-1.2 PUE for state-of-the-art data centers [1]. Table 2: Cloud and hosting services Features Company A Company B Company C Facility design modular modular modular - Floor space 5 500 m² 4 000 m² 6 000 m² Cooling system district cooling air cooling district cooling - Free cooling heat pumps, sea cooling airside economizers heat pumps - Used medium liquid + air outside air liquid + air - Heat reuse district heating warehouses district heating - Uniqueness accurate power measurements hydroelectric power, no UPS tailored services for enterprises Metrics PUE PUE PUE - Values 1.25 1.15 1.2-1.3 Table 2: Cloud and hosting services 3.5.2 Cooling Systems Based on the interviews, state-of-the-art data centers have similar features (table 2). Due to the substantial scale of the facilities, all three companies have applied modular design to postpone some of the capital expenses and to 81 81 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing ournal of Theoretical and Applied Electronic Commerce Research SSN 0718–1876 Electronic Version OL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-187620130003 minimize the risks related to major facility investments. Furthermore, modular design guarantees sufficient workloads for cooling systems, which then operate more energy efficiently. Two of the data centers use district cooling systems to chill hosted devices and one of the facilities utilizes a fully independent cooling system, which is based on airside economizers that exploit the cool of outside air. Energy-efficient liquid cooling seems to be the perfect combination with district cooling, especially when the waste heat is collected and then utilized. Furthermore, heat pumps are an essential part of district cooling and heating, because they offer both additional cool and heat. The ability to collect heat seems to be one of the features which modern data centers should support nowadays. The energy efficiency of the facilities is around 1.2, which is the same as the estimated 1.1-1.2 PUE for state-of-the-art data centers [1]. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 If end users start to increasingly appreciate and demand more green web services, then service providers need to illustrate their greenness. Web service providers and hosting service providers should negotiate and draw up SLAs, which would determine the quality of services and acceptable ranges for different values. Green SLAs underline the importance of transparent and uniform metrics that illustrate energy efficiencies and CO2 emissions [15]. A widely adopted PUE metric alone does not offer adequate information about the energy efficiency of services. To gain more comprehensive overview on the greenness of services, PUE needs to be used in conjunction with several other efficiency metrics. Energy Reuse Effectiveness (ERE); for instance, illustrates service providers’ ability to utilize waste heat. It is calculated by subtracting total energy consumption with reuse energy and then divided by IT energy. [3] Another useful metric, Technology Carbon Efficiency (TCE), highlights the relation between energy consumption and CO2 emissions. In practice, TCE values are formulated by multiplying existing PUE values with carbon emission rates, which depend on energy sources, such as coal, nuclear, and water. Carbon emission rates vary between 0.1 and 2.3 pounds of carbon dioxide per kilowatt-hour. TCE, however, ignores other environmental impacts caused by services, such as the disposal of equipment [7]. Compute Power Efficiency (CPE) metric measures the computation efficiency of data centers. Its value is defined by dividing IT energy consumption with total energy consumption and then multiplied with average IT equipment utilization. It can also be calculated by dividing the average IT utilization with a previously measured PUE [20]. Transparent and uniform metrics enforce derived demand, because they enable end users to compare different web services and select more green alternatives. Efficiency metrics (table 3) and other certificates should be clarified, illustrated, and promoted for end users in order to have desirable impact on web service providers’ and hosting service providers’ sales and reputations. Table 3: Efficiency metrics for green SLAs Table 3: Efficiency metrics for green SLAs Full name Abbr. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Description Formula Power Usage Effectiveness PUE Illustrates the energy efficiency of data center infrastructure PUE = ∑P PIT Energy Reuse Effectiveness ERE Presents the utilizations of waste heat within data centers ERE = ∑P – PRE PIT Technology Car- bon Efficiency TCE Indicates the link between used energy and emissions TCE = ∑P * CO2 PIT Compute Power Efficiency CPE Emphasizes the computation efficiency inside data centers CPE = PIT * IT ∑P Table 3: Efficiency metrics for green SLAs Because of outdoor conditions, the electricity consumption of cooling systems constantly varies, which is why the values of efficiency metrics are not static. Furthermore, different server workloads also affect the overall electricity consumption of web services. Due to the changing conditions, green SLAs need to have specific ranges for metrics instead of single figures. The proposed metrics have a few identical variables and therefore all of their results vary when outdoor conditions and workloads change. In addition to acceptable ranges for metrics with relative results, green SLAs should also guarantee absolute limits for certain important variables. For example, service providers should ensure that a specific minimum quantity of waste heat will be utilized in all circumstances. Moreover, service providers should state that their CO2 emissions will never exceed a certain maximum quantity. Some providers might even ensure that their IT resources will have high utilization rates, meaning that they do not waste electricity and acquire unnecessary devices. Properly utilized resources were not self-evident even in 2012 [14]. Measuring the accurate amount of energy, waste heat, and IT utilization in data centers could be challenging in practice. To calculate exact results, service providers need to measure their electricity consumption in various places, including near cooling systems, network switches, and rack servers. They also need to know where their electricity is produced and how much CO2 emissions it causes. Furthermore, in many countries kilograms need to be converted into pounds to use globally uniform TCE values. However, if service providers are unable to locate the correct power plants or exact CO2 rates, pre-defined rates for different energy sources can be used instead [7]. Determining the average utilization of all IT equipment could be difficult and therefore typically only the utilization of processor units is measured. Moreover, high utilization rates do not necessarily mean that services are energy efficient. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Poorly coded programs; for instance, could drain all the available resources, but perform very little useful work. Determining the accurate IT asset utilization is one of the major challenges of modern energy efficiency metrics [14]. 3.5.4 Efficiency Metrics Even if all these facilities consist of innovative solutions, all three companies still mainly use traditional PUE values to illustrate their greenness. However, there are several other efficiency metrics which would illustrate the relationship between electricity consumption and emissions and present the ratio of utilized waste heat [3], [7], [20]. As a matter of fact, these metrics are not required by the business customers, such as web service providers, because they only emphasize low costs and therefore there is no demand for green cloud services or green hosting services. Currently, the only incentive for companies to enhance their greenness is to lower their own operational costs and possibly improve their competitiveness by reducing their prices. Companies A and C are major actors in their fields, which is why these findings are significant. If business customers themselves do not require green cloud services or green hosting services, it is up to end users to create derived demand (figure 2) for those services. The end users of web services and their environmental awareness are the incentives which are currently missing. 82 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Figure 2: Derived demand for cloud services Cloud service End users derived demand demand Web service 82 Figure 2: Derived demand for cloud services Cloud service End users derived demand demand Web service End users Web service Cloud service Figure 2: Derived demand for cloud services Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Incentives to Apply Green Cloud Computing Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 The case studies indicate that the relationship between service providers and local energy companies needs to be further developed. Nowadays, service providers do not benefit enough from the waste heat they provide for energy companies. It is of mutual interest that service providers would have more affordable electricity or district cooling in exchange for waste heat. It would also add another incentive for service providers to construct green data centers. After all, the waste heat of data centers replaces other energy sources, which would pollute the climate. At least in the European Union, it is advantageous for energy companies to minimize their CO2 emissions (Site 2). Even though local energy companies certainly have a monopoly over district cooling and heating, unsatisfied service providers can still purchase their electricity elsewhere. Green IT services could be especially beneficial for energy companies, if service providers start to acquire more expensive electricity, which is produced from renewable sources. 4 Conclusions In the best case scenario, well-optimized web services are hosted on highly utilized servers that are located in green data centers. Due to their nature, cloud services should be able to fully exploit their resources, because they support numerous simultaneous end users and multi-tenancy, which then provide economies of scale [1]. Major public cloud services definitely have notable scale and they are a great option for many small and medium companies, who want to host their services on fully outsourced infrastructures. Public cloud services, however, do not suit all companies because of privacy and security issues [4]. They could also be expensive in the long term, if significant amounts of data need to be transferred between services and end users [21]. Furthermore, the amount of data and the distances between different parties mainly constrain the greenness of cloud services [5]. As a consequence, large companies should consider utilizing private cloud services near their employees and most of their business customers, even if private clouds have significant upfront costs. In the simplest arrangement a private cloud is managed and hosted by the company itself [2]. However, if large companies want to reduce some of their upfront costs, they can rely on third parties, which then host private clouds in their novel data center infrastructures [18]. This paper presented a multiple-case-study that was used to collect information about state-of-the-art data centers, and consequently to provide new knowledge about the incentives of green cloud computing and green hosting [24]. The research framework was drawn up based on the relevant literature. Even though the research had its limitations, it provided a few conclusions. Server utilization in cloud services can be high, because several instances are hosted on the same hardware. Then it is up to business customers to fully utilize their instances and further increase the utilization. Cooling systems in cloud services can be significant energy consumers, and therefore service providers should apply energy-efficient methods, such as liquid cooling, to shift heat in their facilities and use energy-saving solutions, such as free cooling, to cool the facilities. Nowadays data centers should be able to utilize their waste heat in order to enhance the greenness of cloud services. Even though greenness is an important feature for a service, it should not compromise the quality of a service. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Table 4: Incentives for different actors Actors Cloud/Hosting  Web services  End users Lower opera- tional costs Improved profit- ability or better competitiveness No effect or lower list prices for cloud services No effect or more affordable or free web services Actors End users  Web services  Cloud/Hosting Greenness as a value Interest to use more environ- mentally friendly web services Need to improve competitiveness by emphasizing green features Need to further improve energy efficiency and to reduce emissions Actors Cloud/Hosting  Web services  End users Achieved greenness Need to prove and guarantee green- ness (green SLA) Need to promote and illustrate greenness (ads) Values must be transformed into actions (use them) Actors End users  Web services  Cloud/Hosting Excessive demand Notable need for green services, long-term trend Opportunity to increase prices & market shares Opportunity to increase prices & market shares Actors End users  Cloud/Hosting  Energy CO Green energy Interest towards web services with zero emissions Need to purchase green power for hosted services Demand for re- newable energy, increased profits Actors Energy CO  Cloud/Hosting  Web services Mutual interest Need to ensure that both elect- ricity and district cooling is bought from their plants Need to gain extra benefits when offering waste heat from the data centers Need for informa- tion, the quantity of waste heat, the amount of CO2 reduction Table 4: Incentives for different actors Table 4: Incentives for different actors 3.5.5 Incentives The case studies reveal that currently, the only incentives for service providers to enhance their greenness are lower operational costs and improved competitiveness. However, if Gartner’s predictions become a reality (Site 1), there will be demand for more green IT services and additional incentives for service providers (table 4). It requires that cloud and hosting service providers will draw up green SLAs and promote their greenness with transparent metrics and certificates, which offer credibility for services. Then they need to compete with greenness and sign contracts with web service providers who then need to illustrate their greenness to current and upcoming end users and try to differentiate themselves from other web service providers. Furthermore, regular end users need to comprehend that every action in the Internet requires electricity. For example, a single Google search uses 0.3 watts of electricity and Google’s daily power draw accounts for 13 million watts (Site 3). Perhaps Google should illustrate this information on search results pages. Hopefully in the future, some end users would even be willing to pay more for services that are proven to be green by favoring; for example, web stores with slightly higher prices, but significantly lower emissions. 83 Tommi Makela Sakari Luukkainen ncentives to Apply Green Cloud Computing 83 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile saving solutions; for example, software that automatically powers down servers or network switches. To guarantee greenness and other features in practice, cloud and hosting service providers need to draw up green SLAs, which determine all the relevant efficiency metrics and acceptable ranges for them. saving solutions; for example, software that automatically powers down servers or network switches. To guarantee greenness and other features in practice, cloud and hosting service providers need to draw up green SLAs, which determine all the relevant efficiency metrics and acceptable ranges for them. The literature implies and the research confirms that the main incentive to improve energy efficiency and to reduce electricity consumption is lower operational costs [1], [5]-[6], [8]. However, there could be more incentives, if the end users of web services started to demand more green services. It would also create derived demand for green cloud services and green hosting services, and it would add extra value to them; for example, better reputations, improved marginal profits and possible larger market shares. Green services would be beneficial for energy companies as well, because they would have more market for renewable energy and energy-efficient district cooling systems. y [15] G.V. Laszewski and L. Wang, Green IT service level agreements, In Grids and Service-Oriented Architectures for Service Level Agreements (P. Wieder et al, Eds.). New York, NY: Springer, 2009, pp- 77-88. [16] Li X Yang and M Zhang Cloud-Cmp: Comparing public cloud providers in Proceedings of the 10th ACM References [1] D. Albano, D. Abood, A. Armstrong, R. Murdoch and J. Whitney, (2010, November) Cloud computing and sustainability: The environmental benefits of moving to the cloud. Accenture. [Online]. Available: http://www.accenture.com/SiteCollectionDocuments/PDF/Accenture_Sustainability_Cloud_Computing_TheEnvi ronmentalBenefitsofMovingtotheCloud.pdf. g p [2] M. Armbrust, A.Fox, R. Griffith, and A.D. Joseph, Above the clouds: A Berkeley view of cloud computing, EECS Department, University of California, Berkeley, CA, Technical Rep. UCB/EECS-2009-28, 2009. C C G [3] Azevedo, M.Blackburn, J. Cooley and M. Patterson. (2011, March) Data Center Efficiency Metrics. The Green Grid. [Online]. Available: www.thegreengrid.org/~/media/TechForumPresentations2011/Data_Center_Efficiency_Metrics_2011.pdf. [4] Balding, J. Howie and D. Hurst. (2010, March) Cloud security alliance. Top Threats to Cloud Computing V1.0. [Online]. Available: https://cloudsecurityalliance.org/topthreats/csathreats.v1.0.pdf. [ ] p y g p p [5] J. Baliga, R.W.A. Ayre, K. Hinton and R.S. Tucker, Green cloud computing: Balancing energy in processing, storage, and transport, in Proceedings of the IEEE, Melbourne, 2011, pp. 149-167. [6] M.T. Chaudhry, T.C. Ling, and A. Manzoor, Considering thermal-aware proactive and reactive scheduling and cooling for green data-centers, in Proceedings 2012 International Conference on Advanced Computer Science Applications and Technologies (ACSAT), Kuala Lumpur, 2012, pp. 87-91. pp g ( ), p , , pp [7] Cook. (2007, September) Technology carbon efficiency - A metric for measuring data center green impact. S3 Amazonas. [Online]. Available: pp g ( ) p pp [7] Cook. (2007, September) Technology carbon efficiency - A metric for measuring data center green impact. S3 Amazonas. [Online]. Available: http://s3.amazonaws.com/zanran_storage/www.datacenterdynamics.com/ContentPages/22802800.pdf. http://s3.amazonaws.com/zanran_storage/www.datacenterdynamics.com/ContentPages/22802800.p p _ g y g p [8] Greenberg, J. Hamilton, D. Maltz and P. Patel, The cost of a cloud: Research problems in data center networks, ACM SIGCOMM CCR, vol. 39, no. 1, pp. 68-73, 2009. pp [9] R. Harms and M. Yamartino. (2010, November) The economics of the cloud. Microsoft Press Release Available: http://www.microsoft.com/en-us/news/presskits/cloud/docs/the-economics-of-the-cloud.pdf [9] R. Harms and M. Yamartino. (2010, November) The economics of the cloud. Microsoft Press Release. [Online]. Available: http://www.microsoft.com/en-us/news/presskits/cloud/docs/the-economics-of-the-cloud.pdf. [10] Heller, P. Mahadevan, and S. Seetharaman, ElasticTree: Saving energy in data center networks, NSDI'10 in Proceedings of the 7th USENIX Conference on Networked Systems Design and Implementation, San José, CA, 2010, pp. 249-264. [11] S Hirsjarvi Tutkimushaastattelu Teemahaastattelun teoria ja kaytanto Helsinki Gaudeamus 2010 pp 34 40 Available: http://www.microsoft.com/en us/news/presskits/cloud/docs/the economics of the cloud.pdf. [10] Heller, P. Mahadevan, and S. Seetharaman, ElasticTree: Saving energy in data center networks, NSDI'10 in Proceedings of the 7th USENIX Conference on Networked Systems Design and Implementation, San José, CA, 2010, pp. 249-264. [15] G.V. Laszewski and L. Wang, Green IT service level agreements, In Grids and Service-Oriented Architectures for Service Level Agreements (P. Wieder et al, Eds.). New York, NY: Springer, 2009, pp- 77-88. [16] Li, X. Yang, and M. Zhang, Cloud-Cmp: Comparing public cloud providers, in Proceedings of the 10th ACM SIGCOMM Conference on Internet Measurement (IMC '10), New York, 2010, pp. 1-14. Websites List Site 1: Gartner press release, CO2 estimates http://www.gartner.com/it/page.jsp?id=503867 Site 2: Emissions trading in European Union http://www.energiamarkkinavirasto.fi/alasivu.asp?gid=199&languageid=826 Site 2: Emissions trading in European Union h // i kki i fi/ l i Site 2: Emissions trading in European Union http://www.energiamarkkinavirasto.fi/alasivu.asp?gid=199&languageid=826 Site 2: Emissions trading in European Union http://www.energiamarkkinavirasto.fi/alasivu.asp?gid=199&languageid=826 Site 3: New York Times, Google Details, and Defends, Its Use of Electricity http://www.nytimes.com/2011/09/09/technology/google-details-and-defends-its-use-of-electricity.html?_r=0 Site 3: New York Times, Google Details, and Defends, Its Use of Electricity http://www.nytimes.com/2011/09/09/technology/google-details-and-defends-i for Service Level Agreements (P. Wieder et al, Eds.). New York, NY: Springer, 2009, pp 77 88. [16] Li, X. Yang, and M. Zhang, Cloud-Cmp: Comparing public cloud providers, in Proceedings of the 10th ACM SIGCOMM Conference on Internet Measurement (IMC '10), New York, 2010, pp. 1-14. 4 Conclusions Service providers should be very careful, when they try new energy- 84 Tommi Makela Sakari Luukkainen centives to Apply Green Cloud Computing 84 Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing urnal of Theoretical and Applied Electronic Commerce Research SN 0718–1876 Electronic Version OL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 saving solutions; for example, software that automatically powers down servers or network switches. To guarantee greenness and other features in practice, cloud and hosting service providers need to draw up green SLAs, which determine all the relevant efficiency metrics and acceptable ranges for them. Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 p g g ( ), , , pp [23] A.T. Velte, T.J, Velte and R. Elsenpeter, Cloud Computing, a Practical Approach, New York: McGraw [24] R. Yin, Case Study Research. Design and Methods. California: Sage Publications. Thousand Oaks, 2 References pp [11] S. Hirsjarvi, Tutkimushaastattelu - Teemahaastattelun teoria ja kaytanto, Helsinki, Gaudeamus, 2010 [11] S. Hirsjarvi, Tutkimushaastattelu - Teemahaastattelun teoria ja kaytanto, Helsinki, Gaudeamus, 2010, pp. 34-40. [12] Huan, A Measurement study of server utilization in public clouds, in Proceedings IEEE the 9th International Conference, on Dependable, Autonomic and Secure Computing (DASC), Sidney, 2011, pp. 435-442. [12] Huan, A Measurement study of server utilization in public clouds, in Proceedings IEEE the 9th International Conference, on Dependable, Autonomic and Secure Computing (DASC), Sidney, 2011, pp. 435-442. p p g ( ) y pp [13] Jarschel and R. Pries, An OpenFlow-based energy-efficient data center approach, in Proceedings of the ACM SIGCOMM Conference on the Applications, Technologies, Architectures, and Protocols for Computer Communication, Helsinki, 2012, pp. 87-88. , , , pp [14] G.B. Kenneth. (2010, August) Revolutionizing Data Center Efficiency. Uptime Institute. [Online]. Available: http://uptimeinstitute.com/component/docman/doc_download/48-update-uptime-institute-revolutionizing-data- center-efficiency. [15] G.V. Laszewski and L. Wang, Green IT service level agreements, In Grids and Service-Oriented Architectures for Service Level Agreements (P. Wieder et al, Eds.). New York, NY: Springer, 2009, pp- 77-88. [16] Li, X. Yang, and M. Zhang, Cloud-Cmp: Comparing public cloud providers, in Proceedings of the 10th ACM SIGCOMM Conference on Internet Measurement (IMC '10), New York, 2010, pp. 1-14. 85 Tommi Makela Sakari Luukkainen Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing Journal of Theoretical and Applied Electronic Commerce Research SSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 Journal of Theoretical and Applied Electronic Commerce Research ISSN 0718–1876 Electronic Version VOL 8 / ISSUE 3 / DECEMBER 2013 / 74-86 © 2013 Universidad de Talca - Chile This paper is available online at www.jtaer.com DOI: 10.4067/S0718-18762013000300006 [17] J. Liu, F. Zhao, X. Liu and W. He, Challenges towards elastic power management in internet data centers, in Proceedings of the 29th IEEE International Conference, on Distributed Computing Systems Workshops, (ICDCS Workshops) Stevenson, 2009, pp.65-72. [17] J. Liu, F. Zhao, X. Liu and W. He, Challenges towards elastic power management in internet data centers, in Proceedings of the 29th IEEE International Conference, on Distributed Computing Systems Workshops, (ICDCS Workshops) Stevenson, 2009, pp.65-72. p p y g p _ _ p [19] Rubenstein, R. Zeighami, R, Lankston and E. References Peterson, Hybrid cooled data center using above ambient liquid cooling, in Proceedings 12th IEEE Intersociety Conference on Thermal and Thermomechanical Phenomena in Electronic Systems (ITherm), Las Vegas, 2010, pp. 2-5. [19] Rubenstein, R. Zeighami, R, Lankston and E. Peterson, Hybrid cooled data center using above ambient liquid cooling, in Proceedings 12th IEEE Intersociety Conference on Thermal and Thermomechanical Phenomena in Electronic Systems (ITherm), Las Vegas, 2010, pp. 2-5. y ( ) g pp [20] J. Rylands. (2010, June) Energy Efficiency in the Datacentre. Questnet. [Online]. Available: https://www.questnet.edu.au/download/attachments/8519738/Jason_Rylands.pdf. p q _ y p [21] Spellman and R. Gimarc, Leveraging the cloud for green IT: Predicting the energy, cost and performance of cloud computing, in Proceedings of CMG International Conference, Dallas, 2009, Paper 9138. p g, g , , , p [22] Uchechukwu, K. Li, and Y. Shen, Improving cloud computing energy efficiency, in Proceedings of the Cloud Computing Congress (APCloudCC), IEEE Asia, 2012, pp. 53-58. p g g p [22] Uchechukwu, K. Li, and Y. Shen, Improving cloud computing energy efficiency, in Proceedings of the Cloud Computing Congress (APCloudCC), IEEE Asia, 2012, pp. 53-58. p g g ( ) pp [23] A.T. Velte, T.J, Velte and R. Elsenpeter, Cloud Computing, a Practical Approach, New York: McGraw-Hill, 2010 [24] R. Yin, Case Study Research. Design and Methods. California: Sage Publications. Thousand Oaks, 2003. 86 Tommi Makela Sakari Luukkainen Tommi Makela Sakari Luukkainen Incentives to Apply Green Cloud Computing
https://openalex.org/W4287634794
https://discovery.ucl.ac.uk/10117258/1/ijms-21-09151-v2.pdf
English
null
Photoimmunotherapy using cationic and anionic photosensitizer-antibody conjugates against HIV Env-expressing cells
Zenodo (CERN European Organization for Nuclear Research)
2,020
cc-by
11,464
Received: 22 October 2020; Accepted: 21 November 2020; Published: 1 December 2020 Abstract: Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; “Click” conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and “Lysine” conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells. Keywords: HIV immunotherapy; photoimmunotherapy; photodynamic therapy; porphyrin; phthalocyanine; HIV-infected cell; monoclonal antibody Photoimmunotherapy Using Cationic and Anionic Photosensitizer-Antibody Conjugates against HIV Env-Expressing Cells Mohammad Sadraeian 1,† , Calise Bahou 2, Edgar Ferreira da Cruz 3, Luíz Mário Ramos Janini 3, Ricardo Sobhie Diaz 3, Ross W. Boyle 4 , Vijay Chudasama 2,* and Francisco Eduardo Gontijo Guimarães 1,* 1 São Carlos Institute of Physics, University of São Paulo, São Carlos, SP 13566-590, Brazil; msadraeian@usp.br 2 Department of Chemistry, University College London, London WC1H 0AJ, UK; calise.bahou.12@ucl.ac.uk 3 Laboratório de Retrovirologia, Disciplina de Microbiologia, Departamento de Microbiologia Imunologia Parasitologia, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil; edgar.cruz@unifesp.br (E.F.d.C.); janini@unifesp.br (L.M.R.J.); rsdiaz@catg.com.br (R.S.D.) 4 Department of Chemistry, University of Hull, Cottingham Road, Hull HU6 7RX, UK; R.W.Boyle@hull.ac.uk * Correspondence: v.chudasama@ucl.ac.uk (V.C.); guimaraes@ifsc.usp.br (F.E.G.G.); Tel.: +44-02076792077(V.C.); +55-(16)33739792 (F.E.G.G.) São Carlos Institute of Physics, University of São Paulo, São Carlos, SP 13566-590, Brazil; msadraeian@usp.br 2 Department of Chemistry, University College London, London WC1H 0AJ, UK; calise.bahou.12@ucl.ac.uk 3 Laboratório de Retrovirologia, Disciplina de Microbiologia, Departamento de Microbiologia Imunologia Parasitologia, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil; edgar.cruz@unifesp.br (E.F.d.C.); janini@unifesp.br (L.M.R.J.); rsdiaz@catg.com.br (R.S.D.) 4 Department of Chemistry, University of Hull, Cottingham Road, Hull HU6 7RX, UK; R.W.Boyle@hull.ac.uk * Correspondence: v.chudasama@ucl.ac.uk (V.C.); guimaraes@ifsc.usp.br (F.E.G.G.); Tel.: +44-02076792077(V.C.); +55-(16)33739792 (F.E.G.G.) ( ) ( ) ( ) † Current addresses: Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, São Carlos, SP 13566-590, Brazil. † Current addresses: Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, São Carlos, SP 13566-590, Brazil. International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Antibody-based therapies have become important clinical tools in treating chronic diseases. Different immunotherapeutic strategies, with limited success, have been investigated to target and eliminate viral-infected cells by using armed antibodies specific to viral proteins [1]. This can be achieved by linking a drug to monoclonal antibodies (MAbs), to form immunotoxins (IT) [2], radio-labeled antibodies [3,4] or other cell-binding conjugates [5,6] capable of specifically killing infected cells, such as HIV infected cells [1,7]. Photoimmunotherapy (PIT) is a targeted photodynamic therapy (PDT) that uses photosensitizer-loaded MAbs. PIT has certain advantages over immunotoxins or Int. J. Mol. Sci. 2020, 21, 9151; doi:10.3390/ijms21239151 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9151 2 of 15 RIT to eradicate infected cells [8]. In immunotherapy based on immunotoxins, a MAb is conjugated to an immunogenic toxin such as ricin [9], pulchellin [10,11] or shiga toxin [12], which can elicit an immunogenicity exemplified by the anti-toxin response [3,13]. PIT is a minimally invasive treatment, which is safer and cheaper than immunotoxins or RIT [14]. While HIV RIT is drawing the attention of researchers [4], there are no studies on the possibility of HIV treatment by PIT. In this paper, we propose HIV immunotherapy via arming HIV monoclonal antibodies with photosensitizers (PSs). Anti-HIV immunoconjugates must be targeted to the HIV envelope spike (Env) that consists of gp160 [9,15], gp120 [16], and gp41 glycoproteins [11,16]. Visible-light activated PSs initially form the excited singlet state, which can decay by emitting fluorescence or, alternatively, it reaches the more stable excited triplet state. [17]. This triplet state can involve electron or hydrogen transfer via Type I photochemical mechanisms to yield superoxide, hydrogen peroxide, and hydroxyl radicals (electron transfer process), typical of phenothiazinium dyes [18], or Type II mechanisms (energy transfer process), typical of porphyrins [19,20], and Rose Bengal [21,22]. Reactive oxygen species (ROS) can damage cell membrane via oxidization of lipids, proteins, and nucleic acids, leading to cell necrosis where cells swell and lyse to release their intracellular content causing inflammation, or alternatively cells can undergo apoptosis [23,24]. The conventional treatment using only PS and light is called photodynamic therapy (PDT), which can destroy cells nonspecifically when exposed to light [23]. Related to this, PIT is the targeted form of conventional PDT, achieved through the conjugation of PS with MAbs targeting specific cell surface receptors [14,25]. Two generations of PICs were produced; Porphyrin-7B2 PIC w the strain-promoted azide−alkyne cycloaddition (SPAAC) method 2.1. Production and Characterization of Photoimmunoconjugates PICs p y y gy p y insertion of a strained alkyne harboring pyridazinediones (PD) into native interchain disulfide bonds in humanized IgG1 MAb (7B2), then, the antibody-alkyne reacted with azide-bearing water-soluble porphyrin. As a side by side comparison, a well-known lysine-reactive and relatively hydrophilic dye, IRD 700DX i diff t l t ti l tl j t d ith i i Two generations of PICs were produced; Porphyrin-7B2 PIC was produced through the use of the strain-promoted azide−alkyne cycloaddition (SPAAC) methodology in two steps: firstly, the insertion of a strained alkyne harboring pyridazinediones (PD) into native interchain disulfide bonds in humanized IgG1 MAb (7B2), then, the antibody-alkyne reacted with azide-bearing water-soluble porphyrin. IRDye 700DX, in different molar concentrations, was covalently conjugated with primary amine groups on the structure of 7B2 MAb using a N-hydroxysuccinimide (NHS) ester of the photosensitizer (Figure 1B). Chemical characterization by UV-Vis spectrophotometry showed a PD loading of 4.3 for the rebridged antibody and a DAR of 4.1 for the final porphyrin-PIC species. IR700-PICs showed DARs As a side by side comparison, a well-known lysine-reactive and relatively hydrophilic dye, IRDye 700DX, in different molar concentrations, was covalently conjugated with primary amine groups on the structure of 7B2 MAb using a N-hydroxysuccinimide (NHS) ester of the photosensitizer (Figure 1B). g y p p y p of 2.1, 3.0 and 4.4, which were equal to molar ratios of 3.5, 7, and 14, before conjugation, respectively (Figure 2A). The DLS results showed that the attachment of small molecule drugs to the 7B2 MAb slightly increases the molecule’s average size (hydrodynamic radius, Rh) and polydispersity (%Pd), indicating the presence of some high molecular weight species in this region (Figure 2A,D). 7B2- porphyrin showed the highest %Pd and Rh in the main peak. The zeta potential of 7B2 antibody changed very slightly from +0.05 mV towards +0.01 and −0.04 for cationic porphyrin and anionic IR- 700 DAR4, respectively (Figure 2A). The binding of the 7B2 based-PICs to gp41 loop peptide antigen was examined within the conjugation. ELISA results showed that an increase of IR700-antibody-ratio decreases the binding ability, as 7B2-IR700 DAR4 showed the lowest binding to the gp41. In contrast, the conjugation of porphyrin with 7B2 MAb did not significantly affect the immunologic specificity of the antibody (Figure 2B). 1. Introduction PIT can also reduce other issues, such as dark toxicity and PS aggregation in aqueous media which reduce PDT efficacy [14]. The conjugation of a photosensitizer to antibodies can be achieved through chemical modification of the ε-amino group present on lysine residues [26] or through reactions with the thiol moiety present on cysteine residues (generated via reduction of interchain disulfide bonds). However, neither of these conjugation strategies is ideal [27,28]. Lysine modification can yield heterogeneous products with a broad distribution of drug loading, and conventional cysteine modification methods utilize interchain disulfide reduction, and result in the permanent loss of structural disulfide bonds, which has been shown to potentially impact Fc function [29] and negatively affect the stability of the antibody in vivo [30,31]. Recently, we have employed pyridazinediones (PDs) to functionally re-bridge the interchain disulfide bonds antibodies (PDs) bearing orthogonal ‘clickable’ handles into the native inter-strand disulfide bonds of trastuzumab full antibody [32–35]. By utilizing PDs harboring orthogonal ‘clickable’ handles, functional groups have been added to antibodies whilst retaining the covalent interchain linkage. This technology was then combined with mild bio-orthogonal conjugation of porphyrin photosensitizers and the conjugate targeted Her2 positive breast cancer cells [36,37]. In this study, we utilized lysine and disulfide modifications to identify an appropriate photo-immunoconjugate (PIC) for our suggested PIT. Briefly, a human anti-gp41 antibody (7B2) [38] was conjugated with two different photosensitizers, cationic porphyrin with a net charge of 3+ and anionic IR700 with a net charge of 4-. We employed two different strategies for conjugation to the antibody; lysine conjugation by using phthalocyanine IRDye700DX dye [26], and “Click” conjugation by using an azide-bearing porphyrin with a strained alkyne attached via a disulfide bridge linker [27]. The comparison between PICs is of interest with regard to the dosimetry and solubility of PICs and mechanism of in vitro cytotoxicity (Figure 1A). 3 of 15 16 Int. J. Mol. Sci. 2020, 21, 9151 J Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2- porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. 1. Introduction The presence of CD4 may increase the binding ability of 7B2-porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2- porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. 2. Results and Discussion Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2-porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. 1. Introduction Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. 2. Results and Discussion Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2-porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2- porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4. 2. Results and Discussion Figure 1. Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). Two generations of PICs were produced; Porphyrin-7B2 PIC w the strain-promoted azide−alkyne cycloaddition (SPAAC) method 2.1. Production and Characterization of Photoimmunoconjugates PICs 924 MAb was used as an isotype control. (C) Microcapillary electrophoresis of reduced and non-reduced 7B2 MAb and PICs, before and after irradiation. Unlike 7B2 1 2 Figure 2. Characterization of photo-immunoconjugates (PICs). (A) The table shows products of 7B2 MAb conjugated with different molar concentrations of IR700, rebridged antibody with pyridazinedione and porphyrin. Chemical characterization by UV-Vis spectrophotometry shows the antibody to PS ratio (DAR). In DLS studies, the zeta potential (ζ) was estimated according to the Huckel equation. The increase in the molecular weights (MWs) of conjugations, measured by microcapillary electrophoresis, are in confirmation of determined DARs. (B) ELISA plates coated with gp41 antigen, as a peptide representing 7B2′s epitope. Results are representative of means of triplicate values with three individual experiments. 924 MAb was used as an isotype control. (C) Microcapillary electrophoresis of reduced and non-reduced 7B2 MAb and PICs, before and after irradiation. Unlike 7B2 and 7B2-IR700, the reduced 7B2-porphyrin did not show the separation of light and heavy chains, confirming the accuracy of click chemistry with DAR4. Size standards are indicated on the side of each “gel”. (D) DLS Histograms of hydrodynamic radius (Rh) for 7B2 MAb and PICs, monitoring how the Rh preserved during the conjugation process. When IR700 was conjugated with an antibody molecule, ligand dissociation from IR700 affected the shape and solubility of the antibody to which it is attached as well as antibody−antigen complexes. 2 Figure 2. Characterization of photo-immunoconjugates (PICs). (A) The table shows products of 7B2 MAb conjugated with different molar concentrations of IR700, rebridged antibody with pyridazinedione and porphyrin. Chemical characterization by UV-Vis spectrophotometry shows the antibody to PS ratio (DAR). In DLS studies, the zeta potential (ζ) was estimated according to the Huckel equation. The increase in the molecular weights (MWs) of conjugations, measured by microcapillary electrophoresis, are in confirmation of determined DARs. (B) ELISA plates coated with gp41 antigen, as a peptide representing 7B2′s epitope. Results are representative of means of triplicate values with three individual experiments. 924 MAb was used as an isotype control. (C) Microcapillary 1 2 Figure 2. Characterization of photo-immunoconjugates (PICs). (A) The table shows products of 7B2 MAb conjugated with different molar concentrations of IR700, rebridged antibody with pyridazinedione and porphyrin. Chemical characterization by UV-Vis spectrophotometry shows the antibody to PS ratio (DAR). In DLS studies, the zeta potential (ζ) was estimated according to the Huckel equation. Two generations of PICs were produced; Porphyrin-7B2 PIC w the strain-promoted azide−alkyne cycloaddition (SPAAC) method 2.1. Production and Characterization of Photoimmunoconjugates PICs Results of microcapillary electrophoresis of non-reduced antibody (H and L chains) and PICs demonstrated the molecular weights (MWs) with 151.4, 166.1 and 166.9 kDa for naked antibody, rebridged antibody and clicked porphyrin-antibody. The increase in the MWs of Chemical characterization by UV-Vis spectrophotometry showed a PD loading of 4.3 for the rebridged antibody and a DAR of 4.1 for the final porphyrin-PIC species. IR700-PICs showed DARs of 2.1, 3.0 and 4.4, which were equal to molar ratios of 3.5, 7, and 14, before conjugation, respectively (Figure 2A). The DLS results showed that the attachment of small molecule drugs to the 7B2 MAb slightly increases the molecule’s average size (hydrodynamic radius, Rh) and polydispersity (%Pd), indicating the presence of some high molecular weight species in this region (Figure 2A,D). 7B2-porphyrin showed the highest %Pd and Rh in the main peak. The zeta potential of 7B2 antibody changed very slightly from +0.05 mV towards +0.01 and −0.04 for cationic porphyrin and anionic IR-700 DAR4, respectively (Figure 2A). The binding of the 7B2 based-PICs to gp41 loop peptide antigen was examined within the conjugation. ELISA results showed that an increase of IR700-antibody-ratio decreases the binding ability, as 7B2-IR700 DAR4 showed the lowest binding to the gp41. In contrast, the conjugation of porphyrin with 7B2 MAb did not significantly affect the immunologic specificity of the antibody (Figure 2B). Results of microcapillary electrophoresis of non-reduced antibody (H and L chains) and PICs demonstrated the molecular weights (MWs) with 151.4, 166.1 and 166.9 kDa for naked antibody, rebridged antibody and clicked porphyrin-antibody. The increase in the MWs of 4 of 15 Int. J. Mol. Sci. 2020, 21, 9151 IR700-PICs were in agreement of determined DARs, with 154.1, 154.7 and 156.8 kDa for DARs 2, 3 and 4, respectively (Figure 2A,C). 4, respectively (Figure 2A,C). 2 Figure 2. Characterization of photo-immunoconjugates (PICs). (A) The table shows products of 7B2 MAb conjugated with different molar concentrations of IR700, rebridged antibody with pyridazinedione and porphyrin. Chemical characterization by UV-Vis spectrophotometry shows the antibody to PS ratio (DAR). In DLS studies, the zeta potential (ζ) was estimated according to the Huckel equation. The increase in the molecular weights (MWs) of conjugations, measured by microcapillary electrophoresis, are in confirmation of determined DARs. (B) ELISA plates coated with gp41 antigen, as a peptide representing 7B2′s epitope. Results are representative of means of triplicate values with three individual experiments. 2.2. Flow Cytometry Analysis for water-soluble homogen structural bridges on the a By using a Cary Eclipse UV-Vis-NIR spectroscopy, we analyzed the excitation and emission spectra before flow cytometry study without using a secondary antibody. We showed the excitation of porphyrin by blue laser at 488 nm would not interfere with the emission intensity. The excitation by violet laser at 405 nm caused red emission at 725 nm with 1.163 (a. u.) intensity, while the maximum red fluorescent intensity (3.779 a. u.) can be observed by the excitation of Soret band of porphyrin at 432 nm. The results were in agreement with the theoretical analysis of UV-Vis spectra (Supplementary Figure S1). 2.2. Flow Cytometry analysis By using a Cary Eclipse UV-Vis-NIR spectroscopy, we analyzed the excitation and emission spectra before flow cytometry study without using a secondary antibody. We showed the excitation of porphyrin by blue laser at 488 nm would not interfere with the emission intensity. The excitation by violet laser at 405 nm caused red emission at 725 nm with 1.163 (a. u.) intensity, while the maximum red fluorescent intensity (3.779 a. u.) can be observed by the excitation of Soret band of porphyrin at 432 nm The results were in agreement with the theoretical analysis of UV-Vis spectra Specific cell binding to native Env was examined by flow cytometry. Without using a secondary antibody, the red fluorescent emission from IR700-PICs and porphyrin-PIC were directly detected on the Env-transfected cells, by filter Qdot 705 and filter Qdot 655, respectively (Figure 3A). By using FITC-secondary antibody, PICs showed equal binding ability into Env-transfected cells, while 7B2-IR700 DAR4 showed somewhat unspecific binding into the control 293T cells (Figure 3B). Not surprisingly, the presence of soluble CD4 (sCD4) showed a synergic effect on the binding ability of porphyrin-7B2, in comparison to the absence of sCD4 (Figure 3C). This result is in agreement with our previous studies on immunotoxins [11,16], as we showed sCD4 enhances epitope exposure. porphyrin at 432 nm. The results were in agreement with the theoretical analysis of UV Vis spectra (Supplementary Figure S1). Specific cell binding to native Env was examined by flow cytometry. Without using a secondary antibody, the red fluorescent emission from IR700-PICs and porphyrin-PIC were directly detected on the Env-transfected cells, by filter Qdot 705 and filter Qdot 655, respectively (Figure 3A). Two generations of PICs were produced; Porphyrin-7B2 PIC w the strain-promoted azide−alkyne cycloaddition (SPAAC) method 2.1. Production and Characterization of Photoimmunoconjugates PICs The increase in the molecular weights (MWs) of conjugations, measured by microcapillary electrophoresis, are in confirmation of determined DARs. (B) ELISA plates coated with gp41 antigen, as a peptide representing 7B2′s epitope. Results are representative of means of triplicate values with three individual experiments. 924 MAb was used as an isotype control. (C) Microcapillary electrophoresis of reduced and non-reduced 7B2 MAb and PICs, before and after irradiation. Unlike 7B2 and 7B2-IR700, the reduced 7B2-porphyrin did not show the separation of light and heavy chains, confirming the accuracy of click chemistry with DAR4. Size standards are indicated on the side of each “gel”. (D) DLS Histograms of hydrodynamic radius (Rh) for 7B2 MAb and PICs, monitoring how the Rh preserved during the conjugation process. When IR700 was conjugated with an antibody molecule, ligand dissociation from IR700 affected the shape and solubility of the antibody to which it is attached as well as antibody−antigen complexes. In the presence of reducing agent (TCEP), the light and heavy chains of either 7B2 or 7B2-IR700 were observed, due to the reduction of all available interchain disulfide bridges. The reduced 7B2-porphyrin did not show the separation of chains, confirming the accuracy of click chemistry with DAR4 (Figure 2C). Int. J. Mol. Sci. 2020, 21, 9151 molecule, ligand d is attached as wel 5 of 15 In total, the characterization of PICs showed that the shape, solubility and binding ability of IR700-PICs was affected by increasing the drug-to-antibody ratio (DAR), while they were preserved for water-soluble homogeneous porphyrin-7B2 with a constant DAR of 4, due to the retention of rigid structural bridges on the antibody. p g g g y were observed, due to the reduction of all available interchain disulfide bridges. The reduced 7B2- porphyrin did not show the separation of chains, confirming the accuracy of click chemistry with DAR4 (Figure 2C). In total, the characterization of PICs showed that the shape, solubility and binding ability of IR700-PICs was affected by increasing the drug-to-antibody ratio (DAR) while they were preserved 2.2. Flow Cytometry Analysis for water-soluble homogen structural bridges on the a By using FITC-secondary antibody, PICs showed equal binding ability into Env-transfected cells, while 7B2- IR700 DAR4 showed somewhat unspecific binding into the control 293T cells (Figure 3B). Not surprisingly, the presence of soluble CD4 (sCD4) showed a synergic effect on the binding ability of porphyrin-7B2, in comparison to the absence of sCD4 (Figure 3C). This result is in agreement with our previous studies on immunotoxins [11,16], as we showed sCD4 enhances epitope exposure. Figure 3. The flow cytometry histogram of PICs by using 293T control cells and Env-transfected 293T cells incubated in PBS + 1% BSA + sodium azide (0.2%). (A) PIC binding to 293T cells transfected with 92UG037.8 gp160 (293T/92UG) cells was directly detected through red fluorescent emission from Figure 3. The flow cytometry histogram of PICs by using 293T control cells and Env-transfected 293T cells incubated in PBS + 1% BSA + sodium azide (0.2%). (A) PIC binding to 293T cells transfected with 92UG037.8 gp160 (293T/92UG) cells was directly detected through red fluorescent emission from IR700-PICs and porphyrin-PIC, by filter Qdot 705 and filter Qdot 655, respectively. (B) By using FITC-secondary immunofluorescence, PICs showed equal binding ability into Env-transfected cells, while 7B2-IR700 DAR4 showed somewhat unspecific binding into the control 293T cells. Results are representative of at least three independent experiments. Isotype control (chimeric RAC18) is shown as blue shaded histogram. (C) Studying the binding ability of porphyrin-7B2, in the presence and absence of soluble CD4 (sCD4). Figure 3. The flow cytometry histogram of PICs by using 293T control cells and Env-transfected 293T cells incubated in PBS + 1% BSA + sodium azide (0.2%). (A) PIC binding to 293T cells transfected with 92UG037.8 gp160 (293T/92UG) cells was directly detected through red fluorescent emission from Figure 3. The flow cytometry histogram of PICs by using 293T control cells and Env-transfected 293T cells incubated in PBS + 1% BSA + sodium azide (0.2%). (A) PIC binding to 293T cells transfected with 92UG037.8 gp160 (293T/92UG) cells was directly detected through red fluorescent emission from IR700-PICs and porphyrin-PIC, by filter Qdot 705 and filter Qdot 655, respectively. (B) By using FITC-secondary immunofluorescence, PICs showed equal binding ability into Env-transfected cells, while 7B2-IR700 DAR4 showed somewhat unspecific binding into the control 293T cells. Results are representative of at least three independent experiments. Isotype control (chimeric RAC18) is shown as blue shaded histogram. 2.3. ROS Generation In cellular studies, to have an equal irradiation for both porphyrin-PIC and IR700-PIC, for a side by side comparison, a custom-made LED device with a broad spectrum of light (380−780 nm) was utilized, as the blue spectra can excite the Soret band of porphyrin at 432 nm, meanwhile, red spectra excites IR700-7B2 at 689 nm (Figure 4A). 4 Figure 4. ROS generation and apoptosis assay by PICs. (A) The diagram demonstrates a broad spectrum of light (380−780 nm) by a homemade LED device (30 mW/cm2) for irradiation of both IR700 and porphyrin PICs, at the same time. Blue spectra can excite the Soret band of porphyrin at 432 nm, meanwhile red spectra excite 7B2-IR700 at 689 nm. (B) Samples were kept in the dark (represented by grey) or were irradiated with 50 J/cm2. (C) As a positive control, 293T cells were treated with 80 µM H2O2 for 24 h in dark. Control group (without H2O2) was incubated under the same conditions. (D) In apoptosis assay by Annexin V - FITC/PI, the Env-transfected cells were incubated with PICs for 4 h, then irradiated. PICs showed cell death due to late apoptosis (purple color in subset Upper Right) after 4 h incubation. Control 7B2 antibody showed neither cell death nor apoptosis sign. (n = 3). 4 Figure 4. ROS generation and apoptosis assay by PICs. (A) The diagram demonstrates a broad spectrum of light (380−780 nm) by a homemade LED device (30 mW/cm2) for irradiation of both IR700 and porphyrin PICs, at the same time. Blue spectra can excite the Soret band of porphyrin at 432 nm, meanwhile red spectra excite 7B2-IR700 at 689 nm. (B) Samples were kept in the dark (represented by grey) or were irradiated with 50 J/cm2. (C) As a positive control, 293T cells were treated with 80 µM H2O2 for 24 h in dark. Control group (without H2O2) was incubated under the same conditions. (D) In apoptosis assay by Annexin V - FITC/PI, the Env-transfected cells were incubated with PICs for 4 h, then irradiated. PICs showed cell death due to late apoptosis (purple color in subset Upper Right) after 4 h incubation. Control 7B2 antibody showed neither cell death nor apoptosis sign. (n = 3). To detect the ROS generation, the incubated cells by PICs and H2 DCF-DA fluorescent probe were irradiated with 50 J/cm2. 2.2. Flow Cytometry Analysis for water-soluble homogen structural bridges on the a (C) Studying the binding ability of porphyrin-7B2, in the presence and absence of soluble CD4 (sCD4). Int. J. Mol. Sci. 2020, 21, 9151 6 of 15 Interestingly, IR700-PICs with different DARs (Figure 2A) and different binding abilities (Figure 2B) showed equal fluorescence intensity, not only with the direct red emission by filter Qdot 705 (Figure 3A), also with indirect emission by FITC-secondary antibody (Figure 3B). This can be due to the quenching of chromophores, as some that are too close to each other will prevent the chromophores from being excited by laser light and then bleaching [39]. There is no linear relationship between the number of chromophores per antibody and fluorescence intensity [40], while there is a correlation between the trends in fluorescence quenching and efficacy of heterogeneous IR700-PICs [41]. We assume this quenching would not happen in homogeneous porphyrin-PIC with disulfide modification. 2.5. Photo-Cytotoxicity Assay 2 5 Photo Cytoto i ity A ay Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with a serial dilution of PICs starting at 500 nM in the presence of sCD4 (anti-gp120) for 4 h then were assayed by photoimmunotherapy (PIT) with 20 J/cm2. IR700-PIC with a DAR of 3 appeared more cytotoxic than porphyrin-PIC with a constant DAR of 4. PICs showed neither dark cytotoxicity on transfected cells nor nonspecific killing on 293T control cells. The cell growth inhibition was observed due to the irradiation on the cells without PIC treatment (green stars in Figure 5A). 2.5. Photo Cytotoxicity Assay Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with a serial dilution of PICs starting at 500 nM in the presence of sCD4 (anti-gp120) for 4 h then were assayed by photoimmunotherapy (PIT) with 20 J/cm2. IR700-PIC with a DAR of 3 appeared more cytotoxic than porphyrin-PIC with a constant DAR of 4. PICs showed neither dark cytotoxicity on transfected cells nor nonspecific killing on 293T control cells. The cell growth inhibition was observed due to the irradiation on the cells without PIC treatment (green stars in Figure 5A). Figure 5. Comparing the photo-cytotoxicity and targeting of PICs by direct cytotoxicity assay. (A) Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with 7B2-IR700 (DAR 3), 7B2-porphyrin and an equal molarity mixture of both PICs in the presence of sCD4 (anti-gp120) for 4 h then were assayed by PIT. Data are ± means S.E.M. (n = 3) with three individual experiments. Where no error bars are visible, they are obscured by the symbol. (B) An equal molarity mixture of porphyrin-PIC with IR700-PIC may increase the cytotoxicity in compare to porphyrin-PIC or the average cytotoxicity of both PICs. Data are ± means S.E.M. (n = 3). (C) Incubation of transfected cells Figure 5. Comparing the photo-cytotoxicity and targeting of PICs by direct cytotoxicity assay. (A) Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with 7B2-IR700 (DAR 3), 7B2-porphyrin and an equal molarity mixture of both PICs in the presence of sCD4 (anti-gp120) for 4 h then were assayed by PIT. Data are ± means S.E.M. (n = 3) with three individual experiments. Where no error bars are visible, they are obscured by the symbol. 2.3. ROS Generation H2DCF-DA fluorescent probe was applied to detect ROS production in PIT-treated cells by using flow cytometry. The Env-transfected cells incubated with PICs exhibited a significant increase in ROS levels, whereas control 293T cells did not show ROS production (Figure 4B). In parallel, in the positive control group, we observed ROS production in 293T cells which were treated with 80 µM H2O2 for 24 h in the dark (Figure 4C). The irradiated cells, with either no incubation or incubated with 7B2 antibody, showed an increase in the fluorescence intensity in comparison to the unstained cells in darkness. Int. J. Mol. Sci. 2020, 21, 9151 H2O2 for 24 h in d In apoptosis assay 7 of 15 7 of 15 2.4. Apoptosis Assay after 4 hr incu For apoptosis assays, the Env-transfected cells were incubated by PICs for 4 h, irradiated, then Annexin V - FITC/PI dye added before flow cytometry analysis. No features related to early stages of apoptosis, such as extracellular exposure of phosphatidylserine, was observed in response to PICs (Figure 4D, purple color in the lower right subset). Both porphyrin-PIC and IR700-PIC showed cell death due to late apoptosis (FITC emission) after 4 h incubation, and signs of necrosis (PI emission). Control 7B2 antibody showed neither cell death nor signs of apoptosis (Figure 4D). The non-irradiated cells did not show sign of apoptosis. 2.4. Apoptosis Assay For apoptosis assays, the Env-transfected cells were incubated by PICs for 4 h, irradiated, then Annexin V - FITC/PI dye added before flow cytometry analysis. No features related to early stages of apoptosis, such as extracellular exposure of phosphatidylserine, was observed in response to PICs (Figure 4D, purple color in the lower right subset). Both porphyrin-PIC and IR700-PIC showed cell death due to late apoptosis (FITC emission) after 4 h incubation, and signs of necrosis (PI emission). Control 7B2 antibody showed neither cell death nor signs of apoptosis (Figure 4D). The non- i di d ll did h i f i 2.6. Confocal Microscopy Study The cells were treated with IR700-7B2 or 7B2-porphyrin for 1 h, then washed and irradiated by 2-photons, 800 nm, 2.2 mW/cm. Red fluorescence emission from IR700-7B2 was directly detected (Figure 6A and Supplementary Video S1). As 7B2-porphyrin did not show strong red fluorescent emission, FITC anti-human IgG secondary antibody was applied to detect PIC indirectly (Figure 6B and Supplementary Video S2). After 10 min of adding porphyrin-7B2, the cellular swelling and bleb formation were observed as signs of necrotic cell death. The same necrotic signs were observed by adding IR700-7B2, but in 5 min of irradiation. After 30 min irradiation, Acridine Orange and Ethidium Bromide (AO/EB) were added as Live-dead dyes. Interestingly, the irradiated and non-irradiated regions were clearly distinguishable in red and green colors, respectively, confirming no dark toxicity by PICs (Figure 6C). In conclusion, we compared the mechanism of two photosensitizers in the formation of PICs regarding the induction of cell death in HIV Env-transfected cells. We also addressed some of the main issues associated with conventional PDT including low selectivity, controlled drug dosimetry, dark toxicity and low water solubility of photosensitizer. Finally, we studied the possibility of PIT application to directly killing HIV-1 Env-expressing cells. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the antigen on the cell membrane or HIV Envelope, whilst being independent of the PS type. Considering the fundamental restriction of light penetration, we suggest optimizing the excitation of PSs in deep tissue that may give us a highly flexible theranostic platform for HIV researches. This inevitably issue dictates the use of high-power irradiation [42]. The other alternatives for light delivery to the PICs in deep tissue could be the application of cellular-scale inorganic light-emitting diode (µ-ILED) arrays [43], or upconversion nanoparticles (UCNPs) in the field of PIT [44]. Approaches that focus on the combination of the PIT strategy with “shock and kill” technique could be a cornerstone of future research efforts towards an HIV cure [45]. The photo-cytotoxicity on the cell membrane triggered by the presence of HIV proteins can be an interesting to be proven strategy aiming the elimination of HIV latent reservoir in vivo. Previously, we observed different levels of surface HIV Env on the transfected and infected cells which may reflect differences due to laboratory-adapted versus clinical isolates [11]. 2.5. Photo-Cytotoxicity Assay 2 5 Photo Cytoto i ity A ay (B) An equal molarity mixture of porphyrin-PIC with IR700-PIC may increase the cytotoxicity in compare to porphyrin-PIC or the average cytotoxicity of both PICs. Data are ± means S.E.M. (n = 3). (C) Incubation of transfected cells in two different times (1 and 4 h) before PIT showed the internalization-dependency of Mab-porphyrin to have more photo-cytotoxicity, while the membrane binding of 7B2-IR700 was sufficient to induce cell death. Data are ± means S.E.M. (n = 3). Figure 5. Comparing the photo-cytotoxicity and targeting of PICs by direct cytotoxicity assay. (A) Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with 7B2-IR700 (DAR 3), 7B2-porphyrin and an equal molarity mixture of both PICs in the presence of sCD4 (anti-gp120) for 4 h then were assayed by PIT. Data are ± means S.E.M. (n = 3) with three individual experiments. Where no error bars are visible, they are obscured by the symbol. (B) An equal molarity mixture of porphyrin-PIC with IR700-PIC may increase the cytotoxicity in compare to porphyrin-PIC or the average cytotoxicity of both PICs. Data are ± means S.E.M. (n = 3). (C) Incubation of transfected cells Figure 5. Comparing the photo-cytotoxicity and targeting of PICs by direct cytotoxicity assay. (A) Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with 7B2-IR700 (DAR 3), 7B2-porphyrin and an equal molarity mixture of both PICs in the presence of sCD4 (anti-gp120) for 4 h then were assayed by PIT. Data are ± means S.E.M. (n = 3) with three individual experiments. Where no error bars are visible, they are obscured by the symbol. (B) An equal molarity mixture of porphyrin-PIC with IR700-PIC may increase the cytotoxicity in compare to porphyrin-PIC or the average cytotoxicity of both PICs. Data are ± means S.E.M. (n = 3). (C) Incubation of transfected cells in two different times (1 and 4 h) before PIT showed the internalization-dependency of Mab-porphyrin to have more photo-cytotoxicity, while the membrane binding of 7B2-IR700 was sufficient to induce cell death. Data are ± means S.E.M. (n = 3). As a prospect of this approach in an in vivo study, the doses of irradiation can be increased to maximum energy fluence of 100 J/cm2, depending on the selected wavelength, however, visible light Int. J. Mol. Sci. 2020, 21, 9151 8 of 15 irradiation is unlikely to be toxic [42]. 2.5. Photo-Cytotoxicity Assay 2 5 Photo Cytoto i ity A ay Optical microscopic observations before and after PIT demonstrated the bleb formation as signs of necrotic cell death. Cells PIT-treated with IR700-7B2 showed not only rapid bleb formation but also cell debris (Supplementary Figure S2). To study how the combination of both PICs may affect the cytotoxicity, an equal molarity of porphyrin-PIC was mixed with IR700-PIC. Interestingly, this combination showed a significant increase in the cytotoxicity in compare to the average cytotoxicity of both PICs, due to the synergic effect of two PICs with two different mechanism (Figure 5A,B). Furthermore, the photo-cytotoxicity of porphyrin-7B2 increased twice by increasing the time of incubation from 1 h to 4 h, indicating the internalization-dependency of porphyrin-mAb. In contrast, the photo-cytotoxicity of IR700-7B2 did not change significantly on increasing the time of incubation at 37 ◦C (Figure 5C). 2.6. Confocal Microscopy Study However, it is still unclear how these discrepancies may relate to the expressed levels of HIV Env on the surface of HIV-infected cells, especially in the context of HIV-1 latency. We therefore recognize that further experimental killing approach on HIV-1 latently infected cells would add fundamental knowledge to the proposed PIT strategy. To the best of our knowledge, this is the first report to demonstrate the utility of photoimmunotherapy for HIV Env-expressing cells. The results of this strategy can potentially translate to viral photo-immunotherapy against other enveloped viruses. 9 of 15 Int. J. Mol. Sci. 2020, 21, 9151 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 16 Figure 6. Target-specific cell death in response to PIT in HIV Env-expressing 293T cells. The cells were treated with IR700-7B2 (A) or 7B2-porphyrin (B) in the presence of sCD4 for 1 hour before laser irradiation by microscope. (A) The emitted red fluorescent from IR700-7B2 was directly detected. (B) 7B2-porphyrin was detected indirectly by using FITC anti-human IgG secondary antibody. After 30 minutes irradiation, the cell viability was studied by using Acridine Orange and Ethidium Bromide (Live/dead dye). White arrows indicate the bleb formation caused the necrotic cell death. The white bar indicates 10 µm. (C) The red square shows the irradiated region that the cells are dead (red color). The non-irradiated region (green color) signifies live cells, confirming no dark toxicity by PIC. The white bar indicates 20 µm. In conclusion, we compared the mechanism of two photosensitizers in the formation of PICs regarding the induction of cell death in HIV Env-transfected cells. We also addressed some of the main issues associated with conventional PDT including low selectivity, controlled drug dosimetry, Figure 6. Target-specific cell death in response to PIT in HIV Env-expressing 293T cells. The cells were treated with IR700-7B2 (A) or 7B2-porphyrin (B) in the presence of sCD4 for 1 h before laser irradiation by microscope. (A) The emitted red fluorescent from IR700-7B2 was directly detected. (B) 7B2-porphyrin was detected indirectly by using FITC anti-human IgG secondary antibody. After 30 min irradiation, the cell viability was studied by using Acridine Orange and Ethidium Bromide (Live/dead dye). White arrows indicate the bleb formation caused the necrotic cell death. The white bar indicates 10 µm. (C) The red square shows the irradiated region that the cells are dead (red color). 3.1.2. Cell Lines 293T/92UG cell lines stably express clade A clinical isolate 92UG037.8 gp160 as native trimers of HIV gp120/gp41. In this paper, Env-transfected cells refer to 293T/92UG cell lines [46], and HEK 293T cells refer to non-transfected control cells. The transfected and non-transfected 293T cells were incubated at 37 ◦C in 5% CO2 in DMEM medium with 10% fetal calf serum (Gibco Invitrogen, Grand Island, NY, USA). 2.6. Confocal Microscopy Study The non-irradiated region (green color) signifies live cells, confirming no dark toxicity by PIC. The white bar indicates 20 µm. M t i l d M th d Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Figure 6. Target-specific cell death in response to PIT in HIV Env-expressing 293T cells. The cells were treated with IR700-7B2 (A) or 7B2-porphyrin (B) in the presence of sCD4 for 1 hour before laser irradiation by microscope. (A) The emitted red fluorescent from IR700-7B2 was directly detected. (B) 7B2-porphyrin was detected indirectly by using FITC anti-human IgG secondary antibody. After 30 minutes irradiation, the cell viability was studied by using Acridine Orange and Ethidium Bromide (Live/dead dye). White arrows indicate the bleb formation caused the necrotic cell death. The white bar indicates 10 µm. (C) The red square shows the irradiated region that the cells are dead (red color). The non-irradiated region (green color) signifies live cells, confirming no dark toxicity by PIC. The white bar indicates 20 µm. In conclusion, we compared the mechanism of two photosensitizers in the formation of PICs regarding the induction of cell death in HIV Env-transfected cells. We also addressed some of the Figure 6. Target-specific cell death in response to PIT in HIV Env-expressing 293T cells. The cells were treated with IR700-7B2 (A) or 7B2-porphyrin (B) in the presence of sCD4 for 1 h before laser irradiation by microscope. (A) The emitted red fluorescent from IR700-7B2 was directly detected. (B) 7B2-porphyrin was detected indirectly by using FITC anti-human IgG secondary antibody. After 30 min irradiation, the cell viability was studied by using Acridine Orange and Ethidium Bromide (Live/dead dye). White arrows indicate the bleb formation caused the necrotic cell death. The white bar indicates 10 µm. (C) The red square shows the irradiated region that the cells are dead (red color). The non-irradiated region (green color) signifies live cells, confirming no dark toxicity by PIC. The white bar indicates 20 µm. penetration, we flexible theranos 3.1.1. Chemical Reagents irradiation [42]. The other alternatives for light delivery to the PICs in deep tissue could be the application of cellular-scale inorganic light-emitting diode (µ-ILED) arrays [43], or upconversion nanoparticles (UCNPs) in the field of PIT [44]. All reagents are from ThermoFisher Scientific (Waltham, MA, USA) unless a statement to the contrary is made. main issues assoc dark toxicity and 3. Materials and Methods application to d primarily depen Envelope whilst 3.1. Consumable Materials 3.1.3. Antibodies MAb 7B2 (GenBank accession numbers JX188438 and JX188439) is an Anti-gp41 hIgG1 binding at amino acids 598–604 (CSGKLIC) in the helix-loop-helix region [38,47]. HY (Genbank accession numbers JX188440 and JX188441), is hIgG1/kappa, an affinity matured version of the anti-CD4 binding site Ab b12 [48]. MAbs were purified from supernatant media of hybridoma after passing Protein 10 of 15 Int. J. Mol. Sci. 2020, 21, 9151 A agarose beads (Invitrogen, Carlsbad, CA, USA). Two types of soluble CD4 were utilized to study CD4-mediated effects [47]; CD4-IgG2 is a hIgG2 containing a tetrameric fusion protein in which the Fv portions of both H and L chains have been substituted by the V1 and V2 domains of hCD4. CD4-183 is a soluble fragment of human CD4 that binds the HIV gp120. Goat anti-human IgG (Invitrogen) was conjugated to either fluorescein isothiocyanate (FITC) or alkaline phosphatase (AP). 3.4. UV-Vis Spectroscopy UV-Vis spectroscopy was used to measure protein concentrations and photosensitizer-antibody ratios (DAR), firstly by using Nanodrop 1000 UV-Visible spectrophotometer (ThermoFisher Scientific) and then a Cary 100 Bio UV-Visible spectrophotometer (Varian, CA, USA) operating at 21 ◦C. Blank was the sample buffer to correct base line with extinction coefficients; ε280 = 250,440 M−1 cm−1 for antibodies, ε335 = 9100 M−1 cm−1 for pyridazinedione scaffolds, ε422 = 165,175 M−1 cm−1 for porphyrin and ε689 = 165,000 M−1 cm−1 for IRDye 700DX. A Correction Factor at 280 nm of 0.25 (at A335) was applied for pyridazinedione scaffolds, as described elsewhere [36]. 3.6. ELISA Purified MAbs and photo-immunoconjugates (PICs) were analyzed for antigen-binding specificity and titration by using ELISA, in wells coated with gp41 antigen (1 µg/mL), as described elsewhere [16]. The gp41 antigen is a linear peptide sequence [LGIWGCSGKLICTT] representing the epitope of MAb 7B2. AP-conjugated secondary antibody (goat anti-human IgG) (Zymed Laboratories, South San Francisco, CA, USA) were used to detect the primary antibodies binding to the antigen. Data are represented as optical density at 405 nm and signify means of triplicate values with three independent experiments. 3.3. Conjugation and Optimization of IR700-Antibody by Lysine Modification 3.3. Conjugation and Optimization of IR700-Antibody by Lysine Modification IRDye 700DX (IR700, LI-COR Biosciences, Lincoln, NE, USA) was covalently linked to purified MAb 7B2 through an N-hydroxysuccinimide reactive group, according to the manufacturer’s instructions [26]. The process of conjugation and optimization are described in detail in the supplementary methods. 3.5. Electrophoresis Molecular size, purity and accuracy of the conjugation of products were determined using Non/reducing glycine-SDS-PAGE and then confirmed by microcapillary electrophoresis in the presence or absence of TCEP·HCl (6.0 µL, 20 mM in d.d water, 12 eq.), as a reductive agent (Agilent Bioanalyzer, GE Healthcare, Piscataway, NJ, USA), following standard lab procedures. 3.2. Porphyrin-Antibody Conjugation by Click Chemistry Conjugation between azide porphyrin and mAb 7B2 was carried out in two steps: antibody functionalization, then click chemistry conjugation. The process is a modification of our protocol described previously [35,49,50] and described in detail in the supplementary methods. 3.7. Dynamic Light Scattering (DLS) and Zeta Potential Hydrodynamic radii, electrophoretic mobility, zeta potential, and polydispersity of naked antibody and PICs were measured. Samples with 70 µL volume at 1 mg/mL in UV-transparent 96-well plates were measured by using a DLS Wyatt Möbius (Wyatt Technologies, Dernbach, Germany) with incident light at 532 nm, at an angle of 163.5◦. Samples were equilibrated at 25 ± 0.1 ◦C for 600 s before the measurements, with the constant temperature during the experiments. All samples represent triplicate values with 10 acquisitions and a 5 s acquisition time. The change in cumulant fitted hydrodynamic radius in nanometers was tracked throughout the storage period. Results were analyzed using the 11 of 15 Int. J. Mol. Sci. 2020, 21, 9151 Dynamics software version 7.1.7 (Wyatt Technologies, Santa Barbara, CA, USA). By using an Agilent Technologies Cary 5000 Cary Series UV-VIS-NIR spectrofluorimeter, we showed the incident laser beam at 532 nm is not within the fluorescent sample’s (IR700-7B2 and porphyrin-7B2) band of excitation; as the analysis would not be disrupted or tainted. 3.10. Apoptosis/Cell Death Assay Annexin V-FITC/PI cell staining was performed following 6 h of standard photoimmunotherapy. Briefly, cells were incubated with Annexin V binding buffer (10 mM HEPES, pH 7.4; 2.5 mM CaCl2; 140 mM NaCl) and stained with recombinant annexin V conjugated to green-fluorescent FITC dye (ThermoFisher Scientific, San Jose, CA, USA) for 15 min. After three washes, cells were incubated with 2 µg/mL of propidium iodide (PI) (Life Technologies, Carlsbad, CA, USA) for 15 min. The mean fluorescence intensity was measured for 10,000 events. The data were collected using linear amplification for two optical detectors, FSC and SSC, and log amplification for related filters, and analyzed by associated software as mentioned above. 3.8. Direct and Indirect Immunofluorescence Using Flow Cytometry Direct and indirect immunofluorescence were applied using flow cytometry to analyze the binding of photo-immunoconjugates (PICs) to Env-transfected 293T cells. 8 × 104 cells were incubated with PBS/BSA/0.01% sodium azide (PBA) for 30 min in RT, and then PICs added to reach the final concentrations of 10 µg/mL in the presence of 5 µg/mL soluble CD4. Cells were incubated with PICs for 1 h at room temperature. For indirect assay, half of the samples were washed, and then stained with FITC conjugated goat anti-human IgG secondary antibody (2 µg/mL) for 1 h. All the samples, for direct and indirect assays, were washed twice and fixed in 100 µL of 2% paraformaldehyde. After 4 h, 150 µL of PBS was added to the samples. Cells with 10,000 events were evaluated on BD LSRFortessa (Becton-Dickson, Mountain View, CA, USA), analyzed by Flow-Jo software version 7.5 (Tree Star Inc., Ashland, OR, USA). Mean fluorescence of the gated cell population labelled with immunoconjugate was calculated in relation to the mean fluorescence of cells labelled with MAb 7B2. Direct immunofluorescence without adding FITC conjugated secondary antibody was applied to analyze the binding of PICs to Env-transfected 293T cells. For IR700-PICs, the cells were excited by either violet laser at 405 nm or red laser at 640 nm, and the red fluorescence emission was detected by 710 ± 50 band-pass filter (Qdot 705). For porphyrin-PICs, the cells were excited by violet laser at 405 nm, and the red fluorescence emission was detected by 660 ± 20 band-pass filter (Qdot 655). 3.9. ROS Detection ROS generation in the cell lines was detected with the fluorescent probe H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate). 2 × 105 cells were incubated with 1 µM of native antibody, porphyrin-PIC, or IR700-PIC in 96 well plates in phenol-free DMEM for 45 min. The cells were washed and incubated with 1 µM H2DCF-DA solution prepared in PBS, and washed again and incubated with phenol-free DMEM before irradiation with 50 J/cm2. A mirror dark-plate, submitted to the same procedure, except for light exposure, contained the dark control groups. The cells incubated with 40 mM H2O2, without irradiation, were used as positive control. Fluorescence intensity (Ex/Em = 485/535 nm) was measured with the flow cytometry, with the procedure as mentioned above. 3.13. Statistical Analyses Statistical analyses were performed with GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data are shown as mean and standard error of mean (SEM) of the indicated number of replicate values. If no error bar appears signifying that the error bars are smaller than, and obscured by, the symbol. The unpaired two-tailed Student’s t-test was applied for statistical comparison, unless specifically stated otherwise. 3.12. Live Imaging by Two-Photon Confocal Microscopy Live cell imaging was performed using an inverted LSM 780 multiphoton laser scanning confocal microscope (Zeiss, Jena, Germany) equipped with a Chameleon laser (Ti:sapphire, Coherent, Santa Clara, CA, USA) as a source for two-photons (2P) excitation at 800 nm. The images were obtained by the average of 2 scans and no appreciate variation was observed. The spatial resolution was approximately 350 nm considering the numerical aperture and the excitation wavelength, as described previously [53]. The occurrence of cell death (irradiated region) was compared with live cells (non-irradiated region) by acridine orange-ethidium bromide (AO/EB) double-staining. Detailed conditions and settings are described in the supplementary methods. 3.11. Photo-Cytotoxicity Assay Naked 7B2 antibody, IR700-7B2, porphyrin-7B2 and a mixture of both PICs with equal molarity were diluted in the phenol-free DMEM medium, but without FCS, to reach a range of five final concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM and 31.2 nM into 5 wells of a 96-well plate. HIV Env-transfected cells and 293T cells were adjusted to a concentration of 1 × 106 cells/mL. Soluble CD4 was added to each well in a final concentration of 500 ng/mL. The plate incubated for 4 h at 37 ◦C and 5% CO2. Identical plates were kept protected from light for the same period 12 of 15 Int. J. Mol. Sci. 2020, 21, 9151 to obtain the proper dark control groups. Afterwards, the cells were washed with a 4X excess of phenol-free serum-free DMEM to remove any unbound PICs. The plates were irradiated with 20 J/ cm2 of light (380−780 nm) by a homemade LED array (30 mW/cm2) in two equal doses separated by 10 min, as described elsewhere [51,52]. After irradiation, 10 µL of FCS was added to each well and the plates were kept in the incubator for 3 days. The cell growth in each group was also monitored and imaged under a microscope (IX81, Olympus America, Center Valley, PA, USA) at each time point, before and after irradiation. MTS/PMS substrate (Promega, Madison, WI, USA) was added to cultures and absorbance at 490 nm was recorded at 1–4 h of incubation. Results represent the mean and SEM of triplicate samples for three independent experiments, and plotted as A490 with a subtraction from background. Data are represented as % of cell viability versus PIC concentration. Conflicts of Interest: V.C. is a Director of ThioLogics. Other authors declare no competing interests. Acknowledgments: We also thank NIH AIDS Research and Reference Reagent Program for supporting the reagent related to HIV. We thank Seth Pincus, for his supports in the Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT. We thank Juliana Galinskas (Unifesp) and Bruno Andrade Ono (IFSC) for their valuable expertise in quantification assay and fluorescence microscopy, respectively. References 1. Caskey, M.; Klein, F.; Nussenzweig, M.C. Broadly neutralizing anti-HIV-1 monoclonal antibodies in the clinic. Nat. Med. 2019, 25, 547–553. [CrossRef] [PubMed] 1. Caskey, M.; Klein, F.; Nussenzweig, M.C. Broadly neutralizing anti-HIV-1 monoclonal antibodies in the clinic. Nat. Med. 2019, 25, 547–553. [CrossRef] [PubMed] 2. Polito, L.; Bortolotti, M.; Battelli, M.G.; Calafato, G.; Bolognesi, A. Ricin: An ancient story for a timeless plant toxin. Toxins 2019, 11, 324. [CrossRef] [PubMed] 2. Polito, L.; Bortolotti, M.; Battelli, M.G.; Calafato, G.; Bolognesi, A. Ricin: An ancient story for a timeless plant toxin. Toxins 2019, 11, 324. [CrossRef] [PubMed] 3. Larson, S.M.; Carrasquillo, J.A.; Cheung, N.K.V.; Press, O.W. Radioimmunotherapy of human tumours. Nat. Rev. Cancer 2015, 15, 347–360. [CrossRef] [PubMed] 3. Larson, S.M.; Carrasquillo, J.A.; Cheung, N.K.V.; Press, O.W. Radioimmunotherapy of human tumours. Nat. Rev. Cancer 2015, 15, 347–360. [CrossRef] [PubMed] 4. Helal, M.; Dadachova, E. Radioimmunotherapy as a Novel Approach in HIV, Bacterial, and Fungal Infectious Diseases. Cancer Biother. Radiopharm. 2018, 33, 330–335. [CrossRef] 5. Ventola, C.L. Cancer Immunotherapy, Part 3: Challenges and Future Trends. Pharm. Ther. 2017, 42, 5 6. Biteghe, F.A.N.; Chalomie, N.E.T.; Mungra, N.; Vignaux, G.; Gao, N.; Vergeade, A.; Okem, A.; Naran, K.; Ndong, J.C.; Barth, S. Antibody-based immunotherapy: Alternative approaches for the treatment of metastatic melanoma. Biomedicines 2020, 8, 327. [CrossRef] 7. Ferrari, G.; Haynes, B.F.; Koenig, S.; Nordstrom, J.L.; Margolis, D.M.; Tomaras, G.D. Envelope-specific antibodies and antibody-derived molecules for treating and curing HIV infection. Nat. Rev. Drug Discov. 2016, 15, 823–834. [CrossRef] 8. Tsukrov, D.; Dadachova, E. The potential of radioimmunotherapy as a new hope for HIV patients. Expert. Rev. Clin. Immunol. 2014, 10, 553–555. [CrossRef] 9. Pincus, S.H.; Song, K.; Maresh, G.A.; Hamer, D.H.; Dimitrov, D.S.; Chen, W.; Zhang, M.Y.; Ghetie, V.F.; Chan-Hui, P.Y.; Robinson, J.E.; et al. Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins. J. Virol. 2017, 91, e01955-16. [CrossRef] 10. Sadraeian, M. Production and Characterization of Pulchellin A Chain Conjugated to HIV mAbs, and Study Its Selective Cytotoxicity Against Cells Expressing HIV Envelope. Ph.D. Thesis, Universidade de São Paulo, São Carlos, Brazil, 2017. 11. Sadraeian, M.; Guimarães, F.E.G.; Araújo, A.P.U.; Worthylake, D.K.; LeCour, L.J.; Pincus, S.H. Selective cytotoxicity of a novel immunotoxin based on pulchellin A chain for cells expressing expressing HIV envelope. Sci. Rep. 2017, 7, 7579. [CrossRef] 12. Sadraeian, M.; Mansoorkhani, M.J.K.; Mohkam, M.; Rasoul-Amini, S.; Hesaraki, M.; Ghasemi, Y. 3.14. Data Availability All data generated or analyzed during this research are included in this published article and its Supplementary Information file. The detailed method of live microscopy with extra videos can be found at https://docs.google.com/forms/d/e/1FAIpQLSdOC3q1uWNIpVj_iPcz5G8rLrQRjuffNowE2U- DcUrKI3CAw/viewform?usp=sf_link.Password:USP4030. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/23/ 9151/s1. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21 151/s1 Author Contributions: F.E.G.G. and M.S. wrote the main manuscript text and prepared figures. F.E.G.G., R.W.B., V.C. and M.S. designed and performed the biochemical, immunological and microscopy experiments. R.S.D., E.F.d.C., M.S. and L.M.R.J. designed and performed the viral assays. C.B. performed the small molecule synthesis and performed the bioconjugation experiments. F.E.G.G. and V.C. supervised the study. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the support provided by: FAPESP (Sao Paulo Research Foundation)—grant numbers: 2017/10910-5 (M. Sadraeian-Pós-Doutorado-Fluxo Contínuo), 2013/07276-1(CEPOF—CEPID Program), 2009/54035-4 (EMU). The reagents related to HIV were supported by NIH AIDS Research and Reference Reagent Program. We also gratefully acknowledge the Leverhulme Trust (RPG-2020-010) for funding C.B. Acknowledgments: We also thank NIH AIDS Research and Reference Reagent Program for supporting the reagent related to HIV. We thank Seth Pincus, for his supports in the Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT. We thank Juliana Galinskas (Unifesp) and Bruno Andrade Ono (IFSC) for their valuable expertise in quantification assay and fluorescence microscopy, respectively. 13 of 15 Int. J. Mol. Sci. 2020, 21, 9151 Abbreviations DAR Drug-Antibody-Ratio MAb Monoclonal Antibody PDT Photodynamic Therapy PIT Photoimmunotherapy PIC Photo-immunoconjugate Abbreviations DAR Drug-Antibody-Ratio MAb Monoclonal Antibody PDT Photodynamic Therapy PIT Photoimmunotherapy PIC Photo-immunoconjugate References Prevention and Inhibition of TC-1 Cell Growth in Tumor Bearing Mice by HPV16 E7 Protein in Fusion with Shiga Toxin B-Subunit from shigella dysenteriae. Cell J. 2013, 15, 176–181. [PubMed] Med, A.N.; Hanaoka, Y.H.H. Applying near-infrared photoimmunotherapy to B-cell lymphoma: Comparative evaluation with radioimmunotherapy in tumor xenografts. Ann. Nucl. Med. 2017, 31, 669–677. 14. Sandland, J.; Boyle, R.W. Photosensitizer Antibody-Drug Conjugates: Past, Present, and Future. Bioconjug. Chem. 2019, 30, 975–993. [CrossRef] [PubMed] 15. Pincus, S.H.; Song, K.; Maresh, G.A.; Frank, A.; Worthylake, D.; Chung, H.; Polacino, P.; Hamer, D.H.; Coyne, C.P.; Rosenblum, M.G.; et al. Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160. J. Virol. 2017, 91, e01360-16. [CrossRef] 16. Craig, R.B.; Summa, C.M.; Corti, M.; Pincus, S.H. Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates. PLoS ONE 2012, 7, 1–13. [CrossRef] 17. Buzzá, H.H.; Silva, A.P.D.; Vollet Filho, J.D.; Ramirez, D.P.; Trujillo, J.R.; Inada, N.M.; Moriyama, L.T.; Kurachi, C.; Bagnato, V.S. Photodynamic therapy: Progress toward a scientific and clinical network in Latin America. Photodiagnosis Photodyn. Ther. 2016, 13, 261–266. [CrossRef] 18. Wainwright, M.; O’Kane, C.; Rawthore, S. Phenothiazinium photosensitisers XI. Improved toluidine blue photoantimicrobials. J. Photochem. Photobiol. B Biol. 2016, 160, 68–71. [CrossRef] Int. J. Mol. Sci. 2020, 21, 9151 14 of 15 19. Auwärter, W.; Écija, D.; Klappenberger, F.; Barth, J.V. Porphyrins at interfaces. Nat. Chem. 2015, 7, 105–120. [CrossRef] 20. Fujita, A.K.L.; Rodrigues, P.G.S.; Requena, M.B.; Escobar, A.; da Rocha, R.W.; Nardi, A.B.; Kurachi, C.; de Menezes, P.F.; Bagnato, V.S. Fluorescence evaluations for porphyrin formation during topical PDT using ALA and methyl-ALA mixtures in pig skin models. Photodiagnosis Photodyn. Ther. 2016, 15, 236–244. [CrossRef] 21. Valkov, A.; Nakonechny, F.; Nisnevitch, M. Polymer-immobilized photosensitizers for continuous eradication of bacteria. Int. J. Mol. Sci. 2014, 15, 14984–14996. [CrossRef] 22. Nakonechny, F.; Barel, M.; David, A.; Koretz, S.; Litvak, B.; Ragozin, E.; Etinger, A.; Livne, O.; Pinhasi, Y.; Gellerman, G.; et al. Dark antibacterial activity of rose Bengal. Int. J. Mol. Sci. 2019, 20, 3196. [CrossRef] [PubMed] 23. Dougherty, T.J.; Gomer, C.J.; Henderson, B.W.; Jori, G.; Kessel, D. Photodynamic Therapy. J. Natl. Cancer Inst. 1998, 17, 889–905. [CrossRef] [PubMed] 24. Bacellar, I.O.L.; Tsubone, T.M.; Pavani, C.; Baptista, M.S. Photodynamic efficiency: From molecular photochemistry to cell death. Int. J. Mol. Sci. 2015, 16, 20523–20559. [CrossRef] [PubMed] 5. Crous, A.; Abrahamse, H. References Effective gold nanoparticle-antibody-mediated drug delivery for photodyna therapy of lung cancer stem cells. Int. J. Mol. Sci. 2020, 21, 3742. [CrossRef] [PubMed] 6. Caudle, A.S.; Yang, W.T.; Mittendorf, E.A.; Kuerer, H.M. Cancer Cell-Selective In Vivo Near Infra Photoimmunotherapy Targeting Specific Membrane Molecules. Nat. Med. 2016, 150, 137–143. 27. Bryden, F.; Maruani, A.; Savoie, H.; Chudasama, V.; Smith, M.E.B.; Caddick, S.; Boyle, R.W. Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment. Bioconjug. Chem. 2014, 25, 611–617. [CrossRef] [PubMed] 28. Maruani, A.; Smith, M.E.B.; Miranda, E.; Chester, K.A.; Chudasama, V.; Caddick, S. A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat. Commun. 2015, 6, 1–9. [CrossRef] [PubMed] 29. Bahou, C.; Love, E.A.; Leonard, S.; Spears, R.J.; Maruani, A.; Armour, K.; Baker, J.R.; Chudasama, V. Disulfide Modified IgG1: An Investigation of Biophysical Profile and Clinically Relevant Fc Interactions. Bioconjug. Chem. 2019, 30, 1048–1054. [CrossRef] [PubMed] 30. Chester, K.A.; Baker, R.; Caddick, S. Next generation maleimides enable the controlled assembly. R. So 2014, 10, 1–52. 31. Jackson, D.Y. Processes for Constructing Homogeneous Antibody Drug Conjugates. Org. Process. Res. Dev. 2016, 20, 852–866. [CrossRef] 32. Bryden, F.; Boyle, R.W. A mild, facile, one-pot synthesis of zinc azido porphyrins as substrates for use in click chemistry. Synlett. 2013, 24, 1978–1982. 33. Cheng, M.H.Y.; Maruani, A.; Savoie, H.; Chudasama, V.; Boyle, R.W. Synthesis of a novel HER2 targeted aza-BODIPY-antibody conjugate: Synthesis, photophysical characterisation and: In vitro evaluation. Org. Biomol. Chem. 2018, 16, 1144–1149. [CrossRef] [PubMed] 34. Lee, M.T.W.; Maruani, A.; Richards, D.A.; Baker, J.R.; Caddick, S.; Chudasama, V. Enabling the controlled assembly of antibody conjugates with a loading of two modules without antibody engineering. Chem. Sci. 2017, 8, 2056–2060. [CrossRef] [PubMed] 35. Castañeda, L.; Wright, Z.V.F.; Marculescu, C.; Tran, T.M.; Chudasama, V.; Maruani, A.; Hull, E.A.; Nunes, J.P.; Fitzmaurice, R.J.; Smith, M.E.; et al. A mild synthesis of N-functionalised bromomaleimides, thiomaleimides and bromopyridazinediones. Tetrahedron Lett. 2013, 54, 3493–3495. [CrossRef] [PubMed] py 36. Robinson, E.; Nunes, J.P.M.; Vassileva, V.; Maruani, A.; Nogueira, J.C.F.; Smith, M.E.B.; Pedley, B.; Caddick, S.; Baker, J.R.; Chudasana, V. Pyridazinediones deliver potent, stable, targeted and efficacious antibody-drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody. RSC Adv. 2017, 7, 9073–9077. [CrossRef] 37. Bryden, F.; Maruani, A.; Rodrigues, J.M.M.; Cheng, M.H.Y.; Savoie, H.; Beeby, A.; Chudasana, V.; Boyle, R.W. References A bl f Hi h P t Ph t iti A tib d C j t th h A li ti f D d M lti li 36. Robinson, E.; Nunes, J.P.M.; Vassileva, V.; Maruani, A.; Nogueira, J.C.F.; Smith, M.E.B.; Pedley, B.; Caddick, S.; Baker, J.R.; Chudasana, V. Pyridazinediones deliver potent, stable, targeted and efficacious antibody-drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody. RSC Adv. 2017, 7, 9073–9077. [CrossRef] 37. Bryden, F.; Maruani, A.; Rodrigues, J.M.M.; Cheng, M.H.Y.; Savoie, H.; Beeby, A.; Chudasana, V.; Boyle, R.W. Assembly of High-Potency Photosensitizer-Antibody Conjugates through Application of Dendron Multiplier Technology. Bioconjug. Chem. 2018, 29, 176–181. [CrossRef] 38. Pincus, S.H.; Fang, H.; Wilkinson, R.A.; Marcotte, T.K.; Robinson, J.E.; Olson, W.C. In vivo efficacy of anti-glycoprotein 41, but not anti-glycoprotein 120, immunotoxins in a mouse model of HIV infection. J. Immunol. 2003, 170, 2236–2241. [CrossRef] 39. Kobayashi, H.; Choyke, P.L. Target-Cancer-Cell-Specific Activatable Fluorescence Imaging Probes: Rational Design and in Vivo Applications. Acc. Chem. Res. 2011, 44, 83–90. [CrossRef] Int. J. Mol. Sci. 2020, 21, 9151 15 of 15 15 of 15 40. Kratochvil, H.T.; Ha, D.G.; Zanni, M.T. Counting tagged molecules one by one: Quantitative photoactivation and bleaching of photoactivatable fluorophores. J Chem Phys. 2015, 143, 104201. [CrossRef] 41. Fujimura, D.; Inagaki, F.; Okada, R.; Rosenberg, A.; Furusawa, A.; Choyke, P.L.; Kobayashi, H. Conjugation Ratio, Light Dose, and pH Affect the Stability of Panitumumab–IR700 for Near-Infrared Photoimmunotherapy. ACS Med. Chem. Lett. 2020, 11, 1598–1604. [CrossRef] 42. Pires, L.; Demidov, V.; Wilson, B.C.; Salvio, A.G.; Moriyama, L.; Bagnato, V.S.; Vitkin, I.A.; KurachI, C. Dual-agent photodynamic therapy with optical clearing eradicates pigmented melanoma in preclinical tumor models. Cancers 2020, 12, 1956. [CrossRef] [PubMed] 43. Jeong, J.W.; McCall, J.G.; Shin, G.; Zhang, Y.; Al-Hasani, R.; Kim, M.; Li, S.; Sim, J.Y.; Jang, K.I.; Shi, Y.; et al. Wireless Optofluidic Systems for Programmable In Vivo Pharmacology and Optogenetics. Cell 2015, 162, 662–674. [CrossRef] [PubMed] 44. Ramírez-García, G.; Panikar, S.S.; López-Luke, T.; Piazza, V.; Honorato-Colin, M.A.; Camacho-Villegas, T.; Hernández-Gutiérrez, R.; De la Rosa, E. An immunoconjugated up-conversion nanocomplex for selective imaging and photodynamic therapy against HER2-positive breast cancer. Nanoscale 2018, 10, 10154–10165. [CrossRef] [PubMed] 45. Dashti, A.; Waller, C.; Mavigner, M.; Schoof, N.; Bar, K.J.; Shaw, G.M.; Vanderford, T.H.; Liang, S.; Lifson, J.D.; Dunham, R.M.; et al. SMAC Mimetic Plus Triple-Combination Bispecific HIVxCD3 Retargeting Molecules in SHIV.C.CH505-Infected, Antiretroviral Therapy-Suppressed Rhesus Macaques. J. Virol. 2020, 94, e00793-20. © 2020 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 (http://creativecommons.org/licenses/by/4.0/). References [CrossRef] 46. Kovacs, J.M.; Noeldeke, E.; Ha, H.J.; Peng, H.; Rits-Volloch, S.; Harrison, S.C.; Chen, B. Stable, uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure. Proc. Natl. Acad. Sci. USA 2014, 111, 18542–18547. [CrossRef] 47. Pincus, S.H.; McClure, J. Soluble CD4 enhances the efficacy of immunotoxins directed against gp41 of the human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 1993, 90, 332–336. [CrossRef] 48. Mchugh, L.; Hu, S.; Lee, B.K.; Santora, K.; Kennedy, P.E.; Berger, E.A.; Pastan, I.; Hamer, D.H. Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based Mutagenesis. J. Biol. Chem. 2002, 277, 34383–34390. [CrossRef] 49. Giuntini, F.; Bryden, F.; Daly, R.; Scanlan, E.M.; Boyle, R.W. Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: Towards mild strategies for the labelling of glycans. Org. Biomol. Chem. 2014, 12, 1203–1206. [CrossRef] 50. Bahou, C.; Richards, D.A.; Maruani, A.; Love, E.A.; Javaid, F.; Caddick, S.; Baker, J.R.; Chudasama, V. Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones. Org. Biomol. Chem. 2018, 16, 1359–1366. [CrossRef] 51. Inada, N.M.; da Costa, M.M.; Guimarães, O.C.C.; Ribeiro, E.d.S.; Kurachi, C.; Quintana, S.M.; Lombardi, W.; Bagnato, V.S. Photodiagnosis and treatment of condyloma acuminatum using 5-aminolevulinic acid and homemade devices. Photodiagnosis Photodyn. Ther. 2012, 9, 60–68. [CrossRef] 52. Romano, R.A.; Silva, A.P.; Kurachi, C.; Guimara, F.E.G. Light-driven photosensitizer uptake increases Candida albicans photodynamic inactivation. J. Biophotonics 2017, 10, 1538–1546. [CrossRef] [PubMed] 53. Mello, B.L.; Alessi, A.M.; Riaño-Pachón, D.M.; DeAzevedo, E.R.; Guimarães, F.E.G.; Espirito Santo, M.C.; McQueen-Mason, S.; Bruce, N.C.; Polikarpov, I. Targeted metatranscriptomics of compost-derived consortia l GH11 ti l 1 4 β l ti it Bi t h l Bi f l 2017 10 254 [C R f] 52. Romano, R.A.; Silva, A.P.; Kurachi, C.; Guimara, F.E.G. Light-driven photosensitizer uptake increases Candida albicans photodynamic inactivation. J. Biophotonics 2017, 10, 1538–1546. [CrossRef] [PubMed] 53. Mello, B.L.; Alessi, A.M.; Riaño-Pachón, D.M.; DeAzevedo, E.R.; Guimarães, F.E.G.; Espirito Santo, M.C.; McQueen-Mason, S.; Bruce, N.C.; Polikarpov, I. Targeted metatranscriptomics of compost-derived consortia reveals a GH11 exerting an unusual exo-1,4-β-xylanase activity. Biotechnol. Biofuels. 2017, 10, 254. [CrossRef] [PubMed] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 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 (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3030526793
https://vbn.aau.dk/files/413425951/s41601_020_00156_w.pdf
English
null
Fault protection method of single-phase break for distribution network considering the influence of neutral grounding modes
Protection and control of modern power systems
2,020
cc-by
8,292
Fault protection method of single-phase break for distribution network considering the influence of neutral grounding modes Xiao Yang; Ouyang Jinxin; Xiong Xiaofu; Wang Yutong; Luo Yongjie Xiao, Yang; Ouyang, Jinxin; Xiong, Xiaofu; Wang, Yutong; Luo, Yongjie Published in: Protection and Control of Modern Power Systems DOI (link to publication from Publisher): 10.1186/s41601-020-00156-w Citation for published version (APA): Xiao, Y., Ouyang, J., Xiong, X., Wang, Y., & Luo, Y. (2020). Fault protection method of single-phase break for distribution network considering the influence of neutral grounding modes. Protection and Control of Modern Power Systems, 5(1), Article 10. https://doi.org/10.1186/s41601-020-00156-w General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Aalborg Universitet Aalborg Universitet Citation for published version (APA): Xiao, Y., Ouyang, J., Xiong, X., Wang, Y., & Luo, Y. (2020). Fault protection method of single-phase break for distribution network considering the influence of neutral grounding modes. Protection and Control of Modern Power Systems, 5(1), Article 10. https://doi.org/10.1186/s41601-020-00156-w © The Author(s). 2020 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/. General rights C i h d - Users may download and print one copy of any publication from the public 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 - Users may download and print one copy of any publication from the public 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 - Users may download and print one copy of any publication from the public portal for the purpose of p Y t f th di t ib t th t i l it f fit ki ti it i l i You may not further distribute the material or use it for any profit making activity o - You may freely distribute the URL identifying the publication in the public portal - y y p g y - You may freely distribute the URL identifying the publication in the public portal - Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and w the work immediately and investigate your claim. y this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to ely and investigate your claim. (2020) 5:10 Xiao et al. Protection and Control of Modern Power Systems https://doi.org/10.1186/s41601-020-00156-w Xiao et al. Protection and Control of Modern Power Systems https://doi.org/10.1186/s41601-020-00156-w Protection and Control of Modern Power Systems Open Access Fault protection method of single-phase break for distribution network considering the influence of neutral grounding modes Yang Xiao1, Jinxin Ouyang1*, Xiaofu Xiong1, Yutong Wang2 and Yongjie Luo3 Yang Xiao1, Jinxin Ouyang1*, Xiaofu Xiong1, Yutong Wang2 and Yongjie Luo3 * Correspondence: jinxinoy@163.com 1School of Electrical Engineering, Chongqing University, Chongqing, China Full list of author information is available at the end of the article Abstract With the rapid development of modern distribution network and the access of distributed generation, the network structure is becoming increasingly complex. Frequent single-phase break faults have seriously affected equipment and personal safety and stable operation of the power system. However, with the development and application of the composite neutral grounding modes, the protection of single-phase break fault is facing new challenges. This paper proposes a protection method of single-phase break fault for distribution network considering the influence of neutral grounding modes. The characteristics of neutral voltage and sequence current are analyzed under normal operation and single-phase break fault with different grounding modes. Following this, the protection criterion based on neutral voltage and sequence current variation is constructed. The protection method of single- phase break fault for distribution network is proposed, which is applicable for various neutral grounding modes. Theoretical analysis and simulation results show that the protection method is less affected by system asymmetry, fault location and load distribution. The method has higher sensitivity, reliability and adaptability. Keywords: Distribution network, Single-phase break fault, Protection, Neutral grounding 1 Introduction access of distributed generation and electric vehicles, single-phase break fault will cause voltage to rise beyond the tolerance threshold of sensitive power electronic de- vices and damage it [4–6]. Therefore, the protection of single-phase break fault is especially important. With the expansion of modern distribution network, the demand of power supply reliability is increasing. However, the frequency of single-phase break fault is also increasing because of the complex structure of dis- tribution network, the interlacing of old and new lines, and the influence of natural disasters, external mechan- ical forces or other reasons. Single-phase break fault causes unbalanced voltage between power supply side and load side, seriously affecting the power quality. Phase-deficient operation will also reduce equipment service life and damage motors [1]. If the fault is not dis- covered in time, it is very likely to develop into a short- circuit fault, increasing the severity of the accident and threatening personal safety [2, 3]. In addition, with the For a long time, single-phase break fault has not been paid enough attention because it will not cause high current and voltage. Reference [7–10] deduced the voltage and current characteristics of the ungrounded and grounded break fault of small current grounding system. Reference [11] used the positive sequence current modu- lus maximum to realize the line selection and location of the break fault, but did not consider the difference with the short-circuit fault and the influence of the actual noise on the signal processing. Reference [12] proposed a line selection method based on negative sequence current en- ergy measurement, but its accuracy is greatly affected by the fault location and load distribution. Reference [13] proposed a combined line selection criterion of phase Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 2 of 13 Page 2 of 13 (2020) 5:10 (2020) 5:10 the protection sensitivity, and is applicable to various grounding modes. Firstly, considering the asymmetry of line parameters, this paper respectively deduces the ex- pressions of the neutral voltage and sequence current of the normal operation and single-phase break fault under various grounding modes, and the variation characteristics before and after the fault are analyzed. On this basis, the neutral voltage and sequence current variation are ex- tracted as the protection characteristic parameter and the protection criterion is constructed which is applicable for various grounding modes. Finally, the accuracy of the pro- tection criterion in typical grounding modes is verified by simulation. The results show that the protection method has high reliability and wide applicability. current and phase voltage, but only for ungrounded sys- tems. According to the variation law of break fault voltage of ungrounded distribution network, the fault section lo- cation method based on phase voltage and line voltage is proposed in [14], but the influence of system asymmetry is not considered. Reference [15] used load monitors to collect negative sequence voltage to detect the break fault, but the data processing capacity was large. Reference [16] is based on the zero sequence voltage difference between the power supply side and load side of the fault line, but requires high communication and data synchronization. On the one hand, the existing single-phase break fault protection method is greatly affected by system parame- ters and fault location, with limited accuracy and low reli- ability. On the other hand, since small current grounding mode is mostly adopted in distribution network, the exist- ing methods are only applicable to the specific neutral grounding mode, and the low resistance grounding mode is not considered. However, with the acceleration of urbanization and the widespread use of cables, capacitance current is increasing, and the low resistance grounding mode is widely used. Definitely, there is a certain gap in the study of single-phase break fault in medium voltage distribution networks. And the intelligent multi-mode grounding, arc suppression coil with parallel resistance grounding and other modes are gradually applied to the distribution network [17–19], so that the neutral ground- ing mode may change during actual operation. The break fault protection methods under the specific neutral grounding mode are difficult to meet the requirements. 2.1 Neutral voltage Ignoring line impedance and distributed conductance to ground, there is ð6Þ jω X j i¼1 CiA˙EA þ CiB˙EB þ CiC˙EC þ jωCΣ˙UO þ ˙IO ¼ 0 ½ ð1Þ jω X j i¼1 CiA˙EA þ CiB˙EB þ CiC˙EC þ jωCΣ˙UO þ ˙IO ¼ 0 ½ Where, ˙Iiload is load current of the ith feeder; CiΣ = CiA + CiB + CiC, where CiA、CiB、CiC are each phase capacitance of the ith feeder. ð1Þ Under the three-phase asymmetry of the distribution network, the neutral voltage shifts. At the same time, the negative and zero sequence current appear on feeders. Since the asymmetry of the distribution network is usu- ally quite small, which is about 0.5% to 1.5%, and the cable has a smaller asymmetry than the overhead line. Therefore, the neutral voltage, negative and zero se- quence current generated by the asymmetry can be ignored. Where, CΣ is the sum of the three phase capacitances of the system. Due to the asymmetry of the three-phase capacitance, the unbalanced voltage of the neutral point to ground ˙Uunb can be obtained as follow ˙Uunb ¼ ˙λ˙EA ð2Þ ð2Þ ˙Uunb ¼ ˙λ˙EA Where, ˙λ is system asymmetry, depending on system parameters and grounding modes. 3 Single-phase break fault characteristics of distribution network 3.1 Neutral voltage When the neutral point is ungrounded, the system asymmetry is Suppose the feeder i has a single-phase break fault at the distance d from the bus. Compared with the normal op- erating condition, the single-phase break fault further deepens the asymmetry of the three-phase capacitor, so the neutral voltage will further shift. According to Kirchhoff’s law, the current flowing through the neutral point satisfies ˙λ ¼ CAΣ þ α2CBΣ þ αCCΣ CΣ ð3Þ ð3Þ When the neutral point is grounded by the arc sup- pression coil, the system asymmetry is ˙λ ¼ CAΣ þ α2CBΣ þ αCCΣ CΣ− 1 ω2Lp ð4Þ ˙IA þ ˙IB þ ˙IC þ ˙IO ¼ 0 ð7Þ ð4Þ Where, When the neutral point is grounded by the low resist- ance, the system asymmetry is ( ˙IA ¼ jω xCiA þ X j n ¼ 1 n≠i CnA ! ˙EA þ ˙UO  þ X j n ¼ 1 n≠i ˙InAload ˙IB ¼ jωCBΣ  ˙EB þ ˙UO  þ X j n¼1 ˙InBload ˙IC ¼ jωCCΣ  ˙EC þ ˙UO  þ X j n¼1 ˙InCload ˙IO ¼ ˙UO ZO ð8Þ ˙λ ¼ CAΣ þ α2CBΣ þ αCCΣ CΣ þ 1 jωRd ð5Þ ð5Þ Where, α ¼ ej120∘; ω is angular frequency; CAΣ, CBΣ, CCΣ are each phase capacitance of the system. ð8Þ 2 Distribution network operation characteristics considering three-phase asymmetry g p y y The line parameters are related to not only the material, sec- tional area and insulating medium, but also the arrange- ment of conductors. In practice, the distribution network lines are short, and most of them are arranged horizontally or vertically, so that the phenomenon of three-phase capacitance unbalance is common [20]. The medium voltage distribution net- work generally operates in a radial mode. And the main transformer neutral point is mainly grounded in four ways: ungrounded, arc suppression coil grounded, low resistance grounded and arc suppres- sion coil with parallel resistance grounded, as shown in Fig. 1. CiA, CiB, CiC represent the three-phase ca- pacitances to ground of the ith feeder. ˙EA, ˙EB, ˙EC are transformer three phase induced electromotive force. ˙IA, ˙IB, ˙IC are three phase current flowing into the bus from the upper-level grid. ˙UO is neutral voltage. ˙IO is This paper proposes a protection method of single- phase break fault for distribution network considering the influence of neutral grounding modes. The method is ba- sically not affected by the change of system parameters, fault location and load distribution, effectively improves Fig. 1 Schematic diagram of distribution system Fig. 1 Schematic diagram of distribution system Fig. 1 Schematic diagram of distribution system ms (2020) 5:10 Page 3 of 13 Page 3 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 (2020) 5:10 neutral branch current. Rd is neutral grounding resist- ance. Lp is arc suppression coil inductance. neutral branch current. Rd is neutral grounding resist- ance. Lp is arc suppression coil inductance. ( ˙Ii1 ¼ jω½˙λ CiA þ αCiB þ α2CiC ð Þ þ C1Σ 3 ˙EA þ ˙Iiload ˙Ii2 ¼ jω½ð˙λ þ 1ÞCiA þ αð1 þ α˙λÞCiB þ αð˙λ þ αÞCiC 3 ˙EA ˙Ii0 ¼ jω½ð˙λ þ 1ÞCiA þ ð˙λ þ α2ÞCiB þ ð˙λ þ αÞCiC 3 ˙EA ð6Þ 3.2 Sequence current The single-phase break fault causes the fault phase current at the fracture to be 0, and the non-fault phase voltage is continuous at the front and back of the frac- ture. When phase A is broken, using the symmetrical component method [21], the boundary condition is ð9Þ Where, ZLi is the load impedance of the fault feeder; x = d/Li is the ratio of the distance from the fault point to the bus to the length of the fault feeder Li; k is the ra- tio of total feeders capacitance to fault feeder capacitance. n ˙I 0 A1 þ ˙I 0 A2 þ ˙I 0 A0 ¼ 0 Δ˙U 0 A1 ¼ Δ˙U 0 A2 ¼ Δ˙U 0 A0 ð16Þ n ˙I 0 A1 þ ˙I 0 A2 þ ˙I 0 A0 ¼ 0 Δ˙U 0 A1 ¼ Δ˙U 0 A2 ¼ Δ˙U 0 A0 ð16Þ k ¼ CΣ CiA þ CiB þ CiC ð10Þ ð10Þ Where, ˙I 0 A1, ˙I 0 A2, ˙I 0 A0 are positive, negative and zero se- quence current at the fracture; Δ˙U 0 A1, Δ˙U 0 A2, Δ˙U 0 A0 are positive, negative and zero sequence voltage difference across the fracture. x reflects the degree of capacitance loss of the fault feeder, which is related to the fault location. k measures the degree of impact of a certain feeder single-phase break fault on the system, determined by the line param- eters. Since 0 ≤x ≤1, the high order terms of (1-x) are omitted, and eq. (9) can be simplified as: x reflects the degree of capacitance loss of the fault feeder, which is related to the fault location. k measures the degree of impact of a certain feeder single-phase break fault on the system, determined by the line param- eters. Since 0 ≤x ≤1, the high order terms of (1-x) are omitted, and eq. (9) can be simplified as: The impedance of distribution line and system are very small and can be ignored. The zero-sequence load impedance can be approximated to infinity. According to eq. (16), the composite sequence network in case of phase-A break fault of feeder i can be established as shown in Fig. 2(a). 3.2 Sequence current ˙IA1, ˙IA2, ˙IA0 are positive, negative and zero sequence current at the outlet of the fault feeder; Zeq1, Zeq2 are positive and negative sequence equivalent impedance of all non-fault feeders; Ceq0 is the equivalent zero sequence capacitance for all non-fault feeders; CiA1, CiA2, CiA0 are positive, negative and zero sequence cap- acitance of the fault feeder; ZiM1, ZiM2 are the positive and negative sequence load impedance of the upstream of the fault feeder respectively; ZiN1, ZiN2 are the positive and negative sequence load impedance of the down- stream of the fault feeder. j ˙UO j¼ 1−x 2k j ˙EA j ð11Þ When neutral point is grounded by the arc suppres- sion coil, ZO = jωLp, the neutral voltage after single- phase break fault is ˙UO ¼ −1−x ð Þ−j 1−x ð Þ2ZLiωCiA 2pk þ jωCiAZLi 1−x ð Þ 3kp−x þ 1 ð Þ ˙EA ð12Þ Similarly, eq. (12) can be simplified as j ˙UO j¼ 1−x 2pk j ˙EA j ð13Þ j ˙UO j¼ 1−x 2pk j ˙EA j ð13Þ Figure 2(b) is a simplified circuit of the composite se- quence network. Where Z2 is the equivalent impedance of the negative sequence network; ZM1 and ZN1 are the positive sequence equivalent impedances of the fault feeder at the front and back of the fracture. Where, p is the overcompensation degree of the arc suppression coil. When neutral point is grounded by the low resistance, ZO = 1/Rd, the neutral voltage after single-phase break fault is ZM1 ¼ ZiM1== 1=jωxCiA1 ð Þ ð17Þ ZN1 ¼ ZiN1== 1=jω 1−x ð ÞCiA1 ½  ð18Þ ð17Þ ð18Þ ð17Þ ð17Þ ð18Þ ˙UO ¼ −3 1−x ð Þ2ZLiRdω2CiA2 þ j3 1−x ð ÞRdωCiA 2−jωCiA 6kRd þ 3ZLi 1−x ð Þ ½  ˙EA ˙UO ¼ −3 1−x ð Þ2ZLiRdω2CiA2 þ j3 1−x ð ÞRdωCiA 2−jωCiA 6kRd þ 3ZLi 1−x ð Þ ½  ˙EA ð14Þ Solving the simplified circuit, the sequence current at the fracture is ð14Þ ( ˙I 0 A1 ¼ Z2 þ Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA ˙I 0 A2 ¼ −Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA ˙I 0 A0 ¼ −Z2 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA Similarly, eq. (14) can be simplified as Similarly, eq. (14) can be simplified as j ˙UO j¼ 3RdωCiA 1−x ð Þ 2 j ˙EA j ð15Þ It can be seen from eq. 2.2 Sequence current Where, ˙InAload, ˙InBload, ˙InCload are three phase load cur- rents of the nth feeder, n = 1,2, …,i, …,j; ZO is neutral grounding branch impedance. Where, ˙InAload, ˙InBload, ˙InCload are three phase load cur- rents of the nth feeder, n = 1,2, …,i, …,j; ZO is neutral grounding branch impedance. Asymmetry of the capacitance to ground of system will lead to the asymmetry of three-phase current. The feeder phase current is equal to the sum of the capaci- tance to ground current and load current. Consequently, the positive, negative and zero sequence current on the ith feeder are When the neutral point is ungrounded, ZO is infinity. Substituting eq. (8) into eq. (7), the neutral voltage after the single-phase break fault is (2020) 5:10 Page 4 of 13 Page 4 of 13 Page 4 of 13 (2020) 5:10 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 ˙UO ¼ j3 1−x ð Þ2ZLiωCiA þ 3 1−x ð Þ j3ZLiωCiA 1−x ð Þ x þ 2 ð Þ þ 3 k−1 ð Þ ½  þ 6k ˙EA ð9Þ 3.2 Sequence current (11), (13) and (15) that the neutral voltage is mainly affected by the single-phase break fault location, the grounding mode and parameters of neutral point, and the proportion of the line capacitance. ð19Þ ð19Þ And the sequence current at the outlet of the fault feeder is Page 5 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Fig. 2 Composite sequence network of single-phase break fault (a) Composite sequence network (b) Equivalent circuit Fig. 2 Composite sequence network of single-phase break fault (a) Composite sequence network (b) Equivalent circuit ( ˙IA1 ¼ Z2 þ Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 þ 1 ZM1   ˙EA ˙IA2 ¼ ˙I 0 A2 ¼ −Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA ˙IA0 ¼ 1−3ZO== 1=jωCeq0   == 1=jωxCiA0 ð Þ 1=jωxCiA0 ð Þ  ˙I 0 A0 ð20Þ depends on the system parameters under single-phase break fault. As a result, the sequence current under dif- ferent neutral grounding modes also shows a certain difference. Therefore, in order to improve the applic- ability of protection, it is necessary to consider the in- fluence of the neutral grounding mode. ð20Þ 4 Single-phase break fault protection method for distribution network It can be seen from eq. (19) and (20) that the ampli- tude and phase of the sequence current at the fracture is affected by the single-phase break fault location, and network parameters. And the sequence current at the outlet of the fault feeder is also affected by load distribution. 4.1 Protection characteristic parameter From eq. (3) to (5) and eq. (9) to (15), it can be seen that the neutral voltage and feeder sequence current under normal operation and single-phase break fault of the dis- tribution network are significant different when the grounding mode is different. The neutral voltage is af- fected by x and k after the single-phase break fault, and has a maximum value. The deviation of neutral voltage is different under dif- ferent grounding mode, no matter in normal operation or single-phase break fault. According to eq. (3) to (5), the system asymmetry is minimum when grounded by low resistance, and is maximum when grounded by arc suppression coil. Furthermore, the smaller the over- compensation degree is, the larger the deviation is. The amplitude of the feeder sequence current mainly de- pends on the asymmetry under normal operation, and When the neutral point is ungrounded, combined with eq. (11), the maximum variation of the neutral voltage is UOmax ¼ 1 2kmin Uφ ð21Þ ð21Þ Page 6 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 (2020) 5:10 Xiao et al. Protection and Control of Modern Power Systems Fig. 3 Logic diagram of single-phase break fault protection Fig. 3 Logic diagram of single-phase break fault protection When the neutral point is arc suppression coil grounded, combined with eq. (13), the maximum vari- ation of the neutral voltage is established in various grounding modes, which is inde- pendent of the zero sequence impedance. Before and after the single-phase break fault, the non- fault feeder positive sequence current change is 0, and the negative sequence current change is UOmax ¼ 1 2pkmin Uφ ð22Þ ð22Þ Δ˙I jA2 ¼ ˙I 0 A2 ZS2 Zeq2 þ ZS2 ð25Þ Δ˙I jA2 ¼ ˙I 0 A2 ZS2 Zeq2 þ ZS2 ð25Þ When the neutral point is low resistance grounded, combined with eq. (15), the variation maximum of the neutral voltage is Where, ZS2 is the system negative sequence equivalent impedance. UOmax ¼ 3RdωCmax 2 Uφ ð23Þ ð23Þ The negative sequence current mostly flows from the fault feeder to the upper grid since the negative se- quence equivalent impedance of the medium voltage dis- tribution network system is much smaller than the line negative sequence impedance [11]. As a consequence, the negative sequence current on the non-fault feeder is much lower than that of the fault feeder. 4.1 Protection characteristic parameter The ratio of positive and negative sequence current change of the non-fault feeder is less than 1. Where, kmin is the ratio of system capacitance to max- imum feeder capacitance; Cmax is maximum capacitance to ground in all feeders; Uφ is normal operating phase voltage. Since the sequence current under normal operation is very small, combined with the eq. (19), the positive and negative sequence current changes at the outlet of the fault feeder before and after the single-phase break fault are Therefore, the neutral voltage and the sequence current variation are selected as the protection charac- teristic parameter, which can accurately reflect the oc- currence of the single-phase break fault and distinguish the fault feeder from the non-fault feeder. ( Δ˙IiA1 ¼ ˙I 0 A1− ˙EA ZN1 ¼ − Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA Δ˙IiA2 ¼ ˙I 0 A2−0 ¼ − Z0 ZN1Z0 þ ZN1Z2 þ Z2Z0 ˙EA ð24Þ 4.2 Protection criterion Thi ð24Þ This paper proposes a protection method of single- phase break fault for distribution network considering the influence of neutral grounding modes. In view of the significant change of the neutral voltage before and after the fault, it is selected as the starting criterion, which can quickly reflect the occurrence of the single-phase break fault. The expression is According to eq. (24), the changes of the positive and negative sequence currents are equal before and after the single-phase break fault. Moreover, since the differ- ence in the grounding mode only affects the zero- sequence impedance, the relationship of the positive and negative sequence current variations is always Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 7 of 13 Xiao et al. Protection and Control of Modern Power Systems KrelUunb < UO < K relUO max ð26Þ Where, Krel is reliability coefficient. Nonetheless, some short-circuit faults will also cause the neutral voltage to shift. However, the single-phase break fault does not cause a rise in current. Selecting the bus phase current as the blocking criterion can effect- ively distinguish the single-phase break fault from the short-circuit fault. Considering the light or no load of the line, the expression is Iφ tð Þ−Iφ t−T ð Þ≤0 ð27Þ Where, Iφ(t) is the sample value of bus phase current at the present moment; Iφ(t-T) is the sample value of bus phase current at the previous cycle. Fig. 4 Neutral voltage under different grounding modes (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded Fig. 5 Positive and negative sequence current under different grounding modes (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded y ( ) g Fig. 4 Neutral voltage under different grounding modes (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded Fig. 5 Positive and negative sequence current under different grounding modes (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded Fig. 5 Positive and negative sequence current under different grounding modes (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded KrelUunb < UO < K relUO max ð26Þ ð26Þ short-circuit fault. Considering the light or no load of the line, the expression is Where, Krel is reliability coefficient. Where, Krel is reliability coefficient. Iφ tð Þ−Iφ t−T ð Þ≤0 ð27Þ ð27Þ Nonetheless, some short-circuit faults will also cause the neutral voltage to shift. 4.2 Protection criterion Thi However, the single-phase break fault does not cause a rise in current. Selecting the bus phase current as the blocking criterion can effect- ively distinguish the single-phase break fault from the Where, Iφ(t) is the sample value of bus phase current at the present moment; Iφ(t-T) is the sample value of bus phase current at the previous cycle. Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 8 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 8 of 13 Table 2 Neutral voltage variation with fault location Fault location (km) Neutral voltage (V) Ungrounded Arc suppression coil grounded Low resistance grounded 1 531.69 5176.02 64.96 2 475.16 4631.55 58.05 3 418.64 4101.22 51.15 4 362.12 3528.46 44.24 5 305.61 2941.56 37.34 6 245.59 2390.02 30.00 7 189.92 1845.55 23.20 8 134.30 1311.68 16.40 9 78.79 767.21 9.62 Table 2 Neutral voltage variation with fault location only cooperate with short circuit fault protection, but also realize segment protection of the line. Based on the different variation characteristics, the ra- tio of positive and negative sequence current variation is used to construct the line selection criterion, which can accurately distinguish the fault feeder from the non-fault feeder. The expression is According to the eq. (26) to (28), the single-phase break fault protection method of the distribution net- work is as shown in Fig. 3, including the starting compo- nent, the line selection component, and the blocking component. 1−K set < ni < 1 þ Kset ð28Þ ð28Þ The specific action logic is: when the neutral voltage is higher than the normal unbalanced voltage and less than the maximum voltage of the single-phase break fault, the starting component acts; when the amplitude of the bus phase current increases, the blocking component acts to prevent misjudgment, otherwise, the blocking component does not act, determining that the single-phase break fault occurs; when a certain feeder i satisfies 1 −Kset < ni < 1 + Kset, the line selection component acts and sends the warning or tripping signal, determining that the feeder does have a single-phase break fault. Where, ni is the ratio of the positive and negative se- quence current amplitude variation at the outlet of the feeder i; Kset is the margin, generally 0.1~0.2(It is re- quired that the setting of Kset can clearly identify the fault feeder. 4.2 Protection criterion Thi Taking the influence of asymmetry degree, measurement error and noise into account, it is more appropriate to take Kset = 0.1). In addition, the blocking criterion is mainly used to distinguish short-circuit faults and break faults. The pro- tection of short-circuit fault is generally quick-acting. Therefore, a certain time delay can be set, which can not When the neutral point is grounded in different ways, it is only necessary to adjust the minimum and Fig. 6 Bus phase current variation with fault location Fig. 6 Bus phase current variation with fault location Fig. 7 Positive and negative sequence current amplitude change ratio variation with fault location (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded 0 Page 9 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 9 of 13 Xiao et al. Protection and Control of Modern Power Systems (20 Page 9 of 13 Table 1 Line parameters Feeder number Length (km) Capacitance (μF/km) A B C 1 10 0.26 0.28 0.27 2 8 0.30 0.30 0.31 3 9 0.27 0.27 0.27 4 7 0.32 0.31 0.33 5 6 0.29 0.29 0.28 6 5 0.30 0.30 0.30 maximum values of the neutral voltage corresponding to the starting criterion without changing the protection principle, while the blocking criterion and line selection criterion are fixed. Therefore, this protection method is applicable to any grounding modes. For fault phase selection, it can be achieved in the fol- lowing two ways. First, since the fault phase voltage is larger than the non-fault phase voltage [14, 16], phase selection can be realized by comparing the phase volt- ages. Second, the non-fault phase current is continuous after the single-phase break fault, while the fault phase current is discontinuous, which is equal to the upstream load current and changes more significantly [13]. There- fore, the fault phase selection can be realized by compar- ing the change of the phase current. Fig. 7 Positive and negative sequence current amplitude 5 Simulation and results In order to verify the reliability of the proposed protection method under the ungrounding, arc suppression coil grounding and low resistance grounding modes, the 10 kV typical structural distribution network model shown in Fig. 1 is built in Matlab/Simulink. There are 6 feeders in the distribution network. The neutral low resistance Rd is 10 Ω, and the arc suppression coil is overcompensated by 10%. The line parameters are shown in Table 1. From Table 1, according to the numerical analysis, when the neutral point is ungrounded, the asymmetry is 0.43%, the Uunb is 24.8 V in normal operation, and the UOmax is 600 V in single-phase break fault. When the neutral point is arc suppression coil grounded, the asymmetry is 4.3%, the Uunb is 248.3 V, and the UOmax is 5989 V. When the neutral point is low resistance grounded, the asymmetry is 0.05%, the Uunb is 2.9 V, and the UOmax is 76 V. Fig. 7 Positive and negative sequence current amplitude change ratio variation with fault location (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded Fig. 7 Positive and negative sequence current amplitude change ratio variation with fault location (a) Ungrounded (b) Arc suppression coil grounded (c) Low resistance grounded According to eq. (26) to (28), take Krel = 1.1, Kset = 0.1, and the protection criterion when ungrounded is ( 27 < UO < 660 Iφ tð Þ−Iφ t−T ð Þ≤0 0:9 < ni < 1:1 ð29Þ ( 27 < UO < 660 Iφ tð Þ−Iφ t−T ð Þ≤0 0:9 < ni < 1:1 ð29Þ and when arc suppression coil grounded is Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 10 of 13 Xiao et al. 5 Simulation and results Protection and Control of Modern Power Systems (2020) 5:10 Page 10 of 13 (2020) 5:10 Table 3 Influence of system asymmetry on neutral voltage and sequence current ratio Table 3 Influence of system asymmetry on neutral voltage and sequence current ratio Group Ungrounded Arc suppression coil grounded Low resistance grounded λ(%) Uunb(V) UO(V) n1 λ(%) Uunb(V) UO(V) n1 λ(%) Uunb(V) UO(V) n1 1 0 0.28 410.55 1 0 2.26 4016.37 1 0 0 48.31 1 2 0.29 16.05 412.44 1 2.88 113.27 4058.79 1 0.03 1.97 50.10 1 3 0.43 23.86 418.64 1 4.30 235.21 4101.22 1 0.05 2.91 51.15 1 4 0.83 45.61 437.84 1 8.25 403.05 4115.36 1 0.09 5.59 53.46 1 5 1.10 60.88 454.32 1 10.9 551.54 4131.93 1 0.12 7.42 55.15 1 ( 273 < UO < 6588 Iφ tð Þ−Iφ t−T ð Þ≤0 0:9 < ni < 1:1 ð30Þ ð30Þ The change of bus phase current is shown in Fig. 6. It can be seen that the current of each phase is influenced by load distribution, and the closer the fault location is to the bus, the more the phase current drops. But each phase current is always lower than the normal running phase current. Among them, the phase-A current is the smallest, so it can be determined that the fault phase is the phase A. The blocking criterion does not work. Combined with the starting criterion, it can reliably judge the occurrence of single-phase break fault. and when low resistance grounded is ( 3 < UO < 84 Iφ tð Þ−Iφ t−T ð Þ≤0 0:9 < ni < 1:1 ð31Þ ð31Þ The load of feeder 1 is set at 5 km and 10 km. The feeder 1 has a phase-A single-phase break fault at 3 km and 7 km from the bus respectively when t = 0.7 s. The neutral voltages at different fault locations are shown in Fig. 4. The neutral voltage rises significantly after the single-phase break fault. And the positive and negative sequence currents are shown in Fig. 5. The drop ampli- tude of the positive sequence current is similar to the rise amplitude of the negative sequence current on fault feeder, whereas the sequence current of the non-fault feeder has little change. The ratio of the positive and negative sequence current amplitude changes of each feeder variation with the fault location is shown in Fig. 7. 5 Simulation and results In different ground- ing modes, there are always n1 ≈1 for the fault feeder, and ni < 0.5(i ≠1) for non-fault feeders, which have obvi- ous boundaries. Therefore, the line selection criterion is not affected by the grounding modes and fault location, and the fault line can be accurately and reliably selected. y y At the same fault location(d = 3 km), the neutral volt- age and the positive and negative sequence current amp- litude change ratio of the fault feeder under different system asymmetry can be obtained by changing the three-phase capacitance to ground of feeders, as shown in Table 3. With the increase of the system asymmetry, the neutral voltage tends to increase whether in normal operation or single-phase break fault state, but the change of amplitude is small. All of them are within the Change the fault location from 1 km to 9 km, and the corresponding neutral voltage is shown in Table 2. It can be seen that the neutral voltage is related to the fault location and grounding mode. The closer the fault dis- tance is to the bus, the higher the neutral voltage is. And the voltage is the highest when arc suppression coil grounded, and the lowest when low resistance grounded. In contrast to eq. (29) to (31), the neutral voltage is Table 4 Single-phase break fault with high resistance grounding Grounding resistance(Ω) Ungrounded Arc suppression coil grounded Low resistance grounded n1 UO(V) n1 UO(V) n1 UO(V) MG NG MG NG MG NG MG NG MG NG MG NG Pure resistance 700 1.0 700.1 527.2 1.0 5011.3 4916.5 1.0 83.7 63.8 1000 613.8 476.2 4871.9 4687.9 74.5 57.7 1500 513.4 445.8 4651.3 4435.2 62.4 54.1 2000 473.2 434.5 4539.6 4138.4 57.6 52.8 Arc resistance 700 1.0 656.2 491.4 1.0 4702.3 4625.5 1.0 78.5 56.2 1000 548.4 456.1 4468.9 4302.1 66.1 53.5 1500 480.8 435.7 4256.8 4189.6 58.2 52.0 2000 455.4 428.5 4186.7 4125.9 55.2 51.6 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Page 11 of 13 Xiao et al. 5 Simulation and results Protection and Control of Modern Power Systems (2020) 5:10 Page 11 of 13 Table 5 Comparison of the line selection criterias Fault location (km) Negative sequence current(A) Sequence current changes ratio 0 450.6 1.0 2 350.3 1.0 4 253.5 1.0 6 163.2 1.0 8 78.9 1.0 10 74.6 1.0 range of the starting criterion, and the protection can be reliably initiated. Moreover, the positive and negative se- quence current amplitude change ratio of the fault feeder is basically unaffected. The line selection criterion can still accurately identify the single-phase break fault feeder, which has high reliability. fault distance increases, the negative sequence current decreases. When the fault occurs at the end of the feeder, the negative sequence current is lower than the setting value 77A, causing the negative sequence current protection refuses to act. But n1 is always about 1.0, which is within the action range of the selection criter- ion. Therefore, compared with the negative sequence current criterion, the proposed protection criterion is simpler to set, and can accurately distinguish short cir- cuit fault from the break fault, which has higher reliabil- ity and applicability. The single-phase break fault may also be accompanied by a grounding fault. In order to adapt to a variety of complex break faults, the power supply side and the load side are grounded by resistance and arc resistance re- spectively at the break fault location (d = 3 km). The pro- tection characteristic parameters are shown in Table 4, where MG represents the terminal M (the power supply side) is grounded, and NG represents the terminal N (the load side) is grounded. It can be seen that the add- itional grounding fault exacerbates the deviation of the neutral voltage, the neutral voltage decreases with the increase of the grounding resistance, and gradually ap- proaches the value under the ungrounding break fault. The ratio of the positive and negative sequence current amplitude changes is always 1.0, which guarantees the accuracy of the line selection criteria. Therefore, the pro- tection method is also applicable to the break fault with high resistance grounding. When a single-phase grounding fault, two-phase short circuit fault and the load disappearance occur on feeder 1 respectively, the change of protection characteristic pa- rameters are shown in Table 6. Combining eq. 5 Simulation and results (29) to (31), it can be known that in the case of other fault types or changes in operating conditions, the system neutral voltage and the bus phase current changes cannot meet the starting criterion and the blocking criterion at the same time. The line selection criterion does not work. Therefore, the protection method can accurately distin- guish the single-phase break fault and other conditions. References 1. Campbell, M., & Arce, G. (2018). Effect of motor voltage unbalance on motor vibration: Test and evaluation. IEEE Transactions on Industry Applications, 54(1), 905–911. 1. Campbell, M., & Arce, G. (2018). Effect of motor voltage unbalance on motor vibration: Test and evaluation. IEEE Transactions on Industry Applications, 54(1), 905–911. 1. Campbell, M., & Arce, G. (2018). Effect of motor voltage unbalance on motor vibration: Test and evaluation. IEEE Transactions on Industry Applications, 54(1), 905–911. 2. Singh, M. (2017). Protection coordination in distribution systems with and without distributed energy resources- a review. Protection and Control of Modern Power Systems, 2(1), 27. Acknowledgements Not applicable. Acknowledgements Not applicable. 11. Zhu, L., Zhang, Z., Wang, Z., Zhang, H., Li, C., Zhu, S., & Bai, Y. (2011). Single- phase open-line fault selection and location based on wavelet neural networks. Power System Protection and Control, 39(04), 12–17. Received: 12 November 2019 Accepted: 4 March 2020 Received: 12 November 2019 Accepted: 4 March 2020 Received: 12 November 2019 Accepted: 4 March 2020 Author details 1 1School of Electrical Engineering, Chongqing University, Chongqing, China. 2North China Electric Power University, Beijing, China. 3Aalborg University, Aalborg, Denmark. Aalborg, Denmark. Competing interests Competing interests p g The authors declare that they have no competing interests. Authors’ information Yang Xiao(1997-), female, master student, major in power system protection and control. Email: yangxiao_ee@163.com. Jinxin Ouyang(1984-), male, PHD and Professor, major in analysis, protection, and control of renewable energy integrated power system. Email: jinxinoy@163.com. 14. Zhang, L., Cao, L., Li, L., Xue, Y., Tian, Y., & Feng, G. (2018). Analysis and fault section location of single-phase open fault for ungrounding system. Power System Protection and Control, 46(16), 1–7. 15. You, Y., Liu, D., Li, L., Zheng, S., Wu, A., & Chen, H. (2012). Detection method based on load monitoring for 10 kV overhead line single-phase ungrounded open fault area. Power System Protection and Control, 40(19), 114–149. Xiaofu Xiong(1962-), male, PHD and Professor, major in power system optimal operation and control and smart grid. Email: cquxxf@vip.sina.com. Yutong Wang(2000-), female, student, major in power system automation. Email: 187181713@qq.com. 16. Chang, Z., Song, G., & Wang, X. (2018). Identification and isolation of line breakage fault in distribution network based on zero sequence voltage amplitude differential principle. Automation of Electric Power Systems, 42(06), 135–139. Yongjie Luo(1988-), male, PHD, major in flexible DC transmission control and protection. Email: jieyong@163.com. 7 Conclusion In this paper, the neutral voltage and sequence current variation characteristics of distribution network single- phase break fault under typical grounding modes are stud- ied, and a fault protection method of single-phase break considering the influence of neutral grounding modes is proposed. The protection principle is based on the fact that the neutral voltage change is significant before and after the fault and the positive and negative sequence current amplitude changes of the fault feeder are equal, which have a great difference from non-fault feeders. In addition, the criterion of bus phase current is added to distinguish single-phase break and short-circuit fault, which can reliably identify the occurrence of single-phase break fault and accurately select the fault line. This protec- tion method is simple to set, and is basically not affected by the fault location, load distribution and system asym- metry, which has high sensitivity and reliability, and is ap- plicable for various grounding modes. 3. Yu, S., & Pan, L. (2009). Analysis of not-short circuit faults in neutral indirectly grounding system. Power System Protection and Control, 37(20), 74–78. 4. Telukunta, V., Pradhan, J., Agrawal, A., Singh, M., & Srivani, S. G. (2017). Protection challenges under bulk penetration of renewable energy resources in power systems: A review. CSEE Journal of Power and Energy Systems, 3(4), 365–379. 5. Liu, Y., Meliopoulos, A. P., Sun, L., & Choi, S. (2018). Protection and control of microgrids using dynamic state estimation. Protection and Control of Modern Power Systems, 3(1), 31. 5. Liu, Y., Meliopoulos, A. P., Sun, L., & Choi, S. (2018). Protection and control of microgrids using dynamic state estimation. Protection and Control of Modern Power Systems, 3(1), 31. 6. Singh, M., Vishnuvardhan, T., & Srivani, S. G. (2016). Adaptive protection coordination scheme for power networks under penetration of distributed energy resources. IET Generation Transmission and Distribution, 10(15), 3919– 3929. 6. Singh, M., Vishnuvardhan, T., & Srivani, S. G. (2016). Adaptive protection coordination scheme for power networks under penetration of distributed energy resources. IET Generation Transmission and Distribution, 10(15), 3919– 3929. 7. Shi, F., & Cong, W. (2014). Methodology to differentiate type of single-phase line break fault in 10kV ungrounded distribution networks. In IEEE PES Asia- Pacific power and energy engineering conference (pp. 1–6). 7. Shi, F., & Cong, W. (2014). Methodology to differentiate type of single-phase line break fault in 10kV ungrounded distribution networks. Authors’ contributions Y Xi f d th Yang Xiao performed the study of the method, analyzed the data and draft the manuscript. Jinxin Ouyang and Xiaofu Xiong engaged in modifying the paper and submitted it to the PCMP. Yutong Wang and Yongjie Luo participated in the design and simulation model building. All authors read and approved the final manuscript. 12. Zhu, L., Li, C., Zhang, H., & Zhang, Z. (2009). Negative sequence current distributing and single-phase open-line fault protection in distribution network. Power System Protection and Control, 37(09), 35–38. 13. Kang, Q., Cong, W., Wang, M., Xian, G., Liu, G., Guo, Z., & Yi, R. (2016). Analyses and judgment methods of single-phase broken-line fault for loaded distribution line. In IEEE PES Asia-Pacific power and energy engineering conference (APPEEC) (pp. 482–486). 6 Discussion b The negative sequence current method is the most commonly used existing method, and its setting value needs to avoid the maximum negative sequence current of the line when a single-phase break fault occurs on other lines. However, the negative sequence current is greatly affected by the fault location, the size and distri- bution of the load, and the system impedance. The load is evenly distributed on feeder 1. Make a comparison be- tween the proposed criterion and the negative sequence current criterion. It can be seen from Table 5 that as the It can be seen from the above simulation results that the proposed protection method has the following advan- tages. This method can reliably identify the fault type, accurately select the fault line, and is weakly affected by the fault location and load changes. Compared with the existing methods, it has higher reliability, and is suitable for various neutral grounding modes, which is better adapted to the development of modern distribution net- work. However, this method also has some limitations. Table 6 Protection characteristic parameters in other cases Type Ungrounded Arc suppression coil grounded Low resistance grounded UO(V) ΔIφ(A) n1 UO(V) ΔIφ(A) n1 UO(V) ΔIφ(A) n1 Single phase grounding 5523.9 25.1 0.34 5482.7 5.2 0.86 5345.3 454.5 0.93 Two-phase short circuit 16.3 21,607 1.04 141.7 21,607 1.04 2.0 21,607 1.04 Load disappearance 23.8 −804.1 5426.7 216.4 −804.1 5426.7 2.9 −804.1 5426.7 Page 12 of 13 Page 12 of 13 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Because the non-fault line has a very small negative se- quence current, it requires a higher sensitivity to the current acquisition device. The smaller the line capaci- tance and the farther the fault location is, the weaker characteristics of the neutral voltage change will be. However, with the extensive application of cables, the capacitance of distribution network becomes larger and larger, so the neutral voltage characteristics of the single-phase break fault are stronger. Therefore, the sen- sitivity and applicability of the method can be guaranteed. 7 Conclusion In IEEE PES Asia- Pacific power and energy engineering conference (pp. 1–6). 8. Wang, R., Cong, W., Kang, Q., Liu, G., Wang, H., & Yi, R. (2015). Analysis on single-phase line break fault of 10kV non-grounded distribution network. In International conference on electric utility deregulation and restructuring and power technologies (pp. 1043–1049). 9. Izykowski, J., Rosolowski, E., & Pierz, P. (2015). Location of open conductor failure combined with phase-to-earth fault on power line. In International conference on computational problems of electrical engineering (pp. 56–58). 10. Huang, S., Li, T., Ding, F., & Yang, L. (2011). A new calculation method for open- phase fault based on superposition principle. In International conference on advanced power system automation and protection (pp. 1139–1143). Funding 17. Peng, W., Baichao, C., Cuihua, T., Sun, B., Zhou, M., & Yuan, J. (2017). A novel neutral electromagnetic hybrid flexible grounding method in distribution networks. IEEE Transactions on Power Delivery, 32(3), 1350–1358. This work was supported by the National Natural Science Foundation of China (NO. 51877018). 18. Zeng, H., Yang, P., Cheng, H., Xin, J., Lin, W., Hu, W., & Wu, H. (2019). Research on single-phase to ground fault simulation base on a new type neutral point flexible grounding mode. IEEE Access, 7, 82563–82570. Availability of data and materials Not applicable. 19. Xu, Q., He, J., Huang, S., Zeng, J., Fan, C., & Wang, W. (2019). Researches on grounding modes of the AC/DC hybrid system with various distributed renewable energy. The Journal of Engineering, 2019(16), 3035–3038. 20. Martínez-Molina, M. A., & Ledezma, S. N. (2016). Analysis of three-phase load flow problem in distribution networks with the inclusion of symmetric and asymmetric transformer connections. In IEEE international engineering summit, II cumbre Internacional de las Ingenierias (pp. 1–5). 21. Kadam, S. R., Kale, R. U., Deokar, T. V., & Pawar, P. B. (2018). Fault current & fault voltage analysis of power distribution network. In International conference for convergence in technology (pp. 1–4). Xiao et al. Protection and Control of Modern Power Systems (2020) 5:10 Availability of data and materials Availability of data and materials Not applicable. Page 13 of 13
W2522441022.txt
https://zenodo.org/records/1083367/files/14648.pdf
fr
A Perceptual Image Coding method of High Compression Rate
Zenodo (CERN European Organization for Nuclear Research)
2,007
cc-by
2,681
World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:1, No:9, 2007 A Perceptual Image Coding method of High Compression Rate Fahmi Kammoun, and Mohamed Salim Bouhlel  International Science Index, Computer and Information Engineering Vol:1, No:9, 2007 waset.org/Publication/14648 Abstract—In the framework of the image compression by Wavelet Transforms, we propose a perceptual method by incorporating Human Visual System (HVS) characteristics in the quantization stage. Indeed, human eyes haven’t an equal sensitivity across the frequency bandwidth. Therefore, the clarity of the reconstructed images can be improved by weighting the quantization according to the Contrast Sensitivity Function (CSF). The visual artifact at low bit rate is minimized. To evaluate our method, we use the Peak Signal to Noise Ratio (PSNR) and a new evaluating criteria witch takes into account visual criteria. The experimental results illustrate that our technique shows improvement on image quality at the same compression ratio. Keywords—Contrast Sensitivity Function, System, Image compression, Wavelet transforms. Human Visual I. INTRODUCTION T HE compression is an important area of research in image processing and has been widely studied in the last decades. Compression algorithms can restitute the exact information (Lossless methods) or introduce a little distortion (lossy methods). The lossless image compression is designed to reduce or remove the image’s redundancy. They have weak compression ratio but benefit from an exact reconstruction of the image. The purpose of lossy image compression is to minimize the number of bits needed to represent an image without introducing an important degradation. Natural images have an important redundancy among the space-image pixels. A linear transformation is applied to minimize redundancy in the images since it can decorrelate pixel values in the transform domain. The international standard for still image compression, called Joint Photographic Experts Group or JPEG standard [1]-[2], uses the Discrete Cosine Transform (DCT) [3]-[4]. The JPEG2000 [5] uses The Discrete Wavelet Transforms (DWT). The quantization assigns to each transform coefficient a number of bits according to its position. This step is not conservative and attempts to reduce the number of samples. Finally, an entropy encoder codes Manuscript received Mars 29, 2004. F. Kammoun is with the Research Unit: Sciences and Technologies of Image and Telecommunications, Higher Institute of Biotechnology, Sfax TUNISIA, on leave from: Département de Physique, Faculté des Sciences de Sfax, B.P. 802, 3018 Sfax, TUNISIA, (phone: 216-74-276400; fax: 216-74274437; e-mail: fahmi_kammoun@yahoo.fr). M. S. Bouhlel is with the Research Unit: Sciences and Technologies of Image and Telecommunications Higher Institute of Biotechnology, Road of Soukra km3, 3038 Sfax, TUNISIA. International Scholarly and Scientific Research & Innovation 1(9) 2007 theses samples [6]-[8]. The main objective of the image compression is to achieve the lowest bit rate without loss of the visual information. It implies a distinction between the visible and invisible information contained in an image. This distinction is done by the exploitation of a human visual model incorporated within the compressive process, precisely at the quantization stage. The key element of such a model is the Contrast Sensitivity Function (CSF). This Function describes the human visual sensitivity to different spatial frequencies by varying their contrast. Its curve shows essentially that the sensitivity of the visual system is reduced for structures of high spatial frequencies. The exploitation of the CSF at the quantization phase permits to select the truncation of the information contained in the image. In other words, the truncation affects intensively the spatial high frequency information and assures the visually important spatial frequency preservation. Therefore this approach permits to improve the visual quality of the compression significantly. II. THE HVS CHARACTERISTICS AND IMAGE COMPRESSION A. Presentation The human visual system (HVS) reached during the evolution an important level of complexity. It is capable to execute several tasks that can’t be achieved by the present technology. Nevertheless, it has some limitations concerning the visual perception. These minor imperfections don't make uncomfortable the vision. However these limitations can be exploited in image compression. One of the most important limitations of the HVS system concerns the sensitivity reduced for the spatial high frequency structures. This phenomenon is shown by the contrast sensitivity function (CSF). B. The Contrast Sensitivity Function The function of sensitivity to the contrasts, commonly called Contrast Sensitivity Function (CSF), describes the capacity of the human visual system to detect differences of luminance. The researchers studied the variation of the contrast sensitivity according to the different spatial frequencies. They are represented by a stimulus with a periodic structure formed of alternate strips. The gotten curve constitutes the contrast sensitivity function (CSF) of the examined person considering the conditions of the experimentation: shape of the stimulus, distance of vision, 2891 scholar.waset.org/1307-6892/14648 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:1, No:9, 2007 angle of vision, binocular or monocular vision, etc. 4q+1 5q+1 1.4 1.2 1.0 6q+1 7q+1 0.8 0.6 0.4 Fig. 2: The matrix of quantization 0.2 0.0 International Science Index, Computer and Information Engineering Vol:1, No:9, 2007 waset.org/Publication/14648 0 5 10 15 20 25 30 35 40 III. CSF BASED QUANTIZATION 45 Fig. 1: The contrast sensitivity function Studies have been led to find some analytic formulas close to the experimental results. Among the most known, we mention the formula of Mannos and Sakrison [9]. It is one of the first solutions and it is used in many studies. CSF f 2.6 0.0192  0.114 f e  0.114 f To exploit the CSF in the process of the wavelet transform based quantization, we use the invariable unique weight. In other words, only one factor is affected for every wavelet subband. This factor remains constant during the whole phase of quantization. The set of these quantization factors is called CSF Quantifier. Fig. 3 illustrates the incorporation of the CSF Quantifier in the compression process. 1.1 (1) Original image Other formulas have been proposed [10]-[11]. The most recent is the one of Ngan [12]. Quantization Entropy coding CSF Quantifier Compressed image Dequantization Entropy Decoding DWT C. Quantization and Contrast Sensitivity Function The compression by Discrete Wavelet Transforms (DWT) is accomplished with a quantization and an entropy encoder. Typically, one uses a constant quantifier implemented by a division of the wavelet coefficients by a constant factor Q. The result is approximated to the nearest integer [13]. The factor Q can be different for different intervals of frequency. It is then appropriate to speak of a quantization matrix to make reference to a set of factors. It corresponds to a particular matrix related to a level of decomposition. A matrix that illustrates the increasing quantization is given by the fig. 2. The integer q is the quantum chosen to determine the step of the quantization. The idea of this method consists to reduce the precision of the coefficients of the DWT while moving away of the region of low frequencies (coefficients of the approximation). Indeed, it is not necessary to maintain an important precision on the coefficients of high frequencies because these values are less relevant than the ones of low frequencies. Our aim is to define the terms of a new matrix of quantization by taking into account the HVS characteristics and particularly the Contrast Sensitivity Function. These terms have to increase while moving to the region of high frequencies. 6q+1 q+1 2q+1 4q+1 2q+1 3q+1 International Scholarly and Scientific Research & Innovation 1(9) 2007 IDWT Reconstructed image Fig. 3: The incorporation of the CSF Quantifier IDWT is the inverse DWT. The advantages of this method reside in the simplicity of the determination of the quantization factors and the reduction of the computational time. The quantization factors are inversely proportional to the sensitivity average of the HVS on the frequency band related to each subband of the wavelet transform. Indeed, every subband of the wavelet transform occupies an interval on the spatial frequencies. Therefore, we compute the average of the CSF on every interval of the spatial frequency and we define the CSF quantifier factors as the inverses of these averages. Then they are normalized so that the smallest of these factors is equal to 1 (Fig. 4). On given one interval, more the CSF is raised, more its spatial frequencies are relevant and more the coefficient of quantization is low. For a wavelet decomposition of level 5, this method gives 6 CSF weights. 2892 scholar.waset.org/1307-6892/14648 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:1, No:9, 2007 Quantization factors 10 9 wcsf (1) = 9.90 wcsf (6)=1.68 8 7 wcsf (5)=1.21 6 5 wcsf (4)=1.00 4 3 2 wcsf (3) = 1.02 corresponding to the high spatial frequencies. Indeed, the sensitivity of the SVH system is reduced for these frequencies. On the other hand, our method preserves the perceptible information. In fact, the coefficients of the CSF quantifier related to spatial frequencies of this perceptible information have a value very close to 1. Therefore, an elevation to the power of p nearly preserves the totality of the information after the phase of quantization. Let us define :csf (O) as the CSF weight wcsf (O) optimized by the parameters m and p. They are given by: wcsf (2) = 1.80 :csf (O) = m.(wcsf (O)) p IV. IMAGE QUALITY ASSESSMENT International Science Index, Computer and Information Engineering Vol:1, No:9, 2007 waset.org/Publication/14648 1 Fig. 4: CSF Quantifier Frequency To evaluate our method, we use the Peak Signal to Noise Ratio (PSNR), which is defined as: § 255 2 · ¸ PSNR 10 log 10 ¨ ¨ MSE ¸ © ¹ wcsf (6) wcsf (5) wcsf (4) (3) where MSE is the Mean Square Error: MSE wcsf (3) wcsf (2) wcsf (3) wcsf (3) wcsf (1) wcsf (2) (2) wcsf (2) wcsf (1) 1 M 1 N 1 2 x m,n  x̂ m,n ¦ ¦ MN m 0 n 0 (4) where M and N are the number of lines and columns of the image, x(m,n) and x̂ m,n are the pixels of the original and the processed image. However, the PSNR do not correlate well with subjective quality evaluation. In fact, the HVS is sensitive to the noise on the uniform zones. Its perception on the textured zones is more difficult. To take in account this characteristic of the HVS, we use the weighted PSNR (wPSNR) that use the local variance of the image to ponder the error: wcsf (1) § 255 2 · ¸ wPSNR 10 log 10 ¨ ¨ wMSE ¸ © ¹ (5) where Fig. 5: The matrix of quantization Let us define wcsf (O) as the CSF weight related to the level O of the wavelet decomposition. Fig. 5 shows the new matrix of quantization where the details coefficients of level O{1,2,3,4,5} are pondered by the coefficients wcsf (O), and the approximation coefficients are pondered by the factor wcsf.(6). To optimize our method, we introduce two parameters in the CSF quantifier. The first is noted m. It multiplies the coefficients of the CSF quantifier to adjust the compression ratios. The second is a parameter that raises the coefficients of the CSF quantifier to a power noted p. The main interest of this factor is to increase the truncation of the coefficients International Scholarly and Scientific Research & Innovation 1(9) 2007 wMSE 1 M 1 N 1 x m,n  x̂ m,n MN m¦0 ¦ 1  var m ,n n 0 2 (6) V. RESULTS We applied our method to four images: two medical images, Lena and Bird. The size of these images is of 256u256. We used the 5/3 wavelet of Le Gall. We compare the HVS quantifier and a conventional quantization by using the matrix shown in fig. 2. 2893 scholar.waset.org/1307-6892/14648 International Science Index, Computer and Information Engineering Vol:1, No:9, 2007 waset.org/Publication/14648 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:1, No:9, 2007 (a) Image forearm (b) Reconstructed image with perceptual method at 0.125 bits/pixel (c) Reconstructed image with conventional method at 0.125 bits/pixel (d) Image Lena (e) Reconstructed image with perceptual method at 0.3 bits/pixel (f) Reconstructed image with conventional method at 0.3 bits/pixel Fig. 6: Original and reconstructed images Table 1 shows that the HVS quantifier performs better results than the conventional quantization. This is reasonable since the HVS quantifier allocates less bits to the wavelets TABLE I coefficients related to the frequencies bands which are weakly PERFORMANCE METRICS WITH THE HVS QUANTIFIER AND THE CONVENTIONAL QUANTIZATION perceptible. Subjective testing performed on the Forearm image and Lena image (Fig. 6) shows that the reconstructed conventional HVS quantifier bit quantization Images images by using the perceptual method have a better quality. rate We notice that the reconstructed images by using the PSNR wPSNR PSNR wPSNR conventional method are blurred. Perceptual method preserves 0.417 29.153 78.858 30.475 76.911 the edges better on the reconstructed images. shoulder Forearm Lena bird 0.142 29.153 78.586 28.606 75.529 0.106 27.588 77.792 27.758 74.883 VI. CONCLUSION 0.274 32.466 70.524 30.457 65.95 0.201 31.676 71 .28 29.752 65.960 0.126 29.839 69.263 28.648 65.581 0.638 28.435 87.795 28.229 87.662 0.418 26.765 86.365 26.598 85.673 0.296 25.304 85.21 25.208 84.216 0.450 33.964 83.927 31.098 80.838 0.255 32.502 81.773 30.331 79.671 0.155 30.428 79.228 29.00 78.853 0.09 28.180 77.103 27.341 75 .461 A perceptual image coding method by using the wavelet transform was proposed in this paper. This method takes into account the characteristics of the Human Visual System (HVS) by incorporating the Contrast Sensitivity Function (CSF) in the quantization step. This function describes the capacity of the human visual system to detect differences of luminance according to the different spatial frequencies. To evaluate our method, we use the weighted Peak Signal to Noise Ratio (wPSNR) that uses the local variance of the image to ponder the error. The objectives results showed that the perceptual method outperforms the conventional method. The subjective testing showed that the perceptual method has a better quality. International Scholarly and Scientific Research & Innovation 1(9) 2007 2894 scholar.waset.org/1307-6892/14648 World Academy of Science, Engineering and Technology International Journal of Computer and Information Engineering Vol:1, No:9, 2007 REFERENCES W. Pennebaker, JPEG still image data compression standard. New York: Van Nostrand Reihold, 1993. [2] G. K. Wallace, “Overview of the JPEG (ISO/CCITT) still image compression standard,” in Proc. SPIE/SPSE Symposium on Electronic Imaging Science & Technologies, Feb. 1990. [3] N. Ahmed, T. Natarajan, and K. R. Rao, “Discrete cosine transform,” IEEE Trans. on Computers, vol. C23, no. 1, pp. 90–93, Jan. 1974. [4] W. K. Cham, “Development of integer cosine transforms by the principle of dyadic symmetry,” IEEE Proceedings-I, vol. 136, no. 4, pp. 276–282, Aug. 1989. [5] S. C. Diego, E. Touradj, A. Joel, L. Mathias, and C. Charilaos, “JPEG 2000 still image coding versus other standards,” in Proc. of SPIE, vol..4115, Applications of digital image processing XXIII, Ed. Andrew G. Tesher, Dec. 2000, pp. 446–454. [6] J. Rissanen, and G. Langdon, “Arithmetic coding,” IBM Journal of research and development, vol. 23, no. 2, pp. 35–43, Mar. 1979. [7] D. A. Huffman, “A method for the construction of minimum redundancy codes,” Proc. of the IEEE, vol. 40, no. 9, pp. 1089–1101, Sept. 1952. [8] A. Lempel, and J. Ziv, “Compression of individual sequences via Variable-Rate-Coding,” IEEE Trans. on Information Theory, vol. IT-24, pp. 530–536, 1978. [9] J. Mannos, and D. Sakrison, “The effects of a visual fidelity criterion on the encoding of images,” IEEE Trans. on Information Theory, vol. IT20, pp. 525–536, 1974. [10] A. Watanabe, T. Mori, S. Nagata, and K. Hiwatashi, “Spatial sine-wave responses of the human visual system,” Vision Research, vol. 8, no. 9, pp. 1245–1263, Sep. 1968. [11] F. W. Cambell, and J. G. Robson, “Application of Fourier analysis to the visibility of gratings,” Journal of physiology, of gratings, vol. 197, pp. 551–566, 1966. [12] K. Ngan, K. Rao, and H. Singh, “Cosine transform coding incorporating human visual system model,” in Proc. SPIE fiber'86, 1986. [13] A. B. Watson, G. Y. Yang, J. A. Solomon, and J. Villasenor, “Visibility of wavelet quantization noise,” Ames Research Center, July 1996. International Science Index, Computer and Information Engineering Vol:1, No:9, 2007 waset.org/Publication/14648 [1] International Scholarly and Scientific Research & Innovation 1(9) 2007 2895 scholar.waset.org/1307-6892/14648
https://openalex.org/W2075890718
https://www.mdpi.com/2072-6651/6/4/1325/pdf?version=1397200224
English
null
Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins
Toxins
2,014
cc-by
11,992
Toxins 2014, 6, 1325-1348; doi:10.3390/toxins6041325 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Review Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins Kui Zhu, Richard Dietrich, Andrea Didier, Dominik Doyscher and Erwin Märtlbauer * Institute of Food Science, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Oberschleißheim 85764, Germany; E-Mails: zhukcau@gmail.com (K.Z.); r.dietrich@mh.vetmed.uni-muenchen.de (R.D.); a.didier@mh.vetmed.uni-muenchen.de (A.D.); dominik.doyscher@mh.vetmed.uni-muenchen.de (D.D.) * Author to whom correspondence should be addressed; E-Mail: e.maertlbauer@mh.vetmed.uni-muenchen.de; Tel.: +49-89-2180-78601; Fax: +49-89-2180-78602. Received: 10 February 2014; in revised form: 10 March 2014 / Accepted: 28 March 2014 / Published: 11 April 2014 Abstract: Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS Toxins 2014, 6, 1325-1348; doi:10.3390/toxins6041325 Toxins 2014, 6, 1325-1348; doi:10.3390/toxins6041325 Kui Zhu, Richard Dietrich, Andrea Didier, Dominik Doyscher and Erwin Märtlbauer Institute of Food Science, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Oberschleißheim 85764, Germany; E-Mails: zhukcau@gmail.com (K.Z.); r.dietrich@mh.vetmed.uni-muenchen.de (R.D.); a.didier@mh.vetmed.uni-muenchen.de (A.D.); dominik.doyscher@mh.vetmed.uni-muenchen.de (D.D.) * Author to whom correspondence should be addressed; E-Mail: e.maertlbauer@mh.vetmed.uni-muenchen.de; Tel.: +49-89-2180-78601; Fax: +49-89-2180-78602. Received: 10 February 2014; in revised form: 10 March 2014 / Accepted: 28 March 2014 / Published: 11 April 2014 Abstract: Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. Keywords: bacterial toxins; antibodies; immunoassay; nanomaterials; microfluidics Keywords: bacterial toxins; antibodies; immunoassay; nanomaterials; microfluidics Toxins 2014, 6 Toxins 2014, 6 1326 2. Antibodies and Immunoassays The core principle of immunoassays is the specific recognition of the target of interest by an antibody, which is the key component in any test. Several types of antibodies have been introduced for antibody-based sensing of bacterial toxins [13]. Antibodies (Figure 2A) represent a group of glycoproteins possessing two distinct types of polypeptide chains. Both the light chain and the heavy chain show a variable region of the heavy (VH) and light (VL) chains at their amino terminal end, whereas the remaining part of the polypeptide chain is referred to as the constant region (constant heavy (CH) and constant light (CL) chain). The variable regions of both chains contain a hypervariable part, which represents the antigen binding site (antibody combining site) or “paratope”. After the folding and combining of the light and heavy chains, this hypervariable region of the immunoglobulin shows a structure complementary to the corresponding part of the antigen molecule, which is referred to as the antigenic determinant or “epitope”. The antibodies produced in an animal species are polyclonal in nature (polyclonal antibody, pAb) and synthesized and secreted by plasma cells, derived from different B-lymphocytes. These lymphocytes may be fused with myeloma cells. The myeloma cells provide the genes for continued cell division, whereas the lymphocytes provide the functional immunoglobulin genes. The fused cells are called hybrid cells or hybridomas, and each hybridoma produces identical copies of one antibody, i.e., monoclonal antibodies (mAb). For immunoassays, preferably antibodies of the immunoglobulin G (IgG) class are employed. Whereas each mAb recognizes a single epitope, pAbs may bind to a range of epitopes present on the antigen used for the immunization. For specific applications, enzymes, such as papain and pepsin, are used to digest the intact antibody to generate small antigen binding fragments [14]. A new approach to generate half antibody fragments for biosensor applications by reduction via tris(2-carboxyethyl) phosphine (TCEP) was described recently [15]. Alternatively, recombinant techniques are used to produce the single-chain variable fragment (scFv) containing the variable regions of the VH and VL chains of the original IgG linked by a short peptide [16]. Recombinant antibodies and antibody fragments show distinct advantages to improve the sensitivity of biosensor applications, since they facilitate the loading of more and properly orientated antigen binding fragments onto a limited surface area [17]. Toxins 2014, 6 Toxins 2014, 6 (MALDI-TOF MS) to decipher the toxin profile [10–12]. Detection of bacterial toxins by antibody based assays or immunoassays has also been a successful approach for decades and still gains much attention due to the inherent advantages, such as simplicity, speed and cost-effectiveness. This review is focused on antibody-based techniques for the detection of bacterial toxins. 1. Introduction To avoid potential hazards for human and animal health, accurate and reliable analysis of bacterial toxins is critical in clinical diagnostics, food analysis, water monitoring, as well as for bio-security/defense purposes. Bacterial toxins are generally catalogued into exotoxins (peptides and proteins) produced by both Gram-positive and Gram-negative bacterial pathogens and endotoxins (lipopolysaccharides, LPS) produced by Gram-negative bacteria. These toxins cover a broad range of molecular weights, from less than 1000 Da to more than 100,000 Da (Figure 1A), exhibit different physico-chemical properties and cause a broad variety of clinical symptoms, ranging from mild diarrhea and emesis to severe and fatal neurological disorders [1–4]. According to their preferred targets, bacterial exotoxins may be grouped into toxins acting at the host cell surface and intracellularly active toxins [5]. Pore forming proteins [6,7] and AB-type of toxins (usually consisting of one A- and several B-subunits) [8,9] represent classical examples of the respective groups. Figure 1. (A) Schematic representation of bacterial toxins of low and high molecular weight (MW); and (B) examples for direct or indirect detection methods of bacterial toxins. LPS: lipopolysaccharides; HPLC: high-performance liquid chromatography; HPLC-MS: HPLC-mass spectrometry; MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight; ELISA: enzyme-linked immunosorbent assay; and PCR: polymerase chain reaction. To achieve the selective and sensitive detection of bacterial toxins, many methods have been developed in the past decade, covering direct and indirect approaches (Figure 1B). On the one hand, the toxins are directly captured by antibodies, or measured by mass spectrometry. On the other hand, the functional properties of the toxins are used to trigger effects in animals or cells; also, the bacterial species or the genes involved in toxin production may be detected. These indirect assays can provide valuable information, particularly when searching for unknown bacterial toxins or when direct methods are not available. Direct instrumental assays rely on sophisticated equipment, such as high-performance liquid chromatography (HPLC), HPLC with tandem mass spectrometry (HPLC-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry 1327 2. Antibodies and Immunoassays Furthermore, the recently described technique of the direct pairing of two independent antibody specificities to create new bispecific antibodies [18,19] may enable the enhanced detection of bacterial toxins in the future. An alternative to conventional antibodies, heavy chain antibodies (hcAbs, Figure 2B), found in camelids, have gained considerable attention, because of their unique structure, being composed of heavy chains only [20]. Fragments of hcAb, named nanobody (Nb) or single domain antibody (sdAb), have already been used for the detection of bacterial toxins, such as cholera toxin (CT) [21]. Furthermore, the design and production of recombinant Nbs from hcAbs is simplified, because only the variable region of a single (heavy) chain must be cloned and expressed in E. coli or other organisms [22]. 1328 Toxins 2014, 6 Toxins 2014, 6 Figure 2. Antibodies and assays: (A) types of antibodies used for immunoassay, immunoglobulin G (IgG) and related fragments (VL: variable light chain; VH: variable heavy chain; CL: constant light chain; CH: constant heavy chain; Fab: antigen binding fragment; and scFv: single-chain variable fragment); (B) heavy chain antibody (hcAb) and related fragments (VHH: variable domain of heavy chain antibodies; Nb: nanobody; and sdAb: single domain antibody); (C) the recognition of toxin targets by antibodies in competitive and noncompetitive assays; (D) labeling of primary antibodies for signal generation (Ab: antibody); (E) common readout techniques involved in immunoassays; and (F) examples for indirect assays. The amplification of toxin genes was done by PCR, the enrichment culture of the toxin producing organism and functional assays, such as cytotoxicity testing on mammalian cells. Figure 2. Antibodies and assays: (A) types of antibodies used for immunoassay, immunoglobulin G (IgG) and related fragments (VL: variable light chain; VH: variable heavy chain; CL: constant light chain; CH: constant heavy chain; Fab: antigen binding fragment; and scFv: single-chain variable fragment); (B) heavy chain antibody (hcAb) and related fragments (VHH: variable domain of heavy chain antibodies; Nb: nanobody; and sdAb: single domain antibody); (C) the recognition of toxin targets by antibodies in competitive and noncompetitive assays; (D) labeling of primary antibodies for signal generation (Ab: antibody); (E) common readout techniques involved in immunoassays; and (F) examples for indirect assays. The amplification of toxin genes was done by PCR, the enrichment culture of the toxin producing organism and functional assays, such as cytotoxicity testing on mammalian cells. mmunoassays include several steps: (i) the recognition of toxin targets by antibodies; (ii) subsequent al transduction; and (iii) readout techniques providing qualitative or quantitative results. mpetitive and noncompetitive assays may be employed in the first step, depending on the number of opes available on the toxins (Figure 2C). Competitive methods are based on the competition of and labeled (functionalized) or solid phase-bound antigens for a limited number of antibody bining sites. In most cases, the assay response represents the bound labeled antigen and is efore inversely proportional to the concentration of the free antigen. This type of assay is used for Immunoassays include several steps: (i) the recognition of toxin targets by antibodies; (ii) subsequent signal transduction; and (iii) readout techniques providing qualitative or quantitative results. Toxins 2014, 6 Toxins 2014, 6 the detection of low molecular weight toxins, such as the monocyclic heptapeptide, microcystin, produced by Cyanobacteria, which have only one epitope. Two variants of noncompetitive assays may be used to detect bacterial protein toxins. The so-called sandwich enzyme immunoassay can only be used for the detection of macromolecules, such as protein toxins, having at least two antigenic determinants in suitable steric positions, enabling two antibodies (capture and detection antibody) to bind to the antigen. In the second variant, the solid phase is coated directly with the toxin, and the amount of toxin bound is determined using specific labeled antibodies. In both cases, the assay response is directly proportional to the concentration of the target antigen. In the second step, different protocols can be used to generate the final readout after primary antibody binding. To permit the sensitive observation of the antigen-antibody reaction, antigens or antibodies have to be labeled either directly or indirectly. Protocols for indirect labeling include functionalized secondary antibodies and the biotin-avidin system to bridge the antigen-antibody reaction and signal generation (Figure 2D) [23,24]. Direct modification of the primary antibody can be achieved by biomolecules, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), and may result in decreased affinity and stability induced by unspecific side effects of the coupling chemistry and/or steric hindrance by the attachment of the reporter enzymes. Recently, oligonucleotide-modified primary antibodies have been implemented in immuno-PCR methods to detect Shiga toxin 2 (Stx2) and Stx2 variants [25]. However, the low efficiency of the preparation of the chimera has hindered immuno-PCR from wide acceptance [26]. Alternatively, polymer and “click” chemistry may be useful ways to improve the labeling of the primary antibody. For example, more enzymes can be anchored on the surface of stretch polymers to increase the ratio of enzyme to antibody [27]. Compared to the noncovalent binding involved in protocols utilizing secondary antibodies or biotin-avidin, covalent coupling using “click” chemistry offers several advantages. “Click” chemistry was first described for chemical reactions yielding high amounts of specifically and quickly joined small units; one of the most popular reactions is the azide-alkyne, cycloaddition, with or without catalysis by copper [28,29]. In the third step, the final readout is generated. Toxins 2014, 6 Although label-free methods, such as surface plasmon resonance (SPR) and electrochemical sensors, have been used for the detection of CT and the LPS of Gram-negative bacteria with high sensitivity [30–32], the vast majority of immunoassays utilize labeled immunoreagents. The signal can be amplified by enzymes, which are widely used for colorimetry-based qualitative and quantitative assays. However, the detection of trace amounts of toxin often requires further signal enhancement, and many other methods, such as fluorescence, luminescence, electronic signal and mass spectrometry, have been employed to improve the sensitivity (Figure 2E). In the following section, we will summarize how these approaches enable signal amplification, with special emphasis on the use of nanomaterials. Toxins 2014, 6 Competitive and noncompetitive assays may be employed in the first step, depending on the number of epitopes available on the toxins (Figure 2C). Competitive methods are based on the competition of free and labeled (functionalized) or solid phase-bound antigens for a limited number of antibody combining sites. In most cases, the assay response represents the bound labeled antigen and is therefore inversely proportional to the concentration of the free antigen. This type of assay is used for Immunoassays include several steps: (i) the recognition of toxin targets by antibodies; (ii) subsequent signal transduction; and (iii) readout techniques providing qualitative or quantitative results. Competitive and noncompetitive assays may be employed in the first step, depending on the number of epitopes available on the toxins (Figure 2C). Competitive methods are based on the competition of free and labeled (functionalized) or solid phase-bound antigens for a limited number of antibody combining sites. In most cases, the assay response represents the bound labeled antigen and is therefore inversely proportional to the concentration of the free antigen. This type of assay is used for 1329 3.1. Gold Nanoparticles (AuNPs) AuNPs have had a long history of application in on-site tests since their first use as a colorimetric readout in lateral flow immunoassays (or strips), such as home pregnancy tests. Several recent reviews focus on the synthesis and properties of AuNPs [33–35]. In the following part, we will concentrate on recent applications of AuNPs for the detection of bacterial toxins and on the mechanisms behind the assays. Optical immunoassays are based on direct color generation mediated by AuNPs either on solid surfaces or in solution. Lateral flow immunoassay, for example, is typically performed on hydrophobic nitrocellulose or cellulose acetate membranes. In sandwich-type assays, the red test line reports the presence of the target of interest, due to the accumulation of AuNP-labeled detection antibodies in the test zone. Recently, a dual assay of this type for the simultaneous detection of botulinum neurotoxin serotype A and B on a single strip has been developed [36]. In solution, after the aggregation of AuNPs through an unspecific electrostatic interaction or after specific binding by antibodies, a dramatic shift from red (dispersed particles) to blue (aggregated particles) color is observed, which can be used for visual detection or quantification. This phenomenon is based on localized surface plasmon resonance (LSPR), which is caused by the interaction between AuNPs and the incident light of a larger wavelength than the diameter of the AuNPs [37]. The cholera toxin B subunit (CTB), for example, can selectively bind to AuNPs modified by a lactose derivative and induce the aggregation of AuNPs, allowing the detection of 54 nM (3 µg mL−1) of toxin [38]. A recent exciting example is the chiroplasmonic immunoassay for the detection of microcystin-LR [39] (Figure 3A). Taking advantage of the chiroplasmonic properties of heterodimers of AuNPs and silver nanoparticles (AgNPs) bridged by antibody-antigen complexes, 8 × 10−13 M of microcystin-LR could be detected by circular dichroism (CD) measurement in a competitive assay. This is probably the lowest detection limit achieved up to now for this toxin, and this novel assay holds great promise also for the sensitive analysis of high molecular weight bacterial toxins, such as protein toxins or LPS in sandwich-type assays. Although not used for the detection of toxins so far, a new plasmonic enzyme-linked immunosorbent assay (ELISA) should be mentioned. Toxins 2014, 6 Toxins 2014, 6 Toxins 2014, 6 for different readouts or indirectly as carriers or anchored supports for other labels, such as enzymes and fluorescent probes. for different readouts or indirectly as carriers or anchored supports for other labels, such as enzymes and fluorescent probes. 3. Nanomaterials for Immunoassays As one of the most innovative and attractive technologies, nanotechnology has also entered bioanalysis. Diverse nanomaterials of different sizes, shapes and functional properties have been constructed. Herein, we will focus on the ones that have already been used for the detection of bacterial toxins in the past few years and on those that have the potential to serve this task. Nanomaterials can be used directly 1330 3.1. Gold Nanoparticles (AuNPs) Instead of color generation by the reporter enzyme, enzymatic activity is used to grow AuNP complexes with an ill-defined morphology, but exhibiting an intense blue color [40]. The authors choose a sandwich ELISA model for the ultrasensitive detection of two protein markers, which could be easily adapted as an alternative protocol for the detection of bacterial toxins. The distinctive electronic properties of AuNPs can also be used for electrochemical signal amplification [31]. The fabrication of electrodes implementing AuNPs functionalized with antibodies has considerably improved the selectivity and sensitivity of different electrochemical methods. An electrochemical ELISA platform for the sensitive detection of anthrax protective antigen (PA) utilized nanostructured gold electrodes coated with a binding peptide for the specific capture of PA [41]. The assay exhibits a limit of detection of less than 3 pM, when tested with serum samples. At this point, we would like to mention that beside the detection of toxins, numerous alternative assays based on 1331 3.2. Magnetic Nanoparticles (MNPs) MNPs or magnetic beads (MB), especially iron oxides (Fe3O4), have attracted considerable attention in biomedical and bioanalytical fields [46]. MNPs can be easily separated or enriched from complicated matrices by simply applying a magnetic field. Based on this phenomenon, different functionalized MNPs have been used for the specific or nonspecific capture and detection of bacteria and related products [47–49]. For example, a conductometric immunosensor has been developed for the generic and rapid detection of Gram-negative bacteria [31]. The combination of MNPs functionalized with antibodies for capture and modified AuNPs for detection is widely used for the detection of bacteria [47,50]. The adaption of this method will not only facilitate the enrichment of bacterial toxins at low concentrations from complex sample matrices, but also enable simple readouts, due to the unique properties of the MNPs. Furthermore, based on the dramatic progress made in the synthesis of nanomaterials, new multiple functional core-shell-structured particles consisting of an MNP core and Au or silicon functionalized shells have been prepared [46]. For instance, gentamicin-modified fluorescent MNPs with Fe3O4 cores and fluorescent silica (SiO2) shells were synthesized to capture Gram-negative bacteria [51]. Furthermore, magnetic silica NPs functionalized with antibodies via “click chemistry” have been reported for the detection of microcystin-LR [52]. Most interestingly, MNPs can also be used directly as the readout for the detection of bacterial toxins. Five bacterial toxins, including CT, heat-labile toxin from E. coli, enterotoxins A and B and toxic shock syndrome toxin (TSST) from S. aureus were simultaneously detected in an immunoarray for water, meat and milk samples [24] (Figure 3B). Briefly, the toxins are captured by specific antibodies coated on a microarray and then labeled with biotinylated detection antibodies. The assay signal is generated by scanning the microarray surface with streptavidin-coated MB in a shear flow. This ultrasensitive assay detects 0.1 to 1 pg mL−1 of toxin in less than 10 min, i.e., 100 zeptomoles or 100,000 molecules of CT in a sample volume of 0.1 mL. Furthermore, nonlinear magnetization of MNPs has been used for a novel sandwich immunoassay on a 3D fiber solid phase for the detection of Staphylococcal Enterotoxin A (SEA) and TSST in milk samples [53]. This label-free assay achieves a limit of detection as low as 4 pg mL−1 and 20 pg mL−1 for TSST and SEA, respectively. Toxins 2014, 6 Toxins 2014, 6 1332 Toxins 2014, 6 Toxins 2014, 6 antibody functionalized AuNPs have been developed for the precise and rapid detection of bacteria. Mass spectrometry-based immunosensors using antibody modified AuNPs have been constructed for the quantification of E. coli, Staphylococcus aureus and Salmonella enterica [42]. Furthermore, for the detection of Bacillus cereus, an amperometric immunosensor decorated with AuNPs coated with mAbs against of B. cereus was established, exhibiting an extremely low limit of detection of 10 colony forming units (CFU) mL−1 [43]. Figure 3. (A) Schemes of gold and silver hetero dimer-based chiroplasmonic methods for (a) assaying proteins; (b) microcystin-LR. Reprinted with permission from [39]. Copyright 2013 American Chemical Society; (B) Scheme of streptavidin functionalized magnetic nanoparticles (MNPs) for the simultaneous detection of five bacterial toxins in a microarray. Reprinted with permission from [24]. Copyright 2013 American Chemical Society; (C) Scheme of indirect competitive immunoassay for the detection of microcystin-LR based on the conjugation of quantum dots (QDs) and aminoethyl microcystin-LR. Reprinted with permission from [44]. Copyright 2014 Elsevier; (D) Scheme of Ab-QD-based microarrays for pathogen detection. In the presence of captured pathogen by the Ab-QD probes, the fluorescence of QDs are in the ON state, whereas in the absence of the pathogen, QDs are quenched by fluorescence resonance energy transfer (FRET) interaction between QDs and graphene oxide (GO) (OFF state). Reprinted with permission from [45]. Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. CP: chiroplasmon; NPs: nanoparticles; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid; NHS: N-hydroxysuccinimide; and CD: circular dichroism. 3.3. Quantum Dots (QDs) QDs are semiconductor nanocrystals that have been widely used in many fields, firstly introduced as fluorescent probes for biomedical applications by two independent labs [59,60]. Compared to the traditional chemical fluorescent dyes and proteins, QDs have shown several advantages, such as broad excitation spectra, high quantum yield, large Stokes shifts and high photostability [61]. There are numerous applications of QDs for the detection of bacterial toxins [62]. For example, a fluorescent sandwich immunoassay for the quantification of botulinum neurotoxin serotype A (BoNT/A) employed QDs functionalized with high affinity antibodies for detection [63]. Furthermore, an indirect competitive immunoassay was built for the analysis of microcystin-LR in water [44]. The carboxyl-coated QDs were coupled with aminoethyl-microcystin-LR and used as donors together with Cy5.5-labeled antibodies as receptors to construct a fluorescence resonance energy transfer (FRET) system for the sensitive and rapid detection of microcystin-LR in water samples in a portable optofluidic platform (Figure 3C). FRET or related phenomena are routine methods for the detection of ions, small molecules, proteins or nucleic acids [61,62,64]. For example, another FRET assay has been constructed for the detection of microcystin-LR by monoclonal antibody-coated CdSe-CdS core-shell structured QDs [65]. The fluorescence intensity of QDs is quenched in the presence of microcystin-LR, enabling the detection of 6.9 × 10−11 mol L−1 of toxin in water samples. A unique property of QDs is that they share a broad excitation spectrum, but can emit different narrow spectra, due to the quantum confinement effect [66]. QDs of a different size can be excited by a single wavelength and generate symmetrical emission bands at different wavelengths, exhibiting large Stokes shifts. This allows the detection of multiple targets in a single assay at the same time [64,67]. As the first example, CdSe-ZnS core-shell structured QDs of different sizes coated with antibodies were used for the simultaneous detection of CT, ricin, shiga-like toxin 1 and SEB in a microtiter plate [68]. The wells of the microtiter plate were coated with different capture antibodies, followed by the addition of the toxin mixture and the four corresponding antibody-QDs conjugates. After excitation at 330 nm, the emitted fluorescence was recorded at 510, 555, 590 and 610 nm for the individual QDs. However, the assay showed major drawbacks, such as insufficient sensitivity and high background signals. Toxins 2014, 6 Toxins 2014, 6 3.2. Magnetic Nanoparticles (MNPs) Antibodies coupled to fluorescent magnetic microspheres allowed the multiplex toxin detection of botulinum neurotoxins type A and B and Staphylococcal Enterotoxin B (SEB) and other toxins in a suspension assay [54]. Besides the application of MNPs for sample separation, biological imaging, drug delivery, magnetic sensors and conjugation with enzymes for signal simplification [46,55], Fe3O4 MNPs exhibit intrinsic peroxidase-like activity, which was discovered in 2007 [56]. The enzymatic activity, similar to natural peroxidases, was used to develop novel immunoassays with antibody-modified MNPs for the detection of protein targets in both direct and sandwich formats, integrating three functions (capture, separation and detection). Compared to natural enzymes, such as HRP, MNPs are more stable, easy-to-produce, multi-functional and inexpensive. The enzymatic activity of Fe3O4 MNPs has been used in immunoassays and PCR [57,58] and may also be used as an alternative label for the detection of bacterial toxins, and robust assays could be constructed utilizing multifunctional MNPs. 1333 3.4. Carbon Nanomaterials Carbon nanomaterials have attracted enormous interest in nanotechnology, covering a wide range of applications, from biosensing to drug delivery. Compared to metal-derived nanomaterials, such as AuNPs or cadmium-based fluorescent QDs, carbon nanomaterials show good biocompatibility and are environmental friendly [72]. The most common carbon nanomaterials include fullerenes, carbon nanotubes (CNTs), graphene, carbon dots, nanodiamonds and carbon nanofibers. The distinctive optical and electrical properties of carbon nanomaterials make them good candidates for analytical tasks. For example, different carbon-based nanomaterials have been used as adsorbents for sample preparation, particularly for the nonspecific detection of ions, small molecules and bacterial pathogens [73,74]. With the goal of the ultrasensitive detection of bacterial toxins, specific binding interactions based on CNTs and graphene functionalized by specific antibodies will be summarized here. According to their intrinsic structures, CNTs can be catalogued into single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs) [75]. CNTs have been directly used for the label-free detection of epsilon toxin from Clostridium perfringens with a detection limit of about 2 nM and the genomic DNA of Shiga-toxin from E. coli [76,77]. Furthermore, CNTs modified with plastic antibodies, lactose and peptides were developed for the detection of microcystins, CT and anthrax PA toxin, respectively [78–80]. An electrochemical immunoassay using antibodies immobilized on modified MWNTs was constructed for the sensitive detection of CT [81]. CT first binds to the antibody-coated CNTs and then forms a sandwich complex with cell membrane ligand ganglioside (GM1) functionalized liposomes, from which an electroactive marker is released after treatment with Triton X-100. This assay provides a platform for the ultratrace level detection of CT in a range from 1 × 10−14 g mL−1 to 1 × 10−7 g mL−1. In addition, a rapid, simple and sensitive electrical sensor has been proposed using paper impregnated with SWNTs and antibodies against microcystin LR [82]. This assay relies on the formation of antibody-microcystin LR complexes between CNTs, forming a dense percolation network exhibiting a change in conductivity depending on the presence of the analyte. The performance of this rapid assay is similar to that of ELISA, with a detection limit of 0.6 ng mL−1. It should be mentioned here that, similar to the catalytic activity of MNPs, both CNTs and carboxyl-modified graphene oxides possess intrinsic peroxidase-like activity [75], although there is no report on the analysis of bacterial toxins based on this phenomenon. Toxins 2014, 6 Toxins 2014, 6 versatile applications. It can be envisioned that these new developments could become useful for the detection of bacterial toxins in the near future. 3.3. Quantum Dots (QDs) Later, a waveguide-based immunosensor platform was developed for the analysis of a PA and lethal factor from Bacillus anthracis using QDs as the fluorescent reporters [69]. This assay incorporates multichannel waveguides, enabling the simultaneous detection of the PA (QDs, 605 nm) and lethal factor (QDs, 655 nm) together with an internal standard (QDs, 565 nm) implemented for the optimization of the assay variability. As low as 1 pM of PA and lethal factor, respectively, could be detected in sera. Progress in surface chemistry has further broadened the spectrum of applications of QDs for bioanalytical purposes. For instance, a new series of zwitterionic ligands with enhanced affinity to the surface of CdSe-ZnS QD and the ability to increase water solubility has been synthesized [70]. Such functionalized QDs retain their optical properties and possess a remarkable stability under harsh conditions. Furthermore, a new method has been reported to produce monovalent QDs by “steric exclusion”. A polymer of phosphorothioate DNA with a defined sequence and length is used to treat commercially available CdSe-ZnS QDs [71]. After passivation by polyethylene glycol (PEG) ligands, the phosphorothioate DNA-wrapped QDs show excellent colloidal and optical properties for 1334 4. Micro Total Analysis Systems (μTAS) μTAS, known as lab(oratories)-on-a-chip (LOC) and microfluidic devices, have attracted a lot of attention. These systems integrate chemical and biological labs onto miniaturized chips of the centimeter-scale. The integrated chips can perform sophisticated functions, such as sample separation, signal amplification and detection, to produce “sample-in and answer-out” systems [88]. Excellent recent reviews were focused on design and fabrication of chips [88], as well as the application in immunoassays for point of care (POC) diagnostics [89]. Furthermore, one review summarized the detection of pathogens, including viruses, bacteria and bacterial toxins, using LOC devices [90]. In this section, we will focus on recent achievements in antibody-based microfluidic systems and highlight the combination with nanomaterials for the detection of bacterial toxins. Generally, the assays performed in microfluidic platforms may be categorized into: (i) chips designed for the miniaturization of traditional assays, e.g., ELISA [91,92]; and (ii) chips integrating sample pretreatment, signal amplification and readout techniques [93–97]. For example, sandwich and competitive ELISAs were combined with a microfluidic device for single-cell studies to reliably identify intracellular proteins and metabolites [98]. This platform contains all steps for single-cell analysis from cell lysis to ELISA, including incubation periods, repeated washing steps and fluorescent readouts. Another advantage is that the integration of analyte preconcentration on chips can significantly improve detection limits and improve signal-to-noise ratios. For example, a microfluidic ELISA employed a semipermeable membrane for the preconcentration of the target analyte by electrokinetic means [99]. The local concentration of the analyte in the vicinity of the membrane resulted in a 200-fold enhanced ELISA signal, whereas the background signal increased only two-fold. Furthermore, only 5 μL of the sample was needed compared to 100 μL in ELISA. In the following paragraphs, we will introduce some examples of μTAS used for single and multiplex detection of bacterial toxins. An eight channel chip and an SWNT-based immunoassay were combined for the detection of SEB [100]. Rabbit anti-SEB antibodies are immobilized on SWNTs. The signal generated by enhanced chemiluminescence enabled the detection of 0.1 ng mL−1 of SEB in a 10-μL sample. Later, this microfluidic platform was improved for the label-free detection of SEB and termed “biological semiconductor (BSC)” [101]. A label-free impedimetric immunosensor combined with a microfluidic chip has been constructed for the B subunit of CT [102]. Toxins 2014, 6 Toxins 2014, 6 help of broad-spectrum antibodies for microcystins, 0.5 μg L−1 and 0.3 μg L−1 of microcystin-LR and microcystin-RR, respectively, could be detected. Furthermore, GOs are widely used as fluorescent quenchers to construct different platforms for biosensing. A FRET system that uses an antibody-CdSe/ZnS QD microarray for the detection of E. coli O157:H7 has been reported, with QDs as acceptors of energy transfer [45]. GO interacts with QDs through π–π stacking and quenches the fluorescence in the absence of bacteria, while QDs turn on the fluorescence in the presence of bacteria (Figure 3D). Moreover, GO has been used as a scaffold for the enhanced loading of antibodies, enzymes or other biomolecules onto its large surface. For example, for the detection of Clostridium difficile toxin B, multiple HRP and HRP-secondary antibodies were conjugated to GO to amplify the test signal, allowing the detection of 0.7 pg toxin mL−1 [87]. 3.4. Carbon Nanomaterials Since the first report on electronic properties of graphene in 2004, a variety of applications have been published [73]. For instance, graphene and chitosan were immobilized on electrodes for the detection of microcystin LR, and antibody-carbon nanosphere-HRP conjugates were used for signal amplification [83]. This approach provides a detection limit of 0.016 μg L−1 of microcystin LR in environmental water samples. Graphene oxide (GO), a promising material with superior properties, has been widely used for biomedical applications and analytical purposes [73,75,84,85]. During the process of preparation, GO gains fluorescent properties, a phenomenon that has been directly used to measure microcystins. Antibodies adsorbed on GO sheets specifically capture microcystins attached on AuNPs, which quench the fluorescence of GO by FRET between GO and AuNPs [86]. With the 1335 Toxins 2014, 6 Toxins 2014, 6 Figure 4. (A) (a) Biochip layout; (b) scheme of electrode functionalization; and (c) Nyquist spectra of different cholera toxin (CT) concentrations. (B) (a) Scheme of the competitive immunoassay in the immune-reaction columns; and (b) an illustration of the chip operations for the immunoassay. Reproduced from [102,103] with permission from The Royal Society of Chemistry. Figure 4. (A) (a) Biochip layout; (b) scheme of electrode functionalization; and (c) Nyquist spectra of different cholera toxin (CT) concentrations. (B) (a) Scheme of the competitive immunoassay in the immune-reaction columns; and (b) an illustration of the chip operations for the immunoassay. Reproduced from [102,103] with permission from The Royal Society of Chemistry. For multiplex bacterial toxin detection [24], an antibody-based microarray chip has been proposed for electrochemical detection of Yersinia pestis, Bacillus anthracis and SEB using a super avidin-biotin system [104]. In a new assay for distinguishing and quantifying botulinum neurotoxin type A (BoNT/A), SEB and the plant toxin, ricin, capture antibodies are covalently immunomobilized (printed) by a non-contact microdispensing array printer on a microstructured polymer slide serving also as an incubation chamber. Toxins are detected by biotinylated antibodies and Cy5-labeled streptavidin as the fluorescent probe [105]. Under optimized conditions, 0.5–1.0 ng mL−1 of toxins could be detected in raw milk samples. From the general layout, this chip can be used for the simultaneous detection of up to 28 analytes with six replicates. Another broadly applicable system, an immuno-column microfluidic chip, has been designed and fabricated for bacterial and algal toxin analysis, including microcystin-LR, saxitoxin and cylindrospermopsin [103]. The core principle of this new chip is the competitive immunoassay format applied in seven immuno-columns. Each column is filled with Protein A coated microspheres as the general binding support of the specific antibodies. Different primary anti-toxin antibodies are bound to the surface of the microspheres, and free (sample) toxins and HRP-labeled toxins compete for the antibody binding sites (Figure 4B). The linear range and limit of detection are 0–5.0 ng mL−1 and 0.02 ng mL−1, respectively. Recently, immunomagnetic separation in a microfluidic system and a microflow cytometer have been integrated for the automated and multiplex analysis of pathogenic bacteria [106]. Because this system is of general applicability, we present a short description. 4. Micro Total Analysis Systems (μTAS) The increase of impedance is directly related to the bound toxin on the surface electrode, and as low as 1 ng mL−1 of toxin can be detected (Figure 4A). 1336 5. New Materials and Methods This section will be focused on new materials and methods for signal (readout) generation, the improvement of assay performance and statistics. To increase the sensitivity of immunoassays and to reduce sample handling and processing steps is still a big challenge, and a variety of new materials and assays have been tried for their suitability to fulfill these requirements. New nanomaterials, such as antibody-coated microspheres, have been used for the simultaneous detection of CT, SEB and other target analytes in spiked clinical samples by a microflow cytometer [107,108]. Antibodies conjugated to silica-based nanomaterials, such as fluorescent silica NPs and mesoporous silica, have been broadly applied for the detection of bacterial targets and for the enhanced loading of enzymes or other probes [55,109,110]. It should be emphasized that although the different nanomaterials are discussed separately in this review, the integration of two or more kinds of nanomaterials in one assay is becoming more and more popular, due to their complementary properties [111,112]. The pursuit of new fluorescent probes for potential applications in biological fields has created a series of enhanced materials. Among these, aggregation-induced emission (AIE) fluorescent probes have been developed for the detection of small molecules, DNA and proteins [113,114]. The AIE phenomenon shows fluorescence turn-on responses in the aggregate state in contrast to the common aggregation-caused quenching (ACQ) of fluorescent probes and offers higher sensitivity and accuracy [114]. The combination of AIE probes with nanomaterials, such as GO, and mesoporous materials show excellent performance for sensing specific targets [115,116]. For example, AIE-based aptasensors using aptamers as recognition elements and GO as efficiently adsorptive platforms have been constructed for the detection of targeted DNA and thrombin [115] (Figure 5A). In the presence of complementary single-stranded DNA (ssDNA), the formation of double-stranded DNA from the ssDNA aptamers will result in a reduced FRET effect between GO and the AIE probe-ssDNA aptamer. Therefore, the fluorescence of the AIE probe bound to dsDNA will be enhanced gradually. Similarly, conjugation of AIE probes with specific antibodies and various nanomaterials can offer new opportunities for sensitive and selective immunoassays to detect different bacterial toxins. Metal-organic frameworks (MOFs), a class of microporous materials, offer unique properties, such as a high loading capacity, structural and chemical diversity and biodegradability, and have been used as molecular recognition elements for diverse analytical applications [117]. Toxins 2014, 6 Toxins 2014, 6 coupled to the surface of supraparamagnetic beads, samples, biotinylated antibodies and streptavidin labeled with phycoerythrin are pumped sequentially through and concentrated in microchannels. The fluorescence signal is generated by combinations of streptavidin phycoerythrin with two fluorophores. This automated “sample-in and answer-out” operation can be performed in less than 20 min. Another advantage of this platform is that it possesses the potential to concentrate targets from large volumes, which could improve the detection of low concentrations of toxin. Toxins 2014, 6 Different antibodies For multiplex bacterial toxin detection [24] an antibody-based microarray chip has been For multiplex bacterial toxin detection [24], an antibody-based microarray chip For multiplex bacterial toxin detection [24], an antibody-based microarray chip has been proposed for electrochemical detection of Yersinia pestis, Bacillus anthracis and SEB using a super avidin-biotin system [104]. In a new assay for distinguishing and quantifying botulinum neurotoxin type A (BoNT/A), SEB and the plant toxin, ricin, capture antibodies are covalently immunomobilized (printed) by a non-contact microdispensing array printer on a microstructured polymer slide serving also as an incubation chamber. Toxins are detected by biotinylated antibodies and Cy5-labeled streptavidin as the fluorescent probe [105]. Under optimized conditions, 0.5–1.0 ng mL−1 of toxins could be detected in raw milk samples. From the general layout, this chip can be used for the simultaneous detection of up to 28 analytes with six replicates. Another broadly applicable system, an immuno-column microfluidic chip, has been designed and fabricated for bacterial and algal toxin analysis, including microcystin-LR, saxitoxin and cylindrospermopsin [103]. The core principle of this new chip is the competitive immunoassay format applied in seven immuno-columns. Each column is filled with Protein A coated microspheres as the general binding support of the specific antibodies. Different primary anti-toxin antibodies are bound to the surface of the microspheres, and free (sample) toxins and HRP-labeled toxins compete for the antibody binding sites (Figure 4B). The linear range and limit of detection are 0–5.0 ng mL−1 and 0.02 ng mL−1, respectively. Recently, immunomagnetic separation in a microfluidic system and a microflow cytometer have been integrated for the automated and multiplex analysis of pathogenic bacteria [106]. Because this system is of general applicability, we present a short description. Different antibodies 1337 Toxins 2014, 6 Toxins 2014, 6 Figure 5. (A) (a) Synthesis of a novel aggregation-induced emission (AIE) probe (9,10-distyrylanthracene with two ammonium group, DSAI); and (b) a schematic description of a selective fluorescent aptasensor based on the DSAI/GO probe. Reprinted with permission from [115]. Copyright 2014 American Chemical Society; (B) Scheme of the key steps in the digital microfluidic (DMF) electroimmunoassay. Reproduced from [119] with permission from The Royal Society of Chemistry; (C) The immunochromatographic assay on a thread device assembly and assay protocol. Reprinted with permission from [120]. Copyright 2012 American Chemical Society; (D) The main types of assays for the detection of bacterial toxins in microtiter plates. Reproduced from [121] with permission from The Royal Society of Chemistry. ELISA for a single analyte; parallel detection of multiple analytes with multiple outputs; a single output for the detection of multiple analytes. Compared to immunoassays performed in microfluidic channels, digital microfluidic (DMF) systems are growing in popularity, as these approaches offer alternative open surface droplet-based fluid handling formats. In DMF systems, fluidics is electrostatically controlled in discrete droplets, in the picoliter to microliter range, on an open array of hydrophobic insulator-coated electrodes [122,123]. Separated droplets can be manipulated to mix, split and dispense from different reservoirs, eliminating the need of an oil carrier fluid [122]. These advantages make DMF suitable for both competitive and noncompetitive immunoassays. An automated DMF system was constructed to complete an immunoassay from sample application to optical readout with minimal manual intervention [123]. A three-level factorial design of experiment (DOE) was implemented to optimize the concentration and volume of the sample and incubation time for increased sensitivity in a sandwich assay. Meanwhile, a compact DMF platform integrating immunoassay and electrochemical detection has been developed [119]. Primary antibody conjugated magnetic microparticles are used for target capture, and HRP-labeled secondary antibodies catalyze the substrate for amperometrical measurement (Figure 5B). Compared to immunoassays performed in microfluidic channels, digital microfluidic (DMF) systems are growing in popularity, as these approaches offer alternative open surface droplet-based fluid handling formats. In DMF systems, fluidics is electrostatically controlled in discrete droplets, in the picoliter to microliter range, on an open array of hydrophobic insulator-coated electrodes [122,123]. Separated droplets can be manipulated to mix, split and dispense from different reservoirs, eliminating the need of an oil carrier fluid [122]. These advantages make DMF suitable for both competitive and noncompetitive immunoassays. 5. New Materials and Methods Particularly, the development of nanoscale MOFs (NMOFs) exhibits enormous potential, such as serving as nanocarriers for biomedical imaging and drug delivery [118]. We strongly believe that MOFs or NMOFs functionalized with antibodies will significantly improve sample enrichment and may even facilitate the development of more efficient assay systems. 1338 Toxins 2014, 6 Toxins 2014, 6 μPADs offer several advantages over the widely-used polymer polydimethylsiloxane (PDMS) or poly(methyl methacrylate) (PMMA)-based sticks [125]. They can be easily designed and fabricated by using hydrophobic barriers (wax or air) and are compatible with hydrophilic solutions and small volumes of sample. μPADs provide assay times of a few minutes and require nearly no external equipment. These advantages make μPADs suitable for point-of-care testings (POCTs) and applications in resource-poor situations. In addition, other easily available materials, such as thread and polypropylene, have been used as promising supports for immunoassays to detect multiple proteins [120,126]. The sandwich immunochromatographic assay on thread emulates the principle of a lateral flow chromatographic immunoassay for a single target [120], as shown in the scheme of Figure 5C. The detection limit is in the picomolar range, and the assay performance is similar to that of paper-based and conventional assays. Furthermore, this approach has been applied for the simultaneous detection of three target proteins using three knotted threads coated with various antibodies of different specificities [120]. A promising approach in label-free technologies is the introduction of organic field-effect transistors (OFET), which enable sensitive and selective electronic applications particularly suited for POCTs [127]. OFET sensors are mostly constructed by coating aromatic or otherwise π-electron conjugated organic semiconductors with biological recognition elements, such as antibodies or DNA. One example for the detection of pathogenic E. coli [77] has already been mentioned. Another sensitive and selective OFET immunosensor was developed by directly monitoring the current or conductance changes in OFET to detect E. coli at concentrations as low as 10 CFU mL−1 [128]. In our opinion, systems integrating MOF for sample separation and OFET as the readout technique have the potential to speed up the development of sensitive and miniaturized biosensors for the simultaneous detection of different bacterial toxins. One attractive trend in bacterial toxin analysis is the development of multiplexed analytical methods, for which some examples have been presented. These assays can be categorized into the following groups: (1) spectrally resolved assays, such as immunoassays utilizing different QDs or enzymes generating distinguishable signals; (2) spatially resolved assays, such as SPR and microarrays; (3) temporally resolved assays, such as HPLC; and (4) molecule resolved assays, such as MALDI-TOF mass spectrometry. Toxins 2014, 6 The mathematical theories behind these assays fall into the one-to-one model (M = N), where M means the multiplexing factor and N means the number of targets, as shown in Figure 5D. However, recently, Boolean logic has been introduced for bioanalytical applications. Multiple signals can be processed by Boolean logic biosensors, which contain networks of coupled parallel biochemical reactions, to generate simple “yes or no” answers [129]. As a recent example, an OR gate-based immunoassay was developed for the simultaneous detection of seven protein components of B. cereus toxins [120]. In addition, a novel mathematical theory has been proposed to encode and decode multiplexed assays, which enables a theoretically unlimited number of independent targets to be detected and identified in any combination in the same sample [130]. Toxins 2014, 6 An automated DMF system was constructed to complete an immunoassay from sample application to optical readout with minimal manual intervention [123]. A three-level factorial design of experiment (DOE) was implemented to optimize the concentration and volume of the sample and incubation time for increased sensitivity in a sandwich assay. Meanwhile, a compact DMF platform integrating immunoassay and electrochemical detection has been developed [119]. Primary antibody conjugated magnetic microparticles are used for target capture, and HRP-labeled secondary antibodies catalyze the substrate for amperometrical measurement (Figure 5B). Dipsticks and lateral flow chromatographic immunoassays, such as the pregnancy test, are well-known examples of paper-based diagnostic devices [124]. Recently, paper has been introduced as a substrate to construct microfluidic devices for rapid diagnostic tests, as “microfluidic paper analytical device (μPAD)”. 1339 Toxins 2014, 6 Recent progresses in antibody engineering will further support this progress and facilitate the integration of antibodies or antibody fragments into new assay formats provided by the recent achievements in materials science. Nanomaterials, such as AuNPs, MNPs, QDs, carbon nanomaterials and MOFs, have the potential to improve sample separation, target recognition and signal amplification. The integration of these new materials into conventional immunoassays represents a promising, alternative solution to improve analytical performance. Furthermore, μTAS and microfluidic devices perform sophisticated functions, such as sample separation, signal amplification and detection, and enable “sample-in and answer-out” platforms at the centimeter scale. With these recent advances, all the tools are at hand to realize more robust, rapid and easy to operate immunoassays for the sensitive, high-throughput detection of single and multiple bacterial toxins in the near future. 6. Conclusions The detection of bacterial toxins by antibodies has been a successful approach for decades. Future assays can hardly be imagined without the use of antibodies as recognition elements. 1340 Acknowledgments We have tried our best to identify and report the most interesting publications in this rapidly developing analytical field. Nevertheless, it was not possible to include all the excellent papers published during the last few years, and we apologize for that. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. Toxins 2014, 6 Toxins 2014, 6 10. Stark, T.; Marxen, S.; Rütschle, A.; Lücking, G.; Scherer, S.; Ehling-Schulz, M.; Hofmann, T. Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay. Anal. Bioanal. Chem. 2013, 405, 191–201. 11. Tsilia, V.; Devreese, B.; Baenst, I.; Mesuere, B.; Rajkovic, A.; Uyttendaele, M.; Wiele, T.; Heyndrickx, M. Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group. Anal. Bioanal. Chem. 2012, 404, 1691–1702. 12. Alam, S.I.; Kumar, B.; Kamboj, D.V. Multiplex detection of protein toxins using MALDI-TOF-TOF tandem mass spectrometry: Application in unambiguous toxin detection from bioaerosol. Anal. Chem. 2012, 84, 10500–10507. 13. Byrne, B.; Stack, E.; Gilmartin, N.; O’Kennedy, R. Antibody-based sensors: Principles, problems and potential for detection of pathogens and associated toxins. Sensors 2009, 9, 4407–4445. 14. Akiyoshi, D.E.; Sheoran, A.S.; Rich, C.M.; Richard, L.; Chapman-Bonofiglio, S.; Tzipori, S. Evaluation of Fab and F(ab')2 fragments and isotype variants of a recombinant human monoclonal antibody against Shiga toxin 2. Infect. Immun. 2010, 78, 1376–1382. 15. Sharma, H.; Mutharasan, R. Half antibody fragments improve biosensor sensitivity without loss of selectivity. Anal. Chem. 2013, 85, 2472–2477. 16. Pleckaityte, M.; Mistiniene, E.; Lasickiene, R.; Zvirblis, G.; Zvirbliene, A. Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis. BMC Biotechnol. 2011, 11, doi:10.1186/1472-6750-11-100. 17. Wang, Y.; Tang, L.J.; Jiang, J.H. Surface-enhanced Raman spectroscopy-based, homogeneous, multiplexed immunoassay with antibody-fragments-decorated gold nanoparticles. Anal. Chem. 2013, 85, 9213–9220. 18. Spiess, C.; Merchant, M.; Huang, A.; Zheng, Z.; Yang, N.Y.; Peng, J.; Ellerman, D.; Shatz, W.; Reilly, D.; Yansura, D.G.; et al. Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies. Nat. Biotechnol. 2013, 31, 753–758. 19. Lewis, S.M.; Wu, X.; Pustilnik, A.; Sereno, A.; Huang, F.; Rick, H.L.; Guntas, G.; Leaver-Fay, A.; Smith, E.M.; Ho, C.; et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 2014, 32, 191–198. 20. Muyldermans, S. Nanobodies: Natural single-domain antibodies. Annu. Rev. Biochem. 2013, 82, 775–797. 21. Slocik, J.M.; Kim, S.N.; Auvil, T.; Goldman, E.R.; Liu, J.; Naik, R.R. Single domain antibody templated nanoparticle resistors for sensing. Biosens. Bioelectron. 2010, 25, 1908–1913. 22. References 1. Argudin, M.A.; Mendoza, M.C.; Rodicio, M.R. Food poisoning and Staphylococcus aureus enterotoxins. Toxins 2010, 2, 1751–1773. 2. Senesi, S.; Ghelardi, E. Production, secretion and biological activity of Bacillus cereus enterotoxins. Toxins 2010, 2, 1690–1703. 3. Stenfors Arnesen, L.P.; Fagerlund, A.; Granum, P.E. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Rev. 2008, 32, 579–606. 4. Popoff, M.R.; Poulain, B. Bacterial toxins and the nervous system: Neurotoxins and multipotential toxins interacting with neuronal cells. Toxins 2010, 2, 683–737. 5. Popoff, M.R. Bacterial Exotoxins. Contrib. Microbiol. 2005, 12, 28–54. 6. Alouf, J.E. Molecular features of the cytolytic pore-forming bacterial protein toxins. Folia Microbiol. 2003, 48, 5–16. 6. Alouf, J.E. Molecular features of the cytolytic pore-forming bacterial protein toxins. Folia Microbiol. 2003, 48, 5–16. 7. Iacovache, I.; Bischofberger, M.; van der Goot, F.G. Structure and assembly of pore-forming proteins. Curr. Opin. Struct. Biol. 2010, 20, 241–246. 7. Iacovache, I.; Bischofberger, M.; van der Goot, F.G. Structure and assembly of pore-forming proteins. Curr. Opin. Struct. Biol. 2010, 20, 241–246. 8. Odumosu, O.; Nicholas, D.; Yano, H.; Langridge, W. AB toxins: A paradigm switch from deadly to desirable. Toxins 2010, 2, 1612–1645. 9. Ivarsson, M.E.; Leroux, J.C.; Castagner, B. Targeting bacterial toxins. Angew. Chem. Int. Ed. 2012, 51, 4024–4045. 1341 Toxins 2014, 6 Toxins 2014, 6 24. Shlyapnikov, Y.M.; Shlyapnikova, E.A.; Simonova, M.A.; Shepelyakovskaya, A.O.; Brovko, F.A.; Komaleva, R.L.; Grishin, E.V.; Morozov, V.N. Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins. Anal. Chem. 2012, 84, 5596–5603. 25. He, X.; Qi, W.; Quinones, B.; McMahon, S.; Cooley, M.; Mandrell, R.E. Sensitive detection of Shiga Toxin 2 and some of its variants in environmental samples by a novel immuno-PCR assay. Appl. Environ. Microbiol. 2011, 77, 3558–3564. 26. Zhang, H.; Xu, Y.; Huang, Q.; Yi, C.; Xiao, T.; Li, Q. Natural phage nanoparticle-mediated real-time immuno-PCR for ultrasensitive detection of protein marker. Chem. Commun. 2013, 49, 3778–3780. 27. Broyer, R.M.; Grover, G.N.; Maynard, H.D. Emerging synthetic approaches for protein-polymer conjugations. Chem. Commun. 2011, 47, 2212–2226. 28. Zhu, K.; Zhang, Y.; He, S.; Chen, W.; Shen, J.; Wang, Z.; Jiang, X. Quantification of proteins by functionalized gold nanoparticles using click chemistry. Anal. Chem. 2012, 84, 4267–4270. 29. Zhang, H.; Feng, G.; Guo, Y.; Zhou, D. Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor. Nanoscale 2013, 5, 10307–10315. 30. Liu, Y.; Dong, Y.; Jauw, J.; Linman, M.J.; Cheng, Q. Highly sensitive detection of protein toxins by surface plasmon resonance with biotinylation-based inline atom transfer radical polymerization amplification. Anal. Chem. 2010, 82, 3679–3685. 31. El Ichi, S.; Leon, F.; Vossier, L.; Marchandin, H.; Errachid, A.; Coste, J.; Jaffrezic-Renault, N.; Fournier-Wirth, C. Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria. Biosens. Bioelectron. 2014, 54, 378–384. 32. Loyprasert, S.; Hedstrom, M.; Thavarungkul, P.; Kanatharana, P.; Mattiasson, B. Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensor. Biosens. Bioelectron. 2010, 25, 1977–1983. 33. Alexandridis, P. Gold nanoparticle synthesis, morphology control, and stabilization facilitated by functional polymers. Chem. Eng. Technol. 2011, 34, 15–28. 34. Jiang, S.; Win, K.Y.; Liu, S.; Teng, C.P.; Zheng, Y.; Han, M.Y. Surface-functionalized nanoparticles for biosensing and imaging-guided therapeutics. Nanoscale 2013, 5, 3127–3148. 35. Tiwari, P.; Vig, K.; Dennis, V.; Singh, S. Functionalized gold nanoparticles and their biomedical applications. Nanomaterials 2011, 1, 31–63. 36. Ching, K.H.; Lin, A.; McGarvey, J.A.; Stanker, L.H.; Hnasko, R. Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip. J. Immunol. Methods 2012, 380, 23–29. 37. Willets, K.A.; Van Duyne, R.P. Localized surface plasmon resonance spectroscopy and sensing. Annu. Rev. Phys. Chem. 2007, 58, 267–297. 38. Schofield, C.L.; Field, R.A.; Russell, D.A. Glyconanoparticles for the colorimetric detection of cholera toxin. Anal. Toxins 2014, 6 Wesolowski, J.; Alzogaray, V.; Reyelt, J.; Unger, M.; Juarez, K.; Urrutia, M.; Cauerhff, A.; Danquah, W.; Rissiek, B.; Scheuplein, F.; et al. Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009, 198, 157–174. 23. Skinner, C.; Patfield, S.; Stanker, L.; He, X. Development of monoclonal antibodies and immunoassays for sensitive and specific detection of Shiga toxin Stx2f. PLoS One 2013, 8, doi:10.1371/journal.pone.0076563. 1342 Toxins 2014, 6 Toxins 2014, 6 41. Farrow, B.; Hong, S.A.; Romero, E.C.; Lai, B.; Coppock, M.B.; Deyle, K.M.; Finch, A.S.; Stratis-Cullum, D.N.; Agnew, H.D.; Yang, S.; et al. A chemically synthesized capture agent enables the selective, sensitive, and robust electrochemical detection of anthrax protective antigen. ACS Nano 2013, 7, 9452–9460. 41. Farrow, B.; Hong, S.A.; Romero, E.C.; Lai, B.; Coppock, M.B.; Deyle, K.M.; Finch, A.S.; Stratis-Cullum, D.N.; Agnew, H.D.; Yang, S.; et al. A chemically synthesized capture agent enables the selective, sensitive, and robust electrochemical detection of anthrax protective antigen. ACS Nano 2013, 7, 9452–9460. 42. Tseng, Y.T.; Chang, H.Y.; Huang, C.C. A mass spectrometry-based immunosensor for bacteria using antibody-conjugated gold nanoparticles. Chem. Commun. 2012, 48, 8712–8714. 43. Kang, X.; Pang, G.; Chen, Q.; Liang, X. Fabrication of Bacillus cereus electrochemical immunosensor based on double-layer gold nanoparticles and chitosan. Sens. Actuators B 2013, 177, 1010–1016. 44. Feng, L.; Zhu, A.; Wang, H.; Shi, H. A nanosensor based on quantum-dot haptens for rapid, on-site immunoassay of cyanotoxin in environmental water. Biosens. Bioelectron. 2014, 53, 1–4. 44. Feng, L.; Zhu, A.; Wang, H.; Shi, H. A nanosensor based on quantum-dot haptens for rapid, on-site immunoassay of cyanotoxin in environmental water. Biosens. Bioelectron. 2014, 53, 1–4. 45. Morales-Narvaez, E.; Hassan, A.R.; Merkoci, A. Graphene oxide as a pathogen-revealing agent: 45. Morales-Narvaez, E.; Hassan, A.R.; Merkoci, A. Graphene oxide as a pathogen-revealing agent: Sensing with a digital-like response. Angew. Chem. Int. Ed. 2013, 52, 13779–13783. 46. Guo, J.; Yang, W.; Wang, C. Magnetic colloidal supraparticles: Design, fabrication and biomedical applications. Adv. Mater. 2013, 25, 5196–5214. 47. Sung, Y.J.; Suk, H.J.; Sung, H.Y.; Li, T.; Poo, H.; Kim, M.G. Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk. Biosens. Bioelectron. 2013, 43, 432–439. 48. Clotilde, L.M.; Bernard, C.; Salvador, A.; Lin, A.; Lauzon, C.R.; Muldoon, M.; Xu, Y.; Lindpaintner, K.; Carter, J.M. A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli. J. Microbiol. Methods 2013, 92, 226–230. 49. Huang, Y.-F.; Wang, Y.-F.; Yan, X.-P. Amine-functionalized magnetic nanoparticles for rapid capture and removal of bacterial pathogens. Environ. Sci. Technol. 2010, 44, 7908–7913. 50. Afonso, A.S.; Perez-Lopez, B.; Faria, R.C.; Mattoso, L.H.; Hernandez-Herrero, M.; Roig-Sagues, A.X.; Maltez-da Costa, M.; Merkoci, A. Electrochemical detection of Salmonella using gold nanoparticles. Biosens. Bioelectron. 2013, 40, 121–126. 51. Chen, L.; Razavi, F.S.; Mumin, A.; Guo, X.; Sham, T.-K.; Zhang, J. 55. Syed, M.A. Advances in nanodiagnostic techniques for microbial agents. Biosens. Bioelectron. 2014, 51, 391–400. Toxins 2014, 6 Chem. 2007, 79, 1356–1361. 39. Wu, X.; Xu, L.; Liu, L.; Ma, W.; Yin, H.; Kuang, H.; Wang, L.; Xu, C.; Kotov, N.A. Unexpected chirality of nanoparticle dimers and ultrasensitive chiroplasmonic bioanalysis. J. Am. Chem. Soc. 2013, 135, 18629–18636. 40. De la Rica, R.; Stevens, M.M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat. Nanotechnol. 2012, 7, 821–824. 1343 Toxins 2014, 6 Toxins 2014, 6 Toxins 2014, 6 56. Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577–583. 57. Woo, M.A.; Kim, M.I.; Jung, J.H.; Park, K.S.; Seo, T.S.; Park, H.G. A novel colorimetric immunoassay utilizing the peroxidase mimicking activity of magnetic nanoparticles. Int. J. Mol. Sci. 2013, 14, 9999–10014. 58. Thiramanas, R.; Jangpatarapongsa, K.; Tangboriboonrat, P.; Polpanich, D. Detection of Vibrio cholerae using the intrinsic catalytic activity of a magnetic polymeric nanoparticle. Anal. Chem. 2013, 85, 5996–6002. 59. Chan, W.C.; Nie, S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281, 2016–2018. 60. Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A.P. Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281, 2013–2016. 61. Algar, W.R.; Tavares, A.J.; Krull, U.J. Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction. Anal. Chim. Acta 2010, 673, 1–25. 62. Vinayaka, A.C.; Thakur, M.S. Focus on quantum dots as potential fluorescent probes for monitoring food toxicants and foodborne pathogens. Anal. Bioanal. Chem. 2010, 397, 1445–1455. 63. Warner, M.G.; Grate, J.W.; Tyler, A.; Ozanich, R.M.; Miller, K.D.; Lou, J.; Marks, J.D.; Bruckner-Lea, C.J. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies. Biosens. Bioelectron. 2009, 25, 179–184. 64. Esteve-Turrillas, F.A.; Abad-Fuentes, A. Applications of quantum dots as probes in immunosensing of small-sized analytes. Biosens. Bioelectron. 2013, 41, 12–29. 65. Zhou, X.; Meng, Y.; Ma, H.; Tao, G. Method for determination of microcystin-leucine-arginine in water samples based on the quenching of the fluorescence of bioconjugates between CdSe/CdS quantum dots and microcystin-leucine-arginine antibody. Microchim. Acta 2011, 173, 259–266. 66. Kim, J.Y.; Voznyy, O.; Zhitomirsky, D.; Sargent, E.H. 25th anniversary article: Colloidal quantum dot materials and devices: A quarter-century of advances. Adv. Mater. 2013, 25, 4986–5010. 67. Zhu, K.; Li, J.; Wang, Z.; Jiang, H.; Beier, R.C.; Xu, F.; Shen, J.; Ding, S. Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay. Biosens. Bioelectron. 2011, 26, 2716–2719. 68. Goldman, E.R.; Clapp, A.R.; Anderson, G.P.; Uyeda, H.T.; Mauro, J.M.; Medintz, I.L.; Mattoussi, H. Multiplexed toxin analysis using four colors of quantum dot fluororeagents. Anal. Chem. 2004, 76, 684–688. 69. Toxins 2014, 6 Multifunctional nanoparticles for rapid bacterial capture, detection, and decontamination. RSC Adv. 2013, 3, 2390–2397. 52. Ge, S.; Liu, W.; Ge, L.; Yan, M.; Yan, J.; Huang, J.; Yu, J. In situ assembly of porous Au-paper electrode and functionalization of magnetic silica nanoparticles with HRP via click chemistry for Microcystin-LR immunoassay. Biosens. Bioelectron. 2013, 49, 111–117. 53. Orlov, A.V.; Khodakova, J.A.; Nikitin, M.P.; Shepelyakovskaya, A.O.; Brovko, F.A.; Laman, A.G.; Grishin, E.V.; Nikitin, P.I. Magnetic immunoassay for detection of staphylococcal toxins in complex media. Anal. Chem. 2013, 85, 1154–1163. 54. Pauly, D.; Kirchner, S.; Stoermann, B.; Schreiber, T.; Kaulfuss, S.; Schade, R.; Zbinden, R.; Avondet, M.A.; Dorner, M.B.; Dorner, B.G. Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay. Analyst 2009, 134, 2028–2039. 55. Syed, M.A. Advances in nanodiagnostic techniques for microbial agents. Biosens. Bioelectron. 2014, 51, 391–400. 1344 Toxins 2014, 6 Toxins 2014, 6 71. Farlow, J.; Seo, D.; Broaders, K.E.; Taylor, M.J.; Gartner, Z.J.; Jun, Y.W. Formation of targeted monovalent quantum dots by steric exclusion. Nat. Methods 2013, 10, 1203–1205. 72. Bhunia, S.K.; Saha, A.; Maity, A.R.; Ray, S.C.; Jana, N.R. Carbon nanoparticle-based fluorescent bioimaging probes. Sci. Rep. 2013, 3, doi:10.1038/srep01473. 73. Zhang, B.T.; Zheng, X.; Li, H.F.; Lin, J.M. Application of carbon-based nanomaterials in sample preparation: A review. Anal. Chim. Acta 2013, 784, 1–17. 74. Upadhyayula, V.K.K.; Deng, S.; Mitchell, M.C.; Smith, G.B. Application of carbon nanotube technology for removal of contaminants in drinking water: A review. Sci. Total Environ. 2009, 408, 1–13. 75. Wang, J.; Qu, X. Recent progress in nanosensors for sensitive detection of biomolecules. Nanoscale 2013, 5, 3589–3600. 76. Palaniappan, A.; Goh, W.H.; Fam, D.W.; Rajaseger, G.; Chan, C.E.; Hanson, B.J.; Moochhala, S.M.; Mhaisalkar, S.G.; Liedberg, B. Label-free electronic detection of bio-toxins using aligned carbon nanotubes. Biosens. Bioelectron. 2013, 43, 143–147. 77. Subramanian, S.; Aschenbach, K.H.; Evangelista, J.P.; Najjar, M.B.; Song, W.; Gomez, R.D. Rapid, sensitive and label-free detection of Shiga-toxin producing Escherichia coli O157 using carbon nanotube biosensors. Biosens. Bioelectron. 2012, 32, 69–75. 78. Queirós, R.B.; Guedes, A.; Marques, P.V.S.; Noronha, J.P.; Sales, M.G.F. Recycling old screen-printed electrodes with newly designed plastic antibodies on the wall of carbon nanotubes as sensory element for in situ detection of bacterial toxins in water. Sens. Actuators B 2013, 189, 21–29. 79. Kuramitz, H.; Miyagaki, S.; Ueno, E.; Hata, N.; Taguchi, S.; Sugawara, K. Binding assay for cholera toxin based on sequestration electrochemistry using lactose labeled with an electroactive compound. Analyst 2011, 136, 2373–2378. 80. Tran Ngoc, H.; Ganesh, T.; Han, S.-H.; Yoon, M.-Y.; Chung, H. Sensitive detection of an anthrax biomarker using a glassy carbon electrode with a consecutively immobilized layer of polyaniline/carbon nanotube/peptide. Biosens. Bioelectron. 2011, 26, 4227–4230. 81. Viswanathan, S.; Wu, L.C.; Huang, M.R.; Ho, J.A.A. Electrochemical immunosensor for cholera toxin using liposomes and poly(3,4-ethylenedioxythiophene)-coated carbon nanotubes. Anal. Chem. 2006, 78, 1115–1121. 82. Wang, L.; Chen, W.; Xu, D.; Shim, B.S.; Zhu, Y.; Sun, F.; Liu, L.; Peng, C.; Jin, Z.; Xu, C.; et al. Simple, rapid, sensitive, and versatile SWNT-paper sensor for environmental toxin detection competitive with ELISA. Nano Lett. 2009, 9, 4147–4152. 83. Zhao, H.; Tian, J.; Quan, X. A graphene and multienzyme functionalized carbon nanosphere-based electrochemical immunosensor for microcystin-LR detection. Colloids Surf. B Biointerfaces 2013, 103, 38–44. 84. Toxins 2014, 6 Mukundan, H.; Xie, H.; Price, D.; Kubicek-Sutherland, J.Z.; Grace, W.K.; Anderson, A.S.; Martinez, J.S.; Hartman, N.; Swanson, B.I. Quantitative multiplex detection of pathogen biomarkers on multichannel waveguides. Anal. Chem. 2010, 82, 136–144. 70. Zhan, N.; Palui, G.; Safi, M.; Ji, X.; Mattoussi, H. Multidentate zwitterionic ligands provide compact and highly biocompatible quantum dots. J. Am. Chem. Soc. 2013, 135, 13786–13795. 1345 85. Lin, H.; Huo, J.; Zhang, A.; Liu, Y.; Wang, Q.; Cai, Y.; Ying, W.; Qin, W.; Zhang, Y.; Qian, X. A sensitive dual signal amplification method for western blotting based on antibody-functionalised graphene oxide and gold nanoparticles. Analyst 2012, 137, 3620–3623. Toxins 2014, 6 1346 Toxins 2014, 6 86. Shi, Y.; Wu, J.; Sun, Y.; Zhang, Y.; Wen, Z.; Dai, H.; Wang, H.; Li, Z. A graphene oxide based biosensor for microcystins detection by fluorescence resonance energy transfer. Biosens. Bioelectron. 2012, 38, 31–36. 87. Fang, Y.S.; Chen, S.Y.; Huang, X.J.; Wang, L.S.; Wang, H.Y.; Wang, J.F. Simple approach for ultrasensitive electrochemical immunoassay of Clostridium difficile toxin B detection. Biosens. Bioelectron. 2014, 53, 238–244. 88. Culbertson, C.T.; Mickleburgh, T.G.; Stewart-James, S.A.; Sellens, K.A.; Pressnall, M. Micro total analysis systems: Fundamental advances and biological applications. Anal. Chem. 2014, 86, 95–118. 89. Han, K.N.; Li, C.A.; Seong, G.H. Microfluidic chips for immunoassays. Annu. Rev. Anal. Chem. 2013, 6, 119–141. 90. Foudeh, A.M.; Fatanat Didar, T.; Veres, T.; Tabrizian, M. Microfluidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics. Lab. Chip 2012, 12, 3249–3266. 91. Yoon, J.Y.; Kim, B. Lab-on-a-chip pathogen sensors for food safety. Sensors 2012, 12, 10713–10741. 92. Wang, T.; Zhang, M.; Dreher, D.D.; Zeng, Y. Ultrasensitive microfluidic solid-phase ELISA using an actuatable microwell-patterned PDMS chip. Lab. Chip 2013, 13, 4190–4197. 93. Jiang, X.; Jing, W.; Zheng, L.; Liu, S.; Wu, W.; Sui, G. A continuous-flow high-throughput microfluidic device for airborne bacteria PCR detection. Lab. Chip 2014, 14, 671–676. 94. Aizel, K.; Agache, V.; Pudda, C.; Bottausci, F.; Fraisseix, C.; Bruniaux, J.; Navarro, F.; Fouillet, Y. Enrichment of nanoparticles and bacteria using electroless and manual actuation modes of a bypass nanofluidic device. Lab. Chip 2013, 13, 4476–4485. 95. Ishikawa, T.; Shioiri, T.; Numayama-Tsuruta, K.; Ueno, H.; Imai, Y.; Yamaguchi, T. Separation of motile bacteria using drift velocity in a microchannel. Lab. Chip 2014, 14, 1023–1032. 96. Mach, A.J.; Kim, J.H.; Arshi, A.; Hur, S.C.; Di Carlo, D. Automated cellular sample preparation using a Centrifuge-on-a-Chip. Lab. Chip 2011, 11, 2827–2834. 97. Schumacher, S.; Nestler, J.; Otto, T.; Wegener, M.; Ehrentreich-Forster, E.; Michel, D.; Wunderlich, K.; Palzer, S.; Sohn, K.; Weber, A.; et al. Highly-integrated lab-on-chip system for point-of-care multiparameter analysis. Lab. Chip 2012, 12, 464–473. 98. Eyer, K.; Stratz, S.; Kuhn, P.; Küster, S.K.; Dittrich, P.S. Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells. Anal. Chem. 2013, 85, 3280–3287. 99. Yanagisawa, N.; Dutta, D. Enhancement in the sensitivity of microfluidic enzyme-linked immunosorbent assays through analyte preconcentration. Anal. Chem. 2012, 84, 7029–7036. 100. Yang, M.; Sun, S.; Kostov, Y.; Rasooly, A. Toxins 2014, 6 Pei, X.; Zhang, B.; Tang, J.; Liu, B.; Lai, W.; Tang, D. Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: A review. Anal. Chim. Acta 2013, 758, 1–18. 85. Lin, H.; Huo, J.; Zhang, A.; Liu, Y.; Wang, Q.; Cai, Y.; Ying, W.; Qin, W.; Zhang, Y.; Qian, X. A sensitive dual signal amplification method for western blotting based on antibody-functionalised graphene oxide and gold nanoparticles. Analyst 2012, 137, 3620–3623. Toxins 2014, 6 1347 Toxins 2014, 6 102. Chiriaco, M.S.; Primiceri, E.; D'Amone, E.; Ionescu, R.E.; Rinaldi, R.; Maruccio, G. EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection. Lab. Chip 2011, 11, 658–663. 102. Chiriaco, M.S.; Primiceri, E.; D'Amone, E.; Ionescu, R.E.; Rinaldi, R.; Maruccio, G. EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection. Lab. Chip 2011, 11, 658–663. 103. Zhang, J.; Liu, S.; Yang, P.; Sui, G. Rapid detection of algal toxins by microfluidic immunoassay. Lab. Chip 2011, 11, 3516–3522. 104. Wojciechowski, J.; Danley, D.; Cooper, J.; Yazvenko, N.; Taitt, C.R. Multiplexed electrochemical detection of Yersinia pestis and Staphylococcal Enterotoxin B using an antibody microarray. Sensors 2010, 10, 3351–3362. 105. Weingart, O.G.; Gao, H.; Crevoisier, F.; Heitger, F.; Avondet, M.A.; Sigrist, H. A bioanalytical platform for simultaneous detection and quantification of biological toxins. Sensors 2012, 12, 2324–2339. 106. Verbarg, J.; Plath, W.D.; Shriver-Lake, L.C.; Howell, P.B., Jr.; Erickson, J.S.; Golden, J.P.; Ligler, F.S. Catch and release: Integrated system for multiplexed detection of bacteria. Anal. Chem. 2013, 85, 4944–4950. 107. Kim, J.S.; Anderson, G.P.; Erickson, J.S.; Golden, J.P.; Nasir, M.; Ligler, F.S. Multiplexed detection of bacteria and toxins using a microflow cytometer. Anal. Chem. 2009, 81, 5426–5432. 108. Shriver-Lake, L.C.; Golden, J.; Bracaglia, L.; Ligler, F.S. Simultaneous assay for ten bacteria and toxins in spiked clinical samples using a microflow cytometer. Anal. Bioanal. Chem. 2013, 405, 5611–5614. 109. Zhang, B.; Liu, B.; Liao, J.; Chen, G.; Tang, D. Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers. Anal. Chem. 2013, 85, 9245–9252. 110. Eum, J.Y.; Hwang, S.Y.; Ju, Y.; Shim, J.M.; Piao, Y.; Lee, J.; Kim, H.-S.; Kim, J. A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes. Chem. Commun. 2014, 50, 3546–3548. 111. Liu, D.; Wang, Z.; Jin, A.; Huang, X.; Sun, X.; Wang, F.; Yan, Q.; Ge, S.; Xia, N.; Niu, G.; et al. Acetylcholinesterase-catalyzed hydrolysis allows ultrasensitive detection of pathogens with the naked eye. Angew. Chem. Int. Ed. 2013, 52, 14065–14069. 112. Wang, J.; Han, H.; Jiang, X.; Huang, L.; Chen, L.; Li, N. Quantum dot-based near-infrared electrochemiluminescent immunosensor with gold nanoparticle-graphene nanosheet hybrids and silica nanospheres double-assisted signal amplification. Anal. Chem. 2012, 84, 4893–4899. 113. Shi, H.; Liu, J.; Geng, J.; Tang, B.Z.; Liu, B. Specific detection of integrin αvβ3 by light-up bioprobe with aggregation-induced emission characteristics. J. Am. Chem. Soc. Toxins 2014, 6 Lab-on-a-Chip for carbon nanotubes based immunoassay detection of Staphylococcal Enterotoxin B (SEB). Lab. Chip 2010, 10, 1011–1017. 101. Yang, M.; Sun, S.; Bruck, H.A.; Kostov, Y.; Rasooly, A. Lab-on-a-chip for label free biological semiconductor analysis of Staphylococcal Enterotoxin B. Lab. Chip 2010, 10, 2534–2540. Toxins 2014, 6 2012, 134, 9569–9572. 114. Ding, D.; Li, K.; Liu, B.; Tang, B.Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 2013, 46, 2441–2453. 115. Li, X.; Ma, K.; Zhu, S.; Yao, S.; Liu, Z.; Xu, B.; Yang, B.; Tian, W. Fluorescent aptasensor based on aggregation-induced emission probe and graphene oxide. Anal. Chem. 2014, 86, 298–303. 116. Li, D.; Liu, J.; Kwok, R.T.; Liang, Z.; Tang, B.Z.; Yu, J. Supersensitive detection of explosives by recyclable AIE luminogen-functionalized mesoporous materials. Chem. Commun. 2012, 48, 7167–7169. 1348 Toxins 2014, 6 Toxins 2014, 6 117. Gu, Z.-Y.; Yang, C.-X.; Chang, N.; Yan, X.-P. Metal-organic frameworks for analytical chemistry: From sample collection to chromatographic separation. Acc. Chem. Res. 2012, 45, 734–745. 117. Gu, Z.-Y.; Yang, C.-X.; Chang, N.; Yan, X.-P. Metal-organic frameworks for analytical chemistry: From sample collection to chromatographic separation. Acc. Chem. Res. 2012, 45, 734–745. 118. Della Rocca, J.; Liu, D.; Lin, W. Nanoscale metal-organic frameworks for biomedical imaging and drug delivery. Acc. Chem. Res. 2011, 44, 957–968. 119. Shamsi, M.H.; Choi, K.; Ng, A.H.; Wheeler, A.R. A digital microfluidic electrochemical immunoassay. Lab. Chip 2014, 14, 547–554. 120. Zhou, G.; Mao, X.; Juncker, D. Immunochromatographic assay on thread. Anal. Chem. 2012, 84, 7736–7743. 121. Zhu, K.; Dietrich, R.; Didier, A.; Acar, G.; Maertlbauer, E. Versatile antibody-sensing Boolean logic for the simultaneous detection of multiple bacterial toxins. Chem. Commun. 2013, 49, 9314–9316. 122. Ng, A.H.; Choi, K.; Luoma, R.P.; Robinson, J.M.; Wheeler, A.R. Digital microfluidic magnetic separation for particle-based immunoassays. Anal. Chem. 2012, 84, 8805–8812. 123. Choi, K.; Ng, A.H.; Fobel, R.; Chang-Yen, D.A.; Yarnell, L.E.; Pearson, E.L.; Oleksak, C.M.; Fischer, A.T.; Luoma, R.P.; Robinson, J.M.; et al. Automated digital microfluidic platform for magnetic-particle-based immunoassays with optimization by design of experiments. Anal. Chem. 2013, 85, 9638–9646. 124. Parolo, C.; Merkoci, A. Paper-based nanobiosensors for diagnostics. Chem. Soc. Rev. 2013, 42, 450–457. 125. Yetisen, A.K.; Akram, M.S.; Lowe, C.R. Paper-based microfluidic point-of-care diagnostic devices. Lab. Chip 2013, 13, 2210–2251. 126. Parween, S.; Nahar, P. Image-based ELISA on an activated polypropylene microtest plate—A spectrophotometer-free low cost assay technique. Biosens. Bioelectron. 2013, 48, 287–292. 127. Torsi, L.; Magliulo, M.; Manoli, K.; Palazzo, G. Organic field-effect transistor sensors: A tutorial review. Chem. Soc. Rev. 2013, 42, 8612–8628. 128. Chang, J.; Mao, S.; Zhang, Y.; Cui, S.; Zhou, G.; Wu, X.; Yang, C.H.; Chen, J. Ultrasonic-assisted self-assembly of monolayer graphene oxide for rapid detection of Escherichia coli bacteria. Nanoscale 2013, 5, 3620–3626. 129. Katz, E.; Wang, J.; Privman, M.; Halámek, J. Multianalyte digital enzyme biosensors with built-in Boolean logic. Anal. Chem. 2012, 84, 5463–5469. 130. Rajagopal, A.; Scherer, A.; Homyk, A.; Kartalov, E. Supercolor coding methods for large-scale multiplexing of biochemical assays. Anal. Chem. 2013, 85, 7629–7636. © 2014 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 license (http://creativecommons.org/licenses/by/3.0/).
W2983953714.txt
https://orca.cardiff.ac.uk/140166/1/jcads_2_1_2019_jcads.25.pdf
en
Taxpaying, Importing, Enforcing: Emerging discourse patterns in online newspaper comments about US immigrant education
Journal of corpora and discourse studies
2,019
cc-by
8,940
JOURNAL OF CORPORA AND DISCOURSE STUDIES 2019, 2:94–116 E-ISSN 2515-0251 TAXPAYING, IMPORTING, ENFORCING: EMERGING DISCOURSE PATTERNS IN SHANNON ONLINE NEWSPAPER COMMENTS ABOUT FITZSIMMONS-DOOLAN TEXAS A&M CORPUS CHRISTI U.S. IMMIGRANT EDUCATION CITATION Fitzsimmons-Doolan, S. (2019). Taxpaying, Importing, Enforcing: Emerging Discourse Patterns in Online Newspaper Comments about US Immigrant Education. Journal of Corpora and Discourse Studies, 2:94–116 KEYWORDS applied linguistics, corpus linguistics, discourse analysis, media discourse, online comments, immigration, education DOI 10.18573/jcads.25 ISSUE DOI 10.18573/jcads.v2i1 ABSTRACT Recent scholarship suggests that social norms are becoming more permissive of prejudicial talk about immigrants in the United States (Crandall, Miller, & White, 2018). This is in keeping with trends observed internationally (e.g., Krzyżanowski, 2018; Vollmer & Karakayali, 2018). Past studies of discourse about immigration in the United States have identified a number of patterns — most notably those relating immigrants to threat (e.g., Santa Ana, 2002) — though some have found patterns framing immigration as beneficial (e.g., Strauss, 2012). This study explores discursive patterns about immigrant education in online comments from American newspapers. It focuses on change exhibited by exploring salient lexical and thematic patterns in 2016 comments relative to such patterns in 2009 comments. Using a corpus linguistics design, keyword, thematic, and other analytical techniques were applied to identify and describe the most prominent new patterns in the 2016 comments. Seven themes, through which immigration was framed negatively, emerged as new and salient in 2016. The article also includes discussion of cross-thematic patterns (e.g., a zero-sum framework) identified in the analysis. CONTACT Shannon Fitzsimmons‐Doolan, Department of English, Texas A&M University Corpus Christi, 6300 Ocean Dr, Corpus Christi, TX 78412 , United States. shannon.fitzsimmonsdoolan@tamucc.edu ORCID — LICENSE © The author. Available under the terms of the CC-BY 4.0 license Manuscript accepted 2019-06-20 95 Journal of Corpora and Discourse Studies 2 Taxpaying, Importing, Enforcing: Emerging discourse patterns in online newspaper comments about US immigrant education Shannon Fitzsimmons-Doolan Texas A&M University Corpus Christi 1. Introduction After several decades of relative inertia (Mazza, 2017), numerous recent events indicate that federal policy governing and discourses surrounding immigration to the United States are in a state of flux (Pierce et al., 2018). In addition, recent work in social psychology suggests that social norms in the United States are becoming more permissive of prejudicial talk about immigrants (Crandall et al., 2018). This is in keeping with trends observed internationally — especially in Europe (Krzyżanowski, 2018; Vollmer & Karakayali, 2018). This shifting ground is taking place against the backdrop of a K-12 educational system in which more than 25% of US children have at least one immigrant parent (Migration Policy Institute, 2018). Thus, the education of students from immigrant families will have great economic, political and social consequences in the United States. Such education is affected by discourses about immigration which can (1) transform immigrant students into threatening, inferior entities (Santa Ana, 2002), (2) facilitate educational policies negatively targeting immigrants (Calavita, 1996) and (3) be internalized and brought into learning experiences by immigrant learners themselves (Dabach et al., 2017). Media sources broadly and newspaper sources more particularly have been used in the study of immigration discourses because of their amplified access to the public (Santa Ana, 2002) and their ability to influence the opinions of readership (Gabrielatos & Baker, 2008) through agenda setting and priming mechanisms (Stewart, Pitts, & Osborne, 2011). Online reader comments published in major newspapers are influential because they deliver open-ended, vernacular opinion via the platform of substantial media sources, functioning as both a site of public deliberation (Ruiz et al., 2011) and a measure of public opinion to their readers (Soffer, 2019). This study explores the fluctuating public perceptions and stances surrounding US immigration by investigating emerging patterns in one particular register — online newspaper reader comments — for the domain of immigrant education between 2009 and 2016. Using a mixed-method design grounded in corpus linguistics, this study identifies prominent themes introduced in the 2016 data among top keywords and describes the themes through deployment of those top keywords. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 96 Journal of Corpora and Discourse Studies 2 2. Demographic context and literature review The patterns of actual immigration to the United States during the time of the study are important context for the interpretation of the reader comments. Data presented in Table 1 reveal a complicated picture of immigration patterns that are hard to distill into one narrative. Demographic characteristic Timeframe Trend Rate of total immigration a 2009–2016 steady Percentage of immigrants Latin American a 2009–2016 steady Number of undocumented immigrants b 2010–2016 -6.1% 2010–2016 fluctuation, no trend 2000–2016 +2.4% Percentage residents speaking Spanish at home a 2000–2016 +2.6% Percentage school-aged children with at least one immigrant parent e 2000–2017 +8% Percentage immigrant school-aged children e 2000–2017 -1.1% Removals of non-citizens c Percentage immigrant of total population d Table 1: Salient immigration metrics in the US over the 21th century. Sources: a US Census Bureau, 2018; b Passel and Cohn, 2018; c Baker, 2017; d Migration Policy Institute, 2018; e Migration Policy Institute, 2019 Research focusing on discourses about immigration in the US using newspaper data has consistently found negative representations of immigrants. In his seminal study, Santa Ana (2002) examined Los Angeles Times articles published between 1992 and 1998. He investigated the deployment of metaphors in articles about California Proposition 187, claiming that “[metaphor] is the key element with which discourse constructs the social world” (p. 43) and found that IMMIGRATION AS DANGEROUS WATERS was the dominant metaphor while IMMIGRANTS AS ANIMALS and IMMIGRANTS AS CRIMINALS were also deployed. In an earlier discussion of Proposition 187, Calavita (1996) claimed that rhetoric surrounding the law connected anti-immigrant sentiment to a burden on taxpayers. Examining local Virginia newspaper articles, Stewart et al. (2011) found that articles on the topic of illegal immigration scored lower on lexically-based measures of optimism and communality than articles that merely mentioned the concept. They also found a pattern of one individual immigrant standing for all undocumented immigrants who in turn stood for all Latinos. Cabaniss and Cameron (2017) examined 156 front-page articles of the New York Times about immigration published 1892-1924. Using CDA, the authors found that 75% of the articles referred to immigrants as groups rather than individuals, nearly half of the articles (46.8%) presented immigrants in negative ways, figurative lan- Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 97 guage presented immigrants as animals and immigrants as uncontrollable forces and there was a strong pattern of associating immigrants with disease. Studies of US immigration discourse using vernacular data have identified both positive and negative stances towards immigrants. Marlow (2015) analyzed 1,512 comments made on newspaper and corporate websites about an ad showing the patriotic song “America the Beautiful” sung in multiple languages. Sorting comments into stance types (pro and con), she found that supportive comments outnumbered comments with an opposing stance towards the ad 1,030 to 469. Using interview data from 27 North Carolinians, national immigration surveys and media content, Strauss (2012) identified 24 conventional discourses about immigration. Findings included (1) economic-themed discourses such as “Too Many Immigrants” which focuses on the amount of immigrants indexed by lexical items such as flood and overrun (p. 140) and (2) discourses focused on legality and national security such as “Illegal Is Wrong” indexed by words such as law and broken (p. 155). Among the 24 discourses identified, many framed immigrants in positive ways (e.g., “Nation of Immigrants,” “Immigrants’ Work Ethic”). Also using vernacular data, Dabach et al. (2017) played presidential debate clips to 26 high school students from immigrant families to assess how the students aligned with inclusionary and exclusionary immigration discourses. They found that these immigrant students evoked the theme of fairness to support both exclusionary and inclusionary discourses and identified youth as a category deserving of inclusion and criminals as a category deserving of exclusion. This finding is supported by Epenshade and Belanger (1998) who identified preferred statuses for “children versus adults,” “legal versus illegal immigrants”, “education and health benefits versus welfare benefits” and “citizens versus noncitizens” (p. 379) in US immigration public opinion data. Though several different units of analysis are used across research on immigration discourse, many approaches rely on lexical items as the entry point to analysis, as does this study. Strauss (2012) applied conventional discourses, which have multiple layers of representation including mental schema linking concepts, linguistic chunks and lexical items. Stewart et al. (2011) used categorized lists of lexical items to index texts on five variable dimensions: certainty, optimism, activity, realism and commonality. In their exploration of the semantic scope of the terms refugee, asylum seeker and boat people in a corpus of Australian online comments, Morrissey and Schalley (2017) conducted a lexico-semantic analysis of the use of these lexical items, identifying the underlying models of immigration processes associated with each term in the data. PérezParedes et al. (2017) used the most frequent collocates of the node word MIGRANT in administrative UK texts about immigration to identify priming patterns in the discourse. Finally, in an oft-cited study of 175,139 British newspaper articles about individuals translocating to the UK, Gabrielatos and Baker (2008) used keyword and collocation analysis as well as techniques of CDA to explore how REFUGEE, ASYLUM SEEKER, Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 98 Journal of Corpora and Discourse Studies 2 and MIGRANT were defined and framed. They found that keywords could be grouped into categories such as provenance, number, economic problem, legality, crime and plight. Keyword analysis, the primary technique used in this study, identifies salient lexical items across a corpus of texts by virtue of their unexpected frequency. The idea that strategically repeated lexical items can signal rich points in discourse about immigration is found across study types. In her description of conventional discourses, Strauss (2012) says, “keywords and stock phrases can be a tipoff, signaling conventional discourse” (p. 61). Furthermore, Ädel (2010) reports that metaphors, the unit of analysis in many studies of discourse about immigration, are increasingly identified empirically by keyword analysis. In another study using keyword analysis of texts about immigration, FitzsimmonsDoolan (2009) compared keywords identified in Arizona newspaper corpora focused on immigration to similar corpora focused on language policy. While primarily focused on the overlap among keyword lists differentiated by topic, qualitative analysis found that when the keywords immigrant, immigration and illegal were used in the language policy corpus, illegal was the most frequent collocate of immigrant/immigration, suggesting a strong perception of all immigration activities as unlawful. In sum, the studies above found that discourses negatively framing immigrants by either dehumanization or association with negative economic situations were more common than positive discourses. Discourses asserting overwhelming numbers of immigrants and discourses criminalizing all immigrants were common across the studies surveyed. In addition, while the surveyed studies identified several linguistic devices working to sustain these discourses, lexical items were used across several analyses to identify patterns in immigration discourse. To that end, this study addresses the question, what are emerging discourse patterns in 2016 online newspaper comments about US immigrant education? IMMIGRANT 3. Methods 3.1. Data sets This study explores patterns of discursive emergence in a data set of 2016 reader comments using a comparable set of comments from 2009 as a baseline. The 2009 corpus consists of 892 reader comments (145,652 words) published between 2009-03 and 200912. The 2016 corpus consists of 1,109 reader comments (51,878 words) published in 2016-02. The data sets are comparable in a number of ways. Each comes from major, “prestige” national newspapers in the United States: New York Times (NYT) (2009) and The Washington Post (WP) (2016). Though different newspapers, since 1961 researchers have Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 99 consistently grouped the WP and NYT together in a small cohort of US publications called “the US prestige press” based on their similarities in circulation, readership, aim, scope and rigor of work (e.g., Demont-Heinrich, 2008; Lacey et al., 1991; Stempal, 1961). Comment moderation policies for the two newspapers were matched in that (1) commenters need not have been subscribers, but needed to have registered with the newspaper and (2) comments were moderated according to a similar set of standards, but not edited. While the NYT pre-moderates their comments and the WP post-moderates, the WP comments were captured several months after posting, well after postmoderation would have occurred. Each corpus is a single comment thread. The 2009 data come from one reader comment thread that references (1) an announcement of a debate topic: “the best ways to teach newcomers” to the United States by NYT editors, (2) seven opinion pieces by experts or public figures centered on models for instruction of language learners and (3) an article about a Washington DC metro area high school serving immigrant students who are acquiring English as a second language (Thompson, 2009) — all published as a cohesive set over four days in March of 2009. The 2016 data are also a full reader comment thread. The thread references a single article published in February 2016 about a Washington DC metro area high school serving immigrant students who are acquiring English as a second language (Brown, 2016). To assess topical similarity in the prompting texts, each set of newspaper texts eliciting the corpora analyzed in this study (NYT2009, WP2016) was tagged using the USAS tagger within the Wmatrix corpus analysis tool (Rayson, 2008). After processing the newspaper text sets, the frequency of semantic tags were compared to assess semantic and thematic similarity; eight of the ten most frequent semantic tags for each set were found to overlap (i.e., grammatical bin, pronouns, education in general, unmatched, moving coming and going, existing, geographical names, personal names and speech acts) and accounted for 57.08% of words in the 2009 prompt texts and 58.68% of the words in the 2016 prompt text. Furthermore, 26 of the 30 most frequent semantic tags for each set overlapped, accounting for 73.23% of words in the 2009 prompt texts and 78.47% of the words in the 2016 prompt text. Thus, the two sets of textual prompts which elicited the comment data can be regarded as comparable in terms of semantic topic. A Proquest search specifying the subjects used to code both focal articles eliciting the reader comments (“schools,” “immigration policy,” and “alien”) identified no other articles that matched on both newspaper and article content in the 21st century. Therefore, these two datasets are the closest available match for this research question in either the WP or NYT. The exact time span explored in this study is an artifact of the publication dates of the prompting texts. The reference corpus for the study (82,340 words) consists of reader comments from articles in the NYT and WP published 2009–2016 (distributed evenly across the years and Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 100 Journal of Corpora and Discourse Studies 2 newspapers). The corpus includes comments on a range of US domestic policy issues (i.e., the economy, jobs, racism, global warming, health care, Medicare, abortion, gun control and terrorism) distributed evenly across issues. That is, the reference corpus represents online newspaper comments about major domestic policy issues in the WP and NYT from 2009 to 2016. 3.2. Keyword design Within language studies the term keyword has been used to refer to both cultural and statistical concepts (Ädel, 2010; Culpeper & Demmen, 2015; Fitzsimmons-Doolan, 2009; O’Halloran, 2010). Keywords, in both senses, are posited to reveal underlying properties across a set of texts (Stubbs, 2010). In the case of cultural keywords, Williams (1976) claims that they reveal identifying cultural values. In the case of statistical keywords, they reveal identifying concepts, stylistic features and/or ideologies across a body of texts in relation to a reference body of texts (Gabrielatos, 2018; Kempannen, 2004; Stubbs, 2010). Importantly, the interpretation of the nature of the salience identified by statistical keywords always depends on the texts that comprise the reference corpus (Culpepper and Demmen, 2015; O’Halloran, 2010). Statistical keywords (hereby referred to as keywords) are lexical items that are unusually frequent in a corpus of interest when compared to another, reference corpus (Stubbs, 2010). Thus, the keywords will represent the ways that the corpus of interest is different than the reference corpus. For example, if the two corpora represent the same genre, but different topics, then the keywords will represent the topical differences in the corpus of interest. If the two corpora are about the same topic, but in different registers, then the keywords will represent the ways in which the registers differ in presentation of the topic in the corpus of interest. In this study, a keyword list for each data set (NYT2009, WP2016) relative to the reference corpus was generated in order to identify the keywords in each corpus particular to their year and the topic of immigrant education while controlling for words particular to the register of online newspaper comments about US domestic policy issues from 2009–2016 in the WP and NYT more generally. Although the editorial style of comment sections varies across newspapers, which can affect qualitative aspects of comments (Rowe, 2015), this potential source of variability is addressed in this study by the content of the reference corpus. The keyword lists from each point in time were then used as the basis for further analysis in order to identify patterns of discursive emergence in the 2016 data. 3.3. Analysis Each of the two immigrant education comment corpora (NYT2009, WP2016) was compared to the reference corpus to generate a list of keywords using WordSmith corpus analysis software (Scott, 2016) (α < .005, log ratio > 1.5). To further refine the set of key words, the lists were sorted by log ratio, which is a measure of effect size (Gabrielatos, Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 101 2018). The first distinct break in the log ratio scores of the 2009 data occurred between 135.01 and 9.08 and in the 2016 data it occurred between log ratio scores of 135.64 and 7.04. The keywords from each list with the highest log ratio scores (≥ 135.01, 2009; ≥ 135.64, 2016) that were also statistically significant — top keywords — were extracted for further analysis. The researcher qualitatively examined how each top keyword was used in context within each corpus. The researcher then coded all of the top keywords (both 2009 and 2016) inductively using the contextual analysis to identify themes (Gabrielatos & Baker, 2008; McEnery, 2016). For example, steal was given the thematic code crime and importing was given the thematic codes economy/finances and problem. Next, the researcher identified seven themes that were both new in 2016 and were evoked by top keywords that were also new in 2016. Use-in-context of all top keywords (n = 29) coded as at least one of the seven themes were then investigated qualitatively using concordances and full textual context. 4. Results and discussion In the 2009 corpus, 150 top keywords were identified (p < .005; log ratio ≥ 135.01) and 94 top keywords were identified in the 2016 corpus (p < .005; log ratio ≥ 135.64). (See the Supplemental File for the top keywords and their log ratio scores from each corpus.) A comparison of the two lists reveals that they are primarily non-overlapping. That is, 77% of the top keywords in the 2009 list did not overlap with the 2016 list and 64% of the words in the 2016 list did not overlap with the 2009 list. While some top keywords do overlap across the corpora (e.g., Spanish, bilingual, ESL, taught), this analysis points to sizable change in these register samples during this period of time. The qualitative analysis using the quantitative results identified seven new themes in the 2016 data, all broadly tied to immigration. Table 2 presents these themes (economy/finances, amount/disaster, law, crime, problem, animals and politics) along with the 29 top keywords from 2016 that evoked these themes. None of these 2016 top keywords were keywords in the 2009 data and, furthermore, 18 of these 2016 top keywords (62%) were not used at all in the 2009 corpus. The lack of keyness and, in the majority of cases, even presence of the focal 2016 top keywords in the 2009 corpus confirms that the design of the study did identify salient differences between the 2009 and 2016 data sets even though the corpora were not directly compared. What follows are the results of qualitative analysis of 2016 top keywords evoking the seven emerging themes. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 102 Journal of Corpora and Discourse Studies 2 Theme Economy/Finances Amount/Disaster Law Crime Problem Animals Politics Top keyword Keyness (log ratio) importing 136.78 freebies 136.49 feed 136.49 payers 136.32 handouts 135.92 taxpaying 135.64 budgets 135.64 flood 137.13 surge 135.64 overrun 135.64 infinite 135.64 flooded 135.64 Plyler 136.90 Doe 136.90 deportation 136.32 enforcing 135.64 steal 136.32 incarcerate 136.32 broke 136.13 sneak 135.64 incarcerating 135.64 importing 136.78 fools 136.32 populated 135.90 handouts 135.92 dumbed 135.90 feed 136.49 gophers 136.13 herbs 135.64 Sanders 136.32 Dem 135.64 Bernie 135.64 Table 2: Emerging themes and associated top keywords by log ratio in the 2016 data Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 103 4.1. Economy/finances The use of the top keywords importing, freebies, feed, payers, handouts, taxpaying, and budgets were all categorized as referring to economic or financial concerns. Based on analysis of these top keywords in context, the texts in which they were deployed were overwhelmingly anti-immigration in stance and also evoked other focal themes (i.e., problem, animals, and politics). That is, it was common for this theme to be deployed alongside others. In particular, importing and handouts evoked both the problem and economy/finances themes. (Italicized terms in the following exemplars indicate lexical items that directly relate to the theme and its interpretation, including but not limited to top keywords.) (1) Importing third world poverty and illiteracy into a first world society is only going to produce a substantially larger dependent underclass. And let's face reality here. We're not even importing it. They are importing themselves and then having "American" children as a means of trying to prolong their illegal stays. In the process the American Taxpayer is on the hook for billions in additional educational costs which by and large you might as well flush down a toilet… (2) "to access services…" That's putting a nice spin on getting government handouts. Our schools are pitiful as they are. This will not be helping. In Excerpts 1 and 2 the commenters use the top keywords importing and handouts to not only associate immigrant students with economic concepts (i.e., immigrants as imports, education as valuable benefit), but also to problematize immigrant students at the essential level (i.e., associations with poverty, illiteracy, pitiful schools). The use of payers, taxpaying, budgets, feed, and freebies evoke unearned financial benefits for undocumented immigrants at the financial cost to American citizens, who are presented as taxpayers. (3) so why does America have to babysit this generation of immigrants with handouts, we are being taken advantage of and our Government is really the cause of the flow of illegals by offering them all the freebies paid for by the American Taxpayer, which is depleting the quality of services that is owed to the taxpayer. (4) Flood entire communities with unending hordes, while the taxpaying population supporting it all dwindles and watches their children get pushed to the back of the line. Civilization was fun while it lasted. (5) If the school district's budget is fixed and it has a 10% increase in enrollment due to "immigrant" (read, illegals), who must have special programs like bilingual education, tutoring, etc., what gets cut for the rest of students? In both Excerpts 3 and 5 the commenters associate all immigrants with illegality. In all three excerpts, the financial benefits flowing to immigrant students through the educaFitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 104 Journal of Corpora and Discourse Studies 2 tional system mean that something is getting taken from non-immigrants, presented as taxpayers (Excerpts 3–4). This framing of immigrant education as a zero-sum game is pervasive through the use of 2016 top keywords in this analysis as is the conflation of the categories of immigrant and undocumented immigrant. In fact, illegal is the most frequent collocate of both immigrant(s) and immigration in the 2016 data. In the 2009 data, illegal is the most common collocate of immigration, but is only the 10th most common collocate of immigrant(s). This analysis supports previous identification of the economic theme (Calavita, 1996; Gabrielatos & Baker, 2008; Strauss, 2012). In particular, the data in this study shows evidence of the semantic shift from citizen to taxpayer (e.g., Excerpts 1, 3) described in Calavita (1996). Though Strauss (2012) found both positive and negative conventional discourses about immigrants/immigration and economic concerns, in this data, such comments took a negative stance. 4.2. Amount/disaster The top keywords in this category (flood, flooded, surge, overrun, infinite) refer to increasing amounts of an entity, usually of immigrants and often evoke disaster. The texts using these words were also overwhelmingly anti-immigrant in stance. Flood (7/14 cases) and surge (5/5 cases) are often used in conjunction with historical or quantitative information and deployed with certainty. (6) The flood started after 1982…In June 1982, the Supreme Court issued Plyler v. Doe, a (5-4) landmark disaster decision holding that states cannot constitutionally deny students a free public education on account of their immigration status. This ruling by the courts Court's liberal wing has destroyed my country.… (7) When my daughters were in school in Houston, we had a surge of illegal alien children into our district. They had to attend classes in pods, only had access to textbooks on even/odd days, were in classes with 30+ students, and saw a reduction in extra-curricular activities. We had another surge in 2014 when thousands of illegal minors flooded across the Rio Grande. One school district had to hire teachers in 27 dialects: that's how crazy things are down here. (8) When Will [sic] Americans start protesting against the illegal invasion and Obama illegal immigration amnesty policy allowing the borders to surge? As in Excerpts 6 and 8, comments using flood and surge often tend to tie perceived increases in immigration to political decisions or actors. Use of flooded also employs the metaphor of disaster to refer to increases in negative entities. (9) Additionally, our schools are being flooded with drugs. (10) Of course America's schools are flooded with aliens. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 105 Like flood and surge, the use of overrun refers to increases in immigrants. In addition, it is often deployed with language evoking the problem theme. (11) Also, just an observation: There is a reason Texas schools rank somewhere around 43rd out the 50 states. Here in Texas our schools are overrun with millions of children of illegal migrants. And while it is no fault of those kids, all US citizens should envision THEIR children being denied up to date classroom equipment, computers, facilities, instruction, etc… because that money is used to educate the people sent Northward daily from that worthless, corrupt, cartel-ravaged cesspool called Mexico. In Excerpt 11, the commenter associates immigrant students whose numbers are increasing (“overrun with millions”) with problematic attributes (e.g., “worthless,” “corrupt”). Finally, infinite is not used to describe the amount of immigrant students, rather it is a modifier used to describe resources. In particular, commenters use infinite to make a claim that resources for education are not infinite. (12) The progressive left fringe truly believes that public resources are infinite. The rest of us know that is simply wrong. The "right" to public education is, pure and simple, another claim on limited public resources. Unfortunately, there seems to be a limitless number of economic and political refugees making claims. Excerpt 12 shows this pattern. The commenter claims that others, Americans identifying with the far left, perceive resources as infinite, while a majority, “the rest of us”, rightly understand resources to be limited. The use of infinite and the other amount/ disaster top keywords in this corpus supports the pattern noted earlier of constructing education for immigrant children as a zero-sum game. This theme is one of the most widely documented across the previous literature (Catalano & Fielder, 2018; Gabrielatos & Baker, 2008; Santa Ana, 2002; Strauss, 2012). It is the most prevalent theme identified by both Santa Ana (2002) as a metaphor (i.e., IMMIGRATION AS DANGEROUS WATERS) and Strauss (2012) (i.e., “Too Many Immigrants”). Furthermore, Catalano and Fielder (2018) suggest that use of terms such as surge employ the discursive strategy of proximization which conveys a sense of threat to the addressee. In this data, the threat is to both economic resources (Excerpts 7, 11, 12) and to safety (Excerpts, 9, 10, 11). 4.3. Law As with the reader comments in the previous categories, comments using the top keywords Plyler, Doe, deportation and enforcing almost exclusively presented antiimmigrant sentiments to discuss laws or frameworks of governance. Plyler and Doe were used together to reference the US Supreme Court case prohibiting public K-12 schools from inhibiting access to education based on immigration status. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 106 Journal of Corpora and Discourse Studies 2 (13) Agree totally. I recall being stunned by Plyler v. Doe when in 1982, the U.S. Supreme court, in all its infinite elite sheltered tree lined cul-de-sac wisdom more or less mandated free education for illegal immigrants. The comments referencing the court case had negative evaluations of the outcome. The politics theme was often evoked in comments including deportation (3/8 cases) or enforcing (3/5 cases), alongside the legal theme. (14) NO amnesty. NO work permits. NO nothing but deportation. Pledge to vote for the candidate who will deport all illegals and severely reduce legal immigration. (15) The Obama administration has repeatedly shown it to be a rogue administration when it comes to enforcing US laws. They only enforce those laws they want, and that IS NOT their job description. Obama's legacy will be a Trump presidency… (16) Just start with the law for a second here… at first there is a law that says these people cannot just cross our border and stay, pretty crystal clear. The penalty for doing so is deportation. Simple really. At some point we stopped enforcing this law.… I will vote for Trump just on this issue. I don’t care what else he does. Comments using deportation or enforcing often assume that immigrant students are breaking laws (11/13 cases), following the pattern of conflating the categories of immigrant and undocumented immigrant found across the data. The comments then call for enforcing the law — often through deportation. Somewhere in this subset of comments, there is often a reference to either a perception of President Obama not enforcing laws and/or then presidential candidate Donald Trump being a preferred option based on a perception of his willingness to enforce laws. This analysis is confirmed by Dabach et al. (2017), Gabrielatos and Baker (2008) and Strauss (2012), who all found ways in which legal constructs featured in discourse about immigration. What is marked in this analysis in comparison to previous work is the connection to enforcement and further connection to political choices. 4.4. Crime Related to law, crime comments focused on characterizing undocumented immigrants as criminals. The top keywords in this category were steal, broke, sneak, incarcerate and incarcerating. Steal, broke and sneak were used in comments with anti-immigrant stances, while incarcerate and incarcerating were the only top keywords in the analysis used in some comments that advocated for immigrant education. (17) The Supreme Court needs to revisit its decision that offered free education to anyone who could sneak over our border. The decision itself has offered a prize to any foreign parent who is willing to violate our immigration laws. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 107 (18) I do not owe people that snuck into this country and broke our laws anything whatsoever. I keep hearing that we cannot deport 12 million people from this country. The hell we can’t, one at a time baby!!!! (19) I hope every American who reads this article vote for Trump so that we can begin deporting all illegal aliens who steal from us daily. We can take back our jobs, schools, hospitals, roads, etc. (20) Like it or not illegal aliens steal the education from American children. Excerpts 17 and 18 show how use of sneak and broke first tend to essentialize all immigrant students as undocumented and then attribute flaws to their character. The uses of steal in Excerpts 19 and 20 explicitly claim that immigrant students’ access to education takes away from the education of non-immigrant students. The uses of incarcerate and incarcerating were mostly deployed in a back and forth among a handful of comments attempting to establish the cost of incarcerating an immigrant child versus the cost of educating an immigrant child. (21) So, basically to clarify based on the best numbers we have all come up with it costs about $400 per day to incarcerate a child, and about $60, to educate a child. Some people think it is better to be kind, while putting a lower burden on tax payers. Other folks feel it is more important to consider children, to be like dangerous criminals and incarcerate them even if it costs 600% more to do so . . . . Does that sum it up so far? (22) You can look it up, why not just go to google. On average it $18.6 million or $62 per day to incarcerate 300,000 illegal immigrants. So there you go $62 per day which I guarantee is less than the cost of putting them in school, providing social services, providing medical care, buying tickets to their destination placing them in new homes, etc. etc. (23) Just on a point of clarification. Incarcerating a child costs about $400 per day. Educating a child costs $27 per day. The uses of incarcerate and incarcerating marked a site of negotiation where commenters used the economy/finances theme to justify or resist the criminalization of youth. This is particularly interesting because these are the only top keywords whose use was associated with comments advocating for immigrants in the analysis. The 2016 data evoking crime draws on a robust previously identified discourse history (i.e., Catalano & Fielder, 2018; Dabach et al., 2017; Gabrielatos & Baker, 2008; Santa Ana, 2002; Stewart et al., 2011; and Strauss, 2012) to negatively frame all immigrants. The comments focused on incarceration costs in this data (Excerpts 21, 22, 23) seem to be a place where the identities of criminal (unprivileged status) and child (privileged status) were being negotiated. In this negotiation, some readers argued indirectly that criminal status (as defined by method of entry) superseded age status Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 108 Journal of Corpora and Discourse Studies 2 (Excerpt 22), while others argued indirectly that age status superseded criminal status (Excerpts 21, 23) — though both arguments relied explicitly on economy/finances argumentation. 4.5. Problem The top keywords categorized in the problem theme are importing, handouts, populated, dumbed and fools. Overall, use of these words appears in comments problematizing either immigrants (importing, handouts, populated, dumbed) or liberal immigrant supporters (fools). This more generalized theme was found less explicitly in previous work (Cabaniss & Cameron, 2017; Stewart et al., 2011; Strauss, 2012). The samples below are representative of patterns found in the deployment of these top keywords. (24) Exactly how does America benefit the Third World by becoming the Third World? by importing its poverty and failure? (25) Let's be honest, THEY ARE NOT all fleeing war and oppression, they come seeking taxpayer funded handouts and bring their bad cultural habits with them (26) So we can become as poverty stricken, over populated, living in filth like countries these immigrants are coming from? What is wrong with you? It doesn't make us Nazis if we don't want to take in the entire world. (27) how much are classes slowed down and dumbed down to cater to those who can't understand? What about the large number of gang member that are coming in and creating havoc in our communities? In Excerpt 24 a commenter uses an economic/financial metaphor to characterize immigrants negatively (i.e., poverty, failure). In Excerpt 25, handout, another economic/ financial concept is used to argue that Americans are exchanging financial resources for negative immigrant traits (bad cultural habits). In Excerpt 26, overpopulated is listed with negative traits the commenters associate with immigrants (poverty stricken, filth) and is subtly dehumanizing in this context. Finally, in Excerpt 27 dumbed is attributed to curriculum for immigrant students. Use of dumbed was usually followed by a negative characterization of immigrant students. In this case immigrant students are associated with gang members wreaking havoc. Finally, fools was used to reference liberal politicians or voters who were thought to be pro-immigrant education. (28) I hope the idiots who voted for this President appreciate the fact that their children will receive even less education than the already dumbed down system currently offers. I hope these fools are the ones who contract the diseases which were essentially eradicated in our population The text in Excerpt 28 uses several top keywords to problematize immigrant students. According to the commenter, immigrant students carry diseases, the curriculum for all students will be dumbed down because of such students and supporters of then President Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 109 Obama are characterized as idiots and fools. This comment returns to the zero-sum assumption so prominent in this data, suggesting that curricular modifications are a tool by which the exchange of resources happens — i.e., that in order to access education im migrant students require a simplified curriculum which takes away from the education of non-immigrant students. 4.6. Animals The top keywords feed, gophers and herbs pointed towards an exchange among commenters in which immigrants were likened to animals. Though this theme was identified as very salient in previous studies (Catalano & Fielder, 2018; Santa Ana, 2002) and was identified in this study, it was very isolated in expression in this corpus to the following exchange. (29) If your garden is full of gophers, you have no garden. So, with fencing and an aggressive campaign to remove the gophers, you are able to maintain the garden. I feel sorry for the gophers: we gas them, trap them, neighbors feed them to the dogs. But you probably are starting to understand the metaphor (30) I had a gopher in my garden once, I looked on the internet, planted some herbs, then I never had gophers again. Every problem has a solution, and a solution isn’t more effective because it is brutal. (31) interesting contrary, gophers as people you sound like Herr Eichman proposing the final solution, gas them, trap them, and feed them to the dogs. why don’t you go back to ISIS you maggot (32) I get gophers in my yard occasionally… I shoot them. Sit out there with a can of Bud and a rifle… when they poke their head up, I blow it off. I also have 2 enormous cats who love to sit by a gopher hole all day, it's amazing the patience they have, it has to be a terrible way to go for the gopher… My stupid dog, tries to dig them up… never works. The gophers like my dog, are terrified of my cats and never know that I am there. Excerpts 29, 30 and 32 use the top keywords to characterize immigrants as animals that need to be removed from a territory. In Excerpts 29 and 32, the commenters advocate violence and terror as approaches to removing the gophers. In Excerpt 30, the commenter resists the idea of violence and terror as brutal, but embraces the animals metaphor and the goal of removal. In Excerpt 31, the commenter strongly resists the animals metaphor, associating its deployment to Nazism and then calling the author of Excerpt 29 a terrorist and a highly stigmatized insect — thereby dehumanizing the author of Excerpt 29. 4.7. Politics The three top keywords coded as political were Bernie, Sanders and Dem. Comments using these words focused on arguing that increasing the immigrant population in the United States was politically expedient for the Democratic Party generally with particular Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 110 Journal of Corpora and Discourse Studies 2 reference to then presidential candidate Bernie Sanders and then President Barack Obama. (33) Trouble funding illegal immigrants/"refugees"? Just tax the Bernie Sanders, open-borders, leftists by their proposed 90% rate to pay the way for yet another group to mooch off of the government (productive class) gravy train (34) WaPo, these aren't "immigrants," they're illegal aliens - but to the Left - they're VOTES - which the degenerate Dem Party can't capture legitimately - hence Obama's "browning of America." This Obama Reich is a stain on the fabric of America. Comments deploying Bernie (3/5 cases) or Sanders (5/8 cases) often suggested that the candidate was planning to increase taxes (to 90% was a repeated number) to pay for the cost of supporting such individuals financially (Excerpt 33). Comments deploying Dem (3/5 cases) often suggested that Democrats encourage immigration in order to gain the votes of immigrants in the future (Excerpt 34). Within both types of comments, the assumption that all immigrants are illegal is explicitly repeated. In addition to the comments using these terms, the political theme was also evoked in many of the comments across the other six themes as well — especially the economy/finances and amount/disaster themes. There were two primary patterns in these cases. First, as shown earlier, then President Obama was linked to an increase in immigration rates generally (Excerpt 8) and a deliberate policy of non-enforcement of immigration law (Excerpts 15, 34). Though present across the comments, Obama was not a keyword in the 2016 corpus. This was likely because the reference corpus spanned 2009–2016, years of the Obama presidency and covered high-profile domestic policy issues. Therefore, while frequent in the 2016 corpus (n = 79), use of Obama was not unusually frequent when compared to the reference corpus (n = 110). The second notable politics pattern across themes was the use of Trump, which was a keyword in the 2016 corpus (log ratio 2.78). (35) TRUMP, BUILD THAT WALL AND THEN SEND ALL THOSE WHO BROKE OUR LAWS, AND CONTINUE TO STEAL OUR MONEY, BACK! In conjunction with the top keywords and the emerging themes in 2016, the use of Trump tended to be connected to a claim of immigrants breaking laws (Excerpts 16, 19, 35), a claim of immigrants taking resources (Excerpts 19, 35) and a declaration in favor of then presidential candidate Donald Trump (Excerpts 16, 19, 35). Interestingly, a political theme was not identified in any of the previous literature surveyed for this study. However, the presence of this theme in the 2016 data may point to the effectiveness of Donald Trump’s presidential campaign, for which immigration was a central issue from the outset (Newman et al., 2018). Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 111 5. Conclusion This corpus-driven, keyword investigation of emerging patterns in 2016 reader comments about US immigrant education reveals seven new themes not present in the 2009 data. Grossly, much change was observed, focused on immigration rather than education and almost all adopting a negative stance towards immigrants. Though the themes economy/finances, amount/disaster, law, crime, problem and animals had all been identified in the prior literature, politics was not. The introduction of the politics theme or the nature of this register — online comments — or a combination of both of these factors may have played a role in the overwhelmingly negative focus of the emerging trends. In addition to the local patterns described in the previous section, qualitative investigation of patterns related to the use of top keywords associated with emerging themes found two global patterns: (1) a consistent pattern of metonymy in which all immigrants are equated to illegal immigrants; and (2) an underlying assumption across arguments that immigration policy in the United States is a zero-sum game. The term “illegal immigrant” operates as code to implicitly indicate threat tied to an ethnic/racial identity (Cabaniss & Cameron, 2017; Stewart et al., 2011). This study robustly confirms the presence of this metonymic pattern in the 2016 emerging themes data. Furthermore, the zero-sum game assumption is predicated on an Us vs Them dichotomy which is also found throughout the data. Though Stewart et al. (2011) claimed that this dichotomy is a hallmark of immigration discourse, Strauss (2012) identified several conventional discourses that were not predicated on a zero-sum assumption such as “Jobs Americans Don’t Want,” “Nation of Immigrants,” and “Good People as Individuals.” Thus, what is noteworthy in this analysis is the unequivocal dominance of the zero-sum game assumption across the change data analyzed. Finally, there are strong disconnects between the claims about immigrants in the discourse analyzed and the demographic data from the same time period. For example, there were consistent claims of undocumented immigration increasing, particularly at the southern border. However, estimates of undocumented immigration from Passel and Cohn (2018) indicate that the numbers of undocumented immigrants in the United States decreased by 6.1% between 2010 and 2016. Several possibilities for these disconnects between reader claims and demographic data emerge. One possible explanation is that immigration patterns are not evenly distributed across localities and what is true at the national level (e.g., steady total immigration rates) may not be true at a local level from which a reader comments. Another possible explanation for this discrepancy is that readers are reacting to demographic changes caused by past waves of immigration such as the increase in children of immigrant parents in schools versus the decrease in immigrant students in schools. Finally, the disconnects could be attributed to xenophobia and racism stoked by immigrants as threat narratives promulgated by high profile individuals Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 112 Journal of Corpora and Discourse Studies 2 (Crandall et al., 2018; Pierce et al., 2018) and naturalized through media discourse (Stewart et al., 2011). This study has identified and described emerging patterns in 2016 US-based discourse about immigrant education in an influential social register — online newspaper reader comments. Among the most notable findings is the emergence in the comments of an overwhelmingly negative focus on the immigration status of students deployed using top keywords grouped into seven themes. If this negative focus on immigration status of students is indicative of discursive shifts more broadly, it bodes poorly for the educational context for all American students, but especially those from immigrant families. The window of time explored in this study is important because it fell just prior to notable fluctuation in both public opinion and federal policy about immigration generally in the United States. Future work should investigate discursive changes on this topic within and across other influential registers as well as the nature of the relationship between discursive shifts and shifts in policy and opinion. Acknowledgements The author would like to thank John Lister and Katie Sisk for their help preparing data for analysis, Dr Stephen Doolan and Dr Robert Poole for their insight, and the anonymous reviewers whose questions and feedback greatly improved this manuscript. Funding The author would like to thank Texas A&M Corpus Christi for internal funding supporting this project. Competing interests The author has no competing interests to declare. References Ädel, A. (2010). How to use corpus linguistics in the study of political discourse. In A. O’Keefe & M. McCarthy (Eds.), The Routledge Handbook of Corpus Linguistics (pp. 591– 604). New York: Routledge. Baker, B. (2017). Immigration Enforcement Actions: 2016 Annual Report. Washington, DC: Department of Homeland Security. Brown, E. (2016, February 7). As immigration resurges, US public schools help children find their footing. The Washington Post. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 113 Cabaniss, E. R., & Cameron, A. E. (2017). ‘Unassimilable and undesirable’: News elites’ discursive construction of the American immigrant during the Ellis Island years. Discourse & Society, 28(6), 614–634. doi:10.1177/0957926517710990 Calvita, K. (1996). The new politics of immigration: ‘Balanced-budget conservatism’ and the symbolism of Proposition 187. Social Problems, 43 , 284–305. doi:10.2307/3096979 Catalano, T., & Fielder, G. E. (2018). European spaces and the Roma: Denaturalizing the naturalized in online reader comments. Discourse & Communication, 12(3), 240–257. doi:10.1177/1750481318757772 Crandall, C. S., Miller, J. M., & White, M. H. (2018). Changing norms following the 2016 US presidential election: The Trump effect on prejudice. Social Psychological and Personality Science, 9(2), 186–192. doi:10.1177/1948550617750735 Culpeper, J., & Demmen, J. (2015). Keywords. In D. Biber & R. Reppen (Eds.), The Cambridge Handbook of English Corpus Linguistics (pp. 90–105). Cambridge: Cambridge University Press. Dabach, D. B., Fones, A., Merchant, N. H., & Kim, M. J. (2017). Discourses of exclusion: Immigrant-origin youth responses to immigration debates in an election year. Journal of Language, Identity, & Education, 16, 1–16. doi:10.1080/15348458.2016.1239538 Demont-Heinrich, C. (2008). American ‘prestige-press’ representations of the global hegemony of English. World Englishes, 27, 161–180. doi:10.1111/j.1467971X.2008.00550.x Epenshade, T. J., & Belanger, M. (1998). Immigration and public opinion. In M. M. Suárez-Orozco (Ed.), Crossings: Mexican Immigration in Interdisciplinary Perspectives (pp. 365–403). Cambridge: Harvard University David Rockefeller Center for Latin American Studies. Fitzsimmons-Doolan, S. (2009). Is public discourse about language policy really public discourse about immigration? A corpus-based study. Language Policy, 8, 377–402. doi:10.1007/s10993-009-9147-6 Gabrielatos, C. (2018). Keyness analysis: Nature, metrics and techniques. In C. Taylor & A. Marchi (Eds.), Corpus Approaches to Discourse: A Critical Review (pp. 225–258). New York: Routledge. Gabrielatos, C., & Baker, P. (2008). Fleeing, sneaking, flooding: A corpus analysis of discursive constructions of refugees and asylum seekers in the UK press, 1996–2005. Journal of English Linguistics, 36, 5–38. doi:10.1177/0075424207311247 Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 114 Journal of Corpora and Discourse Studies 2 Kemppanen, H. (2004). Keywords and ideology in translated history texts: A corpus based analysis. Across Language and Cultures, 5(1), 86–106. doi:10.1556/Acr.5.2004.1.5 Krzyżanowski, M. (2018). Discursive shifts in ethno-nationalist politics: On politicization and mediatization of the ‘Refugee Crisis’ in Poland. Journal of Immigrant & Refugee Studies, 16(1–2), 76–96. doi:10.1080/15562948.2017.1317897 Lacey, S., Fico, F., & Simon, T. F. (1991). Fairness and balance in the prestige press. Journalism Quarterly, 68, 363–370. doi:10.1177/107769909106800306 Marlow, M. L. (2015). The American Dream? Anti-immigrant discourse bubbling up from the Coca-Cola ‘It’s Beautiful’ advertisement. Discourse & Communication, 9(6), 625–641. doi:10.1177/1750481315600299 Mazza, J. (2017). The US-Mexico border and Mexican migration to the United States: A 21st century review. SAIS Review of International Affairs, 37(2), 33–47. doi:10.1353/sais.2017.0025 McEnery, T. (2016). Keywords. In P. Baker & J. Egbert (Eds.), Triangulating Methodological Approaches in Corpus Linguistic Research (pp. 20–32). New York: Routledge. Migration Policy Institute. (2018). State Immigration Data Profiles: United States. Retrieved from https://www.migrationpolicy.org/data/state-profiles/state/demographics/US Migration Policy Institute. (2019). State Immigration Data Profiles: United States. Retrieved from https://www.migrationpolicy.org/data/state-profiles/state/demographics/US Morrissey, L., & Schalley, A.C. (2017). A lexical semantics for refugee, asylum seeker and boat people in Australian English. Australian Journal of Linguistics, 37(4), 389–423. doi:https://doi.org/10.1080/07268602.2017.1350130 Newman, B. J., Sha, S., & Collingswood, L. (2018). Race, place, and building a base: Latino population growth and the nascent Trump campaign for president. Public Opinion Quarterly, 82(1), 122–134. doi:10.1093/poq/nfx039 O’Halloran, K. (2010). How to use corpus linguistics in the study of media discourse. In A. O’Keefe & M. McCarthy (Eds.), The Routledge Handbook of Corpus Linguistics (pp. 563–577). New York: Routledge. Passel, J., & Cohn, D. (2018, November 27). U.S. Unauthorized Immigrant Total Dips to Lowest Level in a Decade. Washington, DC: Pew Research Center. Retrieved from Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 115 https://www.pewresearch.org/hispanic/2018/11/27/u-s-unauthorized-immigranttotal-dips-to-lowest-level-in-a-decade/ Pérez-Paredes, P., Jiménez, P.A., & Sánchez Hernández, P. (2017). Constructing immigrants in UK legislation and administration informative texts: A corpus-driven study (2007–2011). Discourse & Society, 28(1), 81–103. doi:https://doi.org/10.1177/0957926516676700 Pierce, S., Bolter, J., & Selee, A. (2018). U.S. Immigration Policy under Trump: Deep Changes and Lasting Impacts. Washington, DC: Migration Policy Institute. Rayson, P. (2008). From key words to key semantic domains. International Journal of Corpus Linguistics, 13(4), 519–549. doi:10.1075/ijcl.13.4.06ray Rowe, I. (2015). Deliberation 2.0: Comparing the deliberative quality of online news user comments across platforms. Journal of Broadcasting & Electronic Media, 59(4), 539–555. doi:10.1080/08838151.2015.1093482. Ruiz, C., Domingo, D., Mico, J. L., Díaz-Noci, J., Meso, K., & Masip, P. (2011). Public sphere 2.0? The democratic qualities of citizen debates in online newspapers. The International Journal of Press/Politics, 16, 463–487. doi:https://doi.org/10.1177/1940161211415849 Santa Ana, O. (2002). Brown Tide Rising: Metaphors of Latinos in Contemporary American Public Discourse. Austin, TX: University of Texas Press. Scott, M. (2016). Wordsmith Tools (Version 7.0) [Computer software]. Oxford: Lexical Analysis and Oxford University Press. Soffer, O. (2019). Assessing the climate of public opinion in the user comments era: A new epistemology. Journalism, 20(6), 772–787. doi:10.1177/1464884917714938 Stempal, G. H. (1961). The prestige press covers the 1960 presidential campaign. Journalism Quarterly, 38:, 157–163. doi:10.1177/107769906103800201 Stewart, C. O., Pitts, M. J., & Osborne, H. (2011). Mediated intergroup conflict: The discursive construction of ‘Illegal Immigrants’ in a regional US newspaper. Journal of Language and Social Psychology, 30(1), 8–27. doi:10.1177/0261927X10387099 Strauss, C. (2012). Making Sense of Public Opinion: American Discourses about Immigration and Social Programs. New York: Cambridge University Press. Stubbs, M. (2010). Three concepts of keywords. In M. Bondi & M. Scott (Eds.), Keyness in Texts (pp. 21–42). Amsterdam: John Benjamins. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25 116 Journal of Corpora and Discourse Studies 2 Thompson, G. (2009, March 15). Where education and assimilation collide. New York Times. United States Census Bureau. (2018). American fact finder: Advanced search. Retrieved from https://factfinder.census.gov/faces/nav/jsf/pages/searchresults.xhtml?refresh=t Vollmer, B., & Karakayali, S. (2018), The volatility of the discourse on refugees in Germany. Journal of Immigrant & Refugee Studies, 16(1–2), 118–139. doi:10.1080/15562948.2017.1288284 Williams, R. (1976). Keywords: A Vocabulary of Culture and Society. Oxford: Oxford University Press. Fitzsimmons-Doolan (2019) Taxpaying, Importing, Enforcing DOI 10.18573/jcads.25
W2131500287.txt
https://bmcpediatr.biomedcentral.com/counter/pdf/10.1186/1471-2431-12-52
en
Hand hygiene instruction decreases illness-related absenteeism in elementary schools: a prospective cohort study
BMC pediatrics
2,012
cc-by
3,813
Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 RESEARCH ARTICLE Open Access Hand hygiene instruction decreases illness-related absenteeism in elementary schools: a prospective cohort study Claudia H Lau1†, Elizabeth E Springston2†, Min-Woong Sohn2,3†, Iyana Mason4†, Emily Gadola4†, Maureen Damitz4† and Ruchi S Gupta1,2*† Abstract Background: Illness-related absences have been shown to lead to negative educational and economic outcomes. Both hand washing and hand sanitizer interventions have been shown to be effective in reducing illness-related absences. However, while the importance of hand hygiene in schools is clear, the role of instruction in use is less obvious. The purpose of this study was to compare absenteeism rates among elementary students given access to hand hygiene facilities versus students given both access and short repetitive instruction in use, particularly during influenza season when illness-related absences are at a peak. Methods: A hand hygiene intervention was implemented from October to May during the 2009/2010 academic year, including peak flu season, in two Chicago Public Elementary Schools among students grades pre-kindergarten to eighth grade (ages 4–14). Classrooms were systematically assigned to an intervention or control group by grade (cluster design). Hand hygiene facilities (sanitizer and soap) were made available to all students. Students in the intervention group also received short repetitive instruction in hand hygiene every 2 months. Only absences as a result of respiratory or gastrointestinal illness were used to establish illness-related absenteeism rates. Percent absent days were calculated and bivariate analyses were performed to compare percent absent days among students given access to hand hygiene facilities versus students given both access and instruction. Prior to the intervention, teachers’ perceptions of students’ hand hygiene were also evaluated. Teacher perceptions were analysed to describe attitudes and beliefs. Results: Data were collected and analysed for 773 students reporting 1,886 absences during the study period (1.73% of total school days). Both the percent total absent days and percent illness-related absent days were significantly lower in the group receiving short instruction during flu season (P = 0.002, P < 0.001, respectively). This difference peaked during the influenza season (when intervention began) and declined in the following months. Teachers (n = 23) agreed that hand hygiene is not performed properly among students and reported time constraints as a barrier to frequent hand washing. Conclusions: Adding hand hygiene instruction to existing hand hygiene practices improved attendance at public elementary schools during the flu season. Standardized and brief repetitive instruction in hand hygiene holds potential to significantly reduce absenteeism. Keywords: Hand hygiene, Education, Elementary school, Illness * Correspondence: RUgupta@childrensmemorial.org † Equal contributors 1 Smith Child Health Research Program, Children’s Memorial Hospital, 2300 Children’s Ave, Box 157, Chicago, IL 60614, USA 2 Institute for Healthcare Studies, Northwestern University Feinberg School of Medicine, 750 N Lake Shore Drive, 10th Fl, Chicago, IL 60611, USA Full list of author information is available at the end of the article © 2012 Lau 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. Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 Background Absenteeism is a major problem among school-aged children, with approximately 75% of all school absences attributed to illness [1,2]. Illness-related absences have been shown to lead to negative educational and economic outcomes. For example, a sick child may fall behind in his or her coursework and suffer academically [3,4]. Teacher absences due to exposure may delay learning in the classroom [5,6]. Finally, schools may lose public funding due to absenteeism [7]. As hands are an important mode of transmission of infectious disease among school-aged children, hand hygiene is critical in reducing illness-related absences [8]. Both hand washing and hand sanitizing have been shown effective to this end [9-14]. Hand washing interventions have been shown to significantly reduce illness-related absences in elementary school students by as much as 26% [9], and significantly reduce a subset of illnessrelated absences (i.e. gastrointestinal illnesses) by as much as 32% [10]. Hand sanitizers have been shown to be an effective alternative to conventional hand washing when hands are not visibly dirty, with one study showing a 51% decrease in illness-related absences compared to usual hand washing practices [11-15]. While the importance of hand hygiene among schoolaged children is clear, the role of instruction in use is less obvious. It is unclear if absenteeism is further reduced when access to sink, soap, and hand sanitizer is coupled with regular instruction in hand hygiene. The purpose of this study was to compare absenteeism rates among elementary students given access to hand hygiene facilities versus students given both access and short repetitive instruction in use, particularly during influenza season when illness-related absences are at a peak. Page 2 of 7 Table 1 Demographic characteristics of study participants Variable No. of Children (%) Alcott (N = 502) Walsh (N = 479) Race/ethnicity White Black 271 (54.0) 0 (0.0) 96 (19.1) 29 (6.0) 101 (20.1) 444 (92.7) 34 (6.7) 6 (1.3) Male 215 (42.9) 246 (51.4) Female 287 (57.1) 233 (48.6) 60 (12.0) 40 (8.4) Hispanic/Latino Others Gender Grade Pre-Kindergarten (ages 4–5) Kindergarten (ages 5–6) 61 (12.2) 47 (9.8) First (ages 6–7) 56 (11.2) 39 (8.1) Second (ages 7–8) 49 (9.8) 48 (10.0) Third (ages 8–9) 48 (9.6) 55 (11.5) Fourth (ages 9–10) 49 (9.8) 59 (12.3) Fifth (ages 10–11) 47 (9.8) 49 (10.2) Sixth (ages 11–12) 48 (9.6) 51 (10.6) Seventh (ages 12–13) 46 (9.2) 34 (7.1) Eighth (ages 13–14) 38 (7.6) 57 (11.9) Low Income Students 157 (31.2) 448 (93.5) 32 (6.4) 102 (21.2) Limited English Learners during the influenza season (when intervention began) and declined in the following months (Figure 1a and b). The peak in percent absent days matched the peak in number of influenza-like illnesses in 2009 (both regular and pandemic i.e. H1N1) reported by the City of Chicago [16]. Results Participant characteristics Teachers’ perceptions A total of 981 students were eligible and participated in the study; no students opted out or discontinued participation during the course of the study (Table 1). Data from grades pre-kindergarten and kindergarten were not used in analyses as a result of inconsistent attendance records, giving a final sample of 773 students. A total 1,913 absences were recorded for students in grades 1 through 8 during the study period. Twenty seven recorded absences were not used in analyses due to missing data (i.e., reason and date of absence), for a total of 1,886 data points. Survey responses were received for 23 of 30 teachers in participating schools (Table 4). The majority of respondents agreed that students wash their hands during the school day, but did not believe that students do so properly. Narrative data suggest that students most often wash their hands after restroom breaks and before lunch. However, responses varied widely, with some teachers encouraging students to wash their hands as needed and others limiting hand washing to scheduled bathroom breaks. Both schools reported time constraints as a barrier to frequent hand washing. In response to the H1N1 outbreak of 2009, both schools were supplied with hand sanitizer by Chicago Public Schools prior to the beginning of this study [17]. Hand sanitizer use was reported to be commonplace in both schools, although half of teachers believed their students used hand sanitizer incorrectly. Walsh teachers observed that students only used hand sanitizer before meals and Absenteeism rates Percent total absent days are listed in Table 2; percent illness-related absent days are listed in Table 3. Both the collapsed total rate and collapsed illness-related rate of absenteeism were significantly lower in the intervention groups during influenza season. This difference peaked Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 Page 3 of 7 Table 2 Total absenteeism during the 2009-2010 academic year Both schools Control Intervention Alcott Control Walsh Intervention Control Intervention During the academic year (October to May) Total absence days Total possible days of attendance* % total absent days 879 1,007 539 519 340 488 52,734 56,259 27,777 25,944 24,957 30,315 1.67 1.79 1.94 2.00 1.361 1.611 During influenza season (October to December) Total absence days 365 309 249 186 116 123 Total possible days of attendance* 18,326 19,551 9,653 9,016 8,673 10,535 % total absent days 1.992 1.582 2.581 2.061 1.34 1.17 *Total number of students multiplied by possible days of attendance. 1 P<0.05 2 P<0.01. after recess while teachers at Alcott reported that most students used hand sanitizer as needed. Discussion This study demonstrates that regular hand hygiene instruction may be useful in reducing illness-related absences during the flu season. Flu season is a critical time for attendance improvement, as illness-related absences are traditionally highest during these months [18,19]. Consistent with past research, our findings demonstrate the importance of instruction in improving efficacy of hand hygiene practices at schools [9-15]. As observed, interventions to change hygiene behaviour are plausible among children [20]. Data from teachers suggest that hand hygiene standards vary greatly from school to school, with one exception: hand hygiene is not performed properly among students. Additional barriers to hand hygiene were consistent with those reported in other studies and included time constraints and limited access to materials/facilities [21,22]. Such findings underscore the importance of compulsory instruction in hand washing and sanitizing techniques as well as uniform distribution and access policies. We accordingly recommend a two-part hand hygiene policy in public elementary schools: (1) We advise schools to ensure that all common areas are well-stocked with hand sanitizer and that all bathrooms are wellstocked with hand washing materials throughout the school day. (2) We urge schools to provide a short hand hygiene lesson for students at the beginning of each academic year, as well as refresher lessons throughout the year. This study is not without limitations. The sample was small and convenience-based, resulting in low statistical power. Small sample size may be the reason for correct directionality without statistical significance until results from both schools were analyzed as a whole. Significant differences detected in outcomes were likely conservative due to limitations in the study design: Per request of school administration, we use alcohol-free hand sanitizer rather than alcohol-based hand sanitizer. We also did not have data on influenza vaccination rates of children in the participating schools, and did not attempt to stop Table 3 Illness-related absenteeism during the 2009-2010 academic year Both schools Control Alcott Walsh Intervention Control Intervention Control Intervention 655 692 450 411 215 281 52,734 56,259 27,777 25,944 24,957 30,315 1.26 1.23 1.62 1.58 0.86 0.93 288 224 208 150 80 74 Total possible days of attendance* 18,326 19,551 9,653 9,016 8,673 10,535 % illness-related absent days 1.572 1.152 2.151 1.661 0.92 0.70 During the academic year (October to May) Illness-related absence days Total possible days of attendance* % illness-related absent days During influenza season (October to December) Illness-related absence days *Total number of students multiplied by possible days of attendance. 1 P<0.05 2 P<0.01. Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 Page 4 of 7 Table 4 Itemized perceptions of hand hygiene among teachers prior to the intervention (n = 23) % Total Absent Days a 3.0% Item 2.5% Students’ hand washing practices 2.0% Students wash hands with soap and water during the school day 82.6 1.0% Students in your classroom wash their hands properly 21.7 Barriers prevent you and your students from proper hand washing 50.0 0.5% 0.0% Students’ hand sanitizer practices Control Intervention 1.5% Month % Illness-Related Absent Days b Responses, % Yes Students use hand sanitizer during the school day 91.3 Students in your classroom use hand sanitizer properly 56.5 Relationship between hygiene and illness 2.5% 2.0% 1.5% Students’ poor hygiene habits impact the amount of illness in your classroom 56.5 Availability of hand sanitizer in your classroom would help decrease the amount of illness in your classroom 86.4 Control Intervention 1.0% and, more importantly, reduce student illness and improve students’ academic performance. 0.5% 0.0% Month Figure 1a Percent total absent days during the 2009-2010 academic year. b Percent illness-related absent days during the 2009-2010 academic year. Shaded area = influenza season. children in the intervention group from passing on hand hygiene instruction to children in the control group. Moreover, the intervention was conducted at a time of heightened hand hygiene awareness following the H1N1 outbreak, which likely resulted in more vigilance in hand hygiene among both control and intervention groups. Finally, our analysis did not correct for clustering at the class level and a simple t-test of absenteeism rates in the two groups at the cluster level (n = 31) did not show any significant associations due to lack of statistical power. Accordingly, our results need to be interpreted cautiously. Conclusions The illness-related absenteeism rate decreased during influenza season when hand hygiene instruction was added to existing hand hygiene practices. As a result, we recommend implementation of standardized hand hygiene policies to ensure access to materials/facilities and regular instruction in use. The proposed policy may reduce loss of funding due to illness-related absenteeism, Methods A hand hygiene intervention was implemented from October to May during the 2009/2010 school year in two Chicago Public Elementary Schools among students’ grades pre-kindergarten to 8. The study period included peak flu season, which occurs from October through December [23]. The study protocol was reviewed and approved by the Institutional Review Board of Children’s Memorial Hospital and the Chicago Public Schools Research Review Board in Chicago, Illinois. Participants Eligible students were enrolled in grades pre-kindergarten through eighth (ages 4 –14) at Louisa May Alcott Elementary School or Walsh Math and Science Academy in Chicago, Illinois. Caregivers were given the opportunity to opt their child out of the intervention, with consent implicit in the absence of a signed opt-out form. The intervention was conducted with the consent and support of participating schools. Study design Systematic sampling was used to assign odd grades to the intervention group (n = 15) and even grades to the control group (n = 16) (cluster design). Hand sanitizer and hand washing facilities were made available to students in both the intervention and control group. Dispensers of alcohol-free hand sanitizer (active ingredient, 0.13% Benzalkonium Chloride) were Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 Page 5 of 7 mounted near the doorway inside every classroom and near entrances to common areas, including the main office, bathrooms, lunchrooms, computer laboratories, and gymnasiums. (Hand sanitizer was not placed in restrooms to avoid disruption of hand washing with soap and water.) Posters describing when to use the hand sanitizer were hung up throughout the schools (Figure 2). Children were instructed to use soap and water in lieu of hand sanitizer when hands were visibly dirty. Study personnel conducted weekly checks to ensure that hand sanitizer and soap were properly stocked and available to all students. In addition to these measures, intervention classrooms were given a protocol for hand sanitizer use and received regular instruction in hand hygiene from study personnel. Grade appropriate curriculum was When to Use Hand Sanitizer When Entering School in the Morning Before and After Lunch After Coughing or Sneezing After Recess Before and After Eating in Class After Using the Gym After a School Field Trip When Leaving School for the Day Figure 2 Hand sanitizer use protocol, distributed and implemented in all classrooms. Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 used to instruct students in proper hand washing with an emphasis on the importance of hand hygiene. The curriculum included an initial 30-minute interactive session, which used a black light experiment with glow-in-the-dark “germ” lotion, trivia games (grades 2 through 8), and a demonstration with finger puppets (pre-kindergarten and kindergarten), as well as three 10-minute review sessions every two months, focusing on when, how, and why to use hand hygiene (Figure 3). At the conclusion of the study, control classrooms also received the 30-minute lesson on hand hygiene. The instructions were all in English and all students had a good grasp on the English language. Teachers and instructors made sure all children participated in and understood the lessons. Statistical analysis Per school policy, data were collected documenting each student absence as reported by parents. If reason for absence was not submitted, study personnel called parents to determine reason for absence. For the purposes of this study, study personnel classified a respiratory or gastrointestinal illness as an illness-related absence. Other illnesses or excuses were documented as non-illness- Page 6 of 7 related. Per school policy, however, we were allowed to use class-level aggregate data on study absences. Prior to the intervention, teachers’ perceptions of students’ hand hygiene were also evaluated. Percent total absent days was calculated as the ratio of total absence days to all possible days of attendance. Percent illness-related absent days was calculated as the ratio of illness-related absence days to all possible days of attendance, illness-related or otherwise. Rates were calculated for the overall academic year (October to May) and for influenza season (October to December) [23]. χ2 tests of independence were performed to determine whether the number of absent student-days (total and illness-related) differed significantly between intervention and control groups. To describe teacher perceptions of student hand hygiene, a brief survey was administered prior to intervention and response categories were collapsed into yes/no. Response frequencies were calculated. Qualitative narrative data were also collected and are summarized in the results section. All statistical analyses were performed using Stata/SE 10.0 (Stata Corp LP, College Station, TX) and Microsoft Excel 2010 (Microsoft Corp, Redmond, WA). Figure 3 Overview of hand hygiene curriculum administered in intervention classrooms. Lau et al. BMC Pediatrics 2012, 12:52 http://www.biomedcentral.com/1471-2431/12/52 Competing interests The authors declare that they have no competing interests. Authors’ contributions CHL and MWS performed the statistical analysis and drafted the manuscript. EES designed the study, carried out the intervention, and assisted with interpretation of data and critical review of the manuscript. IM, EG, and MD assisted with the design of the study, carried out the intervention, and critically reviewed the manuscript. RS conceived and designed the study, and performed interpretation of data and critical review of the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank the Stakeholders Collaboration to Improve Student Health and the Chicago Public Schools, especially Principal David J. Domovic and Assistant Principal Laura Watson of Louisa May Alcott Elementary School and Principal Krish Mohip of Walsh Math and Science Academy, for their support and collaboration. Additionally, special thanks to Woodward Laboratories for providing hand sanitizer for the schools. All authors contributed time and effort in kind. Author details 1 Smith Child Health Research Program, Children’s Memorial Hospital, 2300 Children’s Ave, Box 157, Chicago, IL 60614, USA. 2Institute for Healthcare Studies, Northwestern University Feinberg School of Medicine, 750 N Lake Shore Drive, 10th Fl, Chicago, IL 60611, USA. 3Center for Management of Complex Chronic Case, Hines VA Hospital, 5000 South 5th Avenue, Hines, IL 60141, USA. 4Stakeholders Collaboration to Improve Student Health, Respiratory Health Association of Metropolitan Chicago, 1440 W. Washington Blvd., Chicago, IL 60607, USA. Received: 8 June 2011 Accepted: 27 April 2012 Published: 15 May 2012 References 1. Weitzman M, Klerman LV, Lamb G, Menary J, Alpert JJ: School absence: a problem for the pediatrician. Pediatrics 1982, 69:739–746. 2. Neuzil KM, Hohlbein C, Zhu Y: Illness among schoolchildren during influenza season: effect on school absenteeism, parental absenteeism from work, and secondary illness in families. Arch Pediatr Adolesc Med 2002, 156:986–991. 3. Gottfried M: Evaluating the relationship between student attendance and achievement in urban elementary and middle schools: An instrumental variables approach. Am Educ Res J 2010, 47:434–465. 4. Caldas S: Reexamination of input and process factor effects on public school achievement. J Educ Res 1993, 86:206–214. 5. Miller R, Murnane R, Willett J: Do teacher absences impact student achievement? Longitudinal evidence from one urban school district. Educ Eval Policy Anal 2008, 30:181–200. 6. Woods R, Montagno R: Determining the negative effect of teacher attendance on student achievement. Education 1997, 118:307–317. 7. Chicago Public Schools, Office of school management: Attendance improvement program. [http://www.oism.cps.k12.il.us/dept_sm_ai.shtml]. 8. Goldmann DA: Transmission of infectious diseases in children. Pediatr Rev 1992, 13:283–293. 9. Nandrup-Bus I: Mandatory handwashing in elementary schools reduces absenteeism due to infectious illness among pupils: a pilot intervention study. Am J Infect Control 2009, 37:820–826. 10. Master D, Hess Longe SH, Dickson H: Scheduled hand washing in an elementary school population. Fam Med 1997, 29:336–339. 11. Sandora TJ, Shih MC, Goldmann DA: Reducing absenteeism from gastrointestinal and respiratory illness in elementary school students: a randomized, controlled trial of an infection-control intervention. Pediatrics 2008, 121:e1555–e1562. 12. Dyer DL, Shinder A, Shinder F: Alcohol-free instant hand sanitizer reduces elementary school illness absenteeism. Fam Med 2000, 32:633–638. 13. Hammond B, Ali Y, Fendler E, Dolan M, Donovan S: Effect of hand sanitizer use on elementary school absenteeism. Am J Infect Control 2000, 28:340–346. 14. Guinan M, McGuckin M, Ali Y: The effect of a comprehensive handwashing program on absenteeism in elementary schools. Am J Infect Control 2002, 30:217–220. Page 7 of 7 15. Stebbins A, Cummings DAT, Stark JH, Vukotich C, Mitruka K, Thompson W, Rinaldo C, Roth L, Wagner M, Wisniewski SR, Dato V, Eng H, Burke DS: Reduction in the incidence of Influenza A but not Influenza B associated with use of hand sanitizer and cough hygiene in schools: A randomized control trial. Pedriatr Infect Dis J 2011, 30(11):921–926. 16. City of Chicago, Department of Public Health: Communicable Disease Information, February 2010. [www.cityofchicago.org/content/dam/city/ depts/cdph/infectious_disease/Communicable_Disease/ IP_CDInfo_FEB2010_PandemicFlu.pdf]. 17. Chicago Public Schools, Office of Specialized Services: 2009 H1N1 Flu (Swine Influenza) - Facility Operations and Maintenance. [http://www.cps. edu/Programs/Wellness_and_transportation/Documents/ H1N1FacilitiesOperations.pdf]. 18. Schmidt WP, Pebody R, Mangtani P: School absence data for influenza surveillance: a pilot study in the United Kingdom. Euro Surveill 2010, 15: pii: 19467. 19. Mook P, Joseph C, Gates P, Phin N: Pilot scheme for monitoring sickness absence in schools during the 2006/07 winter in England: can these data be used as a proxy for influenza activity? Euro Surveill 2007, 12:E11–E12. 20. Stebbins S, Stark JH, Vukotich CJ Jr: Compliance with a multilayered nonpharmaceutical intervention in an urban elementary school setting. J Public Health Manag Pract 2010, 16:316–324. 21. Guinan ME, McGuckin-Guinan M, Sevareid A: Who washes hands after using the bathroom? Am J Infect Control 1997, 25:424–425. 22. Pete JM: Handwashing practices among various school age students. Health Educ 1986, 17:37–39. 23. Centers for Disease Control and Prevention: Seasonal influenza Questions & answers - 2009-2010 Influenza (Flu) season. [http://www.cdc. gov/flu/about/season/current-season.htm]. doi:10.1186/1471-2431-12-52 Cite this article as: Lau et al.: Hand hygiene instruction decreases illnessrelated absenteeism in elementary schools: a prospective cohort study. BMC Pediatrics 2012 12:52. 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
https://openalex.org/W4308474869
https://estudiosamericanos.revistas.csic.es/index.php/estudiosamericanos/article/download/954/946
Spanish; Castilian
null
Presentación
Anuario de estudios americanos
2,022
cc-by
2,586
1  Scelle, 1906. Saco, 1938. Mellafe, 1959. Cortés López, 2004. 2  Vila Vilar, 1977. 3  Elbl, 1997. 41 Copyright: © 2022 CSIC. Este es un artículo de acceso abierto distribuido bajo los términos de la licen­cia de uso y distribución Creative Commons Reconocimiento 4.0 Internacional (CC BY 4.0). Anuario de Estudios Americanos, 79, 2, Sevilla (España), julio-diciembre, 2022, 413-418 ISSN: 0210-5810 / eISSN: 1988-4273. https://doi.org/10.3989/aeamer.2022.2.01 Anuario de Estudios Americanos, 79, 2, Sevilla (España), julio-diciembre, 2022, 413-418 ISSN: 0210-5810 / eISSN: 1988-4273. https://doi.org/10.3989/aeamer.2022.2.01 4  Wheat, 2011.   5  Otte y Ruiz-Berruecos, 1963; Otte, 1965. Garcia, 1962. Ramos Pérez, 1976. Vila Vilar, 1977. Ventura, 1999.   6  Fernández Chaves y Pérez García, 2012.   7  Fernández Chaves, 2022a.   8  Fernández Chaves, 2021; 2018a; 2019.   9  Fernández Chaves, 2018b. 10  Fernández Chaves 2022b. 11  Fernández Chaves y Pérez García, 2016. 12  Ortiz Arza, 2019. Presentation Manuel F. Fernández Chaves Universidad de Sevilla, España / mfernandez6@us.es ORCID iD: https://orcid.org/0000-0002-1030-0555 Rafael M. Pérez García Universidad de Sevilla, España / rperez4@us.es ORCID iD: https://orcid.org/0000-0002-3558-5104 Manuel F. Fernández Chaves Universidad de Sevilla, España / mfernandez6@us.es ORCID iD: https://orcid.org/0000-0002-1030-0555 Manuel F. Fernández Chaves Universidad de Sevilla, España / mfernandez6@us.es ORCID iD: https://orcid.org/0000-0002-1030-0555 Rafael M. Pérez García Universidad de Sevilla, España / rperez4@us.es ORCID iD: https://orcid.org/0000-0002-3558-5104 La historia del tráfico atlántico de esclavos en el siglo XVI ha recibido una atención secundaria en comparación con los siglos posteriores. Desde las ya centenarias obras de Georges Scelle y José Antonio Saco, diversos libros aparecidos en las últimas décadas, desde Rolando Mellafe a José Luis Cortés López, han vuelto a presentar un panorama general de la trata hacia las Indias de Castilla durante dicha centuria, aunque partiendo siempre de un repertorio delimitado de fuentes y pasando por alto numerosos elemen­ tos que son necesarios para conocer sus particularidades.1 Por su parte, la monografía de Enriqueta Vila Vilar constituyó en su día un jalón insoslaya­ ble para el conocimiento del periodo de los asientos portugueses.2 Este monográfico está dedicado a los años que van de 1509 a 1595, y que se conoce tradicionalmente como «periodo de las licencias» por estar regulado el envío de esclavos a América mediante la concesión de licencias por la Corona española. Aún hoy, constituye el tiempo peor conocido de la historia de la trata de esclavos hacia el Nuevo Mundo. En los últimos veinticinco años una serie de trabajos han construido un conocimiento más preciso sobre el siglo XVI. Entre ellos destaca el artículo pionero de Elbl, publicado en 1997, sobre el volumen de las primeras tratas atlánticas por­ tuguesas entre 1450 y 1521, que constituye un referente fundamental para la reconstrucción histórica de esta primera fase esclavista.3 Para el periodo de 1570 a 1640 y la trata hacia Cartagena de Indias, el trabajo seminal de 413 MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA 4  Wheat, 2011.   5  Otte y Ruiz-Berruecos, 1963; Otte, 1965. Garcia, 1962. Ramos Pérez, 1976. Vila Vilar, 1977. Ventura, 1999. 12  Ortiz Arza, 2019. 11  Fernández Chaves y Pérez García, 2016. MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA Wheat aparecido en 2011 ha estudiado con mayor precisión e información documental la transición a la época de los asientos portugueses.4 Otra serie de publicaciones han ido precisando las distintas coyunturas de la trata den­ tro del Quinientos a través del estudio de los principales grupos económicos que se hicieron con su control. Los trabajos iniciales se centraron en el estudio de los hombres de negocios portugueses, como los de Otte, Garcia, Ramos, Vila Vilar y Ventura,5 a los que se han unido últimamente los de Fernández Chaves. A un estudio inicial sobre la penetración de los tratantes portugueses en la economía de Sevilla y la Carrera de Indias a mediados del siglo XVI,6 han seguido otros sobre la persona de Blas Reynel,7 padre del futuro primer asentista de esclavos en 1595, Pedro Gomes Reynel, y sobre la familia de los Caldeira y sus agentes en el Caribe y Sevilla durante las décadas siguientes.8 Asimismo, el estudio de los contratos de los ríos de Guinea9 y Angola10 para los años 1574-1580 y 1594-1600, ha profundizado en la perspectiva desde África para explicar con mayor precisión la transi­ ción desde la fase de las licencias a los asientos. Hasta fechas recientes no se había prestado atención sistemática al rol desempeñado por los mercaderes judeoconversos andaluces en la trata de esclavos. En 2016 pusimos de manifiesto cómo entre las décadas de 1530 y 1550 llegaron a ser dominantes en el control del tráfico de esclavos hacia América, sirviendo de transición entre el momento inicial que comienza en 1518-1519, dirigido por mercaderes italianos y burgaleses, y la fase si­ guiente, que se abre a mediados de la centuria, caracterizada por el predo­ minio portugués.11 Más allá del dominio coyuntural de unos u otros grupos, siempre existieron grandes consorcios y redes internacionales de capita­ listas que sostuvieron la trata, entre los que nunca faltaron los andaluces y portugueses, además de italianos y vascos. Este último grupo ha merecido un estudio monográfico por parte de Ortiz Arza.12 También se ha profundizado en los últimos años en otros aspectos fundamentales de la trata atlántica. Así, el sistema de licencias en tanto 414 Anu. estud. am., 79, 2, jul.-dic., 2022, 413-418. ISSN: 0210-5810 / eISSN: 1988-4273. MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA https://doi.org/10.3989/aeamer.2022.2.01 PRESENTACIÓN que mecanismo de gestión de la trata y como medio para su cuantificación; la fiscalidad sobre la misma; el estudio de los barcos, rutas y destinos; los espacios africanos de consecución de los esclavos; etc.13 En toda esta nueva historiografía se contiene una enorme información que permite asimismo afinar y completar las estimaciones cuantitativas que para el siglo XVI ofre­ ce la Trans-Atlantic Slave Trade Database (TSTD2).14 Los artículos reunidos en este monográfico ayudan a completar el pa­ norama presentado. Rafael M. Pérez García estudia de manera precisa las primeras grandes compañías que inventaron la trata negrera hacia las Indias a partir de 1519. Fueron capitalistas de Génova y Burgos los que con su acción dibujaron una primera ruta inicial desde Arguim al Caribe a partir de 1519, que rápidamente fue sustituida desde 1522 por una nueva desde Santo Tomé llamada a perpetuarse durante el resto de la centuria. Este es­ tudio presenta además una vertiente económica que permite conocer por primera vez la organización, costes y beneficios de la trata transatlántica en sus fases más tempranas. Por su parte, Manuel F. Fernández Chaves estudia un contrato inédi­ to de Santo Tomé firmado por el gran mercader y banquero milanés Juan Bautista Rovelasca para abastecer de esclavos la América española durante los años 1583-1589. A través de su análisis se muestra la gestión de la trata de esclavos y de las rentas de la isla asociadas al contrato, permitiéndonos conocer las mercancías involucradas en la trata, el ritmo de salida de las embarcaciones y los beneficios finales, tanto del contrato como de la activi­ dad esclavista, en el periodo ya de decadencia de dicha actividad en la isla. La crisis del ciclo de Santo Tomé guarda relación con el despegue de Angola como nuevo centro fundamental de captura y aprovisionamiento de esclavos destinados a América. Este proceso es estudiado por David Wheat y Kara Schultz, quienes señalan una serie de etapas (1575-1579, 1579-1587, 1587-1595) a través de las cuales emerge con claridad una nueva geografía de la trata atlántica centrada en Angola, cuyas características son nuevas y diferentes de las de otros centros de aprovisionamiento en África, conside­ rando en su trabajo la demanda tanto de Brasil como de la América española. 13  Entre otros, Pérez García, 2015. Eagle, 2018. Ortiz Arza, 2020. Pérez García 2021. Wolff, 2021. 14  Accesible a través de https://www.slavevoyages.org [Consultado: 20/08/2022]. 415 13  Entre otros, Pérez García, 2015. Eagle, 2018. Ortiz Arza, 2020. Pérez García 2021. Wolff, 2021. 14  Accesible a través de https://www.slavevoyages.org [Consultado: 20/08/2022]. MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA El artículo de Hilario Casado Alonso aporta al conocimiento de la trata hacia la América del siglo XVI la novedad de la utilización de una fuente escasamente empleada para esta cronología: las pólizas de seguros 415 MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA marítimos. En este caso se estudian las contratadas por el mayor asegurador de Amberes, Juan Enríquez, durante los años 1562 y 1563, así como las pólizas consultadas en el archivo del Consulado de Burgos entre los años 1565 a 1573. El análisis de esta documentación muestra otra faceta de la complejidad de la trata atlántica, y pone en evidencia sus interrelaciones profundas con los grandes centros económicos europeos de la época. Por último, el trabajo de Rafael M. Girón Pascual ofrece el estudio de caso de la red comercial del mercader burgalés Diego de Polanco que desde el Cádiz de finales del siglo XVI muestra cómo se conectaban las econo­ mías del norte de Europa, Italia, norte de África, península ibérica y archi­ piélagos atlánticos con América. En este artículo, el tráfico de esclavos se contextualiza en el marco de una larga serie de transacciones y operaciones económicas que envolvían multitud de productos muy diferentes, que iban desde los textiles y manufacturas italianas, el azúcar, los vinos andaluces o la lana. Los artículos de este monográfico representan nuevas aportaciones que completan de manera relevante el panorama historiográfico actual, haciendo hincapié en la recuperación de la especificidad histórica del si­ glo XVI, al insertar la historia de estas tratas esclavistas en el seno de la nueva economía atlántica. PRESENTACIÓN Fernández Chaves, Manuel F., «El contrato de arrendamiento de “los tratos de todos los Ríos de Guinea y las islas de Buan” de 1574-1580. Análisis y edición» en Pérez García, Rafael M.; Fernández Chaves, Manuel F. y Belmonte Postigo, José Luis (coords.), Los negocios de la esclavitud. Tratantes y mercados de esclavos en el Atlántico ibérico, siglos XV-XVIII, Sevilla, Universidad de Se­ villa, 2018b, 93-120. Fernández Chaves, Manuel F., «Capital y confianza. Enrique Freire, factor de los tratantes de esclavos portugueses, 1574-1577», en Fernández Chaves, Ma­ nuel F.; Pérez García, Rafael. M. y Perez, Béatrice (coords.), Mercaderes y redes mercantiles en la Península Ibérica. Siglos XV-XVIII, Lisboa/Sevilla/ París, Editorial Universidad de Sevilla / Cátedra Alberto Benveniste / Éditions Hispaniques, 2019, 303-329. Fernández Chaves, Manuel F., «Manuel Caldeira y la trata de esclavos en el Caribe, 1556-1562», en Naranjo Orovio, Consuelo (ed.), Sometidos a esclavitud: los africanos y sus descendientes en el Caribe hispano, Santa Marta, Unimagda­ lena, 2021, 48-88. Fernández Chaves, Manuel F., «Negocios de cristãos-novos en Sevilla a finales del reinado de Carlos I. El caso de Blas Reynel, 1548-1555», en Andrade, An­ tónio M. L.; Gomes, Saúl A.; Reis, M. Fátima (coords.), Diálogos Luso-Se­ farditas (Suplemento n.º 6 da revista Ágora. Estudos Clássicos em Debate), 2022a, Aveiro, 288-325. Disponible en: https://doi.org/10.48528/d9rp-7a51 Fernández Chaves, Manuel F., «El “trato e avenencia del reino de Angola para el Brasil e Indias de Castilla” de 1594-1600. Gestión y organización de la trata de esclavos en una época de transición», Revista de Indias, 82, Madrid, 2022b, 9-44. Disponible en: https://doi.org/10.3989/revindias.2022.001 Fernández Chaves, Manuel F. y Pérez García, Rafael M., «La penetración econó­ mica portuguesa en la Sevilla del siglo XVI», Espacio, Tiempo y Forma. Se­ rie IV, Historia Moderna, 25, Madrid, 2012, 199-222. Disponible en: https:// doi.org/10.5944/etfiv.25.2012.11953 Fernández Chaves, Manuel F. y Pérez García, Rafael M., «La élite mercantil ju­ deoconversa andaluza y la articulación de la trata negrera hacia las Indias de Castilla, ca. 1518-1560», Hispania, 76, Madrid, 2016, 385-414. Disponible en: https://doi.org/10.3989/hispania.2016.012 Garcia, Rozendo Sampaio, «Contribuição ao estudo do aprovisonamento de escra­ vos negros na America espanhola (1580-1640)», Anais do Museu Paulista, 36, São Paulo, 1962, 1-195. Mellafe, Rolando, La introducción de la esclavitud negra en Chile. Tráfico y rutas, Santiago de Chile, Universidad de Chile, 1959. Ortiz Arza, Javier, La comunidad vasca de Sevilla y la trata de esclavos (s. XVI), tesis doctoral, dirigida por Manuel F. Referencias bibliográficas Cortés López, José Antonio, Esclavo y colono. Introducción y sociología de los ne­ groafricanos en la América española del siglo XVI, Salamanca, Universidad de Salamanca, 2004. Eagle, Marc, «Tiempos contrarios: arribadas de barcos negreros en la isla Españo­ la, siglo XVI», en Pérez García, Rafael M.; Fernández Chaves, Manuel F. y Belmonte Postigo, José Luis (coords.), Los negocios de la esclavitud. Tratan­ tes y mercados de esclavos en el Atlántico ibérico, siglos XV-XVIII, Sevilla, Universidad de Sevilla, 2018, 121-134. Elbl, Ivana, «The Volume of the Early Atlantic Slave Trade, 1450-1521», Journal of African History, 38, Cambridge, 1997, 31-75. Disponible en: https://doi. org/10.1017/s0021853796006810 Fernández Chaves, Manuel F., «La consolidación del capitalismo portugués en Se­ villa. Auge, caída y resurgir político del mercader Bento Váez, 1550-1580», en Iglesias Rodríguez, Juan José; García Bernal, J. Jaime y Díaz Blanco, José Manuel (eds.), Andalucía en el mundo atlántico moderno. Ciudades y redes, Madrid, Sílex, 2018a, 183-238. 416 Anu. estud. am., 79, 2, jul.-dic., 2022, 413-418. ISSN: 0210-5810 / eISSN: 1988-4273. https://doi.org/10.3989/aeamer.2022.2.01 PRESENTACIÓN Fernández Chaves y José Ramón Díaz de Durana Ortiz de Urbina, Vitoria, Universidad del País Vasco, 2019. Dispo­ nible en: http://hdl.handle.net/10810/42816 417 Anu. estud. am., 79, 2, jul.-dic., 2022, 413-418. ISSN: 0210-5810 / eISSN: 1988-4273. https://doi.org/10.3989/aeamer.2022.2.01 MANUEL F. FERNÁNDEZ CHAVES Y RAFAEL M. PÉREZ GARCÍA Ortiz Arza, Javier, «Los libros-registro de esclavos, el sistema de licencias y la trata con Hispanoamérica en el siglo XVI: análisis y aspectos metodológicos a través del caso vasco», Anuario de Estudios Americanos, 77, Sevilla, 2020, 579-611. Disponible en: https://doi.org/10.3989/aeamer.2020.2.07 Otte, Enrique, «Die Negersklavenlizenz des Laurent de Gorrevod. Kastilisch-ge­ nuesiche Wirtschafts- und Finanzinteressen bei der Einführung der Negers­ klaverei in Amerika», Spanische Forschungen der Görresgesellschaft, 22, Münster, 1965, 283-320. Otte, Enrique y Ruiz-Berruecos, Conchita, «Los portugueses en la trata de esclavos negros de las postrimerías del siglo XVI», Moneda y Crédito, 85, Madrid, 1963, 3-40. Pérez García, Rafael M., «Metodología para el análisis y cuantificación de la trata de esclavos hacia la América española en el siglo XVI», en Rey Castelao, Ofe­ lia y Suárez Golán, Francisco (eds.), Los vestidos de Clío. Métodos y tenden­ cias recientes de la historiografía modernista española (1973-2013), Santiago de Compostela, Universidad de Santiago de Compostela, 2015, 823-840. Pérez García, Rafael M., «El Caribe y la génesis del mercado negrero atlántico, ca. 1518-1540», en Naranjo Orovio, Consuelo (ed.), Sometidos a esclavitud: los africanos y sus descendientes en el Caribe hispano, Santa Marta, Edito­ rial Unimagdalena, 2021, 21-46. Ramos Pérez, Demetrio, «El negocio negrero de los Welser y sus habilidades mo­ nopolísticas», Revista de Historia de América, 81, San José de Costa Rica, 1976, 7-81. Saco, José Antonio, Historia de la esclavitud de la raza africana en el Nuevo Mun­ do y en especial en los países américo-hispanos, 4 vols., La Habana, Cultu­ ral, 1938. Scelle, Georges, Histoire politique de la traite négrière aux Indes de Castille, 2 vols., París, 1906. Ventura, Maria da Graça Mateus, Negreiros portugueses na rota das Índias de Cas­ tela: 1541-1556, Lisboa, Edições Colibri, 1999. Vila Vilar, Enriqueta, Hispanoamérica y el comercio de esclavos, Sevilla, Escuela de Estudios Hispano-Americanos, 1977. Wheat, David, «The first Great Waves: African provenance zones for the Transat­ lantic Slave Trade to Cartagena de Indias, 1570-1640», Journal of African History, 52, Cambridge, 2011, 1-22. Disponible en: https://doi.org/10.1017/ s0021853711000119 Wolff, Jennifer, «Enmaranhado: Puerto Rico y el comercio trasatlántico de escla­ vos, 1580-1630», en Naranjo Orovio, Consuelo (ed.), Sometidos a esclavi­ tud: los africanos y sus descendientes en el Caribe hispano, Santa Marta, Editorial Unimagdalena, 2021, 117-159. Recibido, 8 de agosto de 2022 Aceptado, 20 de agosto de 2022 418 Anu. estud. am., 79, 2, jul.-dic., 2022, 413-418. ISSN: 0210-5810 / eISSN: 1988-4273. https://doi.org/10.3989/aeamer.2022.2.01
https://openalex.org/W3203199982
https://periodicos.ifrs.edu.br/index.php/REMAT/article/download/4909/3002
Portuguese
null
O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças
Remat
2,021
cc-by
7,311
1 Currículo sucinto: Licenciada em Pedagogia e mestranda do Programa de Educação da Universidade Federal do Pampa. Contribuição de autoria: Administração do Projeto, Análise Formal, Conceituação, Curadoria de Dados, Escrita – Primeira Redação, Investigação, Metodologia. Contato: lutiele.luna@gmail.com. 2 Currículo sucinto: Licenciado em Matemática pela Universidade Estadual do Norte do Paraná, Licenciado em Pedagogia pela Universidade Estadual de Maringá, Mestre em Educação Científica e Tecnológica pela Universidade Federal de Santa Catarina, Doutor em Educação pela Universidade de São Paulo. Docente da Universidade Federal do Pampa. Contribuição de autoria: Administração do Projeto, Escrita – Revisão e Edição, Supervisão, Validação e Visualização. Contato: joaomoraes@unipampa.edu.br. Keywords: number concept; children; State of Knowledge; Mathematics Education. Data de submissão: 23 de Fevereiro de 2021. Data de aprovação: 27 de Junho de 2021. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. oncept of number in Mathematics Education: a study in research with children Lutieli Rodrigues Botelho1 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0002-0600-7428, http://lattes.cnpq.br/1428630152652865 João Carlos Pereira de Moraes2 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 Lutieli Rodrigues Botelho1 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0002-0600-7428, http://lattes.cnpq.br/1428630152652865 Lutieli Rodrigues Botelho1 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0002-0600-7428, http://lattes.cnpq.br/1428630152652865 João Carlos Pereira de Moraes2 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 João Carlos Pereira de Moraes2 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 1 Currículo sucinto: Licenciada em Pedagogia e mestranda do Programa de Educação da Universidade Federal do Pampa. Contribuição de autoria: Administração do Projeto, Análise Formal, Conceituação, Curadoria de Dados, Escrita – Primeira Redação, Investigação, Metodologia. Contato: lutiele.luna@gmail.com. 2 Currículo sucinto: Licenciado em Matemática pela Universidade Estadual do Norte do Paraná, Licenciado em Pedagogia pela Universidade Estadual de Maringá, Mestre em Educação Científica e Tecnológica pela Universidade Federal de Santa Catarina, Doutor em Educação pela Universidade de São Paulo. Docente da Universidade Federal do Pampa. Contribuição de autoria: Administração do Projeto, Escrita – Revisão e Edição, Supervisão, Validação e Visualização. Contato: joaomoraes@unipampa.edu.br. 1 Currículo sucinto: Licenciada em Pedagogia e mestranda do Programa de Educação da Universidade Federal do Pampa. Contribuição de autoria: Administração do Projeto, Análise Formal, Conceituação, Curadoria de Dados, Escrita – Primeira Redação, Investigação, Metodologia. Contato: lutiele.luna@gmail.com. 2 João Carlos Pereira de Moraes Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 REMAT: Revista Eletrônica da Matemática 2 1. Introdução Neste estudo, realizamos uma revisão sistemática de literatura, visando levantar estudos relacionados ao conceito de número. Nesse sentido, consideramos este estudo um estado de conhecimento, como aponta Romanowski e Ens (2006). Segundo os pesquisadores, o estado do conhecimento diferencia-se do estado da arte por abordar um grupo de setores mais restritos de publicações a respeito de um determinado tema estudado (ROMANOWSKI; ENS, 2006). Embora mais limitado, o estado do conhecimento não impede a visualização de elementos constituintes sobre um determinado assunto, bem como suas lacunas e possibilidades. Mediante ao exposto, o objetivo desse estado de conhecimento consiste em analisar como o debate sobre o conceito de número tem sido abordado em pesquisas com crianças de Educação Infantil e anos iniciais do Ensino Fundamental, a partir de seus objetivos, metodologias de pesquisa e ensino e inferências. A partir disso, elaboramos as seguintes etapas da pesquisa: (1) Levantamento em bases de dados definidas a priori; (2) Seleção de trabalhos relacionados com a temática de pesquisa; (3) Análise e classificação dos trabalhos quanto ao ano, natureza e região do país; (4) Análise dos objetivos, metodologias e inferências dos trabalhos. Essa sequência é apresentada de maneira mais descritiva abaixo. 2. Aspectos metodológicos da composição do Estado de Conhecimento Nesta seção, abordamos a revisão sistemática produzida como forma de organizar e produzir o estado do conhecimento relativo ao trabalho com conceito de número na Educação Infantil e anos iniciais do Ensino Fundamental. João Carlos Pereira de Moraes Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 Universidade Federal do Pampa (UNIPAMPA), Jaguarão, RS, Brasil https://orcid.org/0000-0001-9513-018X, http://lattes.cnpq.br/5624816456388130 Resumo: Essa pesquisa objetiva analisar como o debate sobre o conceito de número tem sido abordado em pesquisas com crianças de Educação Infantil e anos iniciais do Ensino Fundamental, a partir de seus objetivos, metodologias de pesquisa, ensino e inferências. Nesse sentido, produzimos um levantamento em três bancos de dados: BDTD, CTD e ENEM, o que suscitou quinze estudos. A análise dos dados foi feita a partir da metodologia de Análise de Conteúdo (BARDIN, 2011). A partir da análise, levantamos que as pesquisas concentram-se na região Sudeste, em sua maioria são dissertações e há um indício de crescimento dos estudos nos últimos cinco anos. Além disso, quanto aos objetivos de pesquisa, notamos que mesmo as pesquisas envolverem sujeitos crianças, há influência de discussões no campo de ensino. No que se refere às metodologias de ensino e pesquisa, o uso de sequência de atividades e o estudo de caso tem a sua maior evidência. Já no que tange as inferências, notamos que muitas ainda são genéricas e com poucas considerações sobre o conceito de número. Palavras-chave: conceito de número; crianças; Estado de Conhecimento; Educação Matemática. Abstract: This research aims to analyze how the debate on the concept of number has been approached in research with children from early childhood education and early years of elementary school; based on its objectives, research and teaching methodologies and inferences. In this sense, we produced a survey in three databases: BDTD, CTD and ENEM, which led to fifteen studies. Data analysis was performed using Content Analysis methodology (BARDIN, 2011). While analyzing the material, we found out that the research is concentrated in the Southeast region, they are, in most part, dissertations, and there is an indication of growth in these studies in the last five years. In addition, regarding the research objectives, we observed that even though the research involves young subjects such as children, there is an influence of discussions in the teaching field. With regard to teaching and research methodologies, the use of sequence of activities and case study have the greatest role. Concerning the inferences, we noticed that many are still generic and with few considerations with respect to the concept of number. Keywords: number concept; children; State of Knowledge; Mathematics Education. Data de submissão: 23 de Fevereiro de 2021. Data de aprovação: 27 de Junho de 2021. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 2.1. Metodologia da revisão Inicialmente para a pesquisa, elegemos duas bases de dados: (1) Biblioteca Digital de Teses e Dissertações (BDTD) e o Catálogo de Teses e Dissertações da Capes (CTD). Nesse momento, utilizamos os descritores “conceito de número” AND “Educação Matemática”. Ao total, encontramos cinquenta e nove (59) produções nas duas bases. Neste grupo e a partir da leitura de títulos e resumos, realizamos a seleção daqueles com sujeitos de pesquisa crianças da Educação Infantil e/ou alunos dos anos iniciais do Ensino Fundamental, o que configurou num total de doze (12) dissertações e teses. Como forma de complementar e de ampliar o corpus de análise, levantamos pesquisas sobre o conceito de número nos últimos dez anos do Encontro Nacional de Educação Matemática – ENEM, do X ao XIII. A busca foi realizada a partir do descritor “conceito de número”, apenas nas REMAT: Revista Eletrônica da Matemática 3 comunicações orais, uma vez que acreditamos que nelas são encontradas as pesquisas sobre o assunto. Ao final da busca, encontramos somente três (3) artigos. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 2.2. Resultado da pesquisa A seguir, apresentamos a lista dos estudos encontrados a partir da pesquisa elencada anteriormente. Ao final, totalizamos quinze (15) trabalhos (dissertações, teses e artigos). Como mostra a classificação do Quadro 1, levantamos inicialmente as seguintes informações: natureza, título, autor e ano de publicação. Quadro 1 – Trabalhos levantados Termo* Natureza Título Autor Ano T1 Dissertação A aprendizagem do conceito de número de crianças do Infantil V: interações com o flex memo Belo 2018 T2 Dissertação A construção do conceito de número por uma aluna com surdocegueira congênita Aleixo 2018 T3 Dissertação O ensino do conceito de número: objetivações nas proposições davydovianas e formalista moderna Souza 2013 T4 Tese O senso numérico da criança: formação e características Ferrari 2008 T5 Dissertação Número: o conceito a partir de jogos Fonseca 2005 T6 Dissertação Investigações sobre números naturais e processos de ensino e aprendizagem desse tema no início da escolaridade Bonaldo 2007 T7 Dissertação A construção do número pela criança com deficiência intelectual: a percepção entre diferentes ambientes escolares Noleto 2017 T8 Tese Investigação da construção do número em LIBRAS: estudo com crianças surdas Madalena 2017 T9 Tese Uma abordagem multissensorial para o desenvolvimento do conceito de número natural em indivíduos com síndrome de down Yokoyama 2012 T10 Dissertação Representação semiótica: uma perspectiva para a construção do conceito de número na educação infantil Dinis 2018 T11 Dissertação Uma proposta de software de educação matemática para educação infantil Boscariol 2004 T12 Dissertação Uma proposta para utilização do computador no processo de ensino e aprendizagem dos primeiros números do sistema hindu-arábico Silva 1998 T13 Artigo Percepções de crianças do 1º ano do Ensino Fundamental sobre o conceito de números Guillen e Sousa 2010 T14 Artigo Análises no processo de construção do conceito de número de alunos do 1º ano do Ensino Fundamental em uma prática colaborativa Santos, Manfredo e Costa 2019 T15 Artigo Correspondência um a um: uma situação desencadeadora de aprendizagem voltada ao conceito de número Binsfeld, Golin e Klein 2019 *Observação: para otimizar a análise dos dados, cada trabalho recebeu a nomenclatura T e um número, conforme a ordem levantada. Fonte: Dados da pesquisa (2020). Com os dados coletados, elaboramos uma análise mais aprofundada de cada estudo, sendo essa sistemática apresentada a seguir. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 2.2. Resultado da pesquisa 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. REMAT: Revista Eletrônica da Matemática 4 4 3. Resultados e discussões Nesta seção, realizamos a análise dos aspectos gerais e das questões norteadoras, a partir da leitura dos trabalhos. 2.3. Análise dos dados Os trabalhos acima elencados foram analisados em dois momentos. Primeiramente, analisamos os aspectos de modo geral, levando em consideração o ano, natureza e região do país. Em segundo momento, a análise ocorreu a partir da metodologia Análise de Conteúdo. Segundo Bardin (2011), a Análise de Conteúdo é um processo de análise que classifica em categorias e, ao longo do tempo, permite que se realizem inferências, deixando de ser apenas um ato descritivo sobre os dados. Como elemento de análise, utilizaram-se as seguintes perguntas norteadoras como categorias a priori:  P1: Qual é o objetivo do trabalho de pesquisa?  P2: Quais as abordagens metodológicas usadas para o ensino do conceito de  P3: Quais as metodologias de pesquisa utilizadas nos trabalhos de pesquisa?  P4: Quais as principais inferências levantadas nos estudos? A partir dessas perguntas, analisamos os dados em cada trabalho até então encontrado na revisão sistemática, dividindo em quatro categorias: (1) objetivos de pesquisa; (2) abordagens metodológicas de ensino; (3) abordagens metodológicas na pesquisa; e (4) inferências levantadas. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.1. Aspectos gerais REMAT: Revista Eletrônica da Matemática 6 Nesta seção, percebemos por meio da análise realizada que os trabalhos estão concentrados na região Sudeste e são em sua maioria dissertações. A seguir, apresentamos nossas análises a partir das perguntas norteadoras. Nesta seção, percebemos por meio da análise realizada que os trabalhos estão concentrados na região Sudeste e são em sua maioria dissertações. A seguir, apresentamos nossas análises a partir das perguntas norteadoras. 3.1. Aspectos gerais Entre os quinze trabalhos elencados anteriormente, levaremos em consideração alguns aspectos mais gerais como: região, natureza e ano. No Quadro 2, apresentamos as regiões nas quais cada trabalho foi publicado. Quadro 2 – Trabalhos por Regiões de publicação Região Quantidade Trabalhos Norte 1 T13 Nordeste 1 T1 Centro-Oeste 3 T7, T14 e T15 Sudeste 8 T4, T5, T6, T8, T9, T10, T11, T12 Sul 2 T2 e T3 Fonte: Dados da pesquisa (2020). A partir dessa busca, percebemos que existem produções em todas as regiões brasileiras, contudo encontramos um número maior de trabalhos publicados na região Sudeste (8). Para REMAT: Revista Eletrônica da Matemática 5 Fernandes, Moraes e Pereira (2020), essa diferenciação ocorre em decorrência da concentração dos cursos de mestrado e doutorado na região Sudeste, bem como o alto número de pesquisadores. No Quadro 3, distribuímos os trabalhos encontrados por sua natureza. Quadro 3 – Natureza dos trabalhos encontrados Natureza Quantidade Trabalhos Dissertação 9 T1, T2, T3, T5, T6, T7, T10, T11, T12 Tese 3 T4, T8, e T9 Artigo 3 T13, T14 e T15 Fonte: Dados da pesquisa (2020). Fonte: Dados da pesquisa (2020). A partir do levantamento, notamos que os trabalhos elencados são em sua maioria dissertações, totalizando nove (9) ao todo. Consideramos que o maior número de dissertações possa ser reflexo da aproximação da natureza deste tipo de pesquisa com a prática docente na Educação Básica, bem como o número de mestrados de cunho profissional no Brasil. Já no Quadro 4, mostramos a separação dos estudos perante o ano de publicação de cada trabalho. Quadro 4 – Ano de publicação dos trabalhos levantados Ano Quantidade Trabalhos 1998 1 T12 2004 1 T11 2005 1 T5 2007 1 T6 2008 1 T4 2010 1 T13 2012 1 T9 2013 1 T3 2017 2 T7 e T8 2018 3 T1, T2 e T10 2019 2 T14 e T15 Fonte: Dados da pesquisa (2020). Fonte: Dados da pesquisa (2020). Nos bancos de dados, encontramos trabalhos num período de mais de vinte (20) anos, entre 1998 e 2019. Se realizarmos o agrupamento dos anos por quinquênio, perceberemos o aumento de estudos no período de 2016-2020, principalmente no ano de 2018, com três (3) estudos. Nesse sentido, consideramos a possibilidade de uma preocupação maior nos últimos anos com a aprendizagem do conceito de número na Educação Infantil e nos anos iniciais do Ensino Fundamental. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.2. Categoria 1: Objetivos de pesquisas Na primeira categoria ressaltamos os objetivos de pesquisa dos trabalhos encontrados. Para melhor organização, construímos um quadro dividindo os objetivos em dois grupos: (1) o primeiro grupo é dos trabalhos com objetivo voltados para o ensino e (2) o segundo com os objetivos voltados para a aprendizagem dos alunos. Quadro 5 – Objetivos dos trabalhos Grupo Quantidade Trabalhos Objetivos de ensino 8 T1, T3, T5, T10, T11, T12, T14 e T15 Objetivos de aprendizagem 7 T2, T4, T6, T7,T8, T9 e T13, Fonte: Dados da pesquisa (2020). No primeiro grupo, objetivos de ensino, temos aqueles trabalhos mais direcionados para o ensino do conceito de números, bem como o foco nas metodologias de ensino. Neste grupo, temos aqueles voltados para o âmbito geral: Neste grupo, temos aqueles voltados para o âmbito geral: T3: Analisamos duas propostas de ensino: a davydoviana e a formalista moderna, no que se refere à introdução do conceito de número, no primeiro ano do Ensino Fundamental. (SOUZA, 2013, p. 8, grifo nosso). T3: Analisamos duas propostas de ensino: a davydoviana e a formalista moderna, no que se refere à introdução do conceito de número, no primeiro ano do Ensino Fundamental. (SOUZA, 2013, p. 8, grifo nosso). T5: Investigar uma nova abordagem para o conceito de número, com vistas a buscar nela elementos que favoreçam o ensino e consequentemente a aprendizagem. (FONSECA, 2005, p. 9, grifo nosso). T14: Investigar processos cognitivos na construção do conceito de número de alunos do 1º ano do Ensino Fundamental por meio de um trabalho colaborativo com os professores. (SANTOS; MANFREDO; COSTA, p. 7, grifo nosso). A partir dos objetivos, observamos que as propostas são mais genéricas. Enquanto T3 compara duas propostas de ensino, a davydoviana e a formalista moderna, T5 procura abordagens que favoreçam o ensino do conceito de número e T14 enfatiza os processos cognitivos para pensar uma formação docente colaborativa. No interior do primeiro grupo ainda, estão àqueles trabalhos voltados para as questões metodológicas, como: T1: Analisar a contribuição da interação com os jogos de Memória, Segredo da Caixa e Batalha proporcionados pelo Flex memo para a ampliação do conceito de número de crianças do infantil V. (BELO, 2018, p. 10, grifo nosso). REMAT: Revista Eletrônica da Matemática 7 (softwares), que possam auxiliar o processo de ensino e aprendizagem do sistema de numeração hindu-arábico, em crianças na fase de alfabetização. (SILVA, 1998, p. 9, grifo nosso). 3.2. Categoria 1: Objetivos de pesquisas REMAT: Revista Eletrônica da Matemática 8 T7: Analisar os processos mentais associados à construção do número desenvolvidos na sala de aula e na sala de recursos por uma criança com deficiência intelectual em fase de alfabetização. (NOLETO, 2017, p. 9, grifo nosso). T8: Investigar a aquisição de conhecimentos numéricos de crianças surdas que utilizam a Língua Brasileira de Sinais, como primeira Língua, a partir da perspectiva da Psicologia do Desenvolvimento Cognitivo. (MADALENA, 2017, p. 12, grifo nosso). T9: Este estudo considera a evolução do conceito de número natural, e mais especificamente a quantificação de conjuntos discretos de até 10 elementos, por crianças e adolescentes com síndrome de Down. (YOKOYAMA, 2012, p. 11, grifo nosso). As pesquisas citadas acima levantam os processos de aprendizagem de crianças com deficiências, tais como: surdocegueira (T2), deficiência intelectual (T7), surdez (T8) e síndrome de Down (T9). Consideramos que uma possível escolha destes grupos para as pesquisas com o conceito de número refere-se à necessidade de pensarmos práticas inclusivas em Educação Matemática, bem como investigar os processos cognitivos de crianças com deficiência e suas possíveis variações relativas ao descrito nas teorias psicológicas da área. 3.2. Categoria 1: Objetivos de pesquisas T15: Relatar uma situação desencadeadora de aprendizagem (SDA) envolvendo o conceito de número, desenvolvida por participantes do projeto Clube de Matemática da Universidade Federal de Santa Maria (UFSM), em uma turma de 1° ano dos anos iniciais do Ensino Fundamental. (BINSFELD; GOLIN; KLEIN, 2019, p. 10, grifo nosso). T10: Investigar o papel do registro de representação semiótica na construção do conceito de número por crianças da educação infantil. (DINIS, 2018, p. 12, grifo nosso). A partir da leitura dos objetivos, notamos que estes estão mais voltados para os procedimentos metodológicos. Com esses, visualizamos o interesse em elucidar como certas práticas metodológicas podem colaborar com o ensino de números para crianças da Educação Infantil e anos iniciais do Ensino Fundamental. Entre as propostas de intervenção estão: jogos (T1), softwares (T11, T12), situação desencadeadora de aprendizagem (T15) e os registros de representação semiótica (T10). Neste primeiro grupo, consideramos que, mesmo enfatizando as pesquisas com crianças, os estudos intentam atingir a formação de professores que ensinam matemática, trazendo possibilidades de intervenção e reflexão para outros docentes que possam ler e analisar as práticas desenvolvidas. No segundo grupo, denominado objetivos de aprendizagem, vemos aqueles objetivos mais voltados para a aprendizagem. Semelhante ao anterior, há aqueles voltados para as questões mais gerais, tais como: T4: Investigar a formação do conceito de número na criança. (FERRARI, 2008, p. 5, grifo nosso). T6: Investigar o ensino e aprendizagem de números naturais, buscando identificar semelhanças e diferenças entre os resultados e indicações de pesquisas sobre a construção do conceito de números pelas crianças. (BONALDO, 2007, p. 8, grifo nosso). T13: Apresentar os resultados de um estudo, realizado em 2008, o qual tinha como foco a análise das percepções de crianças sobre o conceito numérico. (GUILLEN; SOUSA, 2010, p. 7, grifo nosso). Os objetivos trazidos acima, nos mostram uma visão metodológica mais voltada à aprendizagem dos alunos, porém não oferecem indícios sistemáticos de intervenção. Notamos alguns pontos levantados: formação do conceito de números (T4), ensino e aprendizagem dos números naturais (T6) e percepções das crianças (T13). No segundo grupo, também, encontramos trabalhos voltados para a aprendizagem de crianças com deficiência, como: T2: Investigar a construção do conceito de número por uma aluna com surdocegueira congênita. (ALEIXO, 2018, p. 2, grifo nosso). BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.3. Categoria 2: Quais as abordagens metodológicas usadas para o ensino do conceito de número? Nesta seção foram investigados e separados os trabalhos através de seus procedimentos metodológicos. Criamos seis grupos conforme as abordagens de ensino nos estudos: jogos, tecnologia, análise de materiais e documentos, atividades, testes e história virtual do conceito, conforme quadro abaixo. Quadro 6 – Abordagens metodológicas utilizadas Abordagem Quantidade Trabalhos Jogos 3 T1, T5, e T10 Tecnologia 2 T11 e T12 Análise de materiais e Documentos 2 T3 e T6 Atividades 5 T2, T4, T7, T9 e T14 Testes 2 T8 e T13 História virtual do conceito 1 T15 Fonte: Dados da pesquisa (2020). Fonte: Dados da pesquisa (2020). No grupo 1, jogos, os estudos focaram na utilização de jogos para produzir a intervenção com os alunos. Em T1 foi utilizado o Flex memo, mais especificamente os jogos Memória, Segredo da Caixa e Batalha. T5 apoiou-se no jogo Hackenbush para compor a pesquisa. Por sua vez, T10 utilizou-se dos jogos ressignificados, em que os jogos serviram tanto para intervenção quanto para a análise dos dados. REMAT: Revista Eletrônica da Matemática 9 Conforme Grando (1995), os jogos trazem vantagens para a prática pedagógica, como desenvolvimento de conceitos de difícil compreensão, participação ativa dos alunos e motivação nas aulas. Nesse sentido, as pesquisas que utilizam-se deste recurso podem oportunizar maiores socializações e debates sobre o pensamento matemático das crianças. No grupo 2, tecnologia, foram encontrados dois trabalhos que desenvolveram meios que envolvessem as tecnologias como suporte pedagógico. Em T11, desenvolveu-se um software com a intenção de auxiliar os alunos na construção do conceito de número, enquanto que T12 realizou uma sequência de atividades computadorizadas para auxiliar na aprendizagem dos primeiros números pelas crianças. Segundo Moraes (2017), as tecnologias na Educação Matemática tornam-se mais potentes quando o docente tem compreensão da perspectiva epistemológica que envolve o seu entendimento do ensino, bem como, do papel da tecnologia nesse contexto. Perante a perspectiva deste pesquisador, notamos que há um processo consciente de inserção da tecnologia para o ensino de número, tanto em T11 quanto em T12. No grupo 3, análise de materiais e documentos, encontramos dois trabalhos (T3 e T6). Ambos os estudos envolvem a análise de livros didáticos e análise dos cadernos de alunos para a obtenção de inferências na sua pesquisa. Embora não realizassem intervenção diretamente com alunos, os estudos consideram os mesmos como sujeitos de pesquisas em seus trabalhos. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.3. Categoria 2: Quais as abordagens metodológicas usadas para o ensino do conceito de número? Já no grupo 4, denominado de atividades, é aquele com o maior número de trabalhos (T2, T4, T7, T9, T14). Esse grupo reuniu atividades que não foram categorizados com nenhuma metodologia específica. Encontramos aqui, a aplicação de atividades pautadas nos processos mentais estipulados por Lorenzatto (2008) (T2), atividades inspiradas nos experimentos de Dehaene (1997) (T4) e atividades para compreender os processos do conceito de número (T7, T9, T14). Nesse grupo, percebemos a forte influência dos aspectos da psicologia no ensino do conceito de número, bem como aplicação e adaptação de experimentos didáticos. No grupo 5, testes, dois trabalhos foram categorizados (T8, T13). Enquanto T8 propõe testes de habilidades para o conceito de número com alunos surdos que utilizam a Língua Brasileira de Sinais como sua primeira língua, T13 apresenta testes para suscitar percepções de crianças do primeiro ano do Ensino Fundamental. No último grupo, nomeado como História virtual do conceito, traz apenas T15, que visa à criação de histórias virtuais para o ensino do conceito de número, no sentido de produzir contextos para os alunos refletirem sobre a temática. REMAT: Revista Eletrônica da Matemática 10 BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.5. Categoria 4: principais inferências dos estudos Nesta seção, categorizamos as inferências dos trabalhos, dividindo-as em: (1) inferências genéricas; (2) inferências específicas; e (3) inferências incompletas. Torna-se válido ressaltar que esta categorização pautou-se nos resumos e considerações finais dos estudos. Quadro 8 – Inferências dos estudos Inferências Quantidade Trabalhos Inferências Genéricas 5 T1, T2, T4, T7 e T13 Inferências Específicas 5 T3, T8, T9, T10 e T15 Inferências incompletas 5 T5, T6, T11, T12 e T14 Fonte: Dados da pesquisa (2020). Fonte: Dados da pesquisa (2020). No primeiro grupo de inferências, elencamos aquelas que consideramos genéricas: No primeiro grupo de inferências, elencamos aquelas que consideramos genéricas: T1: Ampliaram seus conhecimentos a respeito do conceito de número. (BELO, 2018, p. 85). T2: Construção de novos conhecimentos. (ALEIXO, 2018, p. 100). T4: Conceito de número é basilar na constituição dos conceitos da Matemática. (FERRARI, 2008, p. 91). T7: Criança insere-se no seu lugar como sujeito de suas aprendizagens e de seu desenvolvimento. (NOLETO, 2017, p. 125). T13 C t ib i ã it i (GUILLEN SOUSA 2010 100) T1: Ampliaram seus conhecimentos a respeito do conceito de número. (BELO, 2018, p. 85). T1: Ampliaram seus conhecimentos a respeito do conceito de número. (BELO, 2018, p. 85). T2: Construção de novos conhecimentos. (ALEIXO, 2018, p. 100). T4: Conceito de número é basilar na constituição dos conceitos da Matemática. (FERRARI, 2008, p. 91). T7: Criança insere-se no seu lugar como sujeito de suas aprendizagens e de seu desenvolvimento. (NOLETO, 2017, p. 125). T7: Criança insere-se no seu lugar como sujeito de suas aprendizagens e de seu desenvolvimento. (NOLETO, 2017, p. 125). T13: Contribuição com os nexos conceituais. (GUILLEN; SOUSA, 2010, p. 100). Perante os excertos acima, visualizamos que todas as pesquisas apresentam que modificações positivas ocorreram nos processos de aprendizagem, tanto no que se refere à relação entre alunos e o conceito de número – ampliação (T1) e novos (T2), por exemplo – quanto do próprio desenvolvimento da criança – sujeito de sua aprendizagem (T7). No grupo de inferências específicas, há explicações mais detalhadas dos resultados obtidos ao longo da pesquisa. Podemos ver as seguintes considerações: No grupo de inferências específicas, há explicações mais detalhadas dos resultados obtidos ao longo da pesquisa. Podemos ver as seguintes considerações: T3: As duas propostas de ensino se distinguem, em método e conteúdo, que tem como consequência: o desenvolvimento do conhecimento empírico, na proposição formalista, e do conhecimento teórico, na proposição davydoviana. (SOUZA, 2013, p. 3.4. Categoria 3: metodologias de pesquisa utilizadas nos trabalhos Nesta seção, dividimos as metodologias de pesquisa dos trabalhos analisados em três categorias, sendo elas: (1) metodologias categorizadas; (2) metodologias não categorizadas; e (3) pesquisas que não apresentaram claramente a sua metodologia. Quadro 7 – Metodologias de pesquisa Metodologia da pesquisa Metodologia de pesquisa Quantidade Trabalhos Metodologia categorizada Pesquisa-ação 1 T14 Estudo de caso 5 T2, T4, T7, T8, T13 Bibliográfica e documental 2 T3, T6 Metodologia não categorizada Descrição de etapas 5 T1, T9, T10, T11, T12 Não apresenta claramente 1 T5 Fonte: Dados da pesquisa (2020). Quadro 7 – Metodologias de pesquisa Fonte: Dados da pesquisa (2020). Fonte: Dados da pesquisa (2020). No primeiro grupo, denominado metodologias categorizadas, tem a subdivisão em três subcategorias que são: pesquisa-ação, estudo de caso e bibliográfica e documental. A primeira subcategoria, encontramos T14 que se utiliza da pesquisa-ação, apoiando num grupo colaborativo de docentes para realizar sua intervenção sobre o ensino de números com as crianças. A segunda subcategoria apresenta trabalhos que utilizam da metodologia do estudo de caso (T2, T4, T7, T8, T13). Todos se apropriam desta abordagem por realizarem suas pesquisas com uma única turma de alunos da Educação Básica, considerando, assim, que a unicidade contextual de uma sala permite pensá-la como um caso para intervenção, passível de produzir generalizações mais amplas sobre o objeto de pesquisa. Na terceira subcategoria, encontram-se as pesquisas bibliográfica e documental (T3, T6), que envolvem as metodologias de análise de documentos bibliográficos, curriculares, cadernos de alunos e manual do professor. A concepção de tais pesquisas consiste de que os materiais permitem levantar novas considerações para a prática pedagógica com o conceito de número. No segundo grupo, metodologias não categorizadas, apresentamos aquelas que definem metodologias para a intervenção, que não necessariamente associam-se a análise dos dados. Este grupo prioriza a descrição das etapas de intervenção do que a associação com metodologias específicas (T1, T9, T10, T11, T12). Os estudos do grupo refletem o entendimento que a descrição sequencial de passos da pesquisa é essencial para o desenvolvimento teórico de uma produção científica. Por fim, o último grupo denomina-se não apresenta claramente a sua metodologia de trabalho, composto apenas por T5. A partir da leitura dos pesquisadores, não encontramos descrições detalhadas que pudessem embasar outra classificação. REMAT: Revista Eletrônica da Matemática 11 BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 3.5. Categoria 4: principais inferências dos estudos 5, grifo nosso). T8: Os resultados mostram a influência da escolaridade na construção das habilidades numéricas, assim como do tempo de exposição e frequência de uso da Língua de Sinais. Os dados também apontam associações existentes entre habilidades de recitação e de contagem com desempenhos em linguagem compreensiva e expressiva e com velocidade de processamento, inteligência não- verbal e memória de trabalho. (MADALENA, 2017, p. 110, grifo nosso). T9: Um aspecto das atividades que parecia particularmente importante para permitir que os participantes fossem além da utilização de procedimento de contagem mecanizada foi a presença de recursos multissensoriais que os participantes poderiam usar para verificar e corrigir suas próprias estratégias. (YOKOYAMA, 2012, p. 98, grifo nosso). T10: Os resultados observados indicaram a utilização da representação da língua materna como ponto de partida para a apresentação do objeto matemático, REMAT: Revista Eletrônica da Matemática 12 como formação das primeiras representações e transformação em outro registro dentro do mesmo sistema semiótico (operações de tratamento). Nessa perspectiva, as representações semióticas contribuíram para a interiorização das representações mentais (noésis) que, por meio das relações estabelecidas, possibilitaram a apreensão conceitual do objeto matemático numa dada interação. (DINIS, 2018, p. 100, grifo nosso). T15: Percebemos que as crianças se envolveram na situação, chegando a uma síntese da solução coletiva, aproximando-se do método de controle de variação de quantidades utilizado historicamente. (BINSFELD; GOLIN; KLEIN, 2019, p. 80, grifo nosso). No grupo, percebemos inferências que avaliam as práticas de pesquisas e os seus efeitos na aprendizagem. Com exceção de T8, que associa tempo de escolaridade, habilidades numéricas e habilidades de linguagem de crianças surdas, as demais apontam impactos e benefícios de propostas metodológicas de ensino ou de recursos educacionais para o conceito de número. Nos escritos, observamos que diferentes abordagens teórico-metodológicas permitem a criação e a elaboração de diferentes produções conceituais sobre o pensamento numérico. No grupo inferências incompletas, notamos que os trabalhos não trazem diferenciações e pormenores sobre o conceito de número. T5 e T6 descrevem como foi o processo de pesquisa. T11 e T12 suscitam o potencial, de modo geral, de práticas interativas com tecnologia e a necessidade de novas práticas com a metodologia. Por fim, T14 ressalta que a pesquisa ainda está em andamento, sem conclusões definitivas. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. 4. Considerações finais Essa pesquisa teve por objetivo analisar como o debate sobre o conceito de número tem sido abordado em pesquisas com crianças de Educação Infantil e anos iniciais do Ensino Fundamental, a partir de seus objetivos, metodologias de pesquisa e ensino e inferências. Nesse sentido, produzimos um levantamento em três bancos de dados: BDTD, CTD e ENEM, o que suscitou quinze estudos. A partir da análise, levantamos que as pesquisas estão concentradas na região Sudeste, são em sua maioria dissertações e há um indício de crescimento dos estudos nos últimos cinco anos. Além disso, quanto aos objetivos de pesquisa, notamos que mesmo as pesquisas envolverem sujeitos crianças, há forte influência de atingir discussões voltadas para o campo de ensino. No que se refere às metodologias de ensino, o uso de atividades com uma perspectiva voltada para análise do pensamento numérico tem a sua maior evidência. Já sobre as metodologias de pesquisa, o estudo de caso apresenta-se como a opção mais frequente de intervenção. No que tange as inferências, notamos que muitas ainda são genéricas e com poucas considerações sobre o conceito de número. REMAT: Revista Eletrônica da Matemática 13 Por fim, esperamos que este estudo possibilite outros pesquisadores estruturarem suas pesquisas e produzirem novas intervenções sobre o conceito de número com crianças da Educação Infantil e Anos Iniciais do Ensino Fundamental. Referências ALEIXO, H. P. A construção do conceito de número por uma aluna com surdocegueira congênita. Orientadora: Thaís Philipsen Grützmann. 2018. 181 f. Dissertação (Mestrado em Educação Matemática) - Programa de Pós-Graduação em Educação Matemática, Universidade Federal de Pelotas, Pelotas, RS, 2018. Disponível em: http://guaiaca.ufpel.edu.br:8080/handle/prefix/6565. Acesso em: 30 set. 2021. , , , p http://guaiaca.ufpel.edu.br:8080/handle/prefix/6565. Acesso em: 30 set. 2021. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. BARDIN, L. Análise de Conteúdo. São Paulo: Edições 70, 2011. ARDIN, L. Análise de Conteúdo. São Paulo: Edições 70, 2011. BELO, P. A. P. A aprendizagem do conceito de número de crianças do Infantil V: interações com o flex memo. Orientador: Paulo Meireles Barguil. 2018. 165f. Dissertação (Mestrado em Educação) - Programa de Pós-Graduação em Educação, Universidade Federal do Ceará, Fortaleza, CE, 2018. Disponível em: http://www.repositorio.ufc.br/handle/riufc/39404. Acesso em: 30 set. 2021. BINSFELD, C. D.; GOLIN, A. L.; KLEIN, M. L. Correspondência um a um: uma situação desencadeadora de aprendizagem voltada ao conceito de número. In: ENCONTRO NACIONAL DE EDUCAÇÃO MATEMÁTICA, 13., 2019, Cuiabá, MT. Anais [...]. SBEM Regional Mato Grosso, 2019. BONALDO, I. M. Investigações sobre números naturais e processos de ensino e aprendizagem desse tema no início da escolaridade. Orientadora: Celia Maria Carolino Pires. 2007. 176 f. Dissertação (Mestrado em Educação) - Pontifícia Universidade Católica de São Paulo, São Paulo, 2007. Disponível em: https://tede2.pucsp.br/handle/handle/11508. Acesso em: 30 set. 2021. BONALDO, I. M. Investigações sobre números naturais e processos de ensino e aprendizagem desse tema no início da escolaridade. Orientadora: Celia Maria Carolino Pires. 2007. 176 f. Dissertação (Mestrado em Educação) - Pontifícia Universidade Católica de São Paulo, São Paulo, 2007. Disponível em: https://tede2.pucsp.br/handle/handle/11508. Acesso em: 30 set. 2021. BOSCARIOL, F. Uma proposta de software de educação matemática para educação infantil. Orientadora: Afira Vianna Ripper. 2004. 73f. Dissertação (Mestrado em Educação) - Universidade Estadual de Campinas, Faculdade de Educação, Campinas, SP, 2004. Disponível em: http://repositorio.unicamp.br/jspui/handle/REPOSIP/253242. Acesso em: 30 set. 2021. BOSCARIOL, F. Uma proposta de software de educação matemática para educação infantil. Orientadora: Afira Vianna Ripper. 2004. 73f. Dissertação (Mestrado em Educação) - Universidade Estadual de Campinas, Faculdade de Educação, Campinas, SP, 2004. Disponível em: http://repositorio.unicamp.br/jspui/handle/REPOSIP/253242. Acesso em: 30 set. 2021. DEHAENE, S. The number sense: How the mind creates mathematics. New York: Oxford University Press, 1997. DEHAENE, S. The number sense: How the mind creates mathematics. New York: Oxford University Press, 1997. DINIS, A. C. Representação Semiótica: uma perspectiva para a construção do conceito de número na Educação Infantil. 2018. 191f. Dissertação (Mestrado em Educação, Cultura e Comunicação) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2018. DINIS, A. C. Representação Semiótica: uma perspectiva para a construção do conceito de número na Educação Infantil. 2018. 191f. Dissertação (Mestrado em Educação, Cultura e Comunicação) - Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2018. FERRARI, A. H. O senso numérico da criança: formação e características. BARDIN, L. Análise de Conteúdo. São Paulo: Edições 70, 2011. Orientadora: Sonia Barbosa Camargo Igliori. 2008. 189 f. Tese (Doutorado em Educação) - Pontifícia Universidade Católica de São Paulo, São Paulo, 2008. Disponível em: https://tede2.pucsp.br/handle/handle/11357. Acesso em: 30 set. 2021. FERRARI, A. H. O senso numérico da criança: formação e características. Orientadora: Sonia Barbosa Camargo Igliori. 2008. 189 f. Tese (Doutorado em Educação) - Pontifícia Universidade Católica de São Paulo, São Paulo, 2008. Disponível em: https://tede2.pucsp.br/handle/handle/11357. Acesso em: 30 set. 2021. 14 REMAT: Revista Eletrônica da Matemática FONSECA, R. F. da. Número: o conceito a partir de jogos. Orientadora: Sonia Barbosa Camargo Igliori. Coorientador: Michael Otte. 2005. 96 f. Dissertação (Mestrado em Educação) - Pontifícia Universidade Católica de São Paulo, São Paulo, 2005. Disponível em: https://tede2.pucsp.br/handle/handle/11001. Acesso em: 30 set. 2021. GRANDO, R.C. O jogo e suas possibilidades metodológicas no processo ensino aprendizagem da matemática. Orientador: Sérgio Lorenzato. 1995. 175f. Dissertação (Mestrado em Educação) - Universidade de Campinas, Campinas, SP, 1995. Disponível em: http://repositorio.unicamp.br/jspui/handle/REPOSIP/253786. Acesso em: 30 set. 2021. GRANDO, R.C. O jogo e suas possibilidades metodológicas no processo ensino aprendizagem da matemática. Orientador: Sérgio Lorenzato. 1995. 175f. Dissertação (Mestrado em Educação) - Universidade de Campinas, Campinas, SP, 1995. Disponível em: http://repositorio.unicamp.br/jspui/handle/REPOSIP/253786. Acesso em: 30 set. 2021. GUILLEN, J. D.; SOUSA, M. C. Percepções de crianças do 1º Ano do Ensino Fundamental sobre o conceito de números. In: ENCONTRO NACIONAL DE EDUCAÇÃO MATEMÁTICA, 10., jul. 2010, Salvador, BH. Anais [...]. Salvador: SBEM, 2010. Disponível em: https://atelierdigitas.net/CDS/ENEM10/artigos/CC/T3_CC163.pdf. LORENZATO, S. Educação Infantil e percepção matemática. Campinas: Autores Associados, 2008. LORENZATO, S. Educação Infantil e percepção matemática. Campinas: Autores Associados, 2008. MADALENA, S. P. Investigação da Construção do Número em LIBRAS: estudo com crianças surdas. Orientadora: Jane Correa. 2017. 232f. Tese (Doutorado em Psicologia) - Universidade do Rio de Janeiro, Rio de Janeiro, 2017. Disponível em: htt // i b / i / bli / lt / l t /t b lh C l / i T b lh C MADALENA, S. P. Investigação da Construção do Número em LIBRAS: estudo com crianças surdas. Orientadora: Jane Correa. 2017. 232f. Tese (Doutorado em Psicologia) - Universidade do Rio de Janeiro, Rio de Janeiro, 2017. Disponível em: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoCo nclusao.jsf?popup=true&id_trabalho=5220602. MORAES, J. C. P. Jogos Abacus e Composição e o sistema posicional para alunos surdos: olhares de professores num curso de libras. Ensino da Matemática em Debate, São Paulo, SP, v. 4, n. 2, p. 87-105, 2017. Disponível em: https://revistas.pucsp.br/index.php/emd/article/view/32879. Acesso em: 30 set. 2021. NOLETO, C. A. S. A construção do número pela criança com deficiência intelectual: a percepção entre diferentes ambientes escolares. Orientador: Cristiano Alberto Muniz. 2017. 148f. Dissertação (Mestrado em Educação) - Universidade de Brasília, Brasília, 2017. Disponível em: https://repositorio.unb.br/handle/10482/23935. Acesso em: 30 set. 2021. NOLETO, C. A. S. A construção do número pela criança com deficiência intelectual: a percepção entre diferentes ambientes escolares. Orientador: Cristiano Alberto Muniz. 2017. 148f. Dissertação (Mestrado em Educação) - Universidade de Brasília, Brasília, 2017. Disponível em: https://repositorio.unb.br/handle/10482/23935. Acesso em: 30 set. 2021. ROMANOWSKI, J. P.; ENS, R. T. As pesquisas denominadas do tipo “estado da arte” em educação. Revista Diálogo Educacional, Curitiba, PR, v. 6, n. 19, p. 37-50, set./dez. 2006. Disponível em: https://periodicos.pucpr.br/index.php/dialogoeducacional/article/view/24176. Acesso em: 30 set. 2021. SANTOS, B. M. dos; MANFREDO, E. C. G; COSTA, E. M. P. da. Análises no processo de construção do conceito de número de alunos do 1º Ano do Ensino Fundamental em uma prática colaborativa. In: ENCONTRO NACIONAL DE EDUCAÇÃO MATEMÁTICA, 13., 2019, Cuiabá, MT. Anais [...]. Cuiabá: SBEM, 2019. Disponível em: https://sbemmatogrosso.com.br/xiiienem/anais.php. SILVA, J. E. F. da. Uma proposta para utilização do computador no processo de ensino e aprendizagem dos primeiros números do sistema hindu-arábico. Orientadora: Maria Lucia Lorenzetti Wodewotzky. 1998. 151f. Dissertação (Mestrado em Educação Matemática) - Programa de Pós-Graduação em Educação Matemática, Universidade Estadual Paulista, Rio Claro, SP, 1998. Disponível em: https://repositorio.unesp.br/handle/11449/124141. Acesso em: 30 set. 2021. SOUZA, M. B. de. BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. LORENZATO, S. Educação Infantil e percepção matemática. Campinas: Autores Associados, 2008. O ensino do conceito de número: objetivações nas proposições davydovianas e formalista moderna. Orientador: Ademir Damazio. 2013. 237f. Dissertação (Mestrado em Educação) - Programa de Pós-Graduação em Educação, Universidade do Extremo BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento SOUZA, M. B. de. O ensino do conceito de número: objetivações nas proposições davydovianas e formalista moderna. Orientador: Ademir Damazio. 2013. 237f. Dissertação (Mestrado em Educação) - Programa de Pós-Graduação em Educação, Universidade do Extremo BOTELHO, Lutieli Rodrigues; MORAES, João Carlos Pereira de. O conceito de número na Educação Matemática: uma incursão em pesquisas com crianças. REMAT: Revista Eletrônica da Matemática, Bento Gonçalves, RS, v. 7, n. 2, p. e2007, 5 de outubro de 2021. https://doi.org/10.35819/remat2021v7i2id4909. REMAT: Revista Eletrônica da Matemática 15 Sul Catarinense, Criciúma, SC, 2013. Disponível em: http://repositorio.unesc.net/handle/1/1761. Acesso em: 30 set. 2021. YOKOYAMA, L. A. Uma Abordagem Multissensorial para o desenvolvimento do conceito de número natural em indivíduos com síndrome de Down. Orientadora: Siobhan Victoria Healy. 2012. 230f. Tese (Doutorado em Educação Matemática) - Pós-Graduação Strictu Sensu em Educação Matemática, Universidade Bandeirante de São Paulo, São Paulo, SP, 2012. Disponível em: https://repositorio.pgsskroton.com//handle/123456789/3484. Acesso em: 30 set. 2021. REMAT: Revista Eletrônica da Matemática 15 Sul Catarinense, Criciúma, SC, 2013. Disponível em: http://repositorio.unesc.net/handle/1/1761. Acesso em: 30 set. 2021. Sul Catarinense, Criciúma, SC, 2013. Disponível em: http://repositorio.unesc.net/handle/1/1761. Acesso em: 30 set. 2021. Sul Catarinense, Criciúma, SC, 2013. Disponível em: http://repositorio.unesc.net/handle/1/1761. Acesso em: 30 set 2021 YOKOYAMA, L. A. Uma Abordagem Multissensorial para o desenvolvimento do conceito de número natural em indivíduos com síndrome de Down. Orientadora: Siobhan Victoria Healy. 2012. 230f. Tese (Doutorado em Educação Matemática) - Pós-Graduação Strictu Sensu em Educação Matemática, Universidade Bandeirante de São Paulo, São Paulo, SP, 2012. Disponível em: https://repositorio.pgsskroton.com//handle/123456789/3484. Acesso em: 30 set. 2021.
https://openalex.org/W2297232337
https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/s13104-016-1989-3
English
null
Methicillin-susceptible Staphylococcus aureus skin infections among military conscripts undergoing basic training in Bangkok, Thailand, in 2014
BMC research notes
2,016
cc-by
4,628
© 2016 Nivesvivat et al. 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. Background On June 4, 2014, 25 conscripts were found to possess abscesses on their arms and elbows. This outbreak of infection resulted in one death. The Clinical Epidemiol- ogy Unit investigated other soldiers at the basic military camp on that day to determine the cause(s) of the infec- tion and recommend appropriate prevention and con- trol strategies. In April each year, approximately 9–10 % of men aged 21 years with a good health profile from all provinces are randomly selected to serve in the army, beginning with 10 weeks of basic military training. The *Correspondence: rrangsin@pmk.ac.th 1 Department of Military and Community Medicine, Phramongkutklao College of Medicine, 315, Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand Full list of author information is available at the end of the article Abstract Background:  Skin and soft tissue infections are common among military conscripts undergoing close-contact training activities. On June 4, 2014, an outbreak of Staphylococcus aureus skin infection was reported among military conscripts undergoing basic training in Bangkok, Thailand. An investigation was performed to verify the outbreak and recommend future prevention and control strategies. Case presentation:  The outbreak resulted in a rate of infection of 19.2 % and a fatality rate of 2.5 % (one death). All were Thai men aged 21.2 ± 1.0 years. Risk factors associated with infection were multiple erythematous papules and training in certain subunits. Randomly selected isolates were evaluated by pulsed field gel electrophoresis to confirm the clonal identity. Conclusions:  This report confirms that S. aureus skin infection can be fatal. Our study highlights the role of military personnel in the early detection, prompt treatment, and containment of outbreaks of skin infection, as well as other health issues among conscripts. Keywords:  Methicillin-susceptible Staphylococcus aureus, Skin infection, Military, Outbreak reported outbreak occurred 2–6  weeks after training started, a time when training becomes more intensive. Methicillin‑susceptible Staphylococcus aureus skin infections among military conscripts undergoing basic training in Bangkok, Thailand, in 2014 Thirapa Nivesvivat1, Dusit Janthayanont2, Mathirut Mungthin3, Julphat Intarasuphit4, Siriwan Paojinda2, Kanya Phanitorn5, Paijit Permpool5, Saowapap Kasinant5, Onuma Pengpinij5, Parichart Yingprasert6, Wanida Thaochelee6 and Ram Rangsin1* BMC Research Notes BMC Research Notes Nivesvivat et al. BMC Res Notes (2016) 9:179 DOI 10.1186/s13104-016-1989-3 Case presentationh Pus were collected from the sites of infection. The clini- cal isolates were sent for bacterial culture at the Division of Microbiology, Department of Clinical Pathology, Phra- mongkutklao Hospital. Standard bacteriological cultur- ing by inoculation of blood agar was performed, as well as antibiotic susceptibility testing using the disk diffusion method. During our investigation, antibiotic suscepti- bilities to oxacillin, gentamicin, ciprofloxacin, levofloxa- cin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, clindamycin, fusidic acid, fosfomycin, lin- ezolid, vancomycin, and erythromycin were determined. Isolates that were positive for S. aureus were forwarded to the Reference Laboratory of the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand, where they were confirmed to be S. aureus by standard culture techniques. The PCR tech- nique adopted by the National Institute of Health accom- panied sensitivity analysis by means of disk diffusion (Kirby Bauer) and E-test (vancomycin) assays to differen- tiate MSSA and methicillin-resistant S. aureus (MRSA) [2]. We randomly selected isolates to be further evaluated by pulsed field gel electrophoresis (PFGE) [3] to confirm the clonal identity. A 21-year-old Thai male conscript was admitted to the hospital after fainting with urinary and stool inconti- nence during combat training on June 4, 2014. He was primarily assumed to be suffering from heat syncope. He was a previously healthy man from subunit number 2, who was 20 years old with a BMI of 21.4 kg/m2. Six days before being admitted, abscesses were detected on both arms and elbows and the military camp prescribed oral amoxicillin. Three days later, he developed a high fever, nausea, and vomiting. The Emergency Department examined his vital signs, revealing a body temperature at 38  °C, dyspnea and tachypnea (30/min), tachycardia (140/min), and a blood pressure of 130/80 mmHg. Later, his blood pressure dropped to 85/50  mmHg. The doc- tor administered normal saline infusion, accessed the central venous line for inotropic drugs to increase his blood pressure, and gave broad-spectrum antibiotics. Ceftriaxone (2 g per day) and clindamycin (600 mg every 8 h) were administered intravenously. The conscript was then sent to the intensive care unit, where he remained conscious. Physical examination revealed multiple rup- tured abscesses on an erythematous edematous base of the left elbow. Laboratory tests revealed a raised level of creatinine phosphokinase (10 times higher than normal), as well as an electrolyte imbalance. Liver and renal pan- els were also found to be abnormal. Urinalysis showed hematuria and pyuria. Environmental investigation Environmental samples, comprising combat uniforms, bathing water, and bath reservoirs shared among recruits were collected for bacterial culture using blood agar. The morning after the investigation was completed, the inves- tigation team prescribed antibiotics for all cases. A case was defined as a person who was clinically diag- nosed with one or more abscesses from mid-May to the first 2 weeks of June. Of 213 recruits, 41 (19.2 %), includ- ing the deceased patient, were documented as contract- ing the infection, which was used as the case rate for the whole unit. The index case is thought to have occurred on May 15, 2014 (Fig. 1). Approximately 2  weeks later, four cases were reported from May 30 to 31. On June 1, Epidemiological investigationh The descriptive data were collected on June 5, 2014. Of 213 recruits, 212 completed the self-reported question- naire. The information in the questionnaire included demographic data, clinical manifestation, date of onset, number of lesions, and location(s) of lesions on their body. Demographic questions included age, sex, edu- cation, underlying diseases, and unit types. Clinical manifestations comprised (1) abscesses (2) multiple erythematous papules and (3) fever. Those reporting abscesses were considered as probable cases. The height and weight of the recruits were obtained from heat stroke Nivesvivat et al. BMC Res Notes (2016) 9:179 Page 2 of 6 screening data to calculate the body mass index (BMI) [1] and identify overweight or obese cases. screening data to calculate the body mass index (BMI) [1] and identify overweight or obese cases. The methodologies employed were univariate and mul- tivariate analyses. Using STATA software, the method- ologies resulted in crude odds ratio and adjusted odds ratio, respectively, and 95  % confidence intervals. The variables with significant p values less than 0.05 were fur- ther considered. A case was defined as a person who was clinically diag- nosed with an infectious skin lesion, including abscesses and furuncles, from mid-May to the first 2 weeks of June. The infecting bacteria were confirmed to be methicillin- susceptible Staphylococcus aureus (MSSA) using stand- ard culturing techniques. Healthcare providers and recruits were interviewed about their daily activities, such as hand-to-hand combat training, the characteris- tics of physical training, and hygiene. Ethical approval was obtained from the Institutional Review Board of the Royal Thai Army Medical Depart- ment. Confidentiality was maintained throughout and after the study. Names were not included on the questionnaire for confidentiality reasons. The written informed consent for publication of the clinical details from the next of kin of the deceased patient was also obtained. In the case of the deceased patient, his medical records, signs and symptoms, laboratory information, treatment, and cause of death were reconsidered. Case presentationh The pus culture collected from his elbow and hemoculture indicated MSSA infection. Despite prompt treatment, the patient passed away.i Statistical analysish The case rates were calculated both within each unit and for the whole battalion. Descriptive data were gathered by means of a questionnaire. These data were used to find an association between cases and risk factors. Case fatal- ity was calculated. Nivesvivat et al. BMC Res Notes (2016) 9:179 Page 3 of 6 Fig. 1  Epidemic curve of skin abscesses in conscripts by date of onset (n = 41) Fig. 1  Epidemic curve of skin abscesses in conscripts by date of onset (n = 41) Table 1  Characteristics of all conscripts during basic mili- tary training in Bangkok, Thailand (n = 212) Demographic data Total (%) Age (mean ± SD) 21.2 (±1.0)  20 19 (8.9)  21 156 (73.2)  22 21 (9.9)  23 10 (4.7)  ≥24 7 (3.3) Education  Bachelor’s 8 (4)  High vocational certificate 14 (7)  Vocational certificate 22 (10.9)  High school 106 (52.7)  Secondary school 51 (25.4) Subunit  1 54 (25.4)  2 51 (23.9)  3 55 (25.8)  4 53 (24.9) Weight (kg) (mean ± SD) 64.3 ± 11.4 Height (cm) (mean ± SD) 170.2 ± 6.0 BMI (kg/m​2) (mean ± SD) 22.2 ± 3.6  Underweight <18.5 27 (12.7)  Normal range (18.5–22.9) 119 (55.9)  At risk of obesity (23–24.9) 29 (13.6)  Obese I (25–29.9) 29 (13.6)  Obese II (≥30) 9 (4.2) Symptoms  Abscess 41 (19.2)  Number of abscess lesions (mean ± SD) 2.2 ± 2  Multiple erythematous papules 86 (39.0) Table 1  Characteristics of all conscripts during basic mili- tary training in Bangkok, Thailand (n = 212) the number of patients abruptly rose to 17 cases (41.5 %) in 1  day, reaching the highest peak for skin infections. The case fatality rate was 2.4 % (1/41). Table 1  Characteristics of all conscripts during basic mili- tary training in Bangkok, Thailand (n = 212) h All conscripts at this facility were male, with a mean age of 21.2  ±  1.0  years. They were divided into four subunits in the same facility. The clinical manifestations included abscesses (19.2 %), multiple erythematous pap- ules (39.0 %), and fever (16.9 %). The lesions were located on elbows (46.3 %), shoulders (24.4 %), and legs (14.6 %).h The percentage of new conscripts who reported mul- tiple erythematous papules were 51.9, 37.3, 32.7, and 39.6 % for subunits 1, 2, 3, and 4, respectively (p = 0.211). Statistical analysish of isolates tested (%) Oxa Gen Cip Co-tri Chlo Tet Cli Van Ery 22 22 (100.0 %) 22 (100.0 %) 22 (100.0 %) 22 (100 %) 14 (63.6 %) 4 (18.1 %) 19 (86.6 %) 22 (100.0 %) 22 (100.0 %) Table 2  Univariate and multivariate analysis for risk factors of acquiring skin abscesses Risk factors Total (%) Case (%) P value Crude OR (95 % CI) P value Adjusted OR (95 % C Age (mean ± SD) 21.2 (±1.0) 21.0 (±1.0) 0.033 0.5 (0.3–0.9) 0.036 0.5 (0.3–1.0) Education  Bachelor 8 (4) 3 (37.5) 1  High vocational certificate 14 (7) 4 (28.6) 0.666 0.7 (0.1–4.2)  Vocational certificate 22 (10.9) 5 (22.7) 0.423 0.5 (0.1–2.8)  High school 106 (52.7) 19 (17.9) 0.191 0.4 (0.1–1.7)  Secondary school 51 (25.4) 7 (13.7) 0.112 0.3 (0.1–1.4) Subunit  1 54 (25.4) 20 (37) 0.001 30.6 (3.9–238.6) 0.001 32.6 (4.1–260.6)  2 51 (23.9) 16 (31.4) 0.003 23.8 (3.0–187.5) 0.002 26.9 (3.3–218.2)  3 55 (25.8) 4 (7.3) 0.216 4.1 (0.4–37.7) 0.178 4.6 (0.5–44.1)  4 53 (24.9) 1 (1.9) 1 BMI (kg/m​2)  Underweight <18.5 27 (12.7) 7 (25.9) 0.275 1.7 (0.6–4.6)  Normal range (18.5–22.9) 119 (55.9) 20 (16.8) 1  At risk of obesity (23–24.9) 29 (13.6) 5 (17.2) 0.955 1.0 (0.4–3.0)  Obese I (25–29.9) 29 (13.6) 7 (24.1) 0.362 1.6 (0.6–4.2)  Obese II (≥30) 9 (4.2) 2 (22.2) 0.679 1.4 (0.3–7.3) Multiple erythematous papules  Yes 86 (39.0) 27 (31.4) <0.01 3.7 (1.8–7.6) <0.01 3.4 (1.5–7.5)  No 127 (59.6) 14 (11) 1 Table 2  Univariate and multivariate analysis for risk factors of acquiring skin abscesses Table 3  Staphylococcus aureus susceptibility test results for all available isolates oxa oxacillin, gen gentamicin, cip ciprofloxacin, co-tri trimethoprim sulfamethoxazole, chlo chloramphenicol, tet tetracycline, cli clindamycin, van vancomycin, ery erythromycin No. of isolates Antimicrobial, no. of isolates tested (%) Oxa Gen Cip Co-tri Chlo Tet Cli Van Ery 22 22 (100.0 %) 22 (100.0 %) 22 (100.0 %) 22 (100 %) 14 (63.6 %) 4 (18.1 %) 19 (86.6 %) 22 (100.0 %) 22 (100.0 %) Table 3  Staphylococcus aureus susceptibility test results for all available isolates dicloxacillin (250 mg per oral qid) was prescribed for all cases. reported fever; 86 % were from subunits 1 or 2, and all had a history of skin abscess (es). In addition, three cul- tures (10.3  %) were positive for Bacillus spp. Statistical analysish BMC Res Notes (2016) 9:179 Page 4 of 6 Table 2  Univariate and multivariate analysis for risk factors of acquiring skin abscesses Risk factors Total (%) Case (%) P value Crude OR (95 % CI) P value Adjusted OR (95 % CI) Age (mean ± SD) 21.2 (±1.0) 21.0 (±1.0) 0.033 0.5 (0.3–0.9) 0.036 0.5 (0.3–1.0) Education  Bachelor 8 (4) 3 (37.5) 1  High vocational certificate 14 (7) 4 (28.6) 0.666 0.7 (0.1–4.2)  Vocational certificate 22 (10.9) 5 (22.7) 0.423 0.5 (0.1–2.8)  High school 106 (52.7) 19 (17.9) 0.191 0.4 (0.1–1.7)  Secondary school 51 (25.4) 7 (13.7) 0.112 0.3 (0.1–1.4) Subunit  1 54 (25.4) 20 (37) 0.001 30.6 (3.9–238.6) 0.001 32.6 (4.1–260.6)  2 51 (23.9) 16 (31.4) 0.003 23.8 (3.0–187.5) 0.002 26.9 (3.3–218.2)  3 55 (25.8) 4 (7.3) 0.216 4.1 (0.4–37.7) 0.178 4.6 (0.5–44.1)  4 53 (24.9) 1 (1.9) 1 BMI (kg/m​2)  Underweight <18.5 27 (12.7) 7 (25.9) 0.275 1.7 (0.6–4.6)  Normal range (18.5–22.9) 119 (55.9) 20 (16.8) 1  At risk of obesity (23–24.9) 29 (13.6) 5 (17.2) 0.955 1.0 (0.4–3.0)  Obese I (25–29.9) 29 (13.6) 7 (24.1) 0.362 1.6 (0.6–4.2)  Obese II (≥30) 9 (4.2) 2 (22.2) 0.679 1.4 (0.3–7.3) Multiple erythematous papules  Yes 86 (39.0) 27 (31.4) <0.01 3.7 (1.8–7.6) <0.01 3.4 (1.5–7.5)  No 127 (59.6) 14 (11) 1 Table 3  Staphylococcus aureus susceptibility test results for all available isolates oxa oxacillin, gen gentamicin, cip ciprofloxacin, co-tri trimethoprim sulfamethoxazole, chlo chloramphenicol, tet tetracycline, cli clindamycin, van vancomycin, ery erythromycin No. of isolates Antimicrobial, no. Statistical analysish and one culture (3.4 %) was positive for the following organisms: Corynebacterium spp., Streptococcus pyogenes, Klebsiella pneumoniae (ESBL-producing strain), Enterococcus fae- calis, Citrobacter spp., Acinetobacter baumannii, and Pseudomonas stutzeri. Statistical analysish While the percentage of conscripts who displayed abscesses were 37.0, 31.4, 7.3, and 1.9 % for subunits 1, 2, 3, and 4, respectively (p < 0.01) (Table 1). Adjusted odds ratios for belonging to subunits 1 and 2 were 32.6 (4.1– 260.6, 95 % CI) and 26.9 (3.3–218.2, 95 % CI) respectively. Multiple erythematous papules were significantly asso- ciated (p < 0.01) with abscesses. The adjusted odds ratio was 3.4 (1.5–7.5, 95 % CI) (Table 2). h The data gathered from interviews with healthcare providers and conscripts revealed that 1–2 weeks before the outbreak, part of the training of subunit 1 and 2 con- scripts involved low crawling on a concrete floor. l A dermatologist from the investigation team was able to identify various kinds of skin lesions including multi- ple erythematous papules, multiple discrete small dermal pustules with surrounding erythema, and skin abscesses. Pus from those suffering from abscesses was collected on June 5, 2014. From 29 clinical cultures, 22 (75.9 %), including one blood culture of the deceased patient, grew MSSA isolates. Among all 22 subjects who were posi- tive for MSSA culture (Table 3): one person died; 50.0 % Nivesvivat et al. Conclusions Prevalence of MSSA nasal colonization among healthy young adults in Thailand has been reported to be 15 %, while prevalence of MRSA nasal carriage was 1  % [4]. Patients infected with MSSA were also reportedly more prevalent than patients infected with MRSA [5]. Among soldiers in the US army, a study reported that 38 % were colonized with MSSA and 3.9  % were colonized with MRSA [6]. A total of 11 randomly selected isolates, including one isolate collected from blood culture of the deceased patient, were evaluated by PFGE to confirm clonal iden- tity revealing four genetic patterns (Fig. 2). Sa 35/37 and Sa 43/57 had 100 % genetic similarity. Sa 34/57 (from the deceased patient), Sa 36/57, Sa 37/57, Sa 38/57, Sa 41/57, Sa 42/57 and Sa 44/57 also showed 100 % genetic similar- ity. Sa 39/57 and Sa 40/57 had their own genetic patterns. All environmental swabs were negative for bacterial cul- tures. The morning after the investigation was completed, Investigations revealed that the pathogen responsible for abscesses in military populations in this present study was MSSA. This organism has generally been reported to be less harmful than MRSA, and more susceptible to antimicrobial therapy [7]. However, here we report a con- script who displayed abscesses and consequently died of Page 5 of 6 Nivesvivat et al. BMC Res Notes (2016) 9:179 Fig. 2  Dendrogram of methicillin-susceptible Staphylococcus aureus, as analyzed by the BioNumerics software Fig. 2  Dendrogram of methicillin-susceptible Staphylococcus aureus, as analyzed by the BioNumerics software MSSA skin infection was associated with the PVL gene. In one study, some MSSA isolates from blood culture or skin lesions were found to be positive for the PVL gene [5]. MSSA septic shock. The cultures from skin tissue and hemoculture were positive for MSSA. Considering the laboratory results and the fact that prompt treatment was initiated but was ineffective, the patient most likely suf- fered septic shock and multiorgan failure. Staphylococcus aureus usually colonizes the anterior nares [16, 17] and may be transmitted to clothes and other parts of the skin by touching. PFGE revealed that MSSA isolated from the conscripts exhibited four DNA pat- terns, one of which was shared by 7 of 11 clinical isolates. Among the other four isolates, two shared an identical DNA pattern. References 1. Razak F, Anand SS, Shannon H, Vuksan V, Davis B, Jacobs R, Teo KK, McQueen M, Yusuf S. Defining obesity cut points in a multiethnic popula- tion. Circulation. 2007;115(16):2111–8. 2. Clinical and Laboratory Standards Institute. Clinical and Laboratory Stand- ards Institute [document]. In: Wayne PA, editor. Clinical and Laboratory Standards Institute; 2005. p. v. 3. Smith CL, Klco SR, Cantor CR. Pulsed-field gel electrophoresis and the technology of large DNA molecules. In: Davies KE, editor. Genome analy- sis: a practical approach. Washington DC: IRL Press; 1988. p. 41–72. 4. Kitti T, Boonyonying K, Sitthisak S. Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand. Southeast Asian J Trop Med Public Health. 2011;42(6):1498–504. 1. Razak F, Anand SS, Shannon H, Vuksan V, Davis B, Jacobs R, Teo KK, McQueen M, Yusuf S. Defining obesity cut points in a multiethnic popula- tion. Circulation. 2007;115(16):2111–8. 2. Clinical and Laboratory Standards Institute. Clinical and Laboratory Stand- ards Institute [document]. In: Wayne PA, editor. Clinical and Laboratory Standards Institute; 2005. p. v. 3. Smith CL, Klco SR, Cantor CR. Pulsed-field gel electrophoresis and the technology of large DNA molecules. In: Davies KE, editor. Genome analy- sis: a practical approach. Washington DC: IRL Press; 1988. p. 41–72. 4. Kitti T, Boonyonying K, Sitthisak S. Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand. Southeast Asian J Trop Med Public Health. 2011;42(6):1498–504. 1. Razak F, Anand SS, Shannon H, Vuksan V, Davis B, Jacobs R, Teo KK, McQueen M, Yusuf S. Defining obesity cut points in a multiethnic popula- tion. Circulation. 2007;115(16):2111–8. 16. Williams RE. Healthy carriage of Staphylococcus aureus: its prevalence and importance. Bacteriol Rev. 1963;27:56–71. 16. Williams RE. Healthy carriage of Staphylococcus aureus: its prevalence and importance. Bacteriol Rev. 1963;27:56–71. 17. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus aureu infections. Lancet Infect Dis. 2005;5(12):751–62. 2. Clinical and Laboratory Standards Institute. Clinical and Laboratory Stand- ards Institute [document]. In: Wayne PA, editor. Clinical and Laboratory Standards Institute; 2005. p. v. 18. Elias AF, Chaussee MS, McDowell EJ, Huntington MK. Community-based intervention to manage an outbreak of MRSA skin infections in a county jail. J Correct Health Care. 2010;16(3):205–15. 4. Kitti T, Boonyonying K, Sitthisak S. Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand. Southeast Asian J Trop Med Public Health. 2011;42(6):1498–504. Consent 5. Nickerson EK, Wuthiekanun V, Wongsuvan G, Limmathurosakul D, Srisa- mang P, Mahavanakul W, Thaipadungpanit J, Shah KR, Arayawichanont A, Amornchai P, et al. Factors predicting and reducing mortality in patients with invasive Staphylococcus aureus disease in a developing country. PLoS One. 2009;4(8):e6512. Patient consent forms were waived because this was an outbreak investigation and the study was approved by the royal Thai army medical department institutional review board. 6. Ellis MW, Griffith ME, Jorgensen JH, Hospenthal DR, Mende K, Patter- son JE. Presence and molecular epidemiology of virulence factors in methicillin-resistant Staphylococcus aureus strains colonizing and infect- ing soldiers. J Clin Microbiol. 2009;47(4):940–5. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 31 May 2015 Accepted: 11 March 2016 Received: 31 May 2015 Accepted: 11 March 2016 14. Early GJ, Seifried SE. Risk factors for community-associated Staphylococ- cus aureus skin infection in children of Maui. Hawaii J Med Public Health. 2012;71(8):218–23. 15. Hsiang MS, Shiau R, Nadle J, Chan L, Lee B, Chambers HF, et al. Epidemio- logic similarities in pediatric community-associated methicillin-resistant and methicillin-sensitive in the San Francisco Bay area. J Pediatric Infect Dis Soc. 2012;1(3):200–11. 15. Hsiang MS, Shiau R, Nadle J, Chan L, Lee B, Chambers HF, et al. Epidemio- logic similarities in pediatric community-associated methicillin-resistant and methicillin-sensitive in the San Francisco Bay area. J Pediatric Infect Dis Soc. 2012;1(3):200–11. Author details 1 1 Department of Military and Community Medicine, Phramongkutklao College of Medicine, 315, Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand. 2 Department of Outpatients, Phramongkutklao Hospital, Bangkok, Thailand. 3 Department of Parasitology, Phramongkutklao College of Medicine, 315, Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand. 4 Division of Derma- tology, Department of Internal Medicine, Phramongkutklao Hospital, Bangkok, Thailand. 5 Infection Control Unit of Phramongkutklao Hospital, Bangkok, 9. May L, Porter C, Tribble D, Armstrong A, Mostafa M, Riddle M. Self- reported incidence of skin and soft tissue infections among deployed US military. Travel Med Infect Dis. 2011;9(4):213–20. 10. Landrum ML, Neumann C, Cook C, Chukwuma U, Ellis MW, Hospenthal DR, et al. Epidemiology of Staphylococcus aureus blood and skin and soft tissue infections in the US military health system, 2005–2010. JAMA. 2012;308(1):50–9. g p g Thailand. 6 Clinical Epidemiology Unit of Phramongkutklao Hospital, Bangkok, Thailand. 11. Miller LG, Perdreau-Remington F, Bayer AS, Diep B, Tan N, Bharadwa K, et al. Clinical and epidemiologic characteristics cannot distinguish community-associated methicillin-resistant Staphylococcus aureus infection from methicillin-susceptible S. aureus infection: a prospective investigation. Clin Infect Dis. 2007;44(4):471–82. Conclusions However, different DNA patterns may be observed for the same strain, and similar DNA patterns may not necessarily indicate the same family of isolates [18]. The varying health status and behavioral activities of individuals in a community mean that some pathogens may be transmitted into the environment and passed on to others. Researchers have advocated that clones identi- fied by PFGE are indistinguishable from outbreak isolates and suggest person-to-person transmission [13]. Previous studies have identified the prevalence and risk factors of community-associated methicillin-resistant S. aureus (CA-MRSA) [8–10]. Although the risk fac- tors associated with acquiring CA-MRSA and MSSA infection are similar, the information regarding MSSA infection is limited [8, 11–13]. Inadequate hygiene and physical trauma are likely to lead to the emergence of MRSA infection among military trainees [12, 13]. The higher case rates observed for subunits 1 and 2 compared with subunits 3 and 4 in our study might be explained by the training activity involving low crawling on a concrete floor prior to the onset of abscess skin lesions among conscripts in subunits 1 and 2, while conscripts in subu- nits 3 and 4 did not undergo any such activity during the same period. Low crawl training often causes skin abra- sions on elbows and 43 % of cases had abscesses on their elbows. In summary, we identified an outbreak of MSSA infection among new military conscripts in a crowded environment during basic training. The risk factors asso- ciated with infection were identified as training exercises that involved crawling on a hard floor often resulting in skin abrasions, and the sharing of towels. Delayed diag- nosis and ineffective antibiotic treatment led to severe infection and fatality, for this normally treatable condi- tion. We recommend that a survey of Staphylococcus carriage among military camps be undertaken by the military medical agency to prevent similar outbreaks in the future. Furthermore, multiple erythematous papules are another important risk factor associated with abscess development. A recent study revealed that control of eczema can reduce the chance of S. aureus infection [14] and compromised skin integrity has been found to be associated with CA-MSSA infection [15]. A report of skin infection caused by MSSA or MRSA revealed the Panton-Valentine leukocidin (PVL) gene as a virulence factor [6]. It is possible that this outbreak of Nivesvivat et al. BMC Res Notes (2016) 9:179 Page 6 of 6 Authors’ contributions TN, DJ, MM, JI, SP, KP, PP, SK, OP, PY, WT, and RR conducted the outbreak inves- tigation. TN analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. 7. Miller LG, Kaplan SL. Staphylococcus aureus: a community pathogen. Infect Dis Clin N Am. 2009;23(1):35–52. 7. Miller LG, Kaplan SL. Staphylococcus aureus: a community pathogen. Infect Dis Clin N Am. 2009;23(1):35–52. 8. Zinderman CE, Conner B, Malakooti MA, LaMar JE, Armstrong A, Bohnker BK. Community-acquired methicillin-resistant Staphylococcus aureus among military recruits. Emerg Infect Dis. 2004;10(5):941–4. 8. Zinderman CE, Conner B, Malakooti MA, LaMar JE, Armstrong A, Bohnker BK. Community-acquired methicillin-resistant Staphylococcus aureus among military recruits. Emerg Infect Dis. 2004;10(5):941–4. Acknowledgements We thank the laboratory at the Division of Microbiology, Department of Clini- cal Pathology, Phramongkutklao Hospital, and the Reference Laboratory of the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand, for participating in this study. 12. Campbell KM, Vaughn AF, Russell KL, Smith B, Jimenez DL, Barrozo CP, et al. Risk factors for community-associated methicillin-resistant Staphy- lococcus aureus infections in an outbreak of disease among military train- ees in San Diego, California, in 2002. J Clin Microbiol. 2004;42(9):4050–3. 1. Razak F, Anand SS, Shannon H, Vuksan V, Davis B, Jacobs R, Teo KK, McQueen M, Yusuf S. Defining obesity cut points in a multiethnic popula- tion. Circulation. 2007;115(16):2111–8. 2. Clinical and Laboratory Standards Institute. Clinical and Laboratory Stand- ards Institute [document]. In: Wayne PA, editor. Clinical and Laboratory Standards Institute; 2005. p. v. 3. Smith CL, Klco SR, Cantor CR. Pulsed-field gel electrophoresis and the technology of large DNA molecules. In: Davies KE, editor. Genome analy- sis: a practical approach. Washington DC: IRL Press; 1988. p. 41–72. 4. Kitti T, Boonyonying K, Sitthisak S. Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand. Southeast Asian J Trop Med Public Health. 2011;42(6):1498–504. Competing interests 13. Aiello AE, Lowy FD, Wright LN, Larson EL. Meticillin-resistant Staphylo- coccus aureus among US prisoners and military personnel: review and recommendations for future studies. Lancet Infect Dis. 2006;6(6):335–41. 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 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
https://openalex.org/W4210720477
https://www.researchsquare.com/article/rs-143488/v1.pdf?c=1631885135000
English
null
The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study
Research Square (Research Square)
2,021
cc-by
5,098
The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study Seyedeh Forough Sajjadi  Tehran University of Medical Sciences Atieh Mirzababaei  Tehran University of Medical Sciences Afsoun Abdollahi  Purdue University Farideh Shiraseb  Tehran University of Medical Sciences Khadijeh Mirzaei  (  mirzaei_kh@tums.ac.ir ) Tehran University of Medical Sciences https://orcid The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study Seyedeh Forough Sajjadi  Tehran University of Medical Sciences Atieh Mirzababaei The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study Seyedeh Forough Sajjadi  Tehran University of Medical Sciences Atieh Mirzababaei The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study Seyedeh Forough Sajjadi  Tehran University of Medical Sciences Atieh Mirzababaei  Tehran University of Medical Sciences Afsoun Abdollahi  Purdue University Farideh Shiraseb  Tehran University of Medical Sciences Khadijeh Mirzaei  (  mirzaei_kh@tums.ac.ir ) Tehran University of Medical Sciences https://orcid.org/0000-0002-7554-8551 The Association Between Deficiency of Nutrient Intake on Resting Metabolic Rate in Overweight and Obese Women: a Cross-sectional Study Energy Expenditure Measurements RMR was measured by indirect calorimetry (spirometer METALYZERR 3B-R3, Cortex Biophysik GmbH, Leipzig, Germany). According to the manufacturer’s instructions, gas ventilation and exchange is calibrated before each test. RMR is evaluated by measuring the amount of O2 consumed and CO2 produced. The RMR was assessed in the morning, after a comfortable night’s sleep, and following a 10-12 hour fast. Participants were asked to avoid caffeine or alcohol consumption and severe exercise for a day before RMR measurements. After reclining in a steady-state and a supine position in a quiet room, the RMR was measured for 30 minutes. The respiratory exchange ratio and oxygen uptake (VO2) were analyzed within the middle 20 min of the resting period. Predictive RMR was determined using the Harris-Benedict equation, which considers the weight, height, and age of participants. Study population This cross-sectional research was performed on 293 adult women aged between 18 and 48 years old who were selected by a multistage cluster random sampling method that had been referred to health centers in Tehran recruited. Participants were enrolled in the study according to inclusion and exclusion criteria. Inclusion criteria were: general health, overweight and obese women with BMI in the range of 25-40 kg/m2. The exclusion criteria were as follows: regular use of medicine, history of hypertension, cardiovascular diseases, and other chronic diseases, alcohol consumption, smoking, pregnancy, lactation period, and menopause. Furthermore, those who had been following an arbitrary special dietary regimen, and also those with any significant body weight fluctuations over the past 1 year and energy intakes lower than 800 kcal/d or higher than 4200 kcal/d were excluded. Abstract Objective: The double burden of malnutrition is an emerging public health concern nowadays which a correlation with Obesity. This study aimed to examine the relationship between resting metabolic rate (RMR) and dietary intake of zinc, vitamin C, and riboflavin in overweight and obese women. Results: the RMR/FFM showed a significant association with riboflavin (β=1.59; 95% CI: 1.04-23.26, P=0.04) and zinc (β=0.78; 95% CI: 1.04-4.61, P=0.03) in the crude model. Moreover, differences in vitamin C and RMR/FFM was marginal significant (β=0.75; 95% CI: 0.95-4.77, P=0.06). After adjusting for confounders the riboflavin association change to marginal significance (β=1.52; 95% CI: 0.91-23.04, P=0.06). After controlling for potential confounders, the associations change between zinc and RMR/FFM (β=0.66; 95% CI: 0.78-4.86, P=0.15) and between RMR/FFM and vitamin C (β=0.48; 95% CI: 0.66-3.96, P=0.28). Our study showed a significant association between dietary intake of zinc, riboflavin, and vitamin C and change in RMR/FFM in overweight and obese women. Research note Keywords: Resting metabolic rate, fat-free mass, obesity, overweight, the double burden of malnutrition, Nutrient Adequacy Ratio Keywords: Resting metabolic rate, fat-free mass, obesity, overweight, the double burden of malnutrition, Nutrient Adequacy Ratio Posted Date: January 15th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/9 Introduction Obesity rates are growing globally (1). More than 26% of the Iranian adult community is currently obese (2) Females were much more affected than males (3, 4). Obesity and its associated metabolic disorders develop when energy intake is more than energy expenditure; this can be caused by diminished physical activity, the disability of the central nervous system to down-regulate the ingestion of high-calorie foods, or appetite (5). Although various factors contribute to the etiology of obesity, sedentary lifestyles, and unhealthy eating habits are among the principal contributors to the world obesity epidemic (6). 60_75% of total energy expenditure is correlated with resting metabolic rate (RMR), which is an important part of daily energy consumption (7). weight gaining and obesity may be associated with low RMR(8). Diet quality can be evaluated to better understand overall eating patterns (9). Poor diet quality is a significant factor in the development of many chronic diseases, including obesity (10). Investigations revealed, however, intake of vegetables and fruit is associated with a lower risk of obesity (11), inappropriate food behavior contributes to obesity and contributes to vitamin deficiency. Studies revealed that most vitamins are inadequate in obese individuals (12). The double burden of malnutrition (DBM) is an emerging public health concern nowadays that happens as an inevitable consequence of nutritional transition (13). The coexistence of overnutrition and undernutrition is often referred to as the DBM (14). Nutrient Adequacy Ratio (NAR) and mean adequacy ratio (MAR), known as healthier diet quality indices. Higher scores in diet quality inversely connected with body mass index (BMI), and obesity (15). Similarly, zinc and vitamin C are closely related to adiposity(16, 17). In particular, some diet with a high score of diet quality like the Mediterranean dietary pattern has been reported to be inversely connected with BMI, and waist circumference (18). To the best of our knowledge, this is the first study to investigate the relationship between deficiency of nutrient intakes such as zinc, vitamin C, and riboflavin and RMR in the adult women population. Accordingly, this study was performed to examine the NAR with RMR/FFM among a group of Iranian adult women. Body Composition Measurement Body composition, including weight, BMI, fat mass, and fat-free mass (FFM) were acquired using a multi-frequency bioelectrical impedance analyzer InBody 770 scanner (Inbody Co., Seoul, Korea According to the manufacturer’s instructions, participants removed their shoes, coats, and sweaters, and stood on the balance scale in bare feet, and grasped the handles of the machine.­ Page 2/9 Page 2/9 Assessment of other covariates International Physical Activity Questionnaire (IPAQ, short form) were obtained by using an interview-based questionnaire from all participants about all the vigorous and moderate elements over the last 7 days, considering the time spent on these activities for height measurements, subjects were in a standing position without shoes, in contact with the wall with their head, shoulders, heels, and hips, and their height was recorded to the nearest 0.1 cm. Dietary intake assessment Dietary intake data of the past year were obtained using a validated semi-quantitative food-frequency questionnaire (FFQ)(21), comprised of 168-item a trained nutritionist administered these FFQ. The FFQ consisted of a list of foods with standard serving sizes. Participants were asked to report their frequency and amount of each food item consumed during the previous year. Portion sizes of the consumed foods were converted to grams using household measurements(22). Nutritionist IV computer software was used for the nutrient analysis of the diets. The database of this software was modified for Iranian foods. Nutrient Adequacy Ratios (NAR) For calculating the NAR, the ratio of daily individual intakes to the standard recommended amounts for the subject's sex and age category was used. The standard recommended amounts are based on RDA (Recommended Daily Allowances) (23). We calculated the NAR for three key nutrients, including zinc, vitamin C, and riboflavin according to the above-mentioned method. The prevalence of nutrient deficiency was estimated using NAR. NAR lower than one is considered as a deficiency. Biochemical Assessment and Hormonal As­say Metabolic health was assessed using the metabolic parameters that measured following standard chemical procedures. A 12-hour fasting venous blood sample was used to measure all biochemical markers. Serum glucose was evaluated by a colorimetric method based on the GOD-PAP method. Serum insulin concentrations were analyzed by enzyme-linked immunosorbent assay (ELISA) method (Human insulin ELISA kit, Monobind Inc., Lake Forest, USA). HOMA and QUICKI Calculations Insulin resistance was estimated by homeostasis model assessment (HOMA). The HOMA was calculated accord­ing to the following equation: HOMA = [Fasting Plasma Glu­cose (mmol/L) × Fasting Plasma Insulin (mIU/L)]/22.5 (19). Insulin sensitivity quantitative insulin sensitivity check index (ISQUICKI) was assessed by: ISQUICKI = 1/[log (fasting insulin) + log (fasting glucose) (20). Statistical analysis All statistical analysis was performed using the IBM SPSS software version 22.0 (SPSS, Chicago, IL, USA), and p-values less than 0.05 were considered statistically significant. Normal distribution of data was checked by the Kolmogorov-Smirnov test. An independent sample t-test was used for assessed differences between groups with the low and standard intake of nutrients. RMR/FFM was analyzed after adjusting for FFM. The differences between RMR/FFM groups and dietary intake of nutrients were assessed by the Binary logistic regression were performed to adjust for confounders effects such as age, energy intake, and physical activity (METs/d). Results were presented as odds ratios (ORs) and 95% con­fidence intervals (CIs) compared with the RMR groups. Study population characteristics A total of 293 healthy overweight and obese women were enrolled. The mean age, height, weight, and BMI of the study participants were 36.39 years (SD =  8.71), 161.84 cm (SD = 5.85), 80.22 kg (SD = 11.28), and 30.77 kg/m2 (SD = 3.79), respectively (Table 1) the mean body composition, RMR components, biochemical and anthropometric characteristics of subjects are shown in table 1. Page 3/9 Table 1 Table 1 Study of population characteristics Parameters Minimum Maximum Mean ± SD Age (years) 18 56 36.39±8.71 Height (cm) 147.50 179.00 161.84±5.85 Weight (kg) 59.50 122.40 80.22±11.28 BMI (kg/m2) 25.00 40.70 30.77±3.79 RMR parameters RMR measure (kcal/day) 952.00 2467.00 1569.60±255.70 RQ 0.73 0.99 0.85±0.04 RMR/FFM (kcal/day/kg) 21.50 45.86 33.74±4.48 V. O2 0.14 0.35 0.22±0.036 V. CO2 0.01 0.30 0.19±0.034 Body composition  parameters Body fat mass (kg) 19.40 59.40 33.52±7.74 Fat free mass (kg) 35.30 67.70 46.68±5.50 Blood parameters FBS (mg/dl) 67.00 137.00 87.39±9.76 Insulin (mIU/ml) 6.67 65.89 15.59±6.02 HOMA 1.29 16.59 3.40±1.52 ISQUICKI 0.39 0.68 0.54±0.04 RMR, resting metabolic rate; RQ, respiratory quotient; FFM, fat-free mass; V. O2, Oxygen consumption; V. CO2, carbon dioxide consumption; FBS, fasting blood sugar; HOMA, homeostasis model assessment; ISQUICKI, insulin sensitivity quantitative insulin sensitivity check index. N=293 Data are indicated as Mean ± SD otherwise indicated Participant’s characteristics between standard and deficiency of daily nutrient intakes Dietary intake of three nutrients including riboflavin, vitamin C, and zinc were categorized based on nutrient adequacy ratios (NAR) and divided into two groups, standard and deficiency (Table 2). The RMR indicated a significant association with zinc (P = 0.001), which demonstrates people who consume higher zinc had higher RMR. Moreover, other factors like RMR/FFM (P = 0.06), V. O2 (P = 0.001), V. CO2 (P = 0.007), body fat mass (P = 0.02), FFM (P = 0.006) height (P = 0.005), and weight (P = 0.006) had a significant relationship with zinc. Besides, riboflavin had a significant association with body fat mass (P =  0.05), however, there were showed no relationship between vitamin C and participant characteristics (P > 0.05). Page 4/9 Table 2 Participants’ Characteristics between Standard and Deficiency of Daily Nutrient Intakes     Parameters                                                                                     Nutrient               Vitamin c                      zinc            Riboflavin NAR <1 NAR ≥1   P- value NAR <1 NAR ≥1   P- value NAR <1 NAR ≥1   P- value n=30 n=250 n=36 n=244 n=11 n=269 10.2% 85.3% 12.3% 83.3% 3.8% 91.8% Age (years) 34.90±8.62 63.60±8.53 0.30 38.05±9.13 36.18±8.44 0.22 31.63±8.41 36.62±8.50 0.08 Height (cm) 161.63±6.27 161.32±5.82 0.78 158.78±6.33 161.74±5.70 0.005 160.27±8.16 161.40±5.76 0.53 Weight (kg) 77.54±9.28 80.32±11.13 0.19 75.35±8.78 80.71±11.10 0.006 75.61±14.41 80.20±10.80 0.17 BMI (kg/m2) 29.78±3.29 30.87±3.78 0.13 29.91±3.40 30.88±3.78 0.15 29.30±4.03 30.81±3.72 0.19 RMR parameters RMR measure (kcal/day) 1500.2±307.28 1580.2±247.49 0.17 1437.5±288.33 1591.7±244.25 0.001 1450.6±301.44 1576.5±252.59 0.10 RQ 0.86±0.03 0.85±0.04 0.16 0.86±0.042 0.85±0.041 0.39 0.858±0.03 0.854±0.04   RMR/FFM (kcal/day/kg) 00.04±5.60 33.92±4.36 0.32 32.50±5.10 34.02±4.39 0.06 31.79±2.93 33.91±4.54 0.12 V. O2 0.21±0.04 0.22±0.03 0.13 0.20±0.04 0.22±0.03 0.001 0.20±0.04 0.22±0.03 0.13 V. CO2 0.18±0.04 0.19±0.03 0.22 0.17±0.04 0.19±0.03 0.007 0.17±0.04 0.19±0.03 0.16 Body composition parameters Body fat mass (kg) 31.80±6.66 33.65±7.77 0.21 30.79±6.66 33.84±7.74 0.02 29.15±7.10 33.63±7.66 0.05 Fat free mass (kg) 45.63±5.26 46.76±5.44 0.28 44.27±4.74 46.98±5.44 0.006 45.55±7.70 46.69±5.33 0.49 Blood parameters FBS (mg/dl) 86.50±10.59 87.51±9.64 0.60 87.93±11.62 87.30±9.34 0.73 83.30±7.33 87.57±9.81 0.17 Insulin (mIU/ml) 14.75±4.77 15.70±6.30 0.44 15.37±4.15 15.62±6.41 0.83 18.28±10.03 15.47±5.93 0.40 HOMA 3.17±1.17 3.42±1.59 0.41 3.35±1.05 3.40±1.61 0.84 3.81±2.33 3.38±1.50 0.38 ISQUICKI 0.55±0.045 0.54±0.049 0.49 0.540±0.03 0.545±0.05 0.58 0.53±0.05 0.54±0.04 0.70 RMR, resting metabolic rate; RQ, respiratory quotient; FFM, fat-free mass; V. O2,  Oxygen consumption; V. CO2,  carbon dioxide consumption; FBS, fasting blood sugar; HOMA, homeostasis model assessment; ISQUICKI, insulin sensitivity quantitative insulin sensitivity check index. N=293 Participants’ Characteristics between Standard and Deficiency of Daily Nutrient Intakes                                                           Nutrient Association of riboflavin, vitamin C, and zinc with RMR/FFM among obese women Table 3 shows multivariate-adjusted models for the prevalence of higher RMR/FFM across the median dietary intake of riboflavin, vitamin C, and zinc. The RMR/FFM showed a significant association with riboflavin, vitamin C, and zinc in the crude model. For riboflavin, in the crude model before adjustment for the confounders, showed a statistically significant relationship (β=1.59; 95% CI: 1.04-23.26, P=0.04). After controlling for the potential confounders, the association change to marginal significance (β=1.52; 95% CI: 0.91-23.04, P=0.06). Moreover, differences in vitamin C and RMR/FFM was marginal significant (β=0.75; 95% CI: 0.95-4.77, P=0.06). But after controlling for the potential confounders, the association disappeared (P=0.28). Differences in zinc and RMR/FFM were also significant in the crude model (β=0.78; 95% CI: 1.04-4.61, P=0.03), but after adjustment for the potential confounders, the association disappeared (P=0.15). Page 5/9 Page 5/9 Table 3 Association of riboflavin, vitamin C and zinc and RMR/FFM among obese women RMR/FFM β OR (95% CI) P-value Crude Model   Riboflavin 1.59 4.93 (1.04-23.26) 0.04 Model1 a 1.75 5.80 (1.21-27.77) 0.02 Model2 b 1.71 5.55 (1.11-27.59) 0.03 Model3 c 1.52 4.58 (0.91-23.04) 0.06 Crude Model   Vitamin C 0.75 2.13 (0.95-4.77) 0.06 Model1 a 0.82 2.27 (1.01-5.13) 0.04 Model2 b 0.79 2.20 (0.93-5.19) 0.07 Model3 c 0.48 1.62 (0.66-3.96) 0.28 Crude Model   Zinc 0.78 2.19 (1.04-4.61) 0.03 Model1 a 0.71 2.04 (0.96-4.33) 0.06 Model2 b 0.71 2.04 (0.87-4.76) 0.09 Model3 c 0.66 1.95 (0.78-4.86) 0.15 P-values are from binary logistic regression Model 1: Adjusted for age. bModel2: Further adjusted for energy intake. cModel3: Further adjusted for physical activity (METs/d). Association of riboflavin, vitamin C and zinc and RMR/FFM among obese women β OR (95% CI) Discussion Furthermore, resting reactive oxygen species production was raised while respiratory burs, a key ingredient of intracellular killing, were destroyed. Considering the significant function of adipocytes in the creation of obesity-related chronic inflammation to be justifiable to verify the influence of riboflavin deficiency on adipocytes function in the context of pro-inflammatory activation (36). Discussion In an in-vitro assay, it was perceived that vitamin C inhibited glucose uptake and lactate production, and decreased glycerol release, and caused a consistent reduction of leptin secretion in a dose-dependent manner. (12). In this study, we also found that women who consumed higher riboflavin were more likely to have higher RMR. Considering the dietary restrictions on food intake or common dietary mistakes perceived among obese people, changes in the micronutrient intake leading to their deficiency are possible. Suitable riboflavin content is necessary to perform the effector function of macrophages with inhibition proliferation, intensification of apoptosis incidence, and also the reduction in phagocytosis efficiency [9]. Furthermore, resting reactive oxygen species production was raised while respiratory burs, a key ingredient of intracellular killing, were destroyed. Considering the significant function of adipocytes in the creation of obesity-related chronic inflammation to be justifiable to verify the influence of riboflavin deficiency on adipocytes function in the context of pro-inflammatory activation (36). This study found a significant association between vitamin C and RMR. It showed women with a higher intake of vitamin C had higher RMR. This result is in line with previous studies which showed that the increase in blood vitamin C concentrations associated with the change in RMR/FFM (32). In contrast, some other studies have shown that vitamin C administration significantly decreased RMR (33). A probable mechanism could be ascorbic acid modulation on lipolysis in the interior of adipocytes and inflammatory response (34). Vitamin C, an antioxidant, regulates glucocorticoid metabolism, and gene expression involved in adipogenesis (35). Ascorbic acid decreased weight gain and adipose tissue correlated with low expression of genes involved in adipogenesis, adipocyte differentiation, and metabolism of glucocorticoids (12). In an in-vitro assay, it was perceived that vitamin C inhibited glucose uptake and lactate production, and decreased glycerol release, and caused a consistent reduction of leptin secretion in a dose-dependent manner. (12). In this study, we also found that women who consumed higher riboflavin were more likely to have higher RMR. Considering the dietary restrictions on food intake or common dietary mistakes perceived among obese people, changes in the micronutrient intake leading to their deficiency are possible. Suitable riboflavin content is necessary to perform the effector function of macrophages with inhibition proliferation, intensification of apoptosis incidence, and also the reduction in phagocytosis efficiency [9]. Discussion The results of this study showed a significant association between vitamin C, zinc, and riboflavin intake and the RMR/FFM. RMR was distinct in standard or deficiency consumption of vitamin C, zinc, and riboflavin. The results of this study showed a significant association between vitamin C, zinc, and riboflavin intake and t deficiency consumption of vitamin C, zinc, and riboflavin. The findings of the current study indicate that the amount of zinc consumed has been associated with RMR which higher intakes of zinc enhance RMR. This finding is in the same line with a study by Maxwell which demonstrates that zinc is an essential factor in several enzymes, involved with energy metabolisms such as pyruvate carboxylase, lactate dehydrogenase, carbonic anhydrase, and aldolase (24). Some studies state that zinc is connected to obesity (25). Zinc deficiency may increase fat deposition and decrease lean mass accrual (26). The association between adiposity and zinc status may be attributed to the correlation between zinc metabolism and leptin (26). Moreover, zinc has different functions in the metabolism of energy and works as a component of several enzymes crucial to the metabolism of carbohydrates, proteins, and lipids and metabolism of hormones that take part in the progress of obesity, especially insulin, and seems to be connected with the mechanisms of insulin resistance usually present among obese people (11, 27, 28). Previous investigations recommend a negative association between RMR and insulin resistance (29, 30). Subjects with obesity and impaired glucose tolerance showed higher RMR levels than those with obesity and normal glucose tolerance (31). This study found a significant association between vitamin C and RMR. It showed women with a higher intake of vitamin C had higher RMR. This result is in line with previous studies which showed that the increase in blood vitamin C concentrations associated with the change in RMR/FFM (32). In contrast, some other studies have shown that vitamin C administration significantly decreased RMR (33). A probable mechanism could be ascorbic acid modulation on lipolysis in the interior of adipocytes and inflammatory response (34). Vitamin C, an antioxidant, regulates glucocorticoid metabolism, and gene expression involved in adipogenesis (35). Ascorbic acid decreased weight gain and adipose tissue correlated with low expression of genes involved in adipogenesis, adipocyte differentiation, and metabolism of glucocorticoids (12). Ethics approval and consent to participate All procedures involving human subjects were approved by the Ethics Commission of Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1395.1597), and all participants signed written informed consent. Consent for publication This is formally to submit the article entitled “The association between deficiency of nutrient on resting metabolic rate in overweight and obese women: a cross-sectional study” prepared by the Tehran University of Medical Sciences for review and, hopefully, publication in your prestigious journal. The authors would like to advise that all authors listed have contributed to the work. All authors have agreed to submit the manuscript to Diabetology & Metabolic Syndrome. No part of the work has been published before. There is no conflict of interest in this paper. Funding This study is funded by grants from the Tehran University of Medical Sciences (TUMS). (Grant ID: 97-03-161-41155). Conclusions In conclusion, we could find a significant association between dietary intake of zinc, riboflavin, and vitamin C and change in RMR/FFM in overweight and obese women, although after controlling for ranges of potential confounding factors the meaningful decreased. Page 6/9 Page 6/9 Limitation The major limitation of this study was the participants in the same-sex sample that it is not possible to generalize the results to men population. Because of the study type, cross-sectional study, we could not determine the causality Another limitation for assessing dietary intakes from FFQ is misclassification. Albeit we controlled for the effect of the potential confounder by the statistical methods, because of unknown confounder cannot be excluded residual confounding will affect. Availability of data and materials Participants in this study did not agree to the public sharing of their data so supporting data is not available. Author’s contributions SFS, AM, AA, and FSh wrote the Manuscript, KhM had full access to all the data in the study and was responsible for the integrity and accuracy of the data. All authors have read and approved the final manuscript. SFS, AM, AA, and FSh wrote the Manuscript, KhM had full access to all the data in the study and was respons All authors have read and approved the final manuscript. Competing interests All authors declared that they have no competing interests Abbreviations RMR: Resting Metabolic Rate; DBM: Double Burden of Malnutrition; NAR: Nutrient Adequacy Ratio; MAR: Mean Adequacy Ratio; BMI: Body Mass Index; FFM: Fat-Free Mass; ELISA: Enzyme-Linked Immuno-Sorbent Assay; HOMA: homeostasis model assessment; ISQUICKI: Insulin Sensitivity Quantitative Insulin Sensitivity Check Index; FFQ: Food Frequency Questionnaire; RDA: Recommended Daily Allowances; IPAQ: International Physical Activity Questionnaire; RQ: Respiratory Quotient; FBS: Fasting Blood Sugar. Acknowledgment We are extremely grateful to all the participants who took part in this study and the school of Nutritional and Dietetics at Tehran University of medical sciences. Sources of Support Tehran University of Medical Sciences Conflicts of Interest Page 7/9 Page 7/9 References Hasan M, Sutradhar I, Shahabuddin A, Sarker M. Double Burden of Malnutrition among Bangladeshi Women: 14. Sekiyama M, Jiang HW, Gunawan B, Dewanti L, Honda R, Shimizu-Furusawa H, et al. Double Burden of Malnutrition in Rural West Java: Household- 14. Sekiyama M, Jiang HW, Gunawan B, Dewanti L, Honda R, Shimizu-Furusawa H, et al. Double Burden of Malnutrition in Rural West Java: Household- Level Analysis for Father-Child and Mother-Child Pairs and the Association with Dietary Intake. Nutrients. 2015;7(10):8376-91. 4. Sekiyama M, Jiang HW, Gunawan B, Dewanti L, Honda R, Shimizu-Furusawa H, et al. Double Burden of Malnu Level Analysis for Father-Child and Mother-Child Pairs and the Association with Dietary Intake. Nutrients. 201 15. Fallaize R, Livingstone KM, Celis-Morales C, Macready AL, San-Cristobal R, Navas-Carretero S, et al. Association between diet-quality scores, adiposity, total cholesterol and markers of nutritional status in european adults: Findings from the Food4Me study. Nutrients. 2018;10(1):49. 6. Hu FB. Dietary pattern analysis: a new direction in nutritional epidemiology. Current opinion in lipidology. 200 16. Hu FB. Dietary pattern analysis: a new direction in nutritional epidemiology. Current opinion in lipidology. 2002;13(1):3-9. 7. Md G, Pac A, KüÇüKbay F, Tayfun M, GüL A. Serum zinc, copper, and magnesium levels in obese children. Ped 18. Bertoli S, Leone A, Vignati L, Bedogni G, Martínez-González MÁ, Bes-Rastrollo M, et al. Adherence to the Mediterranean diet is inversely associated with visceral abdominal tissue in Caucasian subjects. Clinical nutrition. 2015;34(6):1266-72. 19. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28(7):412-9. 20. Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G, et al. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab. 2000;85(7):2402-10. 21. Mirmiran P, Hosseini Esfahani F, Azizi F. Relative validity and reliability of the food frequency questionnaire used to assess nutrient intake: Tehran Lipid and Glucose Study. Iranian Journal of Diabetes and Lipid Disorders. 2009;9(2):185-97. Zabik ME, Stampley GL. The role of breakfast in diet adequacy of the US adult population. J Am Coll Nutr. 1986 22. Morgan KJ, Zabik ME, Stampley GL. The role of breakfast in diet adequacy of the US adult population. J A 22. Morgan KJ, Zabik ME, Stampley GL. The role of breakfast in diet adequacy of the US adult population. References 1. Conklin AI, Ponce NA, Crespi CM, Frank J, Nandi A, Heymann J. Economic policy and the double burden of malnutrition: cross-national longitudinal analysis of minimum wage and women's underweight and obesity. Public Health Nutr. 2018;21(5):940-7. 1. Conklin AI, Ponce NA, Crespi CM, Frank J, Nandi A, Heymann J. Economic policy and the double burden of ma analysis of minimum wage and women's underweight and obesity. Public Health Nutr. 2018;21(5):940-7. 2. WHO. Eastern Mediterranean Region: Framework for health information systems and core indicators for monitoring health situation and health system performance–2015. From: applications emro who int/dsaf/EMROPUB_2015_EN_1904 pdf. 2015. 3. Jafari-Adli S, Jouyandeh Z, Qorbani M, Soroush A, Larijani B, Hasani-Ranjbar S. Prevalence of obesity and overweight in adults and children in Iran; a systematic review. Journal of Diabetes & Metabolic Disorders. 2014;13(1):1. 4. Barak F, Falahi E, Keshteli AH, Yazdannik A, Esmaillzadeh A. Adherence to the Dietary Approaches to Stop Hypertension (DASH) diet in relation to obesity among Iranian female nurses. Public health nutrition. 2015;18(4):705-12. 5. Fukunaka A, Fujitani Y. Role of Zinc Homeostasis in the Pathogenesis of Diabetes and Obesity. Int J Mol Sci. 2018;19(2). 6. Shi Z, Makrides M, Zhou SJ. Dietary patterns and obesity in preschool children in Australia: a cross-sectional study. Asia Pac J Clin Nutr. 2018;27(2):406-12. 7. Kim DK. Accuracy of predicted resting metabolic rate and relationship between resting metabolic rate and cardiorespiratory fitness in obese men. Journal of exercise nutrition & biochemistry. 2014;18(1):25. 8. Buscemi S, Verga S, Caimi G, Cerasola G. Low relative resting metabolic rate and body weight gain in adult Caucasian Italians. International journal of obesity. 2005;29(3):287-91. 9. Ruiz LD, Zuelch ML, Dimitratos SM, Scherr RE. Adolescent Obesity: Diet Quality, Psychosocial Health, and Cardiometabolic Risk Factors. Nutrients. 2019;12(1):43. 10. Bettermann EL, Hartman TJ, Easley KA, Ferranti EP, Jones DP, Quyyumi AA, et al. Higher mediterranean diet quality scores and lower body mass index are associated with a less-oxidized plasma glutathione and cysteine redox status in adults. The Journal of nutrition. 2018;148(2):245-53. 11. Hosseini B, Saedisomeolia A, Allman-Farinelli M. Association Between Antioxidant Intake/Status and Obesity: a Systematic Review of Observational Studies. Biol Trace Elem Res. 2017;175(2):287-97. 12. Thomas-Valdés S, Tostes MdGV, Anunciação PC, da Silva BP, Sant'Ana HMP. Association between vitamin deficiency and metabolic disorders related to obesity. Critical Reviews in Food Science and Nutrition. 2017;57(15):3332-43. Sarker M. Double Burden of Malnutrition among Bangladeshi Women: A Literature Review. Cureus. 2017;9(12). 3. References J Am Coll Nutr. 1986;5(6):551-63. 23. L.K. Mahan SE-s. Krause's food nutrition and diet therapy (10th ed.). WB Saunders, Philadelphia. 2008. 24. Maxwell C, Volpe SL. Effect of zinc supplementation on thyroid hormone function. A case study of two college females. Ann Nutr Metab. 2007;51(2):188-94. 25. Chen M-D, Lin P-Y, Sheu WH-H. Zinc status in plasma of obese individuals during glucose administration. Biological trace element research. 1997;60(1- 2):123-9. 26. Weisstaub G, Hertrampf E, De Romana DL, Salazar G, Bugueño C, Castillo-Duran C. Plasma zinc concentration, body composition and physical activity in obese preschool children. Biological trace element research. 2007;118(2):167-74. 27. do Nascimento Marreiro D, Fisberg M, Cozzolino SMF. Zinc nutritional status and its relationships with hyperinsulinemia in obese children and adolescents. Biological trace element research. 2004;100(2):137-49. 28. Mahawar KK, Bhasker AG, Bindal V, Graham Y, Dudeja U, Lakdawala M, et al. Zinc Deficiency after Gastric Bypass for Morbid Obesity: a Systematic Review. Obes Surg. 2017;27(2):522-9. 29. Alawad AO, Merghani TH, Ballal MA. Resting metabolic rate in obese diabetic and obese non-diabetic subjects and its relation to glycaemic control. BMC Res Notes. 2013;6(382):1756-0500. Page 8/9 Page 8/9 30. Sun MX, Zhao S, Mao H, Wang ZJ, Zhang XY, Yi L. Increased BMR in overweight and obese patients with type 2 diabetes may result from an increased fat-free mass. J Huazhong Univ Sci Technolog Med Sci. 2016;36(1):59-63. 31. Drabsch T, Holzapfel C, Stecher L, Petzold J, Skurk T, Hauner H. Associations Between C-Reactive Protein, Insulin Sensitivity, and Resting Metabolic Rate in Adults: A Mediator Analysis. Front Endocrinol. 2018;9(556). 32. Velthuis-te Wierik EJ, van Leeuwen RE, Hendriks HF, Verhagen H, Loft S, Poulsen HE, et al. Short-term moderate energy restriction does not affect indicators of oxidative stress and genotoxicity in humans. J Nutr. 1995;125(10):2631-9. 33. Owu DU, Antai AB, Udofia KH, Obembe AO, Obasi KO, Eteng MU. Vitamin C improves basal metabolic rate and lipid profile in alloxan-induced diabetes mellitus in rats. J Biosci. 2006;31(5):575-9. 33. Owu DU, Antai AB, Udofia KH, Obembe AO, Obasi KO, Eteng MU. Vitamin C improves basal metabolic rate and lipid profile in alloxan-induced diabetes mellitus in rats. J Biosci. 2006;31(5):575-9. 34. Stenzel AP, Carvalho R, Jesus P, Bull A, Pereira S, Saboya C, et al. Serum Antioxidant Associations with Metabolic Characteristics in Metabolically Healthy and Unhealthy Adolescents with Severe Obesity: An Observational Study. Nutrients. 2018;10(2). 34. References Stenzel AP, Carvalho R, Jesus P, Bull A, Pereira S, Saboya C, et al. Serum Antioxidant Associations with Metabolic Characteristics in Metabolically Healthy and Unhealthy Adolescents with Severe Obesity: An Observational Study. Nutrients. 2018;10(2). 35. Park B, Kim J. Oral contraceptive use, micronutrient deficiency, and obesity among premenopausal females in Korea: The necessity of dietary supplements and food intake improvement. PloS one. 2016;11(6):e0158177. 35. Park B, Kim J. Oral contraceptive use, micronutrient deficiency, and obesity among premenopausal females in Korea: The necessity of dietary supplements and food intake improvement. PloS one. 2016;11(6):e0158177. 36. Mazur-Bialy AI, Pochec E. Vitamin B2 deficiency enhances the pro-inflammatory activity of adipocyte, consequences for insulin resistance and metabolic syndrome development. Life Sci. 2017;178:9-16. 36. Mazur-Bialy AI, Pochec E. Vitamin B2 deficiency enhances the pro-inflammatory activity of adipocyte, consequences for insulin resistance and metabolic syndrome development. Life Sci. 2017;178:9-16. Page 9/9
https://openalex.org/W3125385181
https://hal.sorbonne-universite.fr/hal-03127098/file/children-08-00084-1.pdf
English
null
Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis
Children
2,021
cc-by
6,256
Article Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis Nathalie Duparc Alegria 1, Enora Le Roux 2,3, Artemis Toumazi 2, Anne-Françoise Thiollier 1, Malika Benkerrou 3,4, Sophie Dugue 5 and Berengere Koehl 4,6,* Jeanne Sigalla 1 , Nathalie Duparc Alegria 1, Enora Le Roux 2,3, Artemis Toumazi 2, Anne-Françoise Thiollier 1, Laurent Holvoet 4, Malika Benkerrou 3,4, Sophie Dugue 5 and Berengere Koehl 4,6,* 1 Pain Management Unit, Hôpital Robert Debré, APHP, F-75019 Paris, France; jeanne.sigalla@aphp.fr (J.S.); nathalie.duparc@aphp.fr (N.D.A.); anne.thiollier@aphp.fr (A.-F.T.) p p p p p 2 Unité d’Épidémiologie Clinique, Inserm, CIC 1426, Hôpital Universitaire Robert Debré, AP-HP, Nord-Université de Paris, F-75019 Paris, France; enora.leroux@aphp.fr (E.L.R.); artemis.toumazi@aphp.fr (A.T.) p p 3 Université de Paris, ECEVE UMR 1123, Inserm, F-75010 Paris, France; malika.benkerrou@aphp.fr 4 Université de Paris, ECEVE UMR 1123, Inserm, F-75010 Paris, France; malika.benkerrou@aphp.fr 4 Department of Hematology, Reference Center of Sickle Cell Disease, Hôpital Universitaire Robert Debré, AP-HP, Nord-Université de Paris, F-75019 Paris, France; laurent.holvoet@aphp.fr 5 P i M U i T H i l AP HP S b U i i é F 75012 P i F 4 Department of Hematology, Reference Center of Sickle Cell Disease, Hôpital Universitaire Robert Debré AP-HP, Nord-Université de Paris, F-75019 Paris, France; laurent.holvoet@aphp.fr p p 5 Pain Management Unit, Trousseau Hospital, AP-HP, Sorbonne Université, F-75012 Paris, France; sophie.dugue@aphp.fr p g p p 6 INSERM UMRS 1134, Red Blood Cell Pathophysiology, F 6 INSERM UMRS 1134, Red Blood Cell Pathophysiology, F-75015 Paris, France * Correspondence: berengere.koehl@aphp.fr Abstract: The majority of hospitalizations of patients with sickle cell disease (SCD) are related to painful vaso-occlusive crises (VOCs). Although the pain of VOC is classically nociceptive, neuro- pathic pain (NP) has also been demonstrated in SCD patients. The aim of our study is to specify the prevalence of NP during VOCs in SCD children using a dedicated scale and to measure its characteristics. We performed a prospective study that included SCD children hospitalized for an acute VOC. The presence of NP was sought with the DN4 scale on the second and fourth days of hospitalization. A total of 54 SCD children were included in the study. Overall, 41% of the patients (n = 22) experienced neuropathic pain during the VOC, mostly at an early stage (Day 2). The median age, the sex ratio, the location of the pain, and the morphine consumption were similar for patients with and without NP. Our study shows that neuropathic pain is very common during VOCs in SCD children.   Citation: Sigalla, J.; Duparc Alegria, N.; Le Roux, E.; Toumazi, A.; Thiollier, A.-F.; Holvoet, L.; Benkerrou, M.; Dugue, S.; Koehl, B. Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis. Children 2021, 8, 84. https://doi.org/10.3390/ children8020084 Citation: Sigalla, J.; Duparc Alegria, N.; Le Roux, E.; Toumazi, A.; Thiollier, A.-F.; Holvoet, L.; Benkerrou, M.; Dugue, S.; Koehl, B. Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis. Children 2021, 8, 84. https://doi.org/10.3390/ children8020084 Keywords: sickle cell disease; neuropathic pain; vaso-occlusive crisis; pediatrics; DN-4 Academic Editor: Karel Allegaert Received: 30 December 2020 Accepted: 21 January 2021 Published: 26 January 2021 Academic Editor: Karel Allegaert Received: 30 December 2020 Accepted: 21 January 2021 Published: 26 January 2021 Citation: Sigalla, J.; Duparc Alegria, N.; Le Roux, E.; Toumazi, A.; Thiollier, A.-F.; Holvoet, L.; Benkerrou, M.; Dugue, S.; Koehl, B. Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis. Children 2021, 8, 84. https://doi.org/10.3390/ children8020084 Article Neuropathic Pain in Children with Sickle Cell Disease: The Hidden Side of the Vaso-Occlusive Crisis The absence of identified risk factors should prompt us to be vigilant regardless of the patient’s age, sex, and clinical presentation. children children children 1. Introduction Sickle cell disease (SCD) is a genetic hemoglobinopathy resulting from a unique mutation in the β-globin gene characterized by hemolytic anemia, painful vaso-occlusive crisis (VOC), and progressive organ failure. SCD is the most common hemoglobinopathy worldwide, with approximately 300,000 new cases each year and millions of patients, mainly in Africa, but also in the United States, Europe, and South America [1]. A total of 95% of hospitalizations of patients with SCD are related to acute painful episodes caused by VOCs [2]. Vaso-occlusion is a multifactorial process involving occlusion of small blood vessels by sickled red blood cells (RBCs), polymorphonuclear neutrophils, platelets, and activated endothelial cells. This occlusive process leads to hypoxia and then ischemia of the upstream territories, which is associated with local inflammation [3]. This local inflammation activates the release of the substance P from the nociceptive fibers, generating a nociceptive message and pain by excess nociception [4]. The endothelial and immune systems are also involved, reinforcing the release of pro-algogen molecules. This local inflammation is responsible for an amplification of the nociceptive message and can lead to peripheral hyperalgesia (clinically manifested by a lowering of nociceptive thresholds or Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 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/). https://www.mdpi.com/journal/children Children 2021, 8, 84. https://doi.org/10.3390/children8020084 Children 2021, 8, 84 2 of 9 allodynia) and can lead to central sensitization. Clinical manifestations of the VOC often start with a prodromal phase of 1–2 days, with pain peaking on day 3 and lasting until day 6 or day 7 before resolving [2]. However, the pattern of the crisis can be very heterogeneous from one patient to another, or even from one episode to another for the same patient, with sometimes sudden and violent crises from the outset. The intensity of the pain is extreme, affecting one or more of the joints, limbs, or back, with sensations of crushed bones and torsion of the limbs described by patients from an early age [5]. The intensity of the pain commonly requires intravenous opioid treatments. 1. Introduction Recurrent episodes have a significant impact on quality of life (QOL) [6], so identification, treatment, and prevention of VOCs is crucial for lowering the impact of the disease on the morbidity and the global QOL in these patients. Management of VOCs begins with rapid, effective analgesic treatment adapted to the intensity and the nature of the pain [7]. Although the pain of vaso-occlusive episodes is classically nociceptive, neuropathic pain (NP) has also been demonstrated in SCD patients, both in adults [8] and in children aged 7 years and above, by using quantitative sensory testing (QST) [9]. There is no real consensus on the definition of the criteria for neuropathic pain in children. The International Association for the Study of Pain defines neuropathic pain as “pain initiated or caused by a primary lesion or dysfunction in the nervous system.” The classification proposed by the International Association for the Study of Pain (IASP), either nociceptive or neuropathic pain, leaves out a considerable number of children presenting with symptoms of both; therefore, it is referred to as “mixed pain” by clinicians. As discussed in a recent review, the use of this concept can have therapeutic consequences [10]. The distribution between neuropathic and mixed pain cases in daily practice is around one-third versus two-thirds, as estimated by respondents in the survey of de Leeuw et al. [11]. Trauma and operation are mostly regarded as the first cause of neuropathic and/or mixed pain in children, followed by complex regional pain syndrome, cancer-related pain, and phantom limb pain. Neuropathic pain in SCD has essentially been reported as chronic pain, described as numb, tingling, lancinating, spontaneous, shooting, or paroxysmal pain, and is sometimes associated with a sensation of pins and needles, hyperalgesia, and allodynia [12]. These pains are typically resistant to classical anti-nociceptive treatments, and can be responsible for a high consumption of opioids, which underlines the importance of their specific diagnosis. Nevertheless, NP during VOC remains difficult to specifically diagnose and to differentiate from nociceptive pain, especially in children. The DN4 questionnaire is a clinician-administered questionnaire, which has been validated for adults [13] and is commonly used in children [14], for whom there is a lack of a consensual and validated scale for NP. 1. Introduction It is presented in the form of seven questions (illustrated with images in its pediatric form) and three clinical examination items (hypo- or hyperesthesia to the touch and to the puncture and the sign of allodynia). The aim of our study is to evaluate the prevalence of NP during VOC in SCD children using the DN4 scale and to specify its risk factors and characteristics in order to better understand and manage them. 2. Materials and Methods 2.1. Population and Study Design 2.1. Population and Study Design SCD patients (Hemoglobin SS, SC, or Sβ0) aged 6 to18 years old and hospitalized for an acute VOC were included in a prospective study between September 2018 and December 2019. They were all followed up for their chronic disease in the sickle cell disease center of Robert Debré Hospital in Paris. On the second and fourth days of hospitalization for severe VOC (defined by the need for intravenous morphine treatment), the presence of neuropathic pain was sought with the DN4 scale. Demographic/anamnestic data and prescribed analgesic treatments were collected concomitantly on Day 2 and Day 4. 2.2. Diagnosis of Neuropathic Pain Acute neuropathic pain was diagnosed using the pediatric DN4 scale. The scale consists of seven questions on the sensation of pain (burning, swarming, etc.) illus- Children 2021, 8, 84 3 of 9 trated with age-appropriate pictures and three parameters sought via clinical examination (hypo/hyperalgesia, allodynia). If the patients answered “Yes” to four or more questions of the 10 above, they were considered as having neuropathic pain. The DN4 scale was administred either by a specialized nurse from the pediatric pain center of the hospital or by a senior practitioner. 2.5. Statistical Data Descriptive statistics were used to characterize patients and to determine the preva- lence of NP within the cohort. Qualitative variables were described as numbers (percentage) and quantitative variables as medians (1st–3rd quartiles). To compare the characteristics of patients with and without NP, a Khi2 test was performed on qualitative variables and the Student test (t-test) was performed on quantitative parameters. p-values < 0.05 were consid- ered statistically significant. All analyses were performed with SAS v9.4 (SAS Institute Inc., Cary, NC, USA). 2.3. Ethical Consideration The study was conducted in accordance with the declaration of Helsinki and was approved by the French Ethical Committee (CPP Sud Mediterranée 18.029). The collection of clinical and biological data was registered at the French National Commission for Information Technology and Civil Liberties (CNIL, ref 2139039). The parents or legal guardians of the minor patients were informed and gave their consent for the study. 2.4. Data Collection The data were prospectively collected in a standardized form from medical files and included the following items: demographic data (sex, age, genotype, weight), severity of the chronic disease (frequency of hospitalized VOC > 3 per year, past history of acute chest syndrome), chronic treatment (hydroxyurea, ongoing transfusion program), characteristics of the crisis (location of the pain, intensity of the nociceptive pain quantified by visual analog scale (VAS), Numeric Pain Rating Scale (NPRS), or Face Pain Scale—Revised (FPS- R)), date of the onset of the crisis, DN4 results in detail, and medical treatment of the current VOC (analgesic treatments and their doses, hydration). 3.1. Characteristics of the Patients During the inclusion period, the study was proposed to every SCD patient that was aged 6 to 18 years old and hospitalized for an acute VOC. For logistical reasons, we included only the patients admitted to the hospital between Saturday and Monday to be sure that they could be evaluated on Days 2 and 4, avoiding the weekend days. A total of 100% of the patients proposed for the study agreed to participate. A total of 54 SCD children were then included in the study. The sex ratio (F/M) was 1.7, and the median age was 15.0 years old (Q1:11; Q3:16). A total of 83% of the patients (n = 45) had an SS genotype, 6% (n = 3) had an SC genotype, and 11% (n = 6) had an Sβ◦genotype. A total of 78% (n = 42) had chronic hydroxyurea treatment. A total of 68.5% (n = 37) were considered as having a severe profile of SCD because of a past history of acute chest syndrome and/or the occurrence of at least three hospitalized VOCs in the last 12 months. The demographic and anamnestic data are summarized in Table 1. 4 of 9 Children 2021, 8, 84 Table 1. 3.1. Characteristics of the Patients Demographic data and description of the vaso-occlusive crises (Visual Analog Scale (VAS), Consumption of morphine, and DN4 scale results) All Patients (n = 54) Patients with NP * (n = 22) Patients without NP ** (n = 32) p-Value Age Median (Q1; Q3) 15.0 (11.0; 16.0) 14.0 (10.0; 16.0) 15.0 (13.0; 16.5) 0.11 Sex Ratio F/M 1.7 1.4 1.9 0.63 Genotype SC: 6% (n = 3) SC: 14% (n = 3) SC: 0% - SS: 83% (n = 45) SS: 77% (n = 17) SS: 88% (n = 28) Sbêta◦: 11% (n = 6) Sbêta◦: 9% (n = 2) Sbêta◦: 12% (n = 4) Hydroxyurea treatment 78% (n = 42) 73% (n = 16) 81% (n = 26) 0.517 Severe SCD phenotype *** 68.5% (n = 37) 59% (n = 13) 75% (n = 24) 0.246 Characteristics of the episode Limb pain 69% (n = 37) 73% (n = 16) 66% (n = 21) 0.58 VAS at Day 2 Median (Q1; Q3) 6.0 (5.0; 7.0) 6.0 (5.0; 8.0) 6.0 (4.5; 7.0) 0.72 Consumption of Morphine at Day 2 Median (Q1; Q3) 1.2 (0,9; 1,6) 1.3 (1.0; 1.7) 1.1 (0.9; 1.5) 0.52 DN4 scale ≥4 at Day 2 37% (n = 20/54) 91% (n = 20/22) 0 - VAS at Day Median (Q1; Q3) 5.0 (3.0; 7.0) 5.0 (4.0; 7.0) 5.0 (3.0; 7.0) 0.45 Consumption of Morphine at Day 4 Median (Q1; Q3) 1.2 (0.8; 1.5) 1.3 (0.9; 1.6) 1.1 (0.7; 1.5) 0.49 DN4 scale ≥4 at Day 4 25% (n = 11/44) 50% (n = 11/22) 0 - * Patients were considered to have neuropathic pain (NP) if they had a DN4 score ≥4 on Day 2 and/or on Day 4 of the hospitalization. ** Patients were considered to be without NP if they had a DN4 score < 4 on Day 2 and on Day 4 of the hospitalization. *** Severe phenotype was defined by three or more hospitalized VOCs in the last 12 months and/or past history of Acute Chest Syndrome (ACS). F/M: Female/Male. Table 1. Demographic data and description of the vaso-occlusive crises (Visual Analog Scale (VAS), Consumption of morphine, and DN4 scale results) * Patients were considered to have neuropathic pain (NP) if they had a DN4 score ≥4 on Day 2 and/or on Day 4 of the hospitalization. 3.1. Characteristics of the Patients ** Patients were considered to be without NP if they had a DN4 score < 4 on Day 2 and on Day 4 of the hospitalization. *** Severe phenotype was defined by three or more hospitalized VOCs in the last 12 months and/or past history of Acute Chest Syndrome (ACS). F/M: Female/Male. 3.2. Characteristics of the Vaso-Occlusive Crisis A total of 69% of the patients (n = 37) had pain in one or more limbs, while the remain- ing 31% had abdominal, thoracic, or lumbar pain. On the second day of hospitalization, the average morphine intake was 1.2 mg per kilogram of weight and per day (Q1:0.9; Q3:1.6), with a concomitant prescription of per os (po) paracetamol (15 mg/kg/6 h) and intravenous (iv) hyperhydration (1.5 to 2 L/m2/day) for all the patients. A total of 81% (n = 44) had a concomitant prescription of iv nefopam (1 to 2 mg/kg/day), 18% (n = 10) had a concomitant prescription of po ibuprofen (20 mg/kg/8 h), and 50% (n = 27) had a concomitant prescription of inhaled nitrous oxide (6 to 9 L/mn, 15 mn session, three times per day maximum). We proposed different possibilities to each child for evaluating their own pain: VAS (using a little ruler), NPRS (with numbers from 0 to 10), or FPS (with faces expressing pain). Most of the children aged more than 8 years preferred the NPRS, but we observed a good consistency between the different methods for the nociceptive part of the pain. The mean score of the pain scale (VAS, NPRS, or FPS-R) at Day 2 was 6/10 (Q1:5; Q3:7). A total of 44 patients (81%) were still hospitalized four days after arrival. For these 44 patients, at Day 4, the average morphine intake was still high (1.2 mg/kg/d (Q1:0.8; Q3:1.5)). The mean score of the pain scale (VAS, NPRS or FPS-R) at Day 4 was 5/10 (Q1:3; Q3:7). These data are detailed in Table 1. 3.3. Prevalence and Description of Neuropathic Pain during the Vaso-Occlusive Crisis On Day 2, 20 patients (37%) had a positive DN4 score, indicating the occurrence of an early neuropathic component in the VOC. On Day 4, four of these patients had returned home and could not be reassessed. A total of 16 had a second assessment, highlighting that nine (56%) still had positive DN4 scores on Day 4, while neuropathic pain had disappeared for seven (44%) of them. Among the 34 patients with a negative DN4 score on Day 2, 28 Children 2021, 8, 84 5 of 9 of them had a second evaluation on Day 4. 3.4. Predictive Factor for Neuropathic Pain during VOC We looked more closely at the characteristics of patients with neuropathic pain in order to understand whether there might be risk factors for developing NP. Among the 22 children experiencing NP at least one time during hospitalization, 59% were girls, the median age was 14 years old (Q1:10; Q3:16), and 59% (n = 13) were considered as a severe phenotype of SCD because they had more than three hospitalized VOCs in the last 12 months and/or a past history of acute chest syndrome. A total of 73% had chronic treatment with hydroxyurea. The VOCs affected limbs in 73% of cases, with a median pain scale (VAS, NPRS, or FPS-R) of 6 (Q1:5; Q3:8) at Day 2 and 5 (Q1:4; Q3:7) at Day 4 in this population. The median opioid intake in this group was 1.3 mg/kg/day (Q1:1.0; Q3:1.7) at Day 2 and 1.3 mg/kg/day (Q1:0.9; Q3:1.6) at Day 4. In the group without NP during the VOCs, none of the characteristics described above were statistically different from one group to the other (Table 1). 3.2. Characteristics of the Vaso-Occlusive Crisis A total of 26 (59%) had a negative DN4 score, confirming the absence of NP, while a neuropathic component of the pain had appeared belatedly for two of them (4.5%) with a positive DN4 score (Figure 1). Overall, 41% of the patients (n = 22) experienced neuropathic pain during the VOC, usually at an early stage (91% on Day 2). Among these patients, the symptom most often evoked by the children was the sensation of tingling (89%), but also the sensations of swarming (68%), of numbness (53%), of hypoesthesia (44%) or hyperesthesia (44%), of burning (42%), of electric shocks (42%), of painful cold (37%), of allodynia (35%), and, finally, of itching (21%) (Figure 1). Figure 1. Characteristics of neuropathic symptoms depending on gender. Figure 1. Characteristics of neuropathic symptoms depending on gender. Figure 1. Characteristics of neuropathic symptoms depending on gender. 3.4. Predictive Factor for Neuropathic Pain during VOC 4. Discussion The primary approach to the management of acute VOCs is pain control. The key element in the management of VOCs is the evaluation of pain (its location and intensity, but also its different components) in order to propose an analgesia tailored to the pa- tient, his/her painful experience, and the crisis he/she is going through. Acetaminophen Children 2021, 8, 84 6 of 9 (paracetamol) and Non-Steroids Anti-Inflammatory Drugs (NSAIDs) are widely used ad- juvant therapies with opioids [15,16]. Potential adjuvant treatments include intravenous hyperhydration, anxiolytics, local lidocaine patches, and less common non-pharmacologic approaches, such as hypnosis, relaxation, local hot water bottles, a transcutaneous electrical nerve stimulator (TENS), massage, acupuncture, yoga, and meditation [17]. Neuropathic pain is a direct consequence of a lesion or disease affecting the somatosensory system [18]. In SCD, neuropathic lesions may occur rapidly during VOC due to inflammation and acute ischemic/reperfusion injury [19,20], and are supposed to be resolved a few days after the end of the crisis [21]. However, these descriptions most often concern adult patients, and few are reported in children, mainly due to diagnostic difficulties. Indeed, the diagnosis of neuropathic pain during VOC raises the problem of fine descriptions of the charac- teristics of the pain, which are sometimes difficult in young children, especially during an intensely painful episode where many sensations are mixed, including anxiety, fear of parental separation, and adverse effects of treatment (pain related to infusion devices, opioid-induced pruritus or hyperalgesia, etc.). Moreover, the diagnosis between “pure neuropathic” and “mixed pain” in children and adolescents is very difficult for several reasons: We do not dispose of many diagnostic tools, as questionnaires validated for adults and the electrophysiologic methods, such as quantitative sensory testing (QST) or electromyography (EMG), cannot be easily used in children [22,23]. In a recent survey study among members of learned societies or groups whose members are known to treat pediatric pain, de Leeuw et al. [11] observed that only 45.3% of the respondents used the IASP criteria for establishing the diagnosis of neuropathic pain in children, while 18 (15.4%) explicitly answered that they did not use these criteria; 46 persons (39.3%) left this question unanswered. Most practitioners base the diagnosis of neuropathic pain on a combination of physical examination, history, and/or underlying disease. Otherwise, the underlying pathologies are often different from those encountered in adults. 4. Discussion However, Children 2021, 8, 84 7 of 9 the effect of these factors on NP cannot be completely ruled out; the size of our sample may have limited the statistical power of the tests for demonstrating differences. One can wonder if the initiation of treatments could change the assessment of neuropathic pain in these children and thus introduce a bias in our study. We would answer to this question that there was no significant difference between the subjects of our cohort concerning the analgesic treatments initiated at the beginning of their hospitalization; most of the children received an intravenous infusion of morphine, Néfopam, paracetamol, and sometimes non-steroidal anti-inflammatory drugs. There were no treatments of neuropathic pain prescribed between Day 2 and Day 4. Thus, if there is a treatment bias, it is homogeneous across the entire cohort. Considering the opioid consumption, we also compared the two groups. Indeed, if hyperalgesia is a signature of neuropathic pain, it may also be caused by prolonged opioid use. Increasing the dose of opioids for pain management may paradoxically increase pain sensitivity, lead to higher pain scores, and be accompanied by allodynia [26]. Nevertheless, in our study, patients with NP did not have higher doses of opioids than children without NP, partially excluding the diagnosis of opioid-induced hyperalgesia. The other treatment that could have an impact on NP is the inhaled nitrous oxide. Indeed, nitrous oxide is not described as a treatment for neuropathic pain per se, but it does have an action on NMDA receptors [27] and, therefore, is susceptible to action on the central sensitization mechanisms. Its usefulness in the management of VOC has been demonstrated to be nociceptive and anxiolytic [28], but could also have interesting complementary effects—notably, anti-neuropathic effects—which remain to be studied in the context of VOC. The effectiveness of drugs usually used for neuropathic pain, including gabapentin, pregabalin, amitriptyline, serotonin-noradrenaline reuptake inhibitors (SNRIs), and tricyclic antidepressants, has not been firmly established in SCD patients, let alone in SCD children. Another prospective study may be useful to determine if specific treatment against NP in these patients could reduce the consumption of opioid treatments and/or the length of the hospital stay. g p y One of the limitations of this study is the lack of data concerning the persistence of these pains until the end of the crisis, or even beyond. 4. Discussion Conditions such as postherpetic neuralgia, radiculopathies, and complications of stroke are very rare in young patients. However, some neuropathic conditions can be recognized in children and adolescents, including phantom limb pain, spinal cord injury, trauma, postoperative neuropathic pain, and the consequences of cancer disease processes and treatment. There are also neuropathic pain syndromes that are rare and specific to the pediatric population (toxic and metabolic neuropathies, hereditary neurodegenerative disorders, mitochondrial disorders). In all cases, it is important to consider the possibility of neuropathic pain or a neuropathic component in mixed pain, since the treatment is quite different from that for nociceptive or inflammatory pain. Severe and long-lasting pain problems in childhood might result in persistence of pain or development of other chronic pain states in adult- hood. In the case of VOC, the mechanisms of pain are certainly mixed, including vascular occlusion and the consequent inflammation, leading to mast cell activation, neurogenic inflammation, peripheral nociceptor sensitization, and then to central sensitization and modulation of neural circuits in the brain [4]. The use of the DN4 scale in the form of a picture frame allowed us to take the time to help children describe their pain and characterize its components as well as possible. Thus, we were able to show, for the first time, the high prevalence of neuropathic pain (41% of the patients in our study) during acute VOC in SCD children. This neuropathic component would have gone completely unrecognized and, therefore, under-estimated and under-treated if it had not been researched with adapted tools. We have focused on looking for specific clinical profiles or factors favoring the onset of neuropathic pain in these children. However, the comparison of the groups with and without neuropathic pain showed no statistically different values of phenotypic profiles (no more severe children in the NP group than in the non-NP group), sex ratio, median age, or pain location, contrary to other studies that found a higher prevalence of pain in SCD women and adolescents [24,25]. We also did not find any differences in terms of Hydrocyurea (HU) treatment between these two groups, contrary to the data reported by Brandow et al. [24]. In this study, patients on HU treatment were also older, which may constitute an interpretation bias. 4. Discussion In our study, we reported acute neuropathic pain, which sometimes appears to be transient given that 44% of the patients with NP at Day 2 had no more NP at Day 4. Nevertheless, we did not evaluate the persistence or the recurrence of NP after the resolution of the VOC. It is possible that some of the neuropathic pain may persist after the VOC or even become chronic in some patients, requiring long-term treatment. References y 8. Brandow, A.M.; Bs, R.A.F.; Panepinto, J.A. Early insights into the neurobiology of pain in sickle cell disease: A systematic review of the literature. Pediatr. Blood Cancer 2015, 62, 1501–1511. [CrossRef] 9. Brandow, A.M.; Panepinto, J.A. Clinical Interpretation of Quantitative Sensory Testing as a Measure of Pain Sensitivity in Patients with Sickle Cell Disease. J. Pediatr. Hematol. 2016, 38, 288–293. [CrossRef] 10. Freynhagen, R.; Parada, H.A.; Calderon-Ospina, C.A.; Chen, J.; Emril, D.R.; Fernández-Villacorta, F.J.; Franco, H.; Ho, K.-Y.; Lara-Solares, A.; Li, C.C.-F.; et al. Current understanding of the mixed pain concept: A brief narrative review. Curr. Med. Res. Opin. 2019, 35, 1011–1018. [CrossRef] n Der Zanden, T.; Ravera, S.; Felisi, M.; Bonifazi, D.; Tibboel, D.; Ceci, A.; Kaguelidou, F.; De Wildt, S.N.; The 11. De Leeuw, T.G.; Van Der Zanden, T.; Ravera, S.; Felisi, M.; Bonifazi, D.; Tibboel, D.; Ceci, A.; Kaguelidou, F.; De Wildt, S.N.; The GAPP Consortium. Diagnosis and Treatment of Chronic Neuropathic and Mixed Pain in Children and Adolescents: Results of a Survey Study amongst Practitioners. Children 2020, 7, 208. [CrossRef] [PubMed] y y g 12. Bennett, M.; Attal, N.; Backonja, M.M.; Baron, R.; Bouhassira, D.; Freynhagen, R.; Scholz, J.; Tölle, T.R.; Wittchen, H.-U.; Jensen, T.S. Using screening tools to identify neuropathic pain. Pain 2007, 127, 199–203. [CrossRef] [PubMed] 13. Bouhassira, D.; Attal, N.; Alchaar, H.; Boureau, F.; Brochet, B.; Bruxelle, J.; Cunin, G.; Fermanian, J.; Ginies, P.; Grun- Overdyking, A.; et al. Comparison of pain syndromes associated with nervous or somatic lesions and development of a new neuropathic pain diagnostic questionnaire (DN4). Pain 2005, 114, 29–36. [CrossRef] p p g q 14. Julien-Marsollier, F.; David, R.; Hilly, J.; Brasher, C.; Michelet, D.; Dahmani, S. Predictors of chronic neuropathic pain after scoliosis surgery in children. Scand. J. Pain 2017, 17, 339–344. [CrossRef] [PubMed] 15. Lovett, P.B.; Sule, H.P.; Lopez, B.L. Sickle Cell Disease in the Emergency Department. Hematol. Oncol. Clin. N. Am. 2017, 31, 1061–1079. [CrossRef] [PubMed] 16. Mousa, S.A.; Al Momen, A.; Al Jaouni, S.; Nasrullah, Z.; Al Saeed, H.; Alabdullatif, A.; Al Sayegh, M.; Al Zahrani, H.; Hegazi, M.; Al Mohamadi, A.; et al. Management of Painful Vaso-Occlusive Crisis of Sickle-Cell Anemia: Consensus Opinion. Clin. Appl. Thromb. Hemost. 2010, 16, 365–376. [CrossRef] [PubMed] 17. Lu, K.; Cheng, M.-C.J.; Ge, X.; Berger, A.; Xu, D.; Kato, G.J.; Minniti, C.P. References 1. Piel, F.B.; Steinberg, M.H.; Rees, D.C. Sickle Cell Disease. N. Engl. J. Med. 2017, 376, 1561–1573. [CrossRef] [PubMed] 2 Ballas S K ; Lusardi M Hospital readmission for adult acute sickle cell painful episodes: Frequency etiology and prognostic 1. Piel, F.B.; Steinberg, M.H.; Rees, D.C. Sickle Cell Disease. N. Engl. J. Med. 2017, 376, 1561–1573. [CrossRef] [PubMed] 1. Piel, F.B.; Steinberg, M.H.; Rees, D.C. Sickle Cell Disease. N. Engl. J. Med. 2017, 376, 1561–1573. [CrossRef] [PubMed] 2. Ballas, S.K.; Lusardi, M. Hospital readmission for adult acute sickle cell painful episodes: Frequency, etiology, and prognostic significance Am J Hematol 2005 79 17 25 [CrossRef] [PubMed] i, M. Hospital readmission for adult acute sickle cell painful episodes: Frequency, etiology, and prognostic Hematol. 2005, 79, 17–25. [CrossRef] [PubMed] 3. Manwani, D.; Frenette, P.S. Vaso-occlusion in sickle cell disease: Pathophysiology and novel targeted therapies. Blood 2013, 122, 3892–3898. [CrossRef] [PubMed] [ ] [ ] 4. Michaels, L.A.; Ohene-Frempong, K.; Zhao, H.; Douglas, S.D. Serum levels of substance P are elevated in patients with sickle cell 4. Michaels, L.A.; Ohene-Frempong, K.; Zhao, H.; Douglas, S.D. Serum levels of substance P are elevated in patients with sickle cell disease and increase further during vaso-occlusive crisis. Blood 1998, 92, 3148–3151. [CrossRef] [PubMed] 4. Michaels, L.A.; Ohene-Frempong, K.; Zhao, H.; Douglas, S.D. Serum levels of substance P are elevated in patients with sickle cell disease and increase further during vaso-occlusive crisis. Blood 1998, 92, 3148–3151. [CrossRef] [PubMed] 5. Ballas, S.K.; Gupta, K.; Adams-Graves, P. Sickle cell pain: A critical reappraisal. Blood 2012, 120, 3647–3656. [CrossRef] [PubMed] 6. Van Tuijn, C.F.J.; Van Beers, E.J.; Schnog, J.-J.B.; Biemond, B.J. Pain rate and social circumstances rathe damage determine the quality of life in adults with sickle cell disease. Am. J. Hematol. 2010, 85, 532–53 6. Van Tuijn, C.F.J.; Van Beers, E.J.; Schnog, J.-J.B.; Biemond, B.J. Pain rate and social circumstances rather than cumulative organ damage determine the quality of life in adults with sickle cell disease. Am. J. Hematol. 2010, 85, 532–535. [CrossRef] [PubMed] 7. Yawn, B.P.; John-Sowah, J. Management of Sickle Cell Disease: Recommendations from the 2014 Expert Panel Report. Am. Fam. Physician 2015, 92, 1069–1076. damage determine the quality of life in adults with sickle cell disease. Am. J. Hematol. 2010, 85, 532–535. [CrossRef] [PubMed] 7. Yawn, B.P.; John-Sowah, J. Management of Sickle Cell Disease: Recommendations from the 2014 Expert Panel Report. Am. Fam. Physician 2015, 92, 1069–1076. 5. Conclusions Our study shows that neuropathic pain is very common during VOC in SCD children and needs to be specifically screened and treated. The absence of identified risk factors should prompt us to be vigilant regardless of the patient’s age, sex, and clinical presentation. A better understanding and screening of this painful component would lead to a more efficient management of crises. An evaluation of the efficiency and impact of specific treatments for neuropathic pain seems to be essential in SCD children. Author Contributions: Conceptualization, J.S., N.D.A., S.D., and B.K.; methodology, E.L.R.; inclusion of the patients and collected clinical data: J.S., N.D.A., S.D., and A.-F.T.; formal analysis, A.T. and E.L.R.; writing—original draft preparation, B.K.; writing—review and editing, J.S., N.D.A., L.H., and E.L.R.; supervision, M.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted in accordance with the declaration of Helsinki and was approved by the French Ethical Committee (CPP Sud Mediterranée 18.029).The collection of clinical and biological data was registered at the French National Commission for Information Technology and Civil Liberties (CNIL, ref 2139039). Children 2021, 8, 84 8 of 9 Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The parents or legal guardians of the minor patients were informed and gave their written consent for the study. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References A Retrospective Review of Acupuncture Use for the Treatment of Pain in Sickle Cell Disease Patients: Descriptive Analysis from a Single Institution. Clin. J. Pain 2014, 30, 825–830. [CrossRef] [ ] 18. Jensen, T.S.; Baron, R.; Haanpää, M.; Kalso, E.; Loeser, J.D.; Rice, A.S.; Treede, R.-D. A new definition of neuropathic pain. Pain 2011, 152, 2204–2205. [CrossRef] 19 T H G M G K T i l h i f i i i kl ll di Bl d 2017 130 2377 2385 [C R f] 19. Tran, H.; Gupta, M.; Gupta, K. Targeting novel mechanisms of pain in sickle cell disease. Blood 2017, 130, 2377–2385. [CrossRef] 20. Brandow, A.M.; Zappia, K.J.; Stucky, C.L. Sickle cell disease: A natural model of acute and chronic pain. Pain 2017, 158 (Suppl. 1), 19. Tran, H.; Gupta, M.; Gupta, K. Targeting novel mechanisms of pain in sickle cell disease. Blood 2017, 130, 2377–2385. [CrossRef] 19. Tran, H.; Gupta, M.; Gupta, K. Targeting novel mechanisms of pain in sickle cell disease. Blood 2017, 130, 2377–2385. [CrossRef] 20. Brandow, A.M.; Zappia, K.J.; Stucky, C.L. Sickle cell disease: A natural model of acute and chronic pain. Pain 2017, 158 (Suppl. 1), S79–S84. [CrossRef] 21. Wilkie, D.J.; Molokie, R.; Boyd-Seal, D.; Suarez, M.L.; Kim, Y.O.; Zong, S.; Wittert, H.; Zhao, Z.; Saunthararajah, Y.; Wang, Z.J. Patient-Reported Outcomes: Descriptors of Nociceptive and Neuropathic Pain and Barriers to Effective Pain Management in Adult Outpatients with Sickle Cell Disease. J. Natl. Med. Assoc. 2010, 102, 18–27. [CrossRef] pathic pain in children: Steps towards improved recognition and management. EBioMedicine 2020, 62, 103124 d] alker, S.M. Neuropathic pain in children: Steps towards improved recognition and management. EBioMedicin rossRef] [PubMed] 22. Walker, S.M. Neuropathic pain in children: Steps towards improved recognition and management. EBioMedicine 2020, 62, 103124. [CrossRef] [PubMed] 23. Walco, G.A.; Dworkin, R.H.; Krane, E.J.; Lebel, A.A.; Treede, R.-D. Neuropathic Pain in Children: Special Considerations. Mayo Clin Proc 2010 85 S33–S41 [CrossRef] [PubMed] [ ] [ ] 23. Walco, G.A.; Dworkin, R.H.; Krane, E.J.; Lebel, A.A.; Treede, R.-D. Neuropathic Pain in Children: Special Considerations. Mayo Clin. Proc. 2010, 85, S33–S41. [CrossRef] [PubMed] 24. Brandow, A.M.; Farley, R.A.; Panepinto, J.A. Neuropathic pain in patients with sickle cell disease. Pediatr. Blood Cancer 2014, 61, 512–517. [CrossRef] [PubMed] 25. Antunes, F.D.; Silva, J.C.L.; Cerqueira, K.S.; Faro, M.D.L.; Cipolotti, R. Screening for neuropathic pain in patients with sickle cell disease: Is a single assessment scale sufficient? 26. Stoicea, N.; Russell, D.; Weidner, G.; Durda, M.; Joseph, N.C.; Yu, J.; Bergese, S.D. Opioid-induced hyperalgesia in chronic pain patients and the mitigating effects of gabapentin. Front. Pharmacol. 2015, 6, 104. [CrossRef] [PubMed] p g g g p , , [ ] [ ] 27. Jevtovi´c-Todorovi´c, V.; Todorov´c, S.M.; Mennerick, S.; Powell, S.; Dikranian, K.; Benshoff, N.; Zorumski, C.F.; Olney, J.W. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat. Med. 1998, 4, 460–463. [CrossRef] 26. Stoicea, N.; Russell, D.; Weidner, G.; Durda, M.; Joseph, N.C.; Yu, J.; Bergese, S.D. Opioid-induced hyperalgesia in chronic pain patients and the mitigating effects of gabapentin. Front. Pharmacol. 2015, 6, 104. [CrossRef] [PubMed] 27. Jevtovi´c-Todorovi´c, V.; Todorov´c, S.M.; Mennerick, S.; Powell, S.; Dikranian, K.; Benshoff, N.; Zorumski, C.F.; Olney, J.W. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat. Med. 1998, 4, 460–463. [CrossRef] 28. Aboursheid, T.; Albaroudi, O.; Alahdab, F. Inhaled nitric oxide for treating pain crises in people with sickle cell disease. Cochrane Database Syst. Rev. 2019, 2019, CD011808. [CrossRef] ( g g g ) g p 28. Aboursheid, T.; Albaroudi, O.; Alahdab, F. Inhaled nitric oxide for treating pain crises in people with sickle cell disease. Cochrane Database Syst. Rev. 2019, 2019, CD011808. [CrossRef] 27. Jevtovic-Todorovic, V.; Todorovc, S.M.; Mennerick, S.; Powell, S.; Dikranian, K.; Benshoff, N.; Zorumski, C.F.; Olney, J.W. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat. Med. 1998, 4, 460–463. [CrossRef] 28. Aboursheid, T.; Albaroudi, O.; Alahdab, F. Inhaled nitric oxide for treating pain crises in people with sickle cell disease. Cochrane Database Syst. Rev. 2019, 2019, CD011808. [CrossRef] References Orphanet J. Rare Dis. 2019, 14, 108. [CrossRef] 9 of 9 Children 2021, 8, 84
https://openalex.org/W3005941368
https://www.frontiersin.org/articles/10.3389/fneur.2020.00085/pdf
English
null
Measurement of Platelet Function in an Experimental Stroke Model With Aspirin and Clopidogrel Treatment
Frontiers in neurology
2,020
cc-by
7,223
Measurement of Platelet Function in an Experimental Stroke Model With Aspirin and Clopidogrel Treatment Franziska Lieschke 1,2*, Yi Zheng 1, Jan Hendrik Schaefer 2, Klaus van Leyen 1 and Christian Foerch 2 1 Neuroprotection Research Laboratory, Department of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States, 2 Department of Neurology, University Hospital Frankfurt, Goethe-University, Frankfurt am Main, Germany 1 Neuroprotection Research Laboratory, Department of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States, 2 Department of Neurology, University Hospital Frankfurt, Goethe-University, Frankfurt am Main, Germany Dual antiplatelet treatment (DAPT) increases the risk of tPA-associated hemorrhagic transformation (HT) in ischemic stroke. To investigate the effects of DAPT in rodents, reliable indicators of platelet function utilizing a minimally invasive procedure are required. We here established a fluorescence-based assay to monitor DAPT efficiency in a mouse model of ischemic stroke with HT. Male C57/BL6 mice were fed with aspirin and clopidogrel (ASA+CPG). Venous blood was collected, stimulated with thrombin, labeled with anti-CD41-FITC and anti-CD62P-PE, and analyzed by flow cytometry. Subsequently, animals were subjected to experimental stroke and tail bleeding tests. HT was quantified using NIH ImageJ software. In ASA+CPG mice, the platelet activation marker CD62P was reduced by 40.6 ± 4.2% (p < 0.0001) compared to controls. In vitro platelet function correlated inversely with tail bleeding tests (r = −0.8, p = 0.0033, n = 12). Twenty-four hours after drug withdrawal, platelet activation rates in ASA+CPG mice were still reduced by 20.2 ± 4.1% (p = 0.0026) compared to controls, while tail bleeding volumes were increased by 4.0 ± 1.4 µl (p = 0.004). Conventional tests using light transmission aggregometry require large amounts of blood and thus cannot be used in experimental stroke studies. In contrast, flow cytometry is a highly sensitive method that utilizes small volumes and can easily be incorporated into the experimental stroke workflow. Our test can be used to monitor the inhibitory effects of DAPT in mice. Reduced platelet activation is indicative of an increased risk for tPA-associated cerebral hemorrhage following experimental stroke. The test can be applied to individual animals and implemented flexibly prior and subsequent to experimental stroke. Edited by: Simone Beretta, San Gerardo Hospital, Italy Reviewed by: Barbara Casolla, Centre Hospitalier Regional et Universitaire de Lille, France Luigi Sironi, University of Milan, Italy *Correspondence: Franziska Lieschke franziska.lieschke@ stud.uni-frankfurt.de Edited by: Simone Beretta, San Gerardo Hospital, Italy Reviewed by: Barbara Casolla, Centre Hospitalier Regional et Universitaire de Lille, France Luigi Sironi, University of Milan, Italy *Correspondence: Reviewed by: Barbara Casolla, Centre Hospitalier Regional et Universitaire de Lille, France Luigi Sironi, University of Milan, Italy Specialty section: This article was submitted to Stroke, a section of the journal Frontiers in Neurology Received: 01 November 2019 Accepted: 23 January 2020 Published: 11 February 2020 Keywords: dual antiplatelet therapy, aspirin, clopidogrel, flow cytometry, CD62P, CD41, hemorrhagic transformation, MCAO BRIEF RESEARCH REPORT published: 11 February 2020 doi: 10.3389/fneur.2020.00085 Citation: Lieschke F, Zheng Y, Schaefer JH, van Leyen K and Foerch C (2020) Measurement of Platelet Function in an Experimental Stroke Model With Aspirin and Clopidogrel Treatment. Front. Neurol. 11:85. doi: 10.3389/fneur.2020.00085 The use of dual antiplatelet treatment (DAPT) in the acute phase of stroke is becoming increasingly common (1, 2). Apart from patients prescribed DAPT for other indications (3), this includes the application of DAPT in patients with transient ischemic attacks and minor strokes, as well as DAPT treatment during acute revascularization procedures (4, 5). By irreversibly impairing platelet function, DAPT increases the risk of hemorrhagic complications after thrombolysis with tPA (6–8). February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org Monitoring DAPT in Stroke Mice Lieschke et al. used exclusively male animals to limit variability due to sex differences. The operators performing surgical procedures and the investigators evaluating data were blinded to the treatment groups. At first, we assessed platelet function in vitro using our flow cytometry-based assay (as explained below) in mice treated with ASA+CPG and in controls. Mice were then subjected to 2 h MCAO followed by tPA administration. Twenty-four hours later, standard tail bleeding tests were conducted (14, 20), and mice were sacrificed to quantify HT development. A second in vitro analysis of platelet function was performed at the end of the experiment in order to demonstrate the feasibility of monitoring treatment effects beyond drug withdrawal (Figure 1A). Current European and American guidelines do not recommend to perform platelet function tests during the acute phase of ischemic stroke (9, 10). Although platelet function testing has become widely accessible with the use of point of care devices, investigations whether patients could potentially benefit are still lacking. On the other hand, rapid testing of the platelet count is a well-established standard in the treatment of stroke. Here, low platelet counts are excluded from tPA treatment due to the significantly increased risk of hemorrhagic transformation (HT) in platelet depleted patients (11). However, the question of whether and how platelet function contributes to tPA-associated HT has not been answered to date. To address this gap of knowledge, we recently established a model of tPA-associated hemorrhagic transformation (HT) in mice pretreated with Aspirin and Clopidogrel [ASA+CPG, (12)]. While establishing antiplatelets in rodents, the individual hemostatic status of the single mouse becomes relevant. Antiplatelet Pretreatment Mice were allocated randomly to a treatment or control group. ASA (Bayer Health Care, Morristown, NJ, USA) and CPG (Dr. Reddy’s Laboratories Ltd., Beverley, UK) diluted in drinking water (ASA 0.4 mg/mL, CPG 0.15 mg/mL) were supplied ad libitum for 72 h. A water consumption of 15 ml/100 g per 24 h was assumed, providing an estimated daily intake of 60 mg/kg ASA and 22.5 mg/kg CPG per mouse. These dosages were selected based on previous experimental stroke studies (14, 21). Control mice received regular drinking water. g p Conventional platelet function tests like light transmission aggregometry (LTA) require significant volumes of blood (13), which means either collecting a lethal amount of blood from one mouse or collecting and pooling blood from different animals (14). As a result LTA is too invasive to be used as a measure of platelet status at the time of experimental stroke surgery. A simple-to-conduct but unspecific method is the tail bleeding test, which illustrates platelet adhesion in vivo and corresponds to the bleeding time test formerly used in patients (15, 16). The principle underlying this test is that platelet function directly affects primary hemostasis after tissue damage, resulting in prolonged bleeding time (time to complete cessation of the bleeding) and increased blood volumes during testing in mice. It can only be done once at the end of an experiment, and precludes the evaluation of drug efficacy in advance and repeated measurements. Accordingly, methods are required that utilize small sample volumes. Flow cytometry is a highly sensitive method requiring only small volumes of blood. Platelet activation assays based on flow cytometry have been introduced (17–19) but at present they are not widely used and have not been tested in the context of experimental stroke studies. Flow Cytometry Based Platelet Function Test In vitro platelet function testing was based on the platelet expression of CD41 and CD62P. We used the platelet marker CD41 to distinguish platelets from other events. CD62P is usually located in the membrane of platelet α-granules in the cytoplasm and only translocates to the plasma membrane after platelet activation (22). By detecting the surface co- expression of these antigens following thrombin treatment using a BD LSRII analyzer (Becton-Dickinson, San Jose, CA, USA), we gained an individual platelet activation rate for each single mouse. A high activation rate (indicated by increased CD62P expression) demonstrates sufficient platelet function, whereas a lower activation rate indicates impaired platelet function due to the partial inhibition of platelets, hence also serving as an indicator for an efficient anti-platelet treatment. Briefly, mice were anesthetized with isoflurane (1.25–1.5% in a nitrous oxide/oxygen mixture with spontaneous respiration). Venous blood (10–20 µl) was collected from the left jugular vein and given into sodium citrate (final 0.32%, Sigma) to prevent unintended platelet activation and blood clotting. Five microliters of whole blood were diluted in thirty microliters of PBS containing 0.32% sodium citrate. Fifteen microliters of vehicle or agonist solution were added, where the vehicle was composed of PBS containing 0.32% sodium citrate, and the agonist solution consisted of thrombin (final 2 µ/ml) supplemented with 10 mM GPRP (final 2.5 mM) and 6 mM CaCl2. The samples were then incubated shaking at 37◦C for 5 min. Mouse blood was incubated with 1:100 dilution of CD41- FITC and CD62P-PE monoclonal antibodies (BD Biosciences and eBioscienceTM, respectively) for 15 min at room temperature in the dark. After staining, samples were fixed with 650 µl of fixative solution containing 0.1% formalin, 0.1% dextrose, The aim of this study was to demonstrate the feasibility of incorporating flow cytometry-based platelet function testing in a mouse model of DAPT followed by an ischemic stroke and tPA infusion. In addition to monitoring the efficacy of antiplatelet therapy, we also sought to further investigate the impact of platelet function on the development of HT in order to predict bleeding risks and to define inclusion and exclusion criteria for subsequent experiments. Citation: We therefore looked for a simple platelet function test to check for individual drug response. MATERIALS AND METHODS Animals and Experimental Design g In total 28 male C57/B6 mice (Jackson, Bar Harbor, ME, USA) aged 9–10 weeks with a mean body weight of 26 ± 2g were used in this study. All experiments conformed to an institutionally approved protocol in accordance with the National Institute of Health’s guide for the care and use of laboratory animals. We February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org 2 Lieschke et al. Monitoring DAPT in Stroke Mice FIGURE 1 | (A) Timeline diagram of the experimental procedures: platelet function in control and ASA+CPG mice was tested in vitro using flow cytometry at the end of 3 days pretreatment. Mice were subjected to 2 h MCAO and tPA administration. Twenty-two hours later, tail bleeding tests and a second flow cytometry analysis were performed, followed by sacrifice and histological assessment of hemorrhage. (B–D) Gating strategy: Ungated whole blood (B). Gated cells scored high for platelet identification marker FITC-CD41 (C) and low for the platelet activation marker PE-CD62P (D), consistent with the properties of resting platelets (Analysis performed with FlowJo). FIGURE 1 | (A) Timeline diagram of the experimental procedures: platelet function in control and ASA+CPG mice was tested in vitro using flow cytometry at the end of 3 days pretreatment. Mice were subjected to 2 h MCAO and tPA administration. Twenty-two hours later, tail bleeding tests and a second flow cytometry analysis were performed, followed by sacrifice and histological assessment of hemorrhage. (B–D) Gating strategy: Ungated whole blood (B). Gated cells scored high for platelet identification marker FITC-CD41 (C) and low for the platelet activation marker PE-CD62P (D), consistent with the properties of resting platelets (Analysis performed with FlowJo). and 0.2% BSA in PBS. Labeled, diluted and fixed samples were analyzed. Platelets were gated based on their characteristic forward and side scatter properties (Figure 1B), as well as for antiplatelet immunoreactivity for CD41 (Figure 1C). The third gate was set on a positive staining with monoclonal antibody CD62P-PE (Figure 1D). Analyses were performed with BD FACS DIVA software (BD Biosciences) and FloJo (v10). normothermic mice were anesthetized and placed on a heating pad in prone position. A distal 5 mm segment of the tail was amputated with a razor blade. Tails were immediately inserted into microtubes containing 1 microliter saline placed in a water bath pre-warmed to 37◦C. MCAO Animals were anesthetized, and a 6–0 silicone-coated monofilament was introduced into the right internal carotid artery until the tip occluded the ostium of the MCA. Regional cerebral blood flow was monitored by laser doppler flowmetry with the use of a probe fixed to the intact skull above the territory of the right MCA. Rectal temperature was maintained between 36.5 and 37◦C with a heating pad. After surgery, animals were allowed to recover from anesthesia. Hundred and twenty minutes after MCAO, the filament was withdrawn to initiate reperfusion, and 62.5 µL of tPA (4 mg/ml, final 10 mg/kg BW) were given by intravenous infusion into the right jugular vein over 15 min using a perfusion pump. 0.1 mg/kg BW buprenorphine hydrochloride (Buprenex R⃝, Reckitt Benckiser Healthcare Ltd, UK) were administered at the end of all procedures. MATERIALS AND METHODS After 3 min, the tail was removed from the microtube, and hemostatic measures were taken by electro-cauterization using Bovie R⃝. The microtube was vortexed, and ultrasound was applied for 15 s to lyse erythrocyte cell membranes. 3 × 100 µL were transferred to a 96 well plate containing 40 µL of Drabkin’s reagent and incubated for 15 min. Absorption rates at 540 nm were determined using a SpectraMax M5 photometer. Bleeding volumes were calculated using a standard curve (14, 20, 23). HT Determination For HT measurement in the brain, mice were lethally anesthetized and perfused transcardially with saline. The brains were removed and sectioned into 1 mm thick slices and photographed. HT was assessed as red areas in brain sections, outlined and measured using ImageJ. Hemorrhages were classified according to the ECASS II morphologic definitions (24, 25) adapted to animal models as used in previous publications (12, 26, 27). Therefore, every section was individually scored on a 5 point ordinal scale (1 = no HT; 2 = hemorrhagic infarction type 1; 3 = hemorrhagic infarction type 2; 4 = parenchymal hemorrhage type 1; 5 = parenchymal hemorrhage type 2) and Standard Tail Bleeding Statistics and Data Analysis an overall grade for every brain was determined according to the highest grade occurring among the sections. Pre-stroke Platelet Function Testing Pre-stroke Platelet Function Testing In control mice (n = 12) platelet pre-activation in unstimulated samples was 3.6 ± 2.0% vs. 2.0 ± 0.7% in unstimulated blood obtained from ASA+CPG treated mice (n = 15, p = 0.365, Figure 2A). In vitro stimulation with thrombin platelet activation was 88.0 ± 0.6% in controls compared to 47.5 ± 3.7% in Pre-stroke Platelet Function Testing Post-stroke flow cytometry analysis was performed with blood collected from 5 randomly chosen control mice and 3 ASA+CPG treated mice by cardiac puncture at the time point of sacrifice and conducted as described above. FIGURE 2 | (A) Platelet activation rates in unstimulated samples: Control: 3.6 ± 2.0%, n = 12; ASA+CPG: 2.0 ± 0.7%, n = 15, p = 0.3649 with selected example images of fluorescence intensity for platelet activation marker anti-CD62P-PE in unstimulated blood samples (FACSDiva software). (B) Platelet activation rates in thrombin stimulated samples: Controls: 88.0 ± 0.6%, n = 12; ASA+CPG: 47.5 ± 3.8%, n = 15, p < 0.0001, images of anti-CD62P-PE fluorescence intensity after in vitro thrombin stimulation. (C) Blood volumes in post-stroke tail bleeding testing: Controls: 1.4 ± 0.2 µl/3 min, n = 4; ASA+CPG: 5.4 ± 0.9 µl/3 min, n = 8, p = 0.004. (D) Correlation of in vitro platelet function with in vivo tail bleeding volumes: Linear regression: R2 =0.5518, y = −0.08175 (95% CI: −0.134 to −0.03)*X+8.846). Correlation: r = −0.7902, p = 0.0033, n = 12. **p < 0.01; ****p< 0.0001. FIGURE 2 | (A) Platelet activation rates in unstimulated samples: Control: 3.6 ± 2.0%, n = 12; ASA+CPG: 2.0 ± 0.7%, n = 15, p = 0.3649 with selected example images of fluorescence intensity for platelet activation marker anti-CD62P-PE in unstimulated blood samples (FACSDiva software). (B) Platelet activation rates in thrombin stimulated samples: Controls: 88.0 ± 0.6%, n = 12; ASA+CPG: 47.5 ± 3.8%, n = 15, p < 0.0001, images of anti-CD62P-PE fluorescence intensity after in vitro thrombin stimulation. (C) Blood volumes in post-stroke tail bleeding testing: Controls: 1.4 ± 0.2 µl/3 min, n = 4; ASA+CPG: 5.4 ± 0.9 µl/3 min, n = 8, p = 0.004. (D) Correlation of in vitro platelet function with in vivo tail bleeding volumes: Linear regression: R2 =0.5518, y = −0.08175 (95% CI: −0.134 to −0.03)*X+8.846). Correlation: r = −0.7902, p = 0.0033, n = 12. **p < 0.01; ****p< 0.0001. Exclusion Criteria Death within the 22 h recovery period led to exclusion from further assessment. Post-stroke tail bleeding tests were not performed in surviving animals when the general condition was too severe (indicated by a low body temperature (<32◦C) and slow breathing rate, for details please see Supplementary Material). Standard Tail Bleeding To control for confounders, we first assessed the body temperature: Hypothermic mice were excluded, while February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org 3 Monitoring DAPT in Stroke Mice Lieschke et al. an overall grade for every brain was determined according to the highest grade occurring among the sections. Statistics and Data Analysis Data is presented as mean ± SEM. Values were tested for Gaussian distribution with D’Agostino-Pearson omnibus normality test and Kolmogorov test. Statistical significance was determined for two group comparisons with Welch’s t-test or Mann-Whitney test, and data sets were considered different if p < 0.05. Correlations were calculated with Spearman’s test. All statistics were performed using Prism 7 graphpad software. Pre-stroke Platelet Function Testing February 2020 | Volume 11 | Article 85 4 Frontiers in Neurology | www.frontiersin.org Lieschke et al. Monitoring DAPT in Stroke Mice suggesting platelet inhibition was still effective 24 h after cessation of treatment. ASA+CPG treated mice (p < 0.0001, Figure 2B), indicating a partial platelet inhibition in these animals. Thus, we sufficiently demonstrated the in vitro detection of ASA+CPG drug effects in whole blood collected from mice following 3 days of oral drug administration. In comparing the individual results of pre-stroke platelet function testing with post-stroke tail bleeding volumes (the standard in vivo measure of platelet function), we found that low platelet activation rates upon in vitro thrombin stimulation correlated significantly with increased blood loss in the tail bleeding test and vice versa (r = −0.7902, p = 0.0033, n = 12; Figure 2D), suggesting our method is feasible to depict platelet function in mice. Implementation Into Experimental Stroke Subsequently, we subjected all mice to 2 h MCAO and tPA infusion, followed by a 22 h recovery period before performing tail bleeding tests (Figure 1A). During the recovery period, 4 control mice and 2 ASA+CPG treated mice died. We examined the deceased animals, focusing on intracerebral- or gastrointestinal bleeding or any other obvious hemorrhagic complication. Neither the ASA+CPG mice nor the control mice showed severe hemorrhages, suggesting that mortality was mainly related to the fairly severe ischemia induced by 2 h of MCAO. Tail bleeding volumes measured in 8 ASA+CPG treated mice were on average 5.4 ± 0.9 µl compared to 1.4 ± 0.2 µl measured in 4 control mice (p = 0.004, Figure 2C), Investigating the effect of platelet function on HT development, we found that HT did not occur in mice that demonstrated beforehand high activation rates upon in vitro stimulation with thrombin (CD62P expression >80%), whereas mice showing reduced platelet activation rates (CD62P expression <80%) were at higher risk for HT development (CD62P expression >80%: 0.9 ± 0.3 mm2; CD62P expression <80%: 10.8 ± 4.4mm2, p = 0.0335, Figure 3A). No hemorrhages were found remote from the infarct. The morphological classification of the HT types showed that 0/7 control mice GURE 3 | (A) Hemorrhage area measurements in brain sections: Platelet activation rate >80% CD62P expression: 0.9 ± 0.3 mm2, n = 7; Platelet activation rate 80% CD62P expression: 10.8 ± 4.5 mm2, n = 11, p = 0.0335. DISCUSSION With this study, we successfully developed a practical and straightforward method to test platelet function in vitro using flow cytometry, and we applied this assay to ASA+CPG treated mice in the context of experimental stroke studies in order to monitor antithrombotic treatment efficacy. Furthermore, we demonstrated that our method can be implemented at different time points within the stroke model and that repeated measurements are also feasible. Our method can be used to identify animals that are at a higher risk for developing HT, in order to study the pathophysiology and to explore new therapeutic approaches. The current literature also revealed that despite the higher risk of tPA-associated HT in patients on DAPT, mortality and neurologic outcomes did not differ from patients without DAPT; thus, the benefit of tPA seemed to outweigh its risk (37–39). In order to get a clearer picture, it may be useful to implement platelet function testing during the early phase of acute ischemic stroke through using flow cytometry-based assays or point of care devices, which have already replaced the previous standard screening methods for platelet dysfunction (40). Testing the platelet function rather than the platelet count could potentially make discussion about platelet threshold superfluous (41). Thus, far, tail bleeding has been the only measure that assesses platelet function in an individual mouse without the need to pool blood from different subjects (which is needed for LTA). However, when it comes to its implementation into stroke models, this approach faces serious limitations. First, tail bleeding testing can only be performed at the end of an experiment before sacrifice due to a high risk of severe and uncontrolled bleeding. Secondly, the tail bleeding test is relatively unspecific (20). It can be affected by secondary hemostasis, blood pressure and body temperature. Another disadvantage is that mice cannot be tested repetitively, precluding dynamic measurements. In contrast, our flow cytometry based technique of platelet function analysis requires only 0.5–1% of the total blood volume of a mouse, allowing for repeated sampling. We also need to address the limitations of our study. In conventional aggregation assays, typically ADP is used to demonstrate CPG effects (32). ADP receptor activation in mice leads to the release of platelet dense granules but not to the release of alpha granules (14). We used CD62P which is only located in alpha granules; thus, ADP stimulation could not be illustrated using anti-CD62P. Pre-stroke Platelet Function Testing (B) Morphological classification of the bleeding types: 0/7 control mice were rated HI-2, compared 6/11 mice in the ASA+CPG (p = 0.0125). (C) Platelet activation rates in thrombin stimulated samples (post-stroke): Controls: 91.9 ± 2.1%, n = 5; ASA+CPG: 71.6 4.1%, n = 3, p = 0.0026. (D) Platelet activation following thrombin stimulation pre- and post-stroke in ASA+CPG pretreated mice: Platelet activation rates creased significantly by 22.9 ± 8.9% (p = 0.0204) within 24 h between the two measurements. *p < 0.05; **p < 0.01. GURE 3 | (A) Hemorrhage area measurements in brain sections: Platelet activation rate >80% CD62P expression: 0 9 ± 0 3 mm2 n = 7; Platelet activation rate FIGURE 3 | (A) Hemorrhage area measurements in brain sections: Platelet activation rate >80% CD62P expression: 0.9 ± 0.3 mm2, n = 7; Platelet activation rate <80% CD62P expression: 10.8 ± 4.5 mm2, n = 11, p = 0.0335. (B) Morphological classification of the bleeding types: 0/7 control mice were rated HI-2, compared to 6/11 mice in the ASA+CPG (p = 0.0125). (C) Platelet activation rates in thrombin stimulated samples (post-stroke): Controls: 91.9 ± 2.1%, n = 5; ASA+CPG: 71.6 ± 4.1%, n = 3, p = 0.0026. (D) Platelet activation following thrombin stimulation pre- and post-stroke in ASA+CPG pretreated mice: Platelet activation rates increased significantly by 22.9 ± 8.9% (p = 0.0204) within 24 h between the two measurements. *p < 0.05; **p < 0.01. 5 February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org Lieschke et al. Monitoring DAPT in Stroke Mice were rated HI-2, compared to 6/11 mice in the ASA+CPG (p = 0.0125, Figure 3B, Supplementary Table I). As we had hypothesized, ASA+CPG treatment led to significantly reduced platelet activation rates upon in vitro stimulation with thrombin. These treatment effects occurred in a comparable range to previously reported results in humans (28), although relatively high treatment dosages (of 60 mg/kg ASA and 22.5 mg/kg CPG) were used. The faster metabolism of mice and different administration protocols (crushing and diluting tablets which are designed for enteric resorption in humans) may explain these comparable effects in spite of a higher absolute dose (19, 22, 29, 30). Ultimately, while there was some variability in the response, we did not identify any non-responders, and all ASA+CPG mice showed reduced platelet reactivity. Post-stroke Platelet Function Testing g In analogy to the post-stroke tail bleeding testing, we performed a second in vitro platelet function testing post-stroke in randomly selected mice at the time point of sacrifice. In ASA+CPG pretreated mice, platelet activation upon in-vitro thrombin stimulation 24 h after drug withdrawal was 71.6 ± 4.1% compared to 91.9 ± 2.1% in controls (Figure 3C, p = 0.0026). Although the antiplatelet effect of ASA+CPG pretreatment was still detectable 24 h after stroke and drug withdrawal, platelet function in ASA+CPG pretreated mice already improved significantly compared to the platelet activation rates of the first in vitro testing (Figure 3D platelet activation rates in ASA+CPG mice pre-stroke 47.5 ± 3.8% vs. 71.6 ± 4.1% post-stroke). We here present the feasibility of repeated sampling within a stroke model using our flow cytometry-based platelet function assay. Accordingly, we demonstrated that the impaired platelet function after ASA+CPG pre-treatment is strongly correlated with higher tail bleeding volumes. Thus, our method can be used to identify mice with decreased platelet function. Based on this, cohorts with an overall increased risk of HT could be identified. However, HT prediction on an individual animal level remains difficult. Similar to humans, not every pretreated mouse developed HT following thrombolytic treatment, although mice that showed hemorrhages generally had lower activation rates. These findings are in line with previous attempts to better stratify the risk of tPA-associated HT in human patients (7, 31–36), which aimed to define risk scores and predictors for HT. With only modest predictive power, those models failed to identify common key contributors. More research on pre-stroke platelet function and its impact on later HT development and neurological outcome may help to fill this gap of knowledge. Frontiers in Neurology | www.frontiersin.org DISCUSSION We also considered cross-validation by comparison with aggregometry, but decided against it because (a) Due to the large volume required for the aggregometer used in the MGH pathology core facility, we would have had to pool the blood of several mice, which practically could not be combined with a stroke model; (b) The procedure used to initiate platelet activation differs significantly from our approach. For example, the core facility uses ADP or arachidonic acid to activate the platelets in vitro, while we used thrombin in our study; (c) Measurements with aggregometers are manufacturer and laboratory specific, which implies a restricted comparability between centers. These limitations would have ruled out a quantitative comparison of the two methods. However, the previous study by Lauer et al. demonstrated that the dosages of ASA and CPG used in our study were sufficient to significantly reduce platelet aggregation as measured by aggregometry (14), which is in line with our current results based on FACS measurements. model to better investigate and understand the mechanisms underlying the impaired platelet function contributing to HT. model to better investigate and understand the mechanisms underlying the impaired platelet function contributing to HT. ETHICS STATEMENT The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (MGH). FUNDING This work was supported by the National Institutes of Health (R01NS049430 to KL); the American Heart Association (17GRNT33460100 to KL); and the Faculty of Medicine, Goethe University Frankfurt (FPF doctoral scholarship to FL). ACKNOWLEDGMENTS The authors thank Dr. Xiaoying Wang, Dr. Eng H. Lo, Dr. Ken Arai, and Dr. Gen Hamanaka for helpful discussions, and Kelly Kin Chung for help with the manuscript. We gratefully acknowledge the National Institutes of Health and the American Heart Association for their financial support of this work. In summary, we describe a versatile and easy-to-implement tool to investigate the impact of platelet function in experimental stroke. We demonstrated that ASA+CPG treatment resulted in significantly impaired platelet function contributing to an increased risk of HT after experimental stroke and tPA administration. Our test can be used in animal studies to monitor drug effects. To identify and exclude mice with insufficient platelet inhibition from future experiments, it can be implemented at various times within the model and repeated using dynamic measurements. We here offer an experimental DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. AUTHOR CONTRIBUTIONS CF, KL, and FL conceived the idea, designed the model and analyzed the data. FL carried out the experiments and performed the flow cytometry measurements. YZ performed the MCAO surgery. FL processed the experimental data, performed the statistical analysis, drafted the article, and designed the figures. JS aided in interpreting the results. KL and CF supervised the work. CF and KL made critical revisions of the article for important intellectual content. All authors discussed the results and commented on the article. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2020.00085/full#supplementary-material 3. Levine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, et al. 2011 ACCF/AHA/SCAI guideline for percutaneous coronary intervention. J Am Coll Cardiol. (2011) 58:e44–122. doi: 10.1016/j.jacc.2011.08.007 4. Brott TG, Hobson RW, Howard G, Roubin GS, Clark WM, Brooks W, et al. Stenting versus endarterectomy for treatment of carotid-artery stenosis. N Engl J Med. (2010) 363:11–23. doi: 10.1056/NEJMx100035 5. Valgimigli M, Bueno H, Byrne RA, Collet JP, Costa F, Jeppsson A, et al. 2017 ESC focused update on dual antiplatelet therapy in coronary artery DISCUSSION Another consideration addressing the flow cytometry test relates to our method of quantification. An accepted strategy to quantify platelet aggregation using flow cytometry is the determination of the loss of single platelets due to aggregation upon platelet activation (17, 18, 28, 42, 43). Since we were using two separate samples when comparing unstimulated vs. stimulated blood, we were unable to consistently detect and quantify the loss of single platelets in these samples. A complementary strategy to deal with these limitations for future studies is to use only one sample and repeat measurements before and after platelet stimulation. However, this would require the platelet count to be determined. Which implies another We showed that there was no significant difference in platelet activation in unstimulated blood between ASA+CPG treated mice and control mice prior to stroke, which indicates that our samples were handled equally, resulting in acceptable pre- activation rates. However, pre-activation rates in ASA+CPG treated mice were slightly reduced compared to controls, which is in line with the findings of Kassassir et al. (19), who demonstrated that pre-activation rates in mice intravenously treated with cangrelor (another ADP-receptor antagonist like CPG) were reduced compared to untreated controls (19). February 2020 | Volume 11 | Article 85 6 Lieschke et al. Monitoring DAPT in Stroke Mice limitation to our study, since platelet function can be affected by platelet count (44, 45). Also, we have not tested platelet function following long-term treatment yet, in which other hemostatic factors besides platelets may also be affected. In addition, our study was designed as a single-center study at the Neuroprotection Research Laboratory (Massachusetts General Hospital), and needs to be confirmed by replication in other laboratories. A last but major concern is the validation of our flow cytometry assay by using tail bleeding. Since platelet function in vitro and in vivo may differ (44, 46, 47), it is insufficient to base examinations on either in vitro or in vivo only. By combining the FACS analysis with tail bleeding, we intended to achieve a complementary insight into the hemostatic status of an individual mouse. As expected, tail bleeding volumes were increased in the pretreated mice 24 h after the ischemic event. At the same time, FACS analysis of blood from the same mice showed reduced platelet activation in the DAPT treated mice, and these results correlated well (r = −0.7902, p = 0.0033, Figure 2D). 1. Johnston SC, Easton JD, Farrant M, Barsan W, Conwit RA, Elm JJ, et al. Clopidogrel and aspirin in acute ischemic stroke and high-risk TIA. N Engl J Med. (2018) 379:215–25. doi: 10.1056/NEJMoa1800410 2. Wang Y, Wang Y, Zhao X, Liu L, Wang D, Wang C, et al. Clopidogrel with aspirin in acute minor stroke or transient ischemic attack. N Engl J Med. (2013) 369:11–9. doi: 10.1056/NEJMoa1215340 3. Levine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, et al. 2011 ACCF/AHA/SCAI guideline for percutaneous coronary intervention. J Am Coll Cardiol. (2011) 58:e44–122. doi: 10.1016/j.jacc.2011.08.007 5. Valgimigli M, Bueno H, Byrne RA, Collet JP, Costa F, Jeppsson A, et al. 2017 ESC focused update on dual antiplatelet therapy in coronary artery 4. Brott TG, Hobson RW, Howard G, Roubin GS, Clark WM, Brooks W, et al. Stenting versus endarterectomy for treatment of carotid-artery stenosis. N Engl J Med. (2010) 363:11–23. doi: 10.1056/NEJMx100035 1. Johnston SC, Easton JD, Farrant M, Barsan W, Conwit RA, Elm JJ, et al. Clopidogrel and aspirin in acute ischemic stroke and high-risk TIA. N Engl J Med. (2018) 379:215–25. doi: 10.1056/NEJMoa1800410 2. Wang Y, Wang Y, Zhao X, Liu L, Wang D, Wang C, et al. Clopidogrel with aspirin in acute minor stroke or transient ischemic attack. N Engl J Med. (2013) 369:11–9. doi: 10.1056/NEJMoa1215340 REFERENCES (2014) 27:549–64. doi: 10.3122/jabfm.2014.04.130227 34. Saver JL. Hemorrhage after thrombolytic therapy for stroke: the clinically relevant number needed to harm. Stroke. (2007) 38:2279–83. doi: 10.1161/STROKEAHA.107.487009 16. Peterson P, Hayes TE, Arkin CF, Bovill EG, Fairweather RB, Rock William AJ, et al. The preoperative bleeding time test lacks clinical benefit: college of American Pathologists’ and American Society of Clinical Pathologists’ position article. JAMA Surg. (1998) 133:134–9. doi: 10.1001/archsurg.133.2.134 35. Menon BK, Saver JL, Prabhakaran S, Reeves M, Liang L, Olson DM, et al. Risk score for intracranial hemorrhage in patients with acute ischemic stroke treated with intravenous tissue-type plasminogen activator. Stroke. (2012) 43:2293–9. doi: 10.1161/STROKEAHA.112.660415 17. Armstrong PCJ, Kirkby NS, Chan MV, Finsterbusch M, Hogg N, Nourshargh S, et al. Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction. Blood. (2015) 126:e11–8. doi: 10.1182/blood-2015-01-621656 36. Whiteley WN, Bruins SK, Peter F, Peter S, Joanna W. Risk Factors for intracranial hemorrhage in acute ischemic stroke patients treated with recombinant tissue plasminogen activator. Stroke. (2012) 43:2904– 9. doi: 10.1161/STROKEAHA.112.665331 18. De Cuyper IM, Meinders M, Van De Vijver E, De Korte D, Porcelijn L, De Haas M, et al. A novel flow cytometry – based platelet aggregation assay. Blood. (2013) 121:70–80. doi: 10.1182/blood-2012-06-437723 37. Luo S, Zhuang M, Zeng W, Tao J. Intravenous thrombolysis for acute ischemic stroke in patients receiving antiplatelet therapy: a systematic review and meta-analysis of 19 studies. J Am Heart Assoc. (2016) 5:1– 14. doi: 10.1161/JAHA.116.003242 19. Kassassir H, Siewiera K, Sychowski R, Watała C. Can the antiplatelet effects of cangrelor be reliably studied in mice under in vivo and in vitro conditions using flow cytometry? Pharmacol Rep. (2013) 65:870– 83. doi: 10.1016/S1734-1140(13)71068-5 38. Xian Y, Federspiel JJ, Grau-Sepulveda M, Hernandez AF, Schwamm LH, Bhatt DL, et al. Risks and benefits associated with prestroke antiplatelet therapy among patients with acute ischemic stroke treated with intravenous tissue plasminogen activator. JAMA Neurol. (2016) 73:50– 9. doi: 10.1001/jamaneurol.2015.3106 20. Liu Y, L JN, M DA, Du X-J. Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice. World J Exp Med. (2012) 2:30. doi: 10.5493/wjem.v2.i2.30 39. Tsivgoulis G, Katsanos AH, Mavridis D, Gdovinova Z, Karlinski M, Macleod MJ, et al. Intravenous thrombolysis for ischemic stroke patients on dual antiplatelets. Ann Neurol. (2018) 84:89–97. doi: 10.1002/ana.25269 21. Momi S, Pitchford SC, Alberti PF, Minuz P, Del Soldato P, Gresele P. REFERENCES 1. Johnston SC, Easton JD, Farrant M, Barsan W, Conwit RA, Elm JJ, et al. Clopidogrel and aspirin in acute ischemic stroke and high-risk TIA. N Engl J Med. (2018) 379:215–25. doi: 10.1056/NEJMoa1800410 2. Wang Y, Wang Y, Zhao X, Liu L, Wang D, Wang C, et al. Clopidogrel with aspirin in acute minor stroke or transient ischemic attack. N Engl J Med. (2013) 369:11–9. doi: 10.1056/NEJMoa1215340 February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org 7 Lieschke et al. Monitoring DAPT in Stroke Mice method for designing new anti-thrombotic agents. Int J Exp Pathol. (2016) 97:285–92. doi: 10.1111/iep.12182 method for designing new anti-thrombotic agents. Int J Exp Pathol. (2016) 97:285–92. doi: 10.1111/iep.12182 disease developed in collaboration with EACTS. Eur J Cardio-thoracic Surg. (2018) 53:34–78. doi: 10.1093/eurheartj/ehx419 disease developed in collaboration with EACTS. Eur J Cardio-thoracic Surg. (2018) 53:34–78. doi: 10.1093/eurheartj/ehx419 6. Yang Y, Zhou M, Zhong X, Wang Y, Zhao X, Liu L, et al. Dual versus mono antiplatelet therapy for acute non-cardioembolic ischaemic stroke or transient ischaemic attack : a systematic review and meta-analysis. Stroke Vasc Neurol. (2018) 3:107–16. doi: 10.1136/svn-2018-000168 24. Von Kummer R, Broderick JP, Campbell BCV, Demchuk A, Goyal M, Hill MD, et al. The heidelberg bleeding classification: Classification of bleeding events after ischemic stroke and reperfusion therapy. Stroke. (2015) 46:2981– 6. doi: 10.1161/STROKEAHA.115.010049 7. Mazya M, Egido JA, Ford GA, Lees KR, Mikulik R, Toni D, et al. Predicting the risk of symptomatic intracerebral hemorrhage in ischemic stroke treated with intravenous alteplase: Safe Implementation of Treatments in Stroke (SITS) symptomatic intracerebral hemorrhage risk score. Stroke. (2012) 43:1524– 31. doi: 10.1161/STROKEAHA.111.644815 25. Yaghi S, Willey JZ, Cucchiara B, Goldstein JN, Gonzales NR, Khatri P, et al. Treatment and outcome of hemorrhagic transformation after intravenous alteplase in acute ischemic stroke: a scientific statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. (2017) 48:e343–61. doi: 10.1161/STR.0000000000000152 26. García-Yébenes I, Sobrado M, Zarruk JG, Castellanos M, De La Ossa NP, Dávalos A, et al. A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke. (2011) 42:196–203. doi: 10.1161/STROKEAHA.110.600452 8. Diedler J, Ahmed N, Sykora M, Uyttenboogaart M, Overgaard K, Luijckx GJ, et al. Safety of intravenous thrombolysis for acute ischemic stroke in patients receiving antiplatelet therapy at stroke onset. Stroke. (2010) 41:288– 94. doi: 10.1161/STROKEAHA.109.559724 27. Copin J-C, Gasche Y. REFERENCES Effect of the duration of middle cerebral artery occlusion on the risk of hemorrhagic transformation after tissue plasminogen activator injection in rats. Brain Res. (2008) 1243:161–6. doi: 10.1016/j.brainres.2008.09.025 9. Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, et al. 2018 guidelines for the early management of patients with acute ischemic stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. (2018) 49:46–110. doi: 10.1161/STR.0000000000000163 28. Van Velzen JF, Laros-Van Gorkom BAP, Pop GAM, Van Heerde WL. Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers. Thromb Res. (2012) 130:92–8. doi: 10.1016/j.thromres.2012.02.041 10. The European Stroke Organisation (ESO) Executive Committee and the ESO Writing Committee. Guidelines for Management of Ischaemic Stroke and Transient Ischaemic Attack 2008. Cerebrovasc Dis. (2008) 25:457– 507. doi: 10.1159/000131083 29. Li Y, Landqvist C, Grimm SW. Disposition and metabolism of ticagrelor, a novel P2Y12 receptor antagonist, in mice, rats, and marmosets. Drug Metab Dispos. (2011) 39:1555–67. doi: 10.1124/dmd.111.039669 11. Gensicke H, Al Sultan AS, Strbian D, Hametner C, Zinkstok SM, Moulin S, et al. Intravenous thrombolysis and platelet count. Neurology. (2018) 90:e1–8. doi: 10.1212/WNL.0000000000004982 30. Wientjes MG, Levy G. Nonlinear pharmacokinetics of aspirin in rats. J Pharmacol Exp Ther. (1988) 245:809–15. 12. Zheng Y, Lieschke F, Schaefer JH, Wang X, Foerch C, van Leyen K. Dual antiplatelet therapy increases hemorrhagic transformation following thrombolytic treatment in experimental stroke. Stroke. (2019) 50:3650– 3. doi: 10.1161/STROKEAHA.119.027359 31. Cucchiara B, Tanne D, Levine SR, Demchuk AM, Kasner S. A Risk score to predict intracranial hemorrhage after recombinant tissue plasminogen activator for acute ischemic stroke. J Stroke Cerebrovasc Dis. (2008) 17:331– 3. doi: 10.1016/j.jstrokecerebrovasdis.2008.03.012 13. Hughes CE. How to perform aggregometry and lumi-aggregometry in mouse platelets. Platelets. (2018) 29:638–43. doi: 10.1080/09537104.2018.1478074 32. Lou M, Safdar A, Mehdiratta M, Kumar S, Schlaug G, Caplan L, et al. The HAT score. Neurology. (2008) 71:1417– 23. doi: 10.1212/01.wnl.0000330297.58334.dd 14. Lauer A, Schlunk F, Van Cott EM, Steinmetz H, Lo EH, Foerch C. Antiplatelet pretreatment does not increase hematoma volume in experimental intracerebral hemorrhage. J Cereb Blood Flow Metab. (2011) 31:1736–42. doi: 10.1038/jcbfm.2011.22 33. Strbian D, Engelter S, Michel P, Meretoja A, Sekoranja L, Ahlhelm FJ, et al. Symptomatic intracranial hemorrhage after stroke thrombolysis: the SEDAN Score. Ann Neurol. (2012) 71:634–41. doi: 10.1002/ana.23546 15. Kruse-Jarres R, Singleton TC, Leissinger CA. Identification and basic management of bleeding disorders in adults. J Am Board Fam Med. REFERENCES Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice. Thromb Haemost. (2005) 93:535–43. doi: 10.1160/TH04-07-0464 40. Chen F, Maridakis V, O’Neill EA, Beals C, Radziszewski W, De Lepeleire I, et al. A randomized clinical trial comparing point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel. Platelets. (2012) 23:249–58. doi: 10.3109/09537104.2011.604806 22. Tsakiris DA, Scudder L, Hodivala-Dilke K, Hynes RO, Coller BS. Hemostasis in the mouse (Mus musculus): a review. Thrombosis Haemostasis. (1999) 81:177–88. doi: 10.1055/s-0037-1614439 41. Breuer L, Huttner HB, Kiphuth IC, Ringwald J, Hilz MJ, Schwab S, et al. Waiting for platelet counts causes unsubstantiated delay of thrombolysis therapy. Eur Neurol. (2013) 69:317–20. doi: 10.1159/000345702 23. Saito MS, Lourenço AL, Kang HC, Rodrigues CR, Cabral LM, Castro HC, et al. New approaches in tail-bleeding assay in mice: improving an important February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org 8 Lieschke et al. Monitoring DAPT in Stroke Mice 47. Frelinger AL, Grace RF, Gerrits AJ, Berny-Lang MA, Brown T, Carmichael SL, et al. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP. Blood. (2015) 126:873–9. doi: 10.1182/blood-2015-02-6 28461 42. Ramström S, Södergren A, Tynngård N, Lindahl T, Lindahl TL. Platelet function determined by flow cytometry: new perspectives? platelet function determined by flow cytometry -new perspectives? Semin Thromb Hemost. (2016) 3:268–81. doi: 10.1055/s-0035-1570082 43. Gremmel T, Koppensteiner R, Panzer S. Comparison of aggregometry with flow cytometry for the assessment of agonists’-induced platelet reactivity in patients on dual antiplatelet therapy. PLoS ONE. (2015) 10:e0129666. doi: 10.1371/journal.pone.0129666 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 44. Vinholt PJ. The role of platelets in bleeding in patients with thrombocytopenia and hematological disease. Clin Chem Lab Med. (2019) 57:1808. doi: 10.1515/cclm-2019-0380 Copyright © 2020 Lieschke, Zheng, Schaefer, van Leyen and Foerch. 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. 45. Frontiers in Neurology | www.frontiersin.org REFERENCES Krishnegowda M, Rajashekaraiah V. Platelet disorders: an overview. Blood Coagul Fibrinolysis. (2015) 26:479–91. doi: 10.1097/01.mbc.0000469521.23628.2d 46. Panzer S, Rieger M, Vormittag R, Eichelberger B, Dunkler D, Pabinger I. Platelet function to estimate the bleeding risk in autoimmune thrombocytopenia. Eur J Clin Invest. (2007) 37:814–9. doi: 10.1111/j.1365-2362.2007.01855.x 46. Panzer S, Rieger M, Vormittag R, Eichelberger B, Dunkler D, Pabinger I. Platelet function to estimate the bleeding risk in autoimmune thrombocytopenia. Eur J Clin Invest. (2007) 37:814–9. doi: 10.1111/j.1365-2362.2007.01855.x February 2020 | Volume 11 | Article 85 Frontiers in Neurology | www.frontiersin.org 9
https://openalex.org/W2776005862
https://periodicos.uff.br/midiaecotidiano/article/download/9856/6986
Portuguese
null
Religião e Política: medos sociais e extremismo religioso no Brasil
Mídia.e.Cotidiano
2,017
cc-by
2,240
1 Mestranda do Programa de Pós-Graduação em Mídia e Cotidiano (PPGMC) na Universidade Federal Fluminense (UFF), na linha de pesquisa Política, Discursos e Sociedade. Membra do Laboratório de Pesquisa em Comunicação Comunitária e Publicidade Social (LACCOPS). E-mail: lariissadeoliveira@gmail.com Revista Mídia e Cotidiano Resenha Revista Mídia e Cotidiano Resenha Volume 11, Número 3, dezembro de 2017 Resenha Volume 11, Número 3, dezembro de 2017 Religião e Política: medos sociais, extremismo religioso e as eleições 2014 Larissa de Oliveira Cesar1 Larissa de Oliveira Cesar1 VITAL DA CUNHA, Christina.; LOPES, Paulo Victor Leite.; LUI, Janayna. Religião e Política: medos sociais, extremismo religioso e as eleições 2014. Rio de Janeiro: Fundação Henrique Böll/Instituto de Estudos da Religião, 2017. 196 p. ISBN: 978-85- 62669-21-7. VITAL DA CUNHA, Christina.; LOPES, Paulo Victor Leite.; LUI, Janayna. Religião e Política: medos sociais, extremismo religioso e as eleições 2014. Rio de Janeiro: Fundação Henrique Böll/Instituto de Estudos da Religião, 2017. 196 p. ISBN: 978-85- 62669-21-7. Resumo: “Religião e política: medos sociais, extremismo religioso e as eleições 2014” é um livro desenvolvido com o objetivo de entender o panorama político no Brasil contemporâneo a partir da análise das eleições 2014 nos cenários nacional e estadual. A utilização do conceito de “períodos frios” de Durkheim ajuda a compreender como os medos sociais e a ameaça de valores tradicionais propiciam o estabelecimento de um perfil comportamental extremista, explicitado na esfera pública para preservar tais valores através da manipulação de uma leitura bíblica admitida como fundamentalista. Palavras-chave: Religião; Política; Eleições 2014. O cenário político brasileiro recente tem se mostrado polarizado e instável, o que se intensificou, especialmente, a partir das eleições de 2014. A crise política, que reverberou também nas esferas econômica e social, se estendeu desde então, mergulhando o país em um período difícil. Se, por um lado, crescem as demandas por direitos e conquistas de grupos sociais histórica e culturalmente marginalizados, como as mulheres, os negros e as comunidades LGBT, por outro, avançam pautas caracterizadas como conservadoras, que buscam frear os avanços conquistados estimulados por medos sociais. Esse quadro tem influência direta sobre os representantes políticos eleitos. Atualmente, o país possui a maior Bancada Evangélica já eleita. Em pesquisa feita pelo Grupo de Pesquisa Mídia, Religião e Cultura (MIRE) e divulgada no Portal Mídia, Religião e Política, averiguou-se que a bancada eleita em 2014 possui 87 deputados/as federais e 3 senadores, resultando em número de 90 parlamentares, que estão vinculados Volume 11, Número 3, dezembro de 2017 | Página 269 ou declaram pertencer a uma instituição evangélica. É em torno desse contexto que o livro “Religião e Política: medos sociais, extremismo religioso e as eleições 2014” se organiza, buscando compreender as composições que se fazem na relação entre religioso e político, considerando dois âmbitos das eleições de 2014: a candidatura à presidência do Pastor Everaldo e a disputa ao governo do estado do Rio de Janeiro. Volume 11, Número 3, dezembro de 2017 | Página 271 Larissa de Oliveira Cesar1 O estudo tem como autores os pesquisadores Christina Vital, Paulo Victor Leite Lopes e Janayna Lui e foi desenvolvido pelo Instituto de Estudos da Religião (ISER) em parceria com a Fundação Henrich Böll (HBS). O trabalho aborda a questão religiosa no espaço público e se configura como uma espécie de continuidade de uma pesquisa anterior realizada por Christina Vital e Paulo Victor Leite Lopes intitulada “Religião e Política: uma análise da participação de parlamentares evangélicos sobre o direito de mulheres e de LGBTs no Brasil”, 2012. Vital é professora do Programa de Pós-graduação em Cultura e Territorialidades (PPCULT) e do Departamento de Sociologia da Universidade Federal Fluminense (UFF), além de integrante do grupo Religião, arte, materialidade e espaço público (PPGAS- UFRGS) e da Rede de Pesquisadores Luso-Brasileiros e Artes e Intervenções Urbanas. A autora é colaboradora do Instituto de Estudos da Religião desde 2002 e desenvolveu outros trabalhos como o livro “Religião e Conflito”, 2016, e “Oração de Traficante: uma etnografia”, 2015. Janayna de Alencar Lui é doutora em Antropologia Cultural pelo Programa de Pós-graduação em Sociologia e Antropologia, do Instituto de Filosofia e Ciências Sociais, da Universidade Federal do Rio de Janeiro (PPGSA/IFCS/UFRJ), com a tese “Educação, Laicidade, Religião: controvérsias sobre a implementação do ensino religioso em escolas públicas”, 2011. Tem várias publicações nesse sentido e foi pesquisadora colaboradora do Instituto de Estudos da Religião estudando a temática da religião no espaço público. Já Paulo Victor Leite Lopes é cientista social, formado pela Universidade do Estado do Rio de Janeiro (UERJ). O autor trabalha com pesquisas que envolvem as articulações entre segurança pública, religião e política, estudando as temáticas de religião, gênero, sexualidade, Estado, favela e classe social. É integrante do Centro de Estudos de Segurança e Cidadania da Universidade Cândido Mendes (CESeC/UCAM) e Volume 11, Número 3, dezembro de 2017 | Página 270 do Laboratório Integrado em Diversidade Sexual e de Gênero, Políticas e Direitos (LIDIS/UERJ). do Laboratório Integrado em Diversidade Sexual e de Gênero, Políticas e Direitos (LIDIS/UERJ). do Laboratório Integrado em Diversidade Sexual e de Gênero, Políticas e Direito do Laboratório Integrado em Diversidade Sexual e de Gênero, Políticas e Direitos (LIDIS/UERJ). (LIDIS/UERJ). A proposta da publicação é analisar as candidaturas de Pastor Everaldo (PSC), Marcelo Crivella (PRB) e Anthony Garotinho (PR), buscando compreender o complexo cenário que o Brasil vive de uma maneira mais ampla a partir de casos específicos de candidaturas declaradamente evangélicas, admitindo, assim, a centralidade da ação deste grupo religioso no espaço público brasileiro. Para isso, o livro analisa os jogos políticos que se estabeleceram nas eleições 2014, tanto no cenário nacional, como também no estadual de forma detalhada, utilizando uma linguagem objetiva e direta que permite que até mesmo os leitores mais desacostumados com a temática política entendam as especificidades daquele cenário, bem como a contextualização de valores importantes no contexto evangélico. Logo na introdução do livro, os autores dissertam sobre os acontecimentos que precederam as eleições 2014, marcada pelo que chamam de “atordoamento” provocado pelos fatos que antecederam o pleito, com a morte do presidenciável Eduardo Campos, do Partido Socialista Brasileiro (PSB). Com isso, Marina Silva viria a se tornar a candidata do partido. Para os autores, o “jogo de ocultação” a respeito de sua identidade religiosa culminou na figura de Pastor Everaldo, do Partido Social Cristão (PSC), como possível definidor da campanha eleitoral 2014. A partir disso, se desenrolam os exemplos sobre como a religião tem se explicitado no espaço público, desde as eleições de Fernando Collor até Aécio Neves, em 2014, quando houve uma diminuição do voto evangélico ao Partido dos Trabalhadores (PT), passando pelo primeiro pronunciamento de Michel Temer e a votação de encaminhamento do pedido de impeachment de Dilma Rousseff. A religião seria então utilizada como código de posicionamento dos atores políticos em “jogos de visibilidade e invisibilidade” para as ações de motivação religiosa. Com cautela para não produzir conclusões generalistas e totalizantes, a publicação analisa primeiramente o cenário nacional, trazendo informações e dados importantes sobre a religião no cenário político brasileiro que se compõe, a partir das eleições de 2014 como um divisor de águas na relação entre religião e política. Isso porque, pela primeira vez, houve uma candidatura evangélica confessional ao Executivo do país, revelando o Volume 11, Número 3, dezembro de 2017 | Página 271 anseio dos evangélicos de passarem de “ovelhas” a “players” do jogo político (p. 126) pelo seu quantitativo e aumento de capital político, já que, atualmente, temos a maior Bancada Evangélica eleita e quase ¼ da população autodeclarada evangélica. Volume 11, Número 3, dezembro de 2017 | Página 272 afetados, medos são experimentados por diferentes grupos que se sentem ameaçados. Esses medos motivam ações de manipulação para controlar esse processo, freando a garantia de direitos das minorias, por considerá-los uma afronta aos valores tradicionais reivindicados. Entrariam em ação, portanto ações extremistas explicitadas anteriormente. afetados, medos são experimentados por diferentes grupos que se sentem ameaçados. Esses medos motivam ações de manipulação para controlar esse processo, freando a garantia de direitos das minorias, por considerá-los uma afronta aos valores tradicionais reivindicados. Entrariam em ação, portanto ações extremistas explicitadas anteriormente. Já as ações conservadoras se caracterizam pela resistência às mudanças e omissão diante do extremismo, podendo os atores não se sentirem representados pelos políticos e líderes religiosos que adotam tal comportamento. Entretanto, se silenciam contra essas posições, evitando exposição pública (p. 119). Os atores que se comportam dessa forma são denominados como “amoladores de faca” (p.121), se omitindo perante ações extremistas e silenciando os progressistas. O comportamento progressista define-se como defesa das minorias no espaço público, inclusive fazendo uso do plano político e mídias digitais, além de assumir uma postura crítica em relação às ações extremistas e realizar uma leitura bíblica que considera o contexto histórico. Quanto ao cenário estadual, os pesquisadores trazem o panorama das eleições 2014 no Rio de Janeiro, cuja configuração chegou a ser comparada ao poema “Quadrilha” (p. 78), de Carlos Drummond de Andrade, com alianças e apoios difusos. Para entender o cenário, os autores percorrem um caminho detalhado, explicando a vida dos principais candidatos (Anthony Garotinho, Lindbergh Farias, Luiz Fernando Pezão e Marcelo Crivella) e suas relações com a religião. Para tornar mais claro para o leitor o a configuração que se deu, faz-se necessário o uso de uma tabela (p. 90; 91), apontando de quem eram os apoios de partidos e candidatos à presidência. A campanha se transforma em um jogo de acusações políticas e religiosas retratado no texto de maneira bem esclarecedora nos dois turnos que se sucederam, explicando escolhas e posições que se revelam para além do caráter religioso e moral, porém, assumidas especialmente como motivações de interesses políticos. É o que se revela em relação ao Pastor Everaldo na primeira parte do livro, quando explica as posições do partido em relação à retirada de Dilma Rousseff. Volume 11, Número 3, dezembro de 2017 | Página 273 (LIDIS/UERJ). anseio dos evangélicos de passarem de “ovelhas” a “players” do jogo político (p. 126) pelo seu quantitativo e aumento de capital político, já que, atualmente, temos a maior Bancada Evangélica eleita e quase ¼ da população autodeclarada evangélica. O livro traz abordagens sobre figuras evangélicas conhecidas, como Eduardo Cunha e Marco Feliciano. Como material de pesquisa, é utilizada uma entrevista concedida pelo Pastor Everaldo e outros materiais de divulgação pública, como reportagens, debates públicos, conteúdo digital, dados de órgãos públicos e institutos de pesquisa. As informações levantadas são essenciais para traçar o posicionamento destas figuras atreladas a um perfil definido como liberal conservador religioso. Liberal em relação ao econômico e conservador em relação à moral. Por vezes, as posições defendidas pela Frente Parlamentar Evangélica se apresentam como mais conservadoras que a população evangélica, de acordo com pesquisas utilizadas na publicação, que concernem opiniões sobre pena de morte, sindicatos, armamento pessoal e homossexualidade. A pesquisa explorada revela ainda um posicionamento mais conservador da população católica do que do setor pentecostal. Por isso, os autores apresentam o cuidado de não reforçar estereótipos de atores sociais específicos, mas trabalham com categorias referentes aos comportamentos apresentados pelos grupos religiosos, que independem de perfis socioeconômicos ou da vertente religiosa adotada, seja ela protestante histórica ou de correntes pentecostais. São definidos três tipos de formulações: ações extremistas, conservadoras e extremistas. Os pesquisadores se atentam à diferenciação entre extremismo e fundamentalismo (p. 115). De acordo com a proposição, diferentemente da aplicação recorrente, o conceito de fundamentalismo não estaria relacionado à incitação da violência; antes, estaria relacionado a uma interpretação bíblica literal, preservando as narrativas primordiais. Por sua vez, o extremismo faria uso de medida práticas pra punir os que estariam em desacordo com a interpretação bíblica a partir da manipulação da perspectiva fundamentalista. Os autores fazem uso de reflexão feita por Émile Durkheim sobre “períodos frios” (p. 112) para pensar na sociedade e política no Brasil contemporâneo. Segundo a concepção, em períodos de transformação social, onde o tradicional e histórico podem ser Volume 11, Número 3, dezembro de 2017 | Página 272 Volume 11, Número 3, dezembro de 2017 | Página 274 Tanto no cenário nacional, quanto no estadual, os pesquisadores apontam o posicionamento do pastor neopentecostal Silas Malafaia que, apesar de nunca ter se candidatado a nenhum cargo político, é figura recorrente no assunto, atuando e se posicionando em relação a assuntos, partidos e candidatos. O livro traz, ainda, uma Volume 11, Número 3, dezembro de 2017 | Página 273 entrevista do pastor nos anexos, assim como artigos das pesquisadoras Magali do Nascimento Cunha e Tatiane dos Santos Duarte. entrevista do pastor nos anexos, assim como artigos das pesquisadoras Magali do Nascimento Cunha e Tatiane dos Santos Duarte. A publicação, portanto, apresenta contribuições essenciais para a compreensão do panorama político e social que se estabelece no Brasil contemporâneo, ajudando a lançar um olhar para o futuro a partir das eleições 2014. O primeiro passo dos evangélicos em direção ao executivo já foi dado e se torna um anseio possível em um contexto polarizado e difuso, onde as conquistas de direitos pelas minorias provoca um sentimento de ameaça aos setores conservadores da sociedade. Por isso, o cenário propicia ações extremistas de atores e lideranças a partir do estímulo dos medos sociais. O livro ressalta a importância de não atribuir perfis preestabelecidos a grupos religiosos específicos. Antes, define tipos de ações que são utilizadas por diferentes atores, independente de perfis socioeconômicos, escolhas políticas e opções religiosas. Contudo, os evangélicos estão cada vez mais presentes na sociedade e política brasileiras e já foi dada a largada para a passagem de “ovelha” a “player” do jogo político.
https://openalex.org/W3092341819
https://eprints.whiterose.ac.uk/165782/8/VoR.pdf
English
null
On the estimation of entropy in the FastICA algorithm
Journal of Multivariate Analysis/Journal of multivariate analysis
2,021
cc-by
15,822
∗Corresponding author. E-mail address: mmpws@leeds.ac.uk (P. Smith). a r t i c l e i n f o Article history: Received 3 June 2019 Received in revised form 21 September 2020 Accepted 21 September 2020 Available online 9 October 2020 AMS 2010 subject classifications: primary 62-04 secondary 65C60 Keywords: Approximation Blind source separation Convergence Counterexample FastICA Independent component analysis Projection pursuit Projections Article history: Received 3 June 2019 Received in revised form 21 September 2020 Accepted 21 September 2020 Available online 9 October 2020 The fastICA method is a popular dimension reduction technique used to reveal patterns in data. Here we show both theoretically and in practice that the approximations used in fastICA can result in patterns not being successfully recognised. We demonstrate this problem using a two-dimensional example where a clear structure is immediately visible to the naked eye, but where the projection chosen by fastICA fails to reveal this structure. This implies that care is needed when applying fastICA. We discuss how the problem arises and how it is intrinsically connected to the approximations that form the basis of the computational efficiency of fastICA. AMS 2010 subject classifications: primary 62-04 secondary 65C60 © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). y Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Keywords: Approximation Blind source separation Convergence Counterexample FastICA Independent component analysis Projection pursuit Projections https://doi.org/10.1016/j.jmva.2020.104689 0047-259X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4 0/) E-mail address: mmpws@leeds.ac.uk (P. Smith). https://doi.org/10.1016/j.jmva.2020.104689 0047-259X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). https://doi.org/10.1016/j.jmva.2020.104689 0047-259X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). Journal of Multivariate Analysis 181 (2021) 104689 Journal of Multivariate Analysis 181 (2021) 104689 1. Introduction In particular, we need to understand both how well the contrast function estimates entropy and the performance of the optimisation procedure. The main strength of the fastICA method is its speed, which is considerably higher than many other methods. Furthermore, if the data is a mixture of a small number of underlying factors, fastICA is often able to correctly identify these factors. However, fastICA also has some drawbacks, which have been pointed out in the literature. Learned-Miller and Fisher [16] use test problems from Bach and Jordan [2] with performance measured by the Amari error [1] to compare fastICA to other ICA methods. They find that these perform better than fastICA on many examples. Focussing on a different aspect, Wei [23] investigates issues with the convergence of the iterative scheme employed by fastICA to optimise the contrast function. In Wei [24] it is shown that the two most common fastICA contrast functions fail to de-mix certain bimodal distributions with Gaussian mixtures, although some other contrast function choices (related to classical kurtosis estimation) may give reliable results within the fastICA framework. ) y g In this article we identify and discuss a more fundamental problem with fastICA. We demonstrate that the approxi- mations used in fastICA can lead to a contrast function where the optimal points no longer correspond to directions of low entropy. To illustrate the effect discussed in this paper, we consider the example shown in Fig. 1. In this example, two- dimensional samples are generated from a two-dimensional normal distribution, conditioned on avoiding a collection of parallel bands (see Section 5 for details). This procedure produces a pattern which is obvious to the bare eye, and indeed the projection which minimises entropy (solid lines) exposes this pattern. In contrast, the fastICA contrast function seems unable to resolve the pattern of bands and prefers a direction with higher entropy which does not expose the obvious pattern (dotted lines). We remark that this failure by fastICA to recover the obvious structure is relatively robust in this example. Changing the parameters used in the fastICA method does not significantly change the outcome, and the underlying structure is still lost. It is also worth mentioning here that the example dataset was very simple to obtain and no optimisation was performed to make the fastICA method perform poorly. To obtain the projection indicated by the solid lines in Fig. 1. Introduction Independent Component Analysis (ICA) is a well-established and popular dimension reduction technique that finds an orthogonal projection of data onto a lower-dimensional space, while preserving some of the original structure. ICA is also used as a method for blind source separation and is closely connected to projection pursuit. We refer the reader to Hyvärinen et al. [13], Hyvärinen [12] and Stone [21] for a comprehensive overview of the mathematical principles underlying ICA and its applications in a wide variety of practical examples. In ICA, the projections that are determined to be ‘‘interesting’’ are those that maximise the non-Gaussianity of the data, which can be measured in several ways. One quantity for this measurement that is used frequently in the ICA literature is entropy. For distributions with a given variance, the Gaussian distribution is the one which maximises entropy, and all other distributions have strictly smaller entropy. Therefore, our aim is to find projections which minimise the entropy of the projected data. Different methods are available for both the estimation of entropy and the optimisation procedure, and have different speed–accuracy trade-offs. A widely used method to perform ICA in higher dimensions is fastICA [14]. This method has found applications in areas as wide ranging as facial recognition [6], epileptic seizure detection [26] and fault detection in wind turbines [7]. Recent works on extensions of the algorithm can be seen in Miettinen et al. [18], Ghaffarian and Ghaffarian [8] and He et al. [10]. The fastICA method uses a series of substitutions and approximations of the projected density and its entropy. It then applies an iterative scheme for optimising the resulting contrast function (which is an approximation to negentropy). glio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 Scatter plot of original data with densities of the projected data in the direction obtained by m-spacing ICA (solid line) and fastICA (dotted Kernel density estimation was used to obtain the marginal densities shown. Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Fig. 1. Scatter plot of original data with densities of the projected data in the direction obtained by m-spacing ICA (solid line) and fastICA (dotted line). Kernel density estimation was used to obtain the marginal densities shown. Because of its popularity in many areas, analysis and evaluation of the strengths and weaknesses of the fastICA algorithm is crucially important. 1. Introduction 1 we used the m-spacing method [3] for entropy estimation, combined with a standard optimisation technique. The m-spacing entropy approximation is shown to be consistent in Hall [9] and converges to true entropy for large sample size. While the m-spacing entropy approximation theoretically makes for an excellent contrast function for use in ICA, it is relatively slow to evaluate. 2 2 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 Fig. 2. Plots showing an estimate of the density of the projected data (solid line), and the surrogate density f0 used in the fastICA method (dotted line), for two different projections of the data. Panel 2(a) corresponds to the direction of highest entropy, found using m-spacing, and Panel 2(b) corresponds to the direction found by fastICA. These two directions are shown by the solid line and dotted line respectively in Fig. 1. Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Fig. 2. Plots showing an estimate of the density of the projected data (solid line), and the surrogate density f0 used in the fastICA method (dotted line), for two different projections of the data. Panel 2(a) corresponds to the direction of highest entropy, found using m-spacing, and Panel 2(b) corresponds to the direction found by fastICA. These two directions are shown by the solid line and dotted line respectively in Fig. 1. Our main theoretical contribution helps explain why fastICA in the example in Fig. 1 performs poorly. To obtain the contrast function in the fastICA method a surrogate to the true density is first obtained, and then it is approximated through several steps to increase computational speed. In Section 4 we show convergence results for the approximation steps, and conclude that the accuracy loss occurs at the initial stage where the real density is replaced by the surrogate one. This is highlighted in Fig. 2(a), which shows the estimated density (solid line) along the direction that exposes the pattern (i.e. the solid line in Fig. 1). The dotted line shows the surrogate density in this same direction that the fastICA method uses in its approximation to entropy. Fig. 2(b) shows the analogue for the direction found by fastICA (i.e. the dotted line in Fig. 1). This figure motivates this paper by highlighting the area where the approximations used in fastICA diverge from the true values. 2. Entropy and negentropy The aim of the fastICA method is to efficiently find a projection of given data which minimises entropy. Suppose we have a one-dimensional random variable X with density f : R →[0, ∞). Then the entropy H of the distribution of X is defined to be H[f ] := − ∫ R f (x) log f (x) dx, (1) whenever this integral exists. We use square brackets to indicate that H is a functional, taking the function f as its argument. In the special case of a Gaussian random variable with variance σ 2, the entropy can be calculated explicitly and it takes the value η(σ 2) given by η(σ 2) := 1 2 ( 1 + log(2πσ 2)) . (2) It is known that this is an upper bound for entropy, namely the entropy of any random variable with variance σ 2 will belong to the interval (−∞, η(σ 2)] [see, for example,4]. The negentropy J is defined as It is known that this is an upper bound for entropy, namely the entropy of any random variable with variance σ 2 will belong to the interval (−∞, η(σ 2)] [see, for example,4]. The negentropy J is defined as It is known that this is an upper bound for entropy, namely the entropy of any random variable with variance σ 2 will belong to the interval (−∞, η(σ 2)] [see, for example,4]. The negentropy J is defined as J[f ] := η(σ 2) −H[f ], J[f ] := η(σ 2) −H[f ], where η(σ 2) is given by (2). This implies that J[f ] ∈[0, ∞). Negentropy is zero when the density is Gaussian, and strictly greater than zero otherwise. where η(σ 2) is given by (2). This implies that J[f ] ∈[0, ∞). Negentropy is zero when the density is Gaussian, and strictly greater than zero otherwise. where η(σ 2) is given by (2). This implies that J[f ] ∈[0, ∞). Negentropy is zero when the density is Gaussian, and strictly greater than zero otherwise. As the definition of entropy involves the integral of the density, the estimation of entropy or negentropy from data is non-trivial. For a survey of different methods to estimate entropy from data, see Beirlant et al. [3]. As an example, we consider here the m-spacing estimator, originally given in Vasicek [22]. Suppose we have a sample of one-dimensional points, y1, y2, . . . 1. Introduction The two densities in Fig. 2(a) are very different to one-another, and this error propagates through the fastICA method to the approximation used for entropy. Note that the solid lines in Fig. 2 show the same estimated densities as given in Fig. 1. This paper is structured as follows. In Section 2, entropy and negentropy are introduced alongside associated estimates. In Section 3 we describe the fastICA method. Section 4 contains some proofs which help to understand where errors are introduced in the fastICA method. Section 5 contains details on the example given in Fig. 1. Some concluding remarks can be found in Section 6. The code to produce the figures in this paper can be found at https://github.com/pws3141/fastICA_ code. 2. Entropy and negentropy , yn ∈R, from a distribution with density f , and y(1), y(2), . . . , y(n) is the ordering such that y(1) ≤y(2) ≤ 3 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss · · · ≤y(n). Define the m-spacing difference to be ∆myi = y(i+m) −y(i) for m ∈{3, . . . , n −1} and i ∈{1, 2, . . . , n −m}. The m-spacing approximation for entropy H[f ] for the sample y = (y1, y2, . . . , yn) is given by · · · ≤y(n). Define the m-spacing difference to be ∆myi = y(i+m) −y(i) for m ∈{3, . . . , n −1} and i ∈{1, 2, . . . , n −m}. The m-spacing approximation for entropy H[f ] for the sample y = (y1, y2, . . . , yn) is given by · · · ≤y(n). Define the m-spacing difference to be ∆myi = y(i+m) −y(i) for m ∈{3, . . . , n −1} and i ∈{1, 2, . . . , n −m}. The m-spacing approximation for entropy H[f ] for the sample y = (y1, y2, . . . , yn) is given by Hm,n(y) = 1 n n−m ∑ i=1 log ( n m ∆myi ) −𭟋(m) + log(m), Hm,n(y) = 1 n n−m ∑ i=1 log ( n m ∆myi ) −𭟋(m) + log(m), (3) (3) where 𭟋(x) = −d dxΓ (x) is the digamma function. This is a realisation of the general m-spacing formula given in Hall [9]. This approximation tends to the true value of entropy under certain conditions and so for a ‘‘large enough’’ number of points should be comparable to the true value. This method has been used previously within an ICA method by Learned- Miller and Fisher [16]. While the methods provide consistent estimates for the entropy, it is computationally expensive. The main contribution to computational cost comes from the need to sort the sample y in increasing order. where 𭟋(x) = −d dxΓ (x) is the digamma function. This is a realisation of the general m-spacing formula given in Hall [9]. This approximation tends to the true value of entropy under certain conditions and so for a ‘‘large enough’’ number of points should be comparable to the true value. 2. Entropy and negentropy This method has been used previously within an ICA method by Learned- Miller and Fisher [16]. While the methods provide consistent estimates for the entropy, it is computationally expensive. The main contribution to computational cost comes from the need to sort the sample y in increasing order. The fastICA method provides a more efficient way to estimate negentropy J[f ] by using a series of approximations and substitutions both for f and for J[·] to obtain a surrogate for negentropy J[f ] which is then subsequently maximised. The reason behind these substitutions is to reduce computational cost, but the drawback is that the resulting approximation may be very different from the true contrast function. where ˆJ∗is an approximation to negentropy, given in Eq. (11). where ˆJ∗is an approximation to negentropy, given in Eq. (11). If more than one loading is required, Step (ii) is repeated for each subsequent new direction, with the added constraint that w must be orthogonal to the previously found directions. This can be implemented within the fastICA framework using Gram–Schmidt orthogonalisation [14, Section 6.2]. This is known as the deflation fastICA method. There is also a parallel fastICA method that finds all loadings concurrently, although in this paper we only consider the deflation approach. In the literature regarding fastICA it is often the convergence of the iterative method to solve (4) that is examined. It can be shown, for example in Wei [23], that in certain situations this iterative step fails to find a good approximation for w∗. In contrast, here we consider the mathematical substitutions and approximations used in the derivation of ˆJ∗(Dw). Assumption 1 introduces the technical assumptions given in Hyvärinen [11, Sections 4 and 6], using slightly adjusted notation. Assumption 1. Let Gi, i ∈{1, . . . , I} be functions that do not grow faster than quadratically. Let ϕ(·) denote the density of a standard Gaussian random variable and assume that there are αi, βi, γi, δi, i ∈{1, . . . , I}, such that the functions Ki(x) := Gi(x) + αix2 + βix + γi δi (5) satisfy satisfy ∫ R Ki(x)Kj(x)ϕ(x) dx = 1{i=j}; (6a) ∫ R Ki(x)xkϕ(x) dx = 0, k ∈{0, 1, 2}, (6b) for i, j ∈{1, . . . , I}, where 1{i=j} = 1 if i = j and zero otherwise. ∫ R Ki(x)Kj(x)ϕ(x) dx = 1{i=j}; (6a) ∫ R Ki(x)xkϕ(x) dx = 0, k ∈{0, 1, 2}, (6b) for i, j ∈{1, . . . , I}, where 1{i=j} = 1 if i = j and zero otherwise. ∫ R Ki(x)Kj(x)ϕ(x) dx = 1{i=j}; ∫ R Ki(x)xkϕ(x) dx = 0, k ∈{0, 1, 2}, for i, j ∈{1, . . . , I}, where 1{i=j} = 1 if i = j and zero otherwise. ∫ R Ki(x)Kj(x)ϕ(x) dx = 1{i=j}; (6a) ∫ R Ki(x)xkϕ(x) dx = 0, k ∈{0, 1, 2}, (6b) for i, j ∈{1, . . . , I}, where 1{i=j} = 1 if i = j and zero otherwise. Assumption 1. Let Gi, i ∈{1, . . . , I} be functions that do not grow faster than quadratically. Let ϕ(·) denote the density of a standard Gaussian random variable and assume that there are αi, βi, γi, δi, i ∈{1, . . . , I}, such that the functions 3. The fastICA algorithm In this section we describe the fastICA method of Hyvärinen and Oja [14]. The theory behind this method was originally introduced in Hyvärinen [11], although here we adjust the notation to match the rest of this paper. We will mention explicitly where our notation differs from Hyvärinen [11] and Hyvärinen and Oja [14]. We will write ‘fastICA’ when we are discussing the theoretical method, and ‘fastICA’ when we are discussing the R implementation from the fastICA CRAN package [17]. p g [ ] The fastICA method to obtain the first loading from data ˜D ∈Rnטp follows the steps given below. Following the usual convention, the rows of ˜D denote observations, the columns denote variables. (i) Whiten the data to obtain D ∈Rn×p with p = min(˜p, n −1), such that 1 n−1D⊤D = Ip [14, Se (ii) Iteratively find the optimal projection w∗, given by (ii) Iteratively find the optimal projection w∗, given by (4) w∗= argmax w∈Rp, w⊤w=1 ˆJ∗(Dw), w∗= argmax w∈Rp, w⊤w=1 ˆJ∗(Dw), w∈Rp, w⊤w=1 where ˆJ∗is an approximation to negentropy, given in Eq. (11). where ˆJ∗is an approximation to negentropy, given in Eq. (11). Use Monte-Carlo approximation for the expectations in (10), i.e. use has density f , not f0. In Proposition 11 we will show that J[f0] J[f0]. 4. Use Monte-Carlo approximation for the expectations in (10), i.e. use y f , f0. p J[f0] J[f0]. 4. Use Monte-Carlo approximation for the expectations in (10), i.e. use rlo approximation for the expectations in (10), i.e. use se Monte-Carlo approximation for the expectations in (10), i.e. use ˆJ∗(y) = (1 n n ∑ j=1 G(yj) −1 L L ∑ j=1 G(zj) )2 , ˆJ∗(y) = (1 n n ∑ j=1 G(yj) −1 L L ∑ j=1 G(zj) )2 , (11) (11) where z1, . . . , zL are samples from a standard Gaussian and L is large. Here ˆJ∗(y) ≈CˆJ[ˆf0]. The restriction to I = 1 here removes a summation from Step 1. and Step 2. therefore simplifying Step 3. and the associated estimations in Step 4. Theoretically these steps can be completed for arbitrary I ∈N, although in this case a closed-form version equivalent to Step 3. is much more complicated. q p p The approximation (11) to the negentropy used in fastICA dramatically decreases the computational time needed to find ICA projections. Unlike the m-spacing estimator introduced in Section 2, the approximation ˆJ∗(Dw) is a simple Monte- Carlo estimator and does not require sorting of the data. The algorithm to solve (4) also benefits from the fact that an approximate derivative of w ↦→ˆJ∗(Dw) can be derived analytically. pp J ( ) y y The steps in this chain of approximations are illustrated in Fig. 3 and we will investigate the approximation more formally in Section 4. In Step 1. of the procedure, we do not obtain a proper approximation, but have an inequality instead: f is replaced with a density f0 such that J[f0] ≤J[f ]. As a result, the w which maximises J[f0] can be very different from the one which maximises J[f ]. In contrast, Steps 2. and 3. are proper approximations and in Section 4 we prove convergence of ˆf0 to f0 for Step 2. and of ˆJ[ˆf0] to J[f0] for Step 3. in the limit ∥c∥→0, where c = (c1, . . . , cI). Step 4. is a simple Monte-Carlo approximation exhibiting well-understood behaviour. where ˆJ∗is an approximation to negentropy, given in Eq. (11). ∫ R Ki(x)Kj(x)ϕ(x) dx = 1{i=j}; (6a) (6b) The functions Gi are given as ¯Gi in Hyvärinen [11] and as Gi in Hyvärinen and Oja [14]. The functions Ki are described in Hyvärinen [11, Section 6] and are called Gi there. The fastICA algorithm only implements the case I = 1. In this case, the function G1 can be chosen nearly arbitrarily so long as it does not grow faster than quadratically: It is easy to show that for every G which is not exactly equal to a second order polynomial, a function K1 can be found that satisfies the conditions given in (6) by choosing suitable α1, E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 β1, γ1 and δ1. For general I ∈N, specific Gi, i ∈{1, . . . , I} must be chosen for the conditions (6) to hold. With I = 2, the functions G1(x) = x3 and G2(x) = x4 are proposed in the literature [11, Section 7] and seem to be useful in practice, even though these functions violate the growth condition from Assumption 1. We have not found any examples of specific functions Gi that satisfy (6) for I > 2 in the fastICA literature. β1, γ1 and δ1. For general I ∈N, specific Gi, i ∈{1, . . . , I} must be chosen for the conditions (6) to hold. With I = 2, the functions G1(x) = x3 and G2(x) = x4 are proposed in the literature [11, Section 7] and seem to be useful in practice, even though these functions violate the growth condition from Assumption 1. We have not found any examples of specific functions Gi that satisfy (6) for I > 2 in the fastICA literature. Let w ∈Rp with ∥w∥= 1 and let y = (y1, y2, . . . , yn) = Dw ∈Rn be the data projected onto w. Since the data has been whitened, y has sample mean 0 and sample variance 1. Further, let f : R →R be the unknown density of the population-level-whitened and projected data. Then f satisfies ∫ f (x) dx = 1, ∫ x f (x) dx = 0 and ∫ x2 f (x) dx = 1. We need to estimate the negentropy J[f ] using the data y1, . . . , yn. where ˆJ∗is an approximation to negentropy, given in Eq. (11). Define ci := Ef Ki(X) = ∫ f (x)Ki(x) dx (7) for all i ∈{1, . . . , I}. For I = 1, setting K(x) := K1(x), G(x) := G1(x) and c := c1, the derivation of the contrast function used in the fastICA method then consists of the following steps: for all i ∈{1, . . . , I}. For I = 1, setting K(x) := K1(x), G(x) := G1(x) and c := c1, the derivation of the contrast function used in the fastICA method then consists of the following steps: 1. Replace f by a density f0 given by κx + ζx2 + aK(x) ) , (8) f0(x) = A exp ( κx + ζx2 + aK(x) ) , (8) for all x ∈R. The constants A, κ, ζ and a are chosen to minimise negentropy (and hence maximise entropy) under the constraints ∫ f0(x)K(x) dx = c. In Proposition 3 we will show that J[f0] ≤J[f ]. 2 A i f b ˆf d fi d for all x ∈R. The constants A, κ, ζ and a are chosen to minimise negentropy (and hence maximise entropy) under the constraints ∫ f0(x)K(x) dx = c. In Proposition 3 we will show that J[f0] ≤J[f ]. ˆ ∫ 2. Approximate f0 by ˆf0 defined as ˆf0(x) = ϕ(x)( 1 + cK(x)) (9) for all x ∈R. In Theorem 8 we will show J[ˆf0] ≈J[f0]. 3 A i J[ˆf ] b d d T l i for all x ∈R. In Theorem 8 we will show J[f0] J[f0]. 3. Approximate J[ˆf0] by second order Taylor expansion, ˆJ[ˆf0] = 1 C ( Ef G(Y) −EϕG(Z))2, (10) where Y is a random variable with density f , Z ∼N(0, 1), and C some constant. Note that, maybe surprisingly, Y has density f , not f0. In Proposition 11 we will show that ˆJ[ˆf0] ≈J[ˆf0]. where Y is a random variable with density f , Z ∼N(0, 1), and C some constant. Note that, maybe surprisingly, Y has density f , not f0. In Proposition 11 we will show that ˆJ[ˆf0] ≈J[ˆf0]. 4. Use Monte-Carlo approximation for the expectations in (10), i.e. use where Y is a random variable with density f , Z ∼N(0, 1), and C some constant. Note that, maybe surprisingly, Y has density f , not f0. In Proposition 11 we will show that ˆJ[ˆf0] ≈J[ˆf0]. 4. where ˆJ∗is an approximation to negentropy, given in Eq. (11). From the above discussion, it seems sensible to surmise that the loss of accuracy in fastICA is due to the surrogate used in Step 1. above. y g p We conclude this section with a few simple observations: Using (6), (5) and the fact that X and Z are standardised we find y g p n with a few simple observations: Using (6), (5) and the fact that X and Z are standardised we y g p We conclude this section with a few simple observations: Using (6), (5) and the fact that X nd ci = Ef Ki(X) = Ef Ki(X) −EϕKi(Z) = Ef ( Gi(X) + αiX 2 + βiX + γi δi ) −Eϕ ( Gi(Z) + αiZ2 + βiZ + γi δi ) = Ef Gi(X) + αi1 + βi0 + γi δi − EϕGi(Z) + αi1 + βi0 + γi δi = Ef Gi(X) −EϕGi(Z) δi . δi Thus, the fastICA objective function (ignoring the final Monte Carlo approximation) satisfies ˆJ[ˆf0] ∝c2 for the case I = 1, considered above, and ˆJ[ˆf0] ∝∑I i=1 c2 i in the general case. Thus, fastICA can only see the data through the ci. If the data are approximately Gaussian, we have Ef Gi(X) ≈EϕGi(Z) and ci ≈0 for all i and thus ˆJ[ˆf0] ≈0, but the opposite implication does not hold. This is in contrast to the true negentropy, which satisfies J[f ] = 0 if and only if f is Gaussian. 5 Journal of Multivariate Analysis 181 (2021) 104689 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Fig. 3. Approximations used in fastICA: The fastICA contrast function ˆJ∗(y) is used in place of negentropy J[f ]. Note that the first step involves an inequality rather than an approximation. Fig. 3. Approximations used in fastICA: The fastICA contrast function ˆJ∗(y) is used in place of negentropy J[f ]. Note that the first step involves an inequality rather than an approximation. A first consequence of this argument is that projections where the true distribution is Gaussian will look ‘uninteresting’ to fastICA: for these directions w the objective function ˆJ∗(Dw) will be small and the search for the maximum in (4) will be driven away from these directions. where ˆJ∗is an approximation to negentropy, given in Eq. (11). This is particularly relevant since for high dimensional data, where the search volume is vast, projections along most directions are close to Gaussian [5,25], so fastICA will be able to exclude much of the search volume. Conversely, if ˆJ[ˆf0] and thus ∥c∥is large, the projected density f is not Gaussian and by maximising (an approximation to) ˆJ[ˆf0], the fastICA method will find directions which are ‘interesting’. But the above discussion also shows that optima can be missed when ˆJ[ˆf0] is small, but the projected density f is still far from Gaussian. This is the case we are concerned with in this paper and thus we assume ∥c∥≈0 when we consider the fastICA approximations in detail in the next section. 4. Approximations used in the fastICA method In this section, we investigate the validity of the approximation given in Section 3. We consider Step 1. in Proposition 3, Step 2. in Theorem 8, and Step 3. in Proposition 11. Throughout this section, we consider arbitrary I ∈N for completeness. In this section, we investigate the validity of the approximation given in Section 3. We consider Step 1. in Proposition 3, Step 2. in Theorem 8, and Step 3. in Proposition 11. Throughout this section, we consider arbitrary I ∈N for completeness. We first introduce some assumptions, in addition to Assumption 1, that are required for the mathematics in this section to hold. We first introduce some assumptions, in addition to Assumption 1, that are required for the mathematics in this section to hold. Assumption 2. There exists ε > 0 such that for all h ∈RI with h⊤h < ε, we have Assumption 2. There exists ε > 0 such that for all h ∈RI with h⊤h < ε, we have h⊤K(x) ≥−1 2 (12) for all x ∈R, where K(x) = ( K1(x), K2(x), . . . , KI(x)) . In addition, there exists a function M: R →R such that h⊤K(x) ≥−1 2 (12) for all x ∈R, where K(x) = ( K1(x), K2(x), . . . , KI(x)) . In addition, there exists a function M: R →R such that for all x ∈R, where K(x) = ( K1(x), K2(x), . . . , KI(x)) . In addition, there exists a function M: I∑ i=1 I∑ j=1 I∑ k=1 |Ki(x)Kj(x)Kk(x)| ≤M(x) for all x ∈R, (13a) ∫ R ϕ(x)M(x) dx =: ˜M < ∞. (13b) (13a) ∫ R ϕ(x)M(x) dx =: ˜M < ∞. ∫ R ϕ(x)M(x) dx =: ˜M < ∞. (13b) Note that under the condition that each Gi does not grow faster than quadratically (given in Assumption 1), we can always find some positive constants Bi, i ∈{1, . . . , I} such that (14) |Ki(x)| ≤Bi(1 + x2), for all x ∈R. Note also that for I = 1 the condition given by (12) that there exists an ε > 0 such that for all h ∈[0, ε), we have hK(x) ≥−1/2 is satisfied as follows. Let α, β, γ , δ be parameters for which (6) holds. Then, (6) holds also for α, β, γ , −δ. 4. Approximations used in the fastICA method Moreover, since G does not grow faster than quadratically, αx2 is the dominant term in K(x) as x →±∞. Therefore, to ensure that (12) holds it is enough to choose δ or −δ such that the sign is the same as that of α. 4.1. Step 1 4.1. Step 1 We start our discussion by considering Step 1. of the approximations described in Section 3. We prove that the distribution which maximises entropy for given values of c1, . . . , cI is indeed of the form (8) and thus that we indeed have J[f0] ≤J[f ]. Proposition 3. Let f be the density of the population-level-whitened data projected in some direction (thus with zero mean and unit variance). Recall ci is defined by (7). The density f0 that maximises entropy in the set { g: R →R ; g is a density function, and ∫ R g(x)Ki(x) dx = ci, i ∈{1, . . . , I} } , { g: R →R ; g is a density function, and ∫ R g(x)Ki(x) dx = ci, i ∈{1, . . . , I} } , 6 6 E. Issoglio, P. Smith and J. Voss E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 is given by, f0(x) = A exp ( κx + ζx2 + I∑ i=1 aiKi(x) ) ζx2 + I∑ i=1 aiKi(x) ) (15) f0(x) = A exp ( κx + ζx2 + I∑ i=1 aiKi(x) ) (15) (15) e constants κ, ζ, A and ai, i ∈{1, . . . , I} that depend on ci, i ∈{1, . . . , I}. It follows from this that J for some constants κ, ζ, A and ai, i ∈{1, . . . , I} that depend on ci, i ∈{1, . . . , I}. It follows from this that J[f0] ≤J[f ]. Proof. We use the method of Lagrange multipliers in the calculus of variations [see, for example,15] to find a necessary condition for the density that maximises entropy given the constraints on mean and variance, and in (7). Let F[·]: C2 →R be a functional of the function g: R →R, with g ∈C2, where C2 is the set of all twice continuously differentiable functions. Then, the functional derivative δF/δg: R →R is explicitly defined by ∫ R δF δg (x)φ(x) dx := d dε F[g + εφ] ⏐⏐⏐ ε=0 = lim ε↓0 (F[g + εφ] −F[g] ε ) , (16) (16) for any function φ ∈C2. The right-hand side of (16) is known as the Gâteaux differential dF(g; φ). 4.1. Step 1 Define the inner product of two functions by ⟨g, h⟩:= ∫ R g(x)h(x) dx, with norm ∥g∥L2 := ⟨g, g⟩ 1 2 = (∫ R g(x)2 dx) 1 2 . We want to solve the following system of equations ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ U[g](x) := δ δg H[g] + λ1 δ δg V[g] + λ2 δ δg P[g] + λ3 δ δg Q[g] + ∑I i=1 νi δ δg Ri[g] = 0; V[g] = 0; P[g] = 0; Q[g] = 0; Ri[g] = 0, where λ1, λ2, λ3, νi are some real numbers, i ∈{1, . . . , I}, H[g] is entropy as given in (1), and V[g] := Var[g] −1 = ∫ R g(x)x2 dx − (∫ R g(x)x dx )2 −1; P[g] := ∫ R g(x) dx −1; Q[g] := ∫ R g(x)x dx; Ri[g] := ∫ R g(x)Ki(x) dx −ci. Using (1) and (16) the term with H gives, Using (1) and (16) the term with H gives, ⟨δH δg , φ⟩= −d dε ∫( g(x) + εφ(x)) log( g(x) + εφ(x)) dx ⏐⏐⏐ ε=0 = − ∫( g(x) φ(x) g(x) + εφ(x) + φ(x) log( g(x) + εφ(x)) + εφ(x) φ(x) g(x) + εφ(x) ) dx ⏐⏐⏐ ε=0 = − ∫( 1 + log g(x)) φ(x) dx = ⟨−1 −log g(x), φ⟩. H g , φ⟩= −d dε ∫( g(x) + εφ(x)) log( g(x) + εφ(x)) dx ⏐⏐⏐ ε=0 = − ∫( g(x) φ(x) g(x) + εφ(x) + φ(x) log( g(x) + εφ(x)) + εφ(x) φ(x) g(x) + εφ(x) ) dx ⏐⏐⏐ ε=0 ∫ = − ∫( 1 + log g(x)) φ(x) dx = ⟨−1 −log g(x), φ⟩. Now, looking at V[g] and using the constraint Q[g] = 0 we get, Now, looking at V[g] and using the constraint Q[g] = 0 we get, Now, looking at V[g] and using the constraint Q[g] = 0 we get, ⟨δV δg , φ⟩= d dε (∫( g(x) + εφ(x)) x2 dx − (∫( g(x) + εφ(x)) x dx )2 −1 )⏐⏐⏐⏐ ε=0 = ∫ φ(x)x2 dx −2 (∫ φ(x)x dx · ∫ g(x)x dx ) = ⟨x2, φ⟩−2⟨x, φ⟩· Q[g] = ⟨x2, φ⟩. Let L[·]: C2 →R be of the form L[g] = ∫ g(x)l(x) dx −k for some function l: R →R, and some constant k ∈R. 4.1. Step 1 Then it is easy to check that ⟨δL δg , φ⟩= ⟨l, φ⟩and therefore δP δg = 1, δQ δg = x and δRi δg = Ki. Putting this into the equation for U[g], we have U[g](x) = −1 −log g(x) + λ1 + λ2x2 + λ3x + I∑ i=1 νiKi(x). U[g](x) = −1 −log g(x) + λ1 + λ2x2 + λ3x + I∑ i=1 νiKi(x). Setting U[g] = 0 and solving for g gives, g(x) = f0(x) = exp[λ1 −1 + λ2x2 + λ3x + ∑I i=1 νiKi(x)] which is (15) with A = exp(λ1 −1), κ = λ3, ζ = λ2 and ai = νi, i = 1, . . . , I. Note that the constants A, κ, ζ, and ai depend on ci indirectly through the constraints on the Ki expressed as Ri[g] = 0. □ Remark 4. It is possible to specify a density f such that in some limit, H[f ] →∞whilst H[f0] remains bounded and thus ⏐⏐J[f ] −J[f0] ⏐⏐→∞, with f0 the density given in (8). That is, in Step 1. of the fastICA method given in Section 3, E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Journal of Multiva E. Issoglio, P. Smith and J. Voss the difference between the true negentropy and the surrogate negentropy can be arbitrarily large. For example, set the density f to be a mixture of two independent uniform densities, i.e. the difference between the true negentropy and the surrogate negentropy can be arbitrarily large. For example, set the density f to be a mixture of two independent uniform densities, i.e. f (x) = 1 2 ( g(x; −1 −ε, −1) + g(x; 1, 1 + ε)) f (x) = 1 2 ( g(x; −1 −ε, −1) + g(x; 1, 1 + ε)) f (x) = 1 2 ( g(x; −1 −ε, −1) + g(x; 1, 1 + ε)) f (x) = 1 2 ( g(x; −1 −ε, −1) + g(x; 1, 1 + ε)) where ε ∈R and g(· ; a, b) is the density function of a Uniform distribution in the interval [a, b]. 4.2. Step 2 We now switch our attention to Step 2. of the approximations. As discussed in Section 3, we consider the case where c →0. The first step of our analysis is to identify the behaviour of the constants in the definition of f0 as c →0. We then prove some auxiliary results before concluding our discussion of Step 2. in Theorem 8. Proposition 5. Suppose Assumption 1 is satisfied, and let A, κ, ζ, a1, . . . , aI be defined as in Proposition 3, as functions of c. Then ition 5. Suppose Assumption 1 is satisfied, and let A, κ, ζ, a1, . . . , aI be defined as in Proposition 3, e Assumption 1 is satisfied, and let A, κ, ζ, a1, . . . , aI be defined as in Proposition 3, as functions of c A − 1 √ 2π = O(∥c∥2), κ = O(∥c∥2), ζ + 1 2 = O(∥c∥2), ai −ci = O(∥c∥2), i ∈{1, . . . , I}, A − 1 √ 2π = O(∥c∥2), κ = O(∥c∥2), ζ + 1 2 = O(∥c∥2), ai −ci = O(∥c∥2), i ∈{1, . . . , I}, as ∥c∥→0. as ∥c∥→0. Proof. Define x = (c1, . . . , cI)⊤∈RI and y = (A, κ, ζ, a1, . . . , aI)⊤∈RI+3. Furthermore, let F: R by Proof. Define x = (c1, . . . , cI)⊤∈RI and y = (A, κ, ζ, a1, . . . , aI)⊤∈RI+3. Furthermore, let F: RI × RI+3 →RI+3 be given by F(x, y) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ ∫ f0(x) dx −1 ∫ f0(x)x dx ∫ f0(x)x2 dx −1 ∫ f0(x)K1(x) dx −c1 ... ∫ f0(x)KI(x) dx −cI ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ , F(x, y) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ ∫ f0(x) dx −1 ∫ f0(x)x dx ∫ f0(x)x2 dx −1 ∫ f0(x)K1(x) dx −c1 ... ∫ f0(x)KI(x) dx −cI ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ , F(x, y) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ ∫ f0(x) dx −1 ∫ f0(x)x dx ∫ f0(x)x2 dx −1 ∫ f0(x)K1(x) dx −c1 ... ∫ f0(x)KI(x) dx −cI ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ , where f0 is given in (15) and Ki in (5). Then, for the points x1 = (0, . . . , 0)⊤and y1 = ( 1 √ 2π , 0, −1 2, 0, . . . , 0)⊤, we have F(x1, y1) = 0. where f0 is given in (15) and Ki in (5). 4.1. Step 1 Also, Ef Ki(x) =: ci →1 2 ( Ki(−1) + Ki(1)) , (17) Ef Ki(x) =: ci →1 2 ( Ki(−1) + Ki(1)) , (17) as ε →0. With f0 as in (8), as ε →0. With f0 as in (8), ci = ∫ Ki(x)f0(x) dx, (18) d, ci = ∫ Ki(x)f0(x) dx, (18) and, and, H[f0] = ∫ f0(x) log(A) dx + ∫ f0(x)( ηx + κx2 + I∑ i=1 aiKi(x)) dx = log(A) + ηEf0X + κEf0X 2 + I∑ i=1 aiEf0Ki(x) = log(A) + κ + I∑ i=1 aici. H[f0] = ∫ f0(x) log(A) dx + ∫ f0(x)( ηx + κx2 + I∑ i=1 aiKi(x)) dx = log(A) + ηEf0X + κEf0X 2 + I∑ i=1 aiEf0Ki(x) = log(A) + κ + I∑ i=1 aici. Therefore, for H[f0] to be unbounded from below as ε →0 we would require some κ →−∞, ai →−∞or A →0, as ci is bounded by (17) and Assumption 2. However, this cannot occur whilst f0 satisfies (18). Therefore, for H[f0] to be unbounded from below as ε →0 we would require some κ →−∞, ai →−∞or A →0, as ci is bounded by (17) and Assumption 2. However, this cannot occur whilst f0 satisfies (18). 4.2. Step 2 4.1. Step 1 Then we have expectation and variance given by where ε ∈R and g(· ; a, b) is the density function of a Uniform distribution in the interval [a, b]. Then we have expectation and variance given by Ef X = 0; Varf X = 1 + ε + ε2 3 . As the support of g(· ; −1 −ε, −1) is disjoint from that of g(· ; 1, 1 + ε), the entropy is given by, H[f ] = 1 2 ( H[g(· ; −1 −ε, −1)] + H[g(· ; 1, 1 + ε)]) −log(2). We have H[f ] →−∞as ε →0, since f tends to a pair of Dirac deltas. Also, Ef Ki(x) =: ci →1 2 ( Ki(−1) + Ki(1)) , (17) Ef X = 0; Varf X = 1 + ε + ε2 3 . As the support of g(· ; −1 −ε, −1) is disjoint from that of g(· ; 1, 1 + ε), the entropy is given by, Ef X = 0; Varf X = 1 + ε + ε2 3 . As the support of g(· ; −1 −ε −1) is disjoint from that of g(· ; 1 1 + ε) the entropy is given by = 1 + ε + ε2 3 . −ε, −1) is disjoint from that of g(· ; 1, 1 + ε), the entropy is given by, As the support of g(· ; −1 −ε, −1) is disjoint from that of g(· ; 1, 1 + ε), the entropy is given by, As the support of g(· ; −1 −ε, −1) is disjoint from that of g(· ; 1, 1 + ε), the entropy is given by, H[f ] = 1 2 ( H[g(· ; −1 −ε, −1)] + H[g(· ; 1, 1 + ε)]) −log(2). We have H[f ] →−∞as ε →0, since f tends to a pair of Dirac deltas. Also, H[f ] = 1 2 ( H[g(· ; −1 −ε, −1)] + H[g(· ; 1, 1 + ε)]) −log(2). H[f ] = 1 2 ( H[g(· ; −1 −ε, −1)] + H[g(· ; 1, 1 + ε)]) −log(2). We have H[f ] →−∞as ε →0, since f tends to a pair of Dirac deltas. Also, We have H[f ] →−∞as ε →0, since f tends to a pair of Dirac deltas. 4.2. Step 2 Then, for the points x1 = (0, . . . , 0)⊤and y1 = ( 1 √ 2π , 0, −1 2, 0, . . . , 0)⊤, we have F(x1, y1) = 0. 8 8 E. Issoglio, P. Smith and J. Voss E. Issoglio, P. Smith and J. Voss Assuming F is twice differentiable, we use the Implicit Function Theorem around (x1, y1). First, we need to show DyF(x1, y1) is invertible. We have DyF(x1, y1) = ( M 0 0 −II ) , M = ⎛ ⎝ √ 2 0 1 0 1 0 1 0 4 ⎞ ⎠. Therefore, DyF(x1, y1) is non-singular, and so the Implicit Function Theorem holds. There exist some open set U ⊂RI and a unique continuously differentiable function g: U →RI+3 such that g(x1) = y1 and F( x, g(x)) = 0 for all x ∈U. Then, Therefore, DyF(x1, y1) is non-singular, and so the Implicit Function Theorem holds. There exist some open set U ⊂RI and a unique continuously differentiable function g: U →RI+3 such that g(x1) = y1 and F( x, g(x)) = 0 for all x ∈U. Then, Dg(x) = −DyF( x, g(x))−1DxF( x, g(x)) . Dg(x) = −DyF( x, g(x))−1DxF( x, g(x)) . (19) (19) As g is continuous in the set U, there exists some ε > 0, such that for all c ∈U with ∥c∥< ε, g(x1 + c) = y1 + d for some d ∈RI+3. Using Taylor series we can expand g around x1 = 0 ∈RI to obtain g(x1 + c) = g(x1) + Dg(x1) c + O(∥c∥2), and As g is continuous in the set U, there exists some ε > 0, such that for all c ∈U with ∥c∥< ε, g(x1 + c) = y1 + d for some d ∈RI+3. Using Taylor series we can expand g around x1 = 0 ∈RI to obtain g(x1 + c) = g(x1) + Dg(x1) c + O(∥c∥2), and Dg(x1) = d + O(∥c∥2) c . Dg(x1) = d + O(∥c∥2) c . Dg(x1) = d + O(∥c∥2) c . Putting this together with (19) at x = x1 and rearranging gives, Putting this together with (19) at x = x1 and rearranging gives, d = −DyF(x1, y1)−1DxF(x1, y1) c + O(∥c∥2). Now, since d = −DyF(x1, y1)−1DxF(x1, y1) c + O(∥c∥2). d = −DyF(x1, y1)−1DxF(x1, y1) c + O(∥c∥2). 4.2. Step 2 Now, since DxF(x1, y1) = ⎛ ⎜⎝ 0 · · · 0 0 · · · 0 0 · · · 0 II ⎞ ⎟⎠∈R(I+3)×I, one easily obtains that d = ⎛ ⎜⎝ 0 · · · 0 0 · · · 0 0 · · · 0 II ⎞ ⎟⎠c + O(∥c∥2), d = ⎛ ⎜⎝ 0 · · · 0 0 · · · 0 0 · · · 0 II ⎞ ⎟⎠c + O(∥c∥2), and so, and so, y1 + d = ( 1 √ 2π 0 −1 2 c1 · · · cI )⊤ + O(∥c∥2), as c →0. This completes the proof. □ We now define the following functions y(·) and r(·) for future use. Let y: R →R be given by We now define the following functions y(·) and r(·) for future use. Let y: R →R be given by y(x) := κx + (ζ + 1 2 )x2 + I∑ i=1 aiKi(x), (20) and r: R →R given by r(x) := ex −1 −x. (21) y(x) := κx + (ζ + 1 2 )x2 + I∑ i=1 aiKi(x), (20) and r: R →R given by r: R →R given by r(x) := ex −1 −x. (21) r(x) := ex −1 −x. r(x) := ex −1 −x. Using these definitions, we can write f0, given in Proposition 3, as (22) f0(x) = ϕ(x) · √ 2πAey(x). The following lemmas are two technical results needed in the proof of Theorem 8. Lemma 6. Let g: R →R and l: R →R be any functions and h: R →R+ be convex with h(0) = 0. Then, sup x∈R ⏐⏐l(x)h(εg(x)) ⏐⏐≤ε sup x∈R ⏐⏐l(x)h(g(x)) ⏐⏐ for all ε ∈[0, 1]. Proof. As h is convex, for all λ ∈[0, 1] and for all x, y ∈R, we have h( λx + (1 −λ)y) ≤λh(x) + (1 −λ)h(y). Let ε ∈[0, 1]. Then, substituting λ = ε, x = g(x) and y = 0, we have h( ε g(x)) ≤ε h( g(x)) , for all g(x) ∈R, as h(0) = 0. Noticing that h maps to the positive real line allows to conclude. □ Lemma 7. Let r: R →R+ be given as in (21). Then, r(ε y) ≤ε2r(y), for all y ≥0, and for all ε ∈[0, 1]. Lemma 7. Let r: R →R+ be given as in (21). 4.2. Step 2 Then, r(ε y) ≤ε2r(y), for all y ≥0, and for all ε ∈[0, 1]. (23) 9 9 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 Smith and J. Voss Journal of Multivariate Analysis 1 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss E. Issoglio, P. Smith and J. Voss Moreover, for any function l: R →R, we have sup x∈R ⏐⏐⏐l(x)r( ε(1 + x2))⏐⏐⏐≤ε2 sup x∈R ⏐⏐⏐l(x)r(1 + x2) ⏐⏐⏐. Moreover, for any function l: R →R, we have sup x∈R ⏐⏐⏐l(x)r( ε(1 + x2))⏐⏐⏐≤ε2 sup x∈R ⏐⏐⏐l(x)r(1 + x2) ⏐⏐⏐. sup x∈R ⏐⏐⏐l(x)r( ε(1 + x2))⏐⏐⏐≤ε2 sup x∈R ⏐⏐⏐l(x)r(1 + x2) ⏐⏐⏐. Proof. We will use the Taylor expansion of the exponential around 0 for both the left-hand and right-hand side of (23). The left-hand side gives, r(ε y) = exp(ε y) −1 −ε y = ∞ ∑ n=0 εn n! yn −1 −ε y = ε2 ( ∞ ∑ n=2 εn−2 n! yn) , r(ε y) = exp(ε y) −1 −ε y = ∞ ∑ n=0 εn n! yn −1 −ε y = ε2 ( ∞ ∑ n=2 εn−2 n! yn) , absolutely convergent for all εy ∈R r(ε y) = exp(ε y) −1 −ε y = ∞ ∑ n=0 εn n! yn −1 −ε y = ε2 ( ∞ ∑ n=2 εn−2 n! yn) , absolutely convergent for all εy ∈R and the right-hand side of (23) gives, ε2 r(y) = ε2 ( ∞ ∑ n=0 1 n! yn −1 −y ) = ε2 ( ∞ ∑ n=2 1 n! yn) . Putting these two results together, Putting these two results together, r(ε y) −ε2 r(y) = ε2( ∞ ∑ n=2 1 n! yn(εn−2 −1) ) ≤0, r(ε y) −ε2 r(y) = ε2( ∞ ∑ n=2 1 n! yn(εn−2 −1) ) ≤0, as εn −1 ≤0 for all ε ∈[0, 1] and n ∈N+. This proves (23). Let l: R →R be some function Then as r maps to the positive real line and using (23) with y = 1 + x2 we have as εn −1 ≤0 for all ε ∈[0, 1] and n ∈N+. This proves (23). as ε ≤0 o a ε ∈[0, ] a d n ∈N+. s p o es ( 3). Let l: R →R be some function. 4.2. Step 2 Then, as r maps to the positive real line and using (23) with y = 1 + x2, we have |l(x)r( ε(1 + x2)) | ≤ε2|l(x)r(1 + x2)|, for all x ∈R. Taking the supremum over the real line we conclude. □ We now consider the error term between the density f0 that maximises entropy, and its estimate ˆf0. Theorem 8. Suppose we have functions Ki, i ∈{1, . . . , I} that satisfy Assumptions 1 and 2. Let f0 be given as in Proposition 3, and let ˆf0 be given by ˆf0(x) = ϕ(x) ( 1 + I∑ i=1 ciKi(x) ) . Then for all δ < 1/2 sup x∈R ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= O(∥c∥2) as c →0. ˆf0(x) = ϕ(x) ( 1 + I∑ i=1 ciKi(x) ) . hen for all δ < 1/2 sup x∈R ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= O(∥c∥2) as c →0. sup x∈R ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= O(∥c∥2) as c →0. Proof. Let ϕ(x) = (2π)1/2e−x2/2 be the density of a standard Gaussian random variable and let the function g: R →R be defined by Proof. Let ϕ(x) = (2π)1/2e−x2/2 be the density of a standard Gaussian random variable and let the function g: R →R be defined by g(x) := f0(x) −ˆf0(x) ϕ(x) . g(x) := f0(x) −ˆf0(x) ϕ(x) . Then, with y: R →R as defined in (20) and using (22) we get, Then, with y: R →R as defined in (20) and using (22) we get, g(x) = √ 2πA exp( y(x)) −( 1 + I∑ i=1 ciKi(x)) g(x) = √ 2πA exp( y(x)) −( 1 + I∑ i=1 ciKi(x)) = √ 2πA ( exp( y(x)) −1 −y(x) ) + √ 2πA( 1 + y(x)) −(1 + I∑ i=1 ciKi(x)) + √ 2πA( I∑ i=1 ciKi(x) − I∑ i=1 ciKi(x)) . i=1 = √ 2πA ( exp( y(x)) −1 −y(x) ) + √ 2πA( 1 + y(x)) −(1 + I∑ i=1 ciKi(x)) + √ 2πA( I∑ i=1 ciKi(x) − I∑ i=1 ciKi(x)) . 4.2. Step 2 We now multiply both sides by eδx2 and setting ˜δ = 1 2 −δ, so that eδx2ϕ(x) = (2π)−1/2e−˜δx2, we have ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= (2π)−1/2e−˜δx2⏐⏐⏐ √ 2πAr( y(x)) + √ 2πA ( κx + ( ζ + 1 2 ) x2) + ( √ 2πA −1) I∑ i=1 ciKi(x) √ 2 A I∑ ( )K ( ) ( √ 2 A 1) ⏐ T ( ) T ( ) now multiply both sides by eδx2 and setting ˜δ = 1 2 −δ, so that eδx2ϕ(x) = (2π)−1/2e−˜δx2, we have ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= (2π)−1/2e−˜δx2⏐⏐⏐ √ 2πAr( y(x)) + √ 2πA ( κx + ( ζ + 1 2 ) x2) + ( √ 2πA −1) I∑ i=1 ciKi(x) + √ 2πA I∑ i=1 (ai −ci)Ki(x) + ( √ 2πA −1) ⏐⏐⏐≤T1(x) + T2(x) 1 T ( ) T ( ) 1 T ( ) ⏐⏐eδx2( f0(x) −ˆf0(x))⏐⏐= (2π)−1/2e−˜δx2⏐⏐⏐ √ 2πAr( y(x)) + √ 2πA ( κx + ( ζ + 1 2 ) x2) + ( √ 2πA −1) I∑ i=1 ciKi(x) + √ 2πA I∑ i=1 (ai −ci)Ki(x) + ( √ 2πA −1) ⏐⏐⏐≤T1(x) + T2(x) + 1 √ 2π · T3(x) + T4(x) + 1 √ 2π · T5(x), + 1 √ 2π · T3(x) + T4(x) + 1 √ 2π · T5(x), (24) (24) where, where, T1(x) := ⏐⏐Ae−˜δx2r( y(x))⏐⏐; T2(x) := ⏐⏐Ae−˜δx2( κx + (ζ + 1 2 )x2)⏐⏐; T3(x) := ⏐⏐( √ 2πA −1)e−˜δx2 I∑ i=1 ciKi(x) ⏐⏐; T4(x) := ⏐⏐Ae−˜δx2 I∑ i=1 (ai −ci)Ki(x) ⏐⏐; T5(x) := |e−˜δx2( √ 2πA −1)|. If we show that ∥Ti∥∞is at least of order ∥c∥2 as c →0 for i ∈{1, . . . , 5}, then we can conclude the proof by taking the supremum of (24) over x ∈R, which gives, sup x∈R ⏐⏐eδx2(f0(x) −ˆf0(x)) ⏐⏐= O(∥c∥2), sup x∈R ⏐⏐eδx2(f0(x) −ˆf0(x)) ⏐⏐= O(∥c∥2), as c →0. For T1 first note that as c →0. For T1 first note that ⏐⏐e−˜δx2r(y(x)) ⏐⏐≤ max σ∈{−1,1} ⏐⏐e−˜δx2r(σ · |y(x)|) ⏐⏐, for all x ∈R, ⏐⏐e−˜δx2r(y(x)) ⏐⏐≤ max σ∈{−1,1} ⏐⏐e−˜δx2r(σ · |y(x)|) ⏐⏐, for all x ∈R, and thus, sup x∈R |T1(x)| ≤A · sup x∈R σ∈{−1,1} |e−˜δx2r(σ · |y(x)|)|. (25) Next we choose γ such that, q1 := sup x∈R σ∈{−1,1} ⏐⏐e−˜δx2r( σ · γ (1 + x2))⏐⏐< ∞. q1 := sup x∈R σ∈{−1,1} ⏐⏐e−˜δx2r( σ · γ (1 + x2))⏐⏐< ∞. 4.2. Step 2 = √ 2πA ( exp( y(x)) −1 −y(x) ) + √ 2πA( 1 + y(x)) −(1 + I∑ i=1 ciKi(x)) + √ 2πA( I∑ i=1 ciKi(x) − Rearranging this using the function r: R →R given in (21) and by expanding y(x) gives, Rearranging this using the function r: R →R given in (21) and by expanding y(x) gives, (x) = √ 2πA · r( y(x)) + √ 2πA · ( κx + ( ζ + 1 2 ) x2) + (√ 2πA −1) I∑ i=1 ciKi(x) + √ 2πA I∑ i=1 (ai −ci)Ki( g(x) = √ 2πA · r( y(x)) + √ 2πA · ( κx + ( ζ + 1 2 ) x2) + (√ 2πA −1) I∑ i=1 ciKi(x) + √ 2πA I∑ i=1 (ai −ci)Ki(x) + ( √ 2πA −1). Note that the absolute value of g(x) can be bounded by the following terms, |g(x)| ≤ √ 2πA |r( y(x)) | + √ 2πA |κx| + √ 2πA |ζ + 1 2|x2 + √ 2πA ⏐⏐ I∑ i=1 (ai −ci)Ki(x) ⏐⏐+ | √ 2πA −1| ⏐⏐ I∑ i=1 ciKi(x) ⏐⏐+ | √ 2πA −1|. 10 Journal of Multivariate Analysis 181 (2021) 104689 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss We have, |f0(x) −ˆf0(x)| = |ϕ(x) · g(x)| = ϕ(x) ⏐⏐⏐ √ 2πAr( y(x)) + √ 2πA ( κx + ( ζ + 1 2 ) x2) + ( √ 2πA −1) I∑ i=1 ciKi(x) + √ 2πA I∑ i=1 (ai −ci)Ki(x) + ( √ 2πA −1) ⏐⏐⏐. 4.2. Step 2 Voss By Proposition 5, we have A →1/ √ 2π as c →0 and, ε1 = 1 γ (1 2|κ| + |ζ + 1 2| + I∑ |ai|Bi ) = O(∥c∥), as c →0, By Proposition 5, we have A →1/ √ 2π as c →0 and, By Proposition 5, we have A →1/ √ 2π as c →0 and, By Proposition 5, we have A →1/ √ 2π as c →0 and, ε1 = 1 γ (1 2|κ| + |ζ + 1 2| + I∑ i=1 |ai|Bi ) = O(∥c∥), as c →0, and therefore ε2 1 = O(∥c∥2) as c →0, and ∥T1∥∞= O(∥c∥2) as c →0. F T d i il l f T d l k f [0 1] and therefore ε2 1 = O(∥c∥2) as c →0, and ∥T1∥∞= O(∥c∥2) as c →0. For T2 we proceed similarly as for T1 and look for some ε2 ∈[0 1] such that |κx + (ζ + 1)x2| ≤ε2(1 + x2) We have and therefore ε2 1 = O(∥c∥2) as c →0, and ∥T1∥∞= O(∥c∥2) as c →0. For T2 we proceed similarly as for T1, and look for some ε2 ∈[0, 1] such that |κx + (ζ + 1 2)x2| ≤ε2(1 + x2). We have, 1 (∥∥) , ∥1∥∞ (∥∥) T2 we proceed similarly as for T1, and look for some ε2 ∈[0, 1] such that |κx + (ζ + 1 2)x2| ≤ε2(1 ⏐⏐κx + (ζ + 1 2 )x2⏐⏐≤|κ|(1 + x2 2 ) + |ζ + 1 2|(1 + x2) = (1 2|κ| + |ζ + 1 2|) (1 + x2). Setting ε2 := ( 1 2|κ| + |ζ + 1 2|), by Proposition 5, ε2 = O(∥c∥2) as c →0, and thus we can choose c sufficiently small such that ε2 ≤1. Let, Setting ε2 := ( 1 2|κ| + |ζ + 1 2|), by Proposition 5, ε2 = O(∥c∥2) as c →0, and thus we can choose c sufficiently small such that ε2 ≤1. Let, q2 := sup x∈R ⏐⏐e−˜δx2(1 + x2) ⏐⏐< ∞, 2 := sup x∈R ⏐⏐e−˜δx2(1 + x2) ⏐⏐< ∞, where q2 < ∞since e−˜δx2(1 + x2) is continuous and tends to zero in the tails. From this, for ε2 ∈[0, 1] as above, we can apply Lemma 6 and get where q2 < ∞since e−˜δx2(1 + x2) is continuous and tends to zero in the tails. 4.2. Step 2 (26) (26) This is always possible for some γ ∈(−˜δ, ˜δ), as r(0) = 0, and since e−˜δx2r( ±γ (1 + x2)) is continuous and r( ±γ (1 + x2)) grows no faster that eγ x2 as x →±∞, it is beaten by e−˜δx2 in the tails. g , y For y(x) as given in (20) and using (14) we can find an upper bound by |y(x)| ≤|κ| · (1 + x2 2 ) + |ζ + 1 2| · (1 + x2) + I∑ i=1 |ai|Bi(1 + x2) = γ (1 + x2) · 1 γ (1 2|κ| + |ζ + 1 2| + I∑ i=1 |ai|Bi ) =: γ (1 + x2) · ε1, (27) where γ is such that (26) holds. As c →0, we have by Proposition 5, κ →0, ζ →−1/2 and ai →ci. Therefore, we can choose c small enough (and depending on γ ) such that ε1 ∈[0, 1]. Now, from (25), (27), the fact that r is convex with a minimum at zero, and by Lemma 7 we get where γ is such that (26) holds. As c →0, we have by Proposition 5, κ →0, ζ →−1/2 and ai →ci. Therefore, we can choose c small enough (and depending on γ ) such that ε1 ∈[0, 1]. Now, from (25), (27), the fact that r is convex with a minimum at zero, and by Lemma 7 we get sup x∈R ⏐⏐T1(x) ⏐⏐≤A sup x∈R σ∈{−1,1} ⏐⏐e−˜δx2r( σγ (1 + x2)ε1 )⏐⏐≤ε2 1A sup x∈R σ∈{−1,1} ⏐⏐e−˜δx2r( σγ (1 + x2))⏐⏐= ε2 1 A q1. 11 11 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. 4.2. Step 2 From this, for ε2 ∈[0, 1] as above, we can apply Lemma 6 and get sup x∈R ⏐⏐e−˜δx2(κx + (ζ + 1 2 )x2) ⏐⏐≤A sup x∈R ⏐⏐e˜δx2ε2(1 + x2) ⏐⏐≤ε2 sup x∈R ⏐⏐e−˜δx2(1 + x2) ⏐⏐= ε2 q2. Then, sup x∈R ⏐⏐T2(x) ⏐⏐= A sup x∈R ⏐⏐e˜δx2(κx + (ζ + 1 2 )x2) ⏐⏐≤A ε2 q2. Therefore, we have ∥T2∥∞= O(∥c∥2), as c →0. Therefore, we have ∥T2∥∞= O(∥c∥2), as c →0. Therefore, we have ∥T2∥∞= O(∥c∥2), as c →0. For T3, as with the T2 term we want an ε3 ∈[0, 1], such that |∑I i=1 ciKi(x)| ≤ε3(1+x2), so that we can apply Lemma 6 to show Therefore, we have ∥T2∥∞= O(∥c∥2), as c →0. For T3, as with the T2 term we want an ε3 ∈[0, 1], such that |∑I i=1 ciKi(x)| ≤ε3(1+x2), so that we can apply Lemma 6 to show For T3, as with the T2 term we want an ε3 ∈[0, 1], such that |∑I i=1 ciKi(x)| ≤ε3(1+x2), so t to show sup x∈R ⏐⏐e−˜δx2 I∑ i=1 ciKi(x) ⏐⏐≤ε3 sup x∈R ⏐⏐e−˜δx2(1 + x2) ⏐⏐< ∞. First, note that by (14), First, note that by (14), ⏐⏐ I∑ i=1 ciKi(x) ⏐⏐≤ ⏐⏐ I∑ i=1 ciBi(1 + x2) ⏐⏐= ⏐⏐ I∑ i=1 ciBi ⏐⏐· (1 + x2), and thus we set ε3 := ⏐⏐∑I i=1 ciBi ⏐⏐. Clearly, ε3 = O(∥c∥) as c →0. Now, with c sufficiently small such that ε3 ∈[0, 1], we have by Lemma 6, ⏐⏐ I∑ i=1 ciKi(x) ⏐⏐≤ ⏐⏐ I∑ i=1 ciBi(1 + x2) ⏐⏐= ⏐⏐ I∑ i=1 ciBi ⏐⏐· (1 + x2), and thus we set ε3 := ⏐⏐∑I i=1 ciBi ⏐⏐. Clearly, ε3 = O(∥c∥) as c →0. Now, with c sufficiently small such that ε3 ∈[0, 1], we have by Lemma 6, and thus we set ε3 := ⏐⏐∑I i=1 ciBi ⏐⏐. Clearly, ε3 = O(∥c∥) as c →0. Now, with c sufficiently small such that ε3 ∈[0, 1], we have by Lemma 6, and thus we set ε3 := ⏐⏐∑I i=1 ciBi ⏐⏐. Clearly, ε3 = O(∥c∥) as c →0. Now, with c sufficiently small such that ε3 ∈[0, 1], we have by Lemma 6, sup x∈R ⏐⏐e−˜δx2 I∑ i=1 ciKi(x) ⏐⏐≤sup x∈R ⏐⏐e−˜δx2( | I∑ i=1 ciBi|) (1 + x2) ⏐⏐≤| I∑ i=1 ciBi| · sup x∈R ⏐⏐e−˜δx2(1 + x2) ⏐⏐≤ε3 q2. 4.3. Step 3 4.3. Step 3 We now turn our attention to Step 3. of the approximations, where we find approximations for the entropy and negentropy of ˆf0. For these proofs we require that ˆf0(x) ≥0 for all x ∈R, and thus ˆf0 is a density. Lemma 9 (Approximation of Entropy). Suppose Assumptions 1 and 2 hold, and let ˆf0 be given as in Theorem 8. Suppose also that ˆf0(x) ≥0 for all x ∈R. Then the entropy of ˆf0 satisfies H[ˆf0] = ˆH[ˆf0] + R(ˆf0), H[ˆf0] = ˆH[ˆf0] + R(ˆf0), where, ˆH[ˆf0] := η(1) −1 2∥c∥2, with η(·) given in (2), c = (c1, c2, . . . , cI)⊤, with the ci defined in Proposition 3 and the remainder term bounded by |R(ˆf0)| ≤C ˜M · ∥c∥3, with η(·) given in (2), c = (c1, c2, . . . , cI)⊤, with the ci defined in Proposition 3 and the remainde |R(ˆf0)| ≤C ˜M · ∥c∥3, for some constant C ∈R, and ˜M given in Assumption 2. for some constant C ∈R, and ˜M given in Assumption 2. for some constant C ∈R, and ˜M given in Assumption 2. Proof. Set K(x) = (K1(x), K2(x), . . . , KI(x))⊤, for x ∈R. Now, with ˆf0 as in Theorem 8, expanding H[ˆf0] gives H[ˆf0] = − ∫ ˆf0(x) log ˆf0(x) dx = − ∫ ϕ(x) ( 1 + c⊤K(x) )( log ϕ(x) + log( 1 + c⊤K(x))) = − ∫ ϕ(x) log ϕ(x) dx − ∫ ϕ(x)c⊤K(x) log ϕ(x) dx − ∫ ϕ(x)( 1 + c⊤K(x)) log( 1 + c⊤K(x)) dx = η(1) − ∫ ϕ(x)c⊤K(x)( −1 2 log(2π) −1 2 x2) dx − ∫ ϕ(x)( 1 + c⊤K(x)) log( 1 + c⊤K(x)) dx = η(1) −0 − ∫ ϕ(x)( 1 + c⊤K(x)) log( 1 + c⊤K(x)) dx, K1(x), K2(x), . . . , KI(x))⊤, for x ∈R. Now, with ˆf0 as in Theorem 8, expanding H[ˆf0] gives, = η(1) −0 − ∫ ϕ(x)( 1 + c⊤K(x)) log( 1 + c⊤K(x)) dx, using the constraints given in (6). To obtain the approximation ˆH[ˆf0] and remainder R(ˆf0) terms, we consider the expansion of ( 1 + c⊤K(x)) log( 1 + c⊤K(x)) around c = 0 using the Taylor series. Let q(y) = y log(y), y ∈R. Then, we have using the constraints given in (6). 4.2. Step 2 Therefore, sup x∈R ⏐⏐T3(x) ⏐⏐≤| √ 2πA −1|ε3 q2 Thus, ∥T3∥∞= O(∥c∥3), as c →0, since | √ 2πA −1| = O(∥c∥2) and ε3 = O(∥c∥) as c →0. Similar to the T2 and T3 terms, for T4 we want an ε4 ∈[0, 1] such that ∑I i=1(ai −ci)Ki(x) ≤ε4(1 + x2). Note that Thus, ∥T3∥∞= O(∥c∥3), as c →0, since | √ 2πA −1| = O(∥c∥2) and ε3 = O(∥c∥) as c →0. Similar to the T2 and T3 terms, for T4 we want an ε4 ∈[0, 1] such that ∑I i=1(ai −ci)Ki(x) ≤ε4(1 + x2). Note that ⏐⏐ I∑ i=1 (ai −ci)Ki(x) ⏐⏐≤ ⏐⏐ I∑ i=1 (ai −ci)Bi ⏐⏐· (1 + x2), by (14) and thus we set ε4 := |∑I i=1(ai −ci)Bi|, and by Proposition 5, ε4 = O(∥c∥2) as c →0. Choose c small enough such that ε4 ∈[0, 1]. Then, by Lemma 6, by (14) and thus we set ε4 := |∑I i=1(ai −ci)Bi|, and by Proposition 5, ε4 = O(∥c∥2) as c →0. Choose c small enough such that ε4 ∈[0, 1]. Then, by Lemma 6, sup x∈R |T4(x)| ≤A sup x∈R ⏐⏐e−˜δx2 I∑ i=1 (ai −ci)Bi(1 + x2) ⏐⏐= ε4 A q2, and since ε4 = O(∥c∥2) as c →0, we have ∥T4∥∞= O(∥c∥2), as c →0. For T5 we can use e˜δx2 ≤1 for all x ∈R, and from Proposition 5 we have and since ε4 = O(∥c∥2) as c →0, we have ∥T4∥∞= O(∥c∥2), as c →0. For T5 we can use e˜δx2 ≤1 for all x ∈R, and from Proposition 5 we have 4 (∥∥) ∥4∥∞ (∥∥) For T5 we can use e˜δx2 ≤1 for all x ∈R, and from Proposition 5 we have T5(x) ≤| √ 2πA −1| = O(∥c∥2), as c →0. This completes the proof. □ This completes the proof. □ 12 12 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss E. Issoglio, P. Smith and J. Voss We have therefore shown that for sufficiently small c, the approximation ˆf0 for the density that maximises entropy given the constraints in (7) is ‘close to’ f0. We have also shown that the speed of convergence is of order ∥c∥2. 4.3. Step 3 To obtain the approximation ˆH[ˆf0] and remainder R(ˆf0) terms, we consider the expansion of ( 1 + c⊤K(x)) log( 1 + c⊤K(x)) around c = 0 using the Taylor series. Let q(y) = y log(y), y ∈R. Then, we have q′(y) = log(y) + 1; q′′(y) = 1 y ; q′′′(y) = −1 y2 . 1 2 q′(y) = log(y) + 1; q′′(y) = 1 y ; q′′′(y) = −1 y2 . q′(y) = log(y) + 1; q′′(y) = 1 y ; q′′′(y) = −1 y2 . and thus using Taylor series around y0 gives q(y0+h) = h+ 1 2h2+R1(y0, h), where R1(y0, h) is the remainder term given by R1(y0, h) = ∫y0+h y0 (y0 + h −τ)2 2 (−1 τ 2 ) dτ = −h3 ∫1 0 (1 −t)2 2(1 + th)2 dt us using Taylor series around y0 gives q(y0+h) = h+ 1 2h2+R1(y0, h), where R1(y0, R1(y0, h) = ∫y0+h y0 (y0 + h −τ)2 2 (−1 τ 2 ) dτ = −h3 ∫1 0 (1 −t)2 2(1 + th)2 dt R1(y0, h) = ∫y0+h y0 (y0 + h −τ)2 2 (−1 τ 2 ) dτ = −h3 ∫1 0 (1 −t)2 2(1 + th)2 dt with the change of variables τ = (y0 + th). with the change of variables τ = (y0 + th). h the change of variables τ = (y0 + th). Now let us pick y0 = 1 and h = c⊤K(x) and denote by R2(x) the corresponding remainder R2(x) = R1(1, c⊤K(x)). Then, with the change of variables τ = (y0 + th). Now let us pick y0 = 1 and h = c⊤K(x) and denote by R2(x) the corresponding remainder R2(x) = R1(1, c⊤K(x)). Then, with the change of variables τ = (y0 + th). Now let us pick y0 = 1 and h = c⊤K(x) and denote by R2(x) the corresponding remainder R2(x) = R1(1, c⊤K(x)). Then, es τ = (y0 + th). and h = c⊤K(x) and denote by R2(x) the corresponding remainder R2(x) = R1(1, c⊤K(x)). Then, H[ˆf0] = η(1) − ∫ ϕ(x) ( c⊤K(x) + 1 2 ( c⊤K(x))2 + R2(x) ) dx, (28) where the remainder term R2(x) is given explicitly by where the remainder term R2(x) is given explicitly by R2(x) = −( c⊤K(x))3 ∫1 0 (1 −t)2 2( 1 + tc⊤K(x))2 dt. 4.3. Step 3 From (30) with I = 1, we have ˆJ[ˆf0] = 1 2 c2, hence, ˆJ[ˆf0] = ( EG(Y) −EG(Z))2 2 δ2 . ˆJ[ˆf0] = ( EG(Y) −EG(Z))2 2 δ2 . This completes the proof, with C = 2 δ2 in Step 3. of Section 3. Note that δ can be found by solving the additional constraint ∫ ϕ(x)K(x)2 dx = 1. □ This concludes our discussion of the approximations used in fastICA. We have shown that under certain conditions, the approximations given in Steps 2., 3. and 4. in Section 3 are ‘‘close’’ to the true values. We will now give an example where these approximations are indeed close to one-another, but the surrogate density of the projections, f0 from Step 1. is not close to the true density f . 4.3. Step 3 Therefore, the negentropy equivalent of the entropy approximation given in Lemma 9 is J[ˆf0] = ˆJ[ˆf0] + R(ˆf0) with R(ˆf0) given as in (29) and Therefore, the negentropy equivalent of the entropy approximation given in Lemma 9 is J[ˆf0] = ˆJ[ˆf0] + R(ˆf0) with R(ˆf0) given as in (29) and ˆJ[ˆf0] = 1 2∥c∥2 = 1 2 I∑ i=1 c2 i . (30) ˆJ[ˆf0] = 1 2∥c∥2 = 1 2 I∑ i=1 c2 i . (30) Proposition 11. With the same assumptions as in Lemma 9. Set I = 1. Then, 2 Proposition 11. With the same assumptions as in Lemma 9. Set I = 1. Then Proposition 11. With the same assumptions as in Lemma 9. Set I = 1. Then, a random variable with density f and Z ∼N(0, 1). where Y is a random variable with density f and Z ∼N(0, 1). where Y is a random variable with density f and Z ∼N(0, 1). Proof. By the constraints that need to be satisfied by K, given in Assumption 2, we have ∫ ϕ(x)K(x)xk dx = 0 for k = 0, 1, 2. Substituting (5) for K(x) in (6) and solving these three equations gives an explicit expression for α, β, γ in terms of G, given by, α = 1 2 (∫ ϕ(x)G(x) dx − ∫ ϕ(x)G(x)x2 dx ) ; β = − ∫ ϕ(x)G(x)x dx; γ = 1 2 (∫ ϕ(x)G(x)x2 dx −3 ∫ ϕ(x)G(x) dx ) . α = 1 2 (∫ ϕ(x)G(x) dx − ∫ ϕ(x)G(x)x2 dx ) ; β = − ∫ ϕ(x)G(x)x dx; (31) γ = 1 2 (∫ ϕ(x)G(x)x2 dx −3 ∫ ϕ(x)G(x) dx ) . (31) Recall that c = EK(Y) = 1 δ ( EG(Y) + αEY 2 + βEY + γ ) . Now using (31) and the fact that EY = 0 and EY 2 = 1 (since Y has density f ), we get c = 1 δ ( EG(Y) −EG(Z)) . From (30) with I = 1, we have ˆJ[ˆf0] = 1 2 c2, hence, Recall that c = EK(Y) = 1 δ ( EG(Y) + αEY 2 + βEY + γ ) . Now using (31) and the fact that EY = 0 and EY 2 = 1 (since Y has density f ), we get c = 1 δ ( EG(Y) −EG(Z)) . 4.3. Step 3 Now using (6) and setting R(ˆf0) := − ∫ R ϕ(x) R2(x) dx we get from (28), R(ˆf0) := − ∫ R ϕ(x) R2(x) dx R(ˆf0) := − ∫ R ϕ(x) R2(x) dx we get from (28), (29) H[ˆf0] = η(1) + 0 −1 2 I∑ i=1 c2 i + R(ˆf0) = ˆH[ˆf0] + R(ˆf0), as needed to be shown. It remains to prove the bound for R(ˆf0). as needed to be shown. It remains to prove the bound for R(ˆf0). 13 13 oglio, P. Smith and J. Voss Journal of Multivariate An Journal of Multivariate Analysis 181 (2021) 104689 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss From Assumption 2 there exists some ε > 0 such that c⊤K(x) ≥−1/2 for all c with c⊤c ≤ε for all x ∈R, and therefore, From Assumption 2 there exists some ε > 0 such that c⊤K(x) ≥−1/2 for all c with c⊤c ≤ε for all x ∈R, and therefore, |R2(x)| = ⏐⏐⏐ ( c⊤K(x))3 ∫1 0 (t −1)2 2 · (1 + tc⊤K(x))2 dt ⏐⏐⏐≤ ⏐⏐( c⊤K(x))⏐⏐3 · ⏐⏐⏐ ∫1 0 (t −1)2 2 · (1 −t/2)2 dt ⏐⏐⏐= C · ⏐⏐( c⊤K(x))⏐⏐3, where C ∈R, as the integral is of a continuous function over a compact set. ⊤ Now, there exists some δ > 0 such that for all c⊤c ≤δ, ci ≤∥c∥for all i ∈{1, . . . , I}. Then ve Now, there exists some δ > 0 such that for all c⊤c ≤δ, ci ≤∥c∥for all i ∈{1, . . . , I}. Then, with c⊤c ≤min(ε, δ), we have |R2(x)| ≤C I∑ i,j,k=1 ⏐⏐Ki(x)Kj(x)Kk(x) ⏐⏐· ∥c∥3 ≤C · M(x) · ∥c∥3 having used (13a) from Assumption 2. Putting this all together we obtain the bound for R(ˆf0), ⏐⏐R(ˆf0) ⏐⏐≤ ∫ R ϕ(x)|R2(x)| dx ≤C ˜M∥c∥3, where ˜M is given in (13b), as required. □ where ˜M is given in (13b), as required. □ Remark 10. Note that the density ˆf0 has unit variance. Indeed, by (6), Remark 10. Note that the density ˆf0 has unit variance. Indeed, by (6), ∫ ˆf0(x)x2 dx = ∫ ϕ(x)( 1 + I∑ i=1 ciKi(x)) dx = ∫ ϕ(x)x2 dx + I∑ i=1 ci ∫ ϕ(x)Ki(x)x2 dx = 1. 5. Example We now highlight the approximation steps as explained in Section 3 on a toy example. In this section we use example data as illustrated in Fig. 1, which was intentionally created in a very simplistic manner to further emphasise the ease at 14 Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss which false optima are found using the contrast function ˆJ∗(y). The data was obtained by pre-selecting vertical columns where no data points are allowed. An iterative scheme was then employed, as explained below: which false optima are found using the contrast function ˆJ∗(y). The data was obtained by pre-selecting vertical columns where no data points are allowed. An iterative scheme was then employed, as explained below: which false optima are found using the contrast function ˆJ∗(y). The data was obtained by pre-selecting vertical columns where no data points are allowed. An iterative scheme was then employed, as explained below: 1. Sample n points from a standard two-dimensional Gaussian distribution; 2. Remove all points that lie in the pre-specified columns; 3. Whiten the remaining ˜n points; g p 4. Sample n −˜n points from a standard two-dimensional Gaussian distribution. Repeat 2. - 4. until we have a sample of size n with no points lying in the pre-specified columns. No optimisation was done to the distribution of these points to attempt to force the fastICA contrast function to have a false optimum. Repeat 2. - 4. until we have a sample of size n with no points lying in the pre-specified columns. No optimisation was done to the distribution of these points to attempt to force the fastICA contrast function to have a false optimum. We will use the m-spacing approximation (3) to obtain a contrast function that can be compared to the fastICA contrast function (11). Following Learned-Miller and Fisher [16], we chose m = √ n, where n ∈N is the number of observations. This was chosen so that the condition m/n →0 as n →∞is satisfied [3,22]. This approximation to entropy is a direct approximation to H[f ], and therefore does not involve an equivalent Step 1. from Section 3 where f is substituted by a new density f0. Using the m-spacing method to find the first independent component loading, we want to obtain the direction w∗:= argminw∈Rp,w⊤w=1 Hm,n(Dw). 5. Example In the example of this paper, numerical minimisation is used to obtain w∗and the associated projection Dw∗. The contrast function to compare against the fastICA contrast function (11) is given by the m-spacing negentropy approximation, Jm,n(y) = η(1) −Hm,n(y) for directions w ∈Rn on the half-sphere. Note that w∗= argmaxw∈Rp,w⊤w=1 Jm,n(Dw). In general this contrast function is not very smooth, although a method to attempt to overcome this non-smoothness (and the resulting local optima, which can cause numerical optimisation issues) is given in Learned-Miller and Fisher [16], and involves replicating the data with some added Gaussian noise. ˆ ˆ To illustrate the kind of problems which can occur during the approximation from f to ˆf0 and from J[f ] to ˆJ∗(y), we construct an example where the density f in the direction of maximum negentropy is significantly different to ˆf0 in the same direction. This results in fastICA selecting a sub-optimal projection, as shown below. Here we just consider the case I = 1 in Assumption 1, with one G = G1 and thus one K = K1. Moreover, in fastICA there is a choice of two functions to use, G(x) := (1/α) log cosh(αx), α ∈[1, 2], and G(x) := −exp(−x2/2). We have considered these two functions with varying alpha, as well as the fourth moment contrast function given in Miettinen et al. [19]. Here, the function for Step 3. in Section 3 is |Ef X 4−3|, and the empirical approximation of the expectation is used for Step 4., such that the approximate contrast function is | 1 n ∑n i=1 y4 i −3|. In the example of this paper all choices give very similar results and thus we only show the fastICA contrast function resulting from G(x) = (1/α) log cosh(αx), with α = 1 for simplicity. With the data distributed as in Fig. 1, the negentropy over projections in the directions wθ = (sin(θ), cos(θ)) with θ ∈[0, π) found by the m-spacing approximation and used in the fastICA method is shown in Fig. 4. The contrast function obtained by approximating J[f0] directly is also included as the dashed line. The three contrast functions have been placed below one-another in the order of approximations given in Fig. 3 and so the y-axis is independent for each. 5. Example The search is only performed on the half unit circle, as projections in directions w1 = (sin(θ), cos(θ)) and w2 = (sin(θ +π), cos(θ +π)) for any θ ∈[0, π) have a reflected density with the same entropy. It is clear from Fig. 4 that the fastICA result ˆJ∗is poor, with the fastICA contrast function missing the peak of negentropy that appears when using m-spacing. The contrast function used in the fastICA method clearly differentiates between the direction of the maximum and other directions, and thus in this example it is both confident and wrong (since there is a clear and unique peak). This is also true of the direct approximation to J[f0], showing that issues occur at the first step of approximations, when J[f0] is used instead of J[f ]. ˆ As is shown in Section 4, for sufficiently small c, the approximation for the density ˆf0 (given in (9)) is ‘‘close to’’ f0 (given in (8)), and the speed of convergence is of order c2 for c →0. Therefore, it is our belief (backed up by computational experiments) that the majority of the loss of accuracy occurs in the approximation step where the surrogate f0 is used instead of f , rather than in the later estimation steps for J[ˆf0] and ˆJ∗(y). This can be seen by comparing numerically the contrast functions J[f ], J[f0] and ˆJ∗(y) (shown in Fig. 4), and by comparing the densities f , f0 and ˆf0. Here, J[f0] and ˆJ∗(y) give similar directions for the maximum, and these differ significantly from the location of the maximum of J[f ]. This is a fundamental theoretical problem with the fastICA method, and is not a result of computational or implementation issues with fastICA. In particular, the fact that the dotted vertical line in Fig. 4 is at the maximum of ˆJ∗(y) indicates that the effect is not a convergence problem in the fastICA implementation. 6. Conclusions In this paper we have given an example where the fastICA method misses structure in the data that is obvious to the naked eye. Since this example is very simple, the fastICA result is concerning, and this concern is magnified when working in high dimensions as visual inspection is no longer easy. There is clearly some issue with the contrast function (surrogate negentropy) used in fastICA. Indeed, this surrogate has the property of being an approximation of a lower bound for negentropy, and this does not necessarily capture the actual behaviour of negentropy over varying projections since we want to maximise negentropy. To strengthen the claim that accuracy is lost when substituting the density with the surrogate, we have shown convergence results for all the approximation steps used in the method. 15 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 Fig. 4. Objective functions of m-spacing (solid line), J[f0] (dashed line) and fastICA method (dotted line) for projections of the data given in Fig. 1 in the directions θ ∈[0, π). These correspond to J[f ], J[f0] and ˆJ∗(y) in Fig. 3. The vertical lines give the directions which maximise the contrast functions for m-spacing (solid line) and fastICA (dotted line). Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Fig. 4. Objective functions of m-spacing (solid line), J[f0] (dashed line) and fastICA method (dotted line) for projections of the data given in Fig. 1 in the directions θ ∈[0, π). These correspond to J[f ], J[f0] and ˆJ∗(y) in Fig. 3. The vertical lines give the directions which maximise the contrast functions for m-spacing (solid line) and fastICA (dotted line). To conclude this paper, we ask the following questions which could make for interesting future work: Is there a way, a priori, to know whether fastICA will work? This is especially pertinent when fastICA is used with high dimensional data. The trade-off in accuracy for the fastICA method comes at the point where the density f is substituted with f0. Therefore one could also ask: Are there other methods similar to that of fastICA but that use a different surrogate density which more closely reflects the true projection density? CRediT authorship contribution statement Elena Issoglio: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Supervision. Paul Smith: Conceptualization, Methodology, Software, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Jochen Voss: Conceptualization, Methodology, Software, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization, Supervision. 6. Conclusions y p j y If these two options are not possible, then potentially a completely different method for ‘‘fast’’ ICA is needed, one that either gives a ‘‘good’’ approximation for all distributions, or where it is known when it breaks down. An initial step in this direction can be found in Smith et al. [20]. In this work the authors propose a new ICA method, known as clusterICA, using the m-spacing approximation for entropy discussed in this paper, combined with a clustering procedure. Acknowledgments We thank the Editor, Associate Editor and referees for their helpful comments. P. Smith was funded by NERC DTP SPHERES, Grant Number NE/L002574/1. References [1] S. Amari, A. Cichocki, H. Yang, A new learning algorithm for blind signal separation, in: Adv. Neural Inf. Process. Syst., MIT Press, 1996, pp. 757–763. [1] S. Amari, A. Cichocki, H. Yang, A new learning algorithm for blind signal separation, in: Adv. Neural Inf. Pro 757–763. [1] S. Amari, A. Cichocki, H. Yang, A new learning algorithm for blind signal separation, in: Adv. Neural Inf. Process. Syst., MIT Press, 1996, pp. 757–763. [2] F. Bach, M. Jordan, Kernel independent component analysis, J. Mach. Learn. Res. 3 (Jul) (2002) 1–48. [3] J B i l E J D d i L G ö fi E C V d M l N i i i A i I [2] F. Bach, M. Jordan, Kernel independent component analysis, J. Mach. Learn. Res. 3 (Jul) (2002) 1–48. [3] J. Beirlant, E.J. Dudewicz, L. Györfi, E.C. Van der Meulen, Nonparametric entropy estimation: An overview, Int. J. Math. Stat. Sci. 6 (1) (1997) 17–39. [3] J. Beirlant, E.J. Dudewicz, L. Györfi, E.C. Van der Meulen, Nonparametric entropy estimation: An overview, In 17–39. [4] T. Cover, J. Thomas, Elements of Information Theory, John Wiley & Sons, New York, 2012. [5] P. Diaconis, D. Freedman, Asymptotics of graphical projection pursuit, Ann. Statist. 12 (3) (1984) 793–815. [6] B. Draper, K. Baek, M. Bartlett, J. Beveridge, Recognizing faces with PCA and ICA, Comput. Vis. Image Underst. 91 (1–2) (2003) 115–137. [7] M. Farhat, Y. Gritli, M. Benrejeb, Fast-ICA for mechanical fault detection and identification in electromechanical systems for wind turbine applications, Int. J. Adv. Comput. Sci. Appl. 8 (7) (2017) 431–439. [6] B. Draper, K. Baek, M. Bartlett, J. Beveridge, Recognizing faces with PCA and ICA, Comput. Vis. Image Underst. 91 (1–2) (2003) 115–137. [7] M. Farhat, Y. Gritli, M. Benrejeb, Fast-ICA for mechanical fault detection and identification in electromechanical systems for wind turbine applications, Int. J. Adv. Comput. Sci. Appl. 8 (7) (2017) 431–439. [6] B. Draper, K. Baek, M. Bartlett, J. Beveridge, Recognizing faces with PCA and ICA, Comput. Vis. Image Underst [7] M. Farhat, Y. Gritli, M. Benrejeb, Fast-ICA for mechanical fault detection and identification in electromech applications, Int. J. Adv. Comput. Sci. Appl. 8 (7) (2017) 431–439. [7] M. Farhat, Y. Gritli, M. Benrejeb, Fast-ICA for mechanical fault detection and identification in electromechanical systems for wind turbine applications, Int. J. Adv. Comput. Sci. Appl. 8 (7) (2017) 431–439. [8] S. Ghaffarian, S. ärinen, Fast and robust fixed-point algorithms for independent component analysis, IEEE Trans. Neural Netw. 10 (3 ärinen, J. Karhunen, E. Oja, Independent Component Analysis, John Wiley & Sons, New York, 2001. References Ghaffarian, Automatic building detection based on Purposive FastICA (PFICA) algorithm using monocular high resolution Google Earth images, ISPRS J. Photogramm. Remote Sens. 97 (2014) 152–159. [8] S. Ghaffarian, S. Ghaffarian, Automatic building detection based on Purposive FastICA (PFICA) algorithm using monocular high resolution Google Earth images, ISPRS J. Photogramm. Remote Sens. 97 (2014) 152–159. [9] P. Hall, Limit theorems for sums of general functions of m-spacings, Mathematical Proceedings of the Cambridge (1984) 517–532. ] h l i i i f i h i l li i i [10] X. He, F. He, T. Zhu, Large-scale super-Gaussian sources separation using fast-ICA with rational nonlinearities, Internat. J. Adapt. Control Signal Process. 31 (3) (2017) 379–397. [10] X. He, F. He, T. Zhu, Large-scale super-Gaussian sources separation using fast-ICA with rational nonlinearities, Process. 31 (3) (2017) 379–397. [10] X. He, F. He, T. Zhu, Large-scale super-Gaussian sources separation using fast-ICA with rational nonlinearities, Internat. J. Adapt. Control Signal Process. 31 (3) (2017) 379–397. [11] A. Hyvärinen, New approximations of differential entropy for independent component analysis and projection pursuit, in: Proceedings of the 1997 Conference on Advances in Neural Information Processing Systems 10, MIT Press, 1998, pp. 273–279. [12] A. Hyvärinen, Fast and robust fixed-point algorithms for independent component analysis, IEEE Trans. Neural [13] A. Hyvärinen, J. Karhunen, E. Oja, Independent Component Analysis, John Wiley & Sons, New York, 2001. [ ] d d l l h d l l ( ) ( [14] A. Hyvärinen, E. Oja, Independent component analysis: algorithms and applications, Neural Netw. 13 (4) (2000) 411–430. [14] A. Hyvärinen, E. Oja, Independent component analysis: algorithms and applications, Neural Netw. 13 (4) (2000) 411–430. [15] E. Lawrence, Partial Differential Equations, American Mathematical Society, New York, 1998. A. Hyvärinen, E. Oja, Independent component analysis: al 16 [26] C.-H. Yang, Y.-H. Shih, H. Chiueh, An 81.6 µW fastICA processor for epileptic seizure detection, IEEE Trans. Biomed. Circuits Syst. 9 (1) (2015) 60–71. Journal of Multivariate Analysis 181 (2021) 104689 E. Issoglio, P. Smith and J. Voss Journal of Multivariate Analysis 181 (2021) 104689 [16] E. Learned-Miller, J. Fisher, ICA using spacings estimates of entropy, J. Mach. Learn. Res. 4 (Dec) (2003) 1271–1295. [17] J M hi i C H B Ri l F ICA F ICA l i h f ICA d j i i 2013 R k i 1 2 0 [16] E. Learned-Miller, J. Fisher, ICA using spacings estimates of entropy, J. Mach. Learn. Res. 4 (Dec) (2003) 1271–1295. [17] J. Marchini, C. Heaton, B. Ripley, FastICA: FastICA algorithms to perform ICA and projection pursuit, 2013, R package version 1.2-0. [18] J. Miettinen, K. Nordhausen, H. Oja, S. Taskinen, Deflation-based fastICA with adaptive choices of nonlinearities, IEEE Trans. Signal Process. 62 (21) (2014) 5716 5724 [18] J. Miettinen, K. Nordhausen, H. Oja, S. Taskinen, Deflation-based fastICA with adaptive choices of nonlinearities, IEEE Trans. Signal Process. 62 (21) (2014) 5716–5724. ( ) ( ) J. Miettinen, S. Taskinen, K. Nordhausen, H. Oja, et al., Fourth moments and independent component analysis, Stat. Sc [19] J. Miettinen, S. Taskinen, K. Nordhausen, H. Oja, et al., Fourth moments and independent [20] P. Smith, J. Voss, E. Issoglio, ClusterICA, 2020, in preparation. [20] P. Smith, J. Voss, E. Issoglio, ClusterICA, 2020, in preparation. [21] J. Stone, Independent Component Analysis: A Tutorial Introduction, MIT Press, Massachusetts, 2004. [21] J. Stone, Independent Component Analysis: A Tutorial Introduction, MIT Press, Massachusetts, 2004. [22] O. Vasicek, A test for normality based on sample entropy, J. R. Stat. Soc. Ser. B Stat. Methodol. 38 (1) [23] T. Wei, On the spurious solutions of the fastICA algorithm, in: 2014 IEEE Workshop on Statistical Signal Processing (SSP), 2014, pp. 161–164. [24] T. Wei, A study of the fixed points and spurious solutions of the deflation-based FastICA algorithm, Neural Comput. Appl. 28 (1) (2017) 13–24. [25] H. von Weizsäcker, Sudakov’s typical marginals, random linear functionals and a conditional central limit theorem, Probab. Theory Related Fi ld 107 (3) (1997) 313 324 [23] T. Wei, On the spurious solutions of the fastICA algorithm, in: 2014 IEEE Workshop on Statistical Sig [24] T. Wei, A study of the fixed points and spurious solutions of the deflation-based FastICA algorithm, Neural Comput. Appl. 28 (1) (2017) 13–24. 4] T. Wei, A study of the fixed points and spurious solution ( ) ( ) [26] C.-H. Yang, Y.-H. Shih, H. Vasicek, A test for normality based on sample entropy, J. R. Stat. Soc. Ser. B Stat. Methodol. 38 (1) (1976) 54–59. [16] E. Learned-Miller, J. Fisher, ICA using spacings estimates of entropy, J. Mach. Learn. Res. 4 (Dec) (2003) 1271–1295. [17] J Marchini C Heaton B Ripley FastICA: FastICA algorithms to perform ICA and projection pursuit 2013 R package version 1 2-0 [16] E. Learned-Miller, J. Fisher, ICA using spacings estimates of entropy, J. Mach. Learn. Res. 4 (Dec) (2003) 1271–1295. [17] J. Marchini, C. Heaton, B. Ripley, FastICA: FastICA algorithms to perform ICA and projection pursuit, 2013, R package version 1.2-0. [18] J Miettinen K Nordhausen H Oja S Taskinen Deflation-based fastICA with adaptive choices of nonlinearities IEEE Trans Signal Process 62 25] H. von Weizsäcker, Sudakov’s typical marginals, random linear functionals and a conditional central limit theo Fields 107 (3) (1997) 313–324. Journal of Multivariate Analysis 181 (2021) 104689 Chiueh, An 81.6 µW fastICA processor for epileptic seizure detection, IEEE Trans. Biomed. Circuits Syst. 9 (1) (2015) 60–71. [26] C.-H. Yang, Y.-H. Shih, H. Chiueh, An 81.6 µW fastICA processor for epileptic seizure detection, IEEE Trans. Biomed. Circuits Syst. 9 (1) (2015) 60 71 [26] C.-H. Yang, Y.-H. Shih, H. Chiueh, An 81.6 µW fastICA processor for epileptic seizure detection, IEEE Trans. Biomed. Circuits Syst. 9 (1) (2015) 60–71. 17 17
https://openalex.org/W4292182258
https://lipidworld.biomedcentral.com/counter/pdf/10.1186/s12944-022-01683-1
English
null
Association between four nontraditional lipids and ischemic stroke: a cohort study in Shanghai, China
Lipids in health and disease
2,022
cc-by
7,190
© The Author(s) 2022. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  The correlation between nontraditional lipids and ischemic stroke (IS) is inconsistent and controversial. This study aimed to examine the association of four nontraditional lipids with IS risk in Chinese adults. Methods:  This prospective community-based cohort study was performed in Songjiang District, Shanghai, China. The study began in 2016 and included 34,294 participants without stroke before the investigation. The association between nontraditional lipids (nonhigh-density lipoprotein cholesterol [non-HDL-C], total cholesterol/high-density lipoprotein cholesterol [TC/HDL-C], triglyceride [TG]/HDL-C, and low-density lipoprotein cholesterol [LDL-C]/HDL-C) and IS was studied with multivariate Cox regression models. The dose–response associations between these four serum lipids and IS were explored using restricted cubic spline (RCS) analysis. Results:  There were a total of 458 IS cases with 166,380 person-years of follow-up. Compared with the lowest tertiles, the highest tertiles of the nontraditional blood lipids showed greater IS risk after controlling for potential confound- ers. The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were as follows: TC/HDL-C, 1.63 (1.28–2.07); TG/ HDL-C, 1.65 (1.28–2.13); LDL-C/HDL-C, 1.51 (1.18–1.92); and non-HDL-C, 1.43 (1.13–1.81). The fully adjusted RCS curves presented a nonlinear relationship, and the risk increased when the TC/HDL-C, TG/HDL-C, and LDL-C/HDL-C levels were > 3.47, > 0.92, and > 1.98, respectively. Conclusions:  This community-based cohort study presents a positive association between the four nontraditional lipids and IS incidence. Maintaining relatively low lipid ratios can be beneficial for preventing stroke. Nontraditional lipids can be considered targets for managing blood lipids. Keywords:  Nontraditional lipids, Ischemic stroke, Cohort study high economic burden [2]. Stroke has become the pri- mary reason for disability and mortality among Chinese adults [3]. From 1990 to 2019, the absolute number of global stroke events increased by 70%, and the number of stroke-related deaths increased by 43% [4]. Ischemic stroke (IS) is the most common stroke type, and its inci- dence continues to rise in China [2]. Tang et al. Lipids in Health and Disease (2022) 21:72 https://doi.org/10.1186/s12944-022-01683-1 Open Access Association between four nontraditional lipids and ischemic stroke: a cohort study in Shanghai, China Minhua Tang1,2, Qi Zhao1, Kangqi Yi1, Yiling Wu2, Yu Xiang1, Shuheng Cui1, Xuyan Su2, Yuting Genming Zhao1* and Yonggen Jiang2* Minhua Tang1,2, Qi Zhao1, Kangqi Yi1, Yiling Wu2, Yu Xiang1, Shuheng Cui1, Xuyan Su2, Yuting Yu1, Genming Zhao1* and Yonggen Jiang2* Minhua Tang1,2, Qi Zhao1, Kangqi Yi1, Yiling Wu2, Yu Xiang1, Shuheng Cui1, Xuyan Su2, Yuting Yu1, Genming Zhao1* and Yonggen Jiang2* 1 Department of Epidemiology, School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Fudan University, Shanghai 200032, China 2 Songjiang District Center for Disease Control and Prevention, Shanghai 201600, China Introduction Stroke, a vascular disease that causes acute focal lesions in the central nervous system [1], is characterized by high morbidity, recurrence, disability, mortality, and *Correspondence: gmzhao@shmu.edu.cn; Sjjkzx1106@126.com 1 Department of Epidemiology, School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Fudan University, Shanghai 200032, China 2 Songjiang District Center for Disease Control and Prevention, Shanghai 201600, China Dyslipidemia plays an essential role in the development of stroke [5, 6]. It has been proposed that lowering low- density lipoprotein cholesterol (LDL-C) is a major indicator © The Author(s) 2022. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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 10 Page 2 of 10 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease (2022) 21:72 Methods and materials Study design for preventing and controlling atherosclerotic cardiovascu- lar disease (ASCVD) in lipid management [7, 8]. However, even when LDL-C is within normal limits, cardiovascular events occur due to increased serum triglycerides (TGs) or decreased high-density lipoprotein cholesterol (HDL-C) concentrations, a condition known as residual cardiovas- cular risk [9, 10]. Therefore, exploring the impact of novel lipid markers on cardiovascular disease (CVD) has become a hot topic in cardiovascular research. y g The present data were obtained from the Shanghai Suburban Adult Cohort and Biobank (SSACB) study. The baseline investigation started from June 2016 to December 2017. The details of the cohort profile have been well described previously [18]. Participants aged between 20 and 74 years and living for at least 5 years in the district were recruited from four Songjiang com- munities (Xinqiao, Zhongshan, Maogang, and Sheshan) by stratified multistage random sampling. In this analy- sis, 36,404 participants completed the physical exami- nations, questionnaire interviews, and biochemical tests. After excluding the participants with a stroke history before investigation (n = 1129); missing data on blood lipids (n = 341); and other key variables, such as height, weight, and systolic blood pressure (SBP) (n = 640), 34,294 subjects were deemed included in the analysis. The participant inclusion and exclusion crite- ria are depicted in Fig. 1. All participants signed writ- ten informed consent before the research. The cohort protocol was approved by the Ethics Committee on Medical Research at the School of Public Health, Fudan University (IRB#2016-04-0586). More recently, the evidence supported the idea that nontraditional lipids such as non-HDL-C, total choles- terol (TC)/HDL-C, TG/HDL-C, and LDL-C/HDL-C played a significant role in stroke events and indicated that they were more robust predictors than traditional lipids [11–13]. However, some researchers reported inconsistent results. For example, a cohort study in Paris observed that lipid ratios were not significantly associated with IS [14]. Therefore, the role of nontraditional lipids in stroke requires further study. Moreover, research on asso- ciations between these lipids and IS in Shanghai, China, is scarce, and few studies have reported dose–response rela- tionships to explore the risk threshold. Lipids are an easily accessible modifiable cardiovascular risk factor, and the aforementioned nontraditional lipids are easy to calcu- late [15]. Serum lipids are associated not only with stroke but also with obesity and diabetes, which are all factors related to adverse cardiovascular events [16, 17].h Definition of variablesi and physical activities) was collected by well-trained staff using structured questionnaires through face-to-face interviews. The questionnaire interview was adminis- tered on an Android tablet computer with audio record- ing and paperless data input. Of the recording files, 5% were randomly selected and checked to ensure the qual- ity of the interviews. Anthropometric measurements were conducted with standardized methods by clinicians at the Community Health Service Centers and included weight, height, and blood pressure (BP). BP was meas- ured with a digital sphygmomanometer at least three times after a 5-min rest to document the SBP and dias- tolic BP (DBP). i Hypertension was defined as SBP/DBP ≥ 140/90 mmHg or with a previous diagnosis history [21]. Diabetes was defined as a high level of FPG (≥ 7.0 mmol/L) or HbA1c (≥ 6.5%) or with a previous diagnosis history [22]. The definition of chronic kidney disease (CKD) was per- sistent abnormal kidney function or kidney impair- ment, including an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 ­m2 or hematuria or proteinuria [23]. Hyperuricemia (HUA) was defined as high levels of SUA (in women, ≥ 360 μmol/L; in men, ≥ 420 μmol/L) [24]. The smoking index was assessed by multiplying packets per day by the smoking years [25]. The defini- tion of current alcohol drinking was drinking ≥3 times per week in the past 6 months. Physical activities were assessed as the metabolic equivalent task (MET) value multiplied by the total number of minutes per week [26] and were divided into three categories: low (MET-mins/ week < 2000), moderate (2000 ≤ MET-mins/week < 6000), and high (MET-mins/week ≥6000) [27]. Statistical analysis y The distribution of data was assessed by the Kolmogo- rov–Smirnov method. The mean ± standard deviation or median (P25–P75) are described for continuous data. The frequency with corresponding column per- centages is presented for categorical data. The base- line characteristics were described according to the participants’ sex. Continuous data were analyzed with Student’s t test (normal distribution) or the Wilcoxon- Mann–Whitney U test (nonnormal distribution). The χ2 test was used to analyze categorical data. The inci- dence density trend of IS in different lipid quantile groups was tested by the Cochran-Armitage trend test. The relationship between lipids and the risk of IS was studied with Cox regression models. The pro- portional hazard was tested with the Schoenfeld indi- vidual test. The blood lipid levels and lipid ratios were modeled as tertile categories, and the lowest group was considered the reference. Sex, age, retirement status, education level, body mass index (BMI), alco- hol consumption, smoking index, physical activities, diabetes, CKD, hypertension, and HUA were fully adjusted in the models. When individual lipids were analyzed, other lipids were adjusted accordingly. Sub- group analysis was conducted by sex, age, and hyper- tension status. The multiplicative terms of variables were included in the Cox regression models to test the multiplicative interactions. Restricted cubic spline (RCS) analysis with four knots was applied to study the dose–response association between four nontradi- tional blood lipids and IS. A two-sided P value < 0.05 was considered statistically significant. All data were Questionnaire interview and anthropometric measurement Therefore, to better discuss the problems above, the study investigated the relations between four nontradi- tional blood lipids and IS outcomes among populations in Shanghai communities based on a cohort study. Information on sociodemographic characteristics (sex, age, education level, and retirement status), self-reported chronic disease history (hypertension, diabetes, and stroke), and lifestyle (smoking, alcohol consumption, Fig. 1  Selection process of the participants Fig. 1  Selection process of the participants Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease (2022) 21:72 Page 3 of 10 Laboratory assays Fasting for at least 8 h was required before collecting blood samples in the morning. An automatic biochemi- cal analyzer (Roche Cobas C501) was used to test serum lipids with colorimetry (TG) and enzymatic colorimetry (LDL-C, HDL-C, and TC) methods. A Roche Modular P800 automatic biochemical analyzer was used to test fasting plasma glucose (FPG) with hexokinase meth- ods. A Tosoh G8 automatic glycohemoglobin analyzer was used to measure glycated hemoglobin (HbA1c) with high-pressure liquid chromatography methods. Serum uric acid (SUA) was tested by a Roche Cobas C702 with colorimetry methods. Nontraditional lipids were calcu- lated based on conventional lipids in clinical practice, including non-HDL-C (calculation method: TC minus HDLC) and three lipid ratios: LDL-C/HDL-C, TG/HDL- C, and TC/HDL-C [19]. Baseline characteristics and outcome data The study included 34,294 participants without stroke at baseline. Table  1 demonstrates the participants’ baseline characteristics based on sex. The median age of the participants was 58 (50–65) years. The mean or median nontraditional lipid values at baseline were as follows: TC/HDL-C ratio, 3.71 ± 1.18; TG/ Table 1  Comparison of baseline characteristics of participants according to sex a Categorical data are expressed as the frequency (%). Assessment of stroke and follow‑up Follow-up was performed according to the health infor- mation system. The outcomes were fatal and nonfatal IS events and were collected based on the Cardiovascular and Cerebrovascular Disease Registration and Report- ing System, the electronic medical record (EMR) system, and the cause-of-death surveillance system, which can be matched by a unique identification (ID) number: the ID card number. These online information systems con- tained a detailed record of the name of a disease diagno- sis, date of diagnosis, cause and date of death, and date of onset. The Tenth Revision of the International Clas- sification of Diseases (ICD-10) codes were I63–I64 [20]. Transient ischemic attacks were not included. Partici- pants with self-reported or history of diagnosed stroke events were excluded from the baseline to ensure that the participants had no history of earlier stroke. The earliest onset of IS event or death due to the first IS onset after the baseline was recorded as an outcome. The outcomes were documented by the researchers from the baseline date to December 31, 2021. Page 4 of 10 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease analyzed using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). analyzed using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). HDL-C ratio, 0.96 (0.63–1.55); LDL-C/HDL-C ratio, 2.08 ± 0.79; and non-HDL-C, 3.53 ± 0.95. Male partici- pants were slightly older, had greater diabetes, hyper- tension, and HUA prevalence, and had higher BMI, TG, and lipid ratio levels than female participants (all P <  0.001). A total of 458 incident IS cases were doc- umented after a total of 166,380 person-years of fol- low-up (median follow-up: 4.97 years). The incidence density and 95% CI of IS was 275.27 (250.10–300.45) per 100,000 person-years. Analysis of the incidence density according to blood lipids revealed that the a Categorical data are expressed as the frequency (%). Continuous data are presented as the median and interquartile range (nonnormally distributed data) or mean and standard deviation (normally distributed data) Association of blood lipids with IS The Schoenfeld individual test indicated that the risk of lipids on IS had no significant time trends (P > 0.05). The association of lipids according to tertile category with IS risk is shown in Table  2. After controlling for potential confounders (age, sex, retirement status, edu- cation level, BMI, alcohol consumption, smoking index, physical activities, hypertension, diabetes, CKD, HUA, and other lipids), the highest tertiles were positively related to IS risk when compared with the lowest ter- tiles, except HDL-C, which had negative associations. The adjusted HR increased by blood lipid tertile except for the HDL-C group, which demonstrated a negative trend (all Ptrend <   0.05). The HRs and 95% CIs of the highest tertiles were 1.63 (1.28–2.07) for TC/HDL-C, 1.65 (1.28–2.13) for TG/HDL-C, 1.51 (1.18–1.92) for LDL-C/HDL-C, and 1.43 (1.13–1.81) for non-HDL-C and showed a greater risk for IS than TC (1.32, 1.03– 1.68) and LDL-C (1.36, 1.08–1.71). The analysis was further stratified according to sex (male and female, Fig. 3), age group (< 60 and ≥ 60 years old, Fig. S1), and hypertension status (without and with hypertension, Fig. S2) and determined that the positive association was still statistically significant in males, participants aged ≥60 years, and participants with hypertension. Baseline characteristics and outcome data Continuous data are presented as the median and interquartile range (nonnormally distributed data) or mean and standard deviation (normally distributed data) Characteristicsa Male (N = 13,844) Female (N = 20,450) Total (N = 34,294) P value Age (years) 59 (51–66) 57 (50–64) 58 (50–65) <  0.001 Education level (%)    ≤ 6 education years 891 (6.22) 4010 (19.61) 4871 (14.20) <  0.001   7 ~ 12 education years 11,658 (84.21) 14,656 (71.67) 26,314 (76.73)    ≥ 13 education years 1325 (9.57) 1784 (8.72) 3109 (9.07) Retired (%)   No 6897 (49.82) 7614 (37.23) 14,511 (42.31) <  0.001   Yes 6947 (50.18) 12,836 (62.77) 19,783 (57.69) Smoking index, packet-year (%)   None-smoker 5852 (42.27) 20,390 (99.71) 26,242 (76.52) <  0.001    <  20 2203 (15.91) 38 (0.19) 2241 (6.53)   20~ 3143 (22.70) 17 (0.08) 3160 (9.21)   40~ 2646 (19.11) 5 (0.02) 2651 (7.73) Alcohol consumption (%)   Never 9373 (67.70) 20,299 (99.26) 29,672 (86.52) <  0.001   Former 346 (2.50) 13 (0.06) 359 (1.05)   Current 4125 (29.80) 138 (0.67) 4263 (12.43) Physical activities (%)   Low 6554 (47.34) 4039 (19.75) 10,593 (30.89) <  0.001   Moderate 6599 (47.67) 14,485 (70.83) 21,084 (61.48)   High 691 (4.99) 1926 (9.42) 2617 (7.63) Chronic disease (%)   Diabetes 2027 (14.64) 2723 (13.32) 4750 (13.85) <  0.001   Hypertension 7596 (54.87) 10,015 (48.97) 17,611 (51.35) <  0.001   CKD 929 (6.71) 2660 (13.01) 3589 (10.47) <  0.001   HUA 2300 (16.61) 1794 (8.77) 4094 (11.94) <  0.001 BMI (kg/m2) 24.71 ± 3.20 24.16 ± 3.42 24.38 ± 3.35 <  0.001 TC (mmol/L) 4.79 ± 0.91 5.04 ± 0.94 4.94 ± 0.94 <  0.001 TG (mmol/L) 1.38 (0.99–2.03) 1.32 (0.97–1.85) 1.34 (0.98–1.92) <  0.001 HDL-C (mmol/L) 1.30 ± 0.34 1.48 ± 0.35 1.41 ± 0.36 <  0.001 LDL-C (mmol/L) 2.70 ± 0.81 2.83 ± 0.84 2.78 ± 0.83 <  0.001 Non-HDL-C (mmol/L) 3.49 ± 0.93 3.55 ± 0.95 3.53 ± 0.95 <  0.001 TC/HDL-C ratio 3.92 ± 1.29 3.56 ± 1.08 3.71 ± 1.18 <  0.001 TG/HDL-C ratio 1.08 (0.70–1.82) 0.89 (0.60–1.40) 0.96 (0.63–1.55) <  0.001 LDL-C/HDL-C ratio 2.20 ± 0.84 2.00 ± 0.74 2.08 ± 0.79 <  0.001 Table 1  Comparison of baseline characteristics of participants according to sex Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease Page 5 of 10 incidence density increased by lipid and lipid ratio ter- tiles (all Ptrend <  0.01) (Fig. 2). Baseline characteristics and outcome data Non-HDL-C and LDL-C/HDL-C presented no signifi- cant association with IS risk in the female, < 60 years, and nonhypertension groups. These subgroup variables and lipids demonstrated no interaction effect on IS risk (Pinteraction > 0.05). Dose–response relationship of nontraditional blood lipids with IS Nontraditional lipids were studied for their nonlinear connection with IS (Fig. 4). After controlling for con- founders, the RCS curves presented a nonlinear relation- ship with IS (Pnonlinearity < 0.05) (Fig. 4A–C). When the lipid ratio was > 3.47 (TC/HDL-C), > 0.92 (TG/HDL-C), and > 1.98 (LDL-C/HDL-C), the plot showed increased IS risk. No such nonlinear associations were observed for non-HDL-C (Pnonlinearity > 0.05), but a positive asso- ciation between non-HDL-C and IS was observed (Fig. 4D). Discussionh Lipids in Health and Disease ( Page 6 of 10 Table 2  HRs and 95% CIs of IS by tertile category of lipids a Model 1: adjusted variables: age and sex; Model 2: adjusted variables: age, sex, retirement status, education level, BMI, alcohol consumption, smoking index, Cases/N Ischemic stroke Model ­1a Model ­2a TC, mmol/Lb   T1 (<  4.51) 132/11,358 1.0 (ref) 1.0 (ref)   T2 (4.51–5.24) 144/11,427 1.05 (0.83, 1.32) 1.07 (0.84, 1.36)   T3 (≥ 5.25) 182/11,509 1.31 (1.05, 1.65) 1.32 (1.03, 1.68) Ptrend 0.015 0.022 TG, mmol/Lc   T1 (<  1.09) 107/11,206 1.0 (ref) 1.0 (ref)   T2 (1.09–1.66) 149/11,555 1.32 (1.03, 1.69) 1.22 (0.94, 1.57)   T3 (≥ 1.67) 202/11,533 1.87 (1.48, 2.37) 1.58 (1.22, 2.04) Ptrend <  0.001 <  0.001 HDL-C, mmol/Lc   T1 (<  1.25) 213/11,299 1.0 (ref) 1.0 (ref)   T2 (1.25–1.52) 137/11,292 0.62 (0.50, 0.77) 0.64 (0.51, 0.80)   T3 (≥ 1.53) 108/11,703 0.53 (0.42, 0.68) 0.59 (0.46, 0.76) Ptrend <  0.001 <  0.001 LDL-C, mmol/Lb   T1 (<  2.41) 129/11,330 1.0 (ref) 1.0 (ref)   T2 (2.41–3.06) 137/11,410 1.01 (0.79, 1.28) 1.07 (0.84, 1.36)   T3 (≥ 3.07) 192/11,554 1.32 (1.06, 1.66) 1.36 (1.08, 1.71) Ptrend 0.010 0.007 TC/HDL-C   T1 (<  3.12) 110/11,373 1.0 (ref) 1.0 (ref)   T2 (3.12–3.95) 124/11,446 1.05 (0.82, 1.36) 1.00 (0.77, 1.30)   T3 (≥ 3.96) 224/11,475 1.87 (1.48, 2.35) 1.63 (1.28, 2.07) Ptrend <  0.001 <  0.001 TG/HDL-Cc   T1 (<  0.73) 102/11,228 1.0 (ref) 1.0 (ref)   T2 (0.73–1.29) 147/11,615 1.32 (1.03, 1.70) 1.20 (0.92, 1.55)   T3 (≥ 1.30) 209/11,451 1.98 (1.56, 2.51) 1.65 (1.28, 2.13) Ptrend <  0.001 <  0.001 LDL-C/HDL-Cd   T1 (<  1.70) 113/11,426 1.0 (ref) 1.0 (ref)   T2 (1.70–2.29) 116/11,345 0.99 (0.76, 1.28) 0.93 (0.72, 1.22)   T3 (≥ 2.30) 229/11,523 1.78 (1.42, 2.23) 1.51 (1.18, 1.92) Ptrend <  0.001 <  0.001 Non-HDL-C, mmol/L   T1 (<  3.09) 116/11,276 1.0 (ref) 1.0 (ref)   T2 (3.09–3.84) 146/11,548 1.18 (0.92, 1.50) 1.13 (0.89, 1.45)   T3 (≥ 3.85) 196/11,470 1.58 (1.26, 2.00) 1.43 (1.13, 1.81) Ptrend <  0.001 0.002 Comparisons with other studies and what does the current work add to the existing knowledge Comparisons with other studies and what does the current work add to the existing knowledge work add to the existing knowledge Atherosclerosis is a fundamental cause of stroke and heart disease, and an inflammatory response caused by lipoprotein is a critical initiating event that promotes ath- erosclerosis development [28]. Discussionh The study revealed that the four nontraditional lipids were all positively related to IS risk. The nontraditional lipids demonstrated a stronger association with IS than TC and LDL-C. The subgroup analysis determined a similar positive association for males, older age, and par- ticipants with hypertension. Furthermore, the RCS anal- ysis showed a nonlinear relationship between the lipid ratios and IS. Fig. 2  Incidence density of IS according to lipids and lipid ratios by tertile category Fig. 2  Incidence density of IS according to lipids and lipid ratios by tertile category Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Discussionh 3  HRs† and 95% CIs of IS by tertile category of lipid variables according to sex. †Adjusted variables: age, retirement status, education level, BMI, alcohol consumption, smoking index, physical activities, hypertension, diabetes, CKD, and HUA. For TG/HDL-C, TC and LDL-C were additionally adjusted. For LDL-C/HDL-C, TG was additionally adjusted Fig. 3  HRs† and 95% CIs of IS by tertile category of lipid variables according to sex. †Adjusted variables: age, retirement status, education level, BMI, alcohol consumption, smoking index, physical activities, hypertension, diabetes, CKD, and HUA. For TG/HDL-C, TC and LDL-C were additionally adjusted. For LDL-C/HDL-C, TG was additionally adjusted the present study provided additional knowledge that the IS risk increased when the TC/HDL-C, TG-HDL-C, and LDL-C/HDL-C ratios were > 3.47, > 0.92, and > 1.98, respectively. To date, statins are applied in clinical prac- tice to lower cholesterol concentrations to reduce CVD events and are often combined with cholesterol absorption inhibitors (such as ezetimibe) and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors [8, 43, 44]. Nev- ertheless, the evidence supports the idea that residual inflammatory risk persists when statins are used in com- bination with PCSK9 inhibitors [44]. The mechanism of the relationship of nontraditional lipid markers with stroke and whether these markers can be used as a new therapeu- tic target should be confirmed by further studies. high-risk populations, is an essential step in primary pre- vention [45]. The positive association between nontradi- tional lipids and stroke and the cutoff found in this study may help determine the high-risk groups for stroke pri- mary prevention and provide a reference for stratifying cardiovascular risk by nontraditional lipid parameters. However, the study presents several limitations. First, data on antilipemic drug usage were not collected, and dietary conditions were not analyzed, which were pos- sible confounders that may have affected the results. Second, part of the history of earlier strokes was self- reported and may have led to the inclusion of participants with earlier minor strokes. Furthermore, the outcomes were collected based on information systems; inevitably, there will be omissions. However, loss to follow-up was minimized, and new IS events were identified through data linkages across these online information systems. Third, only people from Songjiang district, Shanghai, were included, so the generalization of these results to other populations is limited. Finally, the lipid profile was measured only once at baseline. Discussionh It has been proven that lowering LDL-C reduces CVD risks [29, 30], but a few studies have reported that residual cardiovascular risk remained and that low LDL-C showed greater risks of all- cause mortality and intracerebral hemorrhage [31, 32]. Recently, nontraditional lipids were proposed as valuable predicting factors rather than individual lipids for CVD events [33–36]. Some researchers have reported that lipo- protein ratios reflect metabolic and clinical interactions among lipid components, demonstrating the balance between antiatherogenic and proatherogenic lipopro- teins, while an imbalance indicates greater cardiovascular risk [37, 38]. As expected, the findings on the correlation between nontraditional lipids and IS risk are consistent with those of other studies [11–13, 35, 39]. Zheng et al. [11] reported that the four nontraditional blood lipids were related to higher IS risk among hypertensive indi- viduals. Zhou et al. [13] reported a positive association of TG/HDL-C with IS risk among Chinese individuals. The Framingham Study [35] reported that high TC/HDL-C was related to higher IS risk in middle-aged and elderly people (HR, 1.47; 95% CI, 1.15–1.87). The National Lipid Association recommended non-HDL-C as the major pre- vention target of ASCVD [40], and Saito et al. [39] con- cluded that non-HDL-C presented a positive association with large-artery occlusive infarction (HR, 2.05; 95% CI, 1.07–3.93). Nevertheless, some inconsistency exists. Ima- mura et al. [41] reported that the association was not sta- tistically significant between non-HDL-C and IS risk (HR, 1.01; 95% CI, 0.89–1.16). The PRIME Study [14] reported that non-HDL-C, TC/HDL-C, and LDL-C/HDL-C were not robust predictors for IS but were for coronary heart disease only. These inconsistent results may be caused by the differences in study design and method, participants’ characteristics, and the covariates adjusted in the multi- variate models.h The subgroup analysis determined that the posi- tive association was statistically significant, especially in the male, older, and hypertension groups. Older age and hypertension are known risk factors for stroke [4], and previous studies reported that the stroke preva- lence and incidence were higher in males than females [2, 42], indicating that lowering traditional lipids may be more beneficial for stroke prevention in these high-risk groups. No previous studies explored nonlinear rela- tions between lipid ratios and stroke but only reported a U-shaped association of non-HDL-C with stroke [39]. In the analysis exploring the dose–response relationship, Tang et al. Lipids in Health and Disease (2022) 21:72 Page 7 of 10 Fig. Discussionh Therefore, future studies should increase the follow-up duration and increase the number of indicator measurements to better understand the association of serum lipids with stroke, especially nontraditional lipids. HDL-C: High-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triglycer- ide; LDL-C: Low-density lipoprotein cholesterol; RCS: Restricted cubic spline; IS: Ischemic stroke; HR: Hazard ratio; CI: Confidence interval; CVD: Cardiovascular disease; ASCVD: Atherosclerotic cardiovascular disease; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BP: Blood pressure; SSACB: Shanghai Suburban Adult Cohort and Biobank; HbA1c: Glycated hemoglobin; FPG: Fasting plasma glucose; SUA: Serum uric acid; HUA: Hyperuricemia; EMR: Conclusion Th d This study provides evidence that four nontraditional blood lipids are positively associated with IS risk. Main- taining a relatively low lipid ratio can be beneficial for preventing IS occurrence. Therefore, it is recommended that these nontraditional blood lipid parameters be considered in the management of blood lipids in clini- cal practice. The findings of this study may help provide guidelines for stroke primary prevention and provide a reference for stratifying cardiovascular risk by nontradi- tional lipid parameters. Strength and limitations A comprehensive analysis of the relationship between nontraditional lipids and IS was conducted based on a prospective cohort study with strict quality control meas- ures. The study determined the risk threshold of the lipid ratios, as there is currently no acknowledged cutoff for clinical practice. This is the first study to show a non- linear relationship between nontraditional serum lipids and IS risk in community residents in China. In China, population screening for stroke, which is conducted in Tang et al. Lipids in Health and Disease (2022) 21:72 Page 8 of 10 Fig. 4  RCS analysis of the relationship between the four nontraditional blood lipid levels and IS (A–D). Adjusted variables: age, sex, retirement status, education level, BMI, alcohol consumption, smoking index, physical activities, hypertension, diabetes, CKD, and HUA. For TG/HDL-C, TC and LDL-C were additionally adjusted. For LDL-C/HDL-C, TG was additionally adjusted Fig. 4  RCS analysis of the relationship between the four nontraditional blood lipid levels and IS (A–D). Adjusted variables: age, sex, retirement status, education level, BMI, alcohol consumption, smoking index, physical activities, hypertension, diabetes, CKD, and HUA. For TG/HDL-C, TC and LDL-C were additionally adjusted. For LDL-C/HDL-C, TG was additionally adjusted Electronic medical record; ID: Identification; ICD-10: The Tenth Revision of the International Classification of Diseases; CKD: Chronic kidney disease; eGFR: Estimated glomerular filtration rate; MET: Metabolic equivalent task; BMI: Body mass index; PCSK9: Proprotein convertase subtilisin/kexin type 9. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12944-​022-​01683-1. Additional file 1: Fig. S1. HRs and 95% CIs of IS by tertile category of lipid variables in different age groups. Fig. S2. HRs and 95% CIs of IS by tertile category of lipid variables in population with or without hypertension. Acknowledgments ld l k k g We would like to acknowledge all the subjects participating in this study and the staff of Shanghai Songjiang District Community Health Service Center. References GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet Neurol. 2021;20:795–820. 5. Hindy G, Engström G, Larsson SC, Traylor M, Markus HS, Melander O, et al. Stroke genetics network (SiGN). Role of blood lipids in the development of ischemic stroke and its subtypes: a mendelian randomization study. Stroke. 2018;49:820–7. 5. Hindy G, Engström G, Larsson SC, Traylor M, Markus HS, Melander O, et al. Stroke genetics network (SiGN). Role of blood lipids in the development of ischemic stroke and its subtypes: a mendelian randomization study. Stroke. 2018;49:820–7. 25. Lei W, He S, Wang H, Zhou L, Yu Y, Liu K, et al. Association of smoking status with incident cardiovascular disease in the middle-aged and older male populations. Chin J Dis Control Prev. 2019;23:774–9. 26. Craig CL, Marshall AL, Sjöström M, Bauman AE, Booth ML, Ainsworth BE, et al. International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc. 2003;35:1381–95. 6. Zhou M, Wang H, Zeng X, Yin P, Zhu J, Chen W, et al. Mortality, morbid- ity, and risk factors in China and its provinces, 1990–2017: a system- atic analysis for the global burden of disease study 2017. Lancet. 2019;394:1145–58. y 27. Lou G, Li S, Gong Q, Zhu Y, Ying Y, Wang Y, et al. Association between physical activity and risk of stroke among adults aged 40 years and above: a prospective cohort study. Chin J Epidemiol. 2021;42:1030–6. 7. Grundy SM, Stone NJ, Bailey AL, Beam C, Birtcher KK, Blumenthal RS, et al. 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APHA/ASPC/NLA/ PCNA guideline on the management of blood cholesterol: a report of the American college of cardiology/American Heart Association task force on clinical practice guidelines. Circulation. 2019;139:e1082–143. 28. Linton MF, Yancey PG, Davies SS, Jerome WG, Linton EF, Song WL, et al. The role of lipids and lipoproteins in atherosclerosis. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, Dungan K, Hershman JM, Hofland J, Kalra S, et al., editors. Endotext. South Dart- mouth: MDText.com, Inc.; 2000. Updated 2019 Jan 3.fi 8. Joint committee issued Chinese guideline for the management of dyslipidemia. Chinese guideline for the management of dyslipidemia in adults. Chin J Gen Pract. 2016;2017(11):7–28. 29. Gencer B, Marston NA, Im K, Cannon CP, Sever P, Keech A, et al. References 1. Sacco RL, Kasner SE, Broderick JP, Caplan LR, Connors JJ, Culebras A, et al. An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2013;44:2064–89. 1. Sacco RL, Kasner SE, Broderick JP, Caplan LR, Connors JJ, Culebras A, et al. An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2013;44:2064–89. 21. Writing Group of 2018 Chinese Guidelines for the Management of Hyper- tension. 2018 Chinese guidelines for the management of hypertension. Chin J Cardiovasc Med. 2019;24:24–56. 22. Chinese Diabetes Society. Guideline for the prevention and treatment of type 2 diabetes mellitus in China (2020 edition). Chin J Diabetes Mellitus. 2021;37:311–98. 2. Report on stroke prevention and treatment in China Writing Group. Brief report on stroke prevention and treatment in China, 2020. Chin J Cerebrovasc Dis. 2022;19:136–44. 2. Report on stroke prevention and treatment in China Writing Group. Brief report on stroke prevention and treatment in China, 2020. Chin J Cerebrovasc Dis. 2022;19:136–44. 23. National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis. 2002;39:S1–266. 3. The writing Committee of the Report on cardiovascular health and dis- eases in China. Report on cardiovascular health and diseases burden in China: an updated summary of 2020. Chin Circ J. 2021;36:521–45. 3. The writing Committee of the Report on cardiovascular health and dis- eases in China. Report on cardiovascular health and diseases burden in China: an updated summary of 2020. Chin Circ J. 2021;36:521–45. 4. GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet Neurol. 2021;20:795–820. 3. The writing Committee of the Report on cardiovascular health and dis- eases in China. Report on cardiovascular health and diseases burden in China: an updated summary of 2020. Chin Circ J. 2021;36:521–45. 4. GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet Neurol. 2021;20:795–820. 24. Borghi C, Domienik-Karłowicz J, Tykarski A, Widecka K, Filipiak KJ, Jag- uszewski MJ, et al. Expert consensus for the diagnosis and treatment of patient with hyperuricemia and high cardiovascular risk: 2021 update. Cardiol J. 2021;28:1–14. 4. Funding 12. Liu X, Yan L, Xue F. The associations of lipids and lipid ratios with stroke: a prospective cohort study. J Clin Hypertens (Greenwich). 2019;21:127–35. g This study was funded by the Local High Level Discipline Construction Project of Shanghai, the National Key Research and Development Program of China (2017YFC0907000), and the Shanghai New Three-year Action Plan for Public Health (Grant No. GWV-10.1-XK16). 13. Zhou L, Mai J, Li Y, Guo M, Wu Y, Gao X, et al. Triglyceride to high-density lipoprotein cholesterol ratio and risk of atherosclerotic cardiovascular dis- ease in a Chinese population. Nutr Metab Cardiovasc Dis. 2020;30:1706–13. 14. Canouï-Poitrine F, Luc G, Bard JM, Ferrieres J, Yarnell J, Arveiler D, et al. Relative contribution of lipids and apolipoproteins to incident coronary heart disease and ischemic stroke: the PRIME study. Cerebrovasc Dis. 2010;30:252–9. Availability of data and materials The dataset used and analyzed during the current study is available from the corresponding author on reasonable request. 15. Lee H, Park JB, Hwang IC, Yoon YE, Park HE, Choi SY, et al. Association of four lipid components with mortality, myocardial infarction, and stroke in statin-naïve young adults: a nationwide cohort study. Eur J Prev Cardiol. 2020;27:870–81. Consent for publication Not applicable. 19. Guo X, Li Z, Sun G, Guo L, Zheng L, Yu S, et al. Comparison of four nontra- ditional lipid profiles in relation to ischemic stroke among hypertensive Chinese population. Int J Cardiol. 2015;201:123–5. Competing interests The authors have no competing interests to declare. 20. Wang YJ, Li ZX, Gu HQ, Zhai Y, Jiang Y, Zhao XQ, et al. China stroke statistics 2019: a report from the National Center for healthcare quality Manage- ment in Neurological Diseases, China National Clinical Research Center for neurological diseases, the Chinese Stroke Association, National Center for chronic and non-communicable disease control and prevention, Chinese Center for Disease Control and Prevention and institute for global neuro- science and stroke collaborations. Stroke Vasc Neurol. 2020;5:211–39. Received: 28 May 2022 Accepted: 28 July 2022 Received: 28 May 2022 Accepted: 28 July 2022 Received: 28 May 2022 Accepted: 28 July 2022 Authors’ contributions MT, QZ, and KY conceived and designed the research; YW, SC, and XS con- tributed to the collection and assembly of data; YX and YY contributed to data analysis and interpretation; MT and SC drafted the manuscript; GZ and YJ supervised the study and revised the manuscript; and all authors read and approved the final version of the manuscript to be submitted. Page 9 of 10 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease Ethics approval and consent to participate 16. Kojta I, Chacińska M, Błachnio-Zabielska A. Obesity, bioactive lipids, and adipose tissue inflammation in insulin resistance. Nutrients. 2020;12:1305. The Medical Research Ethics Committee of the School of Public Health, Fudan University, approved the ethical review of this study (IRB#2016-04-0586). All participants provided written informed consent before the investigation and examination. 17. Neeland IJ, Poirier P, Després JP. Cardiovascular and metabolic heteroge- neity of obesity: clinical challenges and implications for management. Circulation. 2018;137:1391–406. 18. Zhao Q, Chen B, Wang R, Zhu M, Shao Y, Wang N, et al. Cohort profile: protocol and baseline survey for the Shanghai suburban adult cohort and biobank (SSACB) study. BMJ Open. 2020;10:e035430. References Efficacy and safety of lowering LDL cholesterol in older patients: a systematic review and meta-analysis of randomised controlled trials. Lancet. 2020;396:1637–43. 9. Sirimarco G, Labreuche J, Bruckert E, Goldstein LB, Fox KM, Rothwell PM, et al. PERFORM and SPARCL investigators and committees. Atherogenic dyslipidemia and residual cardiovascular risk in statin-treated patients. Stroke. 2014;45:1429–36. 30. Sabatine MS, Wiviott SD, Im K, Murphy SA, Giugliano RP. Efficacy and safety of further lowering of low-density lipoprotein cholesterol in patients starting with very low levels: a Meta-analysis. JAMA Cardiol. 2018;3:823–8. 10. Lim S, Park YM, Sakuma I, Koh KK. How to control residual cardiovascular risk despite statin treatment: focusing on HDL-cholesterol. Int J Cardiol. 2013;166:8–14. 31. Lu JM, Wu MY, Yang ZM, Zhu Y, Li D, Yu ZB, et al. Low LDL-C levels are associated with risk of mortality in a Chinese cohort study. Endocrine. 2021;73:563–72. 11. Zheng J, Sun Z, Zhang X, Li Z, Guo X, Xie Y, et al. Non-traditional lipid profiles associated with ischemic stroke not hemorrhage stroke in hyper- tensive patients: results from an 8.4 years follow-up study. Lipids Health Dis. 2019;18:9. 32. Ma C, Gurol ME, Huang Z, Lichtenstein AH, Wang X, Wang Y, et al. Low- density lipoprotein cholesterol and risk of intracerebral hemorrhage: a prospective study. Neurology. 2019;93:e445–57. Page 10 of 10 Tang et al. Lipids in Health and Disease (2022) 21:72 Tang et al. Lipids in Health and Disease (2022) 21:72 33. Barzi F, Patel A, Woodward M, Lawes CM, Ohkubo T, Gu D, et al. Asia Pacific cohort studies collaboration. A comparison of lipid variables as predic- tors of cardiovascular disease in the Asia Pacific region. Ann Epidemiol. 2005;15:405–13. 34. Wang G, Jing J, Wang A, Zhang X, Zhao X, Li Z, et al. Non-high-density lipoprotein cholesterol predicts adverse outcomes in acute ischemic stroke. Stroke. 2021;52:2035–42. 35. Pikula A, Beiser AS, Wang J, Himali JJ, Kelly-Hayes M, Kase CS, et al. Lipid and lipoprotein measurements and the risk of ischemic vascular events: framingham study. Neurology. 2015;84:472–9. 36. Turak O, Afşar B, Ozcan F, Öksüz F, Mendi MA, Yayla Ç, et al. The role of plasma triglyceride/high-density lipoprotein cholesterol ratio to predict new cardiovascular events in essential hypertensive patients. J Clin Hypertens (Greenwich). 2016;18:772–7. y 37. Millán J, Pintó X, Muñoz A, Zúñiga M, Rubiés-Prat J, Pallardo LF, et al. Lipoprotein ratios: physiological significance and clinical usefulness in cardiovascular prevention. Vasc Health Risk Manag. 2009;5:757–65. 38. References Zhu L, Lu Z, Zhu L, Ouyang X, Yang Y, He W, et al. Lipoprotein ratios are better than conventional lipid parameters in predicting coronary heart disease in Chinese Han people. Kardiol Pol. 2015;73:931–8. 39. Saito I, Yamagishi K, Kokubo Y, Yatsuya H, Iso H, Sawada N, et al. Non-high- density lipoprotein cholesterol and risk of stroke subtypes and coronary heart disease: the Japan public health center-based prospective (JPHC) study. J Atheroscler Thromb. 2020;27:363–74. 40. Jacobson TA, Ito MK, Maki KC, Orringer CE, Bays HE, Jones PH, et al. National lipid association recommendations for patient-centered man- agement of dyslipidemia: Part 1--full report. J Clin Lipidol. 2015;9:129–69. 41. Imamura T, Doi Y, Ninomiya T, Hata J, Nagata M, Ikeda F, et al. Non-high- density lipoprotein cholesterol and the development of coronary heart disease and stroke subtypes in a general Japanese population: the Hisayama study. Atherosclerosis. 2014;233:343–8. 42. Carcel C, Woodward M, Wang X, Bushnell C, Sandset EC. Sex matters in stroke: a review of recent evidence on the differences between women and men. Front Neuroendocrinol. 2020;59:100870. 43. Michos ED, McEvoy JW, Blumenthal RS. Lipid Management for the Prevention of atherosclerotic cardiovascular disease. N Engl J Med. 2019;381:1557–67. 44. Pradhan AD, Aday AW, Rose LM, Ridker PM. Residual inflammatory risk on treatment with PCSK9 inhibition and statin therapy. Circulation. 2018;138:141–9. 45. Chao BH, Yan F, Hua Y, Liu JM, Yang Y, Ji XM, et al. Stroke prevention and control system in China: CSPPC-stroke program. Int J Stroke. 2021;16:265–72. • 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: Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • 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:
https://openalex.org/W2921422638
https://childshealth.zaslavsky.com.ua/index.php/journal/article/download/599/719
Russian
null
Бронхиты у детей и выбор противокашлевой терапии
Zdorovʹe rebenka
2,022
cc-by
6,032
Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician УДК 616.233-002-053.2-08:615.233 ÞËÈØ Å.È., Èçðàèëü ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ Резюме. В работе представлен обзор наиболее часто встречающихся форм бронхитов у детей, их кли- нической симптоматики и подходов к диагностике. Рассмотрены механизмы формирования синдрома кашля и методы его лечения. Ключевые слова: бронхиты, кашель, дети. инфекции оказывает существенное влияние на характер поражения слизистой оболочки. Так, для парагриппа, гриппа, цитомегаловирусной ин- фекции характерны дистрофия и деструкция эпи- телия дыхательных путей с отторжением целых слоев. Для респираторно-синцитиальной (РС) вирусной инфекции — гиперплазия эпителия мельчайших бронхов и бронхиол, подушкообраз- ное разрастание эпителия с нарушением бронхи- альной проводимости. Именно с этим связывают факт развития при РС-вирусной инфекции об- структивного процесса в бронхах. Аденовирусная инфекция сопровождается выраженным экссу- дативным компонентом, нередко наблюдаются слизистые наложения, разрыхление и отторжение эпителия, имеет место образование в стенке брон- ха крупноклеточных инфильтратов. Это способ- ствует формированию ателектазов и обструкции дыхательных путей. Бронхиты — одно из наиболее частых прояв- лений ОРВИ у детей. Это связано с тесным кон- тактом органов дыхания с воздушной средой и находящимися в ней патогенами — инфекцион- ными и неинфекционными. Развивающийся при неблагоприятных условиях воспалительный про- цесс в стенках бронхов с раздражением нервных элементов служит причиной ведущего симптома заболевания — синдрома кашля. Частота бронхитов среди детей грудного воз- раста составляет, по данным различных авторов, от 70 до 220 случаев на 1000 детей. Более половины больных, госпитализированных в респираторные отделения, — это дети с бронхитами. Обструктив- ные формы бронхита наблюдаются с частотой 42,7 случая на 1000 детей первого года жизни [1, 2]. Острым бронхитом, согласно современной классификации, называется острое воспаление слизистой оболочки бронхов без признаков по- ражения легочной ткани. Хронический бронхит диагностируется при поражении всех слоев брон- хиальной стенки. Неинфекционные причины заболевания пред- ставлены ингаляционными аллергенами, раз- личными ирритантами и поллютантами (если ребенок проживает в промышленных зонах) и та- бачным дымом (если курящие члены семьи курят в присутствии ребенка — пассивное курение). Этиология. Инфекционные агенты представ- лены главным образом респираторными виру- сами, вирусно-бактериальными ассоциациями, реже — бактериями. Среди вирусных возбудите- лей наиболее частыми являются вирусы гриппа, парагриппа, аденовирусы, респираторно-синци- тиальный, корона- и риновирус, ECHO- и Кок- саки-вирус. Среди бактериальных возбудителей в настоящее время лидируют Chlamidia pneumonia, Streptococcus pneumoniae, Haemophilus influenzae и Moraxella catarrhalis [3]. Патогенез. Воздействие патогенов вызывает отек слизистой оболочки бронхов, гиперсекре- Адрес для переписки с автором: Юлиш Евгений Исаакович E-mail: yulish.yevgeniy@gmail.com © Юлиш Е.И., 2015 © «Здоровье ребенка», 2015 © Заславский А.Ю., 2015 © Юлиш Е.И., 2015 © «Здоровье ребенка», 2015 © Заславский А.Ю., 2015 Наиболее частой причиной (до 90 %) острого бронхита у детей является вирус. Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician ® Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician ® ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ Вид вирусной 133 ¹ 2 (61) • 2015 www.mif-ua.com Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician цию бронхиальной слизи и в ряде случаев — брон- хоспазм. Если эти процессы имеют место в мелких дыхательных путях, развивается генерализован- ное нарушение бронхиальной проходимости. По- скольку суммарная площадь сечения мелких бронхов велика, нарушается альвеолярная вен- тиляция и развивается дыхательная недостаточ- ность — картина обструктивного бронхита. При поражении крупных бронхов и трахеи признаки дыхательной недостаточности не выражены — острый простой бронхит. цию бронхиальной слизи и в ряде случаев — брон- хоспазм. Если эти процессы имеют место в мелких дыхательных путях, развивается генерализован- ное нарушение бронхиальной проходимости. По- скольку суммарная площадь сечения мелких бронхов велика, нарушается альвеолярная вен- тиляция и развивается дыхательная недостаточ- ность — картина обструктивного бронхита. При поражении крупных бронхов и трахеи признаки дыхательной недостаточности не выражены — острый простой бронхит. ризуется катаральными явлениями. Вирусы раз- множаются и повреждают эпителий, вызывая его дистрофию, нарушение связей между отдельными клетками, их отторжение. В просвете бронхов по- является серозный экссудат с примесью макрофа- гов, лейкоцитов и эпителиальных клеток. Тяжесть инфекционного токсикоза (лихорад- ка, ухудшение состояния и т.д.) при ОБ зависит от токсигенных свойств вируса. Возникновению проявлений ОБ предшествуют признаки ОРВИ: повышение температуры тела, головная боль, слабость, явления ринита, фарингита, иногда ла- рингита, трахеита (саднение и боль за грудиной, сухой болезненный кашель) и конъюнктивита. Из органоспецифических симптомов на первый план выходит кашель, сначала сухой, затем влажный. В тех случаях, когда ребенок умеет откашливать мокроту, она не бывает гнойной. Одышка не ха- рактерна для этой формы бронхита. Отклонения при перкуссии грудной клетки не отмечаются. В легких выслушиваются рассеянные влажные раз- нокалиберные хрипы, постепенно уменьшающи- еся в количестве. Изменения в анализе перифери- ческой крови часто отсутствуют. Повреждение бронхиального эпителия ве- дет к нарушению выработки слизистого секрета, движения ресничек и очищения (мукоцилиарно- го клиренса) бронхов. В результате активируется патогенная флора, обусловливающая появление слизисто-гнойной мокроты. Выздоровление за- висит от регенерации эпителия бронхов и обычно происходит на 3-й неделе заболевания и позднее. Симптомы бронхита могут быть объединены в две группы. При инфекционной этиологии брон- хита имеют место признаки инфекционного ток- сикоза: повышение температуры тела, ухудшение состояния ребенка, головная боль, диспептиче- ские явления (у грудных детей) и др. Вторую груп- пу составляют органоспецифические симптомы: кашель (сухой или влажный) — ведущий симптом заболевания, отхаркивание мокроты (детьми старшего возраста), разнообразные по калибру и звучанию хрипы, выслушиваемые на всем про- странстве обоих легких. ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ Отсутствие дыхательной недостаточности, экспираторной одышки, бронхообструкции, эмфизематоза при 134 ¹ 2 (61) • 2015 ¹ 2 (61) • 2015 Çäîðîâüå ðåáåíêà, ISSN 2224-0551 Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician рентгенологическом обследовании и отягощен- ного аллергологического анамнеза. рентгенологическом обследовании и отягощен- ного аллергологического анамнеза. торной вирусной инфекции. Бронхообструктив- ный синдром обычно развивается на 3–4-й день ОРВИ и проявляется экспираторной одышкой, шумным свистящим дыханием, рассеянными сухи- ми и разнокалиберными влажными хрипами в лег- ких. Рецидивы ООБ связаны с вирусными инфек- циями и обычно прекращаются к возрасту 3–4 лет. Острый стенозирующий ларинготрахеит. От- сутствие стридора, дыхательной недостаточности и инспираторной одышки. ОБ, вызванный Mycoplasma pneumoniae. Наблю- дается чаще у детей школьного возраста, симпто- мы инфекционного токсикоза выражены неярко. Перкуторные изменения не характерны. Аускуль- тативная картина более разнообразна: могут отме- чаться удлинение выдоха, сухие свистящие хри- пы, по-разному выраженные в различных отделах обоих легких. Рентгенологическая картина может напоминать таковую при микоплазменной пнев- монии: значительное усиление легочного рисунка за счет периваскулярного уплотнения интерсти- ция. Этиологическая диагностика возможна по выявлению ДНК микоплазмы методом ПЦР и ре- троспективно — по нарастанию титра специфиче- ских IgM и G, отмечаемых нередко после клини- ческого выздоровления. Этиотропная терапия — с использованием антибиотиков (макролидов). Воспалительные изменения в бронхах, возни- кающие главным образом при вирусной инфек- ции, поражают бронхи мелкого калибра. Отек, ги- персекреция и бронхоспазм вызывают нарушение бронхиальной проходимости и альвеолярной вен- тиляции, следствием чего является дыхательная недостаточность — существенный отличительный признак ООБ. Это, по сути, инфекционный, а не аллергический бронхит. Этиологически ООБ обычно связывают с ре- спираторно-синцитиальными вирусами, группой герпесвирусов, вирусом парагриппа 3-го типа. Кроме того, возможна инфекция вирусами грип- па, адено- и риновирусами. При повторных об- структивных бронхитах в смывах из бронхов часто выявляются ДНК персистирующих инфекций — герпесвирусов, хламидий, микоплазм [5]. ОБ, вызванный Haemophilus influenzae. Редко выступает в качестве заболевания sui generis. Как и другие бактериальные процессы в бронхах, чаще имеет место при анатомически обусловленных поражениях бронхиального дерева: аспирации инородного тела, стенозах гортани, после интуба- ции или декануляции, пороках развития бронхов, нарушении мукоцилиарного клиренса или других патологических состояниях, сопровождающихся нарушением дренажной функции бронхов. ОБ, вызванный Haemophilus influenzae. Редко выступает в качестве заболевания sui generis. Как и другие бактериальные процессы в бронхах, чаще имеет место при анатомически обусловленных поражениях бронхиального дерева: аспирации инородного тела, стенозах гортани, после интуба- ции или декануляции, пороках развития бронхов, нарушении мукоцилиарного клиренса или других патологических состояниях, сопровождающихся нарушением дренажной функции бронхов. ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ В периферической крови изменения или отсутствуют, или имеют характе- ристики, свойственные вирусной или бактери- альной инфекции, если они послужили причиной заболевания ребенка бронхитом. Длительность болезни в большинстве случаев не более 7–10 дней, хотя кашлевой синдром может длиться две- три недели. Клинические критерии диагностики острого простого бронхита: Клинические критерии диагностики острого простого бронхита: — кашель со скудным, а затем более обильным отхождением мокроты; — аускультативные данные — рассеянные су- хие хрипы (реже непостоянные влажные средне- и крупнопузырчатые); — перкуторный звук обычно не изменен; — отсутствие выраженной дыхательной недо- статочности. Параклинические данные: Параклинические данные: — рентгенологические данные — усиление ле- гочного рисунка в прикорневых и базальных от- делах легких; — в анализе крови незначительные воспали- тельные изменения (значительный лейкоцитоз не характерен), умеренное повышение СОЭ. — в анализе крови незначительные воспали- тельные изменения (значительный лейкоцитоз не характерен), умеренное повышение СОЭ. Оценка клинической картины бронхитов не представляет трудности. В ряде случаев необхо- дима дифференциальная диагностика с пневмо- нией. Рентгенологическое исследование грудной клетки не показано, если не возникает сомнений в наличии у ребенка бронхита. Этиологическая диагностика затруднительна. Выявление бакте- рий в верхних дыхательных путях или ротоглотке нельзя считать этиологически значимым, так как они могут быть проявлением носительства. Опре- деление в мокроте ДНК микоплазмы или хлами- дии и нарастание титра специфических антител можно считать свидетельством их этиологической роли в развитии заболевания. Длительность заболевания в неосложненных случаях составляет от 1 до 1,5–2 недель. Более за- тяжным течением отличаются бронхиты, вызван- ные аденовирусной, микоплазменной и хламидо- фильной инфекцией. Длительность заболевания в неосложненных случаях составляет от 1 до 1,5–2 недель. Более за- тяжным течением отличаются бронхиты, вызван- ные аденовирусной, микоплазменной и хламидо- фильной инфекцией. Дифференциальную диагностику ОБ проводят со следующими нозологическими единицами: Пневмония. Основные опорные критерии: от- сутствие локальных перкуторных и аускультатив- ных изменений, дыхательной недостаточности, изменения локализации хрипов после кашля, вос- палительных сдвигов в крови при лабораторном исследовании, очагово-инфильтративных изме- нений в легочной ткани при рентгенографии. При подозрении на пневмонию обязательно проведе- ние рентгенологического исследования. В последние десятилетия признано целесо- образным выделять бронхиты без бронхиальной обструкции — простой бронхит и обструктивный бронхит [4]. Острый (простой) бронхит (ОБ). ОБ в 90 % слу- чаев имеет вирусную этиологию. Возникает, как правило, при воздействии вирусов, имеющих тро- пизм к эпителию дыхательных путей, и характе- Острый обструктивный бронхит. ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ Клиническая картина характеризуется острым началом, складывается из симптомов инфекцион- ного токсикоза и бронхообструктивного синдрома (экспираторная одышка, сухой кашель, коробоч- ный оттенок перкуторного звука над легкими, при аускультации — удлиненный выдох, сухие свистя- щие хрипы на всем протяжении обоих легких). Выраженность и распространенность цианоза кожи зависят от тяжести бронхообструктивного синдрома. В периферической крови может при- сутствовать умеренный лейкоцитоз. СОЭ обычно в норме. Рентгенологическое исследование по- казано только для исключения пневмонии. Если оно производится, то на рентгенограмме опре- деляется усиление легочного рисунка, признаки вздутия легочной ткани (повышенная прозрач- ность, горизонтальное положение ребер, удли- нение легочных полей, сужение срединной тени, высокое стояние и уплощение куполов диафраг- мы) при отсутствии инфильтративных и очаговых теней в легких. Возможно выявление сегментар- ных ателектазов, исчезающих после купирования бронхообструкции. ру р фу р ОБ, вызванный Chlamidia. У детей грудного воз- раста этиологическое значение в большей степени имеет Ch.trachomatis. Признаки тяжелого инфек- ционного токсикоза не характерны. Бронхит от- личается длительным кашлем, продолжающимся несколько недель, нередко приступообразным, коклюшеподобным, но без «реприз». В легких вы- слушиваются разнокалиберные влажные хрипы. Рентгенологически могут выявляться изменения, характерные для пневмонии: усиление легочного рисунка и инфильтратоподобные тени. Инфици- рование детей чаще происходит от матерей, име- ющих урогенитальный хламидиоз, что может быть использовано в этиологической диагностике хла- мидийной инфекции у детей. Острый бронхиолит. Наиболее тяжелая фор- ма острого бронхообструктивного заболевания. Развивается, как правило, у детей первого полу- годия на фоне ОРВИ и проявляется выраженным бронхообструктивным синдромом и дыхательной недостаточностью. Поскольку чаще вызывается малотоксигенными вирусами (РС- и вирусами парагриппа 3-го типа), симптомы инфекцион- ного токсикоза выражены нерезко. Преобладают в клинике заболевания признаки тяжелой дыха- тельной недостаточности. У детей старшего возраста хламидийный брон- хит чаще обусловлен Chlamidia pneumonia и также характеризуется длительным кашлевым перио- дом (до нескольких месяцев), который, однако, в большей степени обусловлен фарингитом, чем поражением бронхов. В ряде случаев отмечается бронхообструктивный компонент в клинической картине бронхита. У детей первых трех лет жизни бронхит может протекать с синдромом бронхиальной обструк- ции — как острый обструктивный бронхит (ООБ), характеризующийся диффузным поражением бронхов разного калибра на фоне острой респира- Патологические изменения локализуются в са- мых мелких бронхах — бронхиолах. Преобладает 135 ¹ 2 (61) • 2015 www.mif-ua.com Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician отек и гиперсекреция, вследствие чего возникает труднокупируемое нарушение их проходимости. Состояние, как правило, очень тяжелое. Харак- терна выраженная одышка экспираторного или смешанного характера с участием вспомогатель- ной мускулатуры, втяжением уступчивых мест грудной клетки, раздуванием крыльев носа, циа- нозом; диффузные влажные мелкопузырчатые и крепитирующие хрипы. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. Течение рецидивирующего бронхита обычно ограничивается 2–3 годами. У трети больных ре- цидивы РБ прекращаются к школьному возрасту, у детей с хронической лор-патологией могут быть несколько дольше — до 7–9 лет. РБ у детей может быть маркером хронического бронхолегочного заболевания. Важным диффе- ренциально-диагностическим признаком в этих случаях должно быть отсутствие клинико-брон- хологических признаков пневмосклероза и де- формации бронхов. Рекуррентный (повторный) обструктивный брон- хит (РОБ). Наличие повторных эпизодов брон- хообструкции связывают с более выраженной гиперреактивностью бронхов у этих больных, что обычно обусловлено персистирующими инфек- циями [6, 7]. Чувствительность к ингаляционным неинфекционным аллергенам у них выражена меньше, чем у детей, больных бронхиальной аст- мой. Вместе с тем без соответствующего этиотроп- ного лечения дети с РОБ входят в группу риска по развитию бронхиальной астмы. В подавляющем большинстве случаев рецидивы бронхиальной об- струкции прекращаются в возрасте 3–5 лет после проведения этиотропной противоинфекционной терапии. Рецидивирующие бронхиты (РБ). Рецидивиру- ющим называют бронхит, эпизоды которого при заболевании ОРВИ наблюдаются 2–3 раза в год в течение 1–2 и более лет подряд. Рецидивы брон- хита связаны с респираторной вирусной инфек- цией. Болеют дети первых 5 лет жизни. Частота РБ у детей 2–3 лет составляет 40–50 случаев на 1000 человек, 4–6 лет — 50–75 на 1000. В экологиче- ски неблагоприятных зонах может достигать 250 на 1000. р Клиника болезни складывается из повторных эпизодов бронхообструкции, обычно несколь- ко раз в год. У больных, как правило, отсутству- ет аллергическая наследственность или кожные проявления аллергии. У детей с аллергической реактивностью эпизоды обструктивного бронхита могут быть чаще. Продолжение бронхообструк- тивных эпизодов, особенно не связанных с респи- раторной вирусной инфекцией и после 3–4-лет- него возраста, следует рассматривать как развитие бронхиальной астмы. При наличии у ребенка ги- периммуноглобулинемии Е частота трансформа- ции рекуррентных форм обструктивного бронхита в бронхиальную астму может достигать 45–50 %. Этиологически рецидивирующие бронхиты связаны чаще всего с адено- и РС-вирусами, ви- русами парагриппа, герпесвирусами, хламидо- фильной инфекцией. Вовлечение бронхов при заболевании ОРВИ, вызванными указанными ви- русами, связывают с повышенной реактивностью бронхов, присущей детям указанной возрастной группы, неблагоприятной экологической средой обитания, с легочной дисплазией при рождении. Гиперреактивность бронхов не достигает степени, приводящей к бронхообструкции, поскольку пре- обладает гиперсекреция и отек слизистой оболоч- ки без спазма гладкой мускулатуры бронхов. Рецидивы обратимой бронхиальной обструк- ции в детском возрасте могут быть связаны с ря- дом заболеваний. Показана дифференциальная диагностика с муковисцидозом, рецидивами аспирации жидкой пищи (у детей грудного воз- раста), с некоторыми пороками развития брон- хов — синдромом Мунье — Куна, синдромом Ви- льямса — Кэмпбелла и др. ÁÐÎÍÕÈÒÛ Ó ÄÅÒÅÉ È ÂÛÁÎÐ ÏÐÎÒÈÂÎÊÀØËÅÂÎÉ ÒÅÐÀÏÈÈ Отмечается коробочный оттенок перкуторного звука над легкими, при аускультации удлиненный выдох, преобладают рассеянные влажные мелкопузырчатые хрипы. Такая картина дает основание нередко ставить таким больным диагноз пневмонии. Однако при остром бронхиолите изменения в перифериче- ской крови соответствуют таковым при вирусной инфекции. Рентгенологическая картина характе- ризуется признаками бронхообструкции, выяв- ляется резкое вздутие легочной ткани, обеднение сосудистого рисунка. Сегментарные ателектазы не характерны. Мелкоочаговость может быть об- условлена, по-видимому, ацинарными ателекта- зами. При исследовании газов крови отмечаются гипоксия и гиперкапния. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. У данной категории детей почти каждое забо- левание ОРВИ сопровождается признаками брон- хита. Частота таких эпизодов может достигать 3–4 и более в год. Ведущими симптомами являют- ся кашель и хрипы в легких — разнокалиберные влажные и сухие. Характерна большая длитель- ность этих симптомов — до 3–4 недель — по срав- нению с ОБ. Нормализация температуры и ликви- дация катаральных явлений со стороны верхних дыхательных путей происходят раньше. Анализы периферической крови соответствуют таковым при ОРВИ. Рентгенологически определяется уси- ление легочного рисунка, которое может сохра- Аспирационный бронхит. Эта разновидность бронхита чаще наблюдается у детей грудного воз- раста и бывает связана с аспирацией жидкой пищи (молоко, жидкие смеси — заменители грудного молока). Поскольку аспирация носит повторный характер, аспирационный бронхит характеризует- 136 ¹ 2 (61) • 2015 Çäîðîâüå ðåáåíêà, ISSN 2224-0551 ¹ 2 (61) • 2015 Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician ся рецидивами бронхита. Причина, как правило, связана либо с нарушением акта глотания (парез мягкого неба, надгортанника), либо с гастро- эзофагеальным рефлюксом (ГЭР). Более редкие причины — трахеопищеводный свищ, халазия или ахалазия пищевода, анатомические дефекты (незаращение) мягкого и твердого неба, диафраг- мальная грыжа, аномалии крупных внутригруд- ных сосудов. ки бронхов и более глубоких слоев бронхиальной стенки, характеризующееся длительным течени- ем с периодическими обострениями. По реко- мендации экспертов ВОЗ «…к больным ХБ отно- сят лиц, у которых имеется кашель с мокротой не менее трех месяцев в году в течение двух лет при исключении других заболеваний верхних дыха- тельных путей, бронхов и легких, которые могли бы вызвать эти симптомы». Установлено, что, на- чавшись в детстве, заболевание продолжается и в зрелом возрасте [8–10]. ки бронхов и более глубоких слоев бронхиальной стенки, характеризующееся длительным течени- ем с периодическими обострениями. По реко- мендации экспертов ВОЗ «…к больным ХБ отно- сят лиц, у которых имеется кашель с мокротой не менее трех месяцев в году в течение двух лет при исключении других заболеваний верхних дыха- тельных путей, бронхов и легких, которые могли бы вызвать эти симптомы». Установлено, что, на- чавшись в детстве, заболевание продолжается и в зрелом возрасте [8–10]. Слизистая оболочка бронхов реагирует на раздражение ее пищей и на кислое содержимое желудка (при ГЭР) воспалением — отеком и ги- персекрецией, а при массивной аспирации — бронхоспазмом (синдром Мендельсона). няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. Если исключить из этого определения ВОЗ за- болевания верхних дыхательных путей (фарин- гиты, воспаления околоносовых пазух и др.), за- болевания бронхов, обусловленные пороками их развития (синдромы Мунье — Куна и Вильям- са — Кэмпбелла, цилиарная дискинезия и др.), хронические патологические процессы в легких (бронхоэктатическая болезнь), то можно говорить только о вторичных хронических воспалительных процессах в бронхах у детей, например при муко- висцидозе, иммунодефицитных состояниях и др. Клиника характеризуется продуктивным каш- лем и появлением влажных разнокалиберных и сухих, иногда свистящих (при бронхоспазме) хри- пов на всем протяжении обоих легких. Появление хрипов при аускультации легких или поперхива- ния после кормления свидетельствует об аспира- ции. Рецидивы бронхита при аспирации жидкой пищи могут начинаться и протекать без повы- шения температуры и признаков инфекционного токсикоза. у ф р Патогенез ХБ у детей сложный. Определяющее значение в формировании заболевания имеют ин- фекционные факторы. При воздействии вирусов на незрелые тканевые структуры развитие хро- нического воспаления в бронхах возможно уже в раннем детском возрасте [11]. Острые респира- торные вирусные инфекции способствуют присо- единению бактериального воспаления. Ведущими возбудителями воспалительного процесса в на- стоящее время считаются Haemophilus influenzae, Strеptococcus pneumoniae и Moraxella catarrhalis [12]. Размножение микроорганизмов способствует дальнейшему прогрессированию воспаления как за счет самостоятельного повреждения структуры бронха, так и вследствие активации энзимов вос- палительных клеток [13, 14]. Для выявления ГЭР как причины аспирации прибегают к pH-метрии пищевода, ниточковой пробе или эзофагоскопии (халазия, ахалазия). Дифференциальную диагностику следует прово- дить с муковисцидозом (легочная форма) и с ран- ним дебютом БА. Пластический бронхит. Синонимы — фибри- нозный бронхит, псевдомембранозный бронхит, бронхиальный круп. Пластический бронхит — редкое заболевание. Может быть первичным — идиопатическим или вторичным — при целом ряде болезней: бронхиальной астме (status asthmaticus), лимфогранулематозе, муковисцидозе, бронхоле- гочном аспергиллезе, бронхоэктатической болез- ни, врожденных пороках сердца. ХБ у детей имеет достаточно четкую клини- ческую симптоматику. Заболевание протекает с ежегодными обострениями воспалительного процесса (2–3 раза в год). Основными клиниче- скими проявлениями являются кашель со сли- зисто-гнойной или гнойной мокротой, стойкие физикальные изменения в легких, которые у большинства больных сохраняются не только при обострении процесса, но и в период ремиссии. На рентгенограммах грудной клетки отмечают- ся диффузные изменения легочного рисунка, его усиление, сетчатая деформация, преимуществен- но в прикорневых и нижних отделах, тяжистость корней и отсутствие их структурности. , р д р рдц В клиническом плане характеризуется откаш- ливанием плотных слепков бронхов. Происходит это при сильном приступообразном кашле. Отме- чается одышка, боли или неприятные ощущения в груди, часто на стороне патологического процес- са. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. Интенсивность и выраженность этих симпто- мов зависят от величины откашливаемого слепка бронхов. Нередко сопровождается образованием ателектазов, размер которых (сегментарный или долевой) определяется тем, какой бронх подверг- ся окклюзии. Протяженность слепков может до- стигать 10–15 см. Длинные слепки могут иметь ветвистость соответственно строению бронха, в котором они образовались. В остром периоде на рентгенограмме могут отмечаться сегментарные и долевые ателектазы, которые исчезают после от- хождения слепков бронхов — спонтанного (при кашле) или после бронхоскопии. Течение болезни рецидивирующее. ХБ у детей свойственны стабильные эндо- бронхиальные изменения, преимущественно ка- тарального или катарально-гнойного характера. При бронхографии выявляется деформация стен- ки бронхов в виде различной степени втяжений и выбуханий, неровность контуров бронхиальных ветвей, их оголенность, обеднение мелкими брон- хиальными разветвлениями, обрывки контраст- Хронический бронхит (ХБ). ХБ представляет собой диффузное воспаление слизистой оболоч- 137 ¹ 2 (61) • 2015 www.mif-ua.com Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician ного вещества (деформирующий бронхит) [9, 15]. Достаточную информацию сегодня можно полу- чить при компьютерной томографии легких. вого каркаса трахеи, вследствие чего возникает дискинезия трахеи с ретенцией слизи в нижних отделах бронхиального дерева); синдром Вильям- са — Кэмпбелла (бронхоэктазы вследствие отсут- ствия или недоразвития хрящевой части бронхов среднего и крупного калибра), бронхиолоэкта- тическая эмфизема (эктазия бронхиол, приводя- щая к гиперинфляции легочной ткани); трахео- и бронхопищеводные свищи. Обязательным условием диагноза ХБ является исключение у больного других форм хронической патологии легких, протекающих с синдромом бронхита (бронхоэктатическая болезнь, врожден- ные пороки развития легких, первичные иммуно- дефицитные состояния, муковисцидоз, первич- ная цилиарная дискинезия и др.). При ряде системных болезней, в частности муковисцидозе, также развивается хроническое воспаление бронхов. К системным заболеваниям можно отнести и цилиарную дискинезию, по- скольку при ней нарушается не только подвиж- ность ресничек респираторного эпителия (при- водит к ретенции слизи и воспалению), но также подвижность сперматозоидов и функция эпите- лия фаллопиевых труб. р р ) Хронический облитерирующий бронхиолит, син- дром Мак-Леода. Многочисленные исследования свидетельствуют, что облитерирующий бронхио- лит может в чрезвычайно короткие сроки привести к формированию у ребенка эмфиземы с прогрес- сирующей легочно-сердечной недостаточностью [16]. Морфологическую основу заболевания со- ставляет концентрическое сужение или полная облитерация просвета бронхиол и артериол при отсутствии изменений в альвеолярных ходах и альвеолах, приводящая к развитию эмфиземы и нарушению легочного кровотока. Эта болезнь яв- ляется осложнением острого облитерирующего бронхиолита, чаще ассоциированного с аденови- русной инфекцией. Облитерация бронхиол при- водит к легочной дистрофии и в ряде случаев — к развитию синдрома сверхпрозрачности одного из легких вследствие нарушения легочного кровото- ка и эмфиземы. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. Все многообразие подходов к лечению бронхи- тов трудно рассмотреть в одной статье, тем более что сегодня в литературе и национальных прото- колах им посвящено множество работ [17–19]. За- дачей данной работы было рассмотрение методов противокашлевой терапии у детей в случаях сухого непродуктивного и мучительного кашля, нередко наблюдаемого при различных формах бронхита, а также при трахеитах, фарингитах, ларингитах. р р , ф р , р Кашлевой рефлекс физиологически направ- лен на удаление из дыхательных путей как ве- ществ, попавших извне (некоторые запахи, хо- лодный или сухой воздух, дым, пыль, различные микроорганизмы, инородные тела и т.п.), так и эндогенных компонентов (частички бронхиаль- ного эпителия, слизь и др.). Вместе с тем кашель не только выполняет физиологически полез- ную дренажную функцию, но может оказывать и вредное воздействие. Частый упорный кашель (особенно в виде длительных приступов) сопро- вождается повышением внутригрудного давле- ния, может способствовать развитию гипертен- зии в малом круге кровообращения, эмфиземы легких, формированию легочного сердца. Повы- шение давления в венах большого круга кровоо- бращения во время приступа кашля в ряде случа- ев сопровождается кровоизлияниями из сосудов склер, в системе бронхиальных вен. Приступ сильного кашля может осложниться потерей со- знания, нарушением ритма сердечной деятель- ности, эпилептиформным припадком, пневмо- тораксом. Длительный сухой непродуктивный кашель изнуряет ребенка, ухудшает самочувствие и даже течение заболевания. Болезнь характеризуется волнообразным тече- нием. В периоде обострения определяются экс- пираторная одышка, малопродуктивный кашель, физикальные изменения в виде крепитации и влажных мелкопузырчатых хрипов, стойкой необ- ратимой обструкции дыхательных путей. В даль- нейшем нарастает дыхательная недостаточность. Пораженное легкое увеличивается в объеме из-за вентиляционной эмфиземы, а затем уменьшается вследствие атрофии альвеолярной ткани и нару- шения легочного кровотока. Для большей части больных характерно в этом периоде развитие аст- моподобного синдрома, устойчивого к лечению бронходилататорами. Рентгенологически определяется мозаичность легочного рисунка за счет множественных об- ластей повышенной прозрачности и сниженной васкуляризации, иногда с пролабированием по- раженного легкого в здоровую половину грудной клетки и смещением срединной тени в том же направлении. Характерен симптом «воздушной ловушки»: на выдохе прозрачность пораженного легкого не уменьшается, вследствие чего оно вы- глядит более прозрачным, чем здоровое легкое. Синдром одностороннего сверхпрозрачного лег- кого (синдром Мак-Леода) представляет собой частный случай данного заболевания. Препараты, обладающие противокашлевым эффектом, делятся на две большие группы: пери- ферического и центрального действия. 1. Противокашлевые средства периферическо- го действия подавляют чувствительность кашле- вых рецепторов или афферентные пути регуляции. 1. Противокашлевые средства периферическо- го действия подавляют чувствительность кашле- вых рецепторов или афферентные пути регуляции. Хронические формы бронхита у детей могут определяться при пороках развития бронхов. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. К ним относятся трахеобронхомегалия-трахеоброн- хомаляция (отсутствие или недоразвитие хряще- р р фф р у р у 2. Противокашлевые средства центрального дей- ствия тормозят или подавляют кашлевой рефлекс, 2. Противокашлевые средства центрального дей- ствия тормозят или подавляют кашлевой рефлекс, 138 Çäîðîâüå ðåáåíêà, ISSN 2224-0551 ¹ 2 (61) • 2015 ¹ 2 (61) • 2015 Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician угнетают кашлевой центр в продолговатом мозгу или связанные с ним высшие нервные центры. угнетают кашлевой центр в продолговатом мозгу или связанные с ним высшие нервные центры. центр, не приводя к угнетению дыхания, разви- тию запоров и лекарственной зависимости, и об- ладающее мягким анальгезирующим, бронхоли- тическим и противовоспалительным действием; Противокашлевые средства периферического действия оказывают влияние либо на афферент- ный, либо на эфферентный компонент кашле- вого рефлекса либо имеют сочетанный эффект. Препараты с афферентным эффектом действуют как мягкие анальгетики или анестетики на слизи- стую оболочку дыхательных путей и уменьшают рефлекторную стимуляцию кашлевого рефлекса. Кроме того, они изменяют образование и вяз- кость секрета, расслабляют гладкую мускулатуру бронхов. Препараты с эфферентным действием повышают подвижность секрета, как бы улучшая его скольжение по слизистой, уменьшают вяз- кость слизи или усиливают эффективность и силу самого кашлевого механизма. — эфедрин: являясь адреномиметиком (сти- мулирует - и -рецепторы, способствует высво- бождению адреналина и норадреналина из депо), обладает бронходилатирующим действием, сти- мулируя адренергические 2-рецепторы и снижая действие гистамина на рецепторы H1, что опреде- ляет его спазмолитическое действие на гладкую мускулатуру бронхов, расширяя их. Кроме того, имеет место и его сосудосуживающий эффект с устранением отека слизистых оболочек дыхатель- ных путей; — базиликовое масло: оказывает седативное (слабое), противомикробное и спазмолитическое действие, а также обладает значительным проти- вовоспалительным эффектом. Противокашлевые препараты центрального действия подавляют функцию кашлевого центра продолговатого мозга или связанные с ним другие нервные центры мозга. К ним относят препара- ты с наркотическим эффектом (кодеин, дионин, морфин, декстрометорфан), редко применяющи- еся у детей, и препараты, оказывающие ненарко- тическое противокашлевое действие в сочетании с обезболивающим, успокаивающим и, как прави- ло, слабым спазмолитическим эффектом. К дан- ной группе препаратов относится Бронхолитин — комбинированный противокашлевой препарат многопланового действия, включающий глауцина гидрохлорид, эфедрин, базиликовое масло и ши- роко применяющийся в педиатрии более 30 лет. Показания для применения Бронхолитина: су- хой, непродуктивный кашель при острых и хро- нических бронхитах, трахеобронхитах, бронхи- альной астме (в комбинации с ингаляционными кортикостероидами), пневмонии. няться длительно, особенно при частых рециди- вах. Исследования функции внешнего дыхания (ФВД) у 15–20 % выявляют признаки скрытого (латентного) бронхоспазма, что свидетельствует о наличии у некоторых больных гиперреактивности бронхов. Назначается Бронхолитин в зависимости от возраста: — детям от 3 до 5 лет — по 5 мл препарата, раз- веденных непосредственно перед применением в 10 мл охлажденной кипяченой воды, 3 раза в день; — детям от 5 до 10 лет — по 5 мл 3 раза в день; — взрослым и детям старше 10 лет — по 10 мл 3–4 раза в день. Препарат принимается после еды в течение 5–7 дней. Препарат принимается после еды в течение 5–7 дней. р р р Препараты ненаркотического действия ис- пользуются более широко, но, к сожалению, ча- сто неправильно и необоснованно. Показанием к назначению Бронхолитина является настоятель- ная необходимость подавления кашля и бронхо- дилатация. У детей раннего возраста необходи- мость подавления кашля возникает при коклюше и в случаях очень интенсивного продуктивного кашля при излишне обильном и жидком брон- хиальном секрете (бронхорея), когда имеется ре- альная угроза аспирации. В старшем возрастном периоде Бронхолитин показан в случаях кашля, ассоциированного с умеренным бронхоспазмом, при трахеитах, сопровождающихся мучительным сухим и болезненным кашлем. Препарат приме- няют самостоятельно или как дополнение к те- рапии, подавляющей аллергическое или иррита- тивное воспаление. Бронхолитин также показан при кашле, связанном с раздражением слизистых верхних (надгортанных) отделов дыхательных путей вследствие инфекционного или иррита- тивного воспаления. В этих случаях результат его применения обычно усиливается при сочетании с препаратами периферического действия с обвола- кивающим эффектом. Терапевтическая эффективность Бронхолити- на и отдельных компонентов, входящих в его со- став, при комплексной терапии острых и хрони- ческих заболеваний дыхательных путей изучена в большом количестве клинических исследований. Клинические исследования терапевтической эффективности препарата Бронхолитин были проведены в трех клиниках Медицинского уни- верситета г. Софии. 36 пациентов, страдающих хроническим обструктивным бронхитом, в ком- плексной терапии получали препарат Бронхоли- тин по 15 мл 3 раза в день. Эффективность про- водимой терапии оценивалась по изменению частоты и интенсивности кашля, количеству и качеству мокроты, дыхательным функциям. По- лученные результаты позволили говорить о вы- сокой эффективности препарата Бронхолитин. К 10-му дню лечения кашель полностью исчез у 15 (42 %) больных, у 21 (58 %) отмечалось уменьше- ние частоты и интенсивности кашля (небольшой кашель). Исчезновение одышки к 10-му дню ле- чения отмечено у 24 (67 %) больных. Данный тера- певтический эффект был подтвержден результа- тами исследования функции внешнего дыхания: отмечалось увеличение жизненной и форсиро- ванной жизненной емкости легких, нормализа- ция кислотно-щелочного состава и газов крови. Составляющие Бронхолитина: — глауцина гидрохлорид: эффективное про- тивокашлевое средство, подавляющее кашлевой 139 ¹ 2 (61) • 2015 www.mif-ua.com Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician 5. Юлиш Е.И., Волосовец А.П., Абатуров А.Е. Хламидиозы у детей. — Донецк; Киев; Днепропетровск, 2009. — 195 с. Клинические исследования препарата Бронхо- литин у детей были проведены в университетской клинике детских легочных заболеваний г. Софии. Препарат принимается после еды в течение 5–7 дней. Таким образом, кашель — это физиологиче- ский рефлекс, направленный, на восстановление проходимости дыхательных путей при поражени- ях органов дыхания, и обычно не требует отдель- ного лечения. При развитии кашлевого синдрома у детей с бронхитами с мучительным сухим и не- продуктивным кашлем, нарушающим самочув- ствие и состояние больного, возникает необходи- мость в противокашлевой терапии, и в частности в использовании патогенетически направленных лекарственных средств. Таким образом, кашель — это физиологиче- ский рефлекс, направленный, на восстановление проходимости дыхательных путей при поражени- ях органов дыхания, и обычно не требует отдель- ного лечения. При развитии кашлевого синдрома у детей с бронхитами с мучительным сухим и не- продуктивным кашлем, нарушающим самочув- ствие и состояние больного, возникает необходи- мость в противокашлевой терапии, и в частности в использовании патогенетически направленных лекарственных средств. 14. Muller K., Schmitz S. Chronic bronchitis-alterations of the bronchial mucosa // Wiad Cek. — 1997. — 50. — 252-266. 14. Muller K., Schmitz S. Chronic bronchitis-alterations of the bronchial mucosa // Wiad Cek. — 1997. — 50. — 252-266. 15. Костюченко М.В. Современные методы рентгеноди- агностики хронических неспецифических заболеваний легких у детей // Рос. вестник перинатологии и педиатрии. — 2002. — 2. — 33-38. 16. Каганов С.Ю., Розинова Н.Н., Дрожжев М.Е., Геп- пе Н.А. Хроническая обструктивная патология легких как проблема пульмонологии детского возраста // Хронические об- структивные болезни легких / Под ред. А.Г. Чучалина. — СПб., 1998. — 475-500. 17. Morice A.H., McGarvey L., Pavord I. On behalf of the British Thoracic Society Cough Guideline Group Recommendations for the man- agement of cough in adults // Thorax. — 2006. — Vol. 61. — Р. 1-24. Ñïèñîê ëèòåðàòóðû 1. Инфекции респираторного тракта у детей раннего воз- раста / Под ред. проф. Г.А. Самсыгиной. — М.: Миклош, 2006. — С. 111-136. 1. Инфекции респираторного тракта у детей раннего воз- раста / Под ред. проф. Г.А. Самсыгиной. — М.: Миклош, 2006. — С. 111-136. 18. Malfertheiner P., Megraud F., O’Morain C.A., Atherton J., Axon A.T., Bazzoli F., Gensini G.F., Gisbert J.P., Graham D.Y., Rokkas T., El-Omar E.M., Kuipers E.J. European Helicobacter Study Group. Management of Helicobacter pylori infection — the Maastricht IV/Florence Consensus Report // Gut. — 2012. — 61 (5). — 646-64. doi: 10.1136/gutjnl-2012–302084. 2. Каганов С.Ю., Розинова Н.Н., Богорад А.Е. Различные формы бронхолегочной патологии в Международной статисти- ческой классификации болезней X пересмотра // Педиатрия. — 2003. — № 4. — С. 42-46. 3. Палеев Н.Р., Ильченко В.А. Хронический бронхит. Болезни органов дыхания // Руководство по внутренним болезням / Под ред. Н.Р. Палеева. — М.: Медицина, 2000. 19. Thakkar K., Boatright R.O., Gilger M.A., El-Serag H.B. Gas- troesophageal reflux and asthma in children: a systematic review // Pediatrics. — 2010. — 125 (4). — e925-930. 4. Смирнова М.О., Сорокина Е.В. Бронхиты у детей: прин- ципы современной терапии // Трудный пациент. — 2009. — № 8, 9. — С. 19-23. Получено 02.01.15 Препарат принимается после еды в течение 5–7 дней. 30 больных детей с диагнозом «острый бронхит» в возрасте от 3 до 14 лет в течение 10 дней получали препарат Бронхолитин амбулаторно, в возраст- ных дозах, 26 из них получали также антибакте- риальную терапию. К 10-му дню лечения только у трех пациентов оставался небольшой кашель. Та- ким образом, было сделано заключение: препарат Бронхолитин является эффективным противо- кашлевым средством в комплексной терапии де- тей с заболеваниями дыхательной системы. 6. Юлиш Е.И., Балычевцева И.В., Гадецкая С.Г., Ваку- ленко С.И. Новый поход к лечению детей с рецидивирующим обструктивным бронхитом, ассоциированным с персистирую- щими внутриклеточными инфекциями // Експериментальна і клінічна медицина. — 2008. — № 4. — С. 75-78. 7. Юлиш Е.И., Сорока Ю.А., Басюк Е.С., Вакуленко С.И. Факторы риска развития бронхообструктивного синдрома у де- тей первого года жизни // Запорожский мед. журнал. — 2009. — Т. 2, № 5. — С. 76-78. 8. Захаров П.П. Клинико-функциональные особенности и ис- ходы различных форм хронических заболеваний легких у детей: Дис... канд. мед. наук. — М., 2002. — 138 с. 9. Klein R.B., Huggins B.W. Chronic bronchitis in children // Semin. Resp. Infect. — 1994. — 9, 1. — 13-22. 9. Klein R.B., Huggins B.W. Chronic bronchitis in children // Semin. Resp. Infect. — 1994. — 9, 1. — 13-22. Согласно проведенным исследованиям, ра- циональное применение Бронхолитина в рамках установленных показаний и доз способствует повышению эффективности и безопасности те- рапии основного заболевания. При применении Бронхолитина три раза в сутки препарат надеж- но подавляет кашель, возникающий в различных рецепторных зонах бронхолегочной системы, уменьшает одышку и обструкцию бронхов. 10. Рабочая классификация основных клинических форм бронхолегочных заболеваний у детей (мод. Геппе Н.А., Розино- ва Н.Н., Мизерницкий Ю.Л. и др.) // Медицинская газета. — 2009. — 9, 11. — 7-9. 11. Hoizmann D., Ott P.M., Felix H. Diagnostic approach to pri- mary ciliary dyskinesia: a review // Eur. J. Pediatr. — 2000. — 156, 1-2. — 95-98. 12. Wilson R., Sykes D.A., Watson D. Measurement of Pseudo- monas aeruginosa phenazin pigment in sputum sol toxicity for respira- tori epithelium // Infect. Immum. — 1988. — 56. — 2515-2517. Бронхолитин можно применять одновременно с антибиотиками и другими противомикробными препаратами, антипиретиками и витаминами. 13. Клинические формы хронических воспалительных заболе- ваний легких у детей: критерии диагностики и принципы тера- пии // Трудный пациент. — 2007. — 10. — С. 21-28. BRONCHITIS IN ÑHILDREN AND ANTITUSSIVE THERAPY SELECTION Резюме. У роботі представлений огляд форм бронхітів у дітей, що найбільш часто зустрічаються, їх клінічна симп- томатика і підходи до діагностики. Розглянуто механізми формування синдрому кашлю та методи його лікування. Summary. The paper presents an overview of the most com- mon forms of bronchitis in children, their clinical symptoms and approaches to diagnosis. The mechanisms of cough syn- drome formation and methods of its treatment are considered. Ключові слова: бронхіти, кашель, діти. Key words: bronchitis, cough, children. 140 ¹ 2 (61) • 2015 Çäîðîâüå ðåáåíêà, ISSN 2224-0551
https://openalex.org/W4390472005
http://www.clausiuspress.com/assets/default/article/2023/12/31/article_1704025544.pdf
English
null
The Meaning of Enjoyment of New Rural Social Pension Insurance Benefits
Social security and administration management
2,023
cc-by
3,051
Social Security and Administration Management (2023) Clausius Scientific Press, Canada Social Security and Administration Management (2023) Clausius Scientific Press, Canada DOI: 10.23977/socsam.2023.040815 ISSN 2523-5796 Vol. 4 Num. 8 Keywords: Over-age migrant workers, Pension insurance benefits, New rural social pension insurance benefits Abstract: If a dispute arises between an employer and a person recruited by the employer who is already entitled to old-age insurance, the dispute shall not be handled in accordance with labor relations. The "old-age insurance treatment" means the basic old-age insurance treatment for unban workers, and the new rural social old-age insurance treatment for over-age migrant workers is not equivalent to it. From the point of view of conforming to the legislative purpose of the Labor Law and helping to safeguard the labor rights and interests of over-age rural workers, it should be considered that the enjoyment of new type of rural social old-age pension insurance treatment does not mean the enjoyment of "old-age pension insurance treatment". The Meaning of Enjoyment of New Rural Social Pension Insurance Benefits Jiajun Li Law School, Guilin University of Electronic Technology, Guilin, China 15129776317@163.com Keywords: Over-age migrant workers, Pension insurance benefits, New rural social pension insurance benefits 2. Origins of the Problem - The Different Decisions of Two Similar Cases 1. Formulation of the Problem Document No. 1 of the Central Committee of the People's Republic of China in 2023 clearly stipulates that the employment rights and interests of over-age migrant workers should be safeguarded. Whether or not a labor relationship exists between over-age migrant workers who are entitled to the new rural social pension insurance and the employer is a question of whether or not their employment rights and interests should be protected. The Interpretation (I) of the Supreme People's Court on the Application of Law to the Trial of Labor Dispute Cases (hereinafter referred to as "Interpretation (I)") stipulates that a labor relationship is deemed to be not formed as a person has enjoyed the pension insurance benefits. Is enjoying the new-type social endowment insurance benefits for rural areas the same as enjoying the endowment insurance benefits? This question determines whether or not the overage migrant workers and employers form a labor relation. Therefore, this paper from the different decisions of two similar cases, combined with the related laws and regulations and the jurisprudence, trys to answer the question. 2. Origins of the Problem - The Different Decisions of Two Similar Cases 2.1. Case I 2.1. Case I At about 10:30 on December 3, 2015, Li Jinqun (born on January 3, 1954, a rural resident of Guangzhou City, a janitor of Shenzhen Xinzirun Property Management Co. Ltd.) died in a traffic 111 accident on his way from work. The court held that the entitlement to new rural social pension insurance was the same as the entitlement to "pension insurance", therefore a labor relationship wasn't formed between Li Jinqun and the employer.[1] accident on his way from work. The court held that the entitlement to new rural social pension insurance was the same as the entitlement to "pension insurance", therefore a labor relationship wasn't formed between Li Jinqun and the employer.[1] 2.2. Case II At about 10:30 on July 28, 2015, Liang Jinxia (born on September 14, 1961, a rural resident of Dongguan City, a cook of Dongguan Fushan Textile Bleaching and Dyeing Co., Ltd.) suddenly died in the company's kitchen when washing rice. 3.1. Pension Insurance System Pension insurance is a social insurance system that provides material assistance from the State and society to meet the basic needs of old age when workers reach a certain age and are unable to earn income from their labor as a source of livelihood. Pension insurance in China consists of three levels, of which basic pension insurance is the core and foundation. According to the different objects covered, it is divided into basic old-age insurance for urban workers, social old-age insurance for urban residents and new rural social old-age insurance. A wide range of people are covered by the basic pension insurance, among them urban enterprise workers are the main object. The payment of pension insurance premiums is compulsory for employees. The new rural social pension insurance covers rural residents aged 16 or over (excluding schoolchildren) who are not covered by the basic pension insurance for urban workers. Farmers can voluntarily choose whether to pay new rural social pension insurance. Social pension insurance for urban residents covers unemployed urban residents who are not covered by basic pension insurance for urban workers, and the payment of insurance premiums is voluntary for urban residents. 1. Formulation of the Problem The court considered that enjoying the new rural social pension insurance benefits does not mean enjoying "pension insurance benefits", so a labor relation was formed between Liang Jinxia and the employer.[2] In the two above cases, Li Jinqun and Liang Jinxia were both over-age migrant workers who had been entitled to the new type of rural social old-age pension insurance, but the courts did not make the same determination as to whether or not they were entitled to "old-age pension insurance benefits". The reason for the different court decisions is that there are different views on the issue of whether enjoying the new rural social pension insurance benefits means enjoying "pension insurance benefits". Accordingly, this issue is analyzed below. 3. New Rural Social Pension Insurance Benefits Are Different From "Pension Insurance Benefits". 3. New Rural Social Pension Insurance Benefits Are Different From "Pensio Benefits". ural Social Pension Insurance Benefits Are Different From "Pension Insuranc 3.2. Old-age Insurance Benefits "Pension insurance benefits" refers to the pension insurance benefits paid to insured persons under the pension insurance system in order to protect their basic living needs. With regard to the basic pension insurance benefits for urban workers, they are funded by the employing organization, individual employee contributions and government subsidies. [3]The basic old-age insurance benefit consists of a centralized pension and an individual account pension. [4]To be eligible for urban workers' basic old-age insurance benefits, one must meet the two conditions of reaching the legal retirement age and having paid old-age insurance premiums for 15 years. As for the new rural social pension insurance benefits, it consists of the basic pension and the individual account pension.[5] The funds come from individual payments, collective assistance and government subsidies.[6] The following conditions need to be met to enjoy rural pension insurance benefits: first, you must be 60 112 years of age or older; second, you must not have enjoyed basic pension insurance benefits for urban workers; and third, you must have a rural household registration. The social pension insurance benefits for urban residents are funded by personal payment and government subsidies, and can be received upon reaching the age of 60. As can be seen, there is a clear gap between the basic pension insurance benefits for urban workers and the new rural social pension insurance benefits in terms of the composition of pension benefits and the conditions for receiving them. p p g As mentioned above, according to Interpretation (I), if an over-age worker is entitled to "old-age insurance benefits", he or she is in a labor relationship with the employer, i.e., the existence of a labor relationship is excluded. According to the Labor Contract Law of the People's Republic of China (hereinafter referred to as the "Labor Contract Law"), if a worker has enjoyed the "basic old-age insurance benefits", the labor contract between the worker and the employer will be terminated, and the labor relationship will no longer exists between the two parties. [7]Using the method of systematic interpretation, both the Labor Contract Law and the Interpretation (I) stipulate that the result is that there is no labor relationship, so it is in line with the inherent consistency of the legal system to regard the "old-age insurance benefits" as the "basic old-age insurance benefits". 3.2. Old-age Insurance Benefits According to the provisions of the Social Insurance Law, employees participate in basic old-age insurance, which is differentiated from the new rural social old-age insurance and the social old-age insurance for urban residents, and therefore the basic old-age insurance is in fact meant to be the basic old-age insurance for urban workers. Thus, the "pension insurance treatment" in Interpretation (I) refers to the basic old-age insurance for urban workers. 3.3. Enjoyment of New Rural Social Pension Insurance Benefits isn't the Same as Enjoyment of "Pension Insurance Benefits". ent of New Rural Social Pension Insurance Benefits isn't the Same as Enjoyment Insurance Benefits". (1) Fulfillment of the Purposes of Labor Law Legislation p g Workers are in a weaker position than their employers, so in order to improve this situation, the Labor Law provides for a certain number of legal provisions that favor the protection of workers and, to a certain extent, limit the rights of the employers. This is the "principle of tilt protection" in labor law. [8]In order to continue to maintain the basic needs of life, over-age rural migrant workers, limited to education, technology and other factors can only choose to work most of the labor intensity, low income, high risk, less protection, resulting in the fact that compared with the ordinary workers of the appropriate age, over-age rural migrant workers' disadvantage is more obvious, thus, there is a greater need for them to be given tilt protection. For this reason, it should be considered that over-age rural migrant workers and employers form a labor relationship, that is, "pension insurance treatment" and the new rural social pension insurance treatment is different. On the contrary, if the new rural social old-age insurance treatment is regarded as equivalent to the "old-age insurance treatment", over-age migrant workers will not be able to enjoy the protection of the labor law because of the formation of no labor relationship with the employer. p p y (2) Help Protect the Labor Rights And Interests of Over-age Migrant Workers h b i i i b fi f b k l i l hi h d h ( ) p g g g The basic pension insurance benefits for urban workers are relatively high, and have basically met the living needs of over-age people. The new rural social pension insurance benefits are very low in comparison, making it difficult to protect the basic livelihood of over-age migrant workers. Taking Guangdong Province as an example, the Guangdong Provincial Department of Human Resources and Social Security stipulates that participants of basic pension insurance for urban workers should pay pension insurance premiums on the basis of their actual salary income, which should not be less than 60% of the average monthly salary of the province's on-the-job workers in the previous year.[9] In 2022, the average monthly salary of the province's full-caliber urban employees in Guangdong Province was RMB 8,807. [1] The First Court of Guangzhou Railway Transportation of Guangdong Province (2017) Yue 7101 Xingchu 2254. [2] The First People's Court of Dongguan City, Guangdong Province (2015) Dongyi Fa Xing Chu Zi No. 448. [3] Article 11 of the Social Insurance Law stipulates: "Basic pension insurance is a combination of social coordination and individual accounts. The basic pension insurance fund consists of contributions from employers and individuals and government subsidies." [4] Article 15 of the Social Insurance Law states: "The basic pension consists of a coordinated pension and an (1) Fulfillment of the Purposes of Labor Law Legislation [10] 113 On the contrary, the new rural social pension insurance treatment, its basic pension standard is set at the central level is 55 yuan per person per month, and the minimum standard at the local level in Guangdong Province is 190 yuan per person per month. For the individual account pension, the minimum individual contribution standard is 180 yuan per year and the maximum is 4,800 yuan per year. [11]Comparing the basic pension insurance benefits for urban workers and the new rural social pension insurance benefits in Guangdong Province, the former amount is significantly higher than the latter. This shows that the level of protection of the new rural social pension insurance benefits are significantly lower than the level of protection of the urban workers' basic pension insurance benefits. Compared with the better protection that can be realized by the latter, the new rural social pension insurance benefits cannot reach the level of protecting the basic life of over-age migrant workers. Therefore, as the two protection level is hugely different, and the treatment level gap can not be bridged in a short period of time, it should be clear that the two can not be equated, and enjoying the new rural social old-age pension insurance treatment does not mean to enjoy the "old-age insurance treatment". This is conducive to the protection of over-age rural migrant workers labor rights and interests, and further to realize the substantive fairness. Otherwise, if it is determined that enjoying the new rural social pension insurance benefits means enjoying the "old-age insurance benefits", it is equivalent to considering that the new rural social pension insurance benefits and the basic old-age insurance benefits for urban workers play the same role in safeguarding the old-age lives of employees and over-age migrant workers, respectively. This is clearly not in line with the nature and level of benefits of the new rural social pension insurance, and is extremely unfair to over-age migrant workers. 15 of the Social Insurance Law states: "The basic pension consists of a coordinated pension and a 4. Conclusion The new rural social pension insurance system combines individual contributions, collective subsidies and government subsidies." g [7] Article 44 stipulates: "A labor contract shall be terminated under any of the following circumstances: (ii) if the worker begins to enjoy basic pension insurance benefits in accordance with the law;" g [7] Article 44 stipulates: "A labor contract shall be terminated under any of the following circumstances: (ii) if the worker begins to enjoy basic pension insurance benefits in accordance with the law;" worker begins to enjoy basic pension insurance benefits in accordance with the law;" [8] Zhang Lin, Study on the Principle of "Inclined Protection" in Labor Law, (2021) Doctoral Dissertation, of Southwest University of Political Science and Law, p. 13. worker begins to enjoy basic pension insurance benefits in accordance with the law;" [8] Zhang Lin, Study on the Principle of "Inclined Protection" in Labor Law, (2021) Doctor worker begins to enjoy basic pension insurance benefits in accordance with the law; [8] Zhang Lin, Study on the Principle of "Inclined Protection" in Labor Law, (2021) Doctoral S h U i i f P li i l S i d L 13 worker begins to enjoy basic pension insurance benefits in accordance with the law; [8] Zhang Lin, Study on the Principle of "Inclined Protection" in Labor Law, (2021) Doctoral Dissertation, of Southwest University of Political Science and Law, p. 13. g j y p f [8] Zhang Lin, Study on the Principle of "Inclined Protection" in Labor Law, (2021) Doctoral Dissertation, of Southwest University of Political Science and Law, p. 13. [9] Official website of the Guangdong Provincial Department of Human Resources and Social Security <http://hrss.gd.gov.cn/zmhd/cjwtk/content/post_1298070.html> (accessed November 15, 2023) [9] Official website of the Guangdong Provincial Department of Human Resources and Social Security <http://hrss.gd.gov.cn/zmhd/cjwtk/content/post_1298070.html> (accessed November 15, 2023) p g g j p ( ) [10] Notice on Issues Relating to the Announcement of the Average Monthly Wages of Employed Persons in Full-Caliber Urban Units of the Province in 2022 and the Upper and Lower Limits of the Basic Pension Insurance Contribution Base for Employees in 2023. [10] Notice on Issues Relating to the Announcement of the Average Monthly Wages of Employed Persons in Full-Caliber Urban Units of the Province in 2022 and the Upper and Lower Limits of the Basic Pension Insurance Contribution Base for Employees in 2023. 4. Conclusion The courts have recognized the employment relationship between persons who have enjoyed "old-age insurance benefits" and their employers as a labour relationship. In judicial practice, the courts have made different determinations as to what kind of employment relationship exists between over-age migrant workers and the employing organization, because of the different viewpoints of the courts on the question of whether or not over-age migrant workers' enjoyment of the new type of rural social old-age pension insurance entitlement means that they are entitled to "old-age pension insurance entitlement". The term "old-age insurance treatment" refers to the basic old-age insurance treatment for urban workers, and there are obvious differences between the new rural social old-age insurance and the basic old-age insurance for urban workers in terms of who they cover, whether they are mandatory or not, the source of funds, and the conditions for receiving treatment. From the point of view of protecting disadvantaged workers and safeguarding the labour rights and interests of over-age migrant workers, it should be considered that the enjoyment of the new rural social pension insurance does not mean that they are entitled to "old-age insurance treatment". The relationship between over-age migrant workers who have enjoyed the new rural social pension insurance and their employers should be recognized as a labour relationship. References 114 individual account pension. The basic pension is determined on the basis of an individual's accumulated years of contributions, his or her contribution wage, the average wage of local employees, the amount of the individual account, and the average life expectancy of the urban population." g f p y f p p [5] Article 21 of the Law on Social Insurance states: "The new rural social pension insurance benefits consist of a basic pension and a personal account pension." g f p y f p p [5] Article 21 of the Law on Social Insurance states: "The new rural social pension insurance benefits consist of a basic pension and a personal account pension." [6] Article 20 of the Social Insurance Law states: "The State shall establish and improve the new rural social pension insurance system. The new rural social pension insurance system combines individual contributions, collective subsidies and government subsidies." [6] Article 20 of the Social Insurance Law states: "The State shall establish and improve the new rural social pension insurance system. 4. Conclusion [11] Official website of the Guangdong Provincial Department of Human Resources and Social Security <https://hrss.gd.gov.cn/zmhd/cjwtk/shbz/content/post_4140000.html> (accessed November 16, 2023) [11] Official website of the Guangdong Provincial Department of Human Resources and Social Security <https://hrss.gd.gov.cn/zmhd/cjwtk/shbz/content/post_4140000.html> (accessed November 16, 2023) 115
https://openalex.org/W2801622893
http://www.jksrr.org/journal/download_pdf.php?doi=10.5792/ksrr.17.058
English
null
A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery
Knee surgery & related research
2,018
cc-by
2,690
A Report of Two Cases of Adventitial Cystic Disease of th P lit l A t Doo Jae Lee, MD 1, Hyun Oh Park, MD 2, Ha Nee Jang, MD 3, Ki Nyun Kim, MD 2, Jun Ho Yang, MD 4, Seong Ho Moon, MD 2, Joung Hun Byun, MD 2, Sung Hwan Kim, MD 2, Jun Young Choi, MD 4, In Seok Jang, MD 4, Jong Woo Kim, MD 2, and Chung Eun Lee, MD 2 g g g 1Division of Foot and Ankle, Department of Orthopedic Surgery, Seoul National University Hospital, Seoul; 2Department of Thoracic and Cardiovascular Surgery, Gyeongsang National University Changwon Hospital, Gyeongsang National University College of Medicine, Changwon; 3Department of Internal Medicine, Gyeongsang National University Hospital, Gyeongsang National University College of Medicine, Jinju; 4Department of Thoracic and Cardiovascular Surgery, Gyeongsang National University Hospital, Gyeongsang National University College of Medicine, Jinju, Korea Two patients were admitted to our department because of recent aggravation of claudication in the leg, which was exacerbated by walking. They were diagnosed as having a Baker cyst or acute thrombosis in the popliteal fossa at another hospital. There was no evidence of ischemia, and the ankle brachial index was normal. Computed tomography and magnetic resonance imaging were performed, revealing a cystic mass of the popliteal artery (PA). Intraoperatively, the cystic lesion was found within the adventitia of the PA; based on the biopsy findings, both patients were diagnosed as having adventitial cystic disease of the PA. Keywords: Popliteal artery, Adventitia, Cyst Keywords: Popliteal artery, Adventitia, Cyst Adventitial cystic disease (ACD) is a rare disease that occurs when adventitial mucoid cysts develop inside of the artery and vein, causing narrowing of the vessel lumen 1). Patients are usu­ ally middle age and do not have risk factors of atherosclerosis 2,3). ACD is a rare disease that mainly affects the popliteal artery (PA) and usually manifests as intermittent claudication during exercise 3). ACD can be often misdiagnosed as atherosclerosis, an aneurysm, traumatic injury, a Backer cyst, or an arterial embolus, and a preoperative examination is important for making an ac­ curate diagnosis 4). There have been several reports of ACD of PA. Previous reports have mainly described the pathogenesis, clinical features, and management of ACD 5,6) or imaging findings 7,8). Herein, we de­ scribe the processes of treating ACD of the PA and demonstrate the importance of an accurate preoperative examination. The first patient was diagnosed as having ACD based on an accurate preoperative examination. Case Reports Case Reports Case Report Knee Surg Relat Res 2018;30(2):167-170 https://doi.org/10.5792/ksrr.17.058 pISSN 2234-0726 · eISSN 2234-2451 A Report of Two Cases of Adventitial Cystic Disease of th P lit l A t However, the second patient was mis­ diagnosed as having a thrombotic occlusion of the PA due to an incomplete preoperative examination but was later diagnosed as having ACD postoperatively. Received July 28, 2017; Revised (1st) August 18, 2017; (2nd) September 22, 2017; (3rd) September 27, 2017; (4th) October 6, 2017; Accepted October 7, 2017 Correspondence to: Chung Eun Lee, MD Department of Thoracic and Cardiovascular Surgery, Institute of Health Science, Gyeongsang National University Hospital, 79 Gangnam-ro, Jinju 52727, Korea Tel: +82-55-750-8124, Fax: +82-55-753-8138 E-mail: ilikerem@hanmail.net Received July 28, 2017; Revised (1st) August 18, 2017; (2nd) September 22, 2017; (3rd) September 27, 2017; (4th) October 6, 2017; Accepted October 7, 2017 Correspondence to: Chung Eun Lee, MD Department of Thoracic and Cardiovascular Surgery, Institute of Health Science, Gyeongsang National University Hospital, 79 Gangnam-ro, Jinju 52727, Korea Tel: +82-55-750-8124, Fax: +82-55-753-8138 E-mail: ilikerem@hanmail.net Knee Surgery & Related Research Knee Surgery & Related Research Case Report Knee Surg Relat Res 2018;30(2):167-170 https://doi.org/10.5792/ksrr.17.058 pISSN 2234-0726 · eISSN 2234-2451 Copyright © 2018 KOREAN KNEE SOCIETY 168 Lee et al. A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery 168 Lee et al. A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery performed to determine the site and extent of stenosis and evalu­ ate the entire circulation system, and we found a cystic lesion with mild peripheral enhancement of the popliteal vessels. There was significant focal eccentric narrowing of the PA (Fig. 1). The possibility of ACD of the PA was high, but it was not completely distinguished from ACD originating from the popliteal vein. Hence, there was a need to determine if it was either ACD origi­ nating from the popliteal vein or PA, so we performed magnetic resonance imaging (MRI). Cystic lesions close to both P walls were seen as areas of low to intermediate signal intensity on T1- weighted MRI (Fig. 2A) and as areas of high signal intensity on T2-weighted MRI (Fig. 2B). On the basis of characteristic imag­ ing findings, we diagnosed him as having a cyst originating from the PA. Intraoperatively, we identified a cyst originating from the PA. We performed cyst evacuation and finished the operation af­ ter confirming that blood flow to the PA had been recovered. On the basis of the biopsy result, we made a final diagnosis of ACD. Periodic follow-up studies showed no local recurrence until 32 months postoperatively. 1. Case 1 A 33-year-old man was referred to our institution because of edema and pain in his left lower extremity that had begun 8 days earlier. His risk factors for atherosclerotic diseases included smoking (1 pack per day) but nothing else remarkable. Based on the results of the patient`s ultrasonography done in another hos­ pital, he was diagnosed as having acute thrombosis in the distal portion of the superficial femoral artery or a Baker cyst in the popliteal fossa. Computed tomography (CT) angiography was This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. www.jksrr.org www.jksrr.org www.jksrr.org 167 2. Case 2 A 66-year-old man visited our institution with the chief com­ plaint of pain and claudication in the right leg that had begun 6 months previously. He was diagnosed as having a Baker cyst or thrombotic occlusion of the PA at another hospital. CT angiog­ raphy was performed, and we diagnosed him as thrombotic oc­ clusion of the PA (Fig. 3A and B). We planned and performed an operation to remove the thrombus of the PA. However, we identi­ fied a cystic mass originating from the PA unlike the preoperative diagnosis (Fig. 4). The diameter of the PA was still narrow after removing the lesion, so we performed graft interposition using a 6-mm polytetrafluoroethylene graft (Terumo Cardiovascular Fig. 1. Case 1: A 33-year-old man. Com­ puted tomography angiogram showing a cystic lesion (white arrow) causing marked eccentric narrowing of the popliteal artery. A B Fig. 2. Case 1: A 33-year-old man. (A) T1- weighted axial magnetic resonance imaging (MRI) scan of the knee showing a cystic lesion (white arrow) with intermediate signal intensity due to the mucin content surrounding the popliteal artery. (B) T2- weighted axial MRI scan of the left knee showing a high signal intensity cystic lesion (black arrow) of the popliteal artery. Fig. 1. Case 1: A 33-year-old man. Com­ puted tomography angiogram showing a cystic lesion (white arrow) causing marked eccentric narrowing of the popliteal artery. Fig. 1. Case 1: A 33-year-old man. Com­ puted tomography angiogram showing a cystic lesion (white arrow) causing marked eccentric narrowing of the popliteal artery. A B Fig. 2. Case 1: A 33-year-old man. (A) T1- weighted axial magnetic resonance imaging (MRI) scan of the knee showing a cystic lesion (white arrow) with intermediate signal intensity due to the mucin content surrounding the popliteal artery. (B) T2- weighted axial MRI scan of the left knee showing a high signal intensity cystic lesion (black arrow) of the popliteal artery. Fig. 2. Case 1: A 33-year-old man. (A) T1- weighted axial magnetic resonance imaging (MRI) scan of the knee showing a cystic lesion (white arrow) with intermediate signal intensity due to the mucin content surrounding the popliteal artery. (B) T2- weighted axial MRI scan of the left knee showing a high signal intensity cystic lesion (black arrow) of the popliteal artery. B Knee Surg Relat Res, Vol. 30, No. 2, Jun. 2018 169 Fig. 4. Case 2: A 66-year-old man. 2. Case 2 Intraoperative finding of the adventi­ tial cyst (white arrow) in the surrounding part of the popliteal artery. Fig. 5. Case 2: a 66-year-old man. Photomicrograph showing the cystic space (asterisk) filled with gelatinous material and the dissected arte­ rial wall (arrow) with collagen bundles via mucoid degeneration (H&E, ×20). A B Fig. 3. Case 2: A 66-year-old man. (A) Ax­ ial computed tomography (CT) angiogram showing a cystic lesion of the popliteal ar­ tery. Cystic lesions are large and eccentric, and they may displace the artery to one side—the so-called scimitar sign (white arrow). (B) Three-dimensional CT recon­ struction image showing partial occlusion of the popliteal artery. A B Fig ial sho tery and sid arr stru of t A Fig. 3. Case 2: A 66-year-old man. (A) Ax­ ial computed tomography (CT) angiogram showing a cystic lesion of the popliteal ar­ tery. Cystic lesions are large and eccentric, and they may displace the artery to one side—the so-called scimitar sign (white arrow). (B) Three-dimensional CT recon­ struction image showing partial occlusion of the popliteal artery. Fig. 3. Case 2: A 66-year-old man. (A) Ax­ ial computed tomography (CT) angiogram showing a cystic lesion of the popliteal ar­ tery. Cystic lesions are large and eccentric, and they may displace the artery to one side—the so-called scimitar sign (white arrow). (B) Three-dimensional CT recon­ struction image showing partial occlusion of the popliteal artery. Fig. 3. Case 2: A 66-year-old man. (A) Ax­ ial computed tomography (CT) angiogram showing a cystic lesion of the popliteal ar­ tery. Cystic lesions are large and eccentric, and they may displace the artery to one side—the so-called scimitar sign (white arrow). (B) Three-dimensional CT recon­ struction image showing partial occlusion of the popliteal artery. A B Fig. 5. Case 2: a 66-year-old man. Photomicrograph showing the cystic space (asterisk) filled with gelatinous material and the dissected arte­ rial wall (arrow) with collagen bundles via mucoid degeneration (H&E, ×20). Fig. 5. Case 2: a 66-year-old man. Photomicrograph showing the cystic space (asterisk) filled with gelatinous material and the dissected arte­ rial wall (arrow) with collagen bundles via mucoid degeneration (H&E, ×20). Fig. 4. Case 2: A 66-year-old man. Intraoperative finding of the adventi­ tial cyst (white arrow) in the surrounding part of the popliteal artery. Systems Corp., Ann Arbor, MI, USA). Postoperatively, we found that the blood flow to the PA was completely recovered. 2. Case 2 Later, we made a final diagnosis of ACD based on the biopsy finding (Fig. 5). Periodic follow-up studies showed no local recurrence until 35 months postoperatively. of their lower extremity or in 1 of 1,000 patients who undergo angiography. It is about 15 times more common in men than in women 3). The average age of diagnosis is the fourth decade of life for men and fifth decade of life for women 3,5). 35 months postoperatively. This disease should be suspected and an imaging examination should be performed when a person with a relatively low risk of developing cardiovascular diseases experiences sudden claudica­ tion unilaterally 4,6). Ultrasonography with color Doppler shows characteristic features of an anechoic smooth-walled mass seen in the wall of the PA. It shows good posterior enhancement and no flow within the cysts 7). CT angiography may demonstrate nar­ rowing of the vessels with an absence of collateral vessels. When cystic lesions are large and eccentric, they may displace the artery 70 Lee et al. A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery the adventitia of the left external iliac artery; case report. Br J Surg. 1947;34:426. the adventitia of the left external iliac artery; case report. Br J Surg. 1947;34:426. 2. Ejrup B, Hiertonn T. Intermittent claudication; three cases treated by free vein graft. Acta Chir Scand. 1954;108:217-30. 3. Flanigan DP, Burnham SJ, Goodreau JJ, Bergan JJ. Summary of cases of adventitial cystic disease of the popliteal artery. Ann Surg. 1979;189:165-75. 4. Wright LB, Matchett WJ, Cruz CP, James CA, Culp WC, Eidt JF, McCowan TC. Popliteal artery disease: diagnosis and treatment. Radiographics. 2004;24:467-79. 5. Zhang H, Zhang Y, Wang Q, Zhao WG, Wang JJ. Cystic ad­ ventitial disease of the popliteal artery: report of two cases. Surg Today. 2014;44:1760-3. 6. Kwon DJ, Lee WY, Kim KI, Min SK, Park SW, Kim YC, Lee KB. Cystic adventitial disease of the popliteal artery: a case report. J Korean Knee Soc. 2005;17:258-61. 7. Franca M, Pinto J, Machado R, Fernandez GC. Case 157: bilateral adventitial cystic disease of the popliteal artery. Ra­ diology. 2010;255:655-60. 8. Peterson JJ, Kransdorf MJ, Bancroft LW, Murphey MD. Im­ aging characteristics of cystic adventitial disease of the pe­ ripheral arteries: presentation as soft-tissue masses. AJR Am J Roentgenol. 2003;180:621-5. to one side—the so-called scimitar sign 4). The finding of MRI varies depending on the distribution and size of the cyst. T1- weighted MRI shows that individual lesions have a variable signal dependent on the mucoid content, and T2-weighted MRI shows that individual lesions have a high signal intensity, confirming the diagnosis 8). When the lesions are large, MRI scans can also show the scimitar sign 4). 2. Ejrup B, Hiertonn T. Intermittent claudication; three cases treated by free vein graft. Acta Chir Scand. 1954;108:217-30. 3. Flanigan DP, Burnham SJ, Goodreau JJ, Bergan JJ. Summary of cases of adventitial cystic disease of the popliteal artery. Ann Surg. 1979;189:165-75. We described two patients with ACD. The first patient was diag­ nosed as having ACD with an accurate preoperative examination, but the second patient was misdiagnosed as having thrombotic occlusion of the PA. ACD can be misdiagnosed as atherosclero­ sis, aneurysm, traumatic injury, a Baker cyst, or arterial embolus. Therefore, ACD should be included in the differential diagnosis when a patient with a relatively low risk of cardiovascular disease experiences sudden claudication, and physicians should perform various examinations to make an accurate diagnosis. 4. Conflict of Interest No potential conflict of interest relevant to this article was re­ ported. 8. Peterson JJ, Kransdorf MJ, Bancroft LW, Murphey MD. Im­ aging characteristics of cystic adventitial disease of the pe­ ripheral arteries: presentation as soft-tissue masses. AJR Am J Roentgenol. 2003;180:621-5. 8. 8. Peterson JJ, Kransdorf MJ, Bancroft LW, Murphey MD. Im­ aging characteristics of cystic adventitial disease of the pe­ ripheral arteries: presentation as soft-tissue masses. AJR Am J Roentgenol. 2003;180:621-5. 70 Lee et al. A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery Wright LB, Matchett WJ, Cruz CP, James CA, Culp WC, Eidt JF, McCowan TC. Popliteal artery disease: diagnosis and treatment. Radiographics. 2004;24:467-79. 5. Zhang H, Zhang Y, Wang Q, Zhao WG, Wang JJ. Cystic ad­ ventitial disease of the popliteal artery: report of two cases. Surg Today. 2014;44:1760-3. 6. Kwon DJ, Lee WY, Kim KI, Min SK, Park SW, Kim YC, Lee KB. Cystic adventitial disease of the popliteal artery: a case report. J Korean Knee Soc. 2005;17:258-61. 7. Franca M, Pinto J, Machado R, Fernandez GC. Case 157: bilateral adventitial cystic disease of the popliteal artery. Ra­ diology. 2010;255:655-60. 7. Franca M, Pinto J, Machado R, Fernandez GC. Case 157: bilateral adventitial cystic disease of the popliteal artery. Ra­ diology. 2010;255:655-60. Conflict of Interest Discussion ACD is a non-atherosclerotic vascular disease that causes lo­ cal stenosis or occlusion of the peripheral vessels. Shortly after Atkins and Key 1) first identified a case of ACD in the right exter­ nal iliac artery in 1947, Ejrup and Hiertonn 2) reported a case of ACD of the PA for the first time in 1954. ACD of the PA is found incidentally in 1 of 1,200 patients complaining of claudication 170 Lee et al. A Report of Two Cases of Adventitial Cystic Disease of the Popliteal Artery 1. Atkins HJ, Key JA. A case of myxomatous tumour arising in References 1. Atkins HJ, Key JA. A case of myxomatous tumour arising in 1. Atkins HJ, Key JA. A case of myxomatous tumour arising in
https://openalex.org/W3001516442
https://www.mdpi.com/1996-1944/13/3/592/pdf?version=1580995941
English
null
Mechanical Evaluation of Titanium Plates for Osteoesynthesis High Neck Condylar Fracture of Mandible
Materials
2,020
cc-by
10,300
Received: 6 January 2020; Accepted: 22 January 2020; Published: 27 January 2020 Abstract: Background: In the literature no information about plates for the high-neck mandibular condylar osteosynthesis could be found despite that 30 plate designs have been published. The main course consider the basal condylar or diacapitular fractures. The aim of the study was to test mechanically all available designs (only 4 of 30 was proper) on polyurethane mandibles using an individually designed clamping system. Methods: Forces required for a 1 mm displacement of fixed fracture fragments and incidents of screw loosening were recorded. Results: It has occured that dedicated plates for fixation are much weaker than set of two straight plates (p < 0.0001). General observation is the bigger plate and more screws, the better rigid stable osteosynthesis of mandibular condyle, however, there are limitations in plates design for high-neck fractures resulted in restricted operation field. Conclusion: Double straight plates occured to be the best mechanical fixation for high-neck fractures of the mandibular condyle. Maybe other existing plates could be used but only after prebending or that fracture required novel dedicated plates design. Keywords: condylar fracture; titanium plates; high neck fractures materials materials Article Mechanical Evaluation of Titanium Plates for Osteoesynthesis High Neck Condylar Fracture of Mandible Rafał Zieli´nski 1,* , Marcin Kozakiewicz 1 , Bartłomiej Konieczny 2, Michał Krasowski 2 and Jakub Okulski 1 ieli´nski 1,* , Marcin Kozakiewicz 1 , Bartłomiej Konieczny 2, Michał Krasowski 2 and Ok l ki 1 1 Department of Maxillofacial Surgery, Medical University of Lodz, 1stGen. J. Hallera Pl., 90-647 Lodz, Poland; marcin.kozakiewicz@umed.lodz.pl (M.K.); jakub.okulski@gmail.com (J.O.) 2 Material Science Laboratory, Medical University of Lodz, 251st Pomorska, 92-213 Lodz, Poland; bartlomiej.konieczny@umed.lodz.pl (B.K.); michal.krasowski@gmail.com (M.K.) * Correspondence: bkost@op.pl   Received: 6 January 2020; Accepted: 22 January 2020; Published: 27 January 2020 Materials 2020, 13, 592; doi:10.3390/ma13030592 1.1. Epidemiological Information Managment of mandibular fractures, especially condylar, are still a debateble issue for maxillofacial surgeons [1–3]. Some researches have prooved that surgical management have better outcomes than non-invasive treatment. Masticatory function in patients who underwent treatment by means of plates and screws were better. Nevertheless, in surgically treated patients choosing of particular plates in open reduction and condylar osteosynthesis is not clear because there are no guidelines in the literature [2–9]. Mandibular fractures are a part of the great majority of facial traumas. Condylar fractures comprise 17.5–52% of mandibular fractures according to the published large series. Most of the fractures are condylar base fractures. [10–14] In the literature there is no study showing which type of osteosynthesis plate should be used for management of low-neck and high-neck mandibular condyle fractures. Some authors admit that application of 2 plates—can be very demanding and is not always possible [15]. Materials 2020, 13, 592; doi:10.3390/ma13030592 www.mdpi.com/journal/materials 2 of 12 Materials 2020, 13, 592 1.5. Aim In high-neck condylar fractures only four plates might be used because of anatomy of condylar head. Aim of the article was to define appropriate plate design among available to high-neck fracture. 1.3. Biomechanics of the Mandible The mandible has the same mechanical construction as lever class III including the fulcrum of rotation the condyle. Masticatory muscles exert forces and the load is transferred to teeth. [18,19] Muscles that play the main role in transmission of forces are following: masseter and medial pterygoid muscles together initate movements superiorly and anteriorly from the angle of the mandible. Temporalis muscle is attached from the coronoid process and directed superiorly and slightly posteriorly. Lateral pterygoid runs from the condyle anterior and medial direction. [20] Above muscles are not alone in playing the role in mandible movements but their role is the most significant in consideration of mandibular condylar fractures. Some other muscles also play the role in the movements of the mandible but they are negligible low. During jaw movements forces generated from masticatory muscles make compression and tension in the bone. When bone fracture appears, it means forces were much higher than physiologically. [21] The aim of diminish fractures is osseosynthesis that could resist to physiologic load and to endure tensions and compressions exerting on the condylar neck. 1.2. Surgical Procedures Described approaches to mandibular condylar region are mainly preauricular and retromandibular. Those methods of dissections in order to treat the condylar neck and base fractures are debated and some authors described the approaches very precisely. [16] Authors claim that the main reason of choosing appropriate plating system is not the mechanical endurance but only restricted visibility and hardships resulted from low accessibility of this region. [17] However, no studies could be found with comparison of osteosynthesis plates dedicated to condylar neck fractures. 1.4. Osseosynthesis Plates The prime aim of the study was to analyze in fatigues tests plates designed for high neck condylar fractures. Authors of this article proved that if an osseosynthesis plate is not available on the market it is not equal that a plate has worse mechanical endurance than those that are easy to buy. A lot of plates manufactured for mandibular condylar fractures fixations are available. In the literature it is impossible to find out which type of designs is proper for high-neck fractures.There is almost no data in the literature about comparison of osteosynthesis plating system. [4–14] Recently, 30 plates design have been compared in basal condylar fracture osseosynthesis. [22] Four plates’ designs among 30 have been concluded as those that could withstand screw pull-out and displacing force. 2.1. Mandibles Mandible model made of solid foam used in the article (Figure 1a,b). Biomechanical testing outcomes depend on different density and elastic modulus of bone. [23,24] In the literature it is shown that polyurethan mandibles have been proved to be material of choice in orthopaedic implant testing, especially in fractures, and has been confirmed by the American Society for Testing and Materials. [25,26] The most natural would appear to be cadaver bone, but those differ from each other so results of biomechanical fatigue tests would not be standardized. [24] Solid polyurethane material has properties comparable to the human cancellous bone and it is widely used as an ideal medium to mimic human cancellous bone. In our study, polyurethane mandibles (Sawbones, Vashon, WA, USA: density 0.16 g/cc, compression modulus 58 MPa) were utilized as models for fatigued mechanical tests [27–30]. 3 of 12 of 12 Materials 2020, 13, 592 Materials 2020, 13, 592 3 of 12 of 12 (a) (b) Figure 1. Cont. (a) a) (a) (b) Figure 1. Cont. (b) (b) Figure 1. Cont. Materials 2020, 13, 592 4 of 12 , , (c) Figure 1.a) Plates dedicated for high-neck fractures fixed to polyurethane mandibles before tests.b) Plates dedicated for high-neck fractures fixed to polyurethane mandibles after tests.c) Design 20 of plates during fatigued test. Figure 1. (a) Plates dedicated for high-neck fractures fixed to polyurethane mandibles before tests. (b) Plates dedicated for high-neck fractures fixed to polyurethane mandibles after tests. (c) Design 20 of plates during fatigued test. (c) Figure 1.a) Plates dedicated for high-neck fractures fixed to polyurethane mandibles before tests.b) Plates dedicated for high-neck fractures fixed to polyurethane mandibles after tests.c) Design 20 of plates during fatigued test. Figure 1. (a) Plates dedicated for high-neck fractures fixed to polyurethane mandibles before tests. (b) Plates dedicated for high-neck fractures fixed to polyurethane mandibles after tests. (c) Design 20 of plates during fatigued test. 2.2. Plates 2.2. Plates Plate  11 (red cells) has been occurred as the weakest whereas plates 20 (green cells) have been the golden  standard  of  golden  standard  of  itting anatomically  condylar fracture. lloy grade 23 Plate golden  standard  of  itting anatomically  golden  standard  of  itting anatomically Figure  2. Design  us Figure  2. Design  us condylar osteosynth to region of interest  Table 1. List of shap Above 1 mm thick p Figure  2. Design  us condylar osteosynth t i f i t t Figure  2. Design  us condylar osteosynth to region of interest d  in  this  study:  screw  syst d  in  this  study:  screw  syst sis. Three other plates desig n mandible. es of plates used in osteosyn ates were cut by laser from m d  in  this  study:  screw  syst sis. Three other plates desig dibl d  in  this  study:  screw  syst sis. Three other plates desig n mandible. em  2.0  mm. Design  20  is  the  em  2.0  mm. Design  20  is  the  n feature by narrow upper part  nthesis of high‐neck mandibula medical certified titanium sheet a em  2.0  mm. Design  20  is  the  n feature by narrow upper part  em  2.0  mm. Design  20  is  the  n feature by narrow upper part study:  s s used in cut by la other pl p e. Design  gh‐neck m ied titani narrow u igure  2 Table 1. Above 1 ondylar y o region andard  o r fracture e 23 Plat tomicall Figure 2. Design used in this study: screw system 2.0 mm. Design 20 is the golden standard of condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically to region of interest in mandible. Figure 2. Design used in this study: screw system 2.0 mm. Design 20 is the golden standard of condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically to region of interest in mandible. condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically  to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. p y y g 11  (red  cells)  has  been  occurred  as  the  weakest  whereas  plates  20  (green  cells)  have  been  the  strongest. Design code Manufacturer Design Height (mm) Width (mm) to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. 2.2. Plates 2.2. Plates for plate design, drill 1.5 mm in width was used before filling plate holes by screws. p g g p y Figure 2. Design used in this study: screw system 2.0 mm. Design 20 is the golden standard of condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically to region of interest in mandible. Figure 2. Design used in this study: screw system 2.0 mm. Design 20 is the golden standard of condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically to region of interest in mandible. Figure  2. Design  used  in  this  study:  screw  system  2.0  mm. Design  20  is  the  golden  standard  of  condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically  to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. Figure  2. Design  used  in  this  study:  screw  system  2.0  mm. Design  20  is  the  golden  standard  of  condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically  to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate  11  (red  cells)  has  been  occurred  as  the  weakest  whereas  plates  20  (green  cells)  have  been  the  strongest. Design code Manufacturer Design Height (mm) Width (mm)   Figure  2. Design  used  in  this  study:  screw  system  2.0  mm. Design  20  is  the  golden  standard  of  condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically  to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate    Figure  2. Design  used  in  this  study:  screw  system  2.0  mm. Design  20  is  the  golden  standard  of  condylar osteosynthesis. Three other plates design feature by narrow upper part fitting anatomically  to region of interest in mandible. Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. 2.2. Plates 2.2. Plates Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate  Table 1. List of shapes of plates used in osteosynthesis of high‐neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate  11 (red cells) has been occurred as the weakest whereas plates 20 (green cells) have been the Table 1. List of shapes of plates used in osteosynthesis of high-neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate 11 (red cells) has been occurred as the weakest whereas plates 20 (green cells) have been the Table 1. List of shapes of plates used in osteosynthesis of high-neck mandibular condylar fracture. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate 11 (red cells) has been occurred as the weakest whereas plates 20 (green cells) have been the strongest. Above 1 mm thick plates were cut by laser from medical certified titanium sheet alloy grade 23. Plate  11  (red  cells)  has  been  occurred  as  the  weakest  whereas  plates  20  (green  cells)  have  been  the  strongest. g g g Plate11  Synthes  13,5  8  11  (red  cells)  has  been  occurred  as  the  weakest  whereas  plates  20  (green  cells)  have  been  the  strongest. Design code  Manufacturer  Design  Height (mm)  Width (mm)  strongest. Design code  Manufacturer  Design  Height (mm)  Width (mm)  Plate11 Synthes 13 5 8 strongest. Design code Manufacturer Design Height (mm) Width (mm) Plate11 Synthes 13,5 8 Plate14 Medartis 15,3 8,8 Plate02 Medartis 15,4 8,8 Plate20 any 16,5 3,4 2.3. Simulation Set Forces on temporomandibular joint were simulated according to the literature. [32] 15° inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. All fatigues tests were used by means of Zwick Roell Z020 universal strength machine (Zwick-Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. 2.2. Plates 2.2. Plates All the compressive forces were pointed to the condyle Special Instron software recorded relationship Design Code Manufacturer Design Height (mm) Width (mm) Plate11 Synthes Design code  Manufacturer  Design  Height (mm)  Width (mm)  Plate11  Synthes    13,5  8  Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. 13,5 8 Plate14 Medartis Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. 15,3 8,8 Plate02 Medartis Plate11  Synthes    13,5  8  Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). 2.2. Plates 2.2. Plates The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. 15,4 8,8 Plate20 any Plate11  Synthes    13,5  8  Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. 16,5 3,4 2.3. Simulation Set Forces on temporomandibular joint were simulated according to the literature. [32] 15◦inferiorly in the saggital and 10◦laterally in the coronal plane mandibles were solid stabilized by screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7 m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. All fatigues tests were used by means of Zwick Roell Z020 universal strength machine (Zwick-Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the compressive forces were pointed to the condyle. Special Instron software recorded relationship between load and displacement, load for permanent deformation, and maximum load at fracture. 2.2. Plates 2.2. Plates Among all available for rigid fixation of condylar process of mandible plates, only four designs can be applied for high-neck condylar fixation due to anatomical structure of head of condylar process. Some of the other plates [22] could be used, however, manual bending would be necesary and it would change physical properties the osteosynthesis. That is why only four plates were included into this study (Figure 2, Table1). All plates were cut by laser from medical certified titanium sheets (grade 23, 1-millimeter thickness). The o dyla p o e a ut i le el of typi al head e k o dyla f a tu e i ea h odel Among all available for rigid fixation of condylar process of mandible plates, only four designs can be applied for high-neck condylar fixation due to anatomical structure of head of condylar process. Some of the other plates [22] could be used, however, manual bending would be necesary and it would change physical properties the osteosynthesis. That is why only four plates were included into this study (Figure 2, Table 1). All plates were cut by laser from medical certified titanium sheets (grade 23, 1-millimeter thickness). The condylar process was cut in level of typical head-neck condylar fracture in each model according to the newest classification. [31] Proximal head and distal ramus fracture segment were The condylar process was cut in level of typical head-neck condylar fracture in each model according to the newest classification. [31] Proximal head and distal ramus fracture segment were fixed 5 of 12 of  12  of  12  bles Materials 2020, 13, 592 Materials 2020 13 592 p g , Materials 2020, 13, 592 fixed by plate an by plate and the same 6-mm length self-tapping screws of 2.0 system. In each of 7 mandibles for plate design, drill 1.5 mm in width was used before filling plate holes by screws. Materials 2020, 13, 592 5 of 12 fixed by plate and the same 6-mm length self-tapping screws of 2.0 system. In each of 7 mandibles f l d i d ill 1 5 i id h d b f filli l h l b fixed by plate and the same 6‐mm length self‐tapping screws of 2.0 system. In each of 7 mandibles  for plate design, drill 1.5 mm in width was used before filling plate holes by screws. ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set All fatigues tests were used by means of Zwick Roell Z020 universal strength machine (Zwick-Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the compressive forces were pointed to the condyle. Special Instron software recorded relationship between load and displacement, load for permanent deformation, and maximum load at fracture. y gg y p y screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. All fatigues tests were used by means of Zwick Roell Z020 universal strength machine on 0.7m   0.6 m tilted Block with 4   M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  p p y p p between load and displacement, load for permanent deformation, and maximum load at fracture. g p , , y All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. All fatigues tests were used by means of Zwick Roell Z020 universal strength machine (Zwick-Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the compressive forces were pointed to the condyle. Special Instron software recorded relationship between load and displacement, load for permanent deformation, and maximum load at fracture. (Zwick-Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the compressive forces were pointed to the condyle. ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set 2.3. Simulation Set Forces on temporomandibular joint were simulated according to the literature. [32] 15° inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by 2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All f ti t t d b f Z i k R ll Z020 i l t th hi Forces on temporomandibular joint were simulated according to the literature. [32] 15◦inferiorly in the saggital and 10◦laterally in the coronal plane mandibles were solid stabilized by screws on individual base plate (Figure 3). 2.2. Plates 2.2. Plates Irreversible change of shape was described as the start point that the load-displacement relationship b li Th t h th hi h t l d d d j t b f dd l d strongest. Design code Manufacturer Design Height (mm) Width (mm) Plate11 Synthes 13,5 8 Plate14 Medartis 15,3 8,8 Plate02 Medartis 15,4 8,8 Plate20 any 16,5 3,4 Design Code Manufacturer Design Height (mm) Width (mm) Plate11 Synthes Design code  Manufacturer  Design  Height (mm)  Width (mm)  Plate11  Synthes    13,5  8  Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  13,5 8 Plate14 Medartis Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  15,3 8,8 Plate02 Medartis Plate11  Synthes    13,5  8  Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2 3 Si l i S 15,4 8,8 Plate20 any a e y e   , Plate14  Medartis    15,3  8,8  Plate02  Medartis    15,4  8,8  Plate20  any    16,5  3,4  2.3. Simulation Set  16,5 3,4 3. Simulation Set ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set The plate was 1 mm trick made in stainless steel screwed on 0.7 m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. 2.3. Simulation Set Forces on temporomandibular joint were simulated according to the literature. [32] 15° inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by 2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All f ti t t d b f Z i k R ll Z020 i l t th hi Forces on temporomandibular joint were simulated according to the literature. [32] 15◦inferiorly in the saggital and 10◦laterally in the coronal plane mandibles were solid stabilized by screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7 m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. y gg y p y screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. All fatigues tests were used by means of Zwick Roell Z020 universal strength machine on 0.7m   0.6 m tilted Block with 4   M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  p p y p p between load and displacement, load for permanent deformation, and maximum load at fracture. g p , , y All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  between load and displacement, load for permanent deformation, and maximum load at fracture. ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set All f ti t t d b f Z i k R ll Z020 i l t th hi Forces on temporomandibular joint were simulated according to the literature. [32] 15◦inferiorly in the saggital and 10◦laterally in the coronal plane mandibles were solid stabilized by screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed on 0.7 m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction forces were generated upwards, forwards, and medially. 2.3. Simulation Set Forces on temporomandibular joint were simulated according to the literature. [32] 15° inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by 2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0 7m × 0 6 m tilted Block with 4 × M6 holes for stabilisation with screws In such a construction screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. All  fatigues  tests  were  used  by  means  of  Zwick  Roell  Z020  universal  strength  machine  (Zwick‐Roell, Ulm, Germany). Loading force was 1N and velocity of piston was 1 mm/min. All the  compressive  forces  were  pointed  to  the  condyle. Special  Instron  software  recorded  relationship  2.3. Simulation Set  Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). The plate was 1 mm trick made in stainless steel screwed  on 0.7m × 0.6 m tilted Block with 4 × M6 holes for stabilisation with screws. In such a construction  forces were generated upwards, forwards, and medially. Forces  on  temporomandibular  joint  were  simulated  according  to  the  literature. [32]  15°  inferiorly in the saggital and 10° laterally in the coronal plane mandibles were solid stabilized by  screws on individual base plate (Figure 3). ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set Special Instron software recorded relationship between load and displacement, load for permanent deformation, and maximum load at fracture. between load and displacement, load for permanent deformation, and maximum load at fracture. Irreversible change of shape was described as the start point that the load-displacement relationship became non-linear. The moment when the highest load was recorded just before suddenly decrease was called maximum load (Figure 4). 6 of 12 ement of  12 Materials 2020, 13, 592 Irreversible c Materials 2020, 13, 59 According to the previous study [22] the Plate Design Factor was calculated: Plate Design Factor = 0.850954 x Height (mm) + 0.846751 × Width (mm)+ 0.936732 × Plate (mm2) + 0.848039 × Total fixing screws. relationship became non-linear. The moment when the highest load was recorded just before suddenly decrease was called maximum load (Figure 4). According to the previous study [22] the Plate Design Factor was calculated: Plate Design Factor = 0.850954 x Height (mm) + 0.846751 × Width (mm)+ 0.936732 × Plate (mm2) + 0.848039 × Total fixing screws. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 4. Fracture line as the maximal force used. Control - intact condyle. The weakest (plate 11) and the strongest (plates 20) osteosynthesis set. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Irreversible  change  of  shape  was  described  as  the  start  point  that  the  load‐displacement  relationship  became  non‐linear. The  moment  when  the  highest  load  was  recorded  just  before  suddenly decrease was called maximum load (Figure 4). According to the previous study [22] the Plate Design Factor was calculated:  Plate Design Factor = 0.850954 x Height (mm) + 0.846751 × Width (mm)+ 0.936732 × Plate (mm2)  + 0.848039 × Total fixing screws. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 4. Fracture line as the maximal force used. Control ‐ intact condyle. The weakest (plate 11) and  the strongest (plates 20) osteosynthesis set. Figure 4. Fracture line as the maximal force used. Control - intact condyle. The weakest (plate 11) and the strongest (plates 20) osteosynthesis set. According to the previous study [22] the Plate Design Factor was calculated: p g j suddenly decrease was called maximum load (Figure 4). g p p p relationship  became  non‐linear. ate 0 inferiorly in the s d d 2 3 Simulation Set 2.3. Simulation Set 2.3. Simulation Set The  moment  when  the  highest  load  was  recorded  just  before  dd l d ll d i l d (Fi 4) According to the previous study [22] the Plate Design Factor was calculated: p g j ddenly decrease was called maximum load (Figure 4). g p p p elationship  became  non‐linear. The  moment  when  the  highest  load  was  recorded  just  before According to the previous study [22] the Plate Design Factor was calculated p g suddenly decrease was called maximum load (Figure 4). relationship  became  non‐linear. The  moment  when  the  highest  load  was  reco dd l d ll d l d ( ) Plate Design Factor = 0.850954 x Height (mm) + 0.846751 × Width (mm)+ 0.936732 × Plate (mm2) + 0.848039 × Total fixing screws. According to the previous study [22] the Plate Design Factor was calculated: Plate Design Factor = 0.850954 x Height (mm) + 0.846751 × Width (mm)+ 0.936732 × Plate (mm2) 0 848039 T l f suddenly decrease was called maximum load (Figure 4). According to the previous study [22] the Plate Design Factor was calculated:  Plate Design Factor 0 850954 x Height (mm) + 0 846751 × Width (mm)+ 0 936732 × Plate (mm2) + 0.848039 × Total fixing screws. Figure 3. Stainless steel clamping system for fatigue tests of mandibles Figure 3 Stainless steel clamping system for fatigue tests of mandibles g g ( ) ( ) ( ) + 0.848039 × Total fixing screws. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 3. Stainless steel clamping system for fatigue tests of mandibles. Figure 4. Fracture line as the maximal force used. Control - intact condyle. The weakest (plate 11) and h ( l ) h Figure 4. Fracture line as the maximal force used. Control ‐ intact condyle. The weakest (plate 11) and  the strongest (plates 20) osteosynthesis set. Figure 4. Fracture line as the maximal force used. Control - intact condyle. The weakest (plate 11) and the strongest (plates 20) osteosynthesis set. 7 of 12 7 of 12 Materials 2020, 13, 592 2.4. Statistical Analysis Height, width, plate surface area, plate design factor required force for one milimeter displacement in fracture line after plate fixation were recorded for interpretation of the experimental data. Software used for statistics was Statgraphics Centurion 18 (Statgraphics Technologies Inc. The Plains, Virginia, USA). Kruskal–Wallis test was applied for between design comparisons. Independence Chi-Square tested categorical variables. Indicating the best plates were done on objective description. 3. Results Only double plate fixation survived to the end of the experiment (Table 2). Three other osteosynthesisplates design lost screws in proximal fragment, as well as in distal fragment. As far as the plate integration was considered, the double plates broke during loading (in 160.6±27.3N) contrary to three other plates. Only two single plates tendP to break after rigid screws fixation. However, double plates fixation endured the highest loading (14.02±1.24 N/mm; p < 0.05) comparing to the three small plates (Figure 4). Plate Design Factor calculation that is valuable numerical description of plate design took into account the characteristics of the plate constructions as dimensions and number of screws possible to use (Table 3; Figure 5). Table 2. Analysis of fractures after solid screws fixation (Chi-Square test = 28, p < 0.0001). Only two single plates (plates 20) broke after rigid fixation. In others screws tend to break. Table 2. Analysis of fractures after solid screws fixation (Chi-Square test = 28, p < 0.0001). Only two single plates (plates 20) broke after rigid fixation. In others screws tend to break. Plates No Broken Endured Row Total Plate 11 0 7 7 0.00% 25.00% 25.00% Plate 14 0 7 7 0.00% 25.00% 25.00% Plate 2 0 7 7 0.00% 25.00% 25.00% Plate 20 7 0 7 25.00% 0.00% 25.00% Column Total 7 21 28 25.00% 75.00% 100.00% Table 3. Design data and summary statistics. Abbreviations: F max/dL – force required for one-millimeter displacement in fracture line after osteosynthesis. One-millimeter Displacement in Fracture Line Height (mm) Width (mm) Plate Surface Area (mm2) Plate Design Factor F max/dL (N/mm) Average±stand. dev. 15.18±1.10 7.25±2.29 179.28±32.15 191.23±30.79 8.60±4.03 Minimum 13.5 3.4 138.13 151.05 2.57 Maximum 16.5 8.8 226.82 236.17 15.65 Range 3.0 5.4 88.69 85.13 13.08 Stnd. skewness −1.08 −2.48 0.68 0.53 0.68 Stnd. kurtosis −0.97 −0.68 −1.02 −1.03 −1.30 Materials 2020, 13, 592 8 of 12 8 of 12 Figure 5. Plate Design Factor: plate 11 is 151, plate 14 is 191, plate 2 is 187 and plate 20 is 236. It  proves plate 20 should be used as gold standard in high‐neck fractures whereas plate 11 should fall  into abeyance. plate 11 plate 14 plate 2 plate 20 Box-and-Whisker Plot 150 170 190 210 230 250 Plate Design Factor Design Figure 5. Plate Design Factor: plate 11 is 151, plate 14 is 191, plate 2 is 187 and plate 20 is 236. 4.1. Plates Combination  4.1. Plates Combination In the paper, double plates (plate design 20] has been proved as osseosynthesis material thanks  to  which  excellent  stability  might  be  achieved. These  plates  have  been  well  known  [22,33]  and  derived  from  combination  of  biomechanical  plates  location  and  up  to  4‐screw  fixation  in  the  proximal  fragment  i.e. upper  fragment  [34]. However,  till  now  it  was  impossible  to  find  in  the  literature that scientifically prove double plate osteosynthesis is gold standard in high‐neck fracture. By means of evidence performed in this study we proved that this thesis is true. Unfortunately, the  main  drawback  of  2  plates  number  20  used  together  is  necessity  of  holding  two  separate  plates  firmly in very hard operating space. In our previous study we proved that total osseosynthesis plate  surface is related with dedicated plate, and relation that the bigger plate is, the higher forces are  required to 1‐millimeter movement in fracture line (p <0.05). The correlation coefficient equalled  0.58, indicating a moderately strong relationship between the plate surface area and the displacing  force,  points  to  the  valuable  feature  of  plates  design  which  plays  significant  role  in  stable  osteosynthesis (p <0.05) [22]. In the paper, double plates (plate design 20] has been proved as osseosynthesis material thanks to which excellent stability might be achieved. These plates have been well known [22,33] and derived from combination of biomechanical plates location and up to 4-screw fixation in the proximal fragment i.e. upper fragment [34]. However, till now it was impossible to find in the literature that scientifically prove double plate osteosynthesis is gold standard in high-neck fracture. By means of evidence performed in this study we proved that this thesis is true. Unfortunately, the main drawback of 2 plates number 20 used together is necessity of holding two separate plates firmly in very hard operating space. In our previous study we proved that total osseosynthesis plate surface is related with dedicated plate, and relation that the bigger plate is, the higher forces are required to 1-millimeter movement in fracture line (p < 0.05). The correlation coefficient equalled 0.58, indicating a moderately strong relationship between the plate surface area and the displacing force, points to the valuable feature of plates design which plays significant role in stable osteosynthesis (p < 0.05) [22]. y (p ) [ ] Very important factor that we decided to include in our criteria was to choose plates dedicated  anatomically for high neck fractures. 3. Results It proves plate 20 should be used as gold standard in high-neck fractures whereas plate 11 should fall into abeyance. plate 11 plate 14 plate 2 plate 20 Box-and-Whisker Plot 150 170 190 210 230 250 Plate Design Factor Design Box-and-Whisker Plot Figure 5. Plate Design Factor: plate 11 is 151, plate 14 is 191, plate 2 is 187 and plate 20 is 236. It  proves plate 20 should be used as gold standard in high‐neck fractures whereas plate 11 should fall  into abeyance. Figure 5. Plate Design Factor: plate 11 is 151, plate 14 is 191, plate 2 is 187 and plate 20 is 236. It proves plate 20 should be used as gold standard in high-neck fractures whereas plate 11 should fall into abeyance. 4.1. Plates Combination  4.1. Plates Combination It means we excluded plates that could be used but only after  bending  intrasurgically  or  before  on  patient‐specific  3D  stereolithographic  condylar  process. [35]  Some additional experiments are needed to prove if other available plates could be strong enough to  endure mastication forces in healing process. Without pre‐bending many of presented plates design  are not feasible for use because they are too big for high neck condylar process. Rigidity of plates is  of paramount importance for every inventor, manufacturer, user or doctor for ORIF (open reduction  and rigid internal fixation). Apart from the design, authors of the study wanted to describe physical  properties of the condylar plates (grade of titanium alloy, annealing process, Young module, etc.),  and asked manufacturers by phone and e‐mails, however, any of the manufacturers did not answer  Very important factor that we decided to include in our criteria was to choose plates dedicated anatomically for high neck fractures. It means we excluded plates that could be used but only after bending intrasurgically or before on patient-specific 3D stereolithographic condylar process. [35] Some additional experiments are needed to prove if other available plates could be strong enough to endure mastication forces in healing process. Without pre-bending many of presented plates design are not feasible for use because they are too big for high neck condylar process. Rigidity of plates is of paramount importance for every inventor, manufacturer, user or doctor for ORIF (open reduction and rigid internal fixation). Apart from the design, authors of the study wanted to describe physical properties of the condylar plates (grade of titanium alloy, annealing process, Young module, etc.), and asked manufacturers by phone and e-mails, however, any of the manufacturers did not answer our requests for information. Our study has not only been written directly doctors, 9 of 12 Materials 2020, 13, 592 surgeons but also by inventors, constructors, and medical designers. Authors want to emphasize the importance of design and present first worldwide comparison of all known designs of plate for high neck fractures. Obviously, it is still the open question: how in annealing the alloy, it may be better to use zirconium-molybdenum alloys or titanium-niobium alloys; is 0.9 mm thickness enough, or is 1.3 mm safer and/or more rigid? Those fascinating questions should answered the future. 4.1. Plates Combination  4.1. Plates Combination Moreover, comparison of new with smaller dimensions of plates with narrower screws with available plates but after bending could be interesting study, especially in high neck fractures where intrasurgical access is restricted. Some additional clinical papers are needed to compare the usage of 2 separate plates with others given that endocranial hardships such as restricted operation vision, time of screwing plates, import and anatomical structures that make screwing two plates harder to perform than one single. 4.2. Finite Element Method (FEM) The rehabilitative dentistry has always paid particular attention to the detailed analysis application of occlusal forces, the distribution of tensional forces, and stress dissipation, asbiomechanical factors influence the prosthetic success substantially. Nowadays it is possible to design patient-specific plates for condylar fractures. [36] High technology in computing and manufacturing processes allowsurgeons to design a patient specific implant (PSI) to fit a particular fracture, including high neck, and to then before manufacturing analyze its performance in finite element analysis before screwing it in the patient. Thus a few different shapes can be quickly evaluated and the plate with the optimal outcome selected. Because any dedicated plate was not confirmed biomechanically proper for osseosynthesis of the high neck fracture, it should be better fixation material that we’re looking for. In the literature it might be found that in high neck condyle fracture the most prone to fixation of the fracture was osseosynthesis by means of 3 screws. The area of stress in condylar head was negligibly low σ = 108 MPa and stress distribution in the rest of mandible was almost the same as in untouched bone in mastication process. Some authors have proved that one screw stabilization was the reason of relatively high displacement of bone ends, 558±245µm, whereas in 2 screws displacement was 218±81µm, and for 3 screws it was 217±144µm [34]. 4.3. Plate Design Factor (PDF) In our previous study where we compared plates systems in basal condylar fractures, probabilistic neural network PNN procedure pointed that plates of construction described by PDF over 300 were the most resistant to screw pull-out as well displacing force. However, in our present article PDF observed in tested fixations are relatively low comparing to results of basal condylar fracture fixations [22]. There was no result over 300, and only the double plate fixation exceeded 200. On one hand, it means that there is no proper dedicated plate for high neck fracture osteosynthesis, on the other hand, it is urgent matter to invent the effective one plate for this challenging fracture. The suggested Plate Design Factor (PDF) should be indicator for forthcoming osseosythesis plates. It can be simply measured and compared with others under the circumstance 1-millimeter movement in fracture line will be considered. The discrimination power of that factor is such high as even very similar plates in this study can reach significant difference from another (and can be individually considered). The experiment cannot prove advantage of the osseosynthesis plate number 11 which was the worst among all 4 plates (the lowest force required for 1-millimetere displacement, easiness in screw pull out, the lowest Plate Design Factor). For practitioners it is crucial to know whether plates must not apply or use only with consideration of reduction of forces in occlusion in high-neck fractures (i.e. plate number 11). It is possible that other osseosynthesis plates that authors of this article examined in another publication in lower condyle fracture could be as good as plater number 20 or even better. The question remains as to if the bending of other plates designed for lower condyle fractures play significant role in mechanical properties of titanium alloy grade 23? In this study any bending of titanium plates were performed in order not to diminish titanium properties but maybe individual Materials 2020, 13, 592 10 of 12 bending of plates will not change significantly endurance of material. Comparison of all available plates after preparation such as changing the shape of plates in order to obtain the highest possible bone surface plate contact should be the next mechanical research valuable for treatment patients as well as to know mechanical properties of titanium alloy grade 23. The next problem are study restrictions. 4.3. Plate Design Factor (PDF) Despite the fact the mechanical properties of the foam models were comparable to real mandible, some discrepancies in the structure of the materials were observed. For example artificial models have an almost homogeneous pore size, whereas in human mandibles consist of complex texture filled with different size of pores. It might play the role in compression efficacy and torque of the screws. The outcomes of our research were made on asingle-density foam polyurethane bone; but the biomechanical properties of the screws changes with the bone density environment. [29] Further fatigue tests and FEM analysies with changed shape of other plates are necessary. Concluding, the double straight plates are the best mechanical fixation for high-neck fractures of the mandibular condyle. The limitation of its application significantly limited area for double 2.0 system plate fixation. Maybe other existing dedicated plates could be used, but only after prebending. That shape manipulation influences the mechanical properties of the material. That is the exploring field for novel dedicated plates designs searching. Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, M.K. (Marcin Kozakiewicz) and R.Z.; methodology, R.Z.; M.K. (Michał Krasowski); J.O.; B.K.; software, M.K. (Michał Krasowski); J.O.; B.K.; validation, R.Z.; formal analysis, M.K. (Michał Krasowski); J.O.; B.K.; investigation, Marcin Kozakieiwcz, M.K. (Michał Krasowski); J.O.; B.K.; resources, M.K. (Michał Krasowski); J.O.; B.K.; data curation, J.O.; writing—original draft preparation, R.Z.; writing—review and editing, R.Z.; visualization, M.K. (Marcin Kozakiewicz), J.O., R.Z.; supervision, R.Z., M.K. (Marcin Kozakiewicz); project administration, M.K. (Marcin Kozakiewicz); funding acquisition, R.Z.”, please turn to the CRediT taxonomy for the term explanation. Authorship must be limited to those who have contributed substantially to the work reported. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Medical University, grant number 503/5-061-02/503-51-001-18, 503/5-061-02/503-51-001-17, 503/5-061-02/503-51-002-18. Acknowledgments: no acknowledgments Acknowledgments: no acknowledgments Conflicts of Interest: The authors declare no conflict of interest. References Asprino, L.; Consani, S.; De Moraes, M. A comparative biomechanical evaluation of mandibular fracture plating techniques. J. Oral Maxillofac. Surg. 2006, 64, 452–456. [CrossRef] . Chrcanovic, B. Surgical versus non-surgical treatment of mandibular condylar fractures: A meta-anal Int. J. Oral Maxillofac. Surg. 2015, 44, 158–179. [CrossRef] [PubMed] 3. Chen, C.-T.; Feng, C.-H.; Tsay, P.-K.; Lai, J.-P.; Chen, Y.-R. Functional outcomes following surgical treatment of bilateral mandibular condylar fractures. Int. J. Oral Maxillofac. Surg. 2011, 40, 38–44. [CrossRef] [PubMed] 3. Chen, C.-T.; Feng, C.-H.; Tsay, P.-K.; Lai, J.-P.; Chen, Y.-R. Functional outcomes following surgical treatment of bilateral mandibular condylar fractures. Int. J. Oral Maxillofac. Surg. 2011, 40, 38–44. [CrossRef] [PubMed] 4. Ehrenfeld, M.; Manson, P.N.; Prein, J. Thieme, Principles of Internal Fixation of the Craniomaxillofacial Skeleton Trauma and Orthognathic Surgery; AO Publishing: Stockholm, Sweden, 2012. [CrossRef] 4. Ehrenfeld, M.; Manson, P.N.; Prein, J. Thieme, Principles of Internal Fixation of the Craniomaxillofacial Skeleton Trauma and Orthognathic Surgery; AO Publishing: Stockholm, Sweden, 2012. [CrossRef] g 5. Abdel-Galil, K.; Loukota, R. Review: Fractures of the mandibular condyle: Evidence base and current concepts management. Br J Oral MaxillofacSurg 2010, 48, 520–526. [CrossRef] [PubMed] 6. Al-Moraissi, E.A.; Ellis, E. Surgical Treatment of Adult Mandibular Condylar Fractures Provides Better Outcomes Than Closed Treatment: A Systematic Review and Meta-Analysis. J. Oral Maxillofac. Surg. 2015, 73, 482–493. [CrossRef] 7. Neff, A.; Chossegros, C.; Blanc, J.; Chanpsaur, P.; Cheynet, F.; Devauchelle, B.; Eckelt, U.; Ferri, J.; Gabrielli, M.F.R.; Guyot, L.; et al. Position paper from the IBRA symposium on surgery of the head-the 2nd international Simposium for condylar fracture Osteosynthesis, Marseille, France 2012. J CraniomaxillofacSurg 2014, 42, 1234–1249. [CrossRef] 8. Bredbenner, T.L.; Haug, R.H. Substitutes for human cadaveric bone in maxillofacial rigid fixation research. Oral Surgery, Oral Med. Oral Pathol. Oral Radiol. Endodontology 2000, 90, 574–580. [CrossRef] Materials 2020, 13, 592 11 of 12 11 of 12 9. Montazem, A.H.; Anastassov, G. Management of Condylar Fractures. Atlas Oral Maxillofac. Surg. Clin. 2009, 17, 55–69. [CrossRef] 9. Montazem, A.H.; Anastassov, G. Management of Condylar Fractures. Atlas Oral Maxillofac. Surg. Clin. 2009, 17, 55–69. [CrossRef] 10. Zachariades, N.; Mezitis, M.; Mourouzis, C. Fractures of themandibular condyle: A reviewof 466 cases. Literature review, reflections on treatment and proposals. J CraniomaxillofacSurg 2006, 34, 421–432. [CrossRef] 11. Silvennoinen, U.; Iizuka, T.; Oikarinen, K. Analysis of possibile factors leading to problems after surgical treatment of condylar fractures. J Oral MaxillofacSurg 1994, 52, 793–799. [CrossRef] 12. References Newman, L. A clinical evaluation of the long-term outcome of patients treated for bilateral fracture of the mandibular condyles. Br. J. Oral Maxillofac. Surg. 1998, 36, 176–179. [CrossRef] 13. De Riu, G.; Gamba, U.; Anghinoni, M. A comparison of open and closed treatment of condylar fractures: A change in philosophy. Int J Oral MaxillofacSurg 2001, 30, 384–389. [CrossRef] [PubMed] 14. Villarreal, P.M.; Monje, F.; Junquera, L.M.; Mateo, J.; Morillo, A.J.; González, C. Mandibular condyle fractures: Determinants of treatment and outcome. J. Oral Maxillofac. Surg. 2004, 62, 155–163. [CrossRef] 15. Achour, A.B.; Meißner, H.; Teicher, U.; Haim, D.; Range, U.; Brosius, A.; Lauer, G. Biomechanical Evaluation of Mandibular Condyle Fracture Osteosynthesis Using the Rhombic Three-Dimensional Condylar Fracture Plate. J. Oral Maxillofac. Surg. 2019, 77, 1868.e1–1868.e15. f g 16. Emam, H.A.; Jatana, C.A.; Ness, G.M. Matching Surgical Approach to Condylar Fracture Type. Atlas Oral Maxillofac. Surg. Clin. 2017, 25, 55–61. [CrossRef] 17. Wagner, A.; Krach, W.; Schicho, K.; Undt, G.; Ploder, O.; Ewers, R. A 3-dimensional finite-element analysis investigating the biomechanical behavior of the mandible and plate osteosynthesis in cases of fractures of the condylar process. Oral Surgery, Oral Med. Oral Pathol. Oral Radiol. Endodontology 2002, 94, 678–686. [CrossRef] 18. Meyer, C.; Kahn, J.L.; Boutemi, P. Photoelastic analysis of bone deformation in the region of the mandibular condyle Turing mastication. J CraniomaxillofacSurg 2002, 30, 160e9. 19. Sikes, J.W., Jr.; Smith, B.R.; Mukherjee, D.P. An in vitro study of the effect of bony buttressing on fixation strength of a fractured atrophic edentulous mandible Model.Including commentary by Tucker MR. J Oral MaxillofacSurg 2000, 58, 56e61. 20. Trainor, P.; McLachlan, K.; McCall, W. Modelling of forces in the human masticatory system with optimization of the angulations of the joint loads. J. Biomech. 1995, 28, 829–843. [CrossRef] 21. Tuchtan, L.; Piercecchi-Marti, M.-D.; Bartoli, C.; Boisclair, D.; Adalian, P.; Leonetti, G.; Behr, M.; Thollon, L. Forces transmission to the skull in case of mandibular impact. Forensic Sci. Int. 2015, 252, 22–28. [CrossRef] 22. Kozakiewicz, M.; Zieli´nski, R.; Krasowski, M.; Okulski, J. Forces causing one-millimeter displacement of bonefragments of condylar base fractures of the mandible after fixation by All available plate designs. Materials (Basel) 2019, 12, 3122. [CrossRef] [PubMed] 23. Goldstein, S. The mechanical properties of trabecular bone: Dependence on anatomic location and function. J. Biomech. 1987, 20, 1055–1061. [CrossRef] 24. Chapman, J.R.; Harrington, R.M.; Lee, K.M.; Anderson, P.A.; Tencer, A.F.; Kowalski, D. References Factors Affecting the Pullout Strength of Cancellous Bone Screws. J. Biomech. Eng. 1996, 118, 391–398. [CrossRef] [PubMed] 25. ASTMF 1839-08. Standard Specification for Rigid Polyurethane Foam for Use as a Standard Material for Testing Orthopaedic Devices and Instruments; ASTM International: West Conshohocken, PA, USA, 2016. 26. Kozakiewicz, M. Comparison of compression screws used for mandible head fracture treatment—Experimentalstudy. Clin. oral Investig. 2019, 23, 4059–4066. [CrossRef] [PubMed] 26. Kozakiewicz, M. Comparison of compression screws used for mandible head fracture treatment—Experimentalstudy. Clin. oral Investig. 2019, 23, 4059–4066. [CrossRef] [PubMed] 27. Assari, S.; Darvish, K.; Ilyas, A.M. Biomechanical analysis of second-generation headless compression screws. Inj. 2012, 43, 1159–1165. [CrossRef] 27. Assari, S.; Darvish, K.; Ilyas, A.M. Biomechanical analysis of second-generation headless compression screws. Inj. 2012, 43, 1159–1165. [CrossRef] 28. Baran, O.; Sagol, E.; Oflaz, H.; Sarikanat, M.; Havitcioglu, H. Abiomechanical study on preloaded compression effect on headless screws. Arch. Orthop. Trauma. Surg. 2009, 129, 1601–1605. [CrossRef] 28. Baran, O.; Sagol, E.; Oflaz, H.; Sarikanat, M.; Havitcioglu, H. Abiomechanical study on preloaded compression effect on headless screws. Arch. Orthop. Trauma. Surg. 2009, 129, 1601–1605. [CrossRef] 29. Ramaswamy, R.; Evans, S.; Kosashvili, Y. Holding power of variable pitch screws in osteoporotic, osteopenic and normal bone: Are all screws created equal? Inj. 2010, 41, 179–183. [CrossRef] 29. Ramaswamy, R.; Evans, S.; Kosashvili, Y. Holding power of variable pitch screws in osteoporotic, osteopenic and normal bone: Are all screws created equal? Inj. 2010, 41, 179–183. [CrossRef] 30. Bailey, C.A.; Kuiper, J.H.; Kelly, C.P. Biomechanical Evaluation of a New Composite Bioresorbable Screw. J. Hand Surg. 2006, 31, 208–212. [CrossRef] 30. Bailey, C.A.; Kuiper, J.H.; Kelly, C.P. Biomechanical Evaluation of a New Composite Bioresorbable Screw. J. Hand Surg. 2006, 31, 208–212. [CrossRef] 31. Kozakiewicz, M. Classification proposal for fractures of the processuscondylaris mandibulae. Clin. Oral Investig. 2018, 23, 485–491. [CrossRef] 32. Choi, B.-H.; Kim, K.-N.; Kim, H.-J.; Kim, M.-K. Evaluation of condylar neck fracture plating techniques. J. Cranio-Maxillofacial Surg. 1999, 27, 109–112. [CrossRef] 12 of 12 12 of 12 Materials 2020, 13, 592 33. Cienfuegos, R.; Cornelius, C.; Ellis, E.I.; Kushner, G.M. Condylar Process and Head; Ellis, E., Figari, M., Aniceto, G., Shimozato, K., Eds.; AO CMF Surgery Reference; AO Foundation: New York, NY, USA; Thieme Publisher: New York, NY, USA, 2014. 34. Kozakiewicz, M.; ´Swiniarski, J. Treatment of high fracture of the neck of the mandibular condylar process by rigid fixation performed by lag screws: Finite element analysis. References Dent Med. Probl. 2017, 54, 223–228. [CrossRef] 5. Adamus, J.; Lacki, P.; Motyka, M. Analysis of titanium sheet bending process. Inz. Mater. 2010, 31, 716– 35. Adamus, J.; Lacki, P.; Motyka, M. Analysis of titanium sheet bending process. Inz. Mater. 2010, 31, 716–719. 36. Aquilina, P.; Parr, W.C.; Chamoli, U.; Wroe, S. Finite element analysis of patient-specific condyle fracture plates: a preliminary study. Craniomaxillofac Trauma Reconstr. 2015, 8, 111–116. [CrossRef] [PubMed] 36. Aquilina, P.; Parr, W.C.; Chamoli, U.; Wroe, S. Finite element analysis of patient-specific condyle fracture plates: a preliminary study. Craniomaxillofac Trauma Reconstr. 2015, 8, 111–116. [CrossRef] [PubMed] © 2020 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 (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2129244987
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0090074&type=printable
English
null
Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Common Carp
PloS one
2,014
cc-by
6,089
Abstract 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: Funding was provided by grants from the Special Fund for Agro-Scientific Research in the Public Interest (No. 200903045), National Natural Science Foundation of China (No. 30972250), and by the Shanghai University Knowledge Service Platform (ZF1206). 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. * E-mail: wangch@shou.edu.cn (CW); glu3@unomaha.edu (GL) Chenghui Wang1,2*, Michael Wachholtz2, Jun Wang2, Xiaolin Liao2,3, Guoqing Lu 1 Key Laboratory of Freshwater Fisheries Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China, 2 Department of Biology, University of Nebraska at Omaha, Omaha, Nebraska, United States of America, 3 Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem, Institute of Hydroecology, Ministry of Water Resources & Chinese Academy of Sciences, Wuhan, China Abstract Background: Body color and coloration patterns are important phenotypic traits to maintain survival and reproduction activities. The Oujiang color varieties of common carp (Cyprinus carpio var. color), with a narrow distribution in Zhejiang Province of China and a history of aquaculture for over 1,200 years, consistently exhibit a variety of body color patterns. The molecular mechanism underlying diverse color patterns in these variants is unknown. To the practical end, it is essential to develop molecular markers that can distinguish different phenotypes and assist selective breeding. Methodology/Principal Findings: In this exploratory study, we conducted Roche 454 transcriptome sequencing of two pooled skin tissue samples of Oujiang common carp, which correspond to distinct color patterns, red with big black spots (RB) and whole white (WW), and a total of 737,525 sequence reads were generated. The reads obtained in this study were co-assembled jointly with common carp Roche 454 sequencing reads downloaded from NCBI SRA database, resulting in 43,923 isotigs and 546,676 singletons. Over 31 thousand (31,445; 71.6%) isotigs were found with significant BLAST matches (E,1e-10) to the nr protein database, which corresponds to 12,597 annotated zebrafish genes. A total of 70,947 isotigs and singletons (transcripts) were annotated with Gene Ontology, and 60,221 transcripts were found with corresponding EC numbers. Out of 145 zebrafish pigmentation genes, orthologs for 117 were recovered in Oujiang color carp transcriptome, including 18 found only among singletons. Our transcriptome analysis revealed over 52,902 SNPs in Oujiang common carp, and identified 63 SNP markers that are putatively unique either for RB or WW. Conclusions: The transcriptome of Oujiang color varieties of common carp obtained through this study, along with the pigmentation genes recovered and the color pattern-specific molecular markers developed, will facilitate future research on the molecular mechanism of color patterns and promote aquaculture of Oujiang color varieties of common carp through molecular marker assisted-selective breeding. Citation: Wang C, Wachholtz M, Wang J, Liao X, Lu G (2014) Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Com e90074. doi:10.1371/journal.pone.0090074 holtz M, Wang J, Liao X, Lu G (2014) Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Common Carp. PLoS ONE 9(3) nal.pone.0090074 Editor: La´szlo´ Orba´n, Temasek Life Sciences Laboratory, Singapore Editor: La´szlo´ Orba´n, Temasek Life Sciences Laboratory, Singapore Received June 6, 2013; Accepted January 29, 2014; Published March 6, 2014 Copyright:  2014 Wang et al. Analysis of the Skin Transcriptome in Two Oujiang Color Varieties of Common Carp Chenghui Wang1,2*, Michael Wachholtz2, Jun Wang2, Xiaolin Liao2,3, Guoqing Lu2* PLOS ONE | www.plosone.org Introduction melanogenesis pathways have been found conserved in vertebrates [8,9,10]. Melanin synthesis takes place within the melanosomes of melanocytes and involves the tyrosine metabolism pathway. The tyrosinases (TYR), tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT) are important enzymes in melanin synthesis [8,11]. The most important regulator of melanogenesis is the MC1 receptor (MC1R) with its melanocortic peptides [10]. A single mutation of this gene was found to be associated with the beach mouse color pattern [12]. MC1R activates the cyclic AMP (cAMP) response-element binding protein (CREB), an important cellular transcription factor. Increased expression of microphthal- mia-associated transcription factor (MITF) and its activation by phosphorylation (P) stimulate the transcription of TYR, TYRP1 and DCT [7,8,13,14]. The biosynthesis of other types of pigments may involve different pathways and regulatory networks, but are in general less studied [6]. Body color and coloration patterns are important phenotypic traits associated with the survival and reproduction activities in many organisms, including their camouflage, mimicry, social communication and selective mating [1,2,3]. Coloration is determined mainly by pigments synthesized by chromatophores, i.e., pigment cells. So far, six types of pigment cells, including melanocytes (black, dark brown), xanthophores (yellow), erythro- phores (red/orange), iridophores (reflecting), leucophores (white), and cyanophores (blue), have been reported in vertebrates [4]. Mammals and birds were found to have only melanocytes in their skin, whereas reptiles also possess xanthophores, erythrophores and iridophores. Interestingly, only teleost fishes were found to have all six types of pigment cells [5,6,7]. The molecular mechanism of melanin biosynthesis has been xextensively studied due to its biomedical significance; the March 2014 | Volume 9 | Issue 3 | e90074 1 PLOS ONE | www.plosone.org Transcriptomes of Oujiang Color Varieties of Common Carp Figure 1. Analysis pipeline of Oujiang color varieties of common carp (RB - red with big black spots and WW – whole white) and gynogenic common carp (CC) transcriptomes, with the number of raw 454 reads, singletons and isotigs denoted. The pictures of RB, WW and CC are shown at the top panel. CC raw sequencing reads were downloaded from NCBI SRA database (SRA 050545). doi:10.1371/journal.pone.0090074.g001 Figure 1. Analysis pipeline of Oujiang color varieties of common carp (RB - red with big black spots and WW – whole white) and gynogenic common carp (CC) transcriptomes, with the number of raw 454 reads, singletons and isotigs denoted. The pictures of RB, WW and CC are shown at the top panel. Introduction CC raw sequencing reads were downloaded from NCBI SRA database (SRA 050545). doi:10.1371/journal.pone.0090074.g001 In our previous study, significant genetic differences were revealed among the different varieties of Oujiang color common carp through the use of microsatellite markers [21]. In addition, our field growth experiment demonstrated that RB grew faster than other body color types of Oujiang color common carp, while WW grew at the slowest rate [22]. Developing more genetic markers will not only facilitate the studies of population genetics of Oujiang color varieties of common carp, but it will also improve its The common carp (Cyprinus carpio L.), a cyprinid belonging to Cyprinidae of ray-finned fishes (teleosts), is considered the most widely distributed and important aquaculture species in the world [15]. During the long history of domestication, numerous strains or variants of common carp have been established through artificial selection from the wild ancestral populations, of which many have diverse colors and coloration patterns [16,17]. The molecular mechanisms underlying these diverse color patterns in common carp remain less well-understood. Oujiang color varieties of common carp (Cyprinus carpio var. color), a unique variant of common carp, has been cultured in paddies and backyard ponds for about 1,200 years in the Oujiang river basin of Zhejiang province in China [18]. It has been found that several body colors and coloration patterns consistently exist, including whole red (WR), red with scattered black spots (RB), whole white (WW), and white with scattered black spots (WB), which provide an excellent model to explore the molecular mechanisms of pigment formation and development [16]. Table 1. Summary statistics of common carp skin transcriptomic sequencing and assembly. Raw Reads RB* 408,884 WW* 328,641 CC** 2,119,118 Total 2,856,643 Number of isotigs 43,923 Number of singletons 546,676 Average contig length (bp) 1,000 Maximal contig length (bp) 11,663 * Oujiang color varieties of common carp: RB - red with big black spots, WW - whole white; ** CC reads are from a gynogenic common carp from 12 tissues downloaded from NCBI (SRA 050545). doi:10.1371/journal.pone.0090074.t001 Table 1. Summary statistics of common carp skin transcriptomic sequencing and assembly. PLOS ONE | www.plosone.org Introduction Raw Reads RB* 408,884 WW* 328,641 CC** 2,119,118 Total 2,856,643 Number of isotigs 43,923 Number of singletons 546,676 Average contig length (bp) 1,000 Maximal contig length (bp) 11,663 * Oujiang color varieties of common carp: RB - red with big black spots, WW - whole white; ** CC reads are from a gynogenic common carp from 12 tissues downloaded from NCBI (SRA 050545). doi:10.1371/journal.pone.0090074.t001 Table 1. Summary statistics of common carp skin transcriptomic sequencing and assembly. In a mating experiment involving whole red (WR) and whole white (WW) Oujiang color common carp varieties, we observed a ratio of three red to one white, providing evidence of Mendelian inheritance in the red and white body colors [19]. Although the black color showed dominant inheritance over red and white, it does not seem to follow the Mendelian law, suggesting that the regulation of this pattern is more complicated than the others (Wang et al., unpublished). In a study of red and black color inheritance with Koi, an ornamental variety developed from the common carp, three phenotypes were found associated with the red color and two phenotypes with the black one [20]. PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e90074 March 2014 | Volume 9 | Issue 3 | e90074 2 Transcriptomes of Oujiang Color Varieties of Common Carp Ethics statement This study was approved by the Institutional Animal Care and Use Committee (IACUS) of Shanghai Ocean University, Shang- hai, China. The collection of fish samples was permitted by the Provincial Farm of Oujiang Color Common Carp in Zhejiang Province, China, and all sampling procedures were complied with the guidelines of IACUS on the care and use of animals for scientific purposes. Sample collection Oujiang color common carp samples (tetraploid) were collected from six siblings, including three whole white (WW) and three red with big black spots (RB), from the Provincial Farm of Oujiang Color Common Carp in Zhejiang Province, China. Fresh skin tissues (,3 g) were collected and preserved immediately in liquid nitrogen for RNA extraction. cDNA synthesis y Double-stranded cDNA was synthesized from mRNA according to Ng’s full-length cDNA synthesis protocol with minor Figure 3. Venn diagram of the number of isotigs found in the skin transcriptome of Oujiang color varieties of common carp (RB and WW) and gynogenic transcriptome of common carp (CC). RB - red with big black spots, WW - whole white. doi:10.1371/journal.pone.0090074.g003 Table 2. The number of common carp transcripts (isotigs and singletons) and enzymes found in each KEGG pathway. mber of common carp transcripts (isotigs and singletons) and enzymes found in each KEGG pathway. Table 2. The number of common carp transcripts (isotigs and singletons) and enzymes found in each Table 2. The number of common carp transcripts (isotigs and singletons) and enzymes found in each KEGG pathway. Pathways No. of isotigs No. of enzymes (isotigs) No. of singletons No. of enzymes (singletons) Purine metabolism 477 55 1,263 55 Glycolysis/Gluconeogenesis 196 16 177 24 Oxidative phosphorylation 175 8 445 7 Pyrimidine metabolism 166 30 387 28 Amino sugar and nucleotide sugar metabolism 147 37 94 22 Fructose and mannose metabolism 122 19 78 12 Phosphatidylinositol signaling system 121 18 284 17 Arginine and proline metabolism 121 28 151 28 Aminoacyl-tRNA biosynthesis 119 21 96 23 Pyruvate metabolism 110 24 110 15 Pentose phosphate pathway 108 17 81 14 Carbon fixation pathways in prokaryotes 106 20 119 16 Starch and sucrose metabolism 98 22 93 21 Methane metabolism 96 19 87 16 Inositol phosphate metabolism 94 20 193 17 Glycerophospholipid metabolism 93 26 171 21 T cell receptor signaling pathway 93 2 167 2 Lysine degradation 93 15 152 14 Citrate cycle (TCA cycle) 92 17 100 16 Cysteine and methionine metabolism 89 25 90 19 Shown only the top 20 pathways and see Table S2 for the complete list of 134 pathways. doi:10.1371/journal.pone.0090074.t002 Shown only the top 20 pathways and see Table S2 for the complete list of 134 pathways. doi:10.1371/journal.pone.0090074.t002 Shown only the top 20 pathways and see Table S2 for the complete list of 134 pathways. doi:10.1371/journal.pone.0090074.t002 manufacturer. The mRNA was dissolved in 100 mL THE RNA Storage Solution, and the final concentration was determined using NanoDrop. Transcriptomes of Oujiang Color Varieties of Common Carp aquaculture production through the identification of coloration patterns at early developmental stages. resource for the discovery of different types of molecular markers [25,26,33,34,35,36,37]. In recent years, the transcriptome analysis via next-generation sequencing technology has brought new insight into the knowledge of whole transcriptomes in many organisms [23,24,25,26,27,28]. This powerful approach allows the detection of significantly expressed genes and important pathways involved in different developmental stages and/or physiological processes [25,27,29,30,31,32]. In addi- tion, the large amount of transcriptomic sequences provides a rich Here, we conducted transcriptome analysis of Oujiang color common carp skin and obtained the first transcriptomic repertoire of this unique common carp strain. We also investigated the pigmentation genes expressed in the skin of Oujiang color varieties of common carp and found a set of candidate SNPs markers that can be used for selective molecular breeding in the future. Figure 2. The number of common carp transcripts in each level-2 Gene Ontology category. The transcriptome was assembled from Oujiang color varieties of common carp (RB - red with big black spots and WW – whole white) and gynogenic common carp (CC). doi:10.1371/journal.pone.0090074.g002 common carp transcripts in each level-2 Gene Ontology category. The transcriptome was assembled from mmon carp (RB - red with big black spots and WW – whole white) and gynogenic common carp (CC). 0074 g002 Figure 2. The number of common carp transcripts in each level-2 Gene Ontology category. The transcriptome was assembled from Oujiang color varieties of common carp (RB - red with big black spots and WW – whole white) and gynogenic common carp (CC). doi:10.1371/journal.pone.0090074.g002 March 2014 | Volume 9 | Issue 3 | e90074 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e90074 3 Transcriptomes of Oujiang Color Varieties of Common Carp Sequence assembly and annotation Sequence assembly, downstream analysis and annotation were conducted following the pipeline illustrated in Figure 1. The raw sequencing reads generated in this study have been deposited to the NCBI Short Read Archive (SRA) database (SRA 291286). The reads from Oujiang color common carp (RB and WW) along with the gynogenic common carp (CC) originating from Roche 454 sequencing reads downloaded from NCBI (SRA050545) were assembled using Newbler 2.6 [39]. Transcriptomes of Oujiang Color Varieties of Common Carp Science) and sequenced on the Roche 454 Genome Sequencer FLX instrument. Science) and sequenced on the Roche 454 Genome Sequencer FLX instrument. modifications [38]. A GsuI-oligo dT primer was used to prime first-strand cDNA synthesis from 10 mg of mRNA, using 1000 units of Superscript II reverse transcriptase (Invitrogen). After incubation at 42uC for 1 hr, the 59-CAP structure of mRNA was oxidized by NaIO4 (Sigma) and ligated to biotin hydrazide, which was used to select complete mRNA/cDNA by binding Dynal M280 beads (Invitrogen). After the second strand cDNA synthesis, the polyA and 59 adaptor were removed through GsuI digestion. RNA extraction and mRNA purification Each frozen sample was ground in mortars with liquid nitrogen, and total RNA was isolated using TRIzol reagent (Invitrogen) following the standard protocol. The concentration of total RNA dissolved in 200 mL RNase-free water was determined by NanoDrop (Thermo Scientific, USA), whereas the RNA integrity value (RIN) was estimated using RNA 6000 Pico LabChip of Agilent 2100 Bioanalyzer (Agilent, USA). Total RNA of each pooled sample of three individuals from the same color pattern was incubated with 10 U DNase I (Ambion) at 37uC for 1 hr, and then nuclease-free water was added to bring the sample volume to 250 mL. Messenger RNA was further purified with MicroPoly(A) Purist Kit (Ambion) as per the recommended protocol by the Figure 3. Venn diagram of the number of isotigs found in the skin transcriptome of Oujiang color varieties of common carp (RB and WW) and gynogenic transcriptome of common carp (CC). RB - red with big black spots, WW - whole white. doi:10.1371/journal.pone.0090074.g003 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e90074 4 Transcriptomes of Oujiang Color Varieties of Common Carp Isolation of SNP markers specific to the RB or WW color varieties Reads from Oujiang color varieties of common carp and from NCBI SRA database were mapped to the assembled de novo common carp transcriptome. Over 11,000 isotigs were mapped with reads from RB, WW, and CC (Figure 3). There were about 1,403 and 635 isotigs uniquely mapped with RB and WW reads, respectively, suggesting that more specific genes were involved in the formation of RB phenotype compared with WW. Many more unique isotigs were mapped with reads only from CC, which may be due to the much larger number of original reads from CC (Figures 1 and 3). A total of 52,902 single nucleotide polymorphism (SNP) markers were found in 9,379 isotigs, including 3,721 single nucleotide insertions or deletions (indels). The frequency distribution of different substitutions or indels is shown in Figure 4. The highest SNP pattern was G/A, accounting for 16% of the total. Compared to other substitution types, the substitution between C and G was found to be the least frequent. Further analysis of Gene Ontology (GO) showed that 25,564 isotigs and 45,383 singletons were successfully mapped to Gene Ontology categories - molecular functions, biological processes, and cellular components (Figure 2). For molecular functions, the majority of the transcripts (including both isotigs and singletons) were associated with binding, protein binding, and catalytic activity. For cellular component, the final products of these transcripts (proteins) were located mainly within cell, which is probably not surprising because the skin tissue was sampled for RNA extraction. For biological processes, most of these transcripts were related to cellular process and metabolic process, which was in agreement with most of other studies [24,44]. A total of 134 KEGG pathways were found in the common carp transcriptome with the top 20 pathways shown in Table 2. The pathways with most genes represented were purine metabolism, glycolysis and gluconeogenesis, and oxidative phosphorylation. Tyrosine metab- olism, a pigmentation related pathway, was found with 37 isotigs and 43 singletons that correspond to 23 proteins. To discover reliable markers that can be used to distinguish RB and WW, we set up the following three criteria: 1) detected with two algorithms; 2) homozygous in either RB or WW; and 3) more than two mapped reads. When these criteria were applied, 63 Table 3. Putative color pattern-specific SNP markers found in Oujiang color varieties of common carp isotig sequences generated from the skin transcriptome*. No. Transcriptome of common carp and Oujiang color varieties The statistics of common carp sequencing and assembly are summarized in Table 1. Altogether, 737,525 raw reads were generated via Roche 454 sequencing technology with an average length of 252 bp and maximal length of 680 bp. The reads generated in this study and the over two million Roche 454 sequencing reads from common carp available at NCBI SRA database (SRA050545) were assembled into 43,923 isotigs, with 546,676 reads unassembled (singletons). A total of 344,319 (63.0%) singletons were from the two color varieties of common carp and 75,260 singletons were found to have significant Blast hits. The N50 value, the average length and the largest length of isotigs were 1,237, 1,000 and 11,663 bp, respectively. The BLAST search resulted in 31,445 isotigs (71.6%) with significant matches to the NCBI nr database and 30,304 isotigs (68.9%) to ENSEMBL zebrafish proteins (E-value,1e-10). In addition, 12,597 zebrafish genes (about half of the zebrafish transcriptome) were found with significant BLAST matches to the assembled common carp transcriptome, indicating that approximately half of the common carp transcriptome can be annotated. For singletons, 128,507 out of 546,676 (23.5%) were found to have significant BLAST hits. Mapping and SNP Identification All reads were mapped to the de novo common carp transcriptome assembled in this study to identify candidate SNP markers using Newbler GS mapper [39]. To find a consensus set of candidate SNPs markers, we also used BWA 0.6.1 [42] for mapping and FaSD [43] for SNPs detection. SNPs detected by the above two approaches and with at least three reads mapped were considered as putative markers specific to RB or WW. Pigmentation genes in Oujiang color varieties of common carp After a BLAST search with 145 zebrafish-derived pigmentation genes against the common carp transcriptome, 593 isotigs were found with hits to 99 pigmentation genes and 501 singletons with matches to 95 pigmentation genes (E-value,1e-10). Of the 501 singletons, 291 were from WW and 210 from RB. A total of 77 pigmentation genes were commonly found in both isotigs and singletons, with 22 occurring solely in isotigs and 18 found only in singletons. When comparing the mapped reads of these pigmen- tation genes in RB and WW, 187 isotigs were found with at least a two-fold change, including 106 higher in WW and 81 in RB. In addition, 134 RB isotigs were not found in WW whereas 100 WW isotigs were not found in RB (Table S1). cDNA sequencing cDNA size fractionation was performed using a cDNA size fractionation column (Agencourt). Each cDNA fraction larger than 800 bp was sonicated to the range of 300–800 bp, and then pooled together with the other cDNA samples within the same range of lengths. The prepared cDNA of each pooled sample of three individuals from the same color pattern was transformed into single-stranded template DNA (sstDNA) libraries by using the GS DNA Library Preparation Kit (Roche Applied Science). The sstDNA libraries were clonally amplified in a bead- immobilized form by using the GS emPCR Kit (Roche Applied The assembled transcripts (isotigs and singletons) were BLAST- ed against NCBI nr protein database as well as zebrafish protein sequences, downloaded from ENSEMBL database, with a cutoff E-value smaller than 1e-10 [40]. Gene Ontology analysis was performed with the BLAST2GO program [41]. The BLAST program was also used to identify fish pigmentation genes [4,6] in Oujiang color common carp transcriptome. Figure 4. The distribution of SNP types in the common carp transcriptome. * indicates single nucleotide polymorphisms other than single nucleotide substitution, including deletion. doi:10.1371/journal.pone.0090074.g004 Figure 4. The distribution of SNP types in the common carp transcriptome. * indicates single nucleotide polymorphisms other than single nucleotide substitution, including deletion. doi:10.1371/journal.pone.0090074.g004 Figure 4. The distribution of SNP types in the common carp transcriptome. * indicates single nucleotide polymorphisms other than single nucleotide substitution, including deletion. doi:10.1371/journal.pone.0090074.g004 Figure 4. The distribution of SNP types in the common carp transcriptome. * indicates single nucleotide polymorphisms other than single nucleotide substitution, including deletion. doi:10.1371/journal.pone.0090074.g004 March 2014 | Volume 9 | Issue 3 | e90074 PLOS ONE | www.plosone.org 5 Transcriptomes of Oujiang Color Varieties of Common Carp approaches such as PRINTS [46], PROSITE [47], and Pfam [48] can be used to predict the functions of isotig/singleton sequences. p ** RB - red with big black spots, WW - whole white. * See Table S3 for the complete list of candidate SNP markers. doi:10.1371/journal.pone.0090074.t003 g p doi:10.1371/journal.pone.0090074.t003 References (2012) Characterization of common carp transcriptome: sequencing, de novo assembly, annotation and comparative genomics. PLoS One 7: e35152. 9. Logan DW, Burn SF, Jackson IJ (2006) Regulation of pigmentation in zebrafish melanophores. Pigment Cell Res 19: 206–213. 10. Richardson J, Lundegaard PR, Reynolds NL, Dorin JR, Porteous DJ, et al. (2008) mc1r Pathway regulation of zebrafish melanosome dispersion. Zebrafish 5: 289–295. 25. Liao X, Cheng L, Xu P, Lu G, Wachholtz M, et al. (2013) Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species. PLoS One 8: e62308. 11. Braasch I, Liedtke D, Volff JN, Schartl M (2009) Pigmentary function and evolution of tyrp1 gene duplicates in fish. Pigment Cell Melanoma Res 22: 839– 850. 26. Salem M, Rexroad CE 3rd, Wang J, Thorgaard GH, Yao J (2010) Characterization of the rainbow trout transcriptome using Sanger and 454- pyrosequencing approaches. BMC Genomics 11: 564. 12. Hoekstra HE, Hirschmann RJ, Bundey RA, Insel PA, Crossland JP (2006) A single amino acid mutation contributes to adaptive beach mouse color pattern. Science 313: 101–104. py q g pp 27. Samanta MP, Tongprasit W, Istrail S, Cameron RA, Tu Q, et al. (2006) The transcriptome of the sea urchin embryo. Science 314: 960–962. 28. Shen GM, Dou W, Niu JZ, Jiang HB, Yang WJ, et al. (2011) Transcriptome analysis of the oriental fruit fly (Bactrocera dorsalis). PLoS One 6: e29127. 13. Sugie A, Terai Y, Ota R, Okada N (2004) The evolution of genes for pigmentation in African cichlid fishes. Gene 343: 337–346. 29. Huan P, Wang H, Liu B (2012) Transcriptomic analysis of the clam Meretrix meretrix on different larval stages. Marine Biotechnology 14: 69–78. 14. Altschmied J, Delfgaauw J, Wilde B, Duschl J, Bouneau L, et al. (2002) Subfunctionalization of duplicate mitf genes associated with differential degeneration of alternative exons in fish. Genetics 161: 259–267. 30. Mu Y, Ding F, Cui P, Ao J, Hu S, et al. (2010) Transcriptome and expression profiling analysis revealed changes of multiple signaling pathways involved in immunity in the large yellow croaker during Aeromonas hydrophila infection. BMC Genomics 11: 506. ˝ 15. Balon EK (1995) Origin and domestication of the wild carp,Cyprinus carpio: from Roman gourmets to the swimming flowers. Aquaculture 129: 3–48. . Balon EK (1995) Origin and domestication of the wild carp,Cyprinus Roman gourmets to the swimming flowers. Aquaculture 129: 3–48 16. References 18. Li S (2003) Aquaculture research and its relation to development in China. Agricultural development and the opportunities for aquatic resources research in China 65: 17. 1. Protas ME, Patel NH (2008) Evolution of coloration patterns. Annu Rev Cell Dev Biol 24: 425–446. 2. Rodgers GM, Kelley JL, Morrell LJ (2010) Colour change and assortment in the western rainbowfish. Animal Behaviour 79: 1025–1030. 19. Wang CH, Xiang SP, Lv YP, Wang J, Pan ZY, et al. (2008) Preliminary study on inheritance of red and white color phenotypes in Oujiang color carp, C. carpio var. color. Journal of Shanghai Fisheries University 4: 004. 3. Steiner CC, Weber JN, Hoekstra HE (2007) Adaptive variation in beach mice produced by two interacting pigmentation genes. PLoS biology 5: 1-e219. 20. David L, Blum S, Feldman MW, Lavi U, Hillel J (2003) Recent Duplication of the Common Carp (Cyprinus carpio L.) Genome as Revealed by Analyses of Microsatellite Loci. Molecular Biology and Evolution 20: 1425–1434. 4. Braasch I, Schartl M, Volff JN (2007) Evolution of pigment synthesis pathways by gene and genome duplication in fish. BMC Evol Biol 7: 74. by gene and genome duplication in fish. BMC Evol Biol 7: 74 5. Bagnara JT, Fernandez PJ, Fujii R (2007) On the blue coloration of vertebrates. Pigment Cell Res 20: 14–26. 21. Ma Y, Wang C, Wang J, Yang X, Bi X, et al. (2011) Genetic differentiation of wild and hatchery Oujiang color common carp: potential application to the identification of escapees. Fisheries Science 77: 591–597. 6. Braasch I, Brunet F, Volff JN, Schartl M (2009) Pigmentation pathway evolution after whole-genome duplication in fish. Genome Biol Evol 1: 479–493. 22. Zhu L, Ma Y, Xiang S, Bi X, Wang J, et al. (2013) Observation and analysis of growth dynamics in Oujiang color common carp with different pigmentation types. Journal of Shanghai Fisheries University In Press. 7. Mellgren EM, Johnson SL (2002) The evolution of morphological complexity in zebrafish stripes. Trends Genet 18: 128–134. 8. Li S, Wang C, Yu W, Zhao S, Gong Y (2012) Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks. PLoS One 7: e36592. 23. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics 10: 57–63. 24. Ji P, Liu G, Xu J, Wang X, Li J, et al. Conclusions Table S3 The complete list of putative SNP markers found in Oujiang color varieties of common carp (RB - red with big black spots, WW - whole white). (DOCX) The de novo skin transcriptome sequencing of Oujiang color common carp varieties were conducted, resulting in 737,525 reads assembled to 43,923 isotigs. Over 10,000 genes were annotated using BLAST, Gene Ontology, and KEGG pathways. The orthologs of approximately 80% (117 out of 145) zebrafish pigmentation genes were recovered. In addition, 52,902 SNPs were detected, with 63 candidate markers predicted. Future research directions include expanding this study to analyze more samples of other color patterns and validating the results on potentially differentially expressed genes between the RB and WW phenotypes. We are also planning to validate color pattern-specific molecular markers, which will presumably lead to a better understanding of the molecular mechanisms underlying diverse color patterns and promote aquaculture of Oujiang color varieties of common carp through the application of molecular markers in selective breeding. Acknowledgments We acknowledge Mr. Songping Xiang and Jian Wang for the assistance in fish culture and sample collection. In addition, we want to thank Mr. Xinxin Yang with DNA content measurement, library construction and data analysis. Finally, we are grateful to Mary Christman and Julian Egger for proofreading the manuscript. We acknowledge Mr. Songping Xiang and Jian Wang for the assistance in fish culture and sample collection. In addition, we want to thank Mr. Xinxin Yang with DNA content measurement, library construction and data analysis. Finally, we are grateful to Mary Christman and Julian Egger for proofreading the manuscript. Supporting Information Table S1 Pigmentation genes found in Oujiang color varieties of common carp (RB - red with big black spots and WW - whole white). (XLSX) Table S1 Pigmentation genes found in Oujiang color varieties of common carp (RB - red with big black spots and WW - whole white). (XLSX) Table S2 The complete list of KEGG pathways identi- fied in the common carp skin transcriptome involving two Oujiang color common carp variants. (DOCX) Isolation of SNP markers specific to the RB or WW color varieties Isotigs Location Reference/Variant RB** WW** Isotig11776 721 C/T 11/0 0/3 Isotig21770 1093 A/T 10/0 0/9 Isotig20802 2,791 G/A 10/0 0/5 Isotig11776 1,203 A/G 9/0 0/3 Isotig23531 771 C/G 8/0 0/5 Isotig12829 341 A/G 7/0 0/3 Isotig23531 483 C/T 7/0 0/3 Isotig21113 1,496 A/G 6/0 0/4 Isotig20807 2,081 A/G 5/0 0/5 Isotig21311 779 C/T 4/0 0/7 * See Table S3 for the complete list of candidate SNP markers. ** RB - red with big black spots, WW - whole white. doi:10.1371/journal.pone.0090074.t003 In our study, approximately 30% of the isotigs and 80% of the singletons were found without significant BLAST matches to known proteins in public databases. In fact, most transcriptome studies showed a portion of isotig and singleton sequences that cannot be annotated [24,25,26,34,35,45]. One possible reason for this is that these non-model organisms possess numerous putative novel genes or transcripts, of which the sequences are not available in public databases. Further analysis and annotation of these putative novel transcripts are necessary, but appear to be challenging. With the availability of transcriptomic sequences in many organisms, the comparative analysis of closely related species may assist in the identification of novel orthologous genes. Besides the widely used BLAST algorithm, the profile- or model-based March 2014 | Volume 9 | Issue 3 | e90074 PLOS ONE | www.plosone.org 6 Transcriptomes of Oujiang Color Varieties of Common Carp putative SNPs markers were found, including 21 SNPs with RB showing the reference nucleotide, while WW showing the variant one and 42 SNPs with the opposite (Table 3). In addition, the analysis of pigmentation-related transcripts revealed only 10 SNPs that may be associated with different coloration phenotypes. One of our future research directions is to validate the predicted markers and evaluate their accuracy in identifying specific Oujiang color varieties of common carp with distinct color patterns [36,37,49]. Author Contributions Conceived and designed the experiments: CW. Performed the experi- ments: CW. Analyzed the data: MW JW XL. Contributed reagents/ materials/analysis tools: CW GL. Wrote the paper: CW GL JW. Conceived and designed the experiments: CW. Performed the experi- ments: CW. Analyzed the data: MW JW XL. Contributed reagents/ materials/analysis tools: CW GL. Wrote the paper: CW GL JW. Transcriptomes of Oujiang Color Varieties of Common Carp Transcriptomes of Oujiang Color Varieties of Common Carp 33. Chouvarine P, Cooksey AM, McCarthy FM, Ray DA, Baldwin BS, et al. (2012) Transcriptome-based differentiation of closely-related Miscanthus lines. PLoS One 7: e29850. 41. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, et al. (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676. a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676. 42. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows- Wheeler transform. Bioinformatics 26: 589–595. 34. Ma K, Qiu G, Feng J, Li J (2012) Transcriptome analysis of the oriental river prawn, Macrobrachium nipponense using 454 pyrosequencing for discovery of genes and markers. PLoS One 7: e39727. 43. Xu F, Wang W, Wang P, Jun Li M, Chung Sham P, et al. (2012) A fast and accurate SNP detection algorithm for next-generation sequencing data. Nat Commun 3: 1258. 35. Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, et al. (2012) High- throughput sequence analysis of turbot (Scophthalmus maximus) transcriptome using 454-pyrosequencing for the discovery of antiviral immune genes. PLoS One 7: e35369. 44. Bai X, Mamidala P, Rajarapu SP, Jones SC, Mittapalli O (2011) Transcrip- tomics of the bed bug (Cimex lectularius). PLoS One 6: e16336. 36. Renaut S, Nolte AW, Bernatchez L (2010) Mining transcriptome sequences towards identifying adaptive single nucleotide polymorphisms in lake whitefish species pairs (Coregonus spp. Salmonidae). Mol Ecol 19 Suppl 1: 115–131. 45. Calduch-Giner JA, Bermejo-Nogales A, Benedito-Palos L, Estensoro I, Ballester- Lozano G, et al. (2013) Deep sequencing for de novo construction of a marine fish (Sparus aurata) transcriptome database with a large coverage of protein- coding transcripts. BMC Genomics 14: 178. 37. Ji P, Zhang Y, Li C, Zhao Z, Wang J, et al. (2012) High throughput mining and characterization of microsatellites from common carp genome. Int J Mol Sci 13: 9798–9807. 46. Attwood TK, Blythe MJ, Flower DR, Gaulton A, Mabey J, et al. (2002) PRINTS and PRINTS-S shed light on protein ancestry. Nucleic acids research 30: 239–241. 38. Ng P, Wei CL, Sung WK, Chiu KP, Lipovich L, et al. (2005) Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation. Nat Methods 2: 105–111. 47. Sigrist CJ, Cerutti L, de Castro E, Langendijk-Genevaux PS, Bulliard V, et al. References Wang C, Li S, Nagy ZT, Lehoczky I, Huang L, et al. (2010) Molecular genetic structure and relationship of Chinese and Hungarian common carp (Cyprinus carpio L.) strains based on mitochondrial sequence. Aquaculture Research 41: 1339–1347. 31. Visser WE, Swagemakers SM, O˝ zgu˝r Z, Schot R, Verheijen FW, et al. (2010) Transcriptional profiling of fibroblasts from patients with mutations in MCT8 and comparative analysis with the human brain transcriptome. Human molecular genetics 19: 4189–4200. 17. David L, Rothbard S, Rubinstein I, Katzman H, Hulata G, et al. (2004) Aspects of red and black color inheritance in the Japanese ornamental (Koi) carp (Cyprinus carpio L.). Aquaculture 233: 129–147. 32. Li C, Zhang Y, Wang R, Lu J, Nandi S, et al. (2012) RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus. Fish & Shellfish Immunology 32: 816–827. PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e90074 March 2014 | Volume 9 | Issue 3 | e90074 Transcriptomes of Oujiang Color Varieties of Common Carp Transcriptomes of Oujiang Color Varieties of Common Carp (2010) PROSITE, a protein domain database for functional characterization and annotation. Nucleic acids research 38: D161–D166. 39. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376– 380. 48. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, et al. (2012) The Pfam protein families database. Nucleic acids research 40: D290–D301. 40. Flicek P, Amode MR, Barrell D, Beal K, Brent S, et al. (2012) Ensembl 2012. Nucleic acids research 40: D84–D90. p 49. Salem M, Vallejo RL, Leeds TD, Palti Y, Liu S, et al. (2012) RNA-Seq identifies SNP markers for growth traits in rainbow trout. PLoS One 7: e36264. March 2014 | Volume 9 | Issue 3 | e90074 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
https://openalex.org/W2811332093
https://link.springer.com/content/pdf/10.1007%2F978-3-319-66981-6_55.pdf
English
null
Using LCA and EPD in Public Procurement Within the Construction Sector
Springer eBooks
2,018
cc-by
1,811
Using LCA and EPD in Public Procurement Within the Construction Sector Kristian Jelse and Kristof Peerens Abstract Life cycle assessments (LCAs) and Environmental Product Declarations (EPDs) have long been pointed to as tools to be used in green public procurement (GPP), but doing so in practice is challenging. How can such information be requested without breaking some of the general principles for public procurement, for example non-discrimination and transparency? This session was meant to give insights from different stakeholders through five presentations providing different perspectives on this issue. The individual presentations were followed up on by a panel discussion. © The Author(s) 2018 E. Benetto et al. (eds.), Designing Sustainable Technologies, Products and Policies, https://doi.org/10.1007/978-3-319-66981-6_55 K. Jelse (&) EPD International AB, Stockholm, Sweden e-mail: kristian@environdec.com K. Jelse (&) EPD International AB, Stockholm, Sweden e-mail: kristian@environdec.com K. Peerens 3 M Belgium bvba/sprl, Diegem, Belgium © The Author(s) 2018 E. Benetto et al. (eds.), Designing Sustainable Technologie Products and Policies, https://doi.org/10.1007/978-3-319-6 2.1 Challenges for Industry LCA is a time and resource intensive undertaking for industry, which requires significant effort in gathering data, creating life cycle models in dedicated software packages, appropriately interpreting the results, and reporting the information in an understandable format. This means that for small and medium sized enterprises, cost is often seen as a barrier to starting such studies [1]. As a result, those com- panies that have limited resources available cannot always justify the cost, and as a result will not have LCA or EPD data available. In addition, there are a number of challenges related to communicating LCA and EPD information: some of the information must be specific to the product appli- cation and use, other data is relevant for different types of audiences (general public versus architects or project developers). Unfortunately, even when only considering the European Union, means of communication, as well as the different LCA and EPD elements differ [1]. When publishing LCA and EPD data for a number of different countries, there may be a need to generate multiple LCA reports or EPDs, have them verified multiple times (in line with the countries’ requirements), and often have them registered locally, each of which comes at a cost. Harmonisation of results can therefore be seen as an absolute must when it comes to promoting the consistent use of LCAs and EPDs [1]. 2 Challenges in Using LCAs and EPDs as a Standard Assessment Method in Green Public Procurement Even though LCA and EPD data is commonly used to quantify the potential environmental impact of construction elements or construction works, reality shows that in GPP, despite environmental considerations being implemented in a variety of European Member States, this data is not used in order to come to conclusions and make decisions related to product or supplier selection [3]. The presentations at the session showed different challenges to different types of stakeholder groups. 1 Introduction With the European Union being responsible for more than a third of global resource consumption, 40% of global energy consumption, 36% of global carbon dioxide emissions, and one third of global waste streams [1], there is an increasing need for this region to take a leading position when it comes to reducing the overall envi- ronmental impact. Specifically for the construction sector, energy efficient housing is one of the key elements in being able to reduce Europe’s overall environmental impact, with studies showing that the embodied energy in construction represents the amount of energy equivalent to more than 75 years of heating [1]. The construction sector overall is more than just housing, and each of the different components have a significant impact on the environment. A rough estimate by the Norwegian authorities has shown that the yearly carbon footprint of the public road system (including construction and maintenance) 499 500 K. Jelse and K. Peerens equates to *2.3 million tons of carbon dioxide equivalents, excluding direct traffic emissions (which add 20–25%) [2]. 2 Challenges in Using LCAs and EPDs as a Standard Assessment Method in Green Public Procurement 3 Tool and Platform Development to Assist in Interpretation of LCA and EPD Data A number of industry sectors, depending on their maturity in the area of sustainability, have built tool sets that help in assessing the overall sustainability impact of their sector’s products, technologies and solutions. As a sector, it has therefore been pos- sible to meet the growing demands related to the availability of data. The schemes as developed and deployed in e.g. the steel industry have therefore resulted in collabo- ration and engagement, increasing transparency and improvements in environmental performance, thus triggering innovation and continuous improvement [4]. Similarly, regional and federal governments (e.g. Germany) as well as building certification schemes are developing databases and software packages that allow for generic and product-specific data to be made available to different stakeholders to allow them to assess the impact of new construction developments or maintenance [5]. Not only have these tools reduced the cost of an LCA, but it ensures that LCA is considered during the construction design and planning [5]. 2.2 Challenges for the Public Sector No matter how it is approached, LCA is a complex topic that requires a certain skill set to be able to interpret information generated by the study. So even if LCA and EPD data was to be available for all products participating in a public tender, there Using LCA and EPD in Public Procurement Within the Construction Sector 501 would need to be quite some knowledge required from the procurement depart- ments in order to identify the best possible product for a certain application [3]. Environmental benchmark data is not readily available, which means that this adds a layer of complexity for public authorities for product differentiation and selection. With the risk of duplicating efforts and lack of consistency, public authorities often use national/regional expertise in order to determine GPP criteria, which can result in different regulatory frameworks, and a lack of harmonisation. For public authorities, sustainability criteria must go beyond environmental impacts, and must include social, humanitarian and economic aspects as well. Weighting and characterisation of environmental criteria based on the significance of the differences in environmental impacts are therefore a must [4]. References 1. Adibi N, Pasquet V, Darul M, Roy A, Salamon A, Challenges to mainstream the use of EPDs i construction Public Procurement in European context, LCM 2017 conference, Luxembourg, 2017 2. Fredriksen K-S, O’Born R, Implementing climate emission demands in road construction contracts: Insights from the KRAKK project and the Norwegian Public Roads Administration, LCM 2017 conference, Luxembourg, 2017. 2. Fredriksen K-S, O’Born R, Implementing climate emission demands in road construction contracts: Insights from the KRAKK project and the Norwegian Public Roads Administration, LCM 2017 conference, Luxembourg, 2017. 3. Ganassali S, Lavagna M, Campioli A, Saporetti S, Green Public Procurement and Construction Sector: EPD and LCA based benchmarks of the whole-building, LCM 2017 conference, Luxembourg, 2017. 3. Ganassali S, Lavagna M, Campioli A, Saporetti S, Green Public Procurement and Construction Sector: EPD and LCA based benchmarks of the whole-building, LCM 2017 conference, Luxembourg, 2017. 4. Brankley L, Tugrul A, Anderson J, Knight D, A Manufacturing Sector’s Experience of integrating EPD alongside product, management system and responsible sourcing certification for public procurement, LCM 2017 conference, Luxembourg, 2017. 4. Brankley L, Tugrul A, Anderson J, Knight D, A Manufacturing Sector’s Experience of integrating EPD alongside product, management system and responsible sourcing certification for public procurement, LCM 2017 conference, Luxembourg, 2017. 5. Kusche O, Brockmann T, Figl H, How LCA and EPD are used for implementing Green Public Procurement in the construction sector in Germany, LCM 2017 conference, Luxembourg, 2017. 5. Kusche O, Brockmann T, Figl H, How LCA and EPD are used for implementing Green Public Procurement in the construction sector in Germany, LCM 2017 conference, Luxembourg, 2017. 5. Kusche O, Brockmann T, Figl H, How LCA and EPD are used for implementing Green Public Procurement in the construction sector in Germany, LCM 2017 conference, Luxembourg, 2017. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. 4 Discussion on Further Efforts to Integrate Data from LCAs and EPDs in GPP When considering the different options for integrating LCA and EPD in GPP criteria setting and purchasing decision consistently within any given sector on European level, the key element that must be considered is harmonisation: • It is a must that schemes operated in different European countries work in the same way and avoid setting national special features in addition to the standards to allow companies to consistently report LCA and EPD data; • It is a must that schemes operated in different European countries work in the same way and avoid setting national special features in addition to the standards to allow companies to consistently report LCA and EPD data; 502 K. Jelse and K. Peerens • Benchmarks must be made available in order to ensure that products that are regarded as true differentiators are considered as such regardless of the country they are sold into; • Benchmarks must be made available in order to ensure that products that are regarded as true differentiators are considered as such regardless of the country they are sold into; • Benchmarks must be made available in order to ensure that products that are regarded as true differentiators are considered as such regardless of the country they are sold into; • GPP award criteria must be harmonised, in order to ensure there is a consistent message from the public sector on what the focus areas are supposed to be, and these should be driven by the European Commission; • For those organisations that are not in a position to generate LCA data them- selves, financial support or publicly available data repositories should be made available, driven by the public sector. Truly implementing science-based product or service selection criteria for pur- chasing materials or services is the single most important element that will drive a true reduction of the environmental impact of the European construction sector. Based on today’s assessment methodologies and data availability, LCAs and EPDs are a critical element in this approach, and should therefore be much more visible in public and private efforts to drive down the environmental impact. References If material is not included in the chapter’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.
https://openalex.org/W2981476927
https://www.mdpi.com/1996-1073/12/21/4066/pdf
English
null
Influence of T-Shape Tip Clearance on Energy Performance and Broadband Noise for a NACA0009 Hydrofoil
Energies
2,019
cc-by
10,232
Received: 28 September 2019; Accepted: 24 October 2019; Published: 25 October 2019 Received: 28 September 2019; Accepted: 24 October 2019; Published: 25 October 2019 Abstract: In the present paper, the effect of the proposed T-shape tip on the energy performance, flow patterns and broadband noise sources of a NACA0009 hydrofoil with tip clearance is investigated. The vortex induced by the gap is simulated by means of the SST k-ω turbulence model, and then, the noise generated by dipoles and quadrupoles are analyzed by using the Curle acoustic analogy and Proudman acoustic analogy, respectively. The numerical simulation results agree well with the experimental measurements. Results indicate that three tip shapes, including the half pressure side T-shape model (MPT), the half suction side T-shape model (MST) and the T-shape model (MT), have complex influence on energy performance of the foil. Only the MST model can promote the energy performance of the hydrofoil at all inlet velocities, with the maximum ratio of lift coefficient to drag coefficient increasing by 4.26%. In addition, the ratio of lift coefficient to drag coefficient for MT obviously increases when the inlet velocity is 7.5 m/s, 10 m/s, 12.5 m/s and 15 m/s, and the maximum promotion is 15.21% at 7.5 m/s. The T-shape tip can effectively suppress the tip clearance leakage vortex, which makes the vortex area decrease with a maximum drop of 5.02%. Furthermore, the MPT and MT have good suppression effect on the hydrofoil dipole noise, and reduce the maximum Curle Acoustic Power (AP) of the foil with 2.64% and 3.03%, respectively. The MST model obviously reduces the isosurface area of the Proudman AP by 6.55% for 55 dB. Keywords: T-shape tip; NACA0009; tip clearance; tip leakage vortex; noise Keywords: T-shape tip; NACA0009; tip clearance; tip leakage vortex; noise Zhenwei Huang 1, Yadong Han 1, Lei Tan 1,* and Chuibing Huang 2,* Zhenwei Huang , Yadong Han , Lei Tan and Chuibing Huang 1 State Key Laboratory of Hydroscience and Engineering, Tsinghua University, Beijing 100084, China; zhenweihuang@mail.tsinghua.edu.cn (Z.H.); hanyadong@mail.tsinghua.edu.cn (Y.H.) 2 College of Marine Power Engineering, Naval University of Engineering, Wuhan 430033, China * Correspondence: tanlei@mail.tsinghua.edu.cn (L.T.); ChuibingHuang@nue.edu.cn (C.H.); Tel.: +86-10-6278-0605 (L.T.) Zhenwei Huang , Yadong Han , Lei Tan and Chuibing Huang 1 State Key Laboratory of Hydroscience and Engineering, Tsinghua University, Beijing 100084, China; zhenweihuang@mail.tsinghua.edu.cn (Z.H.); hanyadong@mail.tsinghua.edu.cn (Y.H.) 2 College of Marine Power Engineering, Naval University of Engineering, Wuhan 430033, China * Correspondence: tanlei@mail.tsinghua.edu.cn (L.T.); ChuibingHuang@nue.edu.cn (C.H.); Tel.: +86-10-6278-0605 (L.T.) 1 State Key Laboratory of Hydroscience and Engineering, Tsinghua University, Beijing 100084, China; zhenweihuang@mail.tsinghua.edu.cn (Z.H.); hanyadong@mail.tsinghua.edu.cn (Y.H.) 2 College of Marine Power Engineering, Naval University of Engineering, Wuhan 430033, China * Correspondence: tanlei@mail.tsinghua.edu.cn (L.T.); ChuibingHuang@nue.edu.cn (C.H.); Tel.: +86-10-6278-0605 (L.T.) Influence of T-Shape Tip Clearance on Energy Performance and Broadband Noise for a NACA0009 Hydrofoil Zhenwei Huang 1, Yadong Han 1, Lei Tan 1,* and Chuibing Huang 2,* energies energies energies 1. Introduction Hydraulic machineries are widely used in modern submarines and warships, such as pumps, duct propulsors and waterjet propulsors, which have attracted the attention of researchers on a worldwide scale [1–5]. However, due to the limitation of the impeller structure, there is inevitably a gap between the rotating blades and the fixed impeller chamber [6,7]. The tip leakage flow (TLF) and tip leakage vortex (TLV) is induced by the existence of the gap, which results in pressure drop and energy loss, and then leads to cavitation, erosion, vibration and noise [8–12]. The radiated noise from these hydraulic machineries will harm the worker health and can be easily detected by the enemy sonar systems. Therefore, the internal mechanism among TLF, energy loss and radiated noise is waiting to be explored. In the past decades, considerable experimental investigations have been conducted on the tip leakage flow, cavitation and noise mechanism. Wu et al. [13,14] experimentally investigated the cavitation within the gap of a waterjet pump. The results indicated that the TLF along the aft side of the blade was strong enough to cause sheet cavitation in the gap starting from the pressure side corner. Energies 2019, 12, 4066; doi:10.3390/en12214066 www.mdpi.com/journal/energies www.mdpi.com/journal/energies 2 of 13 Energies 2019, 12, 4066 Moreover, Zhang et al. [15,16] carried out an experimental investigation in an axial flow pump; the results on TLV cloud cavitation revealed that it keeps a stable triangular flow structure from the leading edge to 80% of the length of the chord, while the cavitation shape becomes unstable near the trailing edge. For the fundamental research, Dreyer et al. [17] experimentally studied the influence of the tip size on the TLV formation and cavitation of a NACA0009 hydrofoil. This work is a classic example that is often used to verify the validation of the Computational Fluid Dynamics (CFD) computation of the tip clearance. Recently, Gopalan et al. [18] conducted experimental work upon the impact of gap width on internal flow mechanism, cavitation formation, corresponding bubble dynamics and induced noise. The results showed that the bubbles will grow up in the vortex core, and deform and break into multiple bubbles, which resulted in a noise peak value. By changing the shape and size of bubbles, its cavitation radiation noise can be effectively suppressed. y pp In addition to experimental measurements, the numerical method is of great significance for the study of TLF and its induced cavitation. 1. Introduction Liu et al. [19] analyzed the vortex features and pressure fluctuation within a mixed flow pump; results showed that the TLV can be divided into the primary TLV, secondary TLV, entangled TLV and dispersed TLV. Hao et al. [20] compared the impact of unsymmetrical and symmetrical gaps on the changes of radial force and cavitation within the mixed flow pump. Results indicated that the unsymmetrical tip clearance is inapplicable for reducing cavitation performance, and its existence influenced the magnitude and direction of the radial force as cavitation developed. Liu et al. [21] investigated the TLV evolution with time in a mixed flow pump, and the TLV split, developed and merged under different stages. Results showed that the varied pressure at blade junction aggravated TLV fluctuation. Guo et al. [22] studied the impact of foil tip shape and size on the flow characteristics and energy performance for a NACA0009 hydrofoil. Results showed that the sharp tip mitigated the TLV loss, and the separation of the boundary layer increased the velocity gradient. Okita et al. [23] focused on the cavitation evolution induced by the tip clearance of an inducer; the flow detail was captured by adopting a detached eddy simulation model. Zhang et al. [24] concentrated on energy performance and flow features within multiphase pump; results showed that the pressure maximum and the area of vortex were enhanced as the gap size increased. Decaix et al. [25,26] simulated the TLV between blade tip and the wall of water tunnel; results revealed that the vortex trajectory approached the suction side, which would aggravated the wall erosion. The above references show that despite the fact that tip clearance is small, the interaction between TLV and main flow in the blade passage, the boundary layer near the wall and the wake near blade will form a complex vortex structure. Additionally, it will cause cavitation, vibration and noise, which seriously affect the operation stability of hydraulic machineries. Consequently, it is necessary to put forward some effective control measures. To suppress the TLF and cavitation, many scholars have studied the influence of tip shape and size on TLV or cavitation. Gearhart et al. [27] experimentally studied the effects of the gap width, boundary layer near the wall and blade load on TLF. Moreover, Laborde et al. [28] revealed that the tip geometry was of great significance on the cavitation and flow patterns. 1. Introduction As the clearance edge becomes round on the pressure side, the optimal tip scheme can eliminate cavitation. Recently, Liu et al. [29] presented interesting work: the C groove method can reduce the TLV formation and enhance energy performance within a NACA0009 hydrofoil. Under all conditions, the C groove method exhibited the ability of promoting energy performance and suppressing TLV. As for the 0.2 and 0.4 tip clearance, the maximum of vortex area was decreased by 66.55% and 67.94%, respectively. In summary, several effective methods suppressing TLV and improving the energy performance have been proposed. However, there is not yet a reliable and effective method on suppressing broadband noise. The objective of the present work is to examine the influence of the proposed T-shape tip on the energy performance, flow patterns and broadband noise sources in a NACA0009 hydrofoil. The main content of the present paper is as follows. Firstly, the research background is introduced and a new designed T-shape tip is proposed. Then the comparison between computational results and 3 of 13 Energies 2019, 12, 4066 experimental data is conducted in order to establish the foundation of the subsequent simulation work. Finally, the energy performance, the detailed flow features in T-shape tip and the broadband noise characteristics are systemically analyzed. work. Finally, the energy performance, the detailed flow features in T-shape tip and the broadband noise characteristics are systemically analyzed. experimental data is conducted in order to establish the foundation of the subsequent simulation work. Finally, the energy performance, the detailed flow features in T-shape tip and the broadband noise characteristics are systemically analyzed. work. Finally, the energy performance, the detailed flow features in T-shape tip and the broadband noise characteristics are systemically analyzed. 2.1. Physical Model of NACA0009 Hydrofoil 2.1. Physical Model of NACA0009 Hydrofoil A NACA0009 hydrofoil [17] with tip clearance (Figure 1) is assumed to have a truncated chord of c = 0.1 m, maximum thickness of h = 0.0099 m and span of b = 0.15 m. The gap size δ, normalized by the maximum thickness h, is 0.2, and the incidence angle is 10◦. The fluid density is 997 kg/m3 and the inlet pressure is 1 bar. A NACA0009 hydrofoil [17] with tip clearance (Figure 1) is assumed to have a truncated chord of c = 0.1 m, maximum thickness of h = 0.0099 m and span of b = 0.15 m. The gap size δ, normalized by the maximum thickness h, is 0.2, and the incidence angle is 10°. The fluid density is 997 kg/m3 and the inlet pressure is 1 bar. Figure 1. Physical model of NACA0009 hydrofoil. (a) Isometric view. (b) The gap. (a) (b) x y Figure 1. Physical model of NACA0009 hydrofoil. (a) Isometric view. (b) The gap. (a) (a) (b) x y (b) Figure 1. Physical model of NACA0009 hydrofoil. (a) Isometric view. (b) The gap. Figure 1. Physical model of NACA0009 hydrofoil. (a) Isometric view. (b) The gap. 3. Numerical Setting a Numerical Method 3.1. Numerical Method 3.1. Numerical Method In the present paper, a steady-state Reynolds averaged Navier-Stokes (RANS) equation and the SST k-ω turbulence model [22,29,30] with all y+ wall treatment were used to calculate the flow field within the gap; then, the acoustics models available in STAR_CCM+ 13.02, including the Curle and Proudman broadband noise models, were adopted to investigate the broadband noise sources. The Segregated flow model was used to compute each of the momentum equations in turn. Moreover, the second-order upwind convection scheme was adopted for the advection scheme. For the boundary conditions, a normal velocity with low turbulence intensity imposed at the inlet and a static pressure was applied at the outlet. The no-slip wall conditions were set at the hydrofoil surface and Numerical Method In the present paper, a steady-state Reynolds averaged Navier-Stokes (RANS) equation and the k-ω turbulence model [22,29,30] with all y+ wall treatment were used to calculate the flow field hin the gap; then, the acoustics models available in STAR_CCM+ 13.02, including the Curle and udman broadband noise models, were adopted to investigate the broadband noise sources. The regated flow model was used to compute each of the momentum equations in turn. Moreover, second-order upwind convection scheme was adopted for the advection scheme. For the ndary conditions, a normal velocity with low turbulence intensity imposed at the inlet and a static ssure was applied at the outlet. The no-slip wall conditions were set at the hydrofoil surface and tu el all In the present paper, a steady-state Reynolds averaged Navier-Stokes (RANS) equation and the SST k-ω turbulence model [22,29,30] with all y+ wall treatment were used to calculate the flow field within the gap; then, the acoustics models available in STAR_CCM+ 13.02, including the Curle and Proudman broadband noise models, were adopted to investigate the broadband noise sources. The Segregated flow model was used to compute each of the momentum equations in turn. Moreover, the second-order upwind convection scheme was adopted for the advection scheme. For the boundary conditions, a normal velocity with low turbulence intensity imposed at the inlet and a static pressure was applied at the outlet. The no-slip wall conditions were set at the hydrofoil surface and the tunnel wall. p pp p y the tunnel wall. It should be noted that the SST k-ω turbulence model has its own limitations, errors and uncertainties [31]. Figure 3. Proposed Numerical Setting and Validation 3. Numerical Setting and Validation igu e p Numerical Setting and Validation 3. Numerical Setting and Validation 2.2. T-shape Tips 2.2. T-Shape Tips 2.2. T-shape Tips 2.2. T-Shape Tips p p As shown in Figure 2, the new proposed T-shaped tip is formed by gradually increasing the thickness from point A to the tip, and the foil profile is symmetrical. The distance between A and the foil end is the tip clearance δ. At point A, the foil thickness equals the original blade thickness t0, and the maximum thickness of the foil end is tm increasing by 0.3t0. Figure 3 illustrates the original tip and three types of T-shape tips, including half pressure side T-shape (MPT), half suction side T-shape (MST) and T-shape (MT). The control curves of the MPT, MST and MT are straight lines. As shown in Figure 2, the new proposed T-shaped tip is formed by gradually increasing the thickness from point A to the tip, and the foil profile is symmetrical. The distance between A and the foil end is the tip clearance δ. At point A, the foil thickness equals the original blade thickness t0, and the maximum thickness of the foil end is tm increasing by 0.3t0. Figure 3 illustrates the original tip and three types of T-shape tips, including half pressure side T-shape (MPT), half suction side T-shape (MST) and T-shape (MT). The control curves of the MPT, MST and MT are straight lines. 4 of 13 14 Energies 2019, 12, 4066 gies 2019 8 x FOR PEER Energies 2019, 8, x FOR PEER REVIEW 4 of 14 Figure 2. Sketch map of T-shape tip. Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. (a) (b) (c) (d) PS SS PS PS PS SS SS SS δ ζ ε tm t0 A δ Figure 2. Sketch map of T-shape tip. Figure 2. Sketch map of T-shape tip. Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. (a) (b) (c) (d) PS SS PS PS PS SS SS SS δ ζ ε tm t0 A δ Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. Figure 2. Sketch map of T-shape tip. δ ζ ε tm t0 A δ Figure 2. Sketch map of T-shape tip. Figure 2. Sketch map of T-shape tip. δ ζ ε tm t0 A δ Figure 2. Sketch map of T-shape tip. δ ζ ε tm t0 A δ Figure 2. Sketch map of T-shape tip. Figure 2. 2.2. T-shape Tips 2.2. T-Shape Tips Sketch map of T-shape tip. δ ζ ε tm t0 A Figure 2. Sketch map of T-shape tip. Figure 2. Sketch map of T-shape tip. ure 2. Sketch map of T-shape tip. Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. (a) (b) (c) (d) PS SS PS PS PS SS SS SS g p p p Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. (a) (b) (c) (d) PS SS PS PS PS SS SS SS Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. Figure 3 Proposed T shape tips (a) M0; (b) MPT; (c) MST; (d) MT ure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. Figure 3. Proposed T-shape tips. (a) M0; (b) MPT; (c) MST; (d) MT. 3.2. Mesh Indepedence ICEM 17.0 was ado ICEM 17.0 was adopted to obtain the structured hexahedral element for the whole computational region. As indicated in Figure 4, both the O-type topology and local mesh refinement were used. mputational region. As indicated in Figure 4, both the O-type topology and local mesh refinement re used. Figure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. (a) (b) Figure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. (b) (a) (b) (a) ure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. Figure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. ure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. Figure 4. Mesh arrangement. (a) Grids on hydrofoil; (b) Local mesh refinement. Three sets of meshes were employed to examine the effect of element size in numerical culation. The drag coefficient CD and lift coefficient CL were adopted to check the impact of mesh e and they are expressed as: Three sets of meshes were employed to examine the effect of element size in numerical calculation. The drag coefficient CD and lift coefficient CL were adopted to check the impact of mesh size and they are expressed as: D L 2 D  , 2 L L C  (1) CD = D 0.5ρW2∞SD , CL = L 0.5ρW2∞SL (1) (1) 2 0.5 D D C W S   , 2 0.5 L L C W S   (1) ere D and L denote the drag and lift forces parallel and normal to the inflow, which are the forces Y and Z axial directions in present simulation SD and SL represent the projected area of the foil in where D and L denote the drag and lift forces parallel and normal to the inflow, which are the forces in Y and Z axial directions in present simulation. SD and SL represent the projected area of the foil in Y and Z axial directions, respectively. Y and Z axial directions in present simulation. SD and SL represent the projected area of the foil in nd Z axial directions, respectively. Table 1 is the mesh independence results (inlet velocity is 10 m/s). It can be seen that the drag fficient CD and lift coefficient CL keep the same when the number of elements becomes 5500833. refore, the configuration of mesh 2 is employed in the following investigation. 3. Numerical Setting a Numerical Method 3.1. Numerical Method However, the SST k-ω turbulence model is widely used to investigate the detail tunnel wall. It should be noted that the SST k-ω turbulence model has its own limitations, errors and certainties [31]. However, the SST k-ω turbulence model is widely used to investigate the detail w field in tip clearance for a NACA0009 hydrofoil [22,29]. Consequently, the SST k-ω turbulence It should be noted that the SST k-ω turbulence model has its own limitations, errors and uncertainties [31]. However, the SST k-ω turbulence model is widely used to investigate the detail flow field in tip clearance for a NACA0009 hydrofoil [22,29]. Consequently, the SST k-ω turbulence model 5 of 13 14 Energies 2019, 12, 4066 gies 2019, 8, x FOR PEER with all y+ wall treatment is employed to evaluate the effect of T-shape tips on energy performance and flow patterns in the gap. formance and flow patterns in the gap. Mesh Indepedence 3.2. Mesh Indepedence ICEM 17.0 was ado The grids on the surface are arranged with 150 nodes along chord direction, 80 nodes along span direction and 20 des along thickness direction Furthermore there are 20 nodes along the span direction in the gap Table 1 is the mesh independence results (inlet velocity is 10 m/s). It can be seen that the drag coefficient CD and lift coefficient CL keep the same when the number of elements becomes 5500833. Therefore, the configuration of mesh 2 is employed in the following investigation. The grids on the foil surface are arranged with 150 nodes along chord direction, 80 nodes along span direction and 20 nodes along thickness direction. Furthermore, there are 20 nodes along the span direction in the gap to capture flow details. The average value of y+ on the foil surface is about 10–40. apture flow details. The average value of y+ on the foil surface is about 10–40. Table 1. Mesh independence. Items Elements CD CL Mesh 1 2715286 0.307 1.233 Mesh 2 5500833 0.23 1.246 Mesh 3 6899538 0.23 1.246 Table 1. Mesh independence. Items Elements CD CL Mesh 1 2715286 0.307 1.233 Mesh 2 5500833 0.23 1.246 Mesh 3 6899538 0.23 1.246 3.3. Validation of Numerical Results Result and Discussion Energy Performance 4.1. Energy Performance 1. Energy Performance T-shape tip geometry will affect the hydrofoil lift force and drag force, which can be analyzed y the drag coefficient (CD) and lift coefficient (CL) defined in Equation (1). Figure 7 shows the lift oefficient, drag coefficient and ratio K of lift coefficient to drag coefficient for different cases. Results how that the lift coefficient gradually increases, the drag coefficient decreases and the lift-drag ratio ses with increase of inlet velocity. Moreover, the impact of the three optimization configurations on he lift coefficient, drag coefficient and K is complex. Firstly, the lift coefficient of the MPT, MST and MT are smaller than that of M0 when the inlet velocity is 5 m/s and 7.5 m/s. As the inlet velocity rises 10 m/s, the lift coefficient of MST and MT are larger than that of M0. However, for MPT, the lift oefficient is larger than that of M0 only at 10 m/s. Secondly, the drag coefficient of MPT and MST is bviously smaller than that of M0 with the velocity 5 m/s and 7.5 m/s. Finally, the ratio K of MST is rger than that of M0 under all inlet velocities. Moreover, compared with M0, the ratio K of MT is bviously increased when the inlet velocity is 7.5 m/s, 10 m/s, 12.5 m/s and 15 m/s, and the maximum Energy Performance T-shape tip geometry will affect the hydrofoil lift force and drag force, which can be analyzed the drag coefficient (CD) and lift coefficient (CL) defined in Equation (1). Figure 7 shows the lift fficient, drag coefficient and ratio K of lift coefficient to drag coefficient for different cases. Results ow that the lift coefficient gradually increases, the drag coefficient decreases and the lift-drag ratio es with increase of inlet velocity. Moreover, the impact of the three optimization configurations on lift coefficient, drag coefficient and K is complex. Firstly, the lift coefficient of the MPT, MST and T are smaller than that of M0 when the inlet velocity is 5 m/s and 7.5 m/s. As the inlet velocity rises 10 m/s, the lift coefficient of MST and MT are larger than that of M0. However, for MPT, the lift fficient is larger than that of M0 only at 10 m/s. Secondly, the drag coefficient of MPT and MST is viously smaller than that of M0 with the velocity 5 m/s and 7.5 m/s. Mesh 3 3.3. Validation of Numerical Results Mesh 3 3.3. Validation of Numerical Results Validation of Numerical Results As illustrated in Figure 5 (inlet velocity is 10 m/s), there is a good agreement between the erimental TLV trajectory [17] and calculated vortex expressed by Q criterion (Q = 2 × 107 s-2). reover, Figure 6 examines the axial velocity contour on the cut-plane of y/c = 1, the numerical ulations agree well with the experiment measurements. The above analysis shows that the mesh As illustrated in Figure 5 (inlet velocity is 10 m/s), there is a good agreement between the experimental TLV trajectory [17] and calculated vortex expressed by Q criterion (Q = 2 × 107 s−2). Moreover, Figure 6 examines the axial velocity contour on the cut-plane of y/c = 1, the numerical simulations agree well with the experiment measurements. The above analysis shows that the mesh and numerical method adopted in the present paper are reasonable. 6 of 13 f 14 f 14 Energies 2019, 12, 4066 i 1 8 FOR PEE rgies 2019, 8, x FOR PEER Figure 5. Comparison of experimental TLV trajectory [17] and numerical simulations. (a) Experimental results [17] ;(b) Numerical results. (a) (b) Figure 5. Comparison of experimental TLV trajectory [17] and numerical simulations. (a) Experimental results [17]; (b) Numerical results. gure 5. Comparison of experimental TLV trajectory [17] and numerical simulations. (a) perimental results [17] ;(b) Numerical results. (a) (b) (a) e 5. Comparison of experimental TLV traje (a) (b) tory [17] and numerical simulations. (a) (b) (b) rical (a) xperim gure 5. Comparison of experimental TLV trajectory [17] and numerical simulations. (a) xperimental results [17] ;(b) Numerical results. Figure 5. Comparison of experimental TLV trajectory [17] and numerical simulations. (a) Experimental results [17]; (b) Numerical results. perimental results [17] ;(b) Numerical results. Figure 6. Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and i l i l ti ( ) E i t l lt [17] (b) N i l lt igure 6. Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and umerical simulations. (a) Experimental results [17] ;(b) Numerical results. Figure 6. Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and numerical simulations. (a) Experimental results [17]; (b) Numerical results. R l d Di i gure 6. Mesh 3 3.3. Validation of Numerical Results Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and ure 6. Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and merical simulations. (a) Experimental results [17] ;(b) Numerical results. Figure 6. Comparison of the axial velocity contour at cut-plane of y/c = 1 between experiment [17] and numerical simulations. (a) Experimental results [17]; (b) Numerical results. Result and Discussion Energy Performance 4.1. Energy Performance Finally, the ratio K of MST is ger than that of M0 under all inlet velocities. Moreover, compared with M0, the ratio K of MT is viously increased when the inlet velocity is 7.5 m/s, 10 m/s, 12.5 m/s and 15 m/s, and the maximum omotion of K is 15.21% at 7.5 m/s. The ratio K of MPT promotes significantly only at 7.5m/s with T-shape tip geometry will affect the hydrofoil lift force and drag force, which can be analyzed by the drag coefficient (CD) and lift coefficient (CL) defined in Equation (1). Figure 7 shows the lift coefficient, drag coefficient and ratio K of lift coefficient to drag coefficient for different cases. Results show that the lift coefficient gradually increases, the drag coefficient decreases and the lift-drag ratio rises with increase of inlet velocity. Moreover, the impact of the three optimization configurations on the lift coefficient, drag coefficient and K is complex. Firstly, the lift coefficient of the MPT, MST and MT are smaller than that of M0 when the inlet velocity is 5 m/s and 7.5 m/s. As the inlet velocity rises to 10 m/s, the lift coefficient of MST and MT are larger than that of M0. However, for MPT, the lift coefficient is larger than that of M0 only at 10 m/s. Secondly, the drag coefficient of MPT and MST is obviously smaller than that of M0 with the velocity 5 m/s and 7.5 m/s. Finally, the ratio K of MST is larger than that of M0 under all inlet velocities. Moreover, compared with M0, the ratio K of MT is obviously increased when the inlet velocity is 7.5 m/s, 10 m/s, 12.5 m/s and 15 m/s, and the maximum promotion of K is 15.21% at 7.5 m/s. The ratio K of MPT promotes significantly only at 7.5m/s with the maximum promotion of 11.40%. Energies 2019, 12, 4066 7 of 13 gies 2019, 8, x FOR PEER REVIEW 7 of 14 Figure 7. Energy performance of foil for different configurations. Figure 7. Energy performance of foil for different configurations. Energies 2019, 8, x FOR PEER REVIEW 7 of 14 Figure 7. Energy performance of foil for different configurations. 7 of 13 f 14 7 of 14 7 of 13 f 14 7 of 14 Energies 2019, 12, 4066 rgies 2019, 8, x FOR PEER Energies 2019, 8, x FOR ure 7. Result and Discussion Energy Performance 4.1. Energy Performance Energy performance of foil for different configurations. Figure 7. Energy performance of foil for different configurations. Figure 7. Energy performance of foil for different configurations. . Flow Patterns in the Gap 4.2. Flow Patterns in the Gap 4.2. Flow Patterns in the Gap . Flow Patterns in the Gap 4.2. Flow Patterns in the Gap 4.2. Flow Patterns in the Gap Energies 2019, 12, 4066 Energies 2019, 8, x FOR rgies 2019, 8, x FOR PEER 8 of 13 8 of 14 f 14 As indicated in Figure 9, in order to investigate the pressure and vorticity along the PTLV trajectory (Q = 3 × 106 s−2), 10 monitoring points of P1-P10 were chosen along the PTLV trajectory from 5%c to 50%c with an interval of 5%c. As indicated in Figure 9, in order to investigate the pressure and vorticity along the PTLV trajectory (Q = 3 × 106 s-2), 10 monitoring points of P1-P10 were chosen along the PTLV trajectory from 5%c to 50%c with an interval of 5%c. As indicated in Figure 9, in order to investigate the pressure and vorticity along the PTLV jectory (Q = 3 × 106 s-2), 10 monitoring points of P1-P10 were chosen along the PTLV trajectory from %c to 50%c with an interval of 5%c. Figure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s-2). Figure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s−2). igure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s-2). Figure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s-2). Figure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s−2). gure 9. The TLV pattern and trajectory of the case MST (Q = 3 × 106 s-2). Figure 10 shows the vorticity and pressure curve along the PTLV trajectory. The vorticity sharply declines and then slowly decreases along the PTLV trajectory. The variation of pressure along the PTLV trajectory is almost opposite to that of vorticity. The pressure curves of four cases vary along the PTLV trajectory, and it is obvious that T-shape tip can increase the pressure along the PTLV trajectory, especially near the leading edge. The MT has the best effect on pressure improvement, which is corresponded to the best energy performance at 10 m/s. The vorticity along the PTLV trajectory is nearly the same for different cases. Figure 10 shows the vorticity and pressure curve along the PTLV trajectory. The vorticity sharply declines and then slowly decreases along the PTLV trajectory. The variation of pressure along the PTLV trajectory is almost opposite to that of vorticity. 4.3. Curle Surface Acoustic Power Characteristic Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic The Curle noise source model is adopted to compute the dipole noise of the turbulent boundary layer flow on the solid surface. The surface acoustic power (SAP) can be written as: . Curle Surface Acoustic Power Characteristic The Curle noise source model is adopted to compute the dipole noise of the turbulent boundary yer flow on the solid surface The surface acoustic power (SAP) can be written as: The Curle noise source model is adopted to compute the dipole noise of the turbulent boundary layer flow on the solid surface. The surface acoustic power (SAP) can be written as:     2 3 0 0 SAP 12 c S A p dS t c            y y (2) p     2 3 0 0 P 12 c S A p dS t c            y y (2) SAP = x S Ac(y)  12ρ0πc3 0  ∂p ∂t !2 dS(y) (2) (2) (2) where c0 is the sound speed in the far field, and Ac(y) denotes the correlation area. The steady-state RANS turbulence models can be employed to obtain the turbulence length scale, turbulence time scale and wall shear stress, and further calculate 2) ( t p   . here c0 is the sound speed in the far field, and Ac(y) denotes the correlation area. The steady-state ANS turbulence models can be employed to obtain the turbulence length scale, turbulence time ale and wall shear stress, and further calculate 2) ( t p   . where c0 is the sound speed in the far field, and Ac(y) denotes the correlation area. The steady-state RANS turbulence models can be employed to obtain the turbulence length scale, turbulence time scale and wall shear stress, and further calculate (∂p/∂t)2. scale and wall shear stress, and furthe The SAP can be expressed in dB: ale and wall shear stress, and further calc The SAP can be expressed in dB: The SAP can be expressed in dB: , ) ( t p   The SAP can be expressed in dB: d wall shear stress, and further calculate ) ( t p   . . Flow Patterns in the Gap 4.2. Flow Patterns in the Gap 4.2. Flow Patterns in the Gap p The flow patterns in the gap are investigated with the inlet velocity of 10 m/s in this section. As own in Figure 8, the TLV can be divided into the primary tip leakage vortex (PTLV) and the condary tip leakage vortex (STLV). In STAR_CMM+ software, the iso-surface of the vortex can be ntal projected to the water tunnel wall near the tip, which can be used to examine the effect of the shape tips on suppressing the TLV. Compared with the original foil M0, the MPT, MST and MT viously reduce the TLV area by 4.01%, 5.02% and 4.91%, respectively. The flow patterns in the gap are investigated with the inlet velocity of 10 m/s in this section. As shown in Figure 8, the TLV can be divided into the primary tip leakage vortex (PTLV) and the secondary tip leakage vortex (STLV). In STAR_CMM+ software, the iso-surface of the vortex can be frontal projected to the water tunnel wall near the tip, which can be used to examine the effect of the T-shape tips on suppressing the TLV. Compared with the original foil M0, the MPT, MST and MT obviously reduce the TLV area by 4.01%, 5.02% and 4.91%, respectively. The flow patterns in the gap are investigated with the inlet velocity of 10 m/s in this section. As shown in Figure 8, the TLV can be divided into the primary tip leakage vortex (PTLV) and the secondary tip leakage vortex (STLV). In STAR_CMM+ software, the iso-surface of the vortex can be frontal projected to the water tunnel wall near the tip, which can be used to examine the effect of the T-shape tips on suppressing the TLV. Compared with the original foil M0, the MPT, MST and MT obviously reduce the TLV area by 4.01%, 5.02% and 4.91%, respectively. (a) M0 (b) MPT. (c) MST (d) MT. STLV PTLV Figure 8 Iso-surface showing TLV at Q = 3 × 107 s-2. (a) M0 (b) MPT. (c) MST (d) MT. STLV PTLV Figure 8. Iso-surface showing TLV at Q = 3 × 107 s−2. LV STLV MPT (b) MPT. ( ) Fi u e 8 I o u fa e ho i TLV at Q 3 × 107 2 Figure 8. Iso-surface showing TLV at Q = 3 × 107 s−2. . Flow Patterns in the Gap 4.2. Flow Patterns in the Gap 4.2. Flow Patterns in the Gap The pressure curves of four cases vary along the PTLV trajectory, and it is obvious that T-shape tip can increase the pressure along the PTLV trajectory, especially near the leading edge. The MT has the best effect on pressure improvement, which is corresponded to the best energy performance at 10 m/s. The vorticity along the PTLV trajectory is nearly the same for different cases. Figure 10 shows the vorticity and pressure curve along the PTLV trajectory. The vorticity sharply clines and then slowly decreases along the PTLV trajectory. The variation of pressure along the LV trajectory is almost opposite to that of vorticity. The pressure curves of four cases vary along PTLV trajectory, and it is obvious that T-shape tip can increase the pressure along the PTLV ectory, especially near the leading edge. The MT has the best effect on pressure improvement, hich is corresponded to the best energy performance at 10 m/s. The vorticity along the PTLV ectory is nearly the same for different cases. Figure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. Figure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. Figure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. Figure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. ure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. Figure 10. Pressure and vorticity distribution on planes 1–5 with different tip shapes. 4 3 Curle Surface Acoustic Power Characteristic 3 Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic Curle surface acoustic power (SAP) of the hydrofoil for different T-shape tips. Figure 11. Curle surface acoustic power (SAP) of the hydrofoil for different T-shape tips. The effect of T-shape tips on Curle SAP of hydrofoil with tip clearance is demonstrated in Table 2. The relative change is defined as ΔSAP = (SAP-SAP0)/SAP0, where SAP0 is the value of M0, and the average SAP denotes the surface average value of the Curle SAP. It can be concluded that the maximum SAP for MPT and MT decreases by 2.64% and 3.03%, respectively, and the average SAP for MPT, MST and MT decreases by 0.50%, 0.04% and 0.97%, respectively. For the foil end surface, the MT model can effectively suppress the dipole noise, the maximum SAP and the average SAP decrease 4.75% and 1.74%, respectively. In order to evaluate the impact of the TLV on the dipole noise of the side wall near tip surface, the data of the side wall is listed in Table 2. The average SAP of the side wall for MPT, MST and MT decreases by 0.12%, 0.01% and 0.12%, respectively. In addition, the maximum SAP for MT decreases by 0.43%. Table 2 Cu le SAP fo diffe e t he e The effect of T-shape tips on Curle SAP of hydrofoil with tip clearance is demonstrated in Table 2. The relative change is defined as ∆SAP = (SAP −SAP0)/SAP0, where SAP0 is the value of M0, and the average SAP denotes the surface average value of the Curle SAP. It can be concluded that the maximum SAP for MPT and MT decreases by 2.64% and 3.03%, respectively, and the average SAP for MPT, MST and MT decreases by 0.50%, 0.04% and 0.97%, respectively. For the foil end surface, the MT model can effectively suppress the dipole noise, the maximum SAP and the average SAP decrease 4.75% and 1.74%, respectively. In order to evaluate the impact of the TLV on the dipole noise of the side wall near tip surface, the data of the side wall is listed in Table 2. The average SAP of the side wall for MPT, MST and MT decreases by 0.12%, 0.01% and 0.12%, respectively. In addition, the maximum SAP for MT decreases by 0.43%. 4.3. Curle Surface Acoustic Power Characteristic Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic e SAP can be expressed in dB: The SAP can be expressed in dB:   SAP dB 10log ref SAP P  (3)   AP dB 10log ref SAP P  (3) SAP(dB) = 10 log SAP Pre f (3) (3) (3) Energies 2019, 12, 4066 9 of 13 where Pref = 1 × 10−12 W/m2 is the reference acoustic power. Energies 2019, 8, x FOR PEER REVIEW 9 of 14 where Pref = 1 × 10−12 W/m2 is the reference acoustic power. Energies 2019, 8, x FOR PEER REVIEW 9 of 14 where Pref = 1 × 10−12 W/m2 is the reference acoustic power. Energies 2019, 8, x FOR PEER REVIEW f For the condition of inlet velocity 10 m/s, the Curle SAP characteristic is studied in this section. Figure 11 shows the Curle SAP on the hydrofoil for different T-shape tips. The areas with higher decibel values of dipole noise are mainly on foil leading edge and foil tip near the leading edge. The main reason is that the vortex is close to the foil surface and has obvious interaction with the foil surface. The decibel values of dipole noise on the foil surface along the downstream direction gradually decrease, and the area of high decibel values (red color) on the tip for MPT and MT is small than that of M0. where Pref = 1 × 10-12 W/m2 is the reference acoustic power. For the condition of inlet velocity 10 m/s, the Curle SAP characteristic is studied in this section. Figure 11 shows the Curle SAP on the hydrofoil for different T-shape tips. The areas with higher decibel values of dipole noise are mainly on foil leading edge and foil tip near the leading edge. The main reason is that the vortex is close to the foil surface and has obvious interaction with the foil surface. The decibel values of dipole noise on the foil surface along the downstream direction gradually decrease, and the area of high decibel values (red color) on the tip for MPT and MT is small than that of M0. Figure 11. Curle surface acoustic power (SAP) of the hydrofoil for different T-shape tips. (a) M0 (b) MPT. (c) MST (d) MT. Figure 11. Curle surface acoustic power (SAP) of the hydrofoil for different T-shape tips. (b) MPT. (b) MPT. (c) MST (d) MT. (d) MT. Figure 11. 4.4. Proudman Acoustic Power Characteristic Energies 2019, 8, x FOR PEER REVIEW The Proudman noise source model can be employed for steady-state RANS turbulence models, and visualizes the acoustic power per unit volume which is generated by the quadrupole sound sources of the flow. The total acoustic power can be described as: 4.4. Proudman Acoustic Power Characteristic The Proudman noise source model can be employed for steady-state RANS turbulence models, and visualizes the acoustic power per unit volume which is generated by the quadrupole sound ou e of the flo The total a ou ti o e a be de ibed a AP = acρU3 Lt U5 c5 0 (4) U = Lt T (5) AP(dB) = 10 log AP Pre f (6) ic power can be described as: 3 5 5 0 c t U U AP a L c   (4) tL U T  (5)   dB 10log AP AP P  (6) (4) (5) (6) where αc = 0.629 denotes a constant value, U represents the turbulence velocity, Lt is the turbulence length scale and T denotes the turbulence time scale. ref P ( ) where αc = 0.629 denotes a constant value, U represents the turbulence velocity, Lt is the turbulence The Proudman acoustic power characteristic was studied under the inlet velocity of 10 m/s. To reveal the quadrupole noise around the foil, five cut-planes 1–5 were chosen at 10%c, 15%c, 20%c, 30%c and 50%c, respectively. Figure 12 shows the Proudman AP on these five cut-planes. The quadrupole noise mainly concentrated near the PTLV and STLV in the tip clearance, and the larger decibel value of the quadrupole noise mainly appeared near the STLV and the area where the STLV flow interacts with the PTLV. In addition, the MT model had a slight suppression effect on the quadrupole noise in planes 1–3. length scale and T denotes the turbulence time scale. The Proudman acoustic power characteristic was studied under the inlet velocity of 10 m/s. To reveal the quadrupole noise around the foil, five cut-planes 1–5 were chosen at 10%c, 15%c, 20%c, 30%c and 50%c, respectively. Figure 12 shows the Proudman AP on these five cut-planes. The quadrupole noise mainly concentrated near the PTLV and STLV in the tip clearance, and the larger decibel value of the quadrupole noise mainly appeared near the STLV and the area where the STLV flow interacts with the PTLV. 4.3. Curle Surface Acoustic Power Characteristic Curle Surface Acoustic Power Characteristic 4.3. Curle Surface Acoustic Power Characteristic Scheme Item MPT MST MT △SAP%( Hydrofoi l) Maximum SAP −2.64 0.03 −3.03 Average SAP −0.50 −0.04 −0.97 △SAP%(Tip) Maximum SAP −2.11 −0.81 −4.75 Average SAP −1.92 0.33 −1.74 △SAP%(Side wall) Maximum SAP 0.20 −0.39 −0.43 Average SAP −0.12 −0.01 −0.12 Table 2. Curle SAP for different schemes. Scheme Item MPT MST MT ∆SAP% (Hydrofoil) Maximum SAP −2.64 0.03 −3.03 Average SAP −0.50 −0.04 −0.97 ∆SAP% (Tip) Maximum SAP −2.11 −0.81 −4.75 Average SAP −1.92 0.33 −1.74 ∆SAP% (Side wall) Maximum SAP 0.20 −0.39 −0.43 Average SAP −0.12 −0.01 −0.12 Scheme Item MPT MST Table 2. Curle SAP for different schemes. Energies 2019, 12, 4066 10 of 13 4.4. Proudman Acoustic Power Characteristic Energies 2019, 8, x FOR PEER REVIEW 10 of 14 4.4. Proudman Acoustic Power Characteristic Energies 2019, 8, x FOR PEER REVIEW 4.4. Proudman Acoustic Power Characteristic Energies 2019, 8, x FOR PEER REVIEW In addition, the MT model had a slight suppression effect on the quadrupole noise in planes 1–3. Figure 12. Proudman AP on five cut-planes. Figure 12. Proudman AP on five cut-planes. Figure 12. Proudman AP on five cut-planes. 11 of 13 Energies 2019, 12, 4066 Some comparisons were made at the power levels of 50 dB and 55 dB. As shown in Figure 13, the isosurfaces represent the value of noise at 50 dB. The main noise was located at the tip clearance leakage vortex and the leading edge. In comparison with M0, the area of noise for the three T-shape cases was not significantly reduced. As shown in Figure 14, the noise at the PTLV of the MST and MT was smaller than that of the original model M0. In STAR_CMM+ software, the iso-surface of the Proudman AP can be frontal projected to the water tunnel wall near the tip, which can be adopted to evaluate the impact of the T-shape tips on suppressing the Proudman AP. In comparison with original foil M0, the MST model obviously reduced the area of Proudman AP by 6.55% for 55 dB. However, the other two optimization configurations of MPT and MT could not effectively reduce the total area of the Proudman AP. Some comparisons were made at the power levels of 50 dB and 55 dB. As shown in Figure 13, the isosurfaces represent the value of noise at 50 dB. The main noise was located at the tip clearance leakage vortex and the leading edge. In comparison with M0, the area of noise for the three T-shape cases was not significantly reduced. As shown in Figure 14, the noise at the PTLV of the MST and MT was smaller than that of the original model M0. In STAR_CMM+ software, the iso-surface of the Proudman AP can be frontal projected to the water tunnel wall near the tip, which can be adopted to evaluate the impact of the T-shape tips on suppressing the Proudman AP. In comparison with original foil M0, the MST model obviously reduced the area of Proudman AP by 6.55% for 55 dB. However, the other two optimization configurations of MPT and MT could not effectively reduce the total area of the Proudman AP. 5. Conclusions Conflicts of Interest: The authors declare no conflicts of interest. 4.4. Proudman Acoustic Power Characteristic Energies 2019, 8, x FOR PEER REVIEW Note that although T-shaped tips can reduce the area of TLV, this does not mean that the Note that although T-shaped tips can reduce the area of TLV, this does not mean that the intensity of quadrupole noise sources can be effectively suppressed. For example, compared with original foil M0, the TLV area of the MPT was reduced by 4.01%, but the area of Proudman AP was not significantly reduced. This is because in the sound source region, quadrupole noise mainly depends on Reynolds stress [32], not the size of vortex. g p p , intensity of quadrupole noise sources can be effectively suppressed. For example, compared with original foil M0, the TLV area of the MPT was reduced by 4.01%, but the area of Proudman AP was not significantly reduced. This is because in the sound source region, quadrupole noise mainly depends on Reynolds stress [32], not the size of vortex. (a) M0 (b) MPT (c) MST (d) MT Figure 13. Isosurfaces showing Proudman AP sound sources at 50 dB. s 2019, 8, x FOR PEER REVIEW 12 of 14 (b) MPT (a) M0 (a) M0 (c) MST 19, 8, x FOR PEER REVIEW (d) MT 12 of 14 (d) MT (c) MST EW Figure 13. Isosurfaces showing Proudman AP sound sources at 50 dB. Figure 13. Isosurfaces showing Proudman AP sound sources at 50 dB. Figure 13. Isosurfaces showing Proudman AP sound sources at 50 dB. Figure 14 Isosurfaces showing Proudman AP sound sources at 55 dB (a) M0 (b) MPT (c) MST (d) MT Figure 14. Isosurfaces showing Proudman AP sound sources at 55 dB. Figure 13. Isosurfaces showing Prou (a) M0 udman AP sound sources at 50 dB. (b) MPT (a) M0 (d) MT (c) MST (c) MST (d) MT Figure 14. Isosurfaces showing Proudman AP sound sources at 55 dB. Energies 2019, 12, 4066 12 of 13 5. Conclusions The effect of T-shaped tip on the energy performance, flow patterns and broadband noise sources for a NACA0009 hydrofoil with tip clearance was carried out in the present paper. The numerical calculation was verified by comparing the flow experimental measurements with the numerical simulation results. The conclusions are summarized as follows: (1) The impact of the T-shape tip on lift coefficient, drag coefficient and ratio of lift coefficient to drag coefficient is complex. Under all inlet velocities, the MST model effectively improves ratio of lift coefficient to drag coefficient with maximum promotion of 4.26%. In addition, the ratio of lift coefficient to drag coefficient for the MT model obviously increases when the inlet velocity is 7.5 m/s, 10 m/s, 12.5 m/s and 15 m/s, and the maximum promotion is 15.21% at 7.5 m/s. (2) The T-shape tip can suppress the TLV, which makes the vortex area decrease with a maximum drop of 5.02%. The three optimization configurations improve the pressure along the PTLV trajectory near the leading edge. (2) The T-shape tip can suppress the TLV, which makes the vortex area decrease with a maximum drop of 5.02%. The three optimization configurations improve the pressure along the PTLV trajectory near the leading edge. (3) The MPT and MT have good suppression effect on the hydrofoil dipole noise, and reduce the maximum Curle AP of the foil with 2.64% and 3.03%, respectively. (4) Compared with the original foil M0, the MST model obviously reduces the area of the Proudman AP by 6.55% for 55 dB. However, the optimization models of the MPT and MT cannot effectively reduce the total area of the Proudman AP. reduce the total area of the Proudman AP. Author Contributions: Z.H., L.T. and C.H. proposed the T-shape method, Z.H. and Y.H. completed the numerical simulations, Z.H. and Y.H. analyzed the data, Z.H., Y.H., L.T. and C.H. wrote the paper. Author Contributions: Z.H., L.T. and C.H. proposed the T-shape method, Z.H. and Y.H. completed the numerical simulations, Z.H. and Y.H. analyzed the data, Z.H., Y.H., L.T. and C.H. wrote the paper. Funding: This research was funded by the National Natural Science Foundation of China [grant number 51809148], the Beijing Natural Science Foundation [grant number 3192015], the Open Research Fund Program of State Key Laboratory of Hydroscience and Engineering [grant number sklhse-2018-E-01], the State Key Laboratory of Hydroscience and Engineering [grant number 2018-KY-02]. References 1. Li, X.; Jiang, Z.; Zhu, Z.; Si, Q.; Li, Y. Entropy generation analysis for the cavitating head-drop characteristic of a centrifugal pump. Proc. Inst. Mech. Eng. Part C J. Mech. Eng. Sci. 2018, 232, 4637–4646. [CrossRef] 1. Li, X.; Jiang, Z.; Zhu, Z.; Si, Q.; Li, Y. Entropy generation analysis for the cavitating head-drop characteristic of a centrifugal pump. Proc. Inst. Mech. Eng. Part C J. Mech. Eng. Sci. 2018, 232, 4637–4646. [CrossRef] 2. Li, X.; Gao, P.; Zhu, Z.; Li, Y. Effect of the blade loading distribution on hydrodynamic performance of a centrifugal pump with cylindrical blades. J. Mech. Sci. Technol. 2018, 32, 1161–1170. [CrossRef] 2. Li, X.; Gao, P.; Zhu, Z.; Li, Y. Effect of the blade loading distribution on hydrodynamic performance of a centrifugal pump with cylindrical blades. J. Mech. Sci. Technol. 2018, 32, 1161–1170. [CrossRef] 3. Liu, M.; Tan, L.; Cao, S. Theoretical model of energy performance prediction and BEP determination for centrifugal pump as turbine. Energy 2019, 172, 712–732. [CrossRef] 4. Tan, L.; Zhu, B.; Cao, S.; Wang, Y.; Wang, B. Numerical simulation of unsteady cavitation flow in a centrifugal pump at off-design conditions. Proc. Inst. Mech. Eng. Part C J. Mech. Eng. Sci. 2014, 228, 1994–2006. 5. Tan, L.; Zhu, B.; Wang, Y.; Cao, S.; Gui, S. Numerical study on characteristics of unsteady flow in a centrifugal pump volute at partial load condition. Eng. Comput. 2015, 32, 1549–1566. [CrossRef] Zhang, W.; Yu, Z.; Li, Y. Application of a non-uniform bubble model in a multiphase rotodynamic pump J. Pet. Sci. Eng. 2019, 173, 1316–1322. [CrossRef] Zhang, W.; Yu, Z.; Li, Y. Application of a non-uniform bubble model in a multiphase rotodynamic pump J. Pet. Sci. Eng. 2019, 173, 1316–1322. [CrossRef] 7. Zhang, W.; Yu, Z.; Li, Y.; Yang, J.; Ye, Q. Numerical analysis of pressure fluctuation in a multiphase rotodynamic pump with air–water two-phase flow. Oil Gas Sci. Technol. 2019, 74, 18. [CrossRef] 7. Zhang, W.; Yu, Z.; Li, Y.; Yang, J.; Ye, Q. Numerical analysis of pressure fluctuation in a multiph rotodynamic pump with air–water two-phase flow. Oil Gas Sci. Technol. 2019, 74, 18. [CrossRef] . Li, X.-S.; Song, Y.; Hao, Z.-R.; Gu, C.-W. Cavitation Mechanism of Oil-Film Bearing and Development New Gaseous Cavitation Model Based on Air Solubility. J. Tribol. 2012, 134, 031701. [CrossRef] 8. Li, X.-S.; Song, Y.; Hao, Z.-R.; Gu, C.-W. References Numerical and experimental investigation of tip leakage vortex trajectory and dynamics in an axial flow pump. Comput. Fluids 2015, 112, 61–71. [CrossRef] 16. Zhang, D.; Shi, W.; Van Esch, B.B.; Shi, L.; Dubuisson, M. Numerical and experimental investigation of tip leakage vortex trajectory and dynamics in an axial flow pump. Comput. Fluids 2015, 112, 61–71. [CrossRef] 17. Dreyer, M.; Decaix, J.; Münch-Alligné, C.; Farhat, M. Mind the gap: A new insight into the tip leakage vortex 17. Dreyer, M.; Decaix, J.; Münch-Alligné, C.; Farhat, M. Mind the gap: A new insight into the tip leakage vortex using stereo-PIV. Exp. Fluids 2014, 55, 1–13. [CrossRef] 18. Gopalan, S.; Katz, J.; Liu, H.L. Effect of Gap Size on Tip Leakage Cavitation Inception, Associated Noise and Flow Structure. J. Fluids Eng. 2002, 124, 994–1004. [CrossRef] 9. Liu, Y.; Tan, L. Tip clearance on pressure fluctuation intensity and vortex characteristic of a mixed flow pu as turbine at pump mode. Renew. Energy 2018, 129, 606–615. [CrossRef] 0. Hao, Y.; Tan, L.; Yue, H.; Lei, T. Symmetrical and unsymmetrical tip clearances on cavitation performance radial force of a mixed flow pump as turbine at pump mode. Renew. Energy 2018, 127, 368–376. [Cross 20. Hao, Y.; Tan, L.; Yue, H.; Lei, T. Symmetrical and unsymmetrical tip clearances on cavitation performance and radial force of a mixed flow pump as turbine at pump mode. Renew. Energy 2018, 127, 368–376. [CrossRef] 21. Liu, Y.; Tan, L. Spatial–Temporal Evolution of Tip Leakage Vortex in a Mixed-Flow Pump with Tip Clearance. J. Fluids Eng. 2019, 141, 081302. [CrossRef] 21. Liu, Y.; Tan, L. Spatial–Temporal Evolution of Tip Leakage Vortex in a Mixed-Flow Pump with Tip Clearance. J. Fluids Eng. 2019, 141, 081302. [CrossRef] 22. Guo, Q.; Zhou, L.; Wang, Z. Numerical evaluation of the clearance geometries effect on the flow field and performance of a hydrofoil. Renew. Energy 2016, 99, 390–397. [CrossRef] 23. Okita, K.; Ugajin, H.; Matsumoto, Y. Numerical Analysis of the Influence of the Tip Clearance Flows on the Unsteady Cavitating Flows in a Three-Dimensional Inducer. J. Hydrodyn. 2009, 21, 34–40. [CrossRef] 24. Zhang, J.; Fan, H.; Zhang, W.; Xie, Z. Energy performance and flow characteristics of a multiphase pump with different tip clearance sizes. Adv. Mech. Eng. 2019, 11, 1–14. [CrossRef] 25. Decaix, J.; Balarac, G.; Dreyer, M.; Farhat, M.; Munch, C. RANS and LES computations of the tip-leakage vortex for different gap widths. J. Turbul. References Cavitation Mechanism of Oil-Film Bearing and Development of a New Gaseous Cavitation Model Based on Air Solubility. J. Tribol. 2012, 134, 031701. [CrossRef] 9. Wang, Y.; Ren, X.; Li, X.; Gu, C. Numerical investigation of air-oil-thermal coupling mechanism in floating ring. J. Tribol. 2018, 140, 031701. 9. Wang, Y.; Ren, X.; Li, X.; Gu, C. Numerical investigation of air-oil-thermal coupling mechanism in floating ring. J. Tribol. 2018, 140, 031701. 10. Liu, M.; Tan, L.; Cao, S. Cavitation-vortex-turbulence interaction and one-dimensional model prediction of pressure for hydrofoil ALE15 by large eddy simulation. ASME J. Fluids Eng. 2019, 141, 021103. [CrossRef] 10. Liu, M.; Tan, L.; Cao, S. Cavitation-vortex-turbulence interaction and one-dimensional model prediction of pressure for hydrofoil ALE15 by large eddy simulation. ASME J. Fluids Eng. 2019, 141, 021103. [CrossRef] 11. Cheng, H.Y.; Bai, X.R.; Long, X.P.; Ji, B.; Peng, X.X.; Farhat, M. Large eddy simulation of the tip-leakage cavitating flow with an insight on how cavitation influences vorticity and turbulence. Appl. Math. Model. 2020, 77, 788–809. [CrossRef] 11. Cheng, H.Y.; Bai, X.R.; Long, X.P.; Ji, B.; Peng, X.X.; Farhat, M. Large eddy simulation of the tip-leakage cavitating flow with an insight on how cavitation influences vorticity and turbulence. Appl. Math. Model. 2020, 77, 788–809. [CrossRef] Energies 2019, 12, 4066 13 of 13 13 of 13 12. Liu, Y.; Tan, L.; Wang, B. A Review of Tip Clearance in Propeller, Pump and Turbine. Energies 2018, 11, 2202. [CrossRef] 13. Wu, H.; Miorini, R.L.; Katz, J. Measurements of the tip leakage vortex structures and turbulence in the meridional plane of an axial water-jet pump. Exp. Fluids 2011, 50, 989–1003. [CrossRef] 14. Wu, H.; Tan, D.; Miorini, R.L.; Katz, J. Three-dimensional flow structures and associated turbulence in the tip region of a waterjet pump rotor blade. Exp. Fluids 2011, 51, 1721–1737. [CrossRef] 15. Zhang, D.; Shi, W.; Pan, D.; Dubuisson, M. Numerical and Experimental Investigation of Tip Leakage Vortex Cavitation Patterns and Mechanisms in an Axial Flow Pump. J. Fluids Eng. 2015, 137, 121103. [CrossRef] 15. Zhang, D.; Shi, W.; Pan, D.; Dubuisson, M. Numerical and Experimental Investigation of Tip Leakage Vortex Cavitation Patterns and Mechanisms in an Axial Flow Pump. J. Fluids Eng. 2015, 137, 121103. [CrossRef] 16 Zhang D ; Shi W ; Van Esch B B ; Shi L ; Dubuisson M Numerical and experimental investigation of tip 16. Zhang, D.; Shi, W.; Van Esch, B.B.; Shi, L.; Dubuisson, M. References 2015, 16, 309–341. [CrossRef] 26. Decaix, J.; Dreyer, M.; Balarac, G.; Farhat, M.; Münch, C. RANS computations of a confined cavitating tip-leakage vortex. Eur. J. Mech.-B/Fluids 2018, 67, 198–210. [CrossRef] 27. Gearhart, W.S. Tip clearance cavitation in shrouded underwater propulsors. J. Aircr. 1966, 3, 185–192. [CrossRef] 28. Laborde, R.; Chantrel, P.; Mory, M. Tip Clearance and Tip Vortex Cavitation in an Axial Flow Pump. J. Fluids Eng. 1997, 119, 680–685. [CrossRef] 29. Liu, Y.; Tan, L. Method of C groove on vortex suppression and energy performance improvement for a NACA0009 hydrofoil with tip clearance in tidal energy. Energy 2018, 155, 448–461. [CrossRef] 30. Menter, F.R. Two-equation eddy-viscosity turbulence models for engineering applications. AIAA J. 1994, 32, 1598–1605. [CrossRef] 31. Mishra, A.A.; Mukhopadhaya, J.; Iaccarino, G.; Alonso, J. Uncertainty estimation module for turbulence model predictions in SU2. AIAA J. 2018, 57, 1066–1077. [CrossRef] 32. Ringwall, E. Aeroacoustic Sound Sources around the Wheels of a Passenger Car-A Computational Fluid Dynamics Study Using Steady State Models to Evaluate Main Sources of Flow Noise. Master’s Thesis, Department of Applied Mechanics Chalmers University of Technology, Gothenburg, Sweden, 2017. © 2019 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 (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4292712319
https://zenodo.org/record/6241163/files/256-259.pdf
English
null
Spectrophotometric Studies on Bisulphitesalicyldebyde Compound
Zenodo (CERN European Organization for Nuclear Research)
1,986
cc-by
2,847
Reference& 1. G. V. SATYAVATI, ''Medtcmal Plants of Indta'', I C.M.R., New Delht, 1976, Vol. 1, pp. 75-76 , , , pp 2. R.N. CHOPRA, "Ind1genous D1ug~ ot Ind1a", Calcutta, 1958, p 577. , p 3. K. R. KtRTJKAR and B. D BAsu, "Indtan Medtcmal Plants", Delh1, 1975, Vol. 1, p 67 , , , , p 4. D. S BHAKUNI, P/tytochermstry, 1976, 18, 1584. , y y, , , 5. H WANGE, M REITER and W. FERSTL, Planta Medrcll, ] 960, 40, 75. 1. INDiAN CBEM. SOC., VOL. LXIii, FEBRUARY 19&6 1. INDiAN CBEM. SOC., VOL. LXIii, FEBRUARY 19&6 1.62, 66. 7%). RRt of sterol acetates were gtven relative to cholesterol. 1.62, 66. 7%). RRt of sterol acetates were gtven relative to cholesterol. constanb were deternuned by Ketp and Bauer2• Regarding spectrophotometnc studte!>, a furfurat- bisulpbite complex have been described earlier 11 • The interesting observations reported eallter that alkanols11 possess antiinflammatory activity comparable wtth P-methasone and P·sttosterol', possesses antnnftammatory activtty, similar to hydrocorti&one and oxyphenabutazone dnd antipy- retic actlVlty stmilar to acetyl salicylic acid, suggest that the presence of sterols and alkanols in the leaves of this plant mtght be responstblc for its medicmal value. The present method may be used for the estima· tion of microgram quanttttes of bisulphite ion. The spectrophotometric studie& can also be used to obtam quantitative mformation of aldehyde-sulphite eqmhbna. EAperimental A 0 095 M stoc.k solutiOn of saticyldehyde (B.D H., England) in ethanol-water (40%, v/v) was used. A 0.0034 M sulphtte &olution was prepared by dtssolvmg reagent grade Na 2S 10 11 (E. Merck, G.R.) in double-dtstdled water. All othet chemicals used were reagent grade. All aqueous solutions were made by using double-disttUed water. Tic (silica gel) plates were developed with l-arbon tetrachloride-ethyl acetate (95 : 5} for determmmg R. values, whereas argentation tic plates (sihca get- 20% stlvet mtrate) were developed five ttmes with methylene chlortde-carbontetrachlortde ( 1 : 5, v fv) for separatton of acetyl derivatives of stetols. Absorbance mea~>ulements were made on .1. Beckman 26 double beam uv-vJstble &pectrophoto- meter eqmpped w1th 1 em quartz ceHs. A Systro· nics 331 expanded scale pH meter equipped with glass electrode was used for pH measurements. Acknowledgement The authors are grateful to Dr. S. C. Gupta, Princtpal, S. V. CoJiege, Altgarh, for providing facihtles. Thanks are due to Prof T. Matsumoto, Japan, for glc analysts, Dr I. S Ahuja for tr spectra and C.S.I,R., New Delhi, for the award of Junior Research FeHowshtp to one of the authors (R.K.S.). Procedure for the determznatwll of sulphite : To an ahquot (0.5-2.0 ml) of the salicyldehyde solution (9.54 x I0- 4 M) were ctdded various amounts (0.5- 6.0 ml) of a solutton contctining 50.0 to 550.0 pg sulphtte. pll of the solutton was adjusted between 3.5 to 6.0 followed by addition of sodtum dihY· drogen phosphate buffer (1.0 ml). The volume of the re~>ulting mtxture was brought up to the mark m a 10 ml volumetril- flask. The absorbance was measured at 256 nm after 30 min agamst the reagent blank. Result~~> and Discussion ] 6. R. N. CHoPRA, .. Supplement of Glossary of Ind1an Medl· ctnal Plants", C SIR., New Delhi, 1974, p. 6. Absorbance [>pectra : The absorbanc;e value of sahcyldehyde was found to decrease due to the formatton of a complex wtth btsulphite 1on. Ftg. 1 shows typtcal absorption curves (>.,.u =-256 nm) for salicyldeh)dc and btsulphite-sahcyldebyde complex. , , , , p 7. M. BEHAR!, C. K ANDHIWAL and M STREIBL, Coli. Czech. Chem. Commun., 1977, 42, 1385. z , , , 8. K. BASU, B DASGUPTA, s K BHATTA(,HARYA, R. LAL and P. K DAs, Curr. Scr, 1971, 40, 86. , , , , 9. M. B. GUPTA, R. NATH, N. SHRIVASfAVA, K. SHANKEll, K. KISHORB and K. P. BHAllGAVA, Planta Medrca, 1980, 3?, 157. JVorkmg 1 ange : The absorbance values ot several sahcyldehyde soluttons containmg known amounts of sulphite were taken. In all cases, blanks were used as references. It was found that the absor· bance values gradually dec.reased with the add1tton of sulphtte. It ts seen from F1g. 2 that such a decrease occurs hnearly upto 55.0 ppm sulphite, and further add1t1on of sulphite causes deviation from hnearity. Thus, the working range for the deter· mmation of sulph1te by using sahcyldehyde has been found to be upto 55.0 ppm with the detection limit' of2.4 ppm. S. P. CHA1TOPADHYAY and ARABJNDA K. DA:)• Ef]ect of pll and diterse ion~ ~ The absorbance values ot solutions were sensitive to pH. Below pH 3.5, no appreciable" absorbance difference between bisulphtte 10n and salicyldehyde was detectable. Above this pH, with the formation of higher amounts of bisulphite-salicyldehyde complex, the absorbance d•fference (i)) remained the same until THE addit1on (.Oropound between carbonyl group and bisulph1te ion has an importance since tt ts a basts for the determination of many aldehydes and ltetones1 • A series of aldehyde-bisulphite com- pounds have been studted and thetr dissoctation T 256 NOTBS !.OrA n~ A\ Fig. 1. Absorbance spectra : (I) salicyldehyde solution only ; in presence of (II) 20.5, (IU) 44.0, (IV) 88.0 and (v) 147.0 ppm sulphite. N The tolerance limits of diverse ions (within an error of ± 2%) were studied. The results indicated that for determination of 27.6 ppm sulphite, no interference was caused by SOO ppm of chloride (as NaCl), nitrate (as NaN0 8 ), sulphate (as (Na 11SO.,); 200 ppm of fluoride (as NaF), tartarate (as Na-K-tartarate), EDTA (as Na-EDTA) 1 100 ppm of Mn11 (as MnSO.,), Cd11 (as CdSO.,), Zn11 (as Zn-acetate). Fe111, iodide, thiosulphate and thiocyanate ions have been found to interfere. Nature of the compou11d: The Job's method of continuous variations" was applied by mixing varying proportions of equimolar solutions of salicyldehyde and sulphite. The difference (D) between the absorbance of each mixture and the value which would have been obtained had no reaction with bisulphite occurred, was calculated. This was plotted against the ratio (R) of the sulphite concen- tration to the total concentration of sulphite and salicyldehyde. A representative result for solutions with total molarity of 1.18 x Io-.. M for sulphite plus salioyldehyde at pH 5 and temperature 34° are shown in Fig. 3. The minimum indicates (bat a Fig. 1. Absorbance spectra : (I) salicyldehyde solution only ; in presence of (II) 20.5, (IU) 44.0, (IV) 88.0 and (v) 147.0 ppm sulphite. 160 200 220 Added Sulftle (ppm)----.- Fig, 2. Working range : {I) 1.669 x to-•, (II) 9.539 x to-•. (III) 4. 769 x Jo-• M salicyldehyde. Fig, 2. Working range : {I) 1.669 x to-•, (II) 9.539 x to-•. (III) 4. 769 x Jo-• M salicyldehyde. pH 6.0 was reached. Above pH 6.0. F1g 4. Plot of y vs x (equation 2). Acknowledgement S. P. CHA1TOPADHYAY and ARABJNDA K. DA:)• with the less availability of HSO;, the concentration of the bisulphite-salicyldehyde compound decreased, as a result the absorbance value would again be nearer to pure salicyldehyde solution. complex containing busulpbite and salicyldehyde is formed in a 1 : 1 ratio. Job's curve of similar nature can also be formed in some other cases7 ' 8 , However, these results show that maximum reaction occurs at equal concentrations of sulphite and 251 J. INDIAN CHEM. SOC., VOL. LXIII, F!!BRUARY 1986 -0020 -o 010 .!Q 12 J! 12 ..2. 12 R .1 12 2 f2 Ftg. 3. Job's plot (D- absorbance of sulph1te sahcydehyde mnc:ture- absorbance of sahcyldehyde solutiOn • R.- sulphite concentrattonftotal concentration of sulphtte + sallcyldehyde I. y = A+BC-B'x (2) Where, y = ;~~~ , C = C~ + CZ t X =d-d~ 1 A= K- 1 / (c0 - t:,.) and B = 1/ (fa - • .. ). (2) -0020 -o 010 .!Q 12 J! 12 ..2. 12 R .1 12 2 f2 Now, x is a hnear funotion of C0 , the equihbnum concentration of the comple"<: and equation (2) gtves a linear plot of y vs x w1th slope, s = - B• and mtercept, i = A+ BC. Finally, K = B/A and fa = 1/B + E..t may be calculated. Using the above graphical procedure, the equilt· brium constant and molar absorptivity values of salicyldehyde-bJsulphtte system have been calculated A representative linear plot of y vs x for this system 1s shown in F1g. 4. The calculated values of f 0 and K are respectively -172 dm 8 mo1-1 cm-t and 5.29X 105 dm8 mol- 1 • The negative ec values for other systems. though not common, have been 1eported earlier 1 '. The value of K obtained by the present method IS comparable to K values for other aldehyde-sulphite compounds determined previously by other method •. Ftg. 3. Job's plot (D- absorbance of sulph1te sahcydehyde mnc:ture- absorbance of sahcyldehyde solutiOn • R.- sulphite concentrattonftotal concentration of sulphtte + sallcyldehyde I. salicyldehyde and that the complex is of the following type8 • OH ft c-H + HSO) = 7 \ \ \ 6 \ \ \ \ 5 \ 2 \ \ \ \ 0 .__...._-,!;----L-~----1---J B t6 Y.. x(I03 ) F1g 4. Plot of y vs x (equation 2). Acknowledgement The authors wish to thank! Dr. B. K. Seal Professor of Chemistry. Burdwan Universtty, for suggestions and helpful discussions. K-1 = CSC~(~c-E.d) - c_g-c~ + d-d~ (1) d-d~ Ec-E.t were, Cg and C~ are the mitial concentrations of the donor and acceptor, respectively. c4 is the molar absorptivity of the acceptor, d~ is the absor- bance of the initial concentration of the acceptor, and d corresponds to the total absorbance at any given wavelength for a cell of 1 em path length. Equation (1) can be written in the form : References 1. I M KoLTHOPF and V. A. STBNOER, "Volumetrtc Ana- lysis'', 2nd ed, lnterscience, New York, J942, Vol 1, 213. 2 W. KBRP and E BAUER, Arb. Ka1ser/. Ges11ndh., 1901, 26,231 3, J F HARRIS and L L. LocH, Anal Chem , 1962, 34, 201 S. P. CHA1TOPADHYAY and ARABJNDA K. DA:)• The addition compound thus formed is an o-hydroxy sulphonic acid or its salt which splits to a certain degree into its components m aqueous solution. The formation of such hydroxy compound has also been reported m connect1on with formalde- hyde·bisulphite add1tion complex9 • Nevertheless. the extent of this decomposition depends upon the stength of the union between the components (which is a function of the temperature}, the concentratiOn and pH of the solution 1 • Molar absorptivity and formation constant ) The negative values of '0 for a complex indicate that accurate evaluation of molar absorptivity and formation constant is a tric'ky one10 • Rose Drago11•u formulated a most generalised equation (equation 1) to determine equihbnum constant spectrophotometrically. This equation (1) contains two unknowns: K and e0 , respectively the equilibrium constant and molar absorptivity of the complex. A straightforward, convenient graphical method. based on the consideration of two linear plots, for solvmg the Rose-Drago equation has been reported by Seal et al.u F1g 4. Plot of y vs x (equation 2). 1. I M KoLTHOPF and V. A. STBNOER, "Volumetrtc Ana- lysis'', 2nd ed, lnterscience, New York, J942, Vol 1, 213. , 3. 2 W. KBRP and E BAUER, Arb. Ka1ser/. Ges11ndh., 1901, 26,231 J L H l h 3, J F HARRIS and L L. LocH, Anal Chem , 1962, 34, 201 References 258 NOTBS NOTBS 4. 1. BASSETT, R. c. DENNEY, G. H. 1BFPERY and ;r. MENDHAM, "Vogel's Text book of Quantitative Inorganic Analysis includmg Elementary Instrumen- tal Analysis .. , 4th ed., E.L.B.S., London 1982, p. 834. priate amount of analytical grade zinc(Jn sulphate heptahydrate in water, standardised complexometri- cally with EDTA", and further diluted as required for working standards. A fresh 1% solution was prepared and kept in amber-coloured bottle. A 0.5 N NaOH solution was prepared. p 5. H. W. ScoGGINS, Anal. Chern, 1970. 41, 1091. , , 6. P. JoB, Anal. Chim •• 1936, 11, 97. 6. P. JoB, Anal. Chim •• 1936, 11, 97. 7. R. K. NANDA and s. ADITYA, J. Indian Chem. Soc.,1963. 40,660. 8 S Standard calibration curve for zinc: To a suitable aliquot containing zinc (2.8-9.0 pg) were added 1% ascorbic acid (0.5 ml) and 1 x 1o-s M QATB (1 ml). A 0.5 N NaOH solution (2.0 ml) was then added and diluted to 10 ml keeping the final concentra- tion of ethanol to 40% (v/v). The absorbance was measured at 560 nm against a reagent blank. , 8. R. C. DAs, R. K. NANDA and S. AoiTYA,I.Indian Chern. Soc., 1963, 40, 739. , , , 9. P. K. DASGUPTA, K. DECESARE and J. C. ULLRBY, Anal. Chern., 1980, 52, 1912. , , , 10. M. B. LASATER and R. C. ANDERSON, J. Am. Chern. Soc., 1952, 74, 1429. 11 , , , 11. N. 1. RosE and R. S. DRAGO, /. Am. Chern. Soc., 1959, 81,6138. , 12. R. S. DRAGO and N.J. Rosa,/. Am. Ch4m. Soc., 1969, 81, 6141. Manuscript received 22 February 1985. revised 6 September 1985, accepted 15 November 1985 ZINC is an essential metal in biological system and its essentiality for human being was recog- nised only recently. Zinc has been found con- siderably in milk samples and in this paper the trace amounts of the metal has been determined spectro- photometrically by 1-(2-quinolylciZ0)-2,4,5-trihy- droxybenzene (QATB)1 • The method is simple and rapid. ZI Effect of foreign ions : Effect of diverse ions on the colour development of zinc complex with QA TB was studied by taking 0.65 pg m1-1 of zinc(IJ), adding varying amounts of diverse ions and follow- ing the recommended procedure. It was observed that fluoride, nitrate, nitrite, sulphate. sulphite, alka- line earths, lanthanides. atumininm(Il[), indium(IH), antimony(III), bismuth{III). chromium(IIJ), plati- num metals (ex:cept paiJadium) and thorium!IV) did not interfere at all. However, sulphide and manganese(II) interfered seriously. Cyanide also interfered but a I 50-fold cyanide concentration is tolerated in presence of 1% formaldehyde solu- tion. This is due to instability of tetracyanozincate ions in presence of formatdehyde 11 and zinc ions are again free to interact with the reagent. Results of tolerance limits, in parts per million (given in paran- thesis) of various ions in solution that caused a devia- tion smaller than ::t- 2% in absorbance are as follows: chlroide (600), bromide (tiOO), iodide (100), thiosulphate (100), phosphate (50), bromate (20), thiocyanate (40), citrate (40), tartarate (40), oxalate (20), EDTA (25), Fe" (56, masked by 1% Determination of Zinc in Milk Samples using 1-(2-Quinolylazo)-2,4,5-trihydroxybenzene as Spectrophotometric Reagent ISHWAR SINGH• and (Mrs.) POONAM Department of Chemistry, Maharshi Dayananda Umvemty, Robtak-124 001 Manuscript received 22 February 1985. revised 6 September 1985, accepted 15 November 1985 Determination of Zinc in Milk Samples using 1-(2-Quinolylazo)-2,4,5-trihydroxybenzene as Spectrophotometric Reagent ISHWAR SINGH• and (Mrs.) POONAM Department of Chemistry, Maharshi Dayananda Umvemty, Robtak-124 001 Manuscript received 22 February 1985. revised 6 September 1985, accepted 15 November 1985 Results and Discussion , 13. B. K. SEAL, A. K. MUKHERJEE and D. C. MUKHERJEE, Bull. Chem. Soc. Jpn., 1979,52,2088. Addition of an ethanolic solution of QA TB to a very dilute solution of zinc(II), in a medium con- taining not less than 30% (v/v) ethanol (otherwise a precipitate was obtained), resulted in a darlO brown colour in alkaline solution having -'m1u at 560 nm. The colour intensity was shown maximum and constant at 0 05-0.25 N NaOH, and 25 molar excess of reagent was required for complete com- plexation. Beer's law validity range with optimum concentration range in ppm is 0.0-1.37 and 0.28-0.9, respectively. Sandell's sensitivity and molar absorp- tivity are 0.0012 tzg Zn/cm9 and 5.4x 10" dm8 moJ- 1 cm- 1 , respectively. The composition (M : L) determined by Job's method is 1 : 2. The method is highly sensitive as well as selective for zinc, and the transition metals either donot form complex: with QATB in high alkaline medium or if formed by a few, are masked by cyanide except manganese. In absence of manganese(II), zinc ions which are also masked by cyanide, are demasked by adding formaldehyde solution, and this procedure was applied to find out trace levels of zinc in milk samples. p , , 14, R. L. MIDDAUGH, R. S. DRAGO and R. 1. NIEDZIELSKI, J. Am. Chern. Soc., 1964, 86, 388. Experimental Synthesis1 of QATB: Stoichiometric amounts of 2-hydrazinoqmnoline• (1.59 g, 0.01 mol, dissol- ved in the minimum amount of dilute hydrochloric auid) and 2,5-dihydroxy-1,4-benzoquinone• (1.4 g, 0.01 rrol in ethanol) were mixed and refluxed for 30 min. The solution was then cooled and made ammoniacal. Excess of ammonia was boiled off and the solution was allowed to stand overnight. The dark red precipitate obtained was recrystallised from ethanol and dried over phosphorus pentoxide. The purity of the compound was checked by thin layer chromatography and elemental analysis (Found 2 C, 64.2: H, 3.8; N, 15.1. C18H11N 80 8 requires : C, 64.05 ; H, 3.91 ; N, 14.9.5%). A 1 X to-• M reagent solution was prepared. Reagent solutions were discarded when a week old. Preparation of solutions : A 1 X IO-• M stock solutionl.of _zinc was prepared by dissolving appro- 259 JICS-10
https://openalex.org/W4318761269
https://e-journal.undikma.ac.id/index.php/jtp/article/download/5978/4124
Indonesian
null
Pengembangan Media Monopoli Berbasis Pelajar Pancasila pada Tema “Kewajiban dan Hakku” Kelas 3 Madrasah Ibtidaiyah
Jurnal teknologi pendidikan/Jurnal Teknologi Pendidikan
2,023
cc-by-sa
6,099
Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Kata Kunci Media Pembelajaran Monopoli, Pelajar Pancasila Abstrak Article History Received: 08-09-22 Reviewed:10-01-22 Published:28-01-23 The background of this research is to develop thematic monopoly media based on Pancasila students on the theme "Obligations and My Rights" for grade 3 in elementary schools.By using the ADDIE development model which consists of five stages, namely Analysis, Design, Development, Implementation, and Evaluation. To develop this thematic monopoly media, its validity and effectiveness have been tested. The results showed that the thematic monopoly media obtained a total average score for all aspects of the validity of university lecturers' material experts, namely 93.33% of teaching materials scores assessed from the material aspect indicating valid learning media, 86.15% material aspect scores from the homeroom. three and 93.33% of media experts who showed the quality of monopoly learning media were valid. When viewed from the feasibility of learning media seen from small group trials the average score is 4.4 (Very Good). And when viewed from the large group scale test or overall students get an average score of 4.1 (Very Good). So it can be concluded that the Pancasila-based monopoly thematic learning media for students that was developed is feasible to be used as teaching material in Civics learning in thematic class III. Key Words Monopoly Learning Media, Pancasila students Key Words Monopoly Learning Media, Pancasila students Pengembangan Media Monopoli Berbasis Pelajar Pancasila pada Tema “Kewajiban dan Hakku” Kelas 3 Madrasah Ibtidaiyah Syifa Fauziah El-Abida*, Saktian Dwi Hartantri, Candra Puspita Rini Universitas Muhammadiyah Tangerang *Corresponding Author Email: syifafauziah0120@gmail.com Syifa Fauziah El-Abida*, Saktian Dwi Hartantri, Candra Puspita Rini Universitas Muhammadiyah Tangerang *Corresponding Author Email: syifafauziah0120@gmail.com Sejarah Artikel Diterima: 08-09-22 Direview:10-01-23 Dipublikasi: 28-01-23 https://doi.org/10.33394/jtp.v8i1.5978 How to Cite: El-Abida, S., Hartantri, S., & Rini, C. (2023). Pengembangan Media Monopoli Berbasis Pelajar Pancasila pada Tema “Kewajiban dan Hakku” Kelas 3 Madrasah Ibtidaiyah. Jurnal Teknologi Pendidikan : Jurnal Penelitian dan Pengembangan Pembelajaran, 8(1), 236-246. doi:https://doi.org/10.33394/jtp.v8i1.5978 This is an open-access article under the CC-BY-SA License. Pendahuluan Pendidikan merupakan usaha sadar dan terancam dalam upaya manusia memanusiakan manusia. Oleh karena itu saat ini manusia semakin sadar bahwa pendidikan memiliki peran yang sangat penting dalam kemajuan umat manusia. Di Indonesia itu pendidikan sangat diutamakan, karena pendidikan disini memiliki peranan yang sangat berpengaruh terhadap terwujudnya peradaban dan bangsa yang bermanfaat. Pendidikan adalah salah satu cara manusia dalam terbentuknya karater dan potensi yang sangat mempengaruhi dari lingkungan tempat tinggal. p g g g p gg Pancasila merupakan ideologi bangsa Indonesia. Sebagai dasar negara, Pancasila dijadikan landasan dalam berkehidupan. Untuk menjadi warga negara yang baik Good Citizen maka nilai- nilai Pancasila perlu mendasari dalam setiap pribadi, hal ini membuktikan peran penting Pancasila sebagai landasan atau pandangan hidup tentang bagaimana seharusnya menjadi warga negara yang baik Good Citizen. Nilai- nilai yang terkandung dalam Pancasila akan mengajarkan cara berpikir dan bertindak sesuai dengan ideologi negara yang seharusnya. Pada jenjang Pendidikan sekolah dasar, penanaman sikap sosial yang nantinya dapat membentuk karakter siswa dituangkan dalam mata pelajaran PKn. PKn merupakan salah satu mata pelajaran yang bertujuan untuk menanamkan nilai- nilai ideologi pancasila yang didalamnya mengandung nilai- nilai kemanusiaan, kepribadian, dan konsep dasar menjadi warga global. Pembelajaran PKn masih banyak menekankan pada aspek kognitif, sehingga kurang mendukung perkembangan aspek afektif kepribadian siswa serta pembelajaran klasikal menyebabkanrendahnya rasa ingin tahu dan semangat siswa dalam mengikuti pembelajaran PKn di kelas. Media erat kaitannya dengan proses pembelajaran. Kata media berasal dari bahasa latin, yaitu medius. Arti kata medius adalah tengah, perantara, atau pengantar. Media juga dapat diartikan sebagai alat bantu yang dapat digunakan sebagai penyampaian pesan untuk mencapai tujuan pembelajaran. (Wati Ega, 2016, h.2). dapat diketahui dari pendapat tersebut dijelaskan bahwasanya media pembelajaran sangat berkaitan dengan proses pembelajaran pada setiap siswa dan dapat diartikan juga sebagai jalan tengah untuk menyampaikan sebuah pesan informasi yang disampaikan oleh guru. Media pembelajaran adalah sarana untuk meningkatkan kegiatan proses belajar mengajar. Dari banyaknya macam media, maka guru dapat berusaha memilah dengan cermat agar media dapat digunakan dengan tepat. Dalam kegiatan belajar mengajar, sering pula pemakaian kata media pembelajaran digantikan dengan istilah seperti bahan pembelajaran (audio-visual communication), alat peraga pandang (visual education), alat peraga dan media penjelas (Kustandy Demawan, 2020, h.6). dari pendapat tersebut peneliti memahami bahwa pengertian dari media pembelajaran adalah suatu hal yang sangat berpengaruh dan memiliki dampak besar dalam proses pembelajaran. Abstrak li i Sejarah Artikel Diterima: 08-09-22 Direview:10-01-23 Dipublikasi: 28-01-23 Penelitian ini dilatar belakangi untuk mengembangkan media monopoli tematik berbasis pelajar pancasila pada tema “Kewajiban dan Hakku” kelas 3 di sekolah dasar. Dengan menggunakan model pengembangan ADDIE yang terdiri dari lima tahap yaitu Analisis, Perancangan, Pengembangan, Implementasi, dan Evaluasi. Untuk mengembangkan media monopoli tematik ini telah melalui uji validitas, kepraktisan, dan keefektifan. Hasil penelitian menunjukan bahwa media monopoli tematik memperoleh skor rata- rata total untuk seluruh aspek validitas ahli materi dosen universitas yaitu 93,33% hasil skor bahan ajar dinilai dari aspek materi menunjukkan media pembelajaran valid, 86,15% hasil skor aspek materi dari wali kelas 3 dan ahli media 93,33% yang menunjukkan kualitan media pembelajaran monopoli valid. Jika dilihat dari kelayakan media pembelajaran dilihat dari uji coba kelompok kecil diperoleh skor dengan rata- rata 4,4(Sangat Baik). Dan jika dilihat dari uji skala kelompok besar atau keseluruhan siswa memperoleh skor rata- rata 4,1 (Sangat baik). Sehingga dapat disimpulkan media pembelajaran monopoli tematik berbasis pelajar pancasila yang dikembangkan layak untuk digunakan sebagai bahan ajar dalam pembelajaran PPKn dalam tematik kelas III. Kata Kunci Media Pembelajaran Monopoli, Pelajar Pancasila How to Cite: El-Abida, S., Hartantri, S., & Rini, C. (2023). Pengembangan Media Monopoli Berbasis Pelajar Pancasila pada Tema “Kewajiban dan Hakku” Kelas 3 Madrasah Ibtidaiyah. Jurnal Teknologi Pendidikan : Jurnal Penelitian dan Pengembangan Pembelajaran, 8(1), 236-246. doi:https://doi.org/10.33394/jtp.v8i1.5978 https://doi.org/10.33394/jtp.v8i1.5978 urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 236 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 237 Pendahuluan Tidak hanya itu media pembelajaran adalah salah satu alat untuk menyampaikan pesan informasi kepada siswa agar pengajar dapat menggunakannya dengan baik dan benar. Perkembangan media pembelajaran merupakan suatu yang hal yang kompleks, dan melibatkan beberapa komponen yang saling berkaitan. Maka dari itu sangatlah penting perkembangan media pembelajaran dalam proses kegiatan belajar disekolah. Sebelum terlaksananya pembelajaran pengajar harus menguasai media pembelajaran dan dapat digunakan dengan sebaik- baiknya, Media pembelajaran dapat mewujudkan alat fisik yang digunakan untuk menyampaikan isi pesan atau informasi pada materi pembelajaran. Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Monopoli merupakan keadaan bisnis yang dipegang penuh oleh satu perusahaan. Hal ini terjadi karena hanya perusahaan yang memiliki layanan yang dibutuhkan oleh banyak orang, yang menjadikan perusahaan tidak memiliki saingan (kompetitor). Dengan melakukan praktik monopoli, perusahaan dapat menguntungkan yang maksimal akan tetapi jika pembelajaran menggunakan monopoli, siswa akan memiliki bentuk interaksi yang baik dari cara metode yang ada pada media monopoli tersebut siswa mudah dan akan terbiasa menghadapi banyak orang. Secara umum monopoli adalah sebuah permainan papan yang terkenal dan familiar didunia. Sudah banyak orang mengetahuinya bahkan disemua kalangan dari anak- anak hingga orang dewasa terutama anak sekolah dasar. Dengan menggunakan media monopoli siswa akan terbiasa berinteraksi dengan banyak orang terutama teman sebayanya, selain itu dari media ini (Kustandi Darmawan, 2020, h.17) mengatakan memiliki tujuan informasi media pembelajaran dapat digunakan dalam rangka penyajian informasi dihadapan sekelompok siswa. Isi dan bentuk penyajian bersifat sangat umum, berfungsi sebagai pengantar, ringkasan laporan, atau pengetahuan latar belakang. Permainan monopoli telah dimodifikasi menjadi media yang dapat dimanfaatkan dalam pembelajaran. Beberapa penelitian bahkan telah mengembangkan media pembelajaran untuk digunakan dalam pembelajaran. Beberapa penelitian bahkan telah mengembangkan media pembelajaran monopoli Zulfitrah Akbar (2020) mengembangkan media monopoli tematik pada tema daerah tempat tinggalku untuk siswa kelas IV, Solekhah (2015) mengembangkan media monopoli tematik pada tema “tempat tinggalku”, dan wahyudin (2021) dengan mengembangkan media pembelajaran berbasis video scribe untuk meningkatkan minat belajar siswa. Dari beberapa media monopoli yang dikemabngakan, terdapat beberapa kesamaan diantaranya terdiri siswa kelas tiga dan untuk meningkatkan motivasi belajar siswa. Hasil presentase akan menjadi kategori penilaian monopoli tematik. Media akan dikatakan valid dan praktis apabila presentase angket validasi lebih daro 61% atau berkategori baik dan presentase angket kepraktisan lebih dari 61% atau berkategori baik. Setelah media dikategorikan valid maka media siap diujicobakan. Media dikatakan efektif apabila hasil uji respon siswa memiliki skor diatas rata- rata, untuk mendapatkan skor tersebut maka peneliti melakukan dua uji yang pertama uji kelompok kecil dan hanya bebrapa siswa untuk percobaan media mnopoli yang kedua uji kelompok besar seluruh siswa kelas tiga, siswa diharapkan mengisi angket setelah pembelajaran menggunakan media monopoli. Metode Penelitian Penelitian ini menggunakan penelitian dan pengembangan atau sering disebut Research and Development (RnD). Menurut (Mawardi, 2019 h. 53) penelitian ini adalah strategi atau metode yang paling ampuh untuk memperbaiki praktek. Penelitian ini memiliki rangkaian proses atau langkah- langkah dalam rangka mengembangkan suatu produk baru atau menyempurnakan produk yang telah ada agar dapat dipertanggung jawabkan. Penelitian dalam bidang Pendidikan pada umumnya jarang diarahkan pada pengembangan suatu produk, tetapiditunjukan untuk menemukan pengetahuan baru berkenaan dengan fenomena- fenomena yang bersifat fundamental, serta praktek- praktek Pendidikan. Penelitian ini memfokuskan pada pengembangan produk media pembelajaran monopoli berbasis pelajar Pancasila dan menentukan materi “Kewajiban dan Hakku” pada siswa kelas IV Sekolah Dasar. Model dalam penelitian dan pengembangan yang akan digunakan adalah model ADDIE. Model ADDIE merupakan model yang dikembangkan oleh Mollenda & Reisser (1990-an). Model ADDIE merupakan model perancangan pembelajaran generic yang menyediakan proses terorganisasi dalam pembangunan bahan- bahan pembelajaran yang dapat digunakan untuk pembelajaran tradisional. Adapun langkah- langkah pengembangan pada model tersebut ialah analisis, desain, pengembangan, implementasi, dan evaluasi. Teknik pengumpulan data yang digunakan yaitu metode observasi awal, dokumentasi, wawancara siswa dan guru, angket.seluruh angket dikategorikan dengan kriteria penilaian yang dibagi menjadi 4 yaitu: STS sangat tidak setuju, TS tidak setuju, S setuju, SS sangat al Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 238 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index setuju. Data angket akan dihitung untuk memperoleh presentase hasil penilaian dengan menggunakan rumus: Kevalidan= x 100% 1. Analisis (Analyze) 1. Analisis (Analyze) Tahap pertama adalah analisis, hasil dari analisis ini akan dijadikan sebagai pedoman untuk penyusunan bahan ajar atau media pembelajaran monopoli tematik berbasis pelajar pancasila. Analisis ini akan diperoleh dengan cara mewawancarai salah satu siswa dan wali kelas III. Berdasarkan wawancara dengan siswa dan wali kelas III bahwasanya siswa kebanyakan siswa yang kurang menyukai pelajaran PPKn karena ketika pembelajaran dilaksankan didalam kelas guru menjelaskan materi masih menggunakan metode ceramah yang monoton sehingga membuat siswa dikelas cenderung bosan dan mengantuk. Hal ini disebabkan karena guru masih menggunakan metode ceramah saat menjelaskan Materi dan hanya menggunakan media atau bahan ajar yang tersedia disekolah oleh karena itu siswa menjadi pasif. Berdasarkan permaslahan tersebut peneliti membuat salah satu produk berupa media pembelajaran monopoli tematik berbasis pelajar pancasila agar dapat digunakan ketika penyampaian materi dilakukan agar siswa dengan mudah memahami materi dan informasi yang disampaikan sehingga dapat membantu dan memenuhi kebutuhan siswa dalam belajar. 1. Analisis (Analyze) Tahap pertama adalah analisis, hasil dari analisis ini akan dijadikan sebagai pedoman untuk penyusunan bahan ajar atau media pembelajaran monopoli tematik berbasis pelajar pancasila. Analisis ini akan diperoleh dengan cara mewawancarai salah satu siswa dan wali kelas III. Berdasarkan wawancara dengan siswa dan wali kelas III bahwasanya siswa kebanyakan siswa yang kurang menyukai pelajaran PPKn karena ketika pembelajaran dilaksankan didalam kelas guru menjelaskan materi masih menggunakan metode ceramah yang monoton sehingga membuat siswa dikelas cenderung bosan dan mengantuk. Hal ini disebabkan karena guru masih menggunakan metode ceramah saat menjelaskan Materi dan hanya menggunakan media atau bahan ajar yang tersedia disekolah oleh karena itu siswa menjadi pasif. Berdasarkan permaslahan tersebut peneliti membuat salah satu produk berupa media pembelajaran monopoli tematik berbasis pelajar pancasila agar dapat digunakan ketika penyampaian materi dilakukan agar siswa dengan mudah memahami materi dan informasi yang disampaikan sehingga dapat membantu dan ( yz ) Tahap pertama adalah analisis, hasil dari analisis ini akan dijadikan sebagai pedoman untuk penyusunan bahan ajar atau media pembelajaran monopoli tematik berbasis pelajar pancasila. Analisis ini akan diperoleh dengan cara mewawancarai salah satu siswa dan wali kelas III. Berdasarkan wawancara dengan siswa dan wali kelas III bahwasanya siswa kebanyakan siswa yang kurang menyukai pelajaran PPKn karena ketika pembelajaran dilaksankan didalam kelas guru menjelaskan materi masih menggunakan metode ceramah yang monoton sehingga membuat siswa dikelas cenderung bosan dan mengantuk. Hasil Penelitian dan Pembahasan Produk pengembangan yang dihasilkan oleh peneliti yaitu bahan ajar berupa permainan monopoli tematik. Penelitian ini merupakan penelitian pengembangan (Research and Development). Model penelitian pengembangan yang digunakan yaitu model pengembangan ADDIE. yang melalui 5 tahap pengembangannya. Adapaun tahapan prosedur pengembangannya adalah sebagai berikut: urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 240 1. Analisis (Analyze) Hal ini disebabkan karena guru masih menggunakan metode ceramah saat menjelaskan Materi dan hanya menggunakan media atau bahan ajar yang tersedia disekolah oleh karena itu siswa menjadi pasif. Berdasarkan permaslahan tersebut peneliti membuat salah satu produk berupa media pembelajaran monopoli tematik berbasis pelajar pancasila agar dapat digunakan ketika penyampaian materi dilakukan agar siswa dengan mudah memahami materi dan informasi yang disampaikan sehingga dapat membantu dan memenuhi kebutuhan siswa dalam belajar. 2. Desain ( Design) Setelah peneliti melakukan observasi awal ke Sekolah Dasar dan melakukan wawancara dengan siswa dan guru kelas III maka peneliti diahrapkan telah memiliki rancangan produk media pembelajaran yang ingin di kembangkan. Selain itu peneliti juga mencari materi yang cocok untuk dikaitkan dengan monopoli dan melakukan cara agar media pembelajaran yang dikembangkan menarik perhatian siswa, dengan ketertarikan mereka pasti ada rasa ingin tahu yang tinggi untuk belajar menggunakan media pembelajaran yang telah dikempangkan oleh peneliti. li 2. Desain ( Design) Setelah peneliti melakukan observasi awal ke Sekolah Dasar dan melakukan wawancara dengan siswa dan guru kelas III maka peneliti diahrapkan telah memiliki rancangan produk media pembelajaran yang ingin di kembangkan. Selain itu peneliti juga mencari materi yang cocok untuk dikaitkan dengan monopoli dan melakukan cara agar media pembelajaran yang dikembangkan menarik perhatian siswa, dengan ketertarikan mereka pasti ada rasa ingin tahu yang tinggi untuk belajar menggunakan media pembelajaran yang telah dikempangkan oleh peneliti. 2. Desain ( Design) Setelah peneliti melakukan observasi awal ke Sekolah Dasar dan melakukan wawancara dengan siswa dan guru kelas III maka peneliti diahrapkan telah memiliki rancangan produk media pembelajaran yang ingin di kembangkan. Selain itu peneliti juga mencari materi yang cocok untuk dikaitkan dengan monopoli dan melakukan cara agar media pembelajaran yang dikembangkan menarik perhatian siswa, dengan ketertarikan mereka pasti ada rasa ingin tahu yang tinggi untuk belajar menggunakan media pembelajaran yang telah dikempangkan oleh peneliti. li a. Papan Monopoli Papan monopoli adalah utama dari sebuah permainan pada monopoli yang bertujuan untuk berjalannya permainan dengan baik dan memikat perhatian siswa. Papan media monopoli tematik yang dibuat peneliti dilihat pada gambar 4.1: a. Papan Monopoli Papan monopoli adalah utama dari sebuah permainan pada monopoli yang bertujuan untuk berjalannya permainan dengan baik dan memikat perhatian siswa. Papan media monopoli tematik yang dibuat peneliti dilihat pada gambar 4.1: eknologi Pendidikan Vol 8. No.1 (Jan. 1. Analisis (Analyze) 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 239 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Gambar 1. Papan Monopoli b. Tampilan Materi Tampilan materi yang digunakan pada media ini baik materi, soal latihan, dan kegiatan pembelajaran monopoli tematik ini disusun menggunakan landasan Pancasila atau PPkn. Dari segi bahasa yang digunakan harus seuai dengan siswa, gambar yang tertera pada papan monopoli pun harus sesuai dengan materi apa yang disampaikan oleh guru kepada siswa. Namun untuk pengembangan materi dimonopoli ini siswa dapat menjawab soal yang tertera pada kartu Hak dan Kewajiban. Berikut adalah salah satu contoh soal yang ada pada kartu Hak dan Kewajiban. Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Gambar 1. Papan Monopoli i Gambar 1. Papan Monopoli b. Tampilan Materi Tampilan materi yang digunakan pada media ini baik materi, soal latihan, dan kegiatan pembelajaran monopoli tematik ini disusun menggunakan landasan Pancasila atau PPkn. Dari segi bahasa yang digunakan harus seuai dengan siswa, gambar yang tertera pada papan monopoli pun harus sesuai dengan materi apa yang disampaikan oleh guru kepada siswa. Namun untuk pengembangan materi dimonopoli ini siswa dapat menjawab soal yang tertera pada kartu Hak dan Kewajiban. Berikut adalah salah satu contoh soal yang ada pada kartu Hak dan Kewajiban. Gambar 2. Kartu Hak, Kewajiban dan perlengkapan pion dan dadu c. Perlengkapan Monopoli Maksud dari perlengkapan monopoli adalah barang yan ada dan dapat digunakan untuk bermain seperti 4 buah pion dengan berbeda warna yang Gambar 1. Papan Monopoli b. Tampilan Materi Tampilan materi yang digunakan pada media ini baik materi, soal latihan, dan kegiatan pembelajaran monopoli tematik ini disusun menggunakan landasan Pancasila atau PPkn. Dari segi bahasa yang digunakan harus seuai dengan siswa, gambar yang tertera pada papan monopoli pun harus sesuai dengan materi apa yang disampaikan oleh guru kepada siswa. Namun untuk pengembangan materi dimonopoli ini siswa dapat menjawab soal yang tertera pada kartu Hak dan Kewajiban. Berikut adalah salah satu contoh soal yang ada pada kartu Hak dan Kewajiban. j Gambar 2. Kartu Hak, Kewajiban dan perlengkapan pion dan dadu Gambar 2. Kartu Hak, Kewajiban dan perlengkapan pion dan dadu c. Perlengkapan Monopoli Maksud dari perlengkapan monopoli adalah barang yan ada dan dapat digunakan untuk bermain seperti 4 buah pion dengan berbeda warna yang c. 1. Analisis (Analyze) Setelah melakukan validasi oleh ahli materi maka komentar dan saran yang didapatkan peneliti digunakan untuk melakukan revisi terhadap materi yang ada pada media pembelajaran, sebelum diuji cobakan kepada siswa. Data hasil validasi dengan ahli materi dapat dilihat dalam lampiran. b Validasi Ahli Media a. Validaasi Ahli Materi Validasi dilakukan oleh salah satu Dosen Fakultas Keguruan dan Ilmu Pendidikan (FKIP) dari Universitas Muhammadiyah Tangerang yang terdiri dari 15 butir pernyataan yang digunakan untuk menilai tingkat kevalidan materi yang ada pada media pembelajaran. Setelah melakukan validasi oleh ahli materi maka komentar dan saran yang didapatkan peneliti digunakan untuk melakukan revisi terhadap materi yang ada pada media pembelajaran, sebelum diuji cobakan kepada siswa. Data hasil validasi dengan ahli materi dapat dilihat dalam lampiran. b V lid i Ahli M di Validasi ini dilakukan oleh salah satu Dosen Fakultas Keguruan dan Ilmu Pendidikan (FKIP) dari Universitas Muhammadiyah Tangerang yang terdiri dari 15 butir pernyataan tentang media pembelajaran yang dikembangkan. Setelah melakukan validasi ke ahli media maka untuk selanjutnya peneliti gunakan sebagai acuan dasar untuk melakukan revisi terhadap produk media pembelajaran monopoli tematik berbasis pelajar pancasila sebelum diuji cobakan kepada siswa. Data hasil validasi dengan ahli media dapat dilihat dalam lampiran. c. Validasi Ahli Materi (Guru) Pada tahap validasi ini dilakukan oleh guru kelas III MI Yanida yang terdiri dari 15 butir pernyataan untuk digunakan sebagai bahan penilaian uji kevalidan tentang penggunaan media pembelajaran dalam proses pembelajaran. Saran yang diberikan oleh Validator digunakan untuk siswa sebelum menggunakan media pembelajaran monopoli diharapkan untuk memahami materi apa yang disampaikan oleh guru selama pembelajaran dilakukan dan siswa harus paham cara bermain permainan monopoli, karena permainan ini memakan waktu yang cukup banyak. p p p y g p y Setelah melakukan melakukan validasi oleh para ahli yaitu validasi ahli materi, ahli materi (Guru), ahli media. Maka komentar dan saran yang diberikan kepada peneliti maka selanjutnya akan digunakan sebagai acuan dasar untuk melakukan revisi tahap awal terhadap media pembelajaran yang bertujuan untuk meningkatkan motivasi belajar siswa dan kualitas media pembelajaran yang dikembangkan. urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 241 1. Analisis (Analyze) Perlengkapan Monopoli Maksud dari perlengkapan monopoli adalah barang yan ada dan dapat digunakan untuk bermain seperti 4 buah pion dengan berbeda warna yang c. Perlengkapan Monopoli Maksud dari perlengkapan monopoli adalah barang yan ada dan dapat digunakan untuk bermain seperti 4 buah pion dengan berbeda warna yang Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 didesain dari bahan kayu, 1 buah dadu ukuran 8 cm x 8 cm. 40 buah karakter point untuk masing- masing warna terdapat 10 karakter, 50 Kartu Hak dan Kewajiban masing – masing memiliki 25 kartu. Salah satu pengembangan dari media monopoli tematik ini dari kartu tersebut yang berisikan soal- soal yang terdiri dari beberaoa materi tentang hak dan kewajiban dan tentang pancasila seperti pengamalan sila pancasila pada kehidupan sehari- hari. didesain dari bahan kayu, 1 buah dadu ukuran 8 cm x 8 cm. 40 buah karakter point untuk masing- masing warna terdapat 10 karakter, 50 Kartu Hak dan Kewajiban masing – masing memiliki 25 kartu. Salah satu pengembangan dari media monopoli tematik ini dari kartu tersebut yang berisikan soal- soal yang terdiri dari beberaoa materi tentang hak dan kewajiban dan tentang pancasila seperti pengamalan sila pancasila pada kehidupan sehari- hari. 3. Pengembangan (Development) Produk awal yang telah dikonsultasikan kepada dosen pembimbing dan telah dilakukannya revisi sesuai saran dan masukan dosen pembimbing atau dosen para ahli. Kemudia dapat melanjutkan ke tahap pengembangan untuk mendapatkan saran atau validasi dari para ahli diantaranya yakni ahli materi, ahli media dan untuk mendapatkan hasil dari produk media monopoli tematik. Dalam metode pengembangan ini ada langkah- langkah yang dilakukan penelitian dalam tahap pengembangan sebagai berikut: 3. Pengembangan (Development) Produk awal yang telah dikonsultasikan kepada dosen pembimbing dan telah dilakukannya revisi sesuai saran dan masukan dosen pembimbing atau dosen para ahli. Kemudia dapat melanjutkan ke tahap pengembangan untuk mendapatkan saran atau validasi dari para ahli diantaranya yakni ahli materi, ahli media dan untuk mendapatkan hasil dari produk media monopoli tematik. Dalam metode pengembangan ini ada langkah- langkah yang dilakukan penelitian dalam tahap pengembangan sebagai berikut: a. Validaasi Ahli Materi Validasi dilakukan oleh salah satu Dosen Fakultas Keguruan dan Ilmu Pendidikan (FKIP) dari Universitas Muhammadiyah Tangerang yang terdiri dari 15 butir pernyataan yang digunakan untuk menilai tingkat kevalidan materi yang ada pada media pembelajaran. 4. Implementasi (Implement) Setelah media pembelajaran dilakukan uji kevalidan oleh para ahli maka untu selanjutnya media pembelajaran dapat diuji cobakan dalam bentuk kelompok kec gi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 241 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 yaitu uji coba dan menggunakan 4 orang siswa dalam bentuk proses pembelajaran yang diawali penyampaian materi setelah itu mbelajar menggunakan media pembelajaran. Uji coba ini dilakukan di MI Yanida yang berada di daerah Kabupaten Tangerang bertepatan di Kecamatan Balaraja. Uji coba skala besar diikuti oleh 25 orang siswa kelas III. Pada media ini peneliti membuat 1 buat monopoli akan tetapi memiliki ukuran yang besar agar satu kelas melihat ketika penggunaan media digunakan, kemudian peneliti menyiapkan media yang akan digunakan. Strategi belajar kelompok yang peneliti lakukan bertujuan agar peserta didik dapat memiliki motivasi yang tinggi untuk mempelajari PPKn dan pancasila pada pengamalan di kehidupan sehari- hari. yaitu uji coba dan menggunakan 4 orang siswa dalam bentuk proses pembelajaran yang diawali penyampaian materi setelah itu mbelajar menggunakan media pembelajaran. Uji coba ini dilakukan di MI Yanida yang berada di daerah Kabupaten Tangerang bertepatan di Kecamatan Balaraja. Uji coba skala besar diikuti oleh 25 orang siswa kelas III. Pada media ini peneliti membuat 1 buat monopoli akan tetapi memiliki ukuran yang besar agar satu kelas melihat ketika penggunaan media digunakan, kemudian peneliti menyiapkan media yang akan digunakan. Strategi belajar kelompok yang peneliti lakukan bertujuan agar peserta didik dapat memiliki motivasi yang tinggi untuk mempelajari PPKn dan pancasila pada pengamalan di kehidupan sehari- hari. Tabel 1. Revisi media berdasarkan kritik dan saran validator No Sebelum Revisi Sesudah Revisi 1. Desain Monopoli Desain Monopoli Tabel 1. Revisi media berdasarkan kritik dan saran validator No Sebelum Revisi Sesudah Revisi 1. Desain Monopoli Desain Monopoli Tabel 1. Revisi media berdasarkan kritik dan saran validator No Sebelum Revisi Sesudah Revisi 1. Desain Monopoli Desain Monopoli 2. Ukuran Monopoli 60 x 60. Ukuran Monopoli 1,5m x 1,5m. 3. Jumlah kartu Hak dan Kewajiban masing- masing 17. Jumlah kartu Hak dan Kewajiban masing- masing menjadi 25. 4. urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 242 5. Evaluasi (Evaluate) Tahap evaluasi merupakan tahap akhir yang dilakukan dalam penelitian pengembangan yang sesuai dalam model pengembangan ADDIE. Pada tahap evaluasi ini meluputi revisi akhir terhadap produk yang telah disusun dan sesuai dengan komentar dan saran dari para ahli materi, ahli media, dan dapat dilihat dari angket respon peserta didik. Dari hasil tersebut dapat dilihat kevalidan produk yang telah dikembangkan oleh peneliti. 4. Implementasi (Implement) Kata START dan GO AWAY. Diubah menjadi MULAI dan JALAN TERUS. 5. Lambang negara tidak boleh ditindih. Posisi lambang diubah. Tabel 1. Revisi media berdasarkan kritik dan saran validator No Sebelum Revisi Sesudah Revisi 1. Desain Monopoli Desain Monopoli 2. Ukuran Monopoli 60 x 60. Ukuran Monopoli 1,5m x 1,5m. 3. Jumlah kartu Hak dan Kewajiban masing- masing 17. Jumlah kartu Hak dan Kewajiban masing- masing menjadi 25. 4. Kata START dan GO AWAY. Diubah menjadi MULAI dan JALAN TERUS. 5. Lambang negara tidak boleh ditindih. Posisi lambang diubah. n kritik dan saran validator Sesudah Revisi Desain Monopoli Desain Monopoli 2. Ukuran Monopoli 60 x 60. Ukuran Monopoli 1,5m x 1,5m. 3. Jumlah kartu Hak dan Kewajiban masing- masing 17. Jumlah kartu Hak dan Kewajiban masing- masing menjadi 25. 4. Kata START dan GO AWAY. Diubah menjadi MULAI dan JALAN TERUS. 5. Lambang negara tidak boleh ditindih. Posisi lambang diubah. 5. Evaluasi (Evaluate) Tahap evaluasi merupakan tahap akhir yang dilakukan dalam penelitian pengembangan yang sesuai dalam model pengembangan ADDIE. Pada tahap evaluasi ini meluputi revisi akhir terhadap produk yang telah disusun dan sesuai dengan komentar dan saran dari para ahli materi, ahli media, dan dapat dilihat dari angket respon peserta didik. Dari hasil tersebut dapat dilihat kevalidan produk yang telah dikembangkan oleh peneliti. 5. Evaluasi (Evaluate) Tahap evaluasi merupakan tahap akhir yang dilakukan dalam penelitian pengembangan yang sesuai dalam model pengembangan ADDIE. Pada tahap evaluasi ini meluputi revisi akhir terhadap produk yang telah disusun dan sesuai dengan komentar dan saran dari para ahli materi, ahli media, dan dapat dilihat dari angket respon peserta didik. Dari hasil tersebut dapat dilihat kevalidan produk yang telah dikembangkan oleh peneliti. 5. Evaluasi (Evaluate) 5. Evaluasi (Evaluate) Tahap evaluasi merupakan tahap akhir yang dilakukan dalam penelitian pengembangan yang sesuai dalam model pengembangan ADDIE. Pada tahap evaluasi ini meluputi revisi akhir terhadap produk yang telah disusun dan sesuai dengan komentar dan saran dari para ahli materi, ahli media, dan dapat dilihat dari angket respon peserta didik. Dari hasil tersebut dapat dilihat kevalidan produk yang telah dikembangkan oleh peneliti. Jurnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 243 Teknik Analisis Data Analisis data dilakukan untuk mendapatkan produk bahan ajar yang dikebangkan layak untuk digunakan dan berkualitas dan diperoleh dari hasil penilaian ahli materi, ahli media, angket respon peserta didik. Berikut ini adalah langkah- langkah dalam menganalisis produk yang dikembangkan dan adapun hasil validasi dari para ahli. Ahli Materi Dari data diatas dapat dilakukan sebagai berikut: Hasil Skoring = Hasil Skoring = Hasil Skoring =4,66 Hasil Skoring = Hasil Skoring = Hasil Skoring =4,66 Hasil Skoring = Hasil Skoring = Hasil Skoring =4,66 Dari hasil perhitungan diatas didapat angka 4,66. Jika dilihat dari tabel klasifikasi maka didapat hasil penelitian dari ahli materi adalah SANGAT BAIK bahwa produk yang dikembangakan untuk diuji coba. Dan apabila dianalisis untuk tingkat kevalidan data yang diperoleh dari hasil penelitian ahli materi maka dapat dilakukan sebagai berikut: Kevalidan= x 100% Kevalidan= x 100% Kevalidan= x 100% Kevalidan= 93,33% Dari hasil perhitungkan diatas maka didapat hasil presentase kevalidan yaitu 93,33%. Maka kesimpulannya hasil penelitian dari ahli materi yang dikembangkan telah VALID untuk diuji coba. j Dari data diatas dapat dilakukan sebagai berikut: p g Hasil Skoring = Hasil Skoring = Hasil Skoring =4,26 Hasil Skoring = Hasil Skoring = Hasil Skoring =4,26 Hasil Skoring = Hasil Skoring = Hasil Skoring =4,26 Dari hasil perhitungan diatas didapat angka 4,26 jika dilihat dari tabel klasifikasi maka didapat hasil penelitian dari ahli materi adalah SANGAT BAIK produk dikembangkan dan diuji coba. Dan apabila dianalisis untuk tingkat kevalidan data yang diperoleh dari hasil penelitian ahli materi maka dapat dilakukan sebagai berikut. = x 100 Kevalidan= x 100% Kevalidan= x 100% Kevalidan= x 100% Kevalidan= x 100% Kevalidan = 86,15% Dari perhitungan diatas maka didapat hasil presentase kevalidan yaitu 86,15%. Maka kesimpulannya hasil penelitian dari ahli materi yang dikembangkan telah VALID untuk di uji coba. Dari data diatas dapat dilakukan sebagai berikut: Dari data diatas dapat dilakukan sebagai berikut: Dari data diatas dapat dilakukan sebagai berikut: Hasil Skoring = Hasil Skoring = Hasil Skoring = 46,66 Dari data diatas dapat dilakukan sebagai berikut: Hasil Skoring = Hasil Skoring = Hasil Skoring = 46,66 p g Hasil Skoring = Hasil Skoring = Hasil Skoring = 46,66 Dari hasil perhitungan di atas terdapat angka 46,66. Jika dilihat dari tabel klasifikasi maka didapat hasil penelitian dari ahli materi adalah SANGAT BAIK bahwa produk yang dikembangkan untuk diuji coba. Dan apabila dianalisis untuk tingkat kevalidan data yang diperoleh dari hasil penelitian ahli materi maka dapat dilakukan sebagai berikut: Kevalidan = x 100% Kevalidan = x 100% Kevalidan = 93,33% Setelah validasi selesai, dan media pembelajaran dinyatakan layak untuk diuji cobakan. Uji coba kelompok kecil menggunakan 4 siswa, dimana guru memilih kemampuan siswa yang memiliki kemampuan yang berbeda- beda. Uji coba ini bertujuan untuk mengetahui tingkat kemenarikan dan keefektifan media pembelajaran untuk dijadikan referensi belajar siswa dengan angket yang telah disediakan, referensi belajar siswa dengan angket yang telah disediakan oleh peneliti. Berdasarkan hal diatas pada respon peserta didik terhadap media pembelajaran monopoli tematik berbasis pelajar pancasila telah mencapai presentase rata- rata seberas 4,1 dan sudah termasuk dalam kategori Sangat Baik. urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 244 Ahli Media Ahli media menilai dari aspek tampilan produk. Ahli media yang menjadi validator pada penelitian ini adalah Boy Dorahman, S.kom, MOS, MM. Berikut adalah table hasil dari penilaian validasi ahli media. Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Pembahasan Media pembelajaran salah satu hal yang dapat mengakselerasi pemahaman siswa agar mencapai tujuan pembelajaran. Namun perlu ditekankan bahwa media ini perlu dikondisikan sesuai dengan kondisi yang ada. Hal ini sebagai mana yang dijelaskan oleh Kustandi dan Sutjipto, (2013) “media pembelajaran adalah alat yang dapat membantu proses belajar mengajar dan berfungsi untuk memperjelas makna pesan yang disampaikan, sehingga dapat mencapai tujuan belajar. Untuk itu penting sekali dalam mengembangkan dan memanfaatkan media sesuai dengan tujuan pembelajaran. Salah satu media yang sesuai dengan tujuan pembelajaran untuk Bahasa Indonesia adalah permainan monopoli. Menuru Suciati,dkk (2015) media monopoli merupakan media pembelajaran yang dapat digunakan dengan cara bermain sehingga memberi siswa situasi- situasi yang menyenangkan, tidak membosankan dan mudah untuk mengekspresikan ide-ide dan perasaan yang tidak akan dapat diterima orang lain, untuk mempermudah siswa dalam menjawab pertanyaan. Sehingga media monopoli ini sangat baik diberikan agar siswa agar pembelajaran lebih menyenangkan dari sebelumnya. Kontribusi penelitian ini tentu memberikan pengetahuan kepada khalayak bahwa media monopoli dapat dimasukkan kedalam pembelajaran agar siswa merasa pembelajaran dapat dilakukan dengan cara menyenangkan dan dikolaborasikan dengan berbagai macam permainan untuk mencapai tujuan dan hasil belajar yang lebih maksimal. Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Saran Penelitian ini telah mengembangkan media pembelajaran monopoli tematik yang valid dan efektif. Oleh karena itu, disarankan bagi guru sekolah dasar agar menggunakan media pembelajaran disetiap pembelajaran agar siswa tidak mudah bosan dengan metode ceramah guru yang monoton dan membuat siswa jebuh ketika pembelajaran. Selain itu, diharapkan peneliti yang lain dapat mengembangkan media berupa monopoli versi yang lain dan memasukkan unsur- unsur lainnya seperti membiasakan diri dengan mengamalkan sila- sila pancasila dan memahami perbedaan hak dan kewajiban. Kesimpulan Hasil penelitian menunjukkan kbahwa uji validasi media dan materi berturut- turut menghasilkan presentase rata- rata sebesar 93,33% (Sangat Baik) dan 93,33% (Sangat Baik). Dengan demikian media pembelajaran monopoli tematik valid untuk digunakan. Dilihat dari hasil respon siswa memiliki rata- rata 4,4 dan dapat dikategorikan (Sangat Baik). Hal ini siswa memberikan respon positif terhadap penggunaan media pembelajaran monopoli dalam pembelajaran. Berdasarkan hasil presentasie penelitian pengembangan yang telah dilaksanakn dapat disimpilkan bahwa media pembelajaran monopoli dinyatakan valid dan efektif untuk digunakan untuk meningkatkat belajar siswa dikelas. Ucapan Terima Kasih Terima kasih kepada Saktian Dwi Hartantri, M.Pd dan Candra Puspita Rini,M.Pd selaku dosen pembimbing yang telah membimbing, memberikan motivasi dan meluangkan banyak waktu untuk membimbing saya. Terutama kedua orang tua saya Drs. H. M. Arsyad, M.Pd dan Hj. Komariah, S.P.d.I dan kakaku tercinta Fathurrahman. Yang telah menjadi support system selama penelitian ini. Daftar Pustaka Antari, L. P. S., & De Liska, L. (2020). Implementasi Nilai Nilai Pancasila dalam Penguatan Karakter Bangsa. Widyadari: Jurnal Pendidian, 21(2), 676- 678. Batubara & Husein. (2020). Media Pembelajaran Efektif. Semarang Jawa Tengah: Fatawa Publushing. Cecep Kustandi, D. D. (2020). Pengembangan Media Pembelajaran. Jakarta: Kenca Charda Ujang (2018) Pendidikan Pancasila Depok: PT Raja Grafindo Tinggi ecep Kustandi, D. D. (2020). Pengembangan Media Pembelajaran. Jakarta: Kencana. Charda Ujang. (2018). Pendidikan Pancasila. Depok: PT Raja Grafindo Tinggi. Durotul Yatimah. (2017). Landasan Pendidikan. Jakarta: CV. Alumgadan Mandir Ega, Wati (2016). Ragam Media Pembelajaran. Yogyakarta: Kata Pena Hamja, B (2017). Pancasila sebagai Dasar Negara dan Ideologi Bangsa. Justisia Jurnal Ilmu Hukum, 3(9), 11- 20. ( ) Khasanah & Hasan. (2021). Pengembangan Media Pembelajaran. Khasanah & Hasan. (2021). Pengembangan Media Pembelajaran. Kustandi & Darmawan. (2021). Pengembangan Media Pembelajaran. Jakarta: Kencana. K di C (2013) M di b l j B Gh li I d i Kustandi & Darmawan. (2021). Pengembangan Media Pembelajaran. Jakarta: Kencana Kustandi, C. (2013). Media pembelajaran.Bogor: Ghalia Indonesia. wardi. (2019). Dasar- dasar Metodologi Penelitian Pendidikan. Yogyakarta: Samudr Biru. Nurlatifah & Awiria. (2019). Pembelajaran PKn SD. Bantul Yogyakarta: Samudra Biru. Qomaruzzaman Bambang. (2021). Pendidikan Karakter Berbasis Pancasila. Bandung: Simbiosa rekatama media. maruzzaman Bambang. (2021). Pendidikan Karakter Berbasis Pancasila. Bandun Simbiosa rekatama media. urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 245 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Januari 2023 Vol 8, No. 1 E-ISSN: 2656-1417 P-ISSN: 2503-0602 Pp. 236-246 Jurnal Teknologi Pendidikan: Jurnal Penelitian dan Pengembangan Pembelajaran https://e-journal.undikma.ac.id/index.php/jtp/index Ramen A Purba dkk. (2021). Media dan Teknologi Pembelajaran. Medan: Yayasan Kita Menulis. Ramen A Purba dkk. (2021). Media dan Teknologi Pembelajaran. Medan: Yayasan Kita Menulis. Sa’adah & Wahyu. (2020). Metode Penelitian R&D. Malang: CV. Literasi Nusantara Abadi. Sa’diyah & Rosyid. (2019). Ragam Media Pembelajaran. Malang: CV. Literasi Nusantara Abadi. Suciati, S., Septiana, I., & Untari, M. (2015). Penerapan Media Monosa (Monopoli Bahasa) Berbasis Kemandirian Dalam Pembelajaran Di Sekolah Dasar. Mimbar Sekolah Dasar, 2(2), 175-188. doi:https://doi.org/10.53400/mimbar-sd.v2i2.1328 ( ), p g Sufyadi Dkk. (2020). Kajian Pengembangan Profil Pelajar Pancasila. Jakarta: Kemdikbud. Wahyudin. (2021). urnal Teknologi Pendidikan Vol 8. No.1 (Jan. 2023) Copyright© 2023 The Author(s) Syifa Fauziah E., et.al 246 Daftar Pustaka Pengembangan Media Pembelajaran Berbasis Vidioscribe Untuk Meningkatkan Belajar Siswa Tema 3 Subtema 3 Kelas 3 Sekolah Dasar. Jurnal pendidikan: Mataram. Yatimah, D. (2017). LANDASAN PENDIDIKAN. Jakarta: CV. Alumgadan Mandiri. , ( ) g Zulfitrah. (2020). Pengembangan media monopoli tematik pada tema daerah tempat tinggalku untuk siswa kelas IV di Madrasah Ibtidaiyah Rahmatullah Kota Jambi. Jurnal Pendidikan: Jambi
https://openalex.org/W3128190426
https://scholarlypublications.universiteitleiden.nl/access/item%3A3309861/view
English
null
The role of myocardial innervation imaging in different clinical scenarios: an expert document of the European Association of Cardiovascular Imaging and Cardiovascular Committee of the European Association of Nuclear Medicine
European heart journal. Cardiovascular imaging
2,021
cc-by
10,227
The role of myocardial innervation imaging in different clinical scenarios: an expert document of the European Association of Cardiovascular Imaging and Cardiovascular Committee of the European Association of Nuclear Medicine Gimelli, A.; Liga, R.; Agostini, D.; Bengel, F.M.; Ernst, S.; Hyafil, F.; ... ; Slart, R.H.J.A. MIBG • cardiac innervation • molecular imaging • nuclear cardiology Keywords * Corresponding author. Tel: þ39 050 3152153. E-mail: gimelli@ftgm.it Published on behalf of the European Society of Cardiology. All rights reserved. V C The Author(s) 2021. For permissions, please email: journals.permissions@oup.com. Citation Gimelli, A., Liga, R., Agostini, D., Bengel, F. M., Ernst, S., Hyafil, F., … Slart, R. H. J. A. (2021). The role of myocardial innervation imaging in different clinical scenarios: an expert document of the European Association of Cardiovascular Imaging and Cardiovascular Committee of the European Association of Nuclear Medicine. European Heart Journal - Cardiovascular Imaging, 22(5), 480-490. doi:10.1093/ehjci/jeab007 Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3279783 Publisher's Version Creative Commons CC BY 4.0 license https://hdl.handle.net/1887/3279783 License: Downloaded from: Note: To cite this publication please use the final published version (if applicable). EACVI DOCUMENT European Heart Journal - Cardiovascular Imaging (2021) 22, 480–490 doi:10.1093/ehjci/jeab007 Alessia Gimelli 1*, Riccardo Liga2, Denis Agostini3, Frank M. Bengel4, Sabine Ernst5, Fabien Hyafil6, Antti Saraste7,8, Arthur J.H.A. Scholte9, Hein J. Verberne10, Derk O. Verschure10,11, and Riemer H.J.A. Slart12,13 Alessia Gimelli 1*, Riccardo Liga2, Denis Agostini3, Frank M. Bengel4, Sabine Ernst5, Fabien Hyafil6, Antti Saraste7,8, Arthur J.H.A. Scholte9, Hein J. Verberne10, Derk O. Verschure10,11, and Riemer H.J.A. Slart12,13 1Department of Imaging, Fondazione Toscana/CNR Gabriele Monasterio1, via Moruzzi n.1, Pisa 56124, Italy; 2Cardiac-Thoracic-Vascular Department, Universita` di Pisa, Pisa, Italy; 3Department of Nuclear Medicine, University Hospital of Normandy, CHU Cote de Nacre, Caen, France; 4Department of Nuclear Medicine, Hannover Medical School (MHH), Hannover, Germany; 5Royal Brompton and Harefield NHS Foundation Trust, National Heart and Lung Institute, Imperial College, London, UK; 6Department of Nuclear Medicine, European Hospital Georges-Pompidou, DMU IMAGINA, Assistance Publique—Hoˆpitaux de Paris, University of Paris, Paris, France; 7Turku PET Centre, University of Turku, Turku, Finland; 8Heart Center, Turku University Hospital, Turku, Finland; 9Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands; 10Department of Radiology and Nuclear Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands; 11Department of Cardiology, Zaans Medical Center, Zaandam, the Netherlands; 12Department of Nuclear Medicine and Molecular Imaging, Medical Imaging Centre, University Medical Center Groningen, Groningen, The Netherlands; and 13Faculty of Science and Technology, Biomedical Photonic Imaging, University of Twente, Enschede, The Netherlands Received 14 December 2020; editorial decision 21 December 2020; accepted 8 January 2021; online publish-ahead-of-print 1 February 2021 Cardiac sympathetic activity plays a key role in supporting cardiac function in both health and disease conditions, and nuclear cardiac imag- ing has always represented the only way for the non-invasive evaluation of the functional integrity of cardiac sympathetic terminals, mainly through the use of radiopharmaceuticals that are analogues of norepinephrine and, in particular, with the use of 123I-mIBG imaging. This technique demonstrates the presence of cardiac sympathetic dysfunction in different cardiac pathologies, linking the severity of sympathet- ic nervous system impairment to adverse patient’s prognosis. This article will outline the state-of-the-art of cardiac 123I-mIBG imaging and define the value and clinical applications in the different fields of cardiovascular diseases. MIBG • cardiac innervation • molecular imaging • nuclear cardiology The role of myocardial innervation imaging in different clinical scenarios: an expert document of the European Association of Cardiovascular Imaging and Cardiovascular Committee of the European Association of Nuclear Medicine The role of myocardial innervation imaging in different clinical scenarios: an expert document of the European Association of Cardiovascular Imaging and Cardiovascular Committee of the European Association of Nuclear Medicine Alessia Gimelli 1*, Riccardo Liga2, Denis Agostini3, Frank M. Bengel4, Sabine Ernst5, Fabien Hyafil6, Antti Saraste7,8, Arthur J.H.A. Scholte9, Hein J. Verberne10, Derk O. Verschure10,11, and Riemer H.J.A. Slart12,13 * Corresponding author. Tel: þ39 050 3152153. E-mail: gimelli@ftgm.it p g þ g @ g Published on behalf of the European Society of Cardiology. All rights reserved. V C The Author(s) 2021. For permissions, please email: journ * Corresponding author. Tel: þ39 050 3152153. E-mail: gimelli@ftgm.it Published on behalf of the European Society of Cardiology. All rights reserved. V C The Author(s) 2021. For permissions, please email: journals.permissions@oup.com. MIBG • cardiac innervation • molecular imaging • nuclear cardiology General concepts of nuclear imaging in the assessment of cardiac sympathetic activity Despite solid prognostic evidence in favour of their implementation in daily clinical routine, planar measures of cardiac sympathetic activ- ity have remained somehow neglected, possibly due to the intrinsic rudimentariness of the technique and the ability to provide only a glo- bal evaluation of myocardial innervation.5–7 Radionuclide imaging interrogates the handling of NE in presynaptic sympathetic nerve terminals. Positron emission tomography (PET) uses short-lived tracers, such as 11Carbon (11C)-hydroxyephedrine, 11C-epinephrine, S-[11C]CGP 12177, or more recently longer living variants, such as 18Fluoride (18F)-LMI1195 or 18F-hydroxyphenetyl- guanidine.12,13 It should be noted that the tracers differ in their affinity to the NE transporter, vesicular storage and metabolism, and in their flow-dependency, leading to distinct differences in their kinetics and specificity. Yet, PET can be used to quantify the integrity of presynap- tic nerve terminals in CHF by absolute means. The most commonly used tracer, however, is the gamma-emitting agent 123I-mIBG, which enables semi-quantitative planar scintigraphy and SPECT over a lon- ger period of time.1 Using 123I-mIBG, several parameters can be derived from planar images (Figure): i.e. the early and late heart-to- mediastinum (H/M) ratio, derived 15min and 4 h post-injection, both considered to be markers of NE uptake. Of note, the 123I-mIBG washout (WO) may then be obtained as a potential marker of NE turnover/sympathetic tone (see formula).1 As a matter of fact, regional differences in myocardial 123I-mIBG uptake may characterize specific cardiac pathologies, such as IHD and left ventricular hypertrophy.7,14 Increasing evidence is available on the possibility to compare, by the use of SPECT, cardiac sympathetic activity, and myocardial perfu- sion on a regional basis,8 deriving indexes of myocardial innervation that might be superior to the standard H/M ratio in predicting morbidity.15 Accordingly, SPECT-derived measures of cardiac innervation cor- relate with major indicators of myocardial (hypo)perfusion, contract- ile (dys)function, and volume overload, possibly predicting adverse cardiac events.9,16,17 Consequently, the tomographic evaluation of cardiac sympathetic activity might be of additional value for clinical stratification in selected subgroups of high-risk patients. Nevertheless, the combined assessment of myocardial innervation and perfusion has never gained wide clinical application, possibly due to the high radiation exposure and long acquisition time of this inte- grated imaging protocol. Pros and cons of myocardial innervation imaging: impact of recent technological innovations Pros and cons of myocardial innervation imaging: impact of recent technological innovations This article will outline the state-of-the-art of cardiac 123I-mIBG imaging and define the value and clinical applications in the different fields of cardiovascular diseases. Introduction regulation and enhanced NE spillover by pre-synaptic re-uptake mechanisms in patients with chronic heart failure (CHF) leads.3–6 On the other hand, in patients with ischaemic heart disease (IHD) dis- crete regions of anatomical myocardial denervation are present, typ- ically exceeding the extent of myocardial fibrosis and representing ideal substrates for cardiac arrhythmogenicity.5,7 However, the evalu- ation of cardiac sympathetic activity has been classically precluded by the regionalization of sympathetic activity, but results obtained in other body regions are difficult to extrapolate to the heart. Cardiac sympathetic activity plays a key role in supporting cardiac function in both health and disease conditions, a mechanism operated by released norepinephrine (NE),1–3 the physiological neurotransmit- ter binding alpha- and beta-adrenoceptors and undergoing reuptake by presynaptic (uptake 1) and extra-neuronal transporters (uptake 2).4 While these mechanisms ensure an optimal NE turnover at a normal neuronal stimulation, they cannot compensate NE overflow at high and prolonged stimulation frequencies. This will lead to diffuse functional denervation due to post-synaptic receptor down- Nuclear cardiac imaging has always represented the only way for the non-invasive evaluation of the functional integrity of cardiac 481 Myocardial innervation imaging sympathetic terminals, mainly through the use of radiopharmaceuti- cals that are analogues of NE.6–8 In this context, 123I-mIBG (metaio- dobenzilguanidine) imaging, a non-metabolized NE analogue undergoing reuptake by the uptake-1 mechanism, represents the ref- erence for the non-invasive evaluation of cardiac adrenergic nervous function. This technique demonstrates the presence of cardiac sym- pathetic dysfunction in different cardiac pathologies, linking the sever- ity of sympathetic nervous system (SNS) impairment to adverse patient’s prognosis. While planar acquisitions have been classically used to evaluate global cardiac 123I-mIBG uptake, the current imaging protocol should also include single-photon emission computed tom- ography (SPECT) imaging that is able to evaluate the regional distri- bution of the radiotracer. More recently, the introduction of dedicated cardiac cameras, equipped with solid-state Cadmium– Zinc–Telluride (CZT) detectors has offered the advantage of repeated studies due to its low radiation exposure, while evaluating at the same time the perfusion of the heart.9–11 collimator septa, degrade image quality and affect calculation of the H/M ratio. The use of a medium energy (ME) collimator limits septum penetration and is preferable to low energy (LE) collimators.2 However, ME collimators are not widely available and therefore LE collimators are most often used. Introduction Collimator choice is one of the most important factors causing variation in myocardial 123I-mIBG- derived parameters.2 A part of this variation can be overcome by assessing a phantom based conversion coefficient for each gamma camera-collimator combination.3,4 This allows for conversion of vari- ous conditions to standardized H/M ratios, and thereby, cross- calibration enables a better comparison between institutions. Such an approach may be of critical importance for identifying appropriate thresholds to differentiate high- and low-risk CHF patients for future applications. (metaio- analogue the ref- nervous iac sym- e sever- adverse lassically imaging ed tom- al distri- ction of dmium– tage of valuating 3I-mIBG different synaptic y (PET) hedrine, ger living henetyl- r affinity Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Myocardial innervation imaging in patients with CHF adverse cardiac events. In fact, it has been demonstrated that a higher 123I-mIBG defect score on SPECT imaging is associated with a signifi- cantly worse prognosis, particularly regarding arrhythmic events.15 Accordingly, based on current evidence, 123I-mIBG scintigraphy might be of value in the risk stratification of patients with CHF, pos- sibly contributing in identifying patients at increased risk that could benefit from more aggressive therapeutic/invasive approaches,28 par- ticularly in the case of patients currently excluded from prophylactic ICD implantation (i.e. borderline EF impairment).29 Moreover, the presence of a relatively preserved cardiac sympathetic tone might help to postpone ICD implantation in patients with otherwise bor- derline indication (i.e. non-ischaemic dilated cardiomyopathy with LVEF 25–35%), in whom the benefit of ICD is disputed.30 Patients with CHF present a condition of cardiac sympathetic hyper- activity coupled with impaired post-synaptic beta-adrenoceptor de- sensitization, impaired NE re-uptake and NE spillover.18 In the past decade, cardiac 123I-mIBG scintigraphy has been shown to be useful in prognostication of patients with CHF. Initially, numerous single- centre and retrospective studies have demonstrated the prognostic value of cardiac 123I-mIBG imaging in CHF patients.19–23 The ADMIRE-HF study was the first large multicentre, prospective study in CHF (n = 961), using a predefined late H/M ratio cut-off value of 1.6.5 The late H/M ratio was, independent of brain natriuretic peptide (BNP) and left ventricular ejection fraction (LVEF), a predictor of the composite endpoint and of each individual component of the com- posite endpoint: progression of CHF, lethal ventricular tachycardia (VT), and sudden cardiac death (SCD). A late H/M ratio >1.6 was associated with incidence of 1% cardiac death per year. In contrast, the annual cardiac mortality in those patients with a late H/M ratio <1.2 was almost 10 times higher (9.6%). Specific interest has been given to the evaluation of cardiac sympa- thetic activity in patients eligible for cardiac resynchronization ther- apy (CRT), showing that the degree of sympathetic impairment predicted the magnitude of LV reverse remodelling and that the pres- ence of sympathetic reserve (increase of late H/M ration short after CRT implantation) could individuate positive responders to CRT.31– 33 However, whether multiparametric nuclear cardiac imaging, com- prising the evaluation of perfusion, dyssynchrony (phase analysis), and innervation, may help to individuate responders to CRT is still a matter of investigation10,34 (Figure 1). Myocardial innervation imaging in patients with CHF Those results were reproduced by pooled analyses of independ- ent studies, confirming the long-term prognostic value of 123I-mIBG imaging in patients with CHF.24,25 However, a number of limitations have prevented the implementation of planar 123I-mIBG scintigraphy in daily clinical routine. First of all as a main reason, no real cut-off of H/M ratio actually exists (i.e. <1.6).26 In fact, the H/M ratio shows a continuous association with patients risk, and the different cut-off val- ues proposed so far have only shown a modest accuracy in risk dis- criminating.25 Interestingly, in contrast to the linear correlation between late H/M ratio and prognosis, a ‘bell shaped’ curve was seen for the late H/M ratio in relation to appropriated implantable cardi- overter defibrillator (ICD) therapy. Patients with intermediate late H/ M ratio (range 1.40–2.10) were more likely to have appropriate ICD therapy compared to patients with low and high late H/M ratios. These findings are in line with previous findings of Agostini et al.19 showing arrhythmia in CHF patients with an intermediate late H/M ratio (1.46–2.17) only. This suggests that the presumption of a mono- tonic increase of arrhythmia with decreasing late H/M ratio may not be correct and suggests that CHF patients with severe increased impaired sympathetic activity probably die due to terminal HF rather than arrhythmia. Incorporating 123I-mIBG scintigraphy into the assess- ment of CHF patients eligible for ICD implantation may result in a re- duction in ICD implantation of 21%.27 According to these suggestions, the number needed to screen to prevent one ICD im- plantation would be 5. Screening with 123I-mIBG scintigraphy could reduce the costs per patient by 5500 and 13 431 US dollars over 2 and 10years, respectively, with <1 day and 2 weeks of life lost over 2 and over 2 and 10 years, respectively.27 However, to date no pro- spective evaluation of the prognostic impact of innervation parame- ters on clinical decision-making and patient management has been provided, with the recent ADMIRE-ICD (NCT02656329) study interrupted prematurely because of low recruitment rate. An additional field of interest could be the assessment of cardiac sympathetic reinnervation following heart transplantation. Myocardial innervation imaging in patients with CHF In fact, while cardiac denervation occurs immediately after a heart trans- plantation, due to axial Wallerian degeneration of post-ganglionic sympathetic nerve fibres, the extent of reinnervation if variable and inhomogeneous.35 Specifically, reinnervation usually appears first in the left ventricle followed by the sinus node region.36–38 Cardiac de- nervation explains the abnormally elevated heart rate of heart trans- plant recipients, as well as the compromised exercise capacity and altered blood pressure control. On the other hand, an incoherent reinnervation may increase the arrhythmic risk.35 Nuclear cardiac imaging has been instrumental for clarifying the pathophysiology of cardiac denervation/reinnervation after transplantation and may be possibly used for advanced risk-stratification of such patients.39 General concepts of nuclear imaging in the assessment of cardiac sympathetic activity The use of new solid-state cardiac cameras with CZT detectors, characterized by a higher photon sensitivity and spatial resolution than standard cameras, could overcome these limi- tations and allow the assessment of myocardial innervation and per- fusion in a single imaging session and with a limited radiation exposure, possibly guiding clinical decision-making.9,10 WO ¼ ðearly H=M  late H=MÞ ðearly H=MÞ    100 ................................ Despite the large number of studies on 123I-mIBG assessed cardiac sympathetic activity in CHF, the lack of standardization in both acqui- sition and semi-quantitative analysis between different institutions may be a significant factor limiting wide scale clinical implementation. Hence, technical considerations are important for standardized imag- ing with 123I-mIBG. This particularly includes the choice of collimator. In addition to the main photopeak of 159keV, 123I emits also high- energy photons. These high-energy photons may penetrate Recent data9,10 indicated that CZT-derived measures of myocar- dial innervation heterogeneity (i.e. 123I-mIBG defect score and innerv- ation/perfusion mismatch score) could better identify, if compared with the classical planar indexes, patients at higher cardiovascular risk. 482 A. Gimelli et al. Myocardial innervation imaging as predictor of ventricular arrhythmias The evaluation of the integrity of cardiac sympathetic with 123I-mIBG scintigraphy has been long proposed as a valuable method for the stratification of patients at risk for ventricular arrhythmias (VA).5 However, despite the value of planar 123I-mIBG assessed myocardial innervation for prediction of adverse outcome in CHF, no dedicated randomized study has shown that outcome in 123I-mIBG-defined cat- egories of patients at risk can be improved by a preventive interven- tion, greatly in limiting the implementation of this technique in daily clinical routine. Also, following technological improvements of nu- clear cardiac imaging, the interest in 123I-mIBG scintigraphy has been recently increasing, with reports showing its possible use in innova- tive clinical scenarios.40 Specifically, the application of SPECT technol- ogy to 123I-mIBG imaging has been instrumental in demonstrating the SPECT 123I-mIBG imaging has been proposed as a possible solution to some of these limitations, providing information on the regional heterogeneity of cardiac sympathetic activity that could improve the diagnostic abilities of planar measures in identifying patients at risk of 483 Myocardial innervation imaging Figure 1 A patient with history of diabetes and recent onset of rest dyspnoea. Normal coronary anatomy. One episode of VT treated with DC shock. The scintigraphic perfusion images show a homogenous perfusion. The innervation images (lower rows) reveal an extensive area of denerv- ation involving the basal to mid-ventricular lateral and the basal inferior walls with relevant innervation/perfusion mismatch. At EP study located the sites of origin of the arrhythmia at the level of the infero-lateral LV walls. Figure 1 A patient with history of diabetes and recent onset of rest dyspnoea. Normal coronary anatomy. One episode of VT treated with D shock. The scintigraphic perfusion images show a homogenous perfusion. The innervation images (lower rows) reveal an extensive area of denerv ation involving the basal to mid-ventricular lateral and the basal inferior walls with relevant innervation/perfusion mismatch. At EP study located th Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Figure 1 A patient with history of diabetes and recent onset of rest dyspnoea. Normal coronary anatomy. One episode of VT treated with DC shock. The scintigraphic perfusion images show a homogenous perfusion. Myocardial innervation imaging as predictor of ventricular arrhythmias The innervation images (lower rows) reveal an extensive area of denerv- ation involving the basal to mid-ventricular lateral and the basal inferior walls with relevant innervation/perfusion mismatch. At EP study located the sites of origin of the arrhythmia at the level of the infero-lateral LV walls. causal link between increased regional 123I-mIBG heterogeneity and arrhythmic risk. It is now well accepted that patients with the highest arrhythmic risk, which could possibly benefit from an ICD implant- ation, are not necessarily those with a homogeneously depressed cardiac sympathetic activity (i.e. severely reduced H/M ratio), but ra- ther patients showing a regionally jeopardized 123I-mIBG uptake that may ultimately predispose to cardiac electrical instability.41 In add- ition to that, consistent evidence suggests that denervated regions having relatively preserved perfusion (the so-called innervation/ perfusion mismatch phenomenon) can represent a specific marker of arrhythmic susceptibility, particularly in patients with IHD.16 Accordingly, the presence of areas of viable but denervated myocar- dium located nearby scarred regions has been identified as a likely source of VA and, as a consequence, an ideal site for targeted thera- peutic intervention, such as endocavitary ablations42 (Figure 2). setting, after initial validation hybrid PET/MR evaluation, combining the high-quality molecular imaging of PET with the insuperable tissue characterization of MR,44,45 has been shown to further increase the clinical value of molecular cardiac imaging, allowing a better charac- terization of patients at risk for VA46,47 possibly candidate to invasive procedures (i.e. cardiac ablations). Myocardial innervation imaging in patients with atrial fibrillation ................................ Atrial fibrillation (AF) occurs when structural and/or electrophysio- logical abnormalities alter atrial tissue to promote abnormal impulse formation and/or propagation. A focal source in the pulmonary veins (PVs) can trigger AF, and for this reason, pulmonary vein isolation (PVI) represents the cornerstone technique for treating patients with AF resistant to standard pharmacological therapies.48 However, the success rate of the first AF ablation remains only  60% at 1 year.49 Also, on PET, the presence of innervation/perfusion mismatch resulted the major predictor of VA, typically co-localizing at the level of the electrically unstable border-zone of a myocardial scar.43 Current data may support myocardial innervation imaging for the stratification of patients at risk for VA, showing a causal role of in- nervation/perfusion mismatch in cardiac arrhythmogenicity. In this More recently, interest has turned to the role of left atrium (LA) innervation in the modulation of AF substrate. As a matter of fact, ex- perimental and clinical studies have shown that the intrinsic cardiac 484 A. Gimelli et al. Figure 2 A patient with ischaemic heart disease and a previous myocardial infarction involving the circumflex coronary artery. 99mTc- Tetrofosmine perfusion images (upper rows) show a mainly inferior-to-inferolateral myocardial scar. 123I-MIBG images (lower rows) show an evident rea of sympathetic denervation at the level of the necrotic myocardium (innervation/perfusion match) and another ample region of impaired sympa- hetic tone at the level of the anterior, anteroseptal-to-apical LV segments despite a preserved perfusion (innervation/perfusion mismatch). Phase nalysis demonstrates the presence of significant LVD with the regions of more delayed activation localized at the level of the mismatched myocardial egions. LVD: left ventricular dyssynchrony. Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Figure 2 A patient with ischaemic heart disease and a previous myocardial infarction involving the circumflex coronary artery. 99mTc- Tetrofosmine perfusion images (upper rows) show a mainly inferior-to-inferolateral myocardial scar. 123I-MIBG images (lower rows) show an evident area of sympathetic denervation at the level of the necrotic myocardium (innervation/perfusion match) and another ample region of impaired sympa- thetic tone at the level of the anterior, anteroseptal-to-apical LV segments despite a preserved perfusion (innervation/perfusion mismatch). Phase analysis demonstrates the presence of significant LVD with the regions of more delayed activation localized at the level of the mismatched myocardial regions. LVD: left ventricular dyssynchrony. outcomes by several investigators51,52 and the presence of residual GP activity after PVI might predict likelihood of recurrence.53 The standard approach is to apply high-frequency stimulation (HFS) to the presumed GP areas to induce their typical vagal reaction (i.e. elicit AV block). As HFS has low specificity and sensitivity, is inva- sive and time consuming, better methods for localization of GPs are needed. Myocardial innervation imaging in diabetic patients Diabetes mellitus (DM) is a worldwide healthcare problem that is largely the result of overweight and physical inactivity.73 Long-term complications include microvascular- and macrovascular disease and also cardiac autonomic neuropathy (CAN), a complication that is proportional to the duration and magnitude of hyperglycaemia.74 CAN results from injury of the autonomic nerve fibres innervating the heart and blood vessels, causing abnormalities in heart rate con- trol and vascular dynamics. Major clinical manifestations of CAN are orthostatic hypotension, impaired heart rate control, and silent myo- cardial ischaemia, ultimately leading to increased arrhythmia suscepti- bility.75,76 CAN has been classically demonstrated non-invasively by assessing heart rate variability (HRV) and baroreflex sensitivity.75 In particular, HRV can be tested simply by recording a Holter ECG registry, evaluating the sympatico-vagal balance at the sinoatrial level, and by measuring the difference in systolic blood pressure between supine and upright position. Despite its prognostic value, the evalu- ation of CAN by means of HRV is limited by its indirect nature and the fact that it evaluates predominantly parasympathetic function, mostly disregarding sympathetic contribution.76 According Stirrup et al.59 reported an initial experience of left- atrial sympathetic innervation imaging with a solid-state SPECT. After feasibility assessment, they described methods for image acquisition and interpretation and thereafter present pilot data in patients with AF, with assessment of inter- and intraobserver variability, inter- study variability in sequential. Despite this pilot study comprised small numbers, it underlines that GP size and distribution are highly individ- ual and a simple anatomical approach to GP ablation may not achieve complete results. The use of an individual GP localization by the use of might allow safer and more time-efficient GP ablation. Myocardial innervation imaging in patients with amyloidosis Cardiac amyloidosis (CA) is a form of restrictive cardiomyopathy commonly resulting from deposition of misfolded immunoglobulin light chain (AL) or transthyretin (ATTR) protein. ATTR has gained increasing attention in recent years and can be divided into a heredi- tary type (ATTRv) and a wild-type (ATTRwt). CA is an underesti- mated cause of heart failure and potential risk on arrhythmia, due to amyloid infiltration of the nerve conduction system and the myocar- dial tissue,60,61 most commonly in ATTR CA, particularly ATTRv.62,63 The prognosis in this progressive disease is poor, probably due to the development of arrhythmias. While AL CA patients less commonly manifest autonomic dysfunction, it may develop as a complication of AL amyloidosis treatment.64 Since the 90’s of the former century cardiac 123I-mIBG scintigraphy has been used to assess diabetic CAN, generally diagnosing the pres- ence of a reduced H/M ratio. It has been suggested that 123I-mIBG scintigraphy may be more sensitive than HRV for the detection of CAN in patients with DM77,78 and a better predictor for major car- diac events, likely showing more precocious alterations of the auto- nomic tone. As in the case of HRV, also the H/M ratio provides only a measure of global myocardial (dys)innervation, that may be ‘paradoxically’ pre- served even in the presence of regional abnormalities. In this setting, SPECT imaging is able to look at regional innervation, demonstrating that the inferior wall and apex of the LV are more prone to show impaired 123I-mIBG uptake,76 a condition that might revert after sus- tained glycaemic control.79 Early detection of myocardial innervation disturbances has be- come of major clinical interest, because its occurrence and severity limit the choice of treatment. 123I-mIBG plays an important role in evaluation of myocardial innervation in CA.63,65–67 However, 123I- mIBG scintigraphy is not able to discriminate between CA subtypes nor differentiate CA from other forms of cardiomyopathy.65,68 In TTR mutation carriers, cardiac sympathetic denervation evidenced by decreased 123I-mIBG uptake is detected earlier than amyloid bur- den evidenced by bone scintigraphy.69 These results raise the possi- bility of a diagnostic role for 123I-mIBG scintigraphy at an early stage of cardiac involvement in TTR-mutated carriers, in addition to its well-established prognostic value. Myocardial innervation imaging in patients with atrial fibrillation More recently, it has been proposed that the non-invasive evalu- ation of LA innervation might identify the GPs areas as discrete up- take areas (DUA) on the LA epicardial surface, potentially guiding catheter ablation procedures. Myocardial innervation imaging in patients with atrial fibrillation outcomes by several investigators51,52 and the presence of residual GP activity after PVI might predict likelihood of recurrence.53 The standard approach is to apply high-frequency stimulation (HFS) to the presumed GP areas to induce their typical vagal reaction (i.e. elicit AV block). As HFS has low specificity and sensitivity, is inva- sive and time consuming, better methods for localization of GPs are needed. autonomic nervous system plays an important role in the initiation and maintenance of AF. In fat pads on the atrial epicardium, large ‘nests’ of nerve cell bodies form the so-called ganglionated plexi (GP) containing both sympa- thetic and parasympathetic nerves. The GP can measure from 5 to 10mm.50 Catheter ablation of GPs as an add-on to PVI or even in iso- lation in patients with AF has been reported to improve clinical 485 Myocardial innervation imaging 123I-mIBG imaging has been performed to assess patients with AF, classically by imaging the indirect deleterious effect of AF on the firing activity of LV sympathetic nervous terminals, showing the ability to prognosticate patients with paroxysmal AF,54,55 predict the outcome of PVI,56,57 and evaluate the patterns of denervation/reinnervation after PVI.57,58 In patients with paroxysmal AF and without structural heart disease, cardiac autonomic nervous system abnormality defined as reduced late H/M ratio was a predictor of vascular events (i.e. myocardial infarction, stroke, or heart failure) during a mean follow- up of 4.5years. Therefore, 123I-mIBG imaging in this group of patients could support clinical risk stratification.54 However, more data of multicentre studies are need to implement this strategy for clinical purpose. An overview of the imaging acquisition parameters and reporting for 123I-mIBG is available in recently published expert consensus document.70 The potential role of PET for myocardial innervation in amyloidosis has not yet been identified. Prospective studies evaluating the diagnostic and prognostic value of 123I-mIBG assessed myocardial innervation in CA should be undertaken. Current treatments are tar- geted at reducing the production of or stabilization of the precursor protein of amyloid deposits, thereby aim to stop or slow down fur- ther accumulation of amyloid.71,72 Molecular imaging, such as 123I- mIBG, may be able to visualize regression of CA infiltration of the nerve conduction system under these new treatment regimens, but data are lacking at this moment. Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 PET imaging has sufficient spatiotemporal resolution and well- validated attenuation correction that allows for quantification of glo- bal and regional abnormalities in cardiac sympathetic activity based on tracer activity concentrations (Bq/mL) in the myocardium and blood pool in dynamic datasets.93 Characterization of 11C-meta- hydroxyephedrine ([11C]-mHED) kinetics is usually performed with quantification of the tracer retention index that is defined as the ratio of the activity in the myocardium in the late uptake image of a 40- or 60-min dynamic scan to the integral of the image-derived arterial blood time-activity curve corrected for radiolabelled metabolites.93 Quantification of volume of distribution in myocardial tissue based on compartment models can also be performed, but robustness of compartmental modelling may be impaired by rapid kinetics of [11C]- mHED.1,2,93,94 In patients with stable heart failure and reduced ejection fraction, test–retest repeatability of global [11C]-mHED re- tention index was 30% and regional defects (percentage of the left ventricle myocardium with reduced [11C]-mHED uptake) 12%.2,94 Recently, nuclear studies were conducted at different phases dur- ing the TTC time course.86,87 In the acute and subacute phases of TTC, similar defects of 123I-mIBG and 18F-Fluorodeoxyglucose uptake, despite only slightly reduced perfusion, have been demon- strated in the dyskinetic LV segments. Subsequently, rapid normaliza- tion of myocardial perfusion, and delayed recovery of both LV glucose metabolism and sympathetic innervation, is observed. In a prospective study, late? H/M ratio was compared in subacute and re- covery phases of patients with TTC, showing a dysfunctional sympa- thetic activity in the early phase that resolved with recovery.88 The observed decreased cardiac 123I-mIBG uptake as expressed as H/M ratio could be due to inhibited 123I-mIBG reuptake by high epineph- rine levels in the synaptic cleft and/or down-regulation of the human NE transport. Moreover, in a study based on an animal model by Paur et al.89 proposed the hypothesis that the apical ballooning in TTC is related to switching of epinephrine signalling. Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 The data showed that there is an increasing gradient of from base to apex in rat and exposed to high levels of circulating epinephrine beta-2- receptors signalling switch from positively inotropic beta-1-mediated Gs stimulation (Gs) coupling to negative inotropic b2-dependent G- inhibitor (Gi) coupling in the apex, resulting in apical ballooning.84,89 This hypothesis is based on the assumption that beta-2-receptors are more expressed in the apical region than the basal area, even if, it is unclear whether the human heart has a similar pattern of beta-2- receptors gradient. In addition to [11C]-mHED, other 11C labelled PET tracers, such as [11C]-epinephrine and [11C]-phenylephrine are available.3,93,95 These tracers show differences to [11C]-mHED in their affinity to intra- neuronal vesicular storage and metabolism. Combination of tracers and pharmacologic challenge may provide mechanistic information about differential effects of disease on anatomical nerve density as well as functional changes in cellular catecholamine uptake, storage, and metabolism.95,96 A limitation of tracers labelled with 11C is that due to short half-life an onsite cyclotron is required for production, which has limited their widespread use in the clinical setting. Tracers labelled with the posi- tron emitter 18F have been introduced recently. [18F]-meta-fluoro- benzylguanidine ([18F]-LMI1195) has similar kinetics to 123I-mIBG and was well tolerated in a phase 1 clinical trial.97,98 Yet, approaches for absolute quantification for this tracer have not yet been widely established. Imaging properties of two other tracers, 18F-meta- hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphene- thylguanidine, have been studied in healthy volunteers.99 Both tracers provided myocardial images of high quality and, owing to slower up- take kinetics when compared with 11C labelled NE analogues, they offer the possibility for reproducible quantification of intraneuronal retention by Patlak kinetic analysis. Myocardial innervation imaging in patients with amyloidosis Accordingly, since 123I-mIBG scintigraphy appears the most accur- ate technique for the detection of CAN, it might be used for the early risk stratification of DM patients, guiding intensive risk factors man- agement even before the onset of micro- and macrovascular compli- cations, and evaluating the effect of novel anti-diabetic drugs, such as SGLT2 inhibitors.80 486 A. Gimelli et al. ......................................................................... Myocardial innervation imaging in Takotsubo Myocardial innervation imaging in Takotsubo mIBG uptake is associated with a subsequent increased risk for the wearing-off phenomenon.91 mIBG uptake is associated with a subsequent increased risk for the wearing-off phenomenon.91 Innovations in the field of myocardial innervation imaging: the role of quantification Takotsubo cardiomyopathy (TTC) is a unique cardiac syndrome characterized by the presence of transient left ventricular wall mo- tion abnormalities without a culprit coronary lesion.81–83 In all cases, a massive catecholamine release is observed.84 The precise patho- physiological mechanism of TTC has not been completely elucidated. Emotional, psychological, or physical stress is frequently, but not al- ways present prior to the onset of TTC and may thus trigger the onset of disease.85 It has been suggested that epinephrine-mediated myocardial stunning in TTC is related to multiple coronary artery spasm and impaired coronary microcirculation. Furthermore, epi- nephrine seems to be an important factor in the pathophysiology. In the acute phase of TCC plasma epinephrine levels are more elevated compared with the acute phase of a myocardial infarction.84 Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 20 Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 Future perspectives to myocardial innervation imaging and its appropriate use. Although cardiac 123I-mIBG scintigraphy has been shown to be useful in prog- nostication of patients with CHF, limitations in tracer’s availability and crude quantification methods has discouraged widespread clinical utility. However, the new CZT cameras for 123I-mIBG on one hand and the new 18F-labelled PET tracers on the other, would take advan- tages of the improved spatial resolution, quantification capabilities, and potential wide distribution. Improving standardization and valid- ation of myocardial innervation imaging is needed and will help to im- prove the quality of myocardial imaging and to facilitate appropriate use. Before this non-invasive imaging modality can become a part of clinical practice, larger prospective, and randomized control trials are required to confirm whether myocardial innervation imaging strat- egies can guide clinical management that can improve clinical out- comes, and constrain medical costs. The consistent improvement that has been taking place in nuclear cardiac imaging in the last years, comprising innovations in both soft- ware and hardware as well as the introduction of novel radiotracers, has allowed a gradual revival of cardiac sympathetic innervation imag- ing, with possible novel fields of applications. Specifically, the combined evaluation of cardiac sympathetic in- nervation and perfusion in the same imaging session allows the as- sessment of cardiac innervation/perfusion mismatch, a parameter of increased prognostic relevance that has proven valuable for the char- acterization of patients with malignant ventricular arrhythmias candi- date to ablation procedures. In this setting, dedicated cardiac CZT cameras, with their increased sensitivity and resolution, have represented a quantum leap in con- ventional nuclear cardiology, allowing to couple a significant com- pression of imaging times together with the possibility to perform dynamic acquisitions, and hence the absolute quantification of data. Liability statement On the other hand, the recent introduction of 18-Fr-labelled PET radiotracers for sympathetic innervation imaging has the potential to increase the clinical application of this techniques, whose image qual- ity and overall accuracy is still unsurpassed. This Expert Document summarizes the views of the EACVI and the EANM. It reflects views for which the EACVI and the EANM cannot be held responsible. This position paper should be taken into context of good practice of nuclear medicine and do not substitute for na- tional and international legal or regulatory provisions. The possible field of application of cardiac sympathetic innervation imaging with either conventional nuclear imaging (planar scintigraphy and SPECT) or PET is reported in Table 1. As shown, while the use of cardiac innervation imaging has been classically limited to patient with HF and severely depressed EF, novel clinical scenarios have been re- cently added, comprising both the characterization of patients with malignant VAs, and the risk stratification of patients with innate or acquired cardiomyopathies, in whom the evaluation of cardiac sym- pathetic innervation may have both diagnostic (i.e. amyloidosis) and prognostic values. Conflict of interest: D.A.: Consultancy for Spectrum Dynamics. F.B.: Speaker honoraria by Pfizer, Alnylam, Abbott, and GE Healthcare, and Research grants by Siemens Healthineers. S.E.: Consultant for Biosense Webster, Stereotaxis Inc., and Spectrum Dynamics. Grants from Catheter Precision, Spectrum Dynamics, and Baylis Medical. A.S.: Speaker honoraria by Abbott, Amgen, Astra Zeneca, and Bayer and fees for consultancy from Amgen and Astra Zeneca. A.J.H.A.S.: Speaker honoraria by GE Healthcare. Myocardial innervation imaging in neurodegenerative diseases Increasing attention has been given recently on the use of myocardial innervation imaging in the clinical characterization and differential diagnosis of major neurodegenerative conditions. In particular, myo- cardial 123I-mIBG uptake is reduced in patients with Lewy body dis- eases such as Parkinson disease (PD) and dementia with Lewy bodies,90,91 helping to differentiate these conditions to other types of parkinsonism.92 Specifically, in patients with PD, 123I-mIBG scintig- raphy may play a central role in the functional characterization of patients, since a relatively preserved 123I-mIBG uptake identifies patients with a small motor burden and a reduced myocardial 123I- The introduction of cardiac dedicated SPECT devices equipped with cadmium-zinc-telluride (CZT) detectors has allowed evaluation of regional myocardial 123I-mIBG kinetics in dynamic 3D data- sets.100,101 Furthermore, improved spatial resolution of CZT SPECT scanners may facilitate evaluation of regional 123I-mIBG uptake in combination with myocardial perfusion.9,16 Yet, this approach requires further validation and cross-comparison of clinical and prog- nostic value to the current standard of semi-quantitative heart-medi- astinum ratios from planar scans. 487 Myocardial innervation imaging .................................................................................................................................................................................................................... Table 1 Impact of Planar and SPECT 123 mIBG images in the evaluation of different clinical scenarios Clinical scenarios for 123I-MIBG imaging Planar imaging SPECT HF and severely depressed LV ejection fraction (<30–35%): advanced risk stratification 111 1 Hypertrophic and infiltrative cardiomyopathies: evaluation of the arrhythmic risk 11 1/- Ventricular arrhythmias candidate to trans-catheter ablation: identification of the ablation targets 2 11 Neurodegenerative disorders: differential diagnosis between Lewy body diseases (i.e. Parkinson disease) and parkinsonisms 11 2 Cardiac channellopaties: stratification of the arrhythmic risk 1 2 Atrial fibrillation before transcatheter ablation: identification of the autonomic ganglia 2 1 Heart transplantation: monitoring reinnervation 1/2 1/2 HF and borderline depressed LV ejection fraction (35–40%): indication for ICD implantation 1/2 2 Diabetes mellitus: long-term risk stratification 1/2 2 References 21. Nakata T, Miyamoto K, Doi A et al. Cardiac death prediction and impaired car- diac sympathetic innervation assessed by MIBG in patients with failing and non- failing hearts. J Nucl Cardiol 1998;5:579–90. 1. Flotats A, Carrio´ I, Agostini D, Le Guludec D, Marcassa C, Schaffers M et al. Proposal for standardization of 123I-metaiodobenzylguanidine (MIBG) cardiac sympathetic imaging by the EANM Cardiovascular Committee and the European Council of Nuclear Cardiology. Eur J Nucl Med Mol Imaging 2010;37: 1802–12. g J 22. Merlet P, Pouillart F, Dubois-Rande´ JL, Delahaye N, Fumey R, Castaigne A et al. Sympathetic nerve alterations assessed with 123I-MIBG in the failing human heart. J Nucl Med 1999;40:224–31. 23. Cohen-Solal A, Esanu Y, Logeart D, Pessione F, Dubois C, Dreyfus G et al. Cardiac metaiodobenzylguanidine uptake in patients with moderate chronic heart failure: relationship with peak oxygen uptake and prognosis. J Am Coll Cardiol 1999;33:759–66. 2. Verberne HJ, Habraken JB, van Eck-Smit BL, Agostini D, Jacobson AF. Variations in 123I-metaiodobenzylguanidine (MIBG) late heart mediastinal ratios in chronic heart failure: a need for standardisation and validation. Eur J Nucl Med Mol Imaging 2008;35:547–53. 24. Nakata T, Nakajima K, Yamashina S, Yamada T, Momose M, Kasama S et al. A pooled analysis of multicenter cohort studies of (123)I-mIBG imaging of sym- pathetic innervation for assessment of long-term prognosis in heart failure. JACC Cardiovasc Imaging 2013;6:772–84. 3. Nakajima K, Verschure DO, Okuda K, Verberne HJ. Standardization of (123)I- meta-iodobenzylguanidine myocardial sympathetic activity imaging: phantom calibration and clinical applications. Clin Transl Imaging 2017;5:255–63. 4. Verschure DO, Poel E, Nakajima K, Okuda K, van Eck-Smit BLF, Somsen GA et al. A European myocardial (123)I-mIBG cross-calibration phantom study. J Nucl Cardiol 2018;25:1191–7. 25. Verschure DO, Veltman CE, Manrique A, Somsen GA, Koutelou M, Katsikis A et al. For what endpoint does myocardial 123I-MIBG scintigraphy have the greatest prognostic value in patients with chronic heart failure? Results of a pooled individual patient data meta-analysis. Eur Heart J Cardiovasc Imaging 2014; 15:996–1003. 5. Jacobson AF, Senior R, Cerqueira MD, Wong ND, Thomas GS, Lopez VA et al. Myocardial iodine-123 meta-iodobenzylguanidine imaging and cardiac events in heart failure. Results of the prospective ADMIRE-HF (AdreView Myocardial Imaging for Risk Evaluation in Heart Failure) study. J Am Coll Cardiol 2010;55: 2212–21. 26. Liga R, Scholte A. Neuro-cardiac imaging has a proven value in patient manage- ment: Con. J Nucl Cardiol 2017;24:1583–1587. 27. References O’Day K, Levy WC, Johnson M, Jacobson AF. Cost-effectiveness analysis of iodine-123 meta-iodobenzylguanidine imaging for screening heart failure patients eligible for an implantable cardioverter defibrillator in the USA. Appl Health Econ Health Policy 2016;14:361–73. 6. Veltman CE, Boogers MJ, Meinardi JE, Al Younis I, Dibbets-Schneider P, Van der Wall EE et al. Reproducibility of planar (123)I-meta-iodobenzylguanidine (MIBG) myocardial scintigraphy in patients with heart failure. Eur J Nucl Med Mol Imaging 2012;39:1599–608. 28. Nakajima K, Nakata T, Yamada T, Yamashina S, Momose M, Kasama S et al. A prediction model for 5-year cardiac mortality in patients with chronic heart failure using (1)(2)(3). Eur J Nucl Med Mol Imaging 2014;41:1673–82. 7. Marini C, Giorgetti A, Gimelli A, Kusch A, Sereni N, L’Abbate A et al. Extension of myocardial necrosis differently affects MIBG retention in heart failure caused by ischaemic heart disease or by dilated cardiomyopathy. Eur J Nucl Med Mol Imaging 2005;32:682–8. 29. Kawasaki M, Yamada T, Morita T et al. Prognostic value of cardiac metaiodo- benzylguanidine imaging and QRS duration in implantable cardioverter defibril- lator patients with and without heart failure. Int J Cardiol 2019;296:164–171. 8. Bax JJ, Kraft O, Buxton AE, Fjeld JG, ParI´zek P, Agostini D et al. 123 I-mIBG scintigraphy to predict inducibility of ventricular arrhythmias on cardiac electro- physiology testing: a prospective multicenter pilot study. Circ Cardiovasc Imaging 2008;1:131–40. 30. Køber L, Thune JJ, Nielsen JC, Haarbo J, Videbæk L, Korup E et al. Defibrillator implantation in patients with nonischemic systolic heart failure. N Engl J Med 2016;375:1221–30. 9. Gimelli A, Liga R, Giorgetti A, Genovesi D, Marzullo P. Assessment of myocar- dial adrenergic innervation with a solid-state dedicated cardiac cadmium-zinc- telluride camera: first clinical experience. Eur Heart J Cardiovasc Imaging 2014;15: 575–85. 31. Moreira RI, Abreu A, Portugal G, Oliveira L, Oliveira M, Rodrigues I et al. Prognostic effect and modulation of cardiac sympathetic function in heart failure patients treated with cardiac resynchronization therapy. J Nucl Cardiol 2020;27: 283–290. 10. Gimelli A, Liga R, Genovesi D, Giorgetti A, Kusch A, Marzullo P. Association between left ventricular regional sympathetic denervation and mechanical dys- synchrony in phase analysis: a cardiac CZT study. Eur J Nucl Med Mol Imaging 2014;41:946–55. 32. Curcio A, Cascini GL, De Rosa S, Pasceri E, Veneziano C, Cipullo S et al. (123)I-mIBG imaging predicts functional improvement and clinical outcome in patients with heart failure and CRT implantation. Int J Cardiol 2016;207:107–9. 33. References Cha Y-M, Chareonthaitawee P, Dong Y-X, Kemp BJ, Oh JK, Miyazaki C et al. Cardiac sympathetic reserve and response to cardiac resynchronization ther- apy. Circ Heart Fail 2011;4:339–44. 11. Gimelli A, Liga R, Avogliero F, Coceani M, Marzullo P. Relationships between left ventricular sympathetic innervation and diastolic dysfunction: the role of myocardial innervation/perfusion mismatch. J Nucl Cardiol 2018;25:1101–9. doi: 10.1007/s12350-016-0753-3. 34. Gimelli A, Liga R, Menichetti F, Soldati E, Bongiorni MG, Marzullo P. Interactions between myocardial sympathetic denervation and left ventricular mechanical dyssynchrony: a CZT analysis. J Nucl Cardiol 2019;26:509–518. 12. Bravo PE, Lautama¨ki R, Carter D, Holt DP, Nekolla SG, Dannals RF et al. Mechanistic insights into sympathetic neuronal regeneration: multitracer mo- lecular imaging of catecholamine handling after cardiac transplantation. Circ Cardiovasc Imaging 2015;8:e003507. 35. Awad M, Czer LS, Hou M et al. Early denervation and later reinnervation of th heart following cardiac transplantation: a review. J Am Heart Assoc 2016;5. 35. Awad M, Czer LS, Hou M et al. Early denervation and later reinnervation of t heart following cardiac transplantation: a review. J Am Heart Assoc 2016;5. 36. Bengel FM, Ueberfuhr P, Ziegler SI, Nekolla S, Reichart B, Schwaiger M. Serial assessment of sympathetic reinnervation after orthotopic heart transplantation. A longitudinal study using PET and C-11 hydroxyephedrine. Circulation 1999;99: 1866–71. 13. Gaemperli O, Liga R, Spyrou N, Rosen SD, Foale R, Kooner JS et al. Myocardial beta-adrenoceptor down-regulation early after infarction is associated with long-term incidence of congestive heart failure. Eur Heart J 2010;31:1722–9. 14. Liga R, Gimelli A, Marzullo P, Ambrosio G, Cameli M, Cerbai E et al.; On Behalf of Societa` Italiana di Cardiologia, Sezione Regionale Tosco-Umbra. Myocardial (123)I-metaiodobenzylguanidine imaging in hypertension and left ventricular hypertrophy. J Nucl Cardiol 2018;25:461–70. 37. Bengel F, Ueberfuhr P, Karja J, Schreiber K, Nekolla S, Reichart B et al. Sympathetic reinnervation, exercise performance and effects of beta-adrenergic blockade in cardiac transplant recipients. Eur Heart J 2004;25:1726–33. 38. Di Carli MF, Tobes MC, Mangner T, Levine AB, Muzik O, Chakroborty P et al. Effects of cardiac sympathetic innervation on coronary blood flow. N Engl J Med 1997;336:1208–15. 15. Boogers MJ, Borleffs CJW, Henneman MM, van Bommel RJ, van Ramshorst J, Boersma E et al. Cardiac sympathetic denervation assessed with 123-iodine metaiodobenzylguanidine imaging predicts ventricular arrhythmias in implant- able cardioverter-defibrillator patients. J Am Coll Cardiol 2010;55:2769–77. 39. Bengel FM, Ueberfuhr P, Hesse T, Schiepel N, Ziegler SI, Scholz S et al. Conclusions This Expert Document was brought to the attention of the relevant EANM Committees and the National Societies of Nuclear Medicine, the EACVI, and the ESC CPG bodies. The comments and suggestions from the EANM Committees, EANM National Societies, the EACVI, and the ESC CPG bodies for their review and contribution are highly appreciated and have been considered for this Expert Document. The interpretation of myocardial innervation imaging requires the knowledge of different topics: neuronal biology, tracer kinetics, and the specific disease state. The possibility to help in understanding the pathophysiology of the different disease could add a new dimension 488 A. Gimelli et al. References Vontobel J, Liga R, Possner M, Clerc OF, Mikulicic F, Veit-Haibach P et al. MR- based attenuation correction for cardiac FDG PET on a hybrid PET/MRI scan- ner: comparison with standard CT attenuation correction. Eur J Nucl Med Mol Imaging 2015;42:1574–80. 66. Nakata T, Shimamoto K, Yonekura S, Kobayashi N, Sugiyama T, Imai K et al. Cardiac sympathetic denervation in transthyretin-related familial amyloidotic polyneuropathy: detection with iodine-123-MIBG. J Nucl Med 1995;36:1040–2. 45. Nensa F, Bamberg F, Rischpler C, Menezes L, Poeppel TD, la Fouge`re C et al.; on behalf of the European Society of Cardiovascular Radiology (ESCR). Hybrid cardiac imaging using PET/MRI: a joint position statement by the European Society of Cardiovascular Radiology (ESCR) and the European Association of Nuclear Medicine (EANM). Eur Radiol 2018;28:4086–4101. uropathy: detection with iodine-123-MIBG. J Nucl Med 1995;36:1040–2 67. Tanaka M, Hongo M, Kinoshita O, Takabayashi Y, Fujii T, Yazaki Y et al. Iodine- 123 metaiodobenzylguanidine scintigraphic assessment of myocardial sympa- thetic innervation in patients with familial amyloid polyneuropathy. J Am Coll Cardiol 1997;29:168–74. Nuclear Medicine (EANM). Eur Radiol 2018;28:4086–4101. 46. Palmisano A, Vignale D, Peretto G et al. Hybrid FDG-PET/MR or FDG-PET/CT to detect disease activity in patients with persisting arrhythmias after myocardi- tis. JACC Cardiovasc Imaging 2021;14,288–92. 68. Jonker DL, Hazenberg BPC, Nienhuis HLA, Slart R, Glaudemans A, Noordzij W. Imaging cardiac innervation in hereditary transthyretin (ATTRm) amyloid- osis: a marker for neuropathy or cardiomyopathy in case of heart failure? J Nucl Cardiol 2020;27:1774–84. 47. Wisenberg G, Thiessen JD, Pavlovsky W, Butler J, Wilk B, Prato FS. Same day comparison of PET/CT and PET/MR in patients with cardiac sarcoidosis. J Nucl Cardiol 2020;27:2118–129. 69. Piekarski E, Chequer R, Algalarrondo V, Eliahou L, Mahida B, Vigne J et al. Cardiac denervation evidenced by MIBG occurs earlier than amyloid deposits detection by diphosphonate scintigraphy in TTR mutation carriers. Eur J Nucl Med Mol Imaging 2018;45:1108–1118. 48. Wazni OM, Marrouche NF, Martin DO, Verma A, Bhargava M, Saliba W et al. Radiofrequency ablation vs antiarrhythmic drugs as first-line treatment of symp- tomatic atrial fibrillation: a randomized trial. JAMA 2005;293:2634–40. Med Mol Imaging 2018;45:1108–1118. 70. Dorbala S, Ando Y, Bokhari S, Dispenzieri A, Falk RH, Ferrari VA, et al. ASNC/ AHA/ASE/EANM/HFSA/ISA/SCMR/SNMMI expert consensus recommenda- tions for multimodality imaging in cardiac amyloidosis: part 2 of 2-Diagnostic criteria and appropriate utilization. J Nucl Cardiol 2019. 49. References Fink T, Schlu¨ter M, Heeger C-H, Lemes C, Maurer T, Reissmann B et al. Stand- alone pulmonary vein isolation versus pulmonary vein isolation with additional substrate modification as index ablation procedures in patients with persistent and long-standing persistent atrial fibrillation: the randomized Alster-Lost-AF Trial (Ablation at St. Georg Hospital for Long-Standing Persistent Atrial Fibrillation). Circ Arrhythm Electrophysiol 2017;10:e005114. criteria and appropriate utilization. J Nucl Cardiol 2019. 71. Slart R, Glaudemans A, Noordzij W, Bijzet J, Hazenberg BPC, Nienhuis HLA. Time for new imaging and therapeutic approaches in cardiac amyloidosis. Eur J Nucl Med Mol Imaging 2019;46:1402–1406. 50. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and vari- ability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259:353–82. 72. Zhang KW, Stockerl-Goldstein KE, Lenihan DJ. Emerging therapeutics for the treatment of light chain and transthyretin amyloidosis. JACC Basic Transl Sci 2019;4:438–448. 51. Po SS, Nakagawa H, Jackman WM. Localization of left atrial ganglionated plexi in patients with atrial fibrillation. J Cardiovasc Electrophysiol 2009;20:1186–9. 73. https://www.who.int/news-room/fact-sheets/detail/diabetes. 74. Dal Canto E, Ceriello A, Ryde´n L, Ferrini M, Hansen TB, Schnell O et al. Diabetes as a cardiovascular risk factor: an overview of global trends of macro and micro vascular complications. Eur J Prev Cardiolog 2019;26:25–32. p J p y 52. Pokushalov E, Romanov A, Artyomenko S, Turov A, Shirokova N, Katritsis DG. 52. Pokushalov E, Romanov A, Artyomenko S, Turov A, Shirokova N, Katritsis DG. Left atrial ablation at the anatomic areas of ganglionated plexi for paroxysmal atrial fibrillation. Pacing Clin Electrophysiol 2010;33:1231–8. Left atrial ablation at the anatomic areas of ganglionated plexi for paroxysmal atrial fibrillation. Pacing Clin Electrophysiol 2010;33:1231–8. and micro vascular complications. Eur J Prev Cardiolog 2019;26:25–32 75. Vinik AI, Ziegler D. Diabetic cardiovascular autonomic neuropathy. Circulation 2007;115:387–97. 22/5/4 53. Kurotobi T, Shimada Y, Kino N, Ito K, Tonomura D, Yano K et al. Features of intrinsic ganglionated plexi in both atria after extensive pulmonary isolation and their clinical significance after catheter ablation in patients with atrial fibrillation. Heart Rhythm 2015;12:470–476. 76. Gerritsen J, Dekker JM, TenVoorde BJ, Kostense PJ, Heine RJ, Bouter LM et al. Impaired autonomic function is associated with increased mortality, especially in subjects with diabetes, hypertension, or a history of cardiovascular disease: the Hoorn Study. Diabetes Care 2001;24:1793–8. 54. Akutsu Y, Kaneko K, Kodama Y, Li H-L, Suyama J, Shinozuka A et al. References Clinical determinants of ventricular sympathetic reinnervation after orthotopic heart transplantation. Circulation 2002;106:831–5. 16. Gimelli A, Menichetti F, Soldati E, Liga R, Vannozzi A, Marzullo P et al. Relationships between cardiac innervation/perfusion imbalance and ventricular arrhythmias: impact on invasive electrophysiological parameters and ablation procedures. Eur J Nucl Med Mol Imaging 2016;43:2383–91. 40. Klein T, Abdulghani M, Smith M, Huang R, Asoglu R, Remo BF et al. Three-di- mensional 123I-meta-iodobenzylguanidine cardiac innervation maps to assess substrate and successful ablation sites for ventricular tachycardia: feasibility study for a novel paradigm of innervation imaging. Circ Arrhythm Electrophysiol 2015;8:583–91. 17. Gimelli A, Menichetti F, Soldati E et al. Correlation between ventricular arrhyth- mias and cardiac innervation: clinical impact on ablation procedure. Eur Heart J 2016;37:691. 41. Travin MI, Henzlova MJ, van Eck-Smit BLF, Jain D, Carrio´ I, Folks RD et al. Assessment of (123)I-mIBG and (99m)Tc-tetrofosmin single-photon emission computed tomographic images for the prediction of arrhythmic events in patients with ischemic heart failure: intermediate severity innervation defects are associated with higher arrhythmic risk. J Nucl Cardiol 2017;24:377–391. 18. Florea VG, Cohn JN. The autonomic nervous system and heart failure. Circ Res 2014;114:1815–26. 19. Agostini D, Verberne HJ, Burchert W, Knuuti J, Povinec P, Sambuceti G et al. I- 123-mIBG myocardial imaging for assessment of risk for a major cardiac event in heart failure patients: insights from a retrospective European multicenter study. Eur J Nucl Med Mol Imaging 2008;35:535–46. 42. Abdulghani M, Duell J, Smith M, Chen W, Bentzen SM, Asoglu R et al. Global and regional myocardial innervation before and after ablation of drug-refractory ventricular tachycardia assessed with 123I-MIBG. J Nucl Med 2015;56:52S–58S. 20. Merlet P, Benvenuti C, Moyse D et al. Prognostic value of MIBG imaging in idio- pathic dilated cardiomyopathy. J Nucl Med 1999;40:917–23. 489 Myocardial innervation imaging 43. Sasano T, Abraham MR, Chang K-C, Ashikaga H, Mills KJ, Holt DP et al. Abnormal sympathetic innervation of viable myocardium and the substrate of ventricular tachycardia after myocardial infarction. J Am Coll Cardiol 2008;51: 2266–75. 64. Dingli D, Tan TS, Kumar SK, Buadi FK, Dispenzieri A, Hayman SR et al. Stem cell transplantation in patients with autonomic neuropathy due to primary (AL) amyloidosis. Neurology 2010;74:913–8. 65. Noordzij W, Glaudemans AWJM, van Rheenen RWJ, Hazenberg BPC, Tio RA, Dierckx RAJO et al. I-Labelled metaiodobenzylguanidine for the evaluation of cardiac sympathetic denervation in early stage amyloidosis. Eur J Nucl Med Mol Imaging 2012;39:1609–17. 44. References Significance of cardiac sympathetic nervous system abnormality for predicting vascular events in patients with idiopathic paroxysmal atrial fibrillation. Eur J Nucl Med Mol Imaging 2010;37:742–9. the Hoorn Study. Diabetes Care 2001;24:1793–8. 77. Scholte AJHA, Schuijf JD, Delgado V, Kok JA, Bus MTJ, Maan AC et al. Cardiac autonomic neuropathy in patients with diabetes and no symptoms of coronary artery disease: comparison of 123I-metaiodobenzylguanidine myocardial scintig- raphy and heart rate variability. Eur J Nucl Med Mol Imaging 2010;37:1698–705. 12 by Unive 55. Akutsu Y, Kaneko K, Kodama Y, Li H-L, Suyama J, Shinozuka A et al. Iodine-123 mIBG Imaging for Predicting the Development of Atrial Fibrillation. JACC Cardiovasc Imaging 2011;4:78–86. raphy and heart rate variability. Eur J Nucl Med Mol Imaging 2010;37:1698–705. 78. Langer A, Freeman MR, Josse RG, Armstrong PW. Metaiodobenzylguanidine imaging in diabetes mellitus: assessment of cardiac sympathetic denervation and its relation to autonomic dysfunction and silent myocardial ischemia. J Am Coll Cardiol 1995;25:610–8. ersiteit Lei 56. Arimoto T, Tada H, Igarashi M, Sekiguchi Y, Sato A, Koyama T et al. High wash- out rate of iodine-123-metaiodobenzylguanidine imaging predicts the outcome of catheter ablation of atrial fibrillation. J Cardiovasc Electrophysiol 2011;22: 1297–304. 79. Mba CM, Nganou-Gnindjio C-N, Azabji-Kenfack M, Mfeukeu-Kuate L, Dehayem MY, Mbanya JC et al. Short term optimization of glycaemic control using insulin improves sympatho-vagal tone activities in patients with type 2 diabetes. Diabetes Res Clin Pract 2019;157:107875. 57. Wenning C, Lange PS, Schu¨lke C, Vrachimis A, Mo¨nnig G, Schober O et al. Pulmonary vein isolation in patients with paroxysmal atrial fibrillation is associ- ated with regional cardiac sympathetic denervation. EJNMMI Res 2013;3:81. 80. Sano M. A new class of drugs for heart failure: SGLT2 inhibitors reduce sympa- thetic overactivity. J Cardiol 2018;71:471–476. MC use 58. Lemery R, Ben-Haim S, Wells G, Ruddy TD. I-123-Metaiodobenzylguanidine imaging in patients with atrial fibrillation undergoing cardiac mapping and abla- tion of autonomic ganglia. Heart Rhythm 2017;14:128–132. 81. Bossone E, Savarese G, Ferrara F, Citro R, Mosca S, Musella F et al. Takotsubo cardiomyopathy: overview. Heart Fail Clin 2013;9:249–66. er on 59. Stirrup J, Gregg S, Baavour R, Roth N, Breault C, Agostini D et al. Hybrid solid- state SPECT/CT left atrial innervation imaging for identification of left atrial gan- glionated plexi: technique and validation in patients with atrial fibrillation. J Nucl Cardiol 2019. 82. Prasad A, Lerman A, Rihal CS. References Apical ballooning syndrome (Tako-Tsubo or stress cardiomyopathy): a mimic of acute myocardial infarction. Am Heart J 2008;155:408–17. 21 June 83. Pilgrim TM, Wyss TR. Takotsubo cardiomyopathy or transient left ventricular apical ballooning syndrome: a systematic review. Int J Cardiol 2008;124:283–92. 60. Maurer MS, Elliott P, Comenzo R, Semigran M, Rapezzi C. Addressing common questions encountered in the diagnosis and management of cardiac amyloidosis. Circulation 2017;135:1357–1377. 84. Wittstein IS, Thiemann DR, Lima JAC, Baughman KL, Schulman SP, Gerstenblith G et al. Neurohumoral features of myocardial stunning due to sudden emotion- al stress. N Engl J Med 2005;352:539–48. 61. Goldstein DS. Cardiac Dysautonomia and Survival in Hereditary Transthyretin Amyloidosis. JACC Cardiovasc Imaging 2016;9:1442–1445. 85. Sharkey SW, Windenburg DC, Lesser JR, Maron MS, Hauser RG, Lesser JN et al. Natural history and expansive clinical profile of stress (tako-tsubo) cardio- myopathy. J Am Coll Cardiol 2010;55:333–41. 62. Coutinho MCA, Cortez-Dias N, Cantinho G, Conceic¸~ao I, Oliveira A, Bordalo e Sa´ A et al. Reduced myocardial 123-iodine metaiodobenzylguanidine uptake: a prognostic marker in familial amyloid polyneuropathy. Circ Cardiovasc Imaging 2013;6:627–36. 86. Yoshida T, Hibino T, Kako N, Murai S, Oguri M, Kato K et al. A pathophysio- logic study of tako-tsubo cardiomyopathy with F-18 fluorodeoxyglucose posi- tron emission tomography. Eur Heart J 2007;28:2598–604. 63. Delahaye N, Dinanian S, Slama MS, Mzabi H, Samuel D, Adams D et al. Cardiac sympathetic denervation in familial amyloid polyneuropathy assessed by iodine- 123 metaiodobenzylguanidine scintigraphy and heart rate variability. Eur J Nucl Med 1999;26:416–24. 87. Cimarelli S, Sauer F, Morel O, Ohlmann P, Constantinesco A, Imperiale A. Transient left ventricular dysfunction syndrome: patho-physiological bases through nuclear medicine imaging. Int J Cardiol 2010;144:212–8. 490 A. Gimelli et al. 88. Christensen TE, Bang LE, Holmvang L, Skovgaard DC, Oturai DB, Søholm H et al. I-MIBG scintigraphy in the subacute state of Takotsubo cardiomyopathy. JACC Cardiovasc Imaging 2016;9:982–90. 95. Zelt JGE, deKemp RA, Rotstein BH, Nair GM, Narula J, Ahmadi A et al. Nuclear imaging of the cardiac sympathetic nervous system: a disease-specific interpretation in heart failure. JACC Cardiovasc Imaging 2019;13:1036–105. 96. Lautamaki R, Sasano T, Higuchi T, Nekolla SG, Lardo AC, Holt DP et al. Multiparametric molecular imaging provides mechanistic insights into sympa- thetic innervation impairment in the viable infarct border zone. J Nucl Med 2015;56:457–63. 89. Paur H, Wright PT, Sikkel MB, Tranter MH, Mansfield C, O’Gara P et al. References High levels of circulating epinephrine trigger apical cardiodepression in a beta2- adrenergic receptor/Gi-dependent manner: a new model of Takotsubo cardio- myopathy. Circulation 2012;126:697–706. 97. Yu M, Bozek J, Lamoy M, Guaraldi M, Silva P, Kagan M et al. Evaluation of LMI1195, a novel 18F-labeled cardiac neuronal PET imaging agent, in cells and animal models. Circ Cardiovasc Imaging 2011;4:435–43. 90. Lebasnier A, Lamotte G, Manrique A, Peyronnet D, Bouvard G, Defer G et al. Potential diagnostic value of regional myocardial adrenergic imaging using (123)I-MIBG SPECT to identify patients with Lewy body diseases. Eur J Nucl Med Mol Imaging 2015;42:1043–51. 98. Sinusas AJ, Lazewatsky J, Brunetti J, Heller G, Srivastava A, Liu Y-H et al. Biodistribution and radiation dosimetry of LMI1195: first-in-human study of a novel 18F-labeled tracer for imaging myocardial innervation. J Nucl Med 2014; 55:1445–51. Downloaded from https://academic.oup.com/ehjcimaging/article/22/5/480/6125312 by Universiteit Leiden / LUMC user on 21 June 2022 91. Lee JE, Kim JS, Ryu DW, Oh YS, Yoo IR, Lee KS. Cardiac sympathetic denerv- ation can predict the wearing-off phenomenon in patients with Parkinson dis- ease. J Nucl Med 2018;59:1728–33. 99. Raffel DM, Jung Y-W, Koeppe RA, Jang KS, Gu G, Scott PJH et al. First-in-human studies of [(18)F] fluorohydroxyphenethylguanidines. Circ Cardiovasc Imaging 2018;11:e007965. 92. De Rosa A, Pellegrino T, Pappata` S, Pellecchia MT, Peluso S, Sacca` F et al. Myocardial (123)I-metaiodobenzylguanidine scintigraphy in patients with homo- zygous and heterozygous parkin mutations. J Nucl Cardiol 2017;24:103–107. 100. Tinti E, Positano V, Giorgetti A, Marzullo P. Feasibility of [(123)I]-meta- iodobenzylguanidine dynamic 3-D kinetic analysis in vivo using a CZT ultrafast camera: preliminary results. Eur J Nucl Med Mol Imaging 2014;41:167–73. 93. Thackeray JT, Bengel FM. Assessment of cardiac autonomic neuronal function using PET imaging. J Nucl Cardiol 2013;20:150–65. 94. Wu KY, Zelt JGE, Wang T, Dinculescu V, Miner R, Lapierre C et al. Reliable quantification of myocardial sympathetic innervation and regional denervation using [(11)C]meta-hydroxyephedrine PET. Eur J Nucl Med Mol Imaging 2020;47: 1722–1735. 101. Giorgetti A, Burchielli S, Positano V, Kovalski G, Quaranta A, Genovesi D et al. Dynamic 3D analysis of myocardial sympathetic innervation: an experimental study using 123I-MIBG and a CZT camera. J Nucl Med 2015;56:464–9.
https://openalex.org/W4392962280
https://www.oejournal.org/data/article/export-pdf?id=65f9506499d881433ef8be86
English
null
Multi-wavelength nanowire micro-LEDs for future high speed optical communication
Opto-electronic advances
2,024
cc-by
2,350
Ayush Pandey  and Zetian Mi* The future of optoelectronics is directed towards small-area light sources, foremost being microLEDs. However, their use has been inhibited so far primarily due to fabrication and integration challenges, which impair efficiency and yield. Re- cently, bottom-up nanostructures grown using selective area epitaxy have garnered attention as a solution to the afore- mentioned issues. Prof. Lan Fu et. al. have used this technique to demonstrate uniform p-i-n core-shell InGaAs/InP nanowire array light emitting diodes. The devices are capable of voltage and geometry-controlled multi-wavelength and high-speed operations. Their publication accentuates the wide capabilities of bottom-up nanostructures to resolve the dif- ficulties of nanoscale optoelectronics. Pandey A, Mi ZT. Multi-wavelength nanowire micro-LEDs for future high speed optical communication. Opto-Electron Adv 7, 240011 (2024). Lately, micron-scale, or smaller, optoelectronic devices have emerged as a frontier technology, particularly mi- cron-scale or smaller light emitting diodes (LEDs), known as microLEDs. These small-area devices are dir- ect upgrades to their larger counterparts in areas such as displays or illumination, while they are also carving out their own unique niches in applications such as biology and communication. volume. Intensive research has been dedicated to healing the damage induced on the surface, through methods such as annealing, wet chemical etching and/or surface passivation, but these have been met with limited success3,4. The concern of etch damage can be completely sidestepped using a bottom-up process to grow device mesas. The process of selective area growth (SAG) in- volves patterning vias to the substrate on a mask layer, and the patterned substrate is then loaded for growth. The growth conditions are tuned such that epitaxy only occurs within the openings defined. This results in the growth of nano(micro)structures, having dimensions and shapes matched exactly to those defined while pat- terning the substrate5,6. Furthermore, these nanostruc- tures would not need to be exposed to any dry etching process to define device mesas, thereby preventing the formation of the surface defects associated with that pro- cess. These advantages are necessary to attain a high effi- ciency for any (sub)micron-scale device. Nanostructures can also be grown on non-native substrates, potentially opening up more new applications7. In addition, To date, however, fabricating devices at micron and submicron scales is not straightforward. During typical top-down device fabrication of epilayers, the device mesa is defined using a reactive-ion etching step. Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI 48109, USA. *Correspondence: ZT Mi, E-mail: ztmi@umich.edu Received: 17 January 2023; Accepted: 19 January 2023; Published online: 20 March 2023 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024. Published by Institute of Optics and Electronics, Chinese Academy of Sciences. 240011-1 News & Views March 2024, Vol. 7, No. 3 News & Views March 2024, Vol. 7, No. 3 Multi-wavelength nanowire micro-LEDs for future high speed optical communication Ayush Pandey  and Zetian Mi* Ayush Pandey  and Zetian Mi* This dry etch process severely damages the sidewalls of the device mesa, leaving a large number of crystalline defects and dangling bonds, which contribute to non-radiative re- combination of charge carriers1,2. These surface states de- grade the light output, and hence efficiency, of ultra small optoelectronic devices, rendering them impractic- al for use. The damage caused by the etching process is especially disadvantageous in devices with a smaller mesa size, due to the increased ratio of surface-area-to- Pandey A et al. Opto-Electron Adv 7, 240011 (2024) https://doi.org/10.29026/oea.2024.240011 previous research has highlighted numerous other bene- fits of nanostructures over conventional epilayer-based devices, including a nearly defect-free crystal, enhanced dopant incorporation and reduced strain8,9. SAG can also be used to grow nanostructures in specific arrangements, such as a photonic crystal, which can be harnessed for uses such as in directional LEDs or ultra-small surface- emitting lasers10,11. different nanowire arrays on a single substrate for multi- pixel applications. To study device performance, the nanowire arrays were fabricated to form microLED devices. A schematic of the final device is shown in Fig. 1(c). The authors demonstrated nanowire array LEDs with rectifying char- acteristics, low turn-on voltage and an improvement in output power by over 6×, as compared to a similar previ- ously reported device15. The current-voltage and light output curves are shown in Fig. 1(d). The electrolumin- escence (EL) observed from the device also shows the presence of multiple and broad peaks, at approximately 1.35 μm and 1.5 μm, which is expected due to the com- plex geometry of the nanowire structure, as well as the myriad current injection paths available for carriers to follow and recombine in. Injection dependent spectra show that the emission blueshifts towards shorter emis- sion wavelengths at higher biases, potentially enabling voltage-tunable multi-wavelength operation. Simula- tions performed by the authors were able to identify the emission at 1.5 μm to originate from the relatively larger volume radial QW which dominates emission, while the shorter peak at 1.35 μm originated in the smaller volume axial QW. At high biases, the band-filling effect starts to become dominant in both the axial and radial QWs, pushing the peak emission to shorter wavelengths. The normalized EL spectra at different biases are plotted in Fig. 1(e). The broad EL emission peak with a FWHM of ~286 nm is well suited for applications such as bio-sens- ing and optical coherence tomography. Recently, Prof. Ayush Pandey  and Zetian Mi* Lan Fu from the Australian National University, and their colleagues, published a work in Opto-Electronic Science, where they grew highly uniform p-i-n core-shell InGaAs/InP single quantum well (QW) nanowires, using metalorganic chemical vapor depos- ition (MOCVD), that were subsequently fabricated into microLED arrays12. A schematic representation of the nanowires is shown in Fig. 1(a). Uniform nanowire ar- rays with regular morphology were grown, and a scan- ning electron microscope (SEM) image is shown in Fig. 1(b). Due to variations in Indium incorporation along different facets, emission in the telecom wavelength range of 1.35 μm — 1.5 μm was measured from the nanowire arrays. The authors also noted a dependence of emission wavelength on the nanowire dimension and spacing — this phenomenon has been observed previ- ously and is attributed to the effect of these parameters on the local supply of adatoms/reactants13,14. Further- more, by selecting for a low temperature, high V/III ra- tio growth regime, the authors have been able to minim- ize height differences between the different nanowires; thereby greatly simplifying the process to integrate the n+ InP p+ InP p+ substrate SiO2 InGaAs InP a 16 14 12 10 Power (μW) 8 6 4 2 0 1.0 500 nm 30 μm 12.0 V 10.0 V 8.0 V 6.0 V 4.0 V 3.0 V 2.0 V 1.5 V 0.8 0.6 0.4 0.2 Normalized EL intensity (a.u.) 0 10001100120013001400150016001700 0 2 4 Voltage (V) Wavelength (nm) 6 8 10 0 10 20 30 Current (mA) 40 50 60 ITO n+ InP p+ InP SiO2 InP Ti/Au SU8 InGaAs p+ substrate n+ InP p+ InP p+ substrate SiO2 InGaAs InP a d e f b c Fig. 1 | (a) Schematic of the nanowire cross-section top and profile view. (b) An SEM image of a nanowire array. (c) Schematic of the fabricated nanowire LED. (d) L-I-V curves for a representative nanowire LED. (e) Normalized voltage-dependent EL spectra. (f) SEM image of nanowire ar- rays arranged to form the letters “ANU”. The inset shows an infrared camera image of the EL emission from the nanowire array LEDs. References Smith JM, Ley R, Wong MS et al. Comparison of size-depend- ent characteristics of blue and green InGaN microLEDs down to 1 μm in diameter. Appl Phys Lett 116, 071102 (2020). 1. Smith JM, Ley R, Wong MS et al. Comparison of size-depend- ent characteristics of blue and green InGaN microLEDs down to 1 μm in diameter. Appl Phys Lett 116, 071102 (2020). 1. Konoplev SS, Bulashevich KA, Karpov SY. From large‐size to micro‐LEDs: scaling trends revealed by modeling. Phys Status Solidi (A) 215, 1700508 (2018). 2. Konoplev SS, Bulashevich KA, Karpov SY. From large‐size to micro‐LEDs: scaling trends revealed by modeling. Phys Status Solidi (A) 215, 1700508 (2018). 2. Yang Y, Cao XA. Removing plasma-induced sidewall damage in GaN-based light-emitting diodes by annealing and wet chemic- al treatments. J Vac Sci Technol B 27, 2337–2341 (2009). 3. Yang Y, Cao XA. Removing plasma-induced sidewall damage in GaN-based light-emitting diodes by annealing and wet chemic- al treatments. J Vac Sci Technol B 27, 2337–2341 (2009). 3. Wong MS, Kearns JA, Lee C et al. Improved performance of Al- GaInP red micro-light-emitting diodes with sidewall treatments. Opt Express 28, 5787–5793 (2020). 4. Wong MS, Kearns JA, Lee C et al. Improved performance of Al- GaInP red micro-light-emitting diodes with sidewall treatments. Opt Express 28, 5787–5793 (2020). 4. Kishino K, Sekiguchi H, Kikuchi A. Improved Ti-mask selective- area growth (SAG) by rf-plasma-assisted molecular beam epi- taxy demonstrating extremely uniform GaN nanocolumn arrays. J Cryst Growth 311, 2063–2068 (2009). 5. This work encapsulates the many distinct advantages of bottom-up nanostructure-based devices over conven- tional top-down etched devices in the context of mi- croLEDs. The major improvements in this work were all contingent on the epitaxy: from the regular and con- trolled nanowire morphology, which is an indicator of defect-free crystal epitaxy, to the excellent electrical and light output characteristics of finished devices. The flex- ibility of the emission from the nanostructure-based ar- rays, with nanostructure geometry, allows for the mono- lithic integration of multiple multi-wavelength LEDs for wavelength division multiplexing, demonstrated as a proof of concept by the authors of this work, as shown by the arrangement of multiple small micro-LED arrays to form the letters “ANU” in Fig. 1(f). The inset of this fig- ure contains an infrared camera image of the EL emis- sion from the nanowire array LED. Pandey A et al. Opto-Electron Adv 7, 240011 (2024) https://doi.org/10.29026/oea.2024.240011 Optical communication and photonic circuits both re- quire high speed operation. To evaluate the ultrafast op- eration of the devices, the authors used time-resolved photoluminescence spectroscopy to measure the minor- ity carrier lifetimes. The measured lifetime of only ~309 ps is nearly 4× lower than bulk InP nanowires, indicat- ing efficient carrier recombination. The measured life- time sets an upper limit of ~3 GHz for the modulation frequency. Time-resolved electroluminescence also showed that the nanowire array LED could be modu- lated at GHz frequencies. The authors suggested that op- timization of the ITO p-contact and removal of the InP substrate could greatly reduce the parasitic capacitance and series resistance of the device, effectively overcom- ing the modulation frequency limitations imposed by the RC constants. and bottom-up approaches to drive improvements in nanoscale light sources operating at any wavelength. Ayush Pandey  and Zetian Mi* 240011-2 500 nm b 16 14 12 10 Power (μW) 8 6 4 2 0 0 2 4 Voltage (V) 6 8 10 0 10 20 30 Current (mA) 40 50 60 d e 1.0 12.0 V 10.0 V 8.0 V 6.0 V 4.0 V 3.0 V 2.0 V 1.5 V 0.8 0.6 0.4 0.2 Normalized EL intensity (a.u.) 0 10001100120013001400150016001700 Wavelength (nm) 30 μm Fig. 1 | (a) Schematic of the nanowire cross-section top and profile view. (b) An SEM image of a nanowire array. (c) Schematic of the fabricated nanowire LED. (d) L-I-V curves for a representative nanowire LED. (e) Normalized voltage-dependent EL spectra. (f) SEM image of nanowire ar- rays arranged to form the letters “ANU”. The inset shows an infrared camera image of the EL emission from the nanowire array LEDs. 240011-2 Fig. 1 | (a) Schematic of the nanowire cross-section top and profile view. (b) An SEM image of a nanowire array. (c) Schematic of the fabricated nanowire LED. (d) L-I-V curves for a representative nanowire LED. (e) Normalized voltage-dependent EL spectra. (f) SEM image of nanowire ar- rays arranged to form the letters “ANU”. The inset shows an infrared camera image of the EL emission from the nanowire array LEDs. 240011-2 References The authors are seek- ing to further extend this idea by creating individually addressable pixels consisting of single nanowire, or small-area array LEDs. Pandey A, Malhotra Y, Wang P et al. N-polar InGaN/GaN nanowires: overcoming the efficiency cliff of red-emitting micro- LEDs. Photonics Res 10, 1107–1116 (2022). 6. Wu YP, Xiao YX, Navid I et al. InGaN micro-light-emitting di- odes monolithically grown on Si: achieving ultra-stable opera- tion through polarization and strain engineering. Light Sci Appl 11, 294 (2022). 7. Guo W, Zhang M, Banerjee A et al. Catalyst-free InGaN/GaN nanowire light emitting diodes grown on (001) silicon by molecu- lar beam epitaxy. Nano Lett 10, 3355–3359 (2010). 8. Nguyen HPT, Zhang S, Cui K et al. p-Type modulation doped InGaN/GaN dot-in-a-wire white-light-emitting diodes monolithic- ally grown on Si (111). Nano Lett 11, 1919–1924 (2011). 9. Liu XH, Wu YP, Malhotra Y et al. Micrometer scale InGaN green light emitting diodes with ultra-stable operation. Appl Phys Lett 117, 011104 (2020). 10. Ra YH, Rashid RT, Liu XH et al. An electrically pumped surface- emitting semiconductor green laser. Sci Adv 6, eaav7523 (2020). 11. Zhang FL, Su ZC, Li Z et al. High-speed multiwavelength In- GaAs/InP quantum well nanowire array micro-LEDs for next generation optical communications. Opto-Electron Sci 2, 230003 (2023). 12. With microLED technology being intensively studied at the present, the authors showcased the numerous be- nefits of SAG to form bottom-up nanostructures for mi- cro and nanoscale optoelectronics. While the authors have used the InGaAs/InP material system, other work has demonstrated similar enhancements to selective area grown microLEDs fabricated from other material sys- tems – this underlines the incredible versatility of SAG Sekiguchi H, Kishino K, Kikuchi A. Emission color control from blue to red with nanocolumn diameter of InGaN/GaN nano- column arrays grown on same substrate. Appl Phys Lett 96, 231104 (2010). 13. Ra YH, Wang RJ, Woo SY et al. Full-color single nanowire pixels for projection displays. Nano Lett 16, 4608–4615 (2016). 14. Yang IS, Kim SJ, Niihori M et al. Highly uniform InGaAs/InP quantum well nanowire array-based light emitting diodes. Nano Energy 71, 104576 (2020). 15. Scan for Article PDF 240011-3
https://openalex.org/W2996479899
https://digital.csic.es/bitstream/10261/197163/1/labeling_fructicola_GFP_host-pathogen_stone_pome_fruit.pdf
English
null
Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
Genes
2,019
cc-by
9,690
Received: 30 October 2019; Accepted: 5 December 2019; Published: 11 December 2019 Abstract: To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples. Keywords: Agrobacterium-mediated transformation; confocal microscopy; thermal asymmetric interlaced PCR (TAIL-PCR); brown rot Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit Silvia Rodríguez-Pires 1 , Eduardo Antonio Espeso 2 , Nuria Baró-Montel 3, Rosario Torres 3, Paloma Melgarejo 1 and Antonieta De Cal 1,* Silvia Rodríguez-Pires 1 , Eduardo Antonio Espeso 2 , Nuria Baró-Montel 3, Rosario Torres 3 Paloma Melgarejo 1 and Antonieta De Cal 1,* 1 Department of Plant Protection, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Ctra. de La Coruña Km. 7, 28040 Madrid, Spain; rpires.silvia@inia.es (S.R.-P.); melgar@inia.es (P.M.) 2 Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain; eespeso@cib.csic.es 3 IRTA, XaRTA-Postharvest, Edifici Fruitcentre, Parc Científic i Tecnologic Agroalimentari de Lleida, 25003 Lleida, Spain; nuria.baro@irta.cat (N.B.-M.); rosario.torres@irta.cat (R.T.) * Correspondence: cal@inia.es p * Correspondence: cal@inia.es genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes T T www.mdpi.com/journal/genes Keywords: Agrobacterium-mediated transformation; confocal microscopy; thermal asymmetric interlaced PCR (TAIL-PCR); brown rot 1. Introduction Monilinia spp. cause brown rot disease, one of the most important diseases on stone and pome fruit [1]. The distribution and prevalence of Monilinia spp. depend on the geographical area under study, the inoculum, and the host [2]. M. fructicola (G Winter) Honey and M. laxa (Aderhold & Ruhland) Honey are the main species causing brown rot in stone fruit currently in Spain, both with the same frequency after the first detection of M. fructicola in 2006 [3,4]. However, M. fructigena has been displacing as a minority species on stone fruit [4]. This displacement is supported by the effects of higher growth rate and aggressiveness factors in M. fructicola on stone fruit than M. laxa and M. fructigena [5]. Hyphal tubes from germinated M. fructicola conidia can penetrate the stone fruit surface through stomata or wounds and by degrading the cuticle of intact surface [1]. In nectarines, first brown-rot symptoms are visible 24 h post-inoculation on fruit surface and epidermal discoloration Genes 2019, 10, 1033; doi:10.3390/genes10121033 www.mdpi.com/journal/genes 2 of 14 Genes 2019, 10, 1033 and brown spots are visible after 48 h [6]. This infection process involves initial cuticle decay, followed by the destruction of epidermal and mesocarp cells. The complete breakdown of cuticle and epidermis is the result of progressive vegetative growth and this infection process concludes with M. fructicola sporulation [6]. M. fructicola and M. laxa are species mostly found on stone fruit and only occasionally on pome fruit [2], or other fruit such as strawberry [7,8]. However, M. fructigena affects mainly apple and pear [9]. M. fructicola was first described as a pathogen on peaches in Spain in 2006 [3], while for the time being it has not been described as a pathogen on pome fruit in Spain; it is, however, present on pome fruit in other countries such as Czech Republic [10], Germany [11], Serbia [12], Italy [13], USA [14], China [15], and Brazil [16]. The higher M. fructicola prevalence on stone fruit than on pome fruit could be due to differences in the infection process described above. The green fluorescent protein (GFP) has proven useful in studies of host-pathogen interactions to detect and visualize the infection process in situ by confocal microscopy [17,18]. Although GFP-tagged fungal transformants have been obtained for a variety of postharvest pathogens [19–21], the use of this tool has not been extended to M. fructicola on stone or pome fruit. 1. Introduction Only Monilinia vaccinii-corymbosi infections on blueberry flowers have been studied using fungal strains, which express GFP by transformation of their protoplasts [22]. Construction of a M. fructicola strain expressing GFP may facilitate the understanding of the less aggressive process of M. fructicola infection on pome fruit by confocal microscopy. However, the GFP insert could modify the fungal pathogenesis or alter any other type of genes essential for its growth. Therefore, once the mutant has been obtained, it is required to investigate where in the genome the construct expressing GFP was inserted and which activities might be affected. The aim of this study was to introduce the hph-gfp chimera into a pathogenic wild-type M. fructicola isolate using Agrobacterium tumefaciens-mediated transformation and resistance to hygromycin B as the selectable marker, as provided by the chimera. Purified Hph-GFP expressing transformants were characterized for maintaining their pathogenicity, germination, and growth capacity, compared to wild type strains. M. fructicola transformants were used to explore the infection process on nectarines and apples by confocal microscopy. 2.1. Fungi and Bacteria Growth Conditions fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each transformant were verified with Coomassie staining and also served as loading control for western blot (right). 2.1. Fungi and Bacteria Growth Conditions Monilinia fructicola (wt38c) is a single spore isolate collected from a latent infection on a peach in Alfarrás (Lleida, Spain). Isolate wt38c showed high ability to infect stone fruit and produce conidia, and high resistance to carbendazim fungicide [23]. Wild type (wt38c) and transformant strains were stored as a conidial suspension in 20% glycerol at −80 ◦C for long-term storage, and as a culture on potato dextrose agar (PDA; Difco, Detroit, MI, USA) at 4 ◦C in darkness for short-term storage. PDA was supplemented with hygromycin B (100 µg/mL) for maintenance of transformants. Agrobacterium tumefaciens strain LBA4404 carrying pPK2-hphgfp [24] provided by Dr. J. Martínez-Cruz (Microbiology Department, Malaga University, Spain) was used in fungal transformation experiments. The pPK2-hphgfp vector contains an in-frame fusion of the hygromycin B phosphotransferase resistance gene (hph) and green fluorescent protein (gfp) under control of Aspergillus nidulans gpdA promoter and trpC terminator (see Results section and Figure 1a). A. tumefaciens LBA4404 carrying pPK2-hphgfp was grown on LB agar containing 50 µg/mL rifampicin and 50 µg/mL kanamycin at 28 ◦C. 3 of 14 6 of 15 Genes 2019, 10, 1033 Genes 2019, 10, x FOR P Figure 1 PCR and western blot analysis of transformants of Monilinia fructicola (a) Schematic Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. 2.1. Fungi and Bacteria Growth Conditions fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each transformant were verified with Coomassie staining and also served as loading control for western blot (right). Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. fructicola transformants, confirming the presence of hph gene (left), gfp gene (center) and the in-frame fusion of both genes (right). (c) Hph-GFP expression was confirmed by Western blot analysis of intracellular protein lysates from M. fructicola transformants using a specific primary antibody against GFP (left). Intracellular protein profiles of each transformant were verified with Coomassie staining and also served as loading control for western blot (right). Figure 1. PCR and western blot analysis of transformants of Monilinia fructicola. (a) Schematic description of the T-DNA region of the pPK2hphgfp vector and primers locations. LB, left border; PgpdA, Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter; hph, hygromycin B phosphotransferase resistance gene; gfp, green fluorescent protein; TtrpC, A. nidulans tryptophan biosynthesis terminator; RB, right border. Primers for hph and gfp presence analysis are indicated by arrows. (b) PCR analysis of M. transformant were verified with Coomassie staining and also served blot (right) 2.2. Agrobacterium tumefaciens-Mediated M. fructicola Transformation transformant were verified with Coomassie staining and also served blot (right) 2.2. Agrobacterium tumefaciens-Mediated M. fructicola Transformation ( g ) Analyses of the flanking regions of the inserted T-DNAs were carried out by TAIL-PCR, demonstrating the random integration of T-DNAs into the M. fructicola genome. To identify the location of the construction insertion point in the genome of each transformant and recover the T- DNA border junctions, TAIL-PCR was performed (see experimental procedures, Table 1 for specific and degenerate oligonucleotides). The efficiency of TAIL-PCR was largely improved when several amplification cycles were included using only the specific LB1 or RB1 primer for each border of the construct. Primary amplification reactions, using LB1 and RB1 primers together with randomized A single colony of A. tumefaciens LBA4404 carrying pPK2-hphgfp was grown overnight in LB broth with rifampicin (50 µg/mL) and kanamycin (50 µg/mL) at 25 ◦C on an orbital shaker at 150 rpm. A. tumefaciens cells were collected by centrifugation and washed twice with induction medium (IM) as described by Michielse et al. [25]. The cell pellet was suspended in 5 mL of IM supplemented with acetosyringone (200 µM) and further cultivated for 4–5 h at 28 ◦C on an orbital shaker at 150 rpm. Next, A. tumefaciens culture was diluted to an OD600 of 0.3 in IM. Concurrently, conidia from 7-day-old M. fructicola (wt38c) colonies grown on PDA were harvested by scratching the surface of the plate and the spore suspension was adjusted to 1 × 106 conidia mL−1 in IM. For transformation, this conidial 4 of 14 Genes 2019, 10, 1033 suspension was incubated to promote germination of spores in IM at room temperature for 4 h at 150 rpm. Then, 100 µL of induced A. tumefaciens (OD600 of 0.3) were mixed with an equal volume of germinated conidia and spread on a 0.45 µm cellulose-nitrate membrane or cellophane sheets. The membranes or cellophane sheets were placed on IM with 1.5% agar and incubated at 22 ◦C for 2–3 days. After co-cultivation, membranes were transferred onto selective PDA plates supplemented with hygromycin B (100 µg/mL) and cefotaxime sodium salt (200 µM) to remove Agrobacterium cells. Hygromycin-resistant colonies of M. fructicola were selected individually and maintained on the selective medium PDA-H until further use. 2.3. Molecular Analyses of M. fructicola Transformants M. fructicola hygromycin-resistant transformants were grown for 3 days at 22 ◦C in potato dextrose broth (PDB) supplemented with hygromycin B (100 µg/mL). The mycelium was harvested by filtration through Miracloth (EMD Millipore Corporation, Billerica, MA, USA) and freeze-dried. Mycelium of potential transformants was ground to a fine powder using a FastPrep-24 (MP Biomedical, Solon, OH, USA; power 4, 20 sec/pulse) and genomic DNA was extracted by adding 1 mL of lysis solution (25 mM Tris-HCl pH 8.0, 0.25 M sucrose, 20 mM EDTA pH 8.0, 1% SDS), heating at 65 ◦C for 15 min, and then performing phenol-chloroform-isoamyl alcohol (25:24:1) extraction. Genomic DNA at the aqueous phase was precipitated using isopropanol, washed with 80% ethanol, and suspended in milli-Q water. To remove traces of RNA, DNA samples were treated with RNAseH for 1 h at 37 ◦C, then samples were stored at −20 ◦C until use. The presence of T-DNA in the genomic DNA of transformants was confirmed by PCR to detect the hygromycin resistance gene (primer pair hphF/hphR), gfp gene (primer pair gfpF/gfpR) (Table 1), and the fragment hph-gfp (primer pair hphF/gfpR) (see the Results section, Figure 1). Table 1. Primers used in this study. Primer Sequence (5′–3′) Description Reference gfpF CCCATGAGTAAAGGAGAAGAACTT gfp gene [18] gfpR CTATTTGTATAGTTCATCCATGCCATGTGTA hphF TTCGATGTAGGAGGGCGTGGATATG hph gene [18] hphR GGTTTCCACTATCGGCGAGTACTTC LB1 TTAATTGCGTTGCGCTCACTGC TAIL-PCR [18] LB2 GCTTTCCAGTCGGGAAACCTGTC LB3 GAGCAATTCGGCGTTAATTCAGT RB1 GGCACTGGCCGTCGTTTTACAAC RB2 AACGTCGTGACTGGGAAAACCCT RB3 CCCTTCCCAACAGTTGCGCA AD1 WGTGNAGWANCANAGA AD2 NGTCGASWGANAWGAA InsLB (Atd9) AATACCCTGTTGAGATTTGG Left/right border junction of Atd9 strain This study InsRB (Atd9) TACTGGCTAGGCAAGAGTCTGG scf005-F TCATCATAACGGCAGGGAGG Amplification of putative deletion on Atd9 strain This study scf005-R GCCACCACAAATTCCAAGCG scf014-F TGTGGCGTCTGCTTGTATCC Amplification of putative duplication on Atd9 strain This study sc014-R TTTGGCGAGGTCATCATAGC Expression of GFP by transformants of M. fructicola was analyzed by western blot using total protein extracts according to Hernández-Ortiz and Espeso [26]. Hph-GFP fusion was detected using a polyclonal mouse anti-GFP (1/5000; Roche, Basel, Switzerland) as primary antibody. As the secondary Table 1. Primers used in this study. Expression of GFP by transformants of M. fructicola was analyzed by western blot using total protein extracts according to Hernández-Ortiz and Espeso [26]. Hph-GFP fusion was detected using a polyclonal mouse anti-GFP (1/5000; Roche, Basel, Switzerland) as primary antibody. As the secondary 5 of 14 Genes 2019, 10, 1033 antibody, a peroxidase-conjugated goat anti-mouse IgG immunoglobulin was used (1/4000; Jackson Immuno Research Laboratories, West Grove, PA, USA). 2.4. Characterization of GFP-M. fructicola Transformants Morphology and colony growth were determined for M. fructicola transformants on PDA-H, where M. fructicola wild type (wt38c) on PDA was used as a control. Plugs (ø 5 mm) of actively growing mycelia were cut from the margins of a 7-day-old colony of each isolate and placed on the center of a sterile PDA-H or PDA Petri dish (ø 9 cm). The plates were then incubated for 14 days at 20–25 ◦C in the dark. The daily growth rate (mm day−1) was calculated from the individual measurements of colony diameter that were made on each day of the 14-day incubation using regression analysis. Three replicate plates were used per medium. Each assay was repeated at least twice. The aggressiveness of GFP-M. fructicola transformants was tested on disinfected wounded and unwounded peach fruit, cv. ‘Merryl O’Henry’. Each fruit was inoculated with 25 µl aqueous conidial suspension (104 conidia ml−1) and incubated for 7 days in humidity (100%, RH) chambers at 20 ◦C. Each fruit was visually examined daily for symptoms of brown rot. The percentage of fruit with brown rot symptoms (%, brown rot incidence), the incubation period (the time interval between inoculation and the onset of symptoms), and latency period (the time interval between inoculation and the onset of sporulation) [5] were recorded for each infected fruit. Twenty fruits were used per type of inoculation point. The complete experiment was repeated twice. Data were statistically analyzed by one-way analysis of variance (ANOVA). When the results of the F-test were significant (p < 0.05), the means were compared using the LSD test (p < 0.05). 2.3. Molecular Analyses of M. fructicola Transformants Thermal asymmetric interlaced PCR (TAIL-PCR) was used to identify the flanking regions to T-DNA. Genomic DNAs, extracted as described above, were used as templates in TAIL-PCR reactions. As an initial step, 15 cycles of single chain amplification were done using only the LB or RB primer. This ssDNA was used as a template for the first round of PCR, including LB/RB primers and the degenerate oligonucleotides (Table 1). The TAIL-PCR cycle setting conditions used were described previously [27] with several modifications (Table S1). Subsequently, second and third rounds were performed using 1 µl of the diluted product (1/10) of the previous PCR. After the third PCR reaction, products showing a reduction of 100 bp in size, due to the distance between LB2/LB3 and RB2/RB3, were excised from the agarose gel and purified using Nucleospin gel and PCR clean-up kits (Macherey-Nagel, Düren, Germany). Purified DNA fragments were sequenced (STAB VIDA, Caparica, Portugal) and compared to the available M. fructicola genome (NGKE01) using BLAST (NCBI, https://blast.ncbi.nlm.nih.gov/). Predictions of coding regions in the M. fructicola genome were done using AUGUSTUS [28] trained with Botrytis cinerea transcriptome. PCR results were compatible with a single integration event of TDNA in each transformant. 3.1. Generating Green-Fluorescent M. fructicola Strains by Transformation The growth of M. fructicola (wt38c), considered hereafter as a wild type strain, was completely inhibited on PDA supplemented with 100 µL/mL hygromycin B (PDA-H). This finding allowed a positive selection method for Agrobacterium-mediated transformation of T-DNA expressing the Hygromycin-B 4-O-kinase, Hph. The A. tumefaciens LBA4404 strain carries the pPK2-hphgfp plasmid, allowing the constitutive gpdA-driven expression of a Hph-GFP chimera, linking tolerance to hygromycin B and green fluorescence emission. Three putative M. fructicola transformants (Atd6, At7, and Atd9) were recovered on PDA-H medium. Atd9 was recovered on cellophane sheets, and At7 and Atd9 on cellulose-nitrate membrane. The stability of the genotypic modification was confirmed after at least five successive generations on PDA-H to all M. fructicola transformants. The presence of T-DNA in the genome of M. fructicola transformants was determined by PCR (Figure 1a). Combinations of oligonucleotide pairs were used to amplify either hph and gfp fragments or both. In all cases, genomic DNA from M. fructicola wild type (wt38c) was included as negative control. Detection of specific PCR products sizing, 877 bp for hph and 720 bp for gfp, as well as fusion of both genes hph-gfp 1632 bp (Figure 1b), confirmed the presence of T-DNA in the three transformants. Moreover, western blot analysis using antibodies against the GFP moiety confirmed the expression of the fusion protein Hph-GFP in protein extracts. The Hph-GFP chimera, with a predicted Mw of 64.9 kDa, was detected close to the 75 kDa marker in SDS-PAGE, as well as truncated forms comprising GFP (Mw: 26.9 kDa), which most likely correspond to degradation products of the chimera (Figure 1c). ( ) y p g p ( g ) Analyses of the flanking regions of the inserted T-DNAs were carried out by TAIL-PCR, demonstrating the random integration of T-DNAs into the M. fructicola genome. To identify the location of the construction insertion point in the genome of each transformant and recover the T-DNA border junctions, TAIL-PCR was performed (see experimental procedures, Table 1 for specific and degenerate oligonucleotides). The efficiency of TAIL-PCR was largely improved when several amplification cycles were included using only the specific LB1 or RB1 primer for each border of the construct. Primary amplification reactions, using LB1 and RB1 primers together with randomized primer, showed similar patterns of multiple bands (Figure S1). Secondary (LB2/RB2) and tertiary (LB3/RB3) nested PCRs (Figure S1) showed more specific amplifications of certain PCR products. 2.5. Microscopy Analysis of M. fructicola GFP-Transformants Positive PCR transformants were analyzed for fluorescence emission. Mycelia samples of M. fructicola transformants grown on PDA-H were deposited onto a slide and fluorescence was imaged using a Leica DMRE microscope (Leica Microsystems, Wetzlar, Germany) with suitable filter system GFP ET, S (No: 11504174, Leica). Images were acquired using a Leica DFC550 camera driven by LAS v4.4. Fruit infection process by GFP-M. fructicola transformants was visualized using a confocal laser scanning microscopy Leica SP2 (Leica Microsystems, Wetzlar, Germany). Nectarine (cv. Venus) and apple (cv. Golden Delicious) were surface disinfected [29] and dried in a laminar flow cabinet. Unwounded fruit surfaces were inoculated with 15 µL of 105 conidia ml−1 of GFP-M. fructicola transformants with hygromycin B (100 µg/mL). Inoculated and unwounded nectarines were placed in individual sterilized boxes and incubated for 15 days at 20–25 ◦C in the darkness. Apples were placed in individual sterilized boxes and wounded with a sterilized scalpel 24 h after fungal inoculation. Wounded apples were incubated 14 days more at the same conditions. The infected tissue of each fruit 6 of 14 Genes 2019, 10, 1033 was excised for the examination of surface and internal colonization at 0 h, 24 h, 5 days, and 15 days of fungal inoculation. Fluorescence of GFP was excited by a 488 nm laser and detected at 500–550 nm. When required, the fruit cell walls and membranes were stained using propidium iodide (10 µg/mL) and FM4-64 (17 µM) following [30,31]. Images were acquired using LAS X software v3.3.0.16799 (Leica Microsystems, Wetzlar, Germany) and further processed using ImageJ v1.48 (National Institutes of Health, Bethesda, MD, USA). 3. Results .1. Generating Green-Fluorescent M. fructicola Strains by Transformation 3.1. Generating Green-Fluorescent M. fructicola Strains by Transformation 3.1. Generating Green-Fluorescent M. fructicola Strains by Transformation Tertiary PCR products with a difference of approximately 100 bp from those obtained in the secondary PCRs were purified and sequenced. As a result, we identified 50–60 bp belonging to T-DNA flanking regions, followed by fungal genomic sequences (Table S2). BLAST searches of sequences matched with high similarity (97–99%) against the available M. fructicola genome (NGKE01). Notably, the flanking regions of the left and right borders of T-DNA for a given transformant were not always in the same scaffold (Figure 2—scaffolds for genomic assembly of LMK125 strain). 7 of 14 5 Genes 2019, 10, 1033 Genes 2019, 10, x Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the genomes of the three positive transformants. Unless indicated with an arrow, the scaffolds represent the DNA sequence orientation (5′–3′) and coordinates, as indicated in genome database. The dotted line in Atd9 corresponds to an unknown location, and dark vertical lines denote gene predictions. (b) Schematic representation of PCR analyses to identify the sequences flanking left and right borders of T-DNA and to verify the presence of remaining regions of scaffolds 014 and 005 in the genome of Atd9 transformant. Primers are listed in Table 1. At the bottom is shown the electrophoretic analysis of the PCR products. Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the genomes of the three positive transformants. Unless indicated with an arrow, the scaffolds represent the DNA sequence orientation (5′–3′) and coordinates, as indicated in genome database. Genes 2019, 10, 1033 Genes 2019, 10, 1033 Genes 2019, 10, 1033 8 of 14 Five of six border junctions are located in intergenic regions and at the At7 right border within an intron of genic sequence. In transformant Atd6, we found the insertion of the left border at 1.2 kb upstream of the LMK125.3541 gene and the right border at 3.6 kb upstream of the LMK125.3699 gene, both into scaffold009. With respect to transformant At7, we found the insertion of the left border at 2 kb upstream of the LMK125.4967 gene in scaffold014 and the right border in the predicted coding region of LMK125.1494 gene, scaffold003. In transformant Atd9, we determined a sequence of 319 bp from scaffold014 at the left border, but of unknown location upstream from this point. However, we located the right border 250 bp upstream of LMK125.6942 gene in the scaffold005 (Figure 2a). Further PCR analysis of the Atd9 transformant showed the effect of T-DNA integration. This transformant with similar characteristics to the wild type carried a duplication of a 319 bp region from scaffold014 at the left border junction and genome reorganization of the right border. Firstly, we verified the integration location by PCR with a specific primer between genome prediction and T-DNA (14F1/LB1 and RB1/5R2). We recovered the right, but not the left border. Secondly, we checked the presence of the beginning of scaffold005 (5F2/5R2) and dismissed the possibility of deletion. Moreover, we proved the absence of amplification when primers located on both regions of the identified insertion point were used (5F2/5R2). Finally, 319 bp were recovered from scaffold014 in the left border, which was confirmed by PCR (14F1/14R1) and sequencing to be a duplication of this region (Figure 2b). 3.1. Generating Green-Fluorescent M. fructicola Strains by Transformation The dotted line in Atd9 corresponds to an unknown location, and dark vertical lines denote gene predictions. (b) Schematic representation of PCR analyses to identify the sequences flanking left and right borders of T-DNA and to verify the presence of remaining regions of scaffolds 014 and 005 in the genome of Atd9 transformant. Primers are listed in Table 1. At the bottom is shown the electrophoretic analysis of the PCR products. Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the Figure 2. Identification of integration sites for T-DNA in transformants. Results from BLASTn searches of the NCBI database against Monilinia fructicola genome (LMK125, GenBank NGKE01). (a) Top, schematic representation of T-DNA. In boxes are interpretations of the integration events at the genomes of the three positive transformants. Unless indicated with an arrow, the scaffolds represent the DNA sequence orientation (5′–3′) and coordinates, as indicated in genome database. The dotted line in Atd9 corresponds to an unknown location, and dark vertical lines denote gene predictions. (b) Schematic representation of PCR analyses to identify the sequences flanking left and right borders of T-DNA and to verify the presence of remaining regions of scaffolds 014 and 005 in the genome of Atd9 transformant. Primers are listed in Table 1. At the bottom is shown the electrophoretic analysis of the PCR products. 3.2. Characterization of GFP-M. fructicola Transformants Two of the three GFP-M. fructicola transformants on PDA-H (Atd6 and At7) presented some phenotypic changes with respect to the wild type strain (wt38c) on PDA (Figure 3a,c,d). Growth rates of Atd6 (3.9 mm day−1) and At7 (4.4 mm day−1) transformants were significantly lower than wt38c (9.2 mm day−1). Colony color, stromata, sporulation, and concentric growth circles were also significantly different among wt38c and Atd6 or At7 (Figure 3e,g,h). However, no difference, neither of colony and sporulation appearance nor of growth rate, were observed between the wild strain (wt38c) on PDA and Atd9-M. fructicola transformant on PDA-H (Figure 3a,b,e,f). Aggressiveness tests were only carried out with the At9-M. fructicola transformant and wild type wt38c on disinfected wounded and unwounded peaches. Brown rot incidence caused by Atd9-M. fructicola transformant on wounded peaches was similar to that caused by the wild type (wt38c) (Figure 4a). Only significant differences were observed between Atd9-M. fructicola transformant and the wild type wt38c on unwounded peaches after five days of incubation at 20 ◦C and 100% RH (Figure 4b). 9 of 14 he wild Genes 2019, 10, 1033 neither of colony Figure 3. Colony growth of M. fructicola wild type (wt38c) on PDA (a) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and Atd7) on hygromycin supplemented PDA (b–d). Conidia and hyphae of M. fructicola wild type (wt38c) on PDA (e, i) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and At7) on hygromycin supplemented PDA (f–h, j–l) under optical microscopy (× 40) (Leica Microsystems, Wetzlar, Germany) with (i–l) or without (e–h) suitable filter system GFP ET, S (No: 11504174, Leica). Colonies of wt38c and Atd9 were incubated for 8 days at 22 °C, while Atd6 and At7 colonies were incubated for 14 days at the same temperature. Scale bars = 100 µm. Figure 3. Colony growth of M. fructicola wild type (wt38c) on PDA (a) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and Atd7) on hygromycin supplemented PDA (b–d). Conidia and hyphae of M. fructicola wild type (wt38c) on PDA (e,i) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and At7) on hygromycin supplemented PDA (f–h,j–l) under optical microscopy (× 40) (Leica Microsystems, Wetzlar, Germany) with (i–l) or without (e–h) suitable filter system GFP ET, S (No: 11504174, Leica). Colonies of wt38c and Atd9 were incubated for 8 days at 22 ◦C, while Atd6 and At7 colonies were incubated for 14 days at the same temperature. Scale bars = 100 µm. 3.2. Characterization of GFP-M. fructicola Transformants nes 2019, 10, x FOR PEER REVIEW 10 of d the wild type wt38c on unwounded peaches after five days of incubation at 20 °C and 100% R gure 4b). Figure 3. Colony growth of M. fructicola wild type (wt38c) on PDA (a) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and Atd7) on hygromycin supplemented PDA (b–d). Conidia and hyphae of M. fructicola wild type (wt38c) on PDA (e, i) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and At7) on hygromycin supplemented PDA (f–h, j–l) under optical microscopy (× 40) (Leica Microsystems, Wetzlar, Germany) with (i–l) or without (e–h) suitable filter system GFP ET, S (No: 11504174, Leica). Colonies of wt38c and Atd9 were incubated for 8 days at 22 °C, while Atd6 and At7 colonies were incubated for 14 days at the same temperature Scale bars = 100 µm Figure 3. Colony growth of M. fructicola wild type (wt38c) on PDA (a) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and Atd7) on hygromycin supplemented PDA (b–d). Conidia and hyphae of M. fructicola wild type (wt38c) on PDA (e,i) and GFP-Monilinia fructicola transformants (Atd9, Atd6, and At7) on hygromycin supplemented PDA (f–h,j–l) under optical microscopy (× 40) (Leica Microsystems, Wetzlar, Germany) with (i–l) or without (e–h) suitable filter system GFP ET, S (No: 11504174, Leica). Colonies of wt38c and Atd9 were incubated for 8 days at 22 ◦C, while Atd6 and At7 colonies were incubated for 14 days at the same temperature. Scale bars = 100 µm. 2019, 10, x FOR PEER REVIEW 10 o the wild type wt38c on unwounded peaches after five days of incubation at 20 °C and 100% R ure 4b). Aggressiveness tests were only carried out with the At9-M. fructicola transformant and w pe wt38c on disinfected wounded and unwounded peaches. Brown rot incidence caused by At fructicola transformant on wounded peaches was similar to that caused by the wild type (wt3 igure 4a). Only significant differences were observed between Atd9-M. fructicola transform Figure 4. Comparison of Monilinia fructicola wild type wt38c (●) and Atd9-M. fructicola transformant (◌) growth rate (mm) in wounded (a) and non-wounded (b) ’Merryl O’Henry’ peaches. 3.2. Characterization of GFP-M. fructicola Transformants Wounded fruit were inoculated with 10 µl of each strain at 104 (100 conidia per inoculation site) and non- wounded fruit were inoculated with 10 µl at 105 conidia ml−1 (1000 conidia per inoculation site) and incubated for 5 and 7 days, respectively, at 20 °C and 100% relative humidity. Each point represents the mean and vertical bars indicate the standard deviation of the mean (n = 40). For each point, asterisks denote significant differences between strains according to analysis of variance (ANOVA) and LSD test (p < 0.05). Figure 4. Comparison of Monilinia fructicola wild type wt38c (•) and Atd9-M. fructicola transformant (◦) growth rate (mm) in wounded (a) and non-wounded (b) ‘Merryl O’Henry’ peaches. Wounded fruit were inoculated with 10 µl of each strain at 104 (100 conidia per inoculation site) and non-wounded fruit were inoculated with 10 µl at 105 conidia ml−1 (1000 conidia per inoculation site) and incubated for 5 and 7 days, respectively, at 20 ◦C and 100% relative humidity. Each point represents the mean and vertical bars indicate the standard deviation of the mean (n = 40). For each point, asterisks denote significant differences between strains according to analysis of variance (ANOVA) and LSD test (p < 0.05). Aggressiveness tests were only carried out e wt38c on disinfected wounded and unwoun fructicola transformant on wounded peaches w gure 4a). Only significant differences were o with the At9-M. fructicola transformant and w ded peaches. Brown rot incidence caused by At as similar to that caused by the wild type (wt3 bserved between Atd9-M. fructicola transform Figure 4. Comparison of Monilinia fructicola wild type wt38c (●) and Atd9-M. fructicola transformant (◌) growth rate (mm) in wounded (a) and non-wounded (b) ’Merryl O’Henry’ peaches. Wounded fruit were inoculated with 10 µl of each strain at 104 (100 conidia per inoculation site) and non- wounded fruit were inoculated with 10 µl at 105 conidia ml−1 (1000 conidia per inoculation site) and incubated for 5 and 7 days, respectively, at 20 °C and 100% relative humidity. Each point represents the mean and vertical bars indicate the standard deviation of the mean (n = 40). For each point, asterisks denote significant differences between strains according to analysis of variance (ANOVA) and LSD test (p < 0 05) Figure 4. Comparison of Monilinia fructicola wild type wt38c (•) and Atd9-M. 3.3. Microscopy Analysis of M. fructicola GFP-Transformants 3.3. Microscopy Analysis of M. fructicola GFP-Transformants 3.3. Microscopy Analysis of M. fructicola GFP-Transformants 3.3. Microscopy Analysis of M. fructicola GFP-Transformants Green autofluorescence background was not observed in cells of the wild type strain (wt38c) (Figure 3i). However, the three recovered GFP-M. fructicola transformants (Atd6, At7, and Atd9) showed GFP fluorescence when they grew on PDA-H (Figure 3j–l), although Atd6 displayed an irregular fluorescence distribution throughout its mycelia (Figure 3k). py y f f f Green autofluorescence background was not observed in cells of the wild type strain (wt38c) (Figure 3i). However, the three recovered GFP-M. fructicola transformants (Atd6, At7, and Atd9) showed GFP fluorescence when they grew on PDA-H (Figure 3j–l), although Atd6 displayed an irregular fluorescence distribution throughout its mycelia (Figure 3k) Atd9-M. fructicola transformant infection process on nectarines and apples at 22 ◦C was studied using confocal microscopy (Figure 5). Visible epidermal discoloration and brown spots were only observed at the inoculum sites on unwounded nectarine surfaces after 24 h incubation, while disease signs were not evident on apple fruit. Confocal microscopy showed that a higher conidial germination of Atd9 could be observed on surfaces of unwounded nectarines than on wounded apples after 24 h of pathogen inoculation (Figure 5b,g). irregular fluorescence distribution throughout its mycelia (Figure 3k). Atd9-M. fructicola transformant infection process on nectarines and apples at 22 °C was studied using confocal microscopy (Figure 5). Visible epidermal discoloration and brown spots were only observed at the inoculum sites on unwounded nectarine surfaces after 24 h incubation, while disease signs were not evident on apple fruit. Confocal microscopy showed that a higher conidial germination of Atd9 could be observed on surfaces of unwounded nectarines than on wounded apples after 24 h of pathogen inoculation (Figure 5b,g). Figure 5. Infection process by Atd9-M. fructicola transformant on nectarines (a–e) and apples (f–j) under confocal laser scanning microscopy Leica SP2 (Leica Microsystems, Wetzlar, Germany) for 14 days of incubation at 22 ◦C. Nectarine’s tissues were stained with propidium iodide and FM4-64 (a–e) and apple tissues were visualized without stain (f–j). Atd9-M. fructicola transformant conidia on nectarine (a) and apple (f) surface show recent inoculation by the pathogen. Scale bars = 100 µm. Conidia and germ tubes on nectarine (b) and apple (g) surface after 1 day of pathogen inoculation. Scale bars = 25 µm. 3.3. Microscopy Analysis of M. fructicola GFP-Transformants 3.3. Microscopy Analysis of M. fructicola GFP-Transformants Colonization of the uppermost layer of epidermal cells in nectarine (c,d; the latter is a magnification of experiment shown in c) and in apple (h and i, the latter is a magnification of experiment shown in h) by Atd9-M. fructicola hyphae after 4 or 14 days of pathogen inoculation, respectively. Scale bars = 75 µm (c,h) and 50 µm (d,i). Colonization and sporulation on nectarine (e) and apple (j) surface, after 4 or 14 days of Atd9-M. fructicola inoculation, respectively. Scale bars = 50 µm. All images represent maximum projections of a z-stack. Figure 5. Infection process by Atd9-M. fructicola transformant on nectarines (a–e) and apples (f–j) under confocal laser scanning microscopy Leica SP2 (Leica Microsystems, Wetzlar, Germany) for 14 days of incubation at 22 ◦C. Nectarine’s tissues were stained with propidium iodide and FM4-64 (a–e) and apple tissues were visualized without stain (f–j). Atd9-M. fructicola transformant conidia on nectarine (a) and apple (f) surface show recent inoculation by the pathogen. Scale bars = 100 µm. Conidia and germ tubes on nectarine (b) and apple (g) surface after 1 day of pathogen inoculation. Scale bars = 25 µm. Colonization of the uppermost layer of epidermal cells in nectarine (c,d; the latter is a magnification of experiment shown in c) and in apple (h and i, the latter is a magnification of experiment shown in h) by Atd9-M. fructicola hyphae after 4 or 14 days of pathogen inoculation, respectively. Scale bars = 75 µm (c,h) and 50 µm (d,i). Colonization and sporulation on nectarine (e) and apple (j) surface, after 4 or 14 days of Atd9-M. fructicola inoculation, respectively. Scale bars = 50 µm. All images represent maximum projections of a z-stack. Notably, four days after inoculation, massive mesocarp colonization and profuse external sporulation by Atd9-M. fructicola were visible on unwounded nectarine (Figure 5c–e). Atd9 was spread into the apoplastic nectarine space (Figure 5e) through thin and branched hyphae. Thick hyphal groups were observed in certain intracellular nectarine spaces four days after inoculation. In contrast, brown rot symptoms and Atd9-M. fructicola transformant colonization was only found in the uppermost layer of epidermal cells in wounded apple after 14 days of pathogen inoculation (Figure 5h,i). 3.2. Characterization of GFP-M. fructicola Transformants fructicola transformant (◦) growth rate (mm) in wounded (a) and non-wounded (b) ‘Merryl O’Henry’ peaches. Wounded fruit were inoculated with 10 µl of each strain at 104 (100 conidia per inoculation site) and non-wounded fruit were inoculated with 10 µl at 105 conidia ml−1 (1000 conidia per inoculation site) and incubated for 5 and 7 days, respectively, at 20 ◦C and 100% relative humidity. Each point represents the mean and vertical bars indicate the standard deviation of the mean (n = 40). For each point, asterisks denote significant differences between strains according to analysis of variance (ANOVA) and LSD test (p < 0.05). Genes 2019, 10, 1033 the mean and asterisks deno 10 of 14 nt, VA) 10 of 14 int, VA) 4. Discussion fructicola transformant expressing GFP in comparison of the interaction of this pathogen with nectarine and apple using confocal microscopy. p g pp g py In this work, three GFP-M. fructicola transformants, confirmed by PCR and western blotting, were recovered using ATM-transformation and germinated conidia. Only transformant Atd9 showed similar growth and pathogenic characteristics to wild type, but a slightly increased virulence on unwounded nectarines was also observed. The presence of morphological and pathogenic defects suggested T-DNA integration effect during the transformation process. Thus, we used a standardized technique to verify the insertion limits of T-DNA integration. The initial 50–60 bp from recovery sequences belong to the pPK2-hphgfp vector and the following sequence aligns with M. fructicola genomes, confirming that genome integration and three independent transformation events have occurred due to their different location. In model plant-pathogen Magnaporthe oryzae, two systematic analyses of T-DNA insertion revealed that most transformation events do not cause extreme chromosomal rearrangements, although they produced deletions, inversions, and translocations [44–46]. We observed that in Atd6 (referenced to the M. fructicola LMK125 genome), an inversion of part of the scaffold might have been generated. ATMT showed integration bias toward the non-coding region and promoter regions in M. oryzae and L. maculans [44–47]. Considering the automatic prediction of genes, in the right border of At7, the ATMT transformation seems to have disrupted a gene. Atd6 and At7 transformants presented a different phenotype to the wild type strain, which can be explained in part by a T-DNA integration effect. PCR analyses of Atd9 transformant suggested that most nuclei carry the construct although we cannot discard the presence of wild type nuclei in the mycelium. In addition, these analyses showed that the integration of the construct resulted in a duplication of a small genomic region in the left border. The broader PCR analysis (Figure 2b) detected regions adjacent to the integration sites in scaffolds 014 and 005, supporting the idea of a reorganization of the original genome of M. fructicola wt38c in the Atd9 transformant. The T-DNA integration effects described for the three transformants may be, in part, the cause of the low number of M. fructicola transformants recovered from ATMT. Here, we used the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) from A. nidulans to drive the expression of the gfp reporter gene fused with the hygromycin B resistance gene. As reporter for other fungal species, the gpdA promoter from A. 4. Discussion The use of an aggressive M. fructicola transformant that expresses a green fluorescent chimera has allowed comparison of the infection process on unwounded nectarine against that on wounded apple over a 15-day incubation period. The M. fructicola infection process for nectarines observed by confocal microscopy in the present study was similar to that reported by García-Benítez et al. [6] by optical 11 of 14 11 of 14 Genes 2019, 10, 1033 microscopy using infected tissue histological cuts, which required a great deal of time and effort to obtain. Confocal microscopy is a potent technique that allows in depth high-resolution analysis from many samples [32]. Thus, it allowed us to quickly study the infection and colonization on different hosts by GFP-Monilinia. Fluorescent-tagged proteins have been successfully used in living tissues to study host-pathogen interactions [17,33]. Other plant-pathogen interactions such as Botrytis cinerea in strawberry [20], Fusarium oxysporum in tomato [34] or Penicillium digitatum in orange [35] have been reported, where the expression of a fluorescent protein did not affect their pathogenic capacity. GFP-M. fructicola transformant germinated on the surface of nectarines and apples, but apple colonization by the pathogen was notably delayed compared with nectarines. This observation is in agreement with M. fructicola being classified as a secondary pathogen on apples, while it is a prominent pathogen on stone fruit [1]. M. fructicola, M. fructigena, and M. laxa are polytrophs and infect a wide range of Rosaceae species, but there is host specialization in some cases [36]. M. fructicola is mainly limited to fruit and blossoms in stone fruit orchards (peach, nectarine, plum, and apricots). However, M. fructicola could affect apples at or near maturity with tan to white zones of sporulation [2,11,16]. We have transformed germinated conidia of M. fructicola using Agrobacterium tumefaciens and expressed a chimera consisting of GFP fused to the protein conferring HygB resistance, Hph. Other authors have reported transformed M. fructicola isolates (Bmpc-7, DL25W and MUK-1) to express genes of interest under control of native promoters [37–40] and also with A. nidulans trpC as an exogenous promoter [41,42]. A preliminary Agrobacterium tumefaciens-mediated transformation of M. fructicola with a binary vector expressing GFP has been reported [43]. However, no GFP-expressing M. fructicola strains have been used on pathogenicity works at the moment [38,39,41,43]. This is the first report describing the use of a M. 5. Conclusions GFP-M. fructicola has allowed to demonstrate certain host specificity by the pathogen. Furthermore, Atd9-M. fructicola had similar properties as the wild type, hence opening a new avenue for future modifications in M. fructicola dedicated to deciphering its pathogenic mechanisms in different fruit stages and host compatibility. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/12/1033/s1, Figure S1: TAIL-PCR analysis of border junctions of M. fructicola transformants. The agarose gel analysis from top to the bottom show products of three rounds of PCR with specific primer for the left (LB1/LB2/LB3) or right (RB1/RB2/RB3) border of T-DNA and degenerate oligonucleotides (AD1/AD2) of each M. fructicola transformant (Atd9, Atd6 and At7). The bands surrounded by dashed squares were excised from the gel and sequenced, Table S1: Conditions used for TAIL-PCR; Table S2. Sequence analysis of the T-DNA border junctions (left and right borders) in the Monilinia fructicola transformants (Atd9, Atd6, and AT7). Author Contributions: Conceptualization, E.A.E. and A.D.C.; Methodology, S.R.-P., N.B.-M., R.T., E.A.E. and A.D.C.; Formal Analysis, S.R.-P., E.A.E.; Investigation, S.R.-P. and N.B.-M.; Data Curation, E.A.E. and A.D.C.; Writing-Original Draft Preparation, S.R.-P.; Writing-Review & Editing, E.A.E., P.M. and A.D.C.; Supervision, E.A.E. and A.D.C.; Project Administration, E.A.E. and A.D.C.; Funding Acquisition, A.D.C., E.A.E., R.T. and P.M. Funding: This study was supported by grants AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R from the Ministry of Science, Innovation and Universities (Spain), Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER, EU). S. Rodríguez-Pires received a PhD fellowship from the Ministry of Science, Innovation and Universities (Spain). Acknowledgments: We thank J. Martínez-Cruz (Microbiology Department, Malaga University, Spain) for providing the Agrobacterium tumefaciens strain LBA4404 carrying pPK2-hphgfp, and R. Castillo for her technical support. Conflicts of Interest: The authors declare no conflict of interest. 4. Discussion nidulans is an efficient genetic tool to constitutively express genes of interest [26,48]. 12 of 14 12 of 14 Genes 2019, 10, 1033 References 1. Byrde, R.J.W.; Willetts, H.J. The Brown Rot Fungi of Fruit: Their Biology and Control; Pergamon Press Ltd.: Oxford, UK, 1977; p. 188. 2. Vasi´c, M.; Vico, I.; Jurick, W.M.; Duduk, N. Distribution and characterization of Monilinia spp. causing apple fruit decay in Serbia. Plant Dis. 2018, 102, 359–369. [CrossRef] [PubMed] 3. De Cal, A.; Gell, I.; Usall, J.; Viñas, I.; Melgarejo, P. First report of brown rot caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain. Plant Dis. 2009, 93, 763. [CrossRef] [PubMed] 4. Villarino, M.; Egüen, B.; Lamarca, N.; Segarra, J.; Usall, J.; Melgarejo, P.; De Cal, A. Occurrence of Monilinia laxa and M. fructigena after introduction of M. fructicola in peach orchards in Spain. Eur. J. Plant Pathol. 2013, 137, 835–845. [CrossRef] 5. Villarino, M.; Melgarejo, P.; De Cal, A. Growth and aggressiveness factors affecting Monilinia spp. survival peaches. Inter. J. Food Microbiol. 2016, 227, 6–12. [CrossRef] [PubMed] 6. Garcia-Benitez, C.; Melgarejo, P.; De Cal, A.; Fontaniella, B. Microscopic analyses of latent and visible Monilinia fructicola infections in nectarines. PLoS ONE 2016, 11, e0160675. [CrossRef] 7. Washington, W.S.; Pascoe, I. First record of Monilinia fructicola on strawberry fruit in Victoria, Australia. Australas. Plant Pathol. 2000, 29, 70. [CrossRef] 8. Janisiewicz, W.J.; Nichols, B.; Takeda, F.; Jurick, W.M. First report of Monilinia fructicola causing postharvest decay of strawberries (Fragaria × ananassa) in the United States. Plant Dis. 2017, 101, 1823. [CrossRef] 9. Bevacqua, D.; Quilot-Turion, B.; Bolzoni, L. A model for temporal dynamics of brown rot spreading in fruit orchards. Phytopathology 2018, 108, 595–601. [CrossRef] 10. Duchoslavová, J.; Širuˇcková, I.; Zapletalová, E.; Navrátil, M.; Šafáˇrová, D. First report of brown rot caused by Monilinia fructicola on various stone and pome fruits in the Czech Republic. Plant Dis. 2007, 91, 907. [CrossRef] 11. Grabke, A.; Hu, M.J.; Luo, C.X.; Bryson, P.K.; Schnabel, G. First report of brown rot of apple caused by Monilinia fructicola in Germany. Plant Dis. 2011, 95, 772. [CrossRef] 12. Vasi´c, M.; Duduk, N.; Ivanovi´c, M.M.; Obradovi´c, A.; Ivanovi´c, M.S. First report of brown rot caused by Monilinia fructicola on stored apple in Serbia. Plant Dis. 2012, 96, 456. [CrossRef] [PubMed] 13 of 14 13 of 14 Genes 2019, 10, 1033 13. Martini, C.; Di Francesco, A.; Lantos, A.; Mari, M. First report of asiatic brown rot (Monilinia polystroma) and brown rot (Monilinia fructicola) on pears in Italy. Plant Dis. 2015, 99, 556. References [CrossRef] 14. Peter, K.A.; Gaskins, V.L.; Lehman, B.; Jurick, W.M. First report of brown rot on apple fruit caused by Monilinia fructicola in Pennsylvania. Plant Dis. 2015, 99, 1179. [CrossRef] 15. Zhu, X.Q.; Niu, C.W.; Chen, X.Y.; Guo, L.Y. Monilinia species associated with brown rot of cultivated apple and pear fruit in China. Plant Dis. 2016, 100, 2240–2250. [CrossRef] 16. Pereira, W.V.; May-De-Mio, L.L. First report of brown rot caused by Monilinia fructicola on apple in Brazil. Plant Dis. 2018, 102, 2657. [CrossRef] 17. Horowitz, S.; Freeman, S.; Sharon, A. Use of green fluorescent protein-transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum. Phytopathology 2002, 92, 743–749. [CrossRef] 17. Horowitz, S.; Freeman, S.; Sharon, A. Use of green fluorescent protein transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum. Phytopathology 2002, 92, 743–749. [CrossRef] 18. Martínez-Cruz, J.; Romero, D.; de Vicente, A.; Pérez-García, A. Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens. New Phytol. 2017, 213, 1961–1973. 19. Isshiki, A.; Ohtani, K.; Kyo, M.; Yamamoto, H.; Akimitsu, K. Green fluorescent detection of fungal colonization and endopolygalacturonase gene expression in the interaction of Alternaria citri with citrus. Phytopathology 2003, 93, 768–773. [CrossRef] 20. Li, X.Z.; Zhou, T.; Yu, H. Transformation of Botrytis cinerea with a Green Fluorescent Protein (GFP) Gene for the Study of Host-pathogen Interactions. Plant Pathol. J. 2006, 6, 134–140. 21. De Silva, A.P.; Bolton, M.D.; Nelson, B.D. Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts. Plant Pathol. 2009, 58, 487–496. [CrossRef] 22. Bansal, K.; Rollins, J. Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity. Phytopathology 2016, 106, 152. 23. Egüen, B.; Melgarejo, P.; De Cal, A. Sensitivity of Monilinia fructicola from Spanish peach orchards to thiophanate-methyl, iprodione, and cyproconazole: Fitness analysis and competitiveness. Eur. J. Plant Pathol. 2015, 141, 789–801. [CrossRef] 24. Michielse, C.B.; van Wijk, R.; Reijnen, L.; Cornelissen, B.J.; Rep, M. Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis. Genome Biol. 2009, 10, R4. [CrossRef] 5. Michielse, C.B.; Hooykaas, P.J.; van den Hondel, C.A.; Ram, A.F. Agrobacterium-mediated transformatio the filamentous fungus Aspergillus awamori. Nat. Protoc. 2008, 3, 1671–1678. [CrossRef] the filamentous fungus Aspergillus awamori. Nat. Protoc. 2008, 3, 1671 1678. [CrossRef] 26. Hernández-Ortiz, P.; Espeso, E.A. References AFLP analysis of intraspecific variation between Monilinia laxa isolates from different hosts. Plant Dis. 2008, 92, 1616–1624. [CrossRef] [PubMed] 37. Chen, S.; Yuan, N.; Schnabel, G.; Luo, C. Function of the genetic element “Mona” associated with fungicide resistance in Monilinia fructicola. Mol. Plant Pathol. 2017, 18, 90–97. [CrossRef] 38. Chou, C.M.; Yu, F.Y.; Yu, P.L.; Ho, J.F.; Bostock, R.M.; Chung, K.R.; Huang, J.-W.; Lee, M.H. Expression of five endopolygalacturonase genes and demonstration that MfPG1 overexpression diminishes virulence in the brown rot pathogen Monilinia fructicola. PLoS ONE 2015, 10, e0132012. [CrossRef] 39. Lee, M.H.; Bostock, R.M. Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola. Curr. Genet. 2006, 49, 309–322. [CrossRef] 40. Lee, M.H.; Chiu, C.M.; Roubtsova, T.; Chou, C.M.; Bostock, R.M. Overexpression of a redox-regulated cutinase gene, MfCUT1, increases virulence of the brown rot pathogen Monilinia fructicola on Prunus spp. Mol. Plant Microbe Interact. 2010, 23, 176–186. [CrossRef] 41. Dai, Q.; Sun, Z.; Schnabel, G. Development of spontaneous hygromycin B resistance in Monilinia fructicola and Its impact on growth rate, morphology, susceptibility to demethylation Inhibitor fungicides, and sporulation. Phytopathology 2003, 93, 1354–1359. [CrossRef] 42. Yu, P.L.; Wang, C.L.; Chen, P.Y.; Lee, M.H. YAP1 homologue-mediated redox sensing is crucial for a successful infection by Monilinia fructicola. Mol. Plant Pathol. 2016, 1, 1–15. 43. Marek, S.M.; Pan, Z.; Ciuffetti, L.M.; Bostock, R.M. Agrobacterium tumefaciens-mediated transformation of Monilinia fructicola with green fluorescent protein. In Proceedings of the APS Annual Meeting, Milkwaukee, WI, USA, 27–31 July 2002. P2002-0673-AMA. 44. Li, G.; Zhou, Z.; Liu, G.; Zheng, F.; He, C. Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Curr. Genet. 2007, 51, 233–243. [CrossRef] [PubMed] 45. Choi, J.; Park, J.; Jeon, J.; Chi, M.H.; Goh, J.; Yoo, S.Y.; Park, J.; Jung, K.; Kim, H.; Rho, H.S.; et al. Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae. Mol. Microbiol. 2007, 66, 371–382. [CrossRef] [PubMed] 46. Meng, Y.; Patel, G.; Heist, M.; Betts, M.F.; Tucker, S.L.; Galadima, N.; Donofrio, N.M.; Brown, D.; Mitchell, T.K.; Xu, J.R.; et al. A systematic analysis of T-DNA insertion events in Magnaporthe oryzae. Fungal Genet. Biol. 2007, 44, 1050–1064. [CrossRef] [PubMed] 47. Bourras, S.; Meyer, M.; Grandaubert, J.; Lapalu, N.; Fudal, I.; Linglin, J.; Ollivier, B.; Blaise, F.; Balesdent, M.-H.; Rouxel, T.; et al. References Phospho-regulation and nucleocytoplasmic trafficking of CrzA in response to calcium and alkaline-pH stress in Aspergillus nidulans. Mol. Microbiol. 2013, 89, 532–551. [CrossRef] 6. Hernández-Ortiz, P.; Espeso, E.A. Phospho-regulation and nucleocytoplasmic trafficking of CrzA in respo to calcium and alkaline-pH stress in Aspergillus nidulans. Mol. Microbiol. 2013, 89, 532–551. [CrossRef] 27. Meirinho, S.; Carvalho, M.; Dominguez, Á.; Choupina, A. Isolation and characterization by asymmetric PCR of the ENDO1 gene for glucan endo-1,3-β-D-glucosidase in Phytophthora cinnamomi associated with the ink disease of Castanea sativa Mill. Braz. Arch. Biol. Technol. 2010, 53, 513–518. [CrossRef] 28. Hoff, K.J.; Stanke, M. WebAUGUSTUS—A web service for training AUGUSTUS and predicting genes in eukaryotes. Nucleic Acids Res. 2013, 41, W123–W128. [CrossRef] 29. Sauer, D.B.; Burroughs, R. Disinfection of seed surfaces with sodium hypochlorite. Phytopathology 1986, 76, 745–749. [CrossRef] 30. Marcel, S.; Sawers, R.; Oakeley, E.; Angliker, H.; Paszkowski, U. Tissue-adapted invasion strategies of the rice blast fungus Magnaporthe oryzae. Plant Cell 2010, 22, 3177–3187. [CrossRef] 31. Jones, K.; Kim, D.W.; Park, J.S.; Khang, C.H. Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae. BMC Plant. Biol. 2016, 16, 69. [CrossRef] 32. Hardham, A.R. Confocal Microscopy in Plant–Pathogen Interactions. In Plant Fungal Pathogens. Methods in Molecular Biology (Methods and Protocols); Bolton, M.T.B., Ed.; Humana Press: Totowa, NJ, USA, 2012; pp. 295–309. 33. Buron-Moles, G.; López-Pérez, M.; González-Candelas, L.; Viñas, I.; Teixidó, N.; Usall, J.; Torres, R. Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples. Inter. J. Food Microbiol. 2012, 160, 162–170. [CrossRef] [PubMed] 34. Lagopodi, A.L.; Ram, A.F.; Lamers, G.E.; Punt, P.J.; Van den Hondel, C.A.; Lugtenberg, B.J.; Bloemberg, G.V. Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker. Mol. Plant Microbe Interact. 2002, 15, 172–179. [CrossRef] [PubMed] 14 of 14 14 of 14 Genes 2019, 10, 1033 35. Vu, T.X.; Ngo, T.T.; Mai, L.T.D.; Bui, T.T.; Le, D.H.; Bui, H.T.V.; Nguyen, H.Q.; Ngo, B.X.; Tran, V.T. A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization. J. Microbiol. Methods. 2018, 144, 134–144. [CrossRef] [PubMed] g g J [CrossRef] [PubMed] 36. Gril, T.; Celar, F.; Munda, A.; Javornik, B.; Jakse, J. References Incidence of genome structure, DNA asymmetry, and cell physiology on T-DNA integration in chromosomes of the phytopathogenic fungus Leptosphaeria maculans. G3 Genes Genomes Genet. 2012, 2, 891–904. [CrossRef] [PubMed] 48. Villarino, M.; Etxebeste, O.; Mendizabal, G.; Garzia, A.; Ugalde, U.; Espeso, E.A. Boron tolerance in Aspergillus nidulans is sustained by the SltA pathway through the SLC-family transporters SbtA and SbtB. Genes 2017, 8, 188. [CrossRef] © 2019 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 (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W1996945852
https://zenodo.org/records/2526241/files/01_PIEB_Vol12_Issue3_2012_Kreczmanska-Gigol_and_Liberadzki_Stepped_coupon_bonds_factoring_pp.5-14.pdf
English
null
Stepped coupon bonds and restructuring factoring in relation to net circulating capital in companies in financial difficulty
Perspectives of Innovations, Economics and Business
2,012
cc-by
4,833
Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 ISSN 1804-0519 (Print), ISSN 1804-0527 (Online) www.academicpublishingplatforms.com Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 ISSN 1804-0519 (Print), ISSN 1804-0527 (Online) www.academicpublishingplatforms.com Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 ISSN 1804-0519 (Print), ISSN 1804-0527 (Online) www.academicpublishingplatforms.com KATARZYNA KRECZMAŃSKA-GIGOL, MARCIN LIBERADZKI Collegium of Management and Finance, Warsaw School of Economics, Poland Collegium of Management and Finance, Warsaw School of Economics, Poland Collegium of Management and Finance, Warsaw School of Economics, Poland JEL Classifications: G32, G34, G33 Introduction Companies in financial difficulty, despite the “healthy business”, have a limited access to the sources of financing and, consequently, problems related to the maintenance of financial liquidity. One of the indications of financial liquidity problems is an ill-adjusted net circulating capital level to the demand for net circulating capital. The problem may be tackled in two ways: either through raising the value of net circulating capital or through the reduction of demand for this capital. The instrument which may be used to raise this level is the stepped coupon bonds, a novel solution non-existent on the Polish market. Restructuring factoring applied sporadically in Poland and Germany is an instrument reducing the demand for the corporate net circulating capital. Although it is not a new instrument, it is very seldom applied. These two instruments may be used by companies in a poor financial situation, the co- operation with which is burdened with high risk and the ones which cannot finance their activities applying standard financial services. corresponding email: kgigol[at]sgh(dot)waw(dot)pl postal address: Warsaw School of Economics, aleja Niepodległości 162, Warszawa, Poland STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY KATARZYNA KRECZMAŃSKA-GIGOL, MARCIN LIBERADZKI JEL Classifications: G32, G34, G33 Keywords: Stepped coupon bonds, factoring, company restructuring, financing Abstract: The instrument to be applied in order to raise net circulating capital is stepped coupon bonds, a novel solution non-existent on the Polish market. Restructuring factoring applied sporadically in Poland and Germany is an instrument reducing the demand for the corporate net circulating capital. The article presents the idea of these instruments and the possibility of using them by a company in financial difficulty. http://dx.doi.org/10.15208/pieb.2012.12 Vol.12 (3), PP.5-14 Source: Kreczmanska-Gigol K., Liberadski M., 2012. "Stepped coupon bonds and restructuring factoring in relation to net circulating capital in companies in financial difficulty", Perspectives of Innovations, Economics & Business, Vol.12(3), pp.5-14, http://dx.doi.org/10.15208/pieb.2012.12 Vol.12 (3), PP.5-14 The idea of high risk bonds (stepped coupon bonds). The question of bond issue cost The standard approach to bond cash flow modeling is the so-called straight bullet bond. It is a fixed interest bond with no option clauses, with interest paid corresponding email: kgigol[at]sgh(dot)waw(dot)pl postal address: Warsaw School of Economics, aleja Niepodległości 162, Warszawa, Poland Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY periodically, repurchased through a one-time payment of the nominal bond value made on the maturity date. This model of security repurchase, standard on financial markets, is relatively advantageous for debtors as it allows for the delay of the repayment of debt (principal) in comparison with a bank loan1. Naturally, it refers to the standard repayment models, i.e. generally applied with regard to all financial instruments. The aim of the modification of the payback construction of a ”classical” bond is raising its credit function, i.e. the longest possible postponement the repayment of principal and interest. One of the solutions is zero-coupon bonds, however with reference to corporate debt, which is burdened with credit risk; the average payback period is too distant for a number of investors so they prefer to be paid periodically before the whole security is repurchased. In search of an in-between solution the so-called stepped coupon bonds were constructed. This article presents the straight bullet bond modification called stepped coupon bond. The modification is aimed at transferring the burden of interest bond payback till later with the simultaneous maintenance of interest payment and debt issue cost control. The bond issue debt costs may be divided into two groups: - “transactional”: cost connected with consultancy, prospectus, issue servicing and also possibly issue guarantee; - “transactional”: cost connected with consultancy, prospectus, issue servicing and also possibly issue guarantee; - the yield to maturity of bonds (YTM) generated for investors results from two sources: the coupon paid to the bond holders by the issuer and the difference between the bond sale price and the nominal value. - the yield to maturity of bonds (YTM) generated for investors results from two sources: the coupon paid to the bond holders by the issuer and the difference between the bond sale price and the nominal value. YTM is the most frequently quoted measure of the bond profitability. It is an internal rate of return on investment in this instrument. 6 1 The standard model of the repayment (amortisation) of a bank loan is its payback in periodical principal/interest instalments. The idea of high risk bonds (stepped coupon bonds). The question of bond issue cost YTM is an interest rate to comply with the following equation: , (1)   1 1 1 ) / 1( 100 / 1          n E B n i i E B M YTM M YTM CFi P (1) Where, P - the bond price, M - the number of coupon payments in annual terms, A - duration (in days) of the current coupon period; B - the number of days to the beginning of the next interest period from the settlement day; E - duration (in days) of the interest period in which the settlement is made; CFi - i cash flow generated by the bond on account of interest, n - the number of remaining monetary flows from the moment of bond estimation till its repurchase. The price of bond contains two elements: rate (K) and accrued interest in the current coupon period (for A days from the last coupon). It is assumed that the accrued interest between coupon periods is calculated with the application of simple interest, thus the rate is the difference between the dirty price and accrued interest: (2) i CF E A P K *   (2) 6 1 The standard model of the repayment (amortisation) of a bank loan is its payback in periodical principal/interest instalments. International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY If one deducts from the dirty bond price some additional costs, connected with the organisation and implementation of the issue, incurred on the day of the issue and deducted from the issue income, the YTM calculated on the basis of the reduced security price will include the total debt cost. Stepped coupon bonds. Structure The stepped-coupon is a fixed interest bond characterised by a predetermined scheme of interest payment. TABLE 1. PARAMETERS OF BONDS ISSUED BY COMPANY A Bond A dates Bond A interest rate Bond B dates Bond B interest rate Bond C dates Bond C interest rate 5 May 2012 (settlement) 0.045 5 May 2012 (settlement) 0.045 5 May 2012 (settlement) 0.025 2 February 2013 (coupon change) 0.07875 2 February 2013 (coupon change) 0.07875 2 February 2013 (coupon change) 0.05 2 August 2013 (coupon change) 0.1025 2 February 2014 (coupon change) 0.1025 2 February 2014 (coupon change) 0.0750 2 February 2014 payback 2 February 2015 (coupon change) 0.14 2 February 2016 (coupon change) 0.14 2 February 2016 Payback 2 February 2017 payback Source: Own material. TABLE 1. PARAMETERS OF BONDS ISSUED BY COMPANY A Let us consider the following example: on 5 May 2012 a company issues bonds in series A, B and C. They pay interest half-yearly. The remaining parameters of the discussed securities are included in Table 1. Bonds in different series have different maturities and interest values. These bonds are characterised by the coupon accruing by a definite value for each series. The scheme of cash flow generated by every security is illustrated in Figures 1, 2 and 3. - 7 - International Cross-Industry Journal FIGURE 1. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES A Source: Own material. 1 Time Relative payment FIGURE 1. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES A Relative payment Time Source: Own material. Source: Own material. International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY 8 FIGURE 2. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES B Source: Own material. FIGURE 3. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES C Source: Own material. The increase in the level of coupons in relation to earlier payments must accompanied by a simultaneous debt cost control. The YTM measure will 1 Time Relative payment 1 Time Relative payment FIGURE 2. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES B Source: Own material. 1 Time Relative payment FIGURE 2. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES B Source: Own material. 1 Time Relative payment FIGURE 2. Stepped coupon bonds. Structure SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES B Time Source: Own material. FIGURE 3. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES C FIGURE 3. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES C FIGURE 3. SCHEME OF CASH FLOW GENERATED BY STEPPED COUPON BONDS SERIES C Source: Own material. 1 Time Relative payment Source: Own material. 1 Time Relative payment Time Source: Own material. - 8 - The increase in the level of coupons in relation to earlier payments must be accompanied by a simultaneous debt cost control. The YTM measure will be The increase in the level of coupons in relation to earlier payments must be accompanied by a simultaneous debt cost control. The YTM measure will be Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY applied in this connection. Table 2 shows bond issue prices reduced by issue costs per one security as well as YTM rates resulting from the calculations and interpreted as debt cost. TABLE 2. BOND ISSUE PRICES INCLUDING THE DEBT COST Bond A Bond B Bond C Price including issue cost 101.23 102.45 103.05 Debt cost 0.06276 0.08403 0.06498 Source: Own material TABLE 2. BOND ISSUE PRICES INCLUDING THE DEBT COST From the perspective of the payment schedule stepped-coupon bonds facilitate raising long-term capital by the issuer. Their structure reflects the desire to postpone the burden of interest payment till later. Thus, from the point of view of bond repayment schedule, it is a compromise between a classical fixed interest bond and zero-coupon bond. Considering the value of money over time, the characteristics of variable coupons may be selected in such a way that the debt cost perceived as its internal rate of return would remain unchanged in relation to classical repayment methods. International Cross-Industry Journal The idea of restructuring factoring - 9 - International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY The factor’s tasks within the restructuring factoring may be divided into two groups: The factor’s tasks within the restructuring factoring may be divided into two groups: - tasks convergent with liability factoring (negotiation of terms of agreement with suppliers, guarantee payment to factoree’s suppliers, financing current factoree’s debts, management of the current and outdated liabilities), - tasks convergent with liability factoring (negotiation of terms of agreement with suppliers, guarantee payment to factoree’s suppliers, financing current factoree’s debts, management of the current and outdated liabilities), - tasks convergent with the factoring of existing and future debts (refinancing of current factoree’s liabilities, management of current and delayed factoree’s liabilities, including debt recovery, financing contract execution, i.e. financing of orders). - tasks convergent with the factoring of existing and future debts (refinancing of current factoree’s liabilities, management of current and delayed factoree’s liabilities, including debt recovery, financing contract execution, i.e. financing of orders). In the process of factoree’s restructuring, the factor is a participant of restructuring (factor’s staff take over the duties connected with factoree’s finance management), which supervises the process of the company restructuring and is a guarantee of payment for factoree’s creditors. Not every company can make use of restructuring factoring. The conditions to be met by the factoree in the transaction of restructuring factoring are as follows: the factoree has already used debt or liability factoring and is known to the factor, factoree’s problems are connected with the company’s finance and not any other sphere (the factoree is a company with a sound core business and it is very likely that the financial restructuring will be effective), the factoree agrees that during restructuring the factor becomes factoree’s co-shareholder and the factoring is secured by the company shares, the factoree agrees that the factor will manage or co-manage the company finance in the course of restructuring. The price of restructuring factoring is higher than the price of other types of factoring (debt factoring and liability factoring). The idea of restructuring factoring Factoring is a complex heterogeneous service. It means a long-term cooperation: - between a factoree, who is goods or services provider or buyer of goods, products and services making use of trade credit granted by its suppliers and a specialized factoring institution called factorer, which results in: - between a factoree, who is goods or services provider or buyer of goods, products and services making use of trade credit granted by its suppliers and a specialized factoring institution called factorer, which results in: - transfer of liabilities by the factoree to factor and a number of services provided by the factorer in favour of factoree connected with the transfer of liabilities as well as additional services not connected with the transfer itself; - or repayment by the factor of the current factoree’s liabilities and extension of the period of repayment of these liabilities as well as rendering services connected or not with the repaid liabilities. - or repayment by the factor of the current factoree’s liabilities and extension of the period of repayment of these liabilities as well as rendering services connected or not with the repaid liabilities. On the market there are many kinds of factoring. We may classify restructuring factoring according to the criterion of factoring object as complex factoring which is composed of different objects or it may be classified within special forms of factoring. It is a kind of factoring that consists in the cooperation of the company in financial difficulty with the factor with regard to the restructuring of the company. The object of the restructuring factoring is the cession of existing liabilities, cession of future liabilities, subrogation in which the factor pays the factoree’s debts to suppliers and enters into the rights of satisfied creditors; as well as a number of services connected with the transferred liabilities, services connected with the repaid debts and well- developed additional services connected neither with the transferred debts nor repaid liabilities. International Cross-Industry Journal The idea of restructuring factoring Factor’s high risk makes risk margin higher, which results in an increased cost of funds acquired from the factor. However, the price in this sort of transaction is not most important as restructuring factoring gives a company a chance to survive despite financial problems the company suffers from. The primary effects of restructuring factoring for factors and factorees are: the application of restructuring factoring allows the factoree to survive despite financial problems, restructuring factoring makes financial costs high but the company liquidity is maintained, the payment for the factor for increased risk as well as the finance management know-how shared with the factoree is a high margin in comparison with other kinds of factoring. The duration of a restructuring factoring agreement is set on individual basis, but it is usually not shorter than 2-3 years. It may be prolonged if need be; the factor may take advantage of the security and take over the company, if the restructuring proves unsuccessful and the company has some liabilities to the factor. The cooperation may be terminated if the restructuring is successful or restructuring factoring may be replaced by debt or liability factoring. From the perspective of the company making use of restructuring factoring, it results in the shortening of liabilities turnover cycle due to payment of liabilities before maturity and the extension of trade liabilities turnover cycle because the factor usually settles factoree’s liabilities to suppliers and prolongs - 10 - International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY the deadlines for the factoree. The effects of restructuring factoring are the sum of effects of debt and liability factoring. the deadlines for the factoree. The effects of restructuring factoring are the sum of effects of debt and liability factoring. International Cross-Industry Journal Net circulating capital management According to the balance perspective, the net circulating capital is a part of circulating assets financed with fixed capitals (Michalski, 2005). From the capital perspective, the net circulating capital is a surplus of fixed capitals (own capitals and long-term liabilities) over fixed capitals (Ostaszewski and Cicirko, 2006). Thus, it is this part of fixed capital which remains after financing fixed assets and is to finance circulating assets (Skowronek-Mielczarek, 2003). The volume of corporate net circulating capital changes under the influence of changes in the level of fixed assets and fixed capitals. The volume of net circulating capital is an important indicator of liquidity as it determines the company liquidity risk level. Apparently, companies which permanently lost the ability to settle their liabilities and as a consequence went bankrupt, had shown a negative level of net circulating capital for a long time (4 years) (Skowronek-Mielczarek, 2003). In practice, the financial liquidity is affected not only by the level of net circulating capital but also the adjustment of the net circulating capital level to the demand for the net circulating capital in a given company. The demand for net circulating capital may be calculated in the following way: PSD ODP DSO OKZ ZKON * ) (    , (3) Where, ZKON - net circulating capital demand; OKZ - period of stock conversion in days; DSO - period of receivables inflow in days; ODP - period of deferred payment in days. If the demand for net circulating capital is higher than the level of net circulating capital, the company financial liquidity may be threatened. In such a situation it is necessary to raise the level of net circulating capital or reduce the demand for net circulating capital (Table 3). Net circulating capital management includes the development of capital structure as well as management of corporate circulating assets which determine the demand for net circulating capital. There are three objectives of the net circulating capital management in a company. They are: rise in activity effectiveness, improvement of the capital structure and release of capital involved in the company activity (Ertl, 2004). Net circulating capital management - 11 - International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY TABLE 3. METHODS OF RAISING THE LEVEL OF NET CIRCULATING CAPITAL AND REDUCTION OF DEMAND FOR NET CIRCULATING CAPITAL Methods of raising the level of net circulating capital 1. The decline in the value of fixed assets without the decline in the value of fixed capitals - Sale of redundant fixed assets - Leaseback - Resignation from investment 2. The rise in the value of fixed capitals without the rise in the value of fixed assets - Acquiring fixed capitals and leaving them in the company - Raising corporate basic capital, for example through raising shares of current shareholders or acquisition of a new investor - Incurring subordinated debt - Rise in long-term indebtedness, including long- term debt securities issue M th d f d ti f d d f t i l ti it l Methods of reduction of demand for net circulating capital 3. Shortening of liabilities turnover cycle - Limitation in granting trade credits - Improvement of liabilities management effectiveness, including improved promptness of liabilities settlement, effective debt recovery, effect accompanying debt restructuring factoring - Debt cession, including debt and restructuring factoring 4. Shortening of stock turnover cycle - Improvement of stock management effectiveness, including just-in-time system - Reduction in sales prices of goods and services, including seasonal sale 5. Extension of trade liabilities turnover cycle - Negotiating longer periods of trade credit with suppliers - Trade liabilities settlement delays - Liability factoring and restructuring factoring Source: Own material The basic goal of corporate net circulating capital management is the maintenance of the optimal volume and structure of circulating assets and most advantageous structure of financing sources. The optimal level of circulating capital is to result in the minimization of the company liquidity risk, and at the same time to allow for possibly high profits. Possibilities of using stepped coupon bonds and restructuring factoring as instruments of net circulating capital management 12 Analysing Table 3, one can notice that the level of net circulating capital is developed in the long-term as it is difficult to increase the value of engaged fixed capitals in the short-term if it is not to be accompanied by the rise in fixed assets. A company can incur a long-term loan most easily, but long-term loans are usually granted to finance investment needs which go together with the rise - 12 - International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY in the value of corporate fixed assets. On the other hand, the rise in the value of own capitals occurring irrespective of the rise or fall in the value of fixed assets results from long-term operations. The sale of fixed assets is seldom possible. As a rule, there is a long time from the selling decision till the moment they have been actually sold and payment received. Leaseback is a perfect way to raise the level of own capitals. However, it is not free from flaws. Namely: a company can make use of it only once, this type of lease is least willingly offered by lessors and the condition of leaseback use is the possession of assets with the clear mortgage established in favour of the company’s creditors. In such a situation, the issue of long-term debt securities, which are a kind of loan, is a perfect way of raising the level of corporate net circulating capital. The company does not have to use the funds acquired from the issue for a particular purpose, which in the case of a long-term bank loan is the purchase of fixed assets. If a company is in financial trouble, financing it by creditors is connected with a higher risk for them. A company with a negative or low level of net circulating capital, if this capital is not enough to meet the demand for it, has a low rating finds it difficult to acquire bank financing. For companies with a low rating looking for a source to raise the level of net circulating capital it is a good solution to issue long-term high risk debt securities. International Cross-Industry Journal Possibilities of using stepped coupon bonds and restructuring factoring as instruments of net circulating capital management On the basis of the survey conducted on a group of Krakow companies in the years 1998-2001 it was found out that the negative value of net circulating capitals (the disobeying of the golden rule of financing) allows for the anticipation of a company collapse threat (Rutkowska, 2005). The decline in the demand for net circulating capital is connected with decisions in relation to the operational activity. They are the decisions of short- term reference. The demand may be reduced through the shortening of the operational cycle, i.e. shortening liabilities turnover cycle or stock turnover cycle, or extension of the settlement cycle of company’s liabilities to suppliers. Factoring may be applied in order to shorten liabilities turnover cycle (debt factoring) or extension of trade liabilities settlement cycle (liability factoring). Restructuring factoring combines the services of debt and liability factoring, apart from well-developed additional services not connected with the financed debts and liabilities. The effect of its application is: the shortening of liabilities turnover cycle as the factor pays to the factoree for the transferred debts before maturity, the improvement of current liabilities management effectiveness as an effect of professional operations of the factor which is a financial institution (lower risk of cooperation with incredible and insolvent debtors, effective monitoring of liabilities, higher pace of undertaken activities), the improvement of debt recovery operations as an effect of help of factor’s debt recovery staff and lawyers, the extension of liabilities payment periods as an effect of factor’s negotiation with suppliers and guarantees granted by it to suppliers, the extension of trade liabilities turnover cycle following the extension of liabilities payment periods to the factor entering into the rights of - 13 - International Cross-Industry Journal Perspectives of Innovations, Economics & Business, Volume 12, Issue 3, 2012 STEPPED COUPON BONDS AND RESTRUCTURING FACTORING IN RELATION TO NET CIRCULATING CAPITAL IN COMPANIES IN FINANCIAL DIFFICULTY satisfied creditors on the basis of subrogation, rating improvement as an effect of liabilities management effectiveness by the factor. satisfied creditors on the basis of subrogation, rating improvement as an effect of liabilities management effectiveness by the factor. Conclusion Net circulating capital management includes two major processes: the development of net circulating capital value and the development of net circulating capital demand. These two processes have to be closely connected. The coordination of these two processes results in the adjustment of the level of net circulating capital to the demand. It results in the maintenance of optimal level of net circulating capital, i.e. the level allowing for safe business operations but at the same time allowing for the generation of profit in the company. In order to adjust the level of net circulating capital to the needs, the company may want to raise the value of net circulating capital. One of the ways to raise it is to issue long-term debt securities of the stepped coupon bond construction. In the case of companies suffering from financial problems, the proper solution would be to issue long-term high risk bonds. The application of restructuring factoring is a method to adjust the demand for net circulating capital to the level of net circulating capital in companies in financial difficulty. Restructuring factoring causes shortening of liabilities turnover cycle and extension of trade liabilities turnover cycle. It results in the shortening of cash conversion cycle and reduction in the demand for net circulating capital. International Cross-Industry Journal References Ertl, M., 2004. Aktives Cashflow-Management. Liquiditätssicherung durch wertorientierte Unternehmensführung und effiziente Innenfinanzierung, Vahlen, München Ertl, M., 2004. Aktives Cashflow-Management. Liquiditätssicherung durch wertorientierte Unternehmensführung und effiziente Innenfinanzierung, Vahlen, München Michalski, G., 2005. Financial liquidity in SMEs [Płynność finansowa w małych i średnich przedsiębiorstwach], in Polish, PWN, Warsaw Michalski, G., 2005. Financial liquidity in SMEs [Płynność finansowa w małych i średnich przedsiębiorstwach], in Polish, PWN, Warsaw Ostaszewski, J., Cicirko T., 2006. Joint-Stock Company’s Finance [Finanse spółki akcyjnej], in Polish, Warszawa: Difin Ostaszewski, J., Cicirko T., 2006. Joint-Stock Company’s Finance [Finanse spółki akcyjnej], in Polish, Warszawa: Difin Rutkowska J., 2005. „Utrata płynności czy wypłacalności? - główna determinanta zagrożenia kontynuowania działalności polskich przedsiębiorstw” (Loss of liquidity or solvency? - the major determinant of the threat to the continuity of operations of Polish companies), in: J. Ostaszewski (ed.), Finanse przedsiębiorstwa (Corporate finance), Oficyna Wydawnicza SGH w Warszawie, Warsaw, pp.351-57 Skowronek-Mielczarek, A., 2003. SMEs. Sources of Financing [Małe i średnie przedsiębiorstwa. Źródła finansowania], in Polish, Warsaw: C.H. Beck Skowronek-Mielczarek, A., 2003. SMEs. Sources of Financing [Małe i średnie przedsiębiorstwa. Źródła finansowania], in Polish, Warsaw: C.H. Beck - 14 - International Cross-Industry Journal
https://openalex.org/W4391720096
https://e-journal.umaha.ac.id/index.php/ikonik/article/download/16466/1469
Indonesian
null
Perancangan Buku Ilustrasi Mengenal Tajwid Sebagai Media Pembelajaran di TPG AL-Kusairi
Ikonik
2,024
cc-by
4,447
Ahsan Muafa1, Uswatun Khasana2, Muhammad Syarif Hidayatulloh3 Ahsan Muafa1, Uswatun Khasana2, Muhammad Syarif Hidayatulloh3 Desain Komunikasi Visual, Fakultas Teknik Universitas Maarif Hasyim Latif, Sidoarjo, Indonesia Email: ahsanmuafa@dosen.umaha.ac.id1, uswatunkhasana@student.umaha.ac.id2 syarif_hidayatulloh@dosen.umaha.ac.id3 Desain Komunikasi Visual, Fakultas Teknik Universitas Maarif Hasyim Latif, Sidoarjo, Indonesia Email: ahsanmuafa@dosen.umaha.ac.id1, uswatunkhasana@student.umaha.ac.id2 syarif_hidayatulloh@dosen.umaha.ac.id3 ABSTRACT TPQ Al-Kusairi is an educational institution engaged in religion, more precisely a place to learn to read the Koran. As a medium and source of information regarding science, economics, social and other aspects. Books are a medium that can open horizons and see new things that add to the horizon of knowledge. In Indonesia, the number of books published is already quite large. In designing the illustration book Knowing Tajwid as a Learning Media at TPQ AL-Kusairi the author uses a qualitative method, with interactive analysis consisting of three stages, namely data reduction, data display, and conclusion drawing and verification. Data reduction. In qualitative research, an observation is made about an ongoing phenomenon with an effort to understand general thinking, collect data, and to present an idea that is framed from data and approaches. The resulting product is an illustration book to learn about tajwid as an interactive learning media, this illustration book uses a hard book cover. And equipped with supporting media in the form of stationery, merchandise, and posters. Through this design, it is hoped that children can easily learn tajwid learning books. ABSTRAK TPQ Al-kusairi merupakan suatu lembaga Pendidikan yang bergerak dibidang keagamaan, lebih tepatnya tempat belajar membaca Alquran. Sebagai media dan sumber informasi mengenai ilmu pengetahuan, ekonomi, sosial maupun aspek lain. Buku merupakan suatu media yang dapat membuka wawasan dan melihat hal-hal baru yang menambah cakrawala pengetahuan. Di indonesia sendiri jumlah buku yang diterbitkan sudah terbilang banyak. Dalam Perancangan buku ilustrasi Mengenal Tajwid Sebagai Media Pembelajaran di TPQ AL-Kusairi penulis menggunakan metode kualitatif, dengan analisis interaktif yang terdiri dari tiga tahapan, yaitu reduksi data, display data, serta penarikan dan verifikasi kesimpulan. Reduksi Data. Dalam penelitian kualitatif dilakukan suatu pengamatan tentang fenomena yang sedang terjadi dengan usaha memahami pemikiran umum, mengumpulkan data, dan untuk mengemukakan suatu ide yang dibingkai dari data dan pendekatan. Produk yang dihasilkan merupakan buku ilustrasi untuk belajar mengenal tajwid sebagai media pembelajaran yang interaktif, buku ilustrrasi ini menggunakan cover hard book. Dan dilengkapi media pendukung berupa antara lain stationery, marchendise, dan poster. Melalui perancangan ini diharapkan anak-anak dapat dengan mudah mempelajari buku belajar imu tajwid. Kata kunci : ilustrasi, mengenal tajwid, buku interaktif E-ISSN : 2685-2780 P-ISSN : 2685-4260 E-ISSN : 2685-2780 P-ISSN : 2685-4260 PERANCANGAN BUKU ILUSTRASI MENGENAL TAJWID SEBAGAI MEDIA PEMBELAJARAN DI TPQ AL-KUSAIRI Volume 4, Nomor 2 Juli 2022 (171-177) 1. Pengertian Ilustrasi lembaga Pendidikan yang bergerak dibidang keagamaan, lebih tepatnya tempat belajar membaca Alquran. Dengan membaca Iqro / At- tartil anak-anak dapat mempelajari huruf-huruf Al-Qur’an dan melafalkan nya sesuai mahkarjul huruf yang ditentukan. Ketika anak-anak belajar membaca Al-Qur’an melalui tahap Iqro / At-tartil secara perlahan anak anak akan mulai memahami bagaimana cara membaca huruf nun sukun dan tanwin, secara prakteknya, namun anak anak belum memahami bacaan tersebut (Rahajeng, 2022). Seiring dengan perkembangan teknologi, beriringan dengan berkurangnya literasi membaca buku yang ada di Indonesia, maka buku dengan ilustrasi adalah media pembelajaran yang sesuai untuk diperkenalkan pada anak anak. Sebagai media pokok dalam perancangan ini berupa buku ilustrasi. Dengan buku ilustrasi mengenal tajwid ini dapat menambah minat baca pada anak untuk lebih jauh mempelajari hukum bacaan Al-quran dengan benar. Menurut (Fauzan, Aji, 2017) dalam perancangan yang berjudul “prancangan buku ilustrasi kumpulan hadist pilihan bagi anak dalam kegiatan sehari hari” untuk menarik minat dan memudahkan anak dalam memahami dan mempelajari hadits maka diperlukan tampilan visual menarik dan unik yang sesuai dengan umur mereka tanpa mengurangi kandungan dari setiap hadits dengan format buku ilustrasi dengan gaya ilustrasi yang sesuai dengan dunia mereka. Lebih jauh anak-anak dapat mengaplikasikan hadits-hadits tersebut dalam kehidupan dan keseharian mereka. (Fauzan, 2017) Diketahui bahwa buku perancangan ini di buat untuk sekedar dimembaca dan memahami tanpa ada mini game , namun berbeda dengan perancangan buku ilustrasi mengenal tajwid di TPQ Al-kusairi yakni dengan menambahkan mini game pada buku ilustrasi tersebut . Dalam buku Exploring Illustration dikatakan bahwa Ilustrasi adalah seni yang menyertai proses produksi atau pembuatan sebuah gambar, foto, atau diagram, bentuknya bisa berupa naskah tercetak, terucap, atau dalam bentuk elektronik Menurut kamus, yang dimaksud ilustrasi adalah gambar, diagram, atau peta yang digunakan untuk menjelaskan atau menghias sesuatu, terutama bagian tertulis dari sebuah karya cetak seperti buku. Ilustrasi digunakan untuk membuat jelas atau menjelaskan sesuatu. Dan ilustrasi merupakan tindakan atau proses yang menggambarkan Sesuatu (Indiria Maharsi, 2016). Berdasarkan hasil penelusuran dari perancangan sebelumnya dapat disimpulkan bahwa buku ilustrasi yang menarik dapat meningkatkan minat belajar anak- anak. Metode yang digunakan dalam perancangan diatas adalah menggunakan metode kualitatif. Berdasarkan hal tersebut jika dihubungkan dengan perancangan yang akan diciptakan, maka memiliki persamaan dalam penyelesaian masalah yang berkaitan dengan penciptaan buku ilustrasi, dan memiliki perbedaan dalam proses atau tahapan yang dilakukan dalam proses penciptaan buku ilustrasi PENDAHULUAN ialah Tajwid Menurut pendapat ustadzt yang ada di TPQ Al-Kusairi membaca Alquran merupakan suatu hal yang harus dipelajari bagi setiap umat muslim, yang mana dalam hal ini sebaiknya dimulai sejak dini. Salah satu lembaga yang menjadi tempat belajar alquran yaitu Taman Pendidikan Al-quran (TPQ). TPQ Al-kusairi berada di Desa Keloposepuluh, Kecamatan Sukodono. TPQ Al-kusairi merupakan suatu Sebagai media dan sumber informasi mengenai ilmu pengetahuan, ekonomi, sosial maupun aspek lain. Buku merupakan suatu media yang dapat membuka wawasan dan melihat hal-hal baru yang menambah cakrawala pengetahuan. Salah satu buku pembelajaran yang mengajarkan tata cara membaca Al-quran 171 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 lembaga Pendidikan yang bergerak dibidang keagamaan, lebih tepatnya tempat belajar membaca Alquran. Dengan membaca Iqro / At- tartil anak-anak dapat mempelajari huruf-huruf Al-Qur’an dan melafalkan nya sesuai mahkarjul huruf yang ditentukan. Ketika anak-anak belajar membaca Al-Qur’an melalui tahap Iqro / At-tartil secara perlahan anak anak akan mulai memahami bagaimana cara membaca huruf nun sukun dan tanwin, secara prakteknya, namun anak anak belum memahami bacaan tersebut (Rahajeng, 2022). Seiring dengan perkembangan teknologi, beriringan dengan berkurangnya literasi membaca buku yang ada di Indonesia, maka buku dengan ilustrasi adalah media pembelajaran yang sesuai untuk diperkenalkan pada anak anak. Sebagai media pokok dalam perancangan ini berupa buku ilustrasi. Dengan buku ilustrasi mengenal tajwid ini dapat menambah minat baca pada anak untuk lebih jauh mempelajari hukum bacaan Al-quran dengan benar. Menurut (Fauzan, Aji, 2017) dalam perancangan yang berjudul “prancangan buku ilustrasi kumpulan hadist pilihan bagi anak dalam kegiatan sehari hari” untuk menarik minat dan memudahkan anak dalam memahami dan mempelajari hadits maka diperlukan tampilan visual menarik dan unik yang sesuai dengan umur mereka tanpa mengurangi kandungan dari setiap hadits dengan format buku ilustrasi dengan gaya ilustrasi yang sesuai dengan dunia mereka. Lebih jauh anak-anak dapat mengaplikasikan hadits-hadits tersebut dalam kehidupan dan keseharian mereka. (Fauzan, 2017) Diketahui bahwa buku perancangan ini di buat untuk sekedar dimembaca dan memahami tanpa ada mini game , namun berbeda dengan perancangan buku ilustrasi mengenal tajwid di TPQ Al-kusairi yakni dengan menambahkan mini game pada buku ilustrasi tersebut . Bagan Perancangan Bagan Perancangan Gambar 1. Bagan Perancangan • Penarikan Kesimpulan Penarikan Kesimpulan Langkah ketiga setelah melakukan reduksi data dan penyajian data adalah penarikan kesimpulan. Kesimpulan dalam penelitian kualitatif merupakan temuan baru yang sebelumnya belum pernah ada. Temuan dapat berupa deskripsi atau gambaran suatu obyek yang sebelumnya masih remang-remang atau gelap sehingga setelah diteliti menjadi jelas. Buku merupakan suatu media yang dapat membuka wawasan dan melihat hal-hal baru yang menambah cakrawala pengetahuan. Seiring dengan perkembangan zaman buku telah hadir dari berbagai bentuk seperti majalah, novel, komik, buku pembelajaran. Salah satu buku pembelajaran yang mengajarkan tata cara membaca Al-quran ialah Tajwid. TPQ Al-kusairi merupakan suatu lembaga Pendidikan yang bergerak dibidang keagamaan, lebih tepatnya tempat belajar membaca Alquran. salah satu dasar ilmu membaca Al-quran yaitu Tajwid. Tajwid merupakan suatu ilmu untuk mengetahui bagaimana cara membaca Al-quran dengan sebaik-baiknya. Selama ini buku tajwid diterbitkan dengan model sederhana seperti menggunakan kertas buram, visual yang Gambar 1. Bagan Perancangan a. Geografis a. Geografis Target audiens menurut tempat tinggal penulis untuk TPQ- Al Kusairi, dan TPQ di desa keloposepuluh. y g • Pendapat dari murid-murid TPQ bahwa buku tajwid yang digunakan selama ini kurang menarik untuk dipahami lebih lanjut METODE PENELITIAN/METODE PERANCANGAN Dalam Perancangan buku ilustrasi Mengenal Tajwid Sebagai Media Pembelajaran di TPQ AL- Kusairi penulis menggunakan metode kualitatif, Penelitian kualitatif adalah suatu kegiatan untuk melakukan eksplorasi dan memahami suatu makna dari sekelompok maupun individu orang, karena adanya suatu permasalahan sosial (Sugiyono, 2013). Dalam penelitian kualitatif dilakukan suatu pengamatan tentang fenomena yang sedang terjadi dengan usaha memahami pemikiran umum, mengumpulkan data, dan untuk mengemukakan suatu ide yang dibingkai dari data dan pendekatan. Berdasarkan latar belakang diatas, maka permasalahan yang akan kami bahas dalam perancangan ini adalah : Bagaimana konsep dalam perancangan buku ilustrasi mengenal tajwid yang menarik untuk anak-anak di TPQ Al-Kusairi ? Analisis data penelitian dilakukan menggunakan model analisis Miles & Huberman (1994) dengan jenis Analisis intrraktif yang terdiri dari tiga tahapan, yaitu reduksi data, display data, serta penarikan dan verifikasi kesimpulan. Bagamaina cara menerapkan konsep dalam buku ilustrasi mengenal tajwid tersebut di TPQ Al-Kusairi ? • Reduksi data adalah proses berfikir sensitif yang memerlukan kecerdasan dan keluasan dan kedalaman wawasan yang tinggi. Data yang diperoleh dari lapangan jumlahnya cukup banyak, untuk itu maka perlu dicatat secara teliti dan rinci. Mereduksi data berarti merangkum, memilih hal-hal yang pokok, memfokuskan pad a hal-hal yang penting, dicari tema dan polanya. Dengan demikian Berdasarkan rumusan masalah di atas, k t j k di i d l Berdasarkan rumusan masalah di atas, maka tujuan yang akan dicapai dalam perancangan ini adalah : Menciptakan konsep buku tajwid dengan Menciptakan konsep buku tajwid dengan ilustrasi yang menarik untuk memudahkan anak anak untuk belajar mengenal tajwid. Menerapkan konsep yang menarik di antara halaman halaman agar mengurangi rasa bosan pada saat anak anak belajar mengenal tajwid. 172 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 kurang menarik (tidak ada gambar dan penggunaan warna), dan sampul buku masih berupa foto copy. Saat ini murid di TPQ AL- Kusairi minatnya mulai berkurang saat belajar mandiri menggunakan buku tajwid dari TPQ, sehingga hal tersebut dapat menurunkan minat anak anak untuk membaca Al-quran dengan hukum bacaan yang sesuai. data yang telah direduksi akan memberikan gambaran yang lebih jelas, dan mempermudah peneliti untuk melakukan pengumpulan data selanjutnya, dan mencarinya bila diperlukan (Sugiyono, 2013). g Penyajian Data Setelah data direduksi, maka langkah selanjutnya ialah mendisplaykan data. Dengan mendisplaykan data, maka akan memudahkan untuk memahami apa yang terjadi, merencanakan kerja selanjutnya berdasarkan apa yang telah difahami terlebih dahulu (Sugiyono, 2013). Demografis Demografis Buku Ilustrasi belajar tajwin ini ditujukan untuk anak-anak yang sudah mengenal huruf dan sudah bisa membaca, untuk usia 5-8 tahun. Demografis Buku Ilustrasi belajar tajwin ini ditujukan untuk anak-anak yang sudah mengenal huruf dan sudah bisa membaca, untuk usia 5-8 tahun. • Hasil wawancara menunjukkan bahwa media pembelajaran yang dapat menunjang pembelajaran mandiri ketika dirumah adalah berupa buku tajwid tanpa visual yang menarik. b. Pesikografis Secara psikologis anak umur 5-8 tahun menyukai buku interaktif dengan ilustrasi yang memiliki warna menarik, dengan karakter lucu dan dapat dimainkan oleh anak-anak, sehingga anak-anak tidak mudah bosan saat membaca buku tersebut. • Walimurid sebagai narasumber mengatakan banyak yang tidak melakukan pembelajaran mandiri dirumah dikarenakan media pembelajaran yang kurang menarik dan lebih banyak tulisan tanpa adanya penyederhanaan materi tajwid pada buku tersebut. HASIL DAN PEMBAHASAN Karya yang dihasilkan merupakan buku ilustrasi untuk belajar mengenal tajwid sebagai media pembelajaran yang interaktif, buku ilustrrasi ini menggunakan cover hard book dengan art paper sebagai kertas isinya. Pada bagian dalam terdapat 30 lembar halaman dengan materi tajwid yang berbeda, mulai dari materi hukum nun sukun dan tanwin sampai materi waqof. Selain itu dalam buku terdapat materi interaktif yang bisa dimainkan anak anak dengan materi yang ada pada buku tersebut. Hasil karya ini ada berbagai macam diantaranya Media utamanya adalah buku ilustrasi mengenal 173 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 terutama yang menjadi tujuan adalah anak anak, hal tersebut dapat membantu proses belajar mengajar menjadi lebih menyenangkan untuk anak anak Guru Agama Sekolah Dasar Media pembelajaran interaktif yang disatukan dengan ilmu tajwid merupakan hal yang jarang ditemukan pada buku buku tajwid yang sudah ada sebelumnya. Wali murid TPQ Menggunakan ilustrasi yang menarik dan interaktif akan menjadikan anak anak lebih cepat memahami materi dan kegiatan belajar menjadi lebih muda di tangkap. Murid- murid TPQ Dengan adanya buku ilustrasi belajar tajwid yang interaktif menjadikan suasana belajar menjadi menyenangkan dimana ada kegiatan bermain sambil belajar Anak anak luar TPQ Kegiatan bermain sambil belajar adalah hal yang diinginkan untuk mengurangi rasa bosan saat belajar yang kemungkinan buku ilustrasi mengenal tajwid yang interaktif dapat diterima untuk umum tajwid, kemudian ada totebag yang terdapat ilustrasi, ada 5 pembatas buku dengan ilustrasi yang berberda, 10 stiker dengan gambar tokoh yang ada di buku, dan bucket had dengan sablonan. Buku ilustrasi mengenal tajwid sendiri mempuanyai tujuan sebagai media pembelajaran yang interaktif. Sasaran yang tuju buku ini adalah anak-anak usia 5- 8 tahun yang ad di TPQ Al-Kusairi desa Kloposepuluh kab. Sidoarjo Jawa Timur. Buku Ilustrasi Dirancang Menurut pandangan ilustrator muda, Evelline Andrya yang menggeluti illustrasi buku anak berpendapat bahwa ilustrasi adalah media untuk berimajinasi. Dimana seorang ilustrator bisa menciptakan karakter dan cerita imajinatif, (Indiria Maharsi, 2016).berbeda hal dengan perancangan buku ilutrasi mengenal tajwid yang dijadikan fungsi sebagai media pembelajaran pada TPQ Al-Kusairi 1. Ide dan tema cerita Yang melatar belakangi ide dari perancangan ini merupakan gagasan dari permaslahan yang ada pada TPQ Al-Kusairi dimana media pembelajaran mengenai tajwid masih menggunakan buku materi yang kurang menarik serta tidak ada unsur pewarnaan. Menurut kamus besar bahasa Indonesia Ide merupakan rancangan yang tersusun dalam fikiran sedangkan, tema adalah dasar cerita yang sering di gunakan sebagai dasar mengarang . Ide yang diangkat pada perancangan ini adalah membuat karakter ilustrasi anak anak yang akan diterapkan pada buku media pembelajaran interaktif mengenal tajwid. Untuk tema ilustrasi pada buku tajwid pada perancangan ini dibuat dengan tema anak ceria dimana menggunakan gambar yang komunikatif dan interaktif serta warna warna yang sesuai dengan anak anak Menurut kamus besar bahasa Indonesia Ide merupakan rancangan yang tersusun dalam fikiran sedangkan, tema adalah dasar cerita yang sering di gunakan sebagai dasar mengarang . Ide yang diangkat pada perancangan ini adalah membuat karakter ilustrasi anak anak yang akan diterapkan pada buku media pembelajaran interaktif mengenal tajwid. Untuk tema ilustrasi pada buku tajwid pada perancangan ini dibuat dengan tema anak ceria dimana menggunakan gambar yang komunikatif dan interaktif serta warna warna yang sesuai dengan anak anak. Menurut kamus besar bahasa Indonesia Ide merupakan rancangan yang tersusun dalam fikiran sedangkan, tema adalah dasar cerita yang sering di gunakan sebagai dasar mengarang . Ide yang diangkat pada perancangan ini adalah membuat karakter ilustrasi anak anak yang akan diterapkan pada buku media pembelajaran interaktif mengenal tajwid. Untuk tema ilustrasi pada buku tajwid pada perancangan ini dibuat dengan tema anak ceria dimana menggunakan gambar yang komunikatif dan interaktif serta warna warna yang sesuai dengan anak anak. 4. Tinjauan fungsi dan peranan buku ilustrasi Tujuan dibuatnya permainan interaktif dalam buku ilustrasi mengenal tajwid ini adalah cara mempermudah anak anak untuk memahami materi tajwid yang ada pada buku tersebut dan mampu mengasah perkembangan motorik pada anak melalui permainan interaktif pada buku tersebut. 4. 2. Tinjauan dari aspek dasar perkembangan ilustrasi Ilustrasi juga berperan dalam membangun kesadaran intelektual, kesadaran teknologi, kesadaran politis, kesadaran edukatif, kesadaran akan perkembangan budaya serta peradaban manusia dalam semangat estetis yang menjadi wadah presentasinya (Indiria Maharsi, 2016) d. Pewarnaan Pewarnaan pada sebua buku ilustrasi anak sangatlah penting karena dapat mendukung semua aspek aspek yang dibutuhkan pada perancangan buku ilustrasi mengenal tajwid untuk anak anak. Warna dapat didefinisikan sebagai sifat cahaya yang dipancarkan, atau secara subjektif/psikologis dari pengalaman indra penglihatan. Warna menjadi sangat penting dalam kehidupan sehari-hari, karena warna membangkitkan perasaan yang spontan kepada orang yang melihatnya. Warna adalah subjek yang dapat mempengaruhi daya tarik suatu karya atau desain karena warna dapat memberikan vibrasi tertentu pada pada sebuah desain juga menambah keefektifan penyampaian pesan untuk dikomunikasikan pada audience.(Monica & Luzar, 2011) e. Teknik visualisasi Proses perancangan karya buku ilustrasi mengenal tajwid ini para tokoh yang ada di buku ilustrasi menggunakan cara manual kemudian di tracing menggunakan aplikasi gambar yakni IbisPaint dimana aplikasi ini lebih memudahkan perancang dalam membuat karakter anak pada buku ilustrasi belajar tajwid, yang kemudian dipindah menggunakan Adobe Ilustrator CC2017 untuk penambahan gambar,background, penataan layout dan element lain yang dibutuhkan. d. Gaya visual Buku yang digunakan sebagai refrensi dalam perancangan ini adalah buku anak yang berjudul “ Aku Senang Menjadi Santri” karya Nayla Damai V.P ,Gaya visual perancangan buku ilustrasi mengenal tajwid ini adalah jenis ilustrasi kartun anak anak islami dimana buku ini diperuntukkan kepada murid TPQ Al-Kusairi dan anak anak usia 5-8 tahun. Buku yang digunakan sebagai refrensi dalam perancangan ini adalah buku anak yang berjudul “ Aku Senang Menjadi Santri” karya Nayla Damai V.P ,Gaya visual perancangan buku ilustrasi mengenal tajwid ini adalah jenis ilustrasi kartun anak anak islami dimana buku ini diperuntukkan kepada murid TPQ Al-Kusairi dan anak anak usia 5-8 tahun. d. Konsep Kreatif M “ Aku Mau Belajar Tajwid”, “Yuk Belajar Tajwid Bersama”, dan “Belajar Ilmu Tajwid Itu Menyenangkan”. Dari beberapa pilihan judul tersebut terpilihlah judul “Belajar Ilmu Tajwid itu Menyenangkan” karena dapat meninterpretasikan isi buku ilustrasi pada perancangan ini, dimana interaktif pada buku ini menjadikan hal menyenangkan dalam pembelajaran ilmu tajwid itu sendiri. Storyline 1. Strategi kreatif a. Siapa pembaca Target audien atau pembaca hasil perancangan ini adalah anak anak usia 5- 8 tahun pada murid TPQ Al-Kusairi dan TPQ sekitar yang di desa Kloposepuluh b. Format dan ukuran buku ilustrasi Penentuan ukuran buku adalah hal penting dalam perancangan ini, yang tentunya penentuan ukuran buku ini akan menjadi salah satu point penting dalam kenyamanan pembaca/pengguna buku ilustrasi tajwid ini, dan perancangan ini buku yang dibuat berukuran 21 cm x 25 cm dengan format Cetak hard book. y Storyline merupakan perancangan awal sebelum membuat alur buku yang diinginkan , storyline dari perancangan buku ini sendiri dibuat sesuai dengan urutan materi mulai dari materi dasar hingga materi atas c. Terdapat beberpa jenis buku ilustrasi yaitu Baby Books (Buku untuk Bayi), Interactive Books (Buku Interaktif), Wordless Books (Buku Minim Kata), Movable Books, Concept Books, Picture Storybooks, Graphic Novels (Komik), Transitional Books. c. Gaya layout Axial layout Tata letak yang memiliki tampilan visual yang kuat di tengah halaman dengan tampilan element pendukung di sekeliling gambar utama biasanya berupa gambar atau tulisan yang berhubungan dengan tampilan di tengah halaman sebagai titik pusatnya. Axial layout dipilih dalam perancangan ini dikarenakan objek utama pada buku bisa lebih jelas dan menjadi fokus utama pada saat membuka buku. d. Konsep Kreatif M Konsep Kreatif Menurut kamus Besar Bahasa Indonesia konsep adalah sebuah ide atau pengertian yang diabstrakkan dari peristiwa konkret. Konsep kreatif merupakan hal yang sangat penting dalam sebuah perancangan . Setiap perancangan sebuah karya wajib menggunakan konsep termasuk dalam perancangan buku ilustrasi ini . Konsep kreatif pada buku ini adalah melakukan pendekatan pada anak-anak usia 5- 8 tahun terutama murid dari TPQ AL-Kusairi dengan karakter lucu pada buku ilustrasi yang berjudul “Ilmu tajwid itu menyenangkan”. Disini gambar ilustrasi pada buku dan materi dibuat lebih menarik sehingga materi pada buku ilustrasi mengenal tajwid ini mudah dipahami oleh anak-anak. Ditunjang dengan Konsep Kreatif Menurut kamus Besar Bahasa Indonesia konsep adalah sebuah ide atau pengertian yang diabstrakkan dari peristiwa konkret. Konsep kreatif merupakan hal yang sangat penting dalam sebuah perancangan . Setiap perancangan sebuah karya wajib menggunakan konsep termasuk dalam perancangan buku ilustrasi ini . ) 3. Tinjauan faktor eksternal Dari beberapa peninjauan yang terdiri dari beberapa sumber mengenai pembuatan buku ilustrasi mengenal tajwid sebagai media pembelajaran, berikut hasil dari tinjauan : Guru ngaji TPQ Membuat media pembelajaran yang menggunakan materi mengaji (agama) adalah suatu hal yang baik untuk dikembangkan, Konsep kreatif pada buku ini adalah melakukan pendekatan pada anak-anak usia 5- 8 tahun terutama murid dari TPQ AL-Kusairi dengan karakter lucu pada buku ilustrasi yang berjudul “Ilmu tajwid itu menyenangkan”. Disini gambar ilustrasi pada buku dan materi dibuat lebih menarik sehingga materi pada buku ilustrasi mengenal tajwid ini mudah dipahami oleh anak-anak. Ditunjang dengan 174 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 menarik minat baca anak-anak usia 5-8 tahun. materi interaktif didalam buku yang bisa dijadikan peremaninan pengasah otak sehingga menjadikan suasana belajar tajwid lebih menyenangkan. tahun. Pemilihan judul berdasarkan beberapa aspek dari beberapa pilihan. Berikut adalah beberapa pilihan untuk judul karya pada perancangan ini. “ Aku Mau Belajar Tajwid”, “Yuk Belajar Tajwid Bersama”, dan “Belajar Ilmu Tajwid Itu Menyenangkan”. Dari beberapa pilihan judul tersebut terpilihlah judul “Belajar Ilmu Tajwid itu Menyenangkan” karena dapat meninterpretasikan isi buku ilustrasi pada perancangan ini, dimana interaktif pada buku ini menjadikan hal menyenangkan dalam pembelajaran ilmu tajwid itu sendiri. Storyline Storyline merupakan perancangan awal sebelum membuat alur buku yang diinginkan , storyline dari perancangan buku ini sendiri dibuat sesuai dengan urutan materi mulai dari materi dasar hingga materi atas Pemilihan judul berdasarkan beberapa aspek dari beberapa pilihan. Berikut adalah beberapa pilihan untuk judul karya pada perancangan ini. Typografi e. 2. Program kreatif a. Judul buku Judul buku merupakan suatu hal yang penting dalam perancangan buku terutama pada buku anak anak yang dirancang guna Tipografi merupakan salah satu elemen desain yang mempunyai dua fungsi, yaitu fungsi sebagai TULISAN yang dibaca dan fungsi sebagai IMAGE (gambar). Banyak 175 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 f. Sampul depan dan belakang Pembuatan sampul pada proses perancangan buku merupakan suatu hal yang sangat penting karena sampul dapat menginterpretasikan sebuah isi buku hasil perancangan ini Perancangan pada bagian sampul depan dilakaukan setelah melakukan pemilihan judul, pembuatan karakter dan pembuatan beberapa opsi background. Berikut adalah desain sampul depan dan belakan yang terpilih : f. Sampul depan dan belakang Pembuatan sampul pada proses perancangan buku merupakan suatu hal yang sangat penting karena sampul dapat menginterpretasikan sebuah isi buku hasil perancangan ini Perancangan pada bagian sampul depan dilakaukan setelah melakukan pemilihan judul, pembuatan karakter dan pembuatan beberapa opsi background. Berikut adalah desain sampul depan dan belakan yang terpilih : yang dapat dikembangkan melalui karya tipografi. Di sini penulis akan mengangkat tipografi sebagai salah satu elemen visual yang dibuat untuk materi edukasi anak- anak, karena memang tipografi itu sendiri mempunyai dua fungsi tersebut. Hal ini dilakukan mengingat pada masa anak-anak juga merupakan masa pembelajaran “membaca” , “menulis”, dan “menggambar”. Dengan demikian, akan lebih mudah dan menarik jika ada buku yang memuat karya atau gambar yang terbuat dari karya tipografi. (Setiautami, 2011) yang dapat dikembangkan melalui karya tipografi. Di sini penulis akan mengangkat tipografi sebagai salah satu elemen visual yang dibuat untuk materi edukasi anak- anak, karena memang tipografi itu sendiri mempunyai dua fungsi tersebut. Hal ini dilakukan mengingat pada masa anak-anak juga merupakan masa pembelajaran “membaca” , “menulis”, dan “menggambar”. Dengan demikian, akan lebih mudah dan menarik jika ada buku yang memuat karya atau gambar yang terbuat dari karya tipografi. (Setiautami, 2011) p y g p Gambar 7. Cover Buku Gambar 7. Cover Buku Gambar 6. font comic sans ms g. Penjaringan ide tokoh g. g. Penjaringan ide tokoh Penjaringan ide tokoh diambil dari aktifitas sehari hari di TPQ Al-Kusairi, dimana aktifitas tersebut melibatkan guru laki laki (ustadz), guru perempuan (ustadzah), murid laki-laki dan perempuan usia 5 tahun sampai murid usia 8 tahun. Kemudian dibuat sketsa kasar lalu menggabungkan dari sumber refrensi desain ilustrasi baik dari internet maupun pengalaman keseharian, yang terakhir menggabungkan gaya perancang. DAFTAR PUSTAKA Gambar 9. Layout Buku Gambar 2 penerapan font fredoka one reguler Gambar 3 penerapan font happy kids • Font yang dipakai pada halaman daftar isi buku menggunakan. “Adobe Arabic” Gambar 4 penerapan font adobe arabic • Untuk font kecil keseluruhan menggunakan “gratefull everyday dan comic sans Ms” Gambar 5 font grateful everyday Gambar 6. font comic sans ms Gambar 2 penerapan font fredoka one reguler Gambar 3 penerapan font happy kids Gambar 3 penerapan font happy kids Proses Pembuatan Ilustrasi Proses Pembuatan Ilustrasi Gambar 8. Proses ilustrasi Gambar 3 penerapan font happy kids • Font yang dipakai pada halaman daftar isi buku menggunakan. “Adobe Arabic” Gambar 4 penerapan font adobe arabic • Font yang dipakai pada halaman daftar isi buku menggunakan. daftar isi buku menggunakan. “Adobe Arabic” Gambar 4 penerapan font adobe arabic “Adobe Arabic” Gambar 8. Proses ilustrasi Gambar 8. Proses ilustrasi Gambar 8. Proses ilustrasi Gambar 8. Proses ilustrasi 176 Ahsan Muafa, dkk / IKONIK : Jurnal Seni dan Desain, Vol. 4, No.2, Juli 2022, 171-177 Layout beberapa bagian buku Gambar 9. Layout Buku untuk membaca Al-quran dengan hukum bacaan yang sesuai. Layout beberapa bagian buku Berdasarkan keadaan tersebut maka perlu diciptakan media pembelajaran yang menarik, salah satunya yaitu membuat media pembelajaran interaktif menggunakan visual berupa buku ilustrasi. Dengan buku ilustrasi mengenal tajwid ini dapat menambah minat baca pada anak untuk lebih jauh mempelajari hukum bacaan Al-quran dengan benar. Dalam Perancangan buku ilustrasi Mengenal Tajwid Sebagai Media Pembelajaran di TPQ AL-Kusairi penulis menggunakan metode kualitatif. Analisis data yang dilakukan terdapat tiga hal utama, yaitu reduksi data, penyajian data, dan penarikan kesimpulan. Konsep kreatif pada buku ini adalah melakukan pendekatan pada anak-anak usia 5-8 tahun terutama murid dari TPQ AL-Kusairi dengan karakter lucu pada buku ilustrasi yang berjudul “Ilmu tajwid itu menyenangkan”. Disini gambar ilustrasi pada buku dan materi dibuat lebih menarik sehingga materi pada buku ilustrasi mengenal tajwid ini mudah dipahami oleh anak-anak. Ditunjang dengan materi interaktif didalam buku yang bisa dijadikan peremaninan pengasah otak sehingga menjadikan suasana belajar tajwid lebih menyenangkan. Gambar 9. Layout Buku PENUTUP Fauzan, Aji, N. (2017). Perancangan Buku Ilustrasi Kumpulan Hadits Pilihan Bagi Anak Dalam Kegiatan Sehari-Hari. Fakultas Seni Rupa Institut Seni Indonesia Yogyakarta, 63(May), 9–57. http://dx.doi.org/10.1016/j.chb.2016.05.008 Buku merupakan suatu media yang dapat membuka wawasan dan melihat hal-hal baru yang menambah cakrawala pengetahuan. Seiring dengan perkembangan zaman buku telah hadir dari berbagai bentuk seperti majalah, novel, komik, buku pembelajaran. Salah satu buku pembelajaran yang mengajarkan tata cara membaca Al-quran ialah Tajwid. TPQ Al-kusairi merupakan suatu lembaga Pendidikan yang bergerak dibidang keagamaan, lebih tepatnya tempat belajar membaca Alquran. salah satu dasar ilmu membaca Al-quran yaitu Tajwid. Tajwid merupakan suatu ilmu untuk mengetahui bagaimana cara membaca Al-quran dengan sebaik-baiknya. Selama ini buku tajwid diterbitkan dengan model sederhana seperti menggunakan kertas buram, visual yang kurang menarik (tidak ada gambar dan penggunaan warna), dan sampul buku masih berupa foto copy. Saat ini murid di TPQ AL-Kusairi minatnya mulai berkurang saat belajar mandiri menggunakan buku tajwid dari TPQ, sehingga hal tersebut dapat menurunkan minat anak anak Indiria Maharsi, Ms. (2016). UPT Perpus ISI Yogyakarta buku ilustrasi. 1–13. Monica, M., & Luzar, L. C. (2011). Efek Warna dalam Dunia Desain dan Periklanan. Humaniora, 2(2), 1084. https://doi.org/10.21512/humaniora.v2i2.3158 Setiautami, D. (2011). Eksperimen Tipografi dalam Visual untuk Anak. Humaniora, 2(1), 311. https://doi.org/10.21512/humaniora.v2i1.3011 Sugiyono. (2013). Metode Penelitian Kuantitatif, Kualitatif, dan RD by Prof. Dr. Sugiyono. Alfabeta, Bandung. 177
https://openalex.org/W2156851406
https://europepmc.org/articles/pmc3848504?pdf=render
English
null
Rapid targeted gene disruption in Bacillus anthracis
BMC biotechnology
2,013
cc-by
8,013
METHODOLOGY ARTICLE Open Access * Correspondence: Thomas.Lamkin@wpafb.af.mil 4Air Force Research Laboratory, Air Force Research Laboratory, 711th HPW/ RHXBC, Molecular Signatures Section, 2510 Fifth Street, Area B, Bldg 840, Room W220, Wright-Patterson AFB, OH 45433-7913, USA Full list of author information is available at the end of the article Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Rapid targeted gene disruption in Bacillus anthracis Roland J Saldanha1, Adin Pemberton1, Patrick Shiflett2, Jiri Perutka3, Jacob T Whitt3, Andrew Ellington3, Alan M Lambowitz3, Ryan Kramer4,5, Deborah Taylor4 and Thomas J Lamkin4* © 2013 Saldanha 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. Abstract Background: Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome. Results: Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion. Conclusions: The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics. © 2013 Saldanha 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. Background However, a complete dissection of Bacillus anthracis is hampered by the difficulty of transforming the bacteria (approximately 102-105 transformants per μg DNA) and by relatively few simple ways to mani- pulate the genome genetically [12-15]. Shatalin and Neyfakh used a temperature sensitive (ts) plasmid to de- liver a selectable marker flanked by upstream and down- stream regions homologous to the gene to be disrupted [14]. Through the manipulation of temperatures and screening of antibiotic markers, a chromosomal inte- grant could be obtained. Janes and Stibitz have created an elegant set of plasmids to make markerless gene dis- ruptions in two distinct steps [12]. In the first step, a plasmid is constructed with an I-SceI site flanking a gen- etic marker to be replaced and delivered on a ts replicon using an erythromycin selection marker. On introduc- tion of this plasmid in Bacillus anthracis, the plasmid can integrate into the chromosome at the low frequency of homologous recombination using the homology of genomic sequences cloned into the plasmid. On shifting to a restrictive temperature the plasmid is cured and it is possible to select for those rare chromosomal integration events. After appropriate steps to verify the success of the first step, a second plasmid expressing I-SceI is in- troduced. This leads to cleavage at the I-SceI site and stimulating recombination via double strand break repair which either leads to the desired recombination product or restoration to the parental chromosomal configur- ation. The desired product must be obtained through physical screening of individual colonies Finally Leppla Many group II introns exhibit the ability to insert into DNA through a now well understood series of biochem- ical reactions [16]. The natural mobility of the Ll.LtrB intron has been harnessed into a highly efficient gene targeting technology to achieve gene disruption through intron insertion in a number of gram positive and gram negative bacterial hosts [17,18]. The ability to engineer a group II intron to insert at desired genomic locations, distinguishes group II introns from other insertional ele- ments like transposons that either integrate randomly at a low frequency or insert at a high frequency but only at a specific genomic locus like the attTn7 site. Frequently, the engineered group II introns insert at their newly pro- gramed insertion sites in the genome at frequencies high enough to be detectable by simple colony screening. Background In binary complexes with PA, EF and LT are referred to as edema toxin (ET) and lethal toxin (LT) respectively. ET is an adenyl-cyclase that enhances cAMP production and disrupts water homeostasis leading to kidney failure and death. LT is a zinc protease that cleaves mitogen- activated protein kinase kinases (MAPKK) and induces pro-inflammatory cytokines and cellular apoptosis [4]. Antibodies predominantly directed against PA are the basis of current vaccines that are used prophylactically [7]. edema factor (EF) from genes encoded on the pXO1 virulence plasmid [6]. PA facilitates toxin entry by bind- ing to receptors TEM8 and CMG2 on the surface of hu- man and animal cells. Binding triggers PA cleavage leading to formation of a heptameric or octameric pore that binds and translocates EF and LF into the cytosol. In binary complexes with PA, EF and LT are referred to as edema toxin (ET) and lethal toxin (LT) respectively. ET is an adenyl-cyclase that enhances cAMP production and disrupts water homeostasis leading to kidney failure and death. LT is a zinc protease that cleaves mitogen- activated protein kinase kinases (MAPKK) and induces pro-inflammatory cytokines and cellular apoptosis [4]. Antibodies predominantly directed against PA are the basis of current vaccines that are used prophylactically [7]. The dramatic lethal consequences of an accidental spore release at Sverdlovsk in the former Soviet Union and the deliberate release of anthrax through letters in the US in 2001 have renewed interest in neutralizing this pathogen [8,9]. The complete sequence of Bacillus anthracis and several of its close relatives is available [10,11]. This potentially allows for systematic study of all the genes contributing to the success of this important pathogen. However, a complete dissection of Bacillus anthracis is hampered by the difficulty of transforming the bacteria (approximately 102-105 transformants per μg DNA) and by relatively few simple ways to mani- pulate the genome genetically [12-15]. Shatalin and Neyfakh used a temperature sensitive (ts) plasmid to de- liver a selectable marker flanked by upstream and down- stream regions homologous to the gene to be disrupted [14]. Through the manipulation of temperatures and screening of antibiotic markers, a chromosomal inte- grant could be obtained. Janes and Stibitz have created an elegant set of plasmids to make markerless gene dis- ruptions in two distinct steps [12]. Background The high efficiency of insertion allows gene disruption with- out the introduction of an antibiotic marker into the chromosome and the efficient sequential creation of multiple knockouts at disparate chromosomal sites. We have adapted the Ll.LtrB intron insertion technology for application to Bacillus anthracis and demonstrate the remarkable simplicity with which gene inactivation through intron insertion can be rapidly achieved. Background explaining the monomorphic nature of the bacterium [3]. Human infection occurs most commonly through accidental exposure to infected animals or animal prod- ucts, such as hides or carcasses. 95% of infections occur via the cutaneous route and more rarely through gastro- intestinal and pulmonary routes [4,5]. The disease is dif- ficult to diagnose because early symptoms are relatively non-specific and, when left untreated without prompt antibiotic intervention, can lead to fatality with rates ranging from 20% for cutaneous exposure to as high as 100% for pulmonary anthrax. The high morbidity is due to a rapid overwhelming of host defenses, fulminant septicemia of vegetative cells and the action of bacterial toxins expressed from vegetative cells. Once inside the host, the spores are engulfed by macrophages where they are transported to lymph nodes and germinate to become vegetative cells that eventually disseminate through blood and lymph causing septicemia and tox- emia. The vegetative cells express three monomeric pro- teins: protective antigen (PA), lethal factor (LF) and In 1876 Robert Koch ushered in microbial pathology by establishing a causal relationship between Bacillus anthracis microbes and the disease anthrax [1]. Distrib- uted worldwide, Bacillus anthracis is a gram positive bacterium capable of infecting the widest range of ani- mals of any known pathogen [2]. The bacterium is nor- mally found as an endospore in soil where a chance encounter with an animal can permit entry through a wound, food or inhalation leading to germination of the spore into a vegetative bacterium. The vegetative phase is relatively short consisting of only 20–40 generations. The bacterium is believed to spend large periods of time in spore form thus slowing evolutionary changes and * Correspondence: Thomas.Lamkin@wpafb.af.mil 4Air Force Research Laboratory, Air Force Research Laboratory, 711th HPW/ RHXBC, Molecular Signatures Section, 2510 Fifth Street, Area B, Bldg 840, Room W220, Wright-Patterson AFB, OH 45433-7913, USA Full list of author information is available at the end of the article Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 2 of 8 Page 2 of 8 edema factor (EF) from genes encoded on the pXO1 virulence plasmid [6]. PA facilitates toxin entry by bind- ing to receptors TEM8 and CMG2 on the surface of hu- man and animal cells. Binding triggers PA cleavage leading to formation of a heptameric or octameric pore that binds and translocates EF and LF into the cytosol. Background In the first step, a plasmid is constructed with an I-SceI site flanking a gen- etic marker to be replaced and delivered on a ts replicon using an erythromycin selection marker. On introduc- tion of this plasmid in Bacillus anthracis, the plasmid can integrate into the chromosome at the low frequency of homologous recombination using the homology of genomic sequences cloned into the plasmid. On shifting to a restrictive temperature the plasmid is cured and it is possible to select for those rare chromosomal integration events. After appropriate steps to verify the success of the first step, a second plasmid expressing I-SceI is in- troduced. This leads to cleavage at the I-SceI site and stimulating recombination via double strand break repair which either leads to the desired recombination product or restoration to the parental chromosomal configur- ation. The desired product must be obtained through h l f d d l l F ll l flanked by the loxP sites of the site-specific recombinase Cre to leave a single loxP site at the site of marker inte- gration which could then direct larger genome scale de- letions through multiple rounds of loxP marker insertion at distant sites and site-specific recombination between them [13]. ( ) g p virulence plasmid [6]. PA facilitates toxin entry by bind- ing to receptors TEM8 and CMG2 on the surface of hu- man and animal cells. Binding triggers PA cleavage leading to formation of a heptameric or octameric pore that binds and translocates EF and LF into the cytosol. In binary complexes with PA, EF and LT are referred to as edema toxin (ET) and lethal toxin (LT) respectively. ET is an adenyl-cyclase that enhances cAMP production and disrupts water homeostasis leading to kidney failure and death. LT is a zinc protease that cleaves mitogen- activated protein kinase kinases (MAPKK) and induces pro-inflammatory cytokines and cellular apoptosis [4]. Antibodies predominantly directed against PA are the basis of current vaccines that are used prophylactically [7]. The dramatic lethal consequences of an accidental spore release at Sverdlovsk in the former Soviet Union and the deliberate release of anthrax through letters in the US in 2001 have renewed interest in neutralizing this pathogen [8,9]. The complete sequence of Bacillus anthracis and several of its close relatives is available [10,11]. This potentially allows for systematic study of all the genes contributing to the success of this important pathogen. Results and discussion Group II intron based gene inactivation In two cases (highlighted by a the LtrA protein [27]. On encountering a suitable target, the RNA component of the RNP can integrate through reverse splicing of the intron RNA into the DNA target, followed by an LtrA directed cleavage of the bottom DNA strand which generates a primer that allows copy- ing of the intron into DNA via the reverse transcriptase activity of LtrA. Results and discussion Group II intron based gene inactivation Group II introns in general, and the Ll.LtrB group II in- tron in particular, have been engineered into an efficient gene disruption system [17,19,20]. We have adapted this system for rapid gene inactivation in Bacillus anthracis. Group II introns are autocatalytic RNA molecules [21]. Some group II introns exhibit a property of “homing” where the intron recognizes the DNA equivalent of its cognate exon junctions and inserts itself via retro- transposition [16]. The biochemical basis of this intron insertion has been deduced [22-24]. For example, the Ll. LtrB intron interrupting a relaxase in Lactococcus lactis is excised from its flanking exons through RNA catalysis assisted by an intron encoded protein LtrA [25]. The LtrA protein acts as a scaffold for efficient RNA directed catalysis but also encodes a reverse transcriptase and DNA endonuclease that are used for intron integration into DNA targets [26]. The spliced Ll.LtrB intron re- mains associated with LtrA to form a RNP particle. The intron component of the RNP can recognize DNA exon sequences resembling exon junctions through a combin- ation of exon recognition elements (EBS1/δ and EBS2) within the intron and DNA recognition preferences of Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 3 of 8 genome (located in ORF 1652, ORF 2876 and ORF 5071). Figure 1A shows general features of the vector and the modified base pairing interactions within EBS1/ δ and EBS2 that allow the intron to recognize and insert itself into IS605 orf B which are shown in Figure 1B. The plasmid shown in Figure 1A was introduced into Bacillus anthracis Sterne by electroporation and kana- mycin resistant colonies were obtained. A single colony was grown overnight and induced with 10 μM cadmium for 90 minutes. Dilutions were plated on LB with- out antibiotic selection and individual colonies were screened for presence of the intron insertion using PCR primers flanking the insertion site. Figure 1C shows that this intron is remarkably efficient, and that every colony screened yielded a PCR product consistent with intron insertion into orf B of IS605. Direct sequencing of the PCR product confirmed that the intron did successfully integrate at the desired locus. Figure 1C also shows that in most cases a PCR product that is consistent with an intron free genomic product is also detectable, which suggests that the intron has not integrated at all three possible insertion sites. A vector permitting group II intron gene inactivation using screening To adapt group II introns for targeted gene disruption in Bacillus anthracis we cloned the Ll.LtrB intron and LtrA protein under the control of a cadmium inducible promoter [28] using a gram positive/gram negative shut- tle vector pRB373 as a backbone [29]. This vector offers a pUB110 origin of replication for gram positives and a ColE1 origin for gram negatives. The selection markers are kanamycin/neomycin in gram positives and ampicil- lin in gram negatives. For proof of principle, we changed the sequences in EBS1/δ and EBS2 within the intron to direct the intron to orf B of the IS605 insertion se- quences which has 3 copies in the Bacillus anthracis A B C Intron insertion Targetron IS605 IS605 IS605 Genomic Genomic Intron insertion Genomic Intron insertion E1 E2 Ll.ltrB Cadmium Promoter M M A B C Intron insertion Targetron IS605 IS605 IS605 Genomic Genomic Intron insertion Genomic Intron insertion E1 E2 Ll.ltrB Cadmium Promoter M M Figure 1 Bacillus anthracis Targetron plasmid. (A) A variant of the Ll.LtrB group II intron designed to insert into orfB of IS605 elements of Bacillus anthracis was cloned into a Bacillus anthracis/E. coli. shuttle vector with a kanamycin resistance marker (bacillus) and ampicillin marker for E. coli. The intron and intron encoded LtrA protein are driven from a Cadmium inducible promoter (Cd). (B) EBS1/δ and EBS2 (shown schematically as black bars in the intron) are sequences within the intron controlling exon recognition via base pairing interactions. Reprogrammed sequences in EBS1/δ and EBS2 for efficient integration into IS605 are shown. (C) Plasmid IS605tt pRB373 was introduced into Bacillus anthracis Sterne via electroporation and selection for kanamycin resistant colonies. PCR screening of individual colonies using primers flanking the potential site of intron insertion show virtually all colonies had an intron insertion. There are 3 genomic copies of IS605 and in the two lanes highlighted by a white arrow all three copies have an intron insertion. Lanes marked M have 1 kb molecular weight markers. B E1 E2 Ll.ltrB Cadmium Promoter A A B C Genomic Figure 1 Bacillus anthracis Targetron plasmid. (A) A variant of the Ll.LtrB group II intron designed to insert into orfB of IS605 elements of Bacillus anthracis was cloned into a Bacillus anthracis/E. coli. shuttle vector with a kanamycin resistance marker (bacillus) and ampicillin marker for E. coli. A vector permitting group II intron gene inactivation using screening The intron and intron encoded LtrA protein are driven from a Cadmium inducible promoter (Cd). (B) EBS1/δ and EBS2 (shown schematically as black bars in the intron) are sequences within the intron controlling exon recognition via base pairing interactions. Reprogrammed sequences in EBS1/δ and EBS2 for efficient integration into IS605 are shown. (C) Plasmid IS605tt pRB373 was introduced into Bacillus anthracis Sterne via electroporation and selection for kanamycin resistant colonies. PCR screening of individual colonies using primers flanking the potential site of intron insertion show virtually all colonies had an intron insertion. There are 3 genomic copies of IS605 and in the two lanes highlighted by a white arrow all three copies have an intron insertion. Lanes marked M have 1 kb molecular weight markers. Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 4 of 8 marker is expressed under its own promoter and is cloned at a known neutral site in domain IV of the intron in an antisense direction relative to the group II intron expres- sion. A td self-splicing group I intron interrupts the kana- mycin resistance coding sequences in the same orientation as the Ll.LtrB group II intron. As a result of this configur- ation, the kanamycin resistance marker in the donor plas- mid is silent. Any leak-through expression from its own promoter leads to a non-functional protein since the group I intron interrupting it cannot be spliced out due to its antisense orientation relative to kanamycin expression. Group II intron RNA transcripts will, however, also have the td intron autocatalytically removed restoring the con- tinuity of the kanamycin coding sequence. If these tran- scripts are used as substrates for site-specific intron integration through reverse splicing into DNA targets and reverse transcription, the kanamycin marker can then be expressed. Thus successful retrotransposition events are marked by a gain of kanamycin resistance which allows for selection of rare intron integration events. As a proof of principle, we have designed introns to insert at BAS4553 (a methionine gamma-lyase) and BAS4597 (a pullulanase). The regions within the intron that interact with DNA insertion sites were changed in order to direct the intron to insert between nucleotides 343:344 of the white arrow) a single PCR product is seen which is con- sistent with intron insertion in all three potential insertion sites. The remarkable efficiency of this intron represents the upper end of intron targeting efficiency [19]; i.e. A vector permitting group II intron gene inactivation using screening 100% insertion efficiency for all colonies screened with 1 in 24 colonies showing an intron insertion into three independ- ent chromosomal insertion targets. For other targets we have examined, the intron efficiency is considerably lower requiring extensive arduous PCR screening to obtain a single insertion. A vector using genetic selection for detection of group II intron gene insertion To address the problem of finding intron insertions for less efficient introns, we have engineered a vector with a retrotransposition activated kanamycin resistance marker that allows selection of intron insertion events [30]. Figure 2 illustrates the main features in this vector. An erythromycin resistance marker was introduced into a pRB373 shuttle vector. Group II intron expression was driven using an Ntr promoter previously identified by using a promoter trap strategy [31]. The kanamycin resist- ance marker that is conditionally expressed only after in- tron insertion was created by total DNA synthesis and used codons optimized for low GC organisms. This BAS 4597 BAS 4553 Intron Insertion Genomic Intron Insertion Genome Genome Kanr Intron Insertion M M Figure 2 Bacillus vector for selection of group II intron insertions. A schematic of the important vector features and selection scheme for group II intron based gene disruption. The plasmid is erythromycin resistant but kanamycin sensitve because a td group I intron interrupts its coding sequence in an antisense orientation. Transcription of the group II intron from the Ntr promoter allows self- splicing of the td intron and generates a substrate that is capable of genomic integration at pre-determined loci due to changes in sequences shown as black bars within the intron. Genomic integrants no longer have the td group I intron interrupting the kanamycin resistance gene and thus successful integrants are kanamycin resistant. For PCR verification of intron insertion, primers (shown as arrows above the genomic DNA schematic) were designed to flank the site of intron insertion in BAS4597 and BAS4553. kanamycin resistant colonies arising in Bacillus anthracis Sterne after transformation of plasmids reengineered to integrate the group II intron at BAS4597 and BAS4553 were screened by colony PCR and analyzed after electrophoresis on a 1% agarose gel in TBE buffer and visualized by staining with ethidium bromide. Lanes marked M have 1 Kb molecular weight markers. BAS 4597 BAS 4553 Intron Insertion Genomic Intron Insertion Genome Genome Kanr Intron Insertion M M BAS 4597 M Figure 2 Bacillus vector for selection of group II intron insertions. A schematic of the important vector features and selection scheme for group II intron based gene disruption. The plasmid is erythromycin resistant but kanamycin sensitve because a td group I intron interrupts its coding sequence in an antisense orientation. Conclusions The vectors described here introduce a new tool for gen- etic manipulation of Bacillus anthracis. The ability to directly select for an insertional inactivation of a gene using very few manipulations will accelerate our ability to probe the role of the numerous uncharacterized genes in the genome of this important pathogen and uncover new targets for vaccination strategies or useful targets for therapeutic intervention. While the vectors were de- veloped and tested in Bacillus anthracis, the plasmid backbone used to build the vector is derived from pUB110 a Staphylococcus aureus plasmid that has been extensively used in Bacillus subtilis [29]. These vectors are therefore likely to be able to be used across the Bacillus genus in- cluding other members of the Bacillus cereus group such as Bacillus cereus and Bacillus thuringiensis. Preparation of plasmid DNA from E. coli, transform- ation of E. coli, and recombinant DNA techniques were carried out by standard procedures [34]. The plasmid pRBEryNtr was constructed in several steps. In the first step an erythromycin resistance marker was PCR ampli- fied using pNL9162 as the template and oligonucleotide primers SphI Ery 5’ GAATGAGGCATGCTACACCTCC GGATA3’ and AgeI Ery 5’ GAT C ACCGGTCACACG AA AAACAAGTTAAGGGATGCAG3’ using NEB PCR mastermix. This yielded a 1.2 Kb fragment which was then purified and digested with SphI and AgeI. It was then cloned into pRB373 (which had been digested with the same enzymes) to yield pRB373Ery. The potent Ba- cillus Ntr promoter identified by Gat et al., [31] was amplified from Bacillus anthracis Sterne genomic DNA using PCR primers 5’ SphI Nitro 5’ GatC GCATGC CTGAGTTGGATCATCATTATATGAAAGGC3’ and 3’ Nitro HindIII XhoI EcoRI 5’Gatc gaattc ctcgag gag AAGCTTTTTTTCA TATGTATAC ATC ATA TTC TGC C3’. The PCR product was digested with SphI and EcoR1 then cloned into pRB373Ery digested with the same enzymes to yield pRB373 EryNtr. The coding se- quence of a select agent compliant kanamycin allele was optimized for expression in low GC organisms and synthe- sized (Genscript, Piscataway, NJ). The synthetic fragment also included a self-splicing td intron in the antisense orientation inserted between codons 15 and 16 of the kanamycin gene and cloned into pRB373. A HindIII-XhoI fragment with the group II intron Targetron was cloned into pRB373 digested with the same enzymes. A vector using genetic selection for detection of group II intron gene insertion Transcription of the group II intron from the Ntr promoter allows self- splicing of the td intron and generates a substrate that is capable of genomic integration at pre-determined loci due to changes in sequences shown as black bars within the intron. Genomic integrants no longer have the td group I intron interrupting the kanamycin resistance gene and thus successful integrants are kanamycin resistant. For PCR verification of intron insertion, primers (shown as arrows above the genomic DNA schematic) were designed to flank the site of intron insertion in BAS4597 and BAS4553. kanamycin resistant colonies arising in Bacillus anthracis Sterne after transformation of plasmids reengineered to integrate the group II intron at BAS4597 and BAS4553 were screened by colony PCR and analyzed after electrophoresis on a 1% agarose gel in TBE buffer and visualized by staining with ethidium bromide. Lanes marked M have 1 Kb molecular weight markers. Figure 2 Bacillus vector for selection of group II intron insertions. A schematic of the important vector features and selection scheme for group II intron based gene disruption. The plasmid is erythromycin resistant but kanamycin sensitve because a td group I intron interrupts its coding sequence in an antisense orientation. Transcription of the group II intron from the Ntr promoter allows self- splicing of the td intron and generates a substrate that is capable of genomic integration at pre-determined loci due to changes in sequences shown as black bars within the intron. Genomic integrants no longer have the td group I intron interrupting the kanamycin resistance gene and thus successful integrants are kanamycin resistant. For PCR verification of intron insertion, primers (shown as arrows above the genomic DNA schematic) were designed to flank the site of intron insertion in BAS4597 and BAS4553. kanamycin resistant colonies arising in Bacillus anthracis Sterne after transformation of plasmids reengineered to integrate the group II intron at BAS4597 and BAS4553 were screened by colony PCR and analyzed after electrophoresis on a 1% agarose gel in TBE buffer and visualized by staining with ethidium bromide. Lanes marked M have 1 Kb molecular weight markers. Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 5 of 8 and kanamycin was used at 50 μg/mL for E. coli and 20 μg/mL for Bacillus anthracis. BAS4553 gene as well as between nucleotides 1794:1795 of the BAS4597 gene. Conclusions Finally, PCR mutagenesis was used to generate the mutations necessary to retarget the intron to BAS4597 & BAS4553 and in- troduced as HindIII-BsrG1 fragments into the vector as described above. Plasmid construction The pRB373 plasmid was purchased from ATCC (cat. no. 77373). The sequence of pRB373 is unpublished but based on restriction enzyme digestion it contains a BsrG1 site. Because of this, a construct designed to retarget the Ll.LtrB intron to insert within IS605 needed to be created in three steps. In the first, a PCR fragment containing the changes necessary to retarget the intron to IS605 was digested with BsrGI and HindIII and cloned into the in- tron donor plasmid pNL9161 of Staphylococcus aureus digested with the same enzymes [33]. To optimize pairing at the +1 site of the target, this intermediate plasmid was modified by mutating C + 1 to G + 1. The resulting plas- mid was then digested with SphI and SfoI, and the frag- ment containing the Cd-inducible promoter, LtrB intron, and LtrA protein was gel purified and cloned between the SphI and SmaI sites of pRB373 to yield plasmid IS605-tt. A vector using genetic selection for detection of group II intron gene insertion The reengineered plasmid vectors were introduced into Bacillus anthracis Sterne by electroporation and selection on erythromycin plates. Transformants were regrown in liquid media containing erythromycin and plated on LB agar with kanamycin to select for putative intron integration events. Kanamycin resistant colonies were screened for successful intron insertion by using PCR primers that flanked the site of in- tron insertion. As can be seen in Figure 2, all kanamycin resistant colonies for BAS4533 show a PCR product con- sistent with group II intron insertion. For BAS4597, only one of the 8 colonies is consistent with intron integration at the desired site. The PCR products were sequenced and indicate precise intron integration at the desired locus. Bacterial strains and culture E. coli strains: DH5α™with genotype F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1 and dam-dcm- (Life Technologies) was used for routine cloning. Since trans- formation efficiency of Bacillus anthracis is sensitive to dam methylation [32], plasmid DNA for transformation into Bacillus anthracis was prepared from a dam methyl- ase deficient host E. coli (New England Biolabs C2925H) with genotype: ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10) TetS endA1 rspL136 (StrR) dam13::Tn9 (CamR) xylA-5 mtl-1 thi-1 mcrB1 hsdR2. Bacillus anthracis (Sterne 34 F2) was obtained from Colorado Serum Co. (Denver, CO). Bacteria were grown in Luria-Bertaini (LB) broth or Brain Heart Infusion (BHI). Antibiotics were added for plasmid maintenance or selection at the following con- centrations: ampicillin at 100 μg/mL; erythromycin at 400 μg/mL for E. coli and 4 μg/mL for Bacillus anthracis Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 6 of 8 Table 1 Targetron insertion sites and scores Gene insertion site -30 to -1 NT upstream LtrB intron +1 to +15 NT downstream Score IS605 435|436 s CCGATTATAGATCATCAAAAATATAGTGTA gtgc…tcac GAAATTATTCGCAAA 9.3 BAS4553 343|344 s GTTCAAATGGATTATACGGATGCACGTACG gtgc…tcac GCTTTTTGGAAGTGT 8.4 BAS4597 1794|1795 s AATCAATTAGATTGGGATCGAAAAGAGAAA gtgc…tcac GAAATAGAGACCGTT 7.3 The gene chosen for intron insertion is shown in the first column with the nucleotide positions at which the intron is designed to insert at within the gene shown beside the gene name. All intron insertions are in the sense orientation relative to gene transcription and designated here with the suffix “s”. The sequence of 30 nucleotides upstream and 15 nucleotides downstream of the intron insertion site are shown together with a numeric score reflecting expected efficiency based on a learning set of Ll.Ltrb intron insertions. Table 1 Targetron insertion sites and scores HindIII and BsrG1 and cloned into the intron donor plasmids described above cut with the same enzymes. Intron design & generation of PCR products for intron retargeting The sequence of Bacillus anthracis genomic DNA to be targeted for disruption was scanned for potential intron insertion sites using a computer algorithm derived from a learning set of successful intron integrations [19]. The resulting insertion sites are shown in Table 1 and the mutations necessary to retarget the intron to a desired site were introduced through PCR mutagenesis using two pairs of partially overlapping primers (Table 2) to assemble a short DNA fragment flanked by HindIII and BsrG1 sites and incorporating the changes within the in- tron required to redirect the intron to a new locus as de- scribed previously [17]. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal pri- mer at a concentration of 100 μM and 10 μM respect- ively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. In a separate tube a similar reaction was performed using mutant specific primers EBS1delta (100 μM) and EBS2 (10 μM) primers. At the end of the first round of asym- metric PCR, the two reactions were combined and allowed to proceed through 20 more PCR amplification cycles. The resulting PCR products incorporating all necessary mutations were gel purified, digested with Bacillus anthracis genomic DNA isolation Bacillus anthracis was grown overnight and 2 mL were harvested for genomic isolation with the Epicentre Bio- technologies DNA & RNA purification kit (cat. no. MC85200). Final yield was 70 μL at ~1.3 mg/mL. Bacillus anthracis genomic DNA isolation Bacillus anthracis was grown overnight and 2 mL were harvested for genomic isolation with the Epicentre Bio- technologies DNA & RNA purification kit (cat. no. MC85200). Final yield was 70 μL at ~1.3 mg/mL. Bacillus anthracis electroporation Electroporation conditions described in Koehler et al. were followed with a few modifications [35]. A culture of Bacillus anthracis Sterne was grown overnight in BHI. The following day, 1 mL of the overnight culture was used to inoculate 100 mL of BHI supplemented with 0.5% glycerol in a 1000 mL baffled Erlenmeyer flask and incubated at 37°C with shaking at 225 rpm until the cul- ture reached an OD600 of ~ 0.6. At this point the culture was chilled on an ice bath and filtered through a filter assembly pre chilled to 4°C and washed with an ice cold electroporation buffer containing 10 mM Hepes pH 7.0 and 10% Glycerol (3×50 mL washes). The washed bac- teria were resuspended from the filter using 10 mL of ice cold electroporation buffer, then aliquoted and flash frozen. Aliquots of electrocompetent Bacillus anthracis (Sterne) were electroporated with 3 μg of dam-dcm- Table 2 Primers used for retargeting Primer name Primer sequence Pairings changed IS605-435|436 s AAAAAAGCTTATAATTATCCTTAAAATACAGTGTAGTGCGCCCAGATAGGGTG IBS1/2 IS605-435|436 s CAGATTGTACAAATGTGGTGATAACAGATAAGTCAGTGTAGATAACTTACCTTTCTTTGT EBS1/δ IS605-435|436 s TGAACGCAAGTTTCTAATTTCGGTTTATTTCCGATAGAGGAAAGTGTCT EBS2 BAS4553-343|344 s AAAAAAGCTTATAATTATCCTTAGATGCCCGTACGGTGCGCCCAGATAGGGTG IBS1/2 BAS4553-343|344 s CAGATTGTACAAATGTGGTGATAACAGATAAGTCCGTACGGCTAACTTACCTTTCTTTGT EBS1/δ BAS4553-343|344 s TGAACGCAAGTTTCTAATTTCGATTGCATCTCGATAGAGGAAAGTGTCT EBS2 BAS4597-1794|1795 s AAAAAAGCTTATAATTATCCTTACGAAACGAGAAAGTGCGCCCAGATAGGGTG IBS1/2 BAS4597-1794|1795 s CAGATTGTACAAATGTGGTGATAACAGATAAGTCGAGAAAGATAACTTACCTTTCTTTGT EBS1/δ BAS4597-1794|1795 s TGAACGCAAGTTTCTAATTTCGGTTTTTCGTCGATAGAGGAAAGTGTCT EBS2 EBS-Universal AACCGAAATTAGAAACTTGCGTTCA None The primers shown were used in PCR to generate a HindIII BsrG1 fragment that was exchanged into the Targetron vectors and retarget the intron to the genomic l ti i di t d i th i Th t l tid th i t i d i d t i t t i d i t d b | t th i l ti i di t d b th Table 2 Primers used for retargeting The primers shown were used in PCR to generate a HindIII BsrG1 fragment that was exchanged into the Targetron vectors and retarget the intron to the genomic location indicated in the primer name. The exact nucleotide the intron is designed to insert at is designated by a | at the genomic location indicated above the primer sequences. Page 7 of 8 Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 45 sec and extension at 65°C for 3 min, with a final extension at 65°C for 10 min. Prod- ucts were visualized using 1% ethidium bromide (Fisher Bioreagents) on a 1% agarose gel. plasmid DNA in a 4 mm gap cuvette. Acknowledgements This work was supported by the Chem-Bio Diagnostics program contract # B102387M from the Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA). J.T. W.’s and A.M.L.’s participation in this work was supported by NIH grant GM037949 and Welch Foundation grant F-1607. Competing interests Competing interests Group II intron gene targeting (“targetron”) technology is subject to U.S. and international patents licensed by The Ohio State University and the University of Texas to InGex, LLC. A.M.L., J.P., the Ohio State University, and the University of Texas are minority equity holders in InGex, LLC. A.M.L. and J.P. may receive royalty payments for commercial use of targetron technology. R.S. has received royalties from The Ohio State University in the past. J.P. is founder of Targetronics, a company sublicensed by InGex, LLC to sell research tools based on targetron technology. Authors’ contributions TJL, RK & DT conceived of the project. RJS designed the experiments along with TJL, JP, AML and AE. RJS, JP, AP, JTW, and PS performed the laboratory work and analyzed the results. RS wrote the manuscript. All authors read and approved the final manuscript. References 1. Koch R: Die Ätiologie der Milzbrand Krankheit begründet auf die Entwicklungsgeschichte des Bacillus anthracis. Beitr Biol Pflanz 1876, 2:277–283. 2. Beyer W, Turnbull PC: Anthrax in animals. Molecular aspects of medicine 2009, 30(6):481–489. 3. Keim P, Van Ert MN, Pearson T, Vogler AJ, Huynh LY, Wagner DM: Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 2004, 4(3):205–213. 4. Cote CK, Welkos SL, Bozue J: Key aspects of the molecular and cellular basis of inhalational anthrax. Microbes and infection / Institut Pasteur 2011, 13(14–15):1146–1155. 4. Cote CK, Welkos SL, Bozue J: Key aspects of the molecular and cellular basis of inhalational anthrax. Microbes and infection / Institut Pasteur 2011, 13(14–15):1146–1155. Generation of targetron insertions A frozen glycerol stock of Bacillus anthracis Sterne transformed with the plasmid vector designed to insert into IS605 was used to inoculate LB augmented with 20 μg/mL kanamycin and shaken at 220 RPM at 37°C overnight. The overnight cultures were used to prepare fresh dilutions by inoculating 100 μL of the overnight culture into 25 mL LB augmented with 20 mg/mL kana- mycin and shaken (220 RPM) at 37°C. When the cul- tures reached an OD600 of 0.6, 25 μL of 10 mM CdCl2 was added in order to induce the cadmium promoter controlling expression of the targetron. Cultures were induced for 90 min. Serial dilutions of the induced cul- tures were prepared and 100 μL aliquots taken from the 10-4 and 10-5 dilutions were plated on LB agar without antibiotic selection. Single colonies were tested for in- tron insertion via colony PCR using primers flanking the site of intron insertion. A frozen glycerol stock of Bacil- lus anthracis Sterne transformed with the plasmid vector designed to insert into BAS4597 or BAS4553 was used to inoculate LB augmented with 4 μg/mL erythromycin and shaken at 220 RPM at 37°C overnight. The overnight cultures were used to prepare fresh dilutions by ino- culating 100 μL into 25 mL LB with 4 μg/mL erythro- mycin and shaken (220 RPM) at 37°C and grown to saturation. 0.2-0.5 mL of the saturated cultures was spread on LB agar plates containing kanamycin at 20 μg/mL and incubated at 37°C for three days. Any kanamycin resistant colony arising reflects a potential intron insertion and this was verified by colony PCR using primers flanking the site of intron insertion. Author details 1UES Inc, 4401 Dayton-Xenia Road, Dayton, OH 45432, USA. 2Henry Jackson Foundation, 6720-A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA. 3Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-0159, USA. 4Air Force Research Laboratory, Air Force Research Laboratory, 711th HPW/RHXBC, Molecular Signatures Section, 2510 Fifth Street, Area B, Bldg 840, Room W220, Wright-Patterson AFB, OH 45433-7913, USA. 5Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA. Received: 13 April 2013 Accepted: 13 September 2013 Published: 18 September 2013 Received: 13 April 2013 Accepted: 13 September 2013 Published: 18 September 2013 Bacillus anthracis electroporation All electroporation was conducted on a BioRad Gene Pulser. Electropor- ation conditions were as follows: 2,500 V, 25 μF, 400 Ω. The time constant for the pulse was (on average) 6.5 ms. After electroporation, the cells were diluted with 1 mL of BHI augmented with 10% glycerol, 0.4% glucose and 10 mM MgCl2. The cells were incubated for one hour before plating on LB or BHI agar plates with 20 μg/mL kanamycin or 4 μg/mL erythromycin. 5. WHO: Anthrax in humans and animals. Geneva 27, Switzerland: WHO Press; 2008. 6. Okinaka RT, Cloud K, Hampton O, Hoffmaster AR, Hill KK, Keim P, Koehler TM, Lamke G, Kumano S, Mahillon J, et al: Sequence and organization of pXO1, Colony PCR screening Colonies were assessed for potential intron insertions by conventional PCR. Specific primers flanking the site of chromosomal insertion were designed and synthesized by Integrated DNA Technologies. DNA was amplified using 1 μL of LongAmp Taq DNA Polymerase (New England Biolabs), 2.5 μL of 5× LongAmp Taq Reaction Buffer, 0.4 μL of 10 mM dNTP Mix (New England Biolabs), nuclease-free water (Ambion), 200 nM of each primer and whole cell template in a final volume of 12.5 μL per reaction. The following cycling conditions were used: initial denaturation at 94°C for 30 sec, 5. WHO: Anthrax in humans and animals. Geneva 27, Switzerland: WHO Press; 2008. 5. WHO: Anthrax in humans and animals. Geneva 27, Switzerland: WHO Press; 2008. 6. Okinaka RT, Cloud K, Hampton O, Hoffmaster AR, Hill KK, Keim P, Koehler TM, Lamke G, Kumano S, Mahillon J, et al: Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol 1999, 181(20):6509–6515. 6. Okinaka RT, Cloud K, Hampton O, Hoffmaster AR, Hill KK, Keim P, Koehler TM, Lamke G, Kumano S, Mahillon J, et al: Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol 1999 181(20):6509–6515 6. Okinaka RT, Cloud K, Hampton O, Hoffmaster AR, Hill KK, Keim P, Koehler TM, Lamke G, Kumano S, Mahillon J, et al: Sequence and organization of pXO1, th l B ill th i l id h b i th th t i 7. Bouzianas DG: Medical countermeasures to protect humans from anthrax bioterrorism. Trends in microbiology 2009, 17(11):522–528. 7. Bouzianas DG: Medical countermeasures to protect humans from anthrax bioterrorism. Trends in microbiology 2009, 17(11):522–528. 8. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, Yampolskaya O: The Sverdlovsk anthrax outbreak of 1979. Science 1994, 266(5188):1202–1208. 8. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, Yampolskaya O: The Sverdlovsk anthrax outbreak of 1979. Science 1994, 266(5188):1202–1208. 9. Atlas RM: Bioterriorism: from threat to reality. Annu Rev Microbiol 2002, 56:167–185. Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Page 8 of 8 Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 Saldanha et al. BMC Biotechnology 2013, 13:72 http://www.biomedcentral.com/1472-6750/13/72 10. Ravel J, Jiang L, Stanley ST, Wilson MR, Decker RS, Read TD, Worsham P, Keim PS, Salzberg SL, Fraser-Liggett CM, et al: The complete genome sequence of Bacillus anthracis Ames “Ancestor”. Colony PCR screening y 22. Zimmerly S, Guo H, Perlman PS, Lambowitz AM: Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 1995, 82(4):545–554. 23. Zimmerly S, Guo H, Eskes R, Yang J, Perlman PS, Lambowitz AM: A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell 1995, 83(4):529–538. 24. Guo H, Zimmerly S, Perlman PS, Lambowitz AM: Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA. The EMBO journal 1997, 16(22):6835–6848. 25. Matsuura M, Saldanha R, Ma H, Wank H, Yang J, Mohr G, Cavanagh S, Dunny GM, Belfort M, Lambowitz AM: A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron. Genes & development 1997, 11(21):2910–2924. 26. Saldanha R, Chen B, Wank H, Matsuura M, Edwards J, Lambowitz AM: RNA and protein catalysis in group II intron splicing and mobility reactions using purified components. Biochemistry 1999, 38(28):9069–9083. 27. Singh NN, Lambowitz AM: Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference. J Mol Biol 2001, 309(2):361–386. 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: • 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: • 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: 28. Colony PCR screening J Bacteriol 2009, 191(1):445–446. 32. Marrero R, Welkos SL: The transformation frequency of plasmids into Bacillus anthracis is affected by adenine methylation. Gene 1995, 152(1):75–78. 33. Yao J, Zhong J, Fang Y, Geisinger E, Novick RP, Lambowitz AM: Use of targetrons to disrupt essential and nonessential genes in staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. Rna 2006, 12(7):1271–1281. 11. Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Tettelin H, Fouts DE, Eisen JA, Gill SR, et al: The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 2003, 423(6935):81–86. 34. Sambrook Russel DW: Molecular cloning: a laboratory manual. NY: Cold Spring Harbor Press; 2001. 12. Janes BK, Stibitz S: Routine markerless gene replacement in Bacillus anthracis. Infection and immunity 2006, 74(3):1949–1953. 35. Koehler TM, Dai Z, Kaufman-Yarbray M: Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters. J Bacteriol 1994, 176(3):586–595. 13. Pomerantsev AP, Sitaraman R, Galloway CR, Kivovich V, Leppla SH: Genome engineering in Bacillus anthracis using Cre recombinase. Infection and immunity 2006, 74(1):682–693. doi:10.1186/1472-6750-13-72 Cite this article as: Saldanha et al.: Rapid targeted gene disruption in Bacillus anthracis. BMC Biotechnology 2013 13:72. 14. Shatalin KY, Neyfakh AA: Efficient gene inactivation in Bacillus anthracis. FEMS Microbiol Lett 2005, 245(2):315–319. 15. Koehler TM: Bacillus anthracis physiology and genetics. Molecular aspects of medicine 2009, 30(6):386–396. 16. Lambowitz AM, Zimmerly S: Group II introns: mobile ribozymes that invade DNA. Cold Spring Harbor perspectives in biology 2011, 3(8):a003616. 17. Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM: Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nature biotechnology 2001, 19(12):1162–1167. 18. Akhtar P, Khan SA: Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis. Plasmid 2012, 67(2):111–117. 19. Perutka J, Wang W, Goerlitz D, Lambowitz AM: Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol 2004, 336(2):421–439. 20. Rawsthorne H, Turner KN, Mills DA: Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl Environ Microbiol 2006, 72(9):6088–6093. 21. Saldanha R, Mohr G, Belfort M, Lambowitz AM: Group I and group II introns. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 1993, 7(1):15–24. Colony PCR screening Charpentier E, Anton AI, Barry P, Alfonso B, Fang Y, Novick RP: Novel cassette-based shuttle vector system for gram-positive bacteria. Appl Environ Microbiol 2004, 70(10):6076–6085. 29. Bruckner R: A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 1992, 122(1):187–192. 30. Zhong J, Karberg M, Lambowitz AM: Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res 2003, 31(6):1656–1664. 31. Gat O, Inbar I, Aloni-Grinstein R, Zahavy E, Kronman C, Mendelson I, Cohen S, Velan B, Shafferman A: Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines. Infection and immunity 2003, 71(2):801–813.
https://openalex.org/W2044040735
https://www.scielo.br/j/pab/a/6DH8KBQ5qc8GCwPVMpmh84N/?lang=pt&format=pdf
Portuguese
null
Distribuição de Carabidae e Staphylinidae em agroecossistemas
Pesquisa Agropecuária Brasileira
2,008
cc-by
3,850
Distribuição de Carabidae e Staphylinidae em agroecossistemas Francisco Jorge Cividanes(1) e Terezinha Monteiro dos Santos-Cividanes(2) Francisco Jorge Cividanes(1) e Terezinha Monteiro dos Santos-Cividanes(2) (1)Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Fitossanidade, Via de Acesso Prof. Paul Donato Castellane, s/no, CEP 14884-900 Jaboticabal, SP. E-mail: fjcivida@fcav.unesp.br (2)Agência Paulista de Tecnologia dos Agronegócios Avenida Bandeirantes, no 2.419, CEP 14030-670 Ribeirão Preto, SP. E-mail: terezinha@apta.sp.gov.br Resumo – O objetivo deste trabalho foi determinar a distribuição da riqueza de espécies e a preferência pelo habitat de Carabidae e Staphylinidae (Coleoptera), em áreas com rotação de soja e milho, em plantio direto e convencional, e em áreas adjacentes a estas com fragmento florestal e povoamento de pínus, respectivamente. Os besouros foram amostrados por meio de armadilhas de solo distribuídas em dois transectos de 100 m de comprimento. A distribuição da riqueza de espécies nas culturas, no fragmento florestal e no pínus foi avaliada por meio de análise de regressão linear. A análise de agrupamento foi empregada para identificar as espécies quanto à preferência pelos habitats: fragmento florestal, pínus, cultura e interface. A distribuição da riqueza de espécies de Carabidae e Staphylinidae não variou em relação à posição no transecto, enquanto a riqueza de espécies observada nas interfaces foi elevada em comparação com a encontrada nos demais habitats.A ocorrência de espécies de Carabidae diferiu conforme o tipo de cobertura vegetal: Megacephala sp. e Scarites sp. preferiram áreas cultivadas em sistema de rotação soja-milho; Odontochila nodicornis (Dejean) preferiu o fragmento florestal e o povoamento de pínus. A espécie Abaris basistriatus Chaudoir caracterizou-se como generalista quanto à preferência pelo habitat. Termos para indexação: Glycine max, Pinus, Zea mays, controle biológico, diversidade, plantio direto. Distribution of Carabidae and Staphylinidae in agroecosystems Abstract – The objective of this work was to determine the distribution of species richness and habitat preference of Carabidae and Staphylinidae (Coleoptera) in two areas cultivated with soybean/corn under no-tillage and conventional tillage systems and in adjacents areas with forest fragment and a Pinus stand, respectively. Beetles were sampled by pitfall traps which were distributed in two transects of 100 m. The distribution of species richness in the crops and forest fragment/Pinus was evaluated by regression analysis. Cluster analysis was used to identify species in relation to preference for the forest fragment, Pinus, crop and interface. The distribution of Carabidae and Staphylinidae species richness did not show variation in relation to the position in transect, while the species richness observed in the interfaces between forest fragment or Pinus stand and crops were higher compared with the ones found in other habitats. The occurrence of Carabidae species differed in relation to the vegetation type: Megacephala sp. and Scarites sp. preferred crop areas under soybean/corn rotation system. The same was observed with Odontochila nodicornis (Dejean) in the forest fragment and Pinus stand. The species Abaris basistriatus Chaudoir was characterized as a generalist species in relation to habitat preference. Index terms: Glycine max, Pinus, Zea mays, biological control, diversity, no-tillage. Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 Introdução para aumentar a sustentabilidade dos agroecossistemas (Kromp, 1999; French et al., 2001). A diversidade e a abundância de insetos predadores nas culturas estão relacionadas com a natureza da vegetação nas adjacências (Thomas et al., 2002). Fragmentos florestais e demais habitats localizados próximo de culturas constituem o refúgio primordial de insetos predadores associados ao solo (Pfiffner & Luka, 2000; Fournier & Loreau, 2001), o que aumenta a ocorrência desses predadores nas culturas e contribui Estudos sobre a distribuição e a composição de espécies de insetos predadores em culturas próximas de fragmentos florestais se mostraram fundamentais para o entendimento da função que esses organismos desempenham nos agroecossistemas (Clark et al., 1997; Crist & Ahern, 1999) e são também importantes para a previsão dos efeitos benéficos desses predadores nas culturas (Levesque & Levesque, 1994). Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 158 F.J. Cividanes e T.M. dos Santos-Cividanes F.J. Cividanes e T.M. dos Santos-Cividanes As famílias Carabidae e Staphylinidae (Coleoptera) incluem importantes espécies predadoras associadas ao solo de culturas (Pfiffner & Luka, 2000), com potencial para reduzir populações de pragas agrícolas (Mundy et al., 2000; Sadej & Nietupski, 2000; Suenaga & Hamamura, 2001). Apesar do enorme potencial que esses besouros apresentam para controlar pragas, são praticamente inexistentes informações, no Brasil, sobre a composição e a distribuição de carabídeos e estafilinídeos em culturas e outros habitats. O carabídeo Calosoma granulatum Perty tem sido a espécie mais estudada em agroecossistemas brasileiros (Chocorosqui & Pasini, 2000) e existem estudos que avaliaram o efeito do plantio direto e da consorciação soja-milho nesse predador (Cividanes, 2002) e a ocorrência desse besouro em fragmento florestal e hortaliças (Cividanes et al., 2003). A ocorrência de estafilinídeos tem sido relatada em estudos de levantamento populacional de insetos (Barbosa & Fonseca, 2002) e manejo integrado de pragas (Motta-Miranda et al., 2005). com cerca de 28 anos de idade. As interfaces entre o fragmento, o povoamento de pínus e as culturas apresentavam uma faixa de terra não cultivada de aproximadamente 2,5 m de largura, com discreta presença de plantas herbáceas e por onde eventualmente trafegavam veículos motorizados. Na safra 2003/2004, a área em sistema de plantio direto foi cultivada com milho híbrido AG-7575 no espaçamento 0,90 m entre linhas. A semeadura ocorreu em 22/12/2003 e a adubação consistiu de 300 kg ha-1 da fórmula 10–20–20 (NPK). Introdução Antes da semeadura foram aplicados os herbicidas glyphosate e 2-4-D e durante a safra, nicosulfuron e atrazina. Na safra 2004/2005, a área foi cultivada com soja, cultivar BRS-133, no espaçamento 0,45 m entre linhas. A semeadura foi efetuada em 23/11/2004 e, na adubação, utilizaram-se 300 kg ha-1 da fórmula 0–20–10 (NPK). Os herbicidas fluazifop-p-butil e fomesafen foram aplicados durante a safra e glyphosate em pré-semeadura. Na safra 2003/2004 da área sob plantio convencional, a semeadura do milho ocorreu em 27/11/2003, utilizando-se o híbrido Máster, espaçamento 0,90 m entre linhas e 300 kg ha-1 do adubo de fórmula 10–20–20 (NPK). Durante o desenvolvimento da cultura, foram aplicados os herbicidas nicosulfuron e atrazina. Na safra 2004/ 2005, a semeadura da soja foi efetuada em 21/11/2004, utilizando-se a cultivar BRS-Macota, no espaçamento 0,45 m entre linhas, e 300 kg ha-1 do adubo de fórmula 0–20–10 (NPK). Antes da semeadura, foram aplicados os herbicidas glyphosate e 2-4-D e, durante a safra, fluazifop-p-butil e fomesafen. Ressalte-se que, durante o período de amostragem, não foram aplicados inseticidas nas imediações das armadilhas instaladas nas áreas experimentais. Neste trabalho, efetuou-se amostragem de Carabidae e Staphylinidae em áreas de fragmento florestal e povoamento de pínus e em áreas adjacentes com rotação soja-milho, em plantio direto e convencional, a fim de determinar a distribuição da riqueza de espécies e a preferência pelo habitat. Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 F.J. Cividanes e T.M. dos Santos-Cividanes Distribuição de Carabidae e Staphylinidae em agroecossistemas 159 direto, esses valores foram: 23 espécies de Carabidae, 4 espécies de Staphylinidae e 326 espécimes. Na instalação, utilizaram-se dois transectos paralelos, separados 10 m entre si. Cada transecto apresentava 100 m de comprimento, com 50 m na cultura anual e 50 m no fragmento florestal ou no povoamento de pínus. O total de 24 armadilhas foi instalado por área, sendo que, duas permaneceram na interface cultura-fragmento florestal e cultura-pínus e as demais distantes 10 m entre si. As armadilhas permaneceram no campo por sete dias, sendo depois encaminhadas ao laboratório para a triagem dos besouros. A distribuição relacionada à riqueza de espécies (número de espécies) de Carabidae ao longo dos transectos na área de plantio convencional foi maior que no povoamento de pínus (Figura 1), enquanto no sistema de plantio direto a riqueza de espécies foi similar à do fragmento florestal. Com relação aos estafilinídeos, a riqueza foi maior no fragmento florestal e na área de pínus comparado à riqueza nas culturas (Figura 1). Não foram capturados estafilinídeos na área de cultivo anual sob plantio convencional. Avaliou-se a distribuição da riqueza de espécies dos besouros, considerando-se as variações do número de espécies encontradas nas culturas e no fragmento florestal ou povoamento de pínus. Esse parâmetro foi determinado por dois processos: plotação do número de espécies capturadas pelas armadilhas nas diferentes posições do transecto, durante todo o período de amostragem (Bedford & Usher, 1994), e utilização de análise de regressão linear, considerando-se a distribuição das espécies da interface para o interior das culturas, fragmento florestal ou pínus.A significância do modelo ajustado foi determinada pelo teste t. Figura 1. Número de espécies de Carabidae e de Staphylinidae plotado de acordo com a posição no transecto. O ponto zero indica a interface entre o fragmento florestal ou o pínus (números positivos) e a cultura soja/milho (números negativos). A análise de agrupamento foi empregada para agrupar as espécies por família (Carabidae ou Staphylinidae) de acordo com a posição das armadilhas no transecto e identificar as espécies quanto à preferência pelos habitats: fragmento florestal, povoamento de pínus, culturas e interfaces. As espécies incluídas na análise foram selecionadas de acordo com metodologia de Bedford & Usher (1994). Uma espécie foi considerada presente se dois ou mais indivíduos foram capturados na armadilha durante todo o período de amostragem. Distribuição de Carabidae e Staphylinidae em agroecossistemas O critério de presença foi considerado a fim de reduzir a possibilidade de que indivíduos que vagassem ao acaso pudessem afetar os resultados. As espécies que ocorreram em menos de cinco armadilhas foram consideradas raras e foram retiradas dos dados a serem analisados. As espécies avaliadas pela análise de agrupamento foram apenas as da família Carabidae. Não foram incluídas espécies de Staphylinidae em razão de nenhuma delas ter sido selecionada pelos critérios adotados na análise. Material e Métodos O trabalho foi conduzido na área experimental da Fazenda de Ensino, Pesquisa e Produção da Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade Estadual Paulista (Unesp), campus de Jaboticabal, SP, de dezembro de 2003 a novembro de 2005. As coordenadas geográficas do local correspondem à latitude 21°15'22"S e longitude 48°18'58"O. Os besouros foram amostrados de 15/12/2003 a 30/11/2005 e esse período abrangeu duas safras e duas entressafras, totalizando 36 datas de amostragem: quinzenais nas safras e mensais nas entressafras. Utilizaram-se armadilhas constituídas de copos de plástico com 8 cm de diâmetro e 14 cm de altura, contendo solução de água e formol 1% e detergente neutro, colocadas no solo. Na instalação das armadilhas, utilizou-se cilindro de PVC com 8,5 cm de diâmetro e 20 cm de altura, enterrado, com a borda externa a 1 cm da superfície do solo. Uma cobertura de plástico foi colocada sobre as armadilhas, a fim de evitar a entrada de água de chuva. Utilizaram-se duas áreas experimentais distantes cerca de 2 km entre si e situadas em solo tipo Latossolo Vermelho distrófico textura argilosa. Uma das áreas era constituída de 15 ha de fragmento de Floresta Tropical Latifoliada Semidecídua adjacente a um campo de 40 ha cultivado com soja e milho em sistemas de plantio direto e rotação de culturas e pousio na entressafra. A outra área apresentava um campo de 12 ha de plantio convencional de soja e milho conduzidos em sistema de rotação e pousio na entressafra. Adjacente a esse campo existia um povoamento de 3 ha de Pinus caribaea var. hondurensis (Sénécl) Barr. & Golf. e, ao lado, uma área de 5 ha de Eucalyptus citriodora Hook, ambos A distribuição das armadilhas foi realizada conforme método indicado por Bedford & Usher (1994). Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 Distribuição de Carabidae e Staphylinidae em agroecossistemas Distribuição de Carabidae e Staphylinidae em agroecossistemas Resultados e Discussão Figura 1. Número de espécies de Carabidae e de Staphylinidae plotado de acordo com a posição no transecto. O ponto zero indica a interface entre o fragmento florestal ou o pínus (números positivos) e a cultura soja/milho (números negativos). Na área experimental sob sistema de plantio convencional, foram capturadas 23 espécies de Carabidae e 6 espécies de Staphylinidae, o que totalizou 405 espécimes. Na área conduzida em sistema de plantio Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 F.J. Cividanes e T.M. dos Santos-Cividanes 160 F.J. Cividanes e T.M. dos Santos-Cividanes interior, o que permite que funcionem como habitat fonte de carabídeos que, ao se dispersarem, recolonizam habitats alterados pelo homem (Magura, 2002). A distribuição da riqueza de espécies dos carabídeos e estafilinídeos não variou significativamente da interface para o interior das culturas anuais, fragmento florestal e do povoamento de pínus (Figura 1, Tabela 1).A exceção foi verificada para Carabidae, no sistema de plantio convencional. Nesse caso, verificou-se que o número de espécies de carabídeos aumentou a partir da interface para o interior da cultura (Figura 1, Tabela 1). De acordo com Usis & Maclean (1998) e Kromp (1999), a distribuição de carabídeos em determinado habitat pode ser influenciada por fatores como: temperatura, umidade do ar e do solo, disponibilidade de alimento, tipo de vegetação, estação do ano e o ciclo de vida do besouro. p g Já foi demonstrado que áreas constituídas por árvores e arbustos foram mais favoráveis à ocorrência de carabídeos em culturas de milho do que áreas com cobertura de gramíneas, consideradas menos diversificadas (Varchola & Dunn, 2001). Segundo os autores, as árvores e arbustos funcionam como abrigo para carabídeos e proporcionam maior atividade e diversidade de espécies desses predadores na fase inicial de desenvolvimento da cultura do milho. Portanto, a presença de habitats alternativos tem sido considerada componente importante dos agroecossistemas, por favorecerem a ocorrência de elevada densidade de carabídeos (French et al., 2001). O estabelecimento desses habitats e a utilização de sistemas agrícolas de baixo impacto podem contribuir para o incremento da diversidade de fauna de carabídeos e o sucesso do controle biológico natural de pragas (Pfiffner & Luka, 2003). Apesar da grande quantidade de fatores que podem influenciar a distribuição das espécies desses besouros, na maioria das vezes as espécies das duas famílias apresentaram comportamento semelhante com relação à dispersão no interior do fragmento florestal, povoamento de pínus e mesmo culturas. Resultados e Discussão De acordo com Pfiffner & Luka (2000), em florestas, a temperatura ambiental apresenta pequena variação. Essa característica microclimática pode ter favorecido a dispersão relativamente homogênea de carabídeos e estafilinídeos no interior do fragmento e do pínus. A análise de agrupamento reuniu as espécies de carabídeos de acordo com a preferência pelo habitat (Figuras 2 e 3). A espécie Odontochila nodicornis (Dejean) foi relacionada como de ocorrência em pínus (Figura 2). As espécies Calosoma granulatum Perty, Megacephala sp., Scarites sp. e Selenophorus alternans Dejean foram associadas às culturas milho- soja, no sistema plantio convencional, enquanto Abaris basistriatus Chaudoir esteve relacionada aos três habitats. As espécies Megacephala brasiliensis Kirby, Megacephala sp. e Scarites sp. foram freqüentes em soja-milho, no sistema de plantio direto; O. nordicornis preferiu o fragmento florestal, enquanto A. basistriatus distribuiu-se igualmente nos três habitats (Figura 3). A riqueza de espécies de carabídeos e estafilinídeos observada nas interfaces foi relativamente elevada quando comparada com a encontrada nos demais habitats (Figura 1). A constatação de elevada riqueza de espécies na interface está de acordo com estudos sobre Carabidae e outros grupos de artrópodes (Alderweireldt, 1989; Bedford & Usher, 1994). As bordas de florestas têm grande importância no manejo ambiental, em razão de apresentarem maior diversidade de espécies de carabídeos que em seu Tabela 1. Equações de regressão para a riqueza de espécies de Carabidae e Staphylinidae, de acordo com a posição no transecto, considerando a ocorrência das espécies no fragmento florestal, no pínus ou na cultura. *Significativo a 5% de probabilidade pelo teste t Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 Distribuição de Carabidae e Staphylinidae em agroecossistemas Distribuição de Carabidae e Staphylinidae em agroecossistemas 161 Figura 2. Diagrama em árvore para agrupamento das espécies de Carabidae, de acordo com a posição da armadilha, pelo método da distância euclidiana, em área de pínus, culturas em plantio convencional e na interface. caracterizar tais locais, existindo espécies que predominam em florestas, outras em culturas agrícolas ou outros habitats (Lövei & Sunderland, 1996). De acordo com Thiele (1977), em razão da preferência por fatores abióticos como temperatura, umidade relativa e intensidade luminosa, os carabídeos são diferenciados como “de floresta”, ao preferirem locais escuros e úmidos, e “de campo”, ao se adaptarem a locais quentes e secos. Resultados e Discussão Outros autores (Fournier & Loreau, 1999; French & Elliott, 1999) classificaram os carabídeos, em relação ao habitat, em espécies que preferem culturas, restritas às cercas vivas e generalistas. Conclusões Figura 2. Diagrama em árvore para agrupamento das espécies de Carabidae, de acordo com a posição da armadilha, pelo método da distância euclidiana, em área de pínus, culturas em plantio convencional e na interface. 1. A ocorrência de espécies de Carabidae difere conforme o tipo de cobertura vegetal. 2. As espécies de Carabidae Megacephala sp. e Scarites sp. prevalecem em áreas cultivadas em sistema de rotação soja/milho, e O. nodicornis prevalece no fragmento florestal e povoamento de pínus. 2. As espécies de Carabidae Megacephala sp. e Scarites sp. prevalecem em áreas cultivadas em sistema de rotação soja/milho, e O. nodicornis prevalece no fragmento florestal e povoamento de pínus. Figura 3. Diagrama em árvore para agrupamento das espécies de Carabidae, de acordo com a posição da armadilha, pelo método da distância euclidiana, em área de fragmento florestal, culturas em plantio direto e na interface. Agradecimentos Ao Prof. Dr. José Carlos Barbosa, do Departamento de Ciências Exatas da Faculdade de Ciências Agrárias e Veterinárias/Universidade Estadual Paulista, pelo auxílio nas análises estatísticas; ao Dr. Sérgio Ide, da Agência Paulista de Tecnologias dos Agronegócios/ Instituto Biológico, pela identificação de Carabidae; ao Conselho Nacional de Desenvolvimento Científico e Tecnológico, pela bolsa concedida. Referências Figura 3. Diagrama em árvore para agrupamento das espécies de Carabidae, de acordo com a posição da armadilha, pelo método da distância euclidiana, em área de fragmento florestal, culturas em plantio direto e na interface. ALDERWEIRELDT, M. An ecological analysis of the spider fauna (Araneae) occurring in maize fields, Italian rye-grass fields and their edge zones, by means of different multivariate techniques. Agriculture, Ecosystem & Environment, v.27, p.293-306, 1989. ALDERWEIRELDT, M. An ecological analysis of the spider fauna (Araneae) occurring in maize fields, Italian rye-grass fields and their edge zones, by means of different multivariate techniques. Agriculture, Ecosystem & Environment, v.27, p.293-306, 1989. BARBOSA, M. das G.V.; FONSECA, C.R.V. da. Coleopterofauna visitante de Theobroma grandiflorum Schum. (Sterculiaceae) de uma plantação nos arredores de Manaus, Amazonas, Brasil. Acta Amazonica, v.32, p.83-100, 2002. Os resultados obtidos nas duas áreas experimentais evidenciam que a maior parte das espécies de carabídeos preferiu os campos cultivados com culturas anuais. Por sua vez, o carabídeo A. basistriatus apresentou distribuição semelhante nos três habitats estudados, caracterizando-se como generalista com relação à preferência pelo habitat. A espécie O. nodicornis preferiu apenas o povoamento de pínus e o fragmento florestal. Os carabídeos são tão específicos na escolha do habitat que freqüentemente são utilizados para BEDFORD, S.E.; USHER, M.B. Distribution of arthropod species across the margins of farm woodlands. Agriculture, Ecosystem & Environment, v.48, p.295-305, 1994. CHOCOROSQUI, V.R.; PASINI,A. Predação de pupas de Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) por larvas e adultos de Calosoma granulatum Perty (Coleoptera: Carabidae) em laboratório. Anais da Sociedade Entomológica do Brasil, v.29, p.65-70, 2000. Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 162 F.J. Cividanes e T.M. dos Santos-Cividanes MAGURA, T. Carabids and forest edge: spatial pattern and edge effect. Forest Ecology and Management, v.157, p.23-37, 2002. CIVIDANES, F.J. Efeitos do sistema de plantio e da consorciação soja-milho sobre artrópodes capturados no solo. Pesquisa Agropecuária Brasileira, v.37, p.15-23, 2002. MOTTA-MIRANDA, M.M.; COUTINHO-PICANÇO, M.; ZANÚNCIO, J.C.; BACCI, L.; SILVA, E.M. da. Impact of integrated pest management on the population of leafminers, fruit borers, and natural enemies in tomato. Ciência Rural, v.35, p.204-208, 2005. CIVIDANES, F.J.; SOUZA, V.P. de; SAKEMI, L.K. Composição faunística de insetos predadores em fragmento florestal e em área de hortaliças na região de Jaboticabal, SP. Acta Scientiarum, v.25, p.315-321, 2003. MUNDY, C.A.; ALLEN-WILLIAMS, L.J.; UNDERWOOD, N.; WARRINGTON, S. Prey selection and foraging behaviour by Pterostichus cupreus L. (Col., Carabidae) under laboratory conditions. Referências Journal of Applied Entomology, v.124, p.349-358, 2000. CLARK, M.S.; GAGE, S.H.; SPENCE, J.R. Habitats and management associated with common ground beetles (Coleoptera: Carabidae) in a CLARK, M.S.; GAGE, S.H.; SPENCE, J.R. Habitats and management associated with common ground beetles (Coleoptera: Carabidae) in a Michigan agricultural landscape. Environmental Entomology, v.26, p.519-527, 1997. Michigan agricultural landscape. Environmental Entomology, v.26, p.519-527, 1997. PFIFFNER, L.; LUKA, H. Effects of low-input farming systems on carabids and epigeal spiders: a paired farm approach. Basic and Applied Ecology, v.4, p.117-127, 2003. CRIST, T.O.; AHERN, R.G. Effects of habitat patch size and temperature on the distribution and abundance of ground beetles (Coleoptera: Carabidae) in an old field. Environmental Entomology, v.28, p.681-689, 1999. PFIFFNER, L.; LUKA, H. Overwintering of arthropods in soils of arable fields and adjacent semi-natural habitats. Agriculture, Ecosystem & Environment, v.78, p.215-222, 2000. FOURNIER, E.; LOREAU, M. Effects of newly planted hedges on ground-beetle diversity (Coleoptera, Carabidae) in an agricultural landscape. Ecography, v.22, p.87-97, 1999. SADEJ, W.; NIETUPSKI, M. Occurrence of pea aphid (Acyrthosiphon pisum Harris) on fabe bean and some biotic factors reducing its numbers. Natural Sciences, v.5, p.73-82, 2000. FOURNIER, E.; LOREAU, M. Respective roles of recent hedges and forest patch remnants in the maintenance of ground-beetle (Coleoptera: Carabidae) diversity in an agricultural landscape. Landscape Ecology, v.16, p.17-32, 2001. SUENAGA, H.; HAMAMURA, T. Occurrence of carabid beetles (Coleoptera: Carabidae) in cabbage fields and their possible impact on lepidopteran pests. Applied Entomology and Zoology, v.36, p.151-160, 2001. FRENCH, B.W.; ELLIOTT, N.C. Temporal and spatial distribution of ground beetle (Coleoptera: Carabidae) assemblages in grasslands and adjacent wheat fields. Pedobiologia, v.43, p.73-84, 1999. FRENCH, B.W.; ELLIOTT, N.C.; BERBERET, R.C.; BURD, J.D. Effects of riparian and grassland habitats on ground beetle (Coleoptera: Carabidae) assemblages in adjacent wheat fields. Environmental Entomology, v.30, p.225-234, 2001. THIELE, H.U. Carabid beetles in their environments: a study on habitat selection by adaptations in physiology and behaviour. Berlin: Springer, 1977. 369p. THOMAS, C.F.G.; HOLLAND J.M.; BROWN N.J. The spatial distribution of carabid beetles in agricultural landscapes. In: HOLLAND, J.M. (Ed.). The agroecology of carabid beetles. Andover: Intercept, 2002. p.305-344. THOMAS, C.F.G.; HOLLAND J.M.; BROWN N.J. The spatial distribution of carabid beetles in agricultural landscapes. In: KROMP, B. Carabid beetles in sustainable agriculture: a review on pest control efficacy, cultivation impacts and enhancement. Agriculture, Ecosystem & Environment, v.74, p.187-228, 1999. HOLLAND, J.M. (Ed.). The agroecology of carabid beetles. Andover: Intercept, 2002. p.305-344. Pesq. agropec. bras., Brasília, v.43, n.2, p.157-162, fev. 2008 Referências LEVESQUE, C.; LEVESQUE, G.Y.Abundance and seasonal activity of ground beetles (Coleoptera: Carabidae) in a raspberry plantation and adjacent sites in Southern Québec (Canada). Journal of the Kansas Entomological Society, v.67, p.73-101, 1994. USIS, J.D.; MACLEAN, D.B. The ground beetles (Coleoptera: Carabidae) of stillfork swamp nature preserve, Carroll County, Ohio. Ohio Journal of Science, v.98, p.66-68, 1998. VARCHOLA, J.M.; DUNN, J.P. Influence of hedgerow and grassy field borders on ground beetle (Coleoptera: Carabidae) activity in fields of corn. Agriculture, Ecosystem & Environment, v.83, p.153-163, 2001. LÖVEI, G.L.; SUNDERLAND, K.D. Ecology and behavior of ground beetles (Coleoptera: Carabidae). Annual Review of Entomology, v.41, p.231-256, 1996. Recebido em 27 de agosto de 2007 e aprovado em 4 de janeiro de 2008
https://openalex.org/W4384490896
https://zenodo.org/records/8152794/files/IMMURDANA%20MBI-V10N8-2016.pdf
Indonesian
null
Pemanduan Wisata Selam Di Bluemarlin Dive Gili Trawangan Lombok
Zenodo (CERN European Organization for Nuclear Research)
2,016
cc-by
5,071
Vol. 10 No. 8 Agustus 2016 PENDIDIKAN – PENELITIAN – OLAHRAGA – TEKNOLOGI – SENI ISSN 1978-3787 Vol. 10 No. 8 Agustus 2016 MEDIA BINA ILMIAH Terbit Setiap Bulan DAFTAR ISI : Artikel Pendidikan 1. Perbedaan Efektifitas Albendazole Bolus 1500 Mg Dengan Ivermectine 1%Terhadap Penurunan Jumlah Telur Cacing Haemonchus Contortus Pada Sapi Bali Di Desa Ubung Lombok Tengah ………………………………………….. 1 Oleh : Supriadi , Muhammad Edi Jayadi 2. Analisis Rasio Kinerja Keuangan KPRI Patut Patuh Patju Kabupaten Lombok Barat Tahun 2012 – 2015 …………………………………………….…...... 4 Oleh : I Gusti Putu Bagus Suastina, I Gusti Ayu Ola Netrawati 3. Upaya Meningkatkan Kompetensi Guru Binaan Dalam Proses Pembelajaran Di Kelas Melalui Supervisi Akademik Semester Dua Tahun 2015/2016 Di SMP Negeri 21 Mataram ………..………..……...........................…………………. 12 Oleh Hj. Srirahayu Suryaningsih 4. Pembelajaran Membudayakan Hidup Sehat Dengan Menggunakan Media Gambar Pada Siswa Kelas III SDN 3 Cakranegara Tahun Pembelajaran 2014/2015 .......... 20 Oleh : Gusti Lanang Adnyana 5. Meningkatkan Prestasi Belajar Ipa Materi Isolator Dan Konduktor Menggunakan Metode Experimen Pada Siswa Kelas VI Semester I SDN 1 Cakranegara Tahun Pelajaran 2015/2016.……………………………………………... 25 Oleh : Hamidah 6. Meningkatkan Hasil Belajar Matematika Menggunakan Bantuan Benda-Benda Sekitar Pada Siswa Kelas 1 SDN I Cakranegara Tahun Pelajaran 2015/2016 …...... 31 Oleh : Luh Sri Suhartini 7. Meningkatkan Hasil Belajar Bahasa Indonesia Materi Unsur Dan Nilai Moral Cerita Rakyat Melalui Metode Value Clarification Techniquekelas V SDN 1 Cakranegara Tahun Pelajaran 2015/2016 ……….……………………...........…. 36 Oleh : Baiq Titik Setiawati 8. Manajemen Kepemimpinan Kepala Sekolah Terhadap Motivasi Belajar Siswa Di SMP Tri Murti Kecamatan Pakisan Kabupaten Malang ……..………..……… 41 Oleh : Made Dwianan Mustawan 9. Membentuk Karakter Dan Membudayakan Nilai-Nilai Pancasilamelalui Penataan Suasana Sekolah Yang Kondusif Di SDN 3 Dasan Tereng………………… 46 Oleh : Nuraini 10. Paragdima Pendidikan Inklusi Sekolah Dasar Di Kecamatan Narmada ……….… 53 Oleh : I Nyoman Suweca 11. Pengaruh Aspek Sosiologi Terhadap Korban Bulliying Di Kota Mataram …..….. 58 Oleh: Yani Rosita Sarlan 12. Ecotourism In Subak Kerdung, Pedungan Takes Part To Support Sustainable Tourism ……………………………………………………………..………….. 62 Oleh: I Ketut Redjasa 13. Pemanduan Wisata Selam Di Bluemarlin Dive Gili Trawangan Lombok ……….. 66 Oleh: Mahsun, I Made Murdana*, Widia Susanti PEMANDUAN WISATA SELAM DI BLUEMARLIN DIVE GILI TRAWANGAN LOMBOK Oleh: Oleh: Mahsun, I Made Murdana*, Widia Susanti DPK Kopertis pada Akademi Pariwisata mataram Abstract: This study was aimed to find out about tourist dive tour with a case study in blue marlin dive centre of Gili Trawangan. The research method is using qualitative methods. The dive master did dive tour with PADI referring to the standard rules and regulation. Standardization and specification of equipment must also comply with the standards of PADI and safety. Assisting dive tour in blue marlin Gili Trawangan Lombok is based on three categories of stages, namely 1) the preparation stage dive ((1) Receiving Consumers who already have orders, (2) Information dive site that will be in the lead, (3) Testing of equipment and preparation, (4) Briefing by the dive master, (5) Preparation boat to the dive site area), 2) the implementation phase of the dive, and 3) the end of stage / terminate the dive. Base of the risk and high danger, The dives are performed approached to procedures comply with the rules and standards regulation of PADI as assure and guaranty. So the dive procedures and guidance to be precise. Key word: Diving, Dive tour, Tourist, Gili Trawangan, Artikel Pendidikan ISSN No. 1978-3787 66 Media Bina Ilmiah PENDAHULUAN atraksi wisata yang cocok di daerahnya untuk dikembangkan. Kurangnya pemahaman tentang biota laut khususnya terumbu karang dalam menopang biomasa laut. Kurang tertariknya masyarakat akan atraksi wisata selam , kurang fahamnya tentang teknik-teknik penyelaman dan keselamatan wisatawan yang menyelam. Hal itu adalah sekelumit fenomena yang terjadi ditengah- tengah pengembangan atraksi wisata bawah laut serta pemanduan wisata bawah laut. Pulau Gili Trawangan merupakan salah satu destinasi wisata unggulan yang ada di Lombok pada khususnya dan Nusa Tenggara Barat pada umumnya. Pengembangan Pulau Gili Trawangan sebagai destinasi unggulan sangat dipengaruhi oleh panorama alam yang masih asli, budaya dan adat yang unik, serta yang tidak kalah adalah panorama bawah laut yang sangat mepesoan. Semua panorama dan kekayaan alam tersebut terkumpul dan disebut sebagai sebuah atraksi wisata. Kesadaran masyarakat akan pentingnya sebuah atraksi wisata bagi pengembangan suatu daerah, telah memberikan kedewasaan berfikir masyarakat Gili Trawangan dalam mengembangkan daerahnya. Masyarakat sangat atosisa dan bahu membahu dalam ikut berpartisifasi pada pengembangan pariwisata pulau. Kultur dan karakter masyarakat pulau Gili Trawangan sangat berbeda dengan masyarakat daratannya. Masyarakat gili trawangan sangat proaktif dan ellcoming terhadap wisatawan. Masyrakat juga sangat peduli terhadap keselamatan dan keamanan wisatawan yang berada di Gili Trawangan. Masyaraka sangat berkontribusi besar terhadap program pengembangan pariwisata Pulau gili Trawangan melalui program konservasi trumbu karang. METODE Pemanfaatan metode dengan wawancara dan dokumentasi terhadap populasi yang diteliti dengan pendekatan deskriptif kualitatif 4. Menjelajah di bawah permukaan air Penyelam harus dapat menguasai peralatan dengan baik dan benar. Peralatan dasar selam dan peralatan scuba merupakan alat bantu kita melakukan kegiatan penyelaman. Latihan yang rutin di kolam sangat membantu agar kita familier dengan semua peralatn tersebut seperti melakukan skin diving, scuba diving, regulator clearing, mask clearing equalization muncul secara lambat (slow ascent) dan bongkar pasang peralatan scuba. c. Back Roll Entry Jika akan masuk ke air dari kapal kecil atau perahu karet, maka back roll entry adalah cara yang terbaik dan termudah. Dengan cara duduk di pinggir perahu karet, posisi kaki rapat, satu tangan di mask dan regulator sedang yang lain di B.C. Gulingkan badan ke belakang. 5. Menguras kaca muka (mask clearing) Tujuan mask clearing adalah untuk menghilangkan pengem bunan pada mask saat penyelaman di kedalaman. Pengembunan akan terjadi karena adanya perbeedaan suhu dalam air. Dengan cara mengisi air ke dalam mask pengembunan akan hilang, dan dengan menekan bagian atas mask serta menghembuskan udara melalui hidung, maka air akan terdorong keluar melalui sela-sela mask, sehingga pandangan akan menjadi jelas kembali. Tujuan mask clearing adalah untuk menghilangkan pengem bunan pada mask saat penyelaman di kedalaman. Pengembunan akan terjadi karena adanya perbeedaan suhu dalam air. Dengan cara mengisi air ke dalam mask pengembunan akan hilang, dan dengan menekan bagian atas mask serta menghembuskan udara melalui hidung, maka air akan terdorong keluar melalui sela-sela mask, sehingga pandangan akan menjadi jelas kembali. d. Said Roll Entry Cara lain untuk masuk ke air dari perahu karet adalah dengan dengan said roll entry. Baringkan badan di pinggiran perahu karet kemudian bergulir ke air. Cara ini lazim digunakan oleh under water demolation team (UDT) atau regu penghancur bawah air, yang harus masuk ke air sementara perahu karet tetap melaju cepat. 2. Muncul ke permukaan (safety ascent) 6. Menguras mouthpiece 2. Muncul ke permukaan (safety ascent) Untuk menghindari cedera pada waktu muncul ke permukaan penyelam harus selalu melihat ke atas, menggapai dan kemudian muncul perlahan-lahan berputar 360o sambil tetap mengawasi permukaan. Manufer ini penting sekali, terutama pada tahap 10 feet terakhir untuk sampai di permukaan. Pada saat melakukan penyelaman kadang terjadi regulator terlepas dari mulut kita. Sering dari kita melakukan kecerobohan dengan segera measukkan mouthpiece ke dalam mulut dan menghisapnya. Akibatnya bukan udara yang kita peroleh melainkan air, sehingga kita mengalami tersedak dan panik. Cara yang benar untuk menguras mouthpiece adalah dengan menghembuskan udara terlebih dahulu untuk menguras air yang ada di dalam mouthpiece kemudian menghisapnya. a. http://www.lpsdimataram.com g 3. Tehnik muncul darurat (esa) 3. Tehnik muncul darurat (esa) Pada saat terjadi udara pada tabung habis, seorang penyelam harus melakukan tehnik muncul darurat dengan cara melepaskan regulator dari mulut, naik ke permukaan secara vertikal dengan perlahan-lahan dan menghembuskan nafas secara terus-menerus hingga mencapai kedalaman 10 feet, pada kedalaman tersebut posisi badan berubah menjadi horizontal, kepala menengadah keatas, tangan mengembang, hingga sampai ke permukaan. Pada keadaan tertentu weight belt dapat dilepas untuk mendapatkan tambahan daya apung positif. b. b. Sitting front entry b. Sitting front entry Cara ini akan sangat berguna jika akan masuk air dari dermaga yang rendah ataupun plat form. Dalam posisi duduk, fins berjuntai keluar, tempatkan tangan di kedua sisi, berputarlah dan masuk ke air, kedua kaki mengayuh agar tidak enyembul ke permukaan. 4. Menjelajah di bawah permukaan air LANDASAN Ada beberapa tahapan yang dialakukan dalam melaksanakan latihan penyelaman (Oven Water Diver Manual, 2011) yaitu; 1. Masuk ke air (Entry) Ada 4 macam cara yang lazim digunakan oleh seorang penyelam untuk masuk ke bawah permukaan air, yaitu : a. Standing Front Entry Seorang penyelam dapat masuk ke air dari geladak kapal, perahu dan dermaga dengan menggunakan posisi masuk berdiri.Ini cara yang paling aman untuk digunakan.Penyelam akan masuk dengan kaki terlebih dahulu, dengan posisi “Melangkahkan kaki (Giant Step)”.Pada waktu kaki menyentuh air, kedua kaki dikatupkan kembali untuk menjaga posisi penyelam agar tetap di permukaan. Satu tangan memegang mask dan regulator agar Berkembangnya dan ikutsertanya masyarakat dalam mensukseskan program konservasi tersebut menandakan bahwa kesadaran akan kebutuhan atraksi wisata pulau yaitu wisata bawah laut sangat besar. Usaha tersebut tidaklah sia-saia saja, terbukti telah banyak dive master-dive master berasal dari pulau tersebut. Lain halnya dengan phenomena pengemabngan pulau lainnya masyarakat cendrung tidak memahami tentang http://www.lpsdimataram.com ___________________________ Volume 10, No. 8,Agustus 2016 ISSN No. 1978-3787 Media Bina Ilmiah67 menengadah, pandangan dan tangan mengarah ke atas. menengadah, pandangan dan tangan mengarah ke atas. tidak lepas pada saat bersentuhan dengan air, sedangkan satu tangan lagi memegang bagian bawah B.C. untuk menjaga supaya tabung tidak mengenai tengkuk seandainya tabung tidak terikat pada B.C. secara sempurna, kemudian kepala menunduk.Jika sudah siap, langkahkan kaki ke depan tanpa adanya gerakan lompatan.Jangan miringkan ke belakang ataupun ke depan. Biarkan kaki tetap terentang hingga menyentuh permukaan air. 3. 4. Menjelajah di bawah permukaan air Tehnik muncul terkendali (slow ascent) Penyelam harus selalu naik ke permukaan dengan lambat. Kecepatan aman untuk naik adalah 60 feet per menit. Cara mengetahui kecepatan yang paling mudah adalah melihat gelembung udara yang paling kecil dan tidak boleh mendahului. Pada snorkel g p y Pada snorkel Pada snorkel Oleh karena itu dunia penyelaman menganut dan mempraktekan prinsip penyelaman yang mengatakan never dive alone. Jadi menyelamlah selalu dalam suatu team dengan sitim mitra (buddy sistim). Keadaan darurat pada penyelaman lazim diidentikkan dengan keadaan tanpa atau kehabisan udara. Salah satu cara yang paling efektif mengantisipasi situasi ini adalah dengan melakukan “buddy breathing” (patungan udara). Jika keadaan masih dapat dikendalikan dan buddy berada di dekat, udara dapat dipakai bersama (patungan ) di dalam air atau ketika naik ke atas. 11. 11. Penanganan peralatan di dasar Seorang penyelam harus dapat seca ra mudah menangani peralatannya di bawah permukaan air apabila mengalami kesulitan. Maka seorang bpenyelam dituntut untuk mampu membongkar pasang peralatan secara benar di bawah air. Cara membongkar peralatan; bawah air. Cara membongkar peralatan; a. Melepaskan ikat pinggang b. Angkat lewat atas kepala c. Atur regulator agar tidak terjepit Cara memasang peralatan : a. Raih regulator, pasang mouth piece ke dalam mulut dan hembuskan udara agar air terkuras; b. Angkat tabung melewati atas kepal, usahakan agar regulator tidak terbelit; c. Atur ikat pinggang/gesper supaya BC terpasang dengan nyaman 8. 8. Pengendalian romphi apung dipermukaan dan di kedalaman Di permukaan Penyelam berada di tempat dalam dengan posisi berdiri dengan mulut meniup penuh BC nya melalui oral inflator. Pengendalian romphi apung dengan cara mengatur udara di BC sampai batas permukaan air berada di mata. Di kedalaman Penyelam di kedalaman mengatur netral buoyancy dengan cara mengisi udara ke BC baik lewat oral inflator maupun mekanikal inflator. Pada snorkel Pada snorkel a. Popping Cara menghilangkan air dari snorkel dengan cara menghembuskan udara ke dalam snorkel sehingga air di dalam snorkel hilang dan kita dapat bernafas lagi. b. Tehnik muncul bebas Penyelam melakukan surface dive, berenang ke dasar kemudian melepaskan snorkel dan naik ke permukaan secara perlahan-lahan sambil menghembuskan nafas terus-menerus hingga muncul ke permukaan. Posisi kepala p g b. Water displacing method b. Water displacing method Cara snorkel clearing dengan metode ini sangat membantu, karena tidak perlu meniup _______________________ Volume 10, No. 8, Agustus 2016 68 Media Bina Ilmiah ISSN No. 1978-3787 68 Media Bina Ilmiah ISSN No. 1978-3787 Setiap penyelam harus mahir mengambil dan memasang kembali peralatan weight belt di bawah permukaan air. Hal ini dikarenakan bisa saja terjadi weight belt terlepas pada saat penyelam melakukan manuver-manuver di bawah air. 7.10. Renang snorkel dengan peralatan lengkap Pada saat penyelam sudah berada di permukaan, namun jauh dari kapal maka penyelam dapat berenang menuju ke kapal. Berhubung penyelam masih menggunakan peralatan selam lengkap, maka cara yang terbaik dan tidak melelahkan adalah dengan mengisi udar a ke BC untuk mengurangi beban tersebut dan berenang dengan menggunakan snorkel menuju ke kapal. udara dengan keras. Pada saat penyelam mendekati permukaan dengan tangan keatas tengadahkan kepala sehingga ujung atas snorkel mengarah ke bawah dengan menghembuskan secara perlahan dan terus- menerus akan mengakibatkan udara yang dihembuskan menahan air masuk ke snorkel sewaktu penyelam menuju ke atas. Setelah penyelam sampai di permukaan dan posisi berenang, maka ia akan dapat menghirup udara tanpa harus meniup snorkel karena snorkel telah bersih dari air Setiap penyelam harus mahir mengambil dan memasang kembali peralatan weight belt di bawah permukaan air. Hal ini dikarenakan bisa saja terjadi weight belt terlepas pada saat penyelam melakukan manuver-manuver di bawah air. 7.10. Renang snorkel dengan peralatan lengkap Pada saat penyelam sudah berada di permukaan, namun jauh dari kapal maka penyelam dapat berenang menuju ke kapal. Berhubung penyelam masih menggunakan peralatan selam lengkap, maka cara yang terbaik dan tidak melelahkan adalah dengan mengisi udar a ke BC untuk mengurangi beban tersebut dan berenang dengan menggunakan snorkel menuju ke kapal. 7. Sistim mitra selam dan patungan udara Sistim mitra selam dan patungan udara p g Menyelam adalah kegiatan yang berisiko tinggi, terlebih-lebih bila penyelaman itu dilakukan seorang diri. Bila terjadi suatu keadaan arurat yang membahayakan keselamatan jiwa dan raga, tidak akan ada orang yang mengetahui dan membantu kesulitan tersebut. http://www.lpsdimataram.com 2.2 Latihan Perairan Terbuka (LPT) 2.4.1. Pengertian c) Ketahui dan pastikan “Gesper Luncur” berfungsi normal. d) Tabung : supplay udara; Katup terbuka sempurna; Regulator berfungsi sempurna; SPG berfungsi sempurna e) Pemeriksaan diakhiri dengan memberikan isyarat OK. Masuk ke bawah permukaan air dengan menelusuri : d. Buoyancy control Melatih penyelam agar mampu memenetukan beberpa pemberat yang ia perlukan, sehingga tidak mengalami kesulitan untuk turun dan naik dari kedalaman. Latihan ini masih dipraktekkan di kolam. a) Lengkung landas b) Tali jangkar kapal / perahu c) Tali yang dijulurkan dari pantai sampai ke dasar d) Tali pemandu vertikal d. Mask Clearing e. Briefing Sebelum atau sesaat suatu penyelaman akan dimulai, hendaknya diberikan penjelasan atau pengarahan tentang segala aspek yang bersangkutan dengan kegiatan selam yang akan dilakukan agar penyelaman itu dapat berlangsung dengan aman, selamat, menyenangkan, berhasil guna dan berdaya guna. e. Snorkel Clearing f. Muncul ke permukaan (Ascent) p 2. Latihan ketrampilan menggunakan peralatan scuba di perairan terbuka. Sebagai bagian terakhir dari rangkaian proses pendidikan dan pelatihan. Latihan ini ditujukan untuk menciptakan seorang penyelam scuba yang berdaulat (mandiri), percaya diri, aman serta mampu secara santai manikmati kenikmatan penyelaman. Yang penting disini adalah, latihan ini mewujudkan tingkat penguasaan ketrampilan selam dalam pengertian : mahir, bukan sekedar selasai mengikuti semua mata acara latihan secara lengkap. Oleh karena itu dalam latihan ini setiap peserta dituntut untuk mampu melaksanakan tehnik dan prosedur penyelaman scuba di medan yang sebenarnya secara urut, lugas dan tepat, yang antara lain meliputi : 2. Latihan ketrampilan menggunakan peralatan scuba di perairan terbuka. Sebagai bagian terakhir dari rangkaian proses pendidikan dan pelatihan. Latihan ini ditujukan untuk menciptakan seorang penyelam scuba yang berdaulat (mandiri), percaya diri, aman serta mampu secara santai manikmati kenikmatan penyelaman. Yang penting disini adalah, latihan ini mewujudkan tingkat penguasaan ketrampilan selam dalam pengertian : mahir, bukan sekedar selasai mengikuti semua mata acara latihan secara lengkap. Oleh karena itu dalam latihan ini setiap peserta dituntut untuk mampu melaksanakan tehnik dan prosedur penyelaman scuba di medan yang sebenarnya secara urut, lugas dan tepat, yang antara lain meliputi : f. f. Entry dan Exit Pada waktu “entry” Tehnik entry senantiasa berbeda-beda tergantung pada lokasi/daerah penyelamannya. Yang penting dalam entry adalah hindari “bertabrakan” dengan air, karena air : a) Selalu pegang mask; b) Maouthpiece selalu ditempatnya; c) Hindarkan tehnik entry dengan berguling, karena dapat menyebabkan disorientasi; a. Persiapan perlengkapan Kemampuan melaksanakan pemeriksaan kelengkapan, pengaturan dan penyetelan peralatan masing-masing untuk selalu berada dalam kondisi “siap pakai” sebelum dimulainya penyelaman. 2.2 Latihan Perairan Terbuka (LPT) 2.4.1. Pengertian Latihan yang diselenggarakan di perairan terbuka misalnya laut, danau dan lain-lain) dan merupakan urutan dari manuver yang sederhana sampai kepada manuver yang paling sulit, yang dilakukan secara berangsur-angsur di kedalaman yang semakin meningkat. LPT harus merupakan latihan ketrampilan lapangan yang wajar, masuk akal dan penerapannya berlaku bagi kondisi medan penyelaman yang aman. Pada dasarnya LPT dibagi dalam 3 (tiga) kategori: 9. Menghadapi masalah di bawah permukaan air Tehnik penyelamatan dengan vest sangat penting dikuasai. Hal ini sangat berguna apabila terjadi masalah di bawah permukaan air pada saat melakukan penyelaman. Dengan tambahan daya apung dari vest tersebut kita dapat mengangkat korban dari dasar menuju kepermukaan dan segera memberikan bantuan pernafasan dari mulut ke mulut sebelum sampai di pantai. a) Latihan awal Berlaku bagi para pemula dengan jenjang / tingkat kemahiran scuba diver 3 (A2) dan scuba diver 2 (A3). b) Latihan pengembangan kecakapan Khusus dilatihkan untuk meningkatkan kemampuan dan pengalaman penyelam dengan jenjang scuba diver 1 (A4). c) Latihan untuk kecakapan khusus Latihan yang dititik beratkan kepada prosedur penanganan keadaan darurat dan c) Latihan untuk kecakapan khusus p p 10. Weight belt 10. Weight belt ___________________________ Volume 10, No. 8,Agustus 2016 http://www.lpsdimataram.com ISSN No. 1978-3787 Media Bina Ilmiah69 memimpin suatu kegiatan selam. Ini berlaku bagi pemegang sertifikat jenjang master scuba diver, sebagai persiapan untuk memasuki kegiatan keinstrukturan. Kemampuan mengenakan peralatan selam dengan lengkap tanpa harus menjadi kelelahan dan kepanasan. Dalam hal ini prinsip ke-mitraan (Buddy System) sudah harus membudaya. g 2.4.2. Latihan awal g 2.4.2. Latihan awal c. Pre Dive Safety Drill Mitra yang telah lengkap mengenakan peralatan selam (kecuali mask, snorkel, fins dan sarung tangan)saling berhadapan dan secara bergantian melakukan pemeriksaan antara lain : c. 1. Latihan ketrampilan menggun akan peralatan dasar selam di perairan terbuka. 1. Latihan ketrampilan menggun akan peralatan dasar selam di perairan terbuka. Pada hakekatnya merupakan penerapan lebih lanjut dari latihan menggunakan peralatan selam di kolam (LKK) yang antara lain meliputi : j gg peralatan selam di kolam (LKK) yang antara lain meliputi : a. Berenang di permukaan (Fins swimming) Berenang di permukaan air dengan peralatan dasar selam a) BC : terpsang dengan sempurna/tidak, Berfungsi dengan baik/tidak b) Weight belt pastikan bebas dari hambatan sehingga dalam kondisi “Quick Release” b. Surface dive Yang dilakukan menukik ke kedalaman dengan kepala lebih dahulu, kemudian mengayuh dengan kaki sampai ke dasar pada kedalaman 10 s/d 15 kaki dan muncul kembali ke permukaaan sambil membawa benda yang dipungut dari dasar. http://www.lpsdimataram.com 2.2 Latihan Perairan Terbuka (LPT) 2.4.1. Pengertian d) Jangan mengadakan penyetelan peralatan pada titik entry. e) Usahakan daya apung yang benar, periksa posisi peralatan, istirahat sejenak baru melanjutkan latihan. y p b. Suiting Up j Pada waktu “exit” Pada waktu “exit” _______________________ Volume 10, No. 8, Agustus 2016 70 Media Bina Ilmiah ISSN No. 1978-3787 ISSN No. 1978-3787 70 Media Bina Ilmiah a) Berhenti dan istirahat sejenak sebelum exit; sebuah tabung udara yang diletakkan dipunggung dan anda hanya menyelam ke dalam air dengan dilindungi seperangkat baju anti air serta tabung tersebut yang membuat tetap dapat bernapas dalam air. Untuk melakukan penyelaman dan beradaptasi dengan lingkungan bawah perairan diperlukan suatu alat yang disebut SCUBA (Self Contained Underwater Breathing Apparatus). Hal-hal yang perlu diperhatikan dalam olahraga pariwisata selam (Dive Guide to Indonesia, 2005) adalah sebagai berikut : b) Saling memperhatikan antara sesama mitra; c) Dalam situasi exit ke kapal / daerah berbatu, terlebih dahulu, lepaskan perlengkapan yang berat-berat. g. Manuver-manuver di bawah permukaan, antara lain meliputi : a) equalzing; b) Mask clearing; 1. Mempersiapkan diri dari segi fisik dan mental. Dari segi fisik, harus dapat berenang, sehat dan tidak memiliki gangguan pernapasan serta kemampuan untuk menyesuaikan keseimbangan. Dari segi mental diharuskan mampu mengatasi kepanikan beradaptasi dengan orang lain serta mengatsi rasa jijik mengahadapi makhluk laut yang berbentuk aneh. c) buddy breathing; d) trimming h. Adaptasi h. Adaptasi Setiap penyelam harus mampu beradaptasi baik secara fisiologis maupun skologis terhadap lingkungan bawah air yang sedang diselaminya, sehingga ia merasa betah dan nyaman waktu melakukan penyelaman itu. 2. i. De-briefing Latihan perairan terbuka diakhiri dengan acara de-briefing di dekat lokasi penyelaman dengan tujuan : 2. Mempersiapkan semua peralatan yang dibutuhkan. Membeli perlengkapan menyelam bisa jadi lebih murah dibandingkan bila harus menyewa setiap kali ingin menyelam. Tapi perlu diketahui bahwa harga peralatan selam cukup mahal sehingga penting untuk terlebih dahulu memastikan bahwa benar-benar berminat dengan olahraga ini dan akan melakukannya dengan teratur. a) Menyimpulkan dan evaluasi atas proses dan detail latihan selagi masih segar dalam ingatan; b) Merupakan tambahan pengalaman melalui pembahasan bersama atas acara latihan. Peralatan dasar yang sebaiknya dimiliki sendiri oleh seorang diver (Taufik, 2002) adalah: j. Perawatan peralatan Membiasakan dan menumbuhkan kesadaran penyelam untuk merawat perlengkapan/peralatannya setiap selesai dari suatu penyelaman, sehingga awet dan selalu siap pakai. 1. Googgle atau kacamata selam, harus pas dengan ukuran dan kontur wajah. 2. Fin atau kaki katak, harus pas dengan ukuran telapak kaki. p 3. 2.2 Latihan Perairan Terbuka (LPT) 2.4.1. Pengertian Weight atau pemberat, setiap orang memiliki sifat buoyancy (kemampuan mengambang di bawah air) yang berbeda- beda sehingga weight yang dipakai harus sesuai dengan sifat buoyancynya tersebut. k. Logging penyelaman Membiasakan penyelam membuat catatan setelah selesai acara latihan, secara cermat tentang : 4. Wet suit, harus pas dengan ukuran tubuh a) kedalaman; b) bottom time; PEMBAHASAN HASIL c) jarak pandang; d) temperature; Blue Marlin Dive Centre sebagai sebuah usaha wisata selam dalam operasional diving nya dilakukan dalam sehari melakukan dua kali penyelaman. Penyelaman dilakukan di pagi hari yaitu pada jam 09:30 dan penyelaman yang dilakukan di sore hari pada jam 13:30 wita. Setiap kali penyelaman seorang dive master memendu 4 orang dalam satu group. Peserta diving tuduk pada semua instruksi dari dive master selama kegiatan dilakukan. Peserta kegiatan wsata selam adalah seseorang yang telah lulus dan memiliki sertifikat diving yang dikeluarkan oleh PADI. Bagi calon peserta yang berminat dan belum memiliki e) repetitive dive e) repetitive dive Catatan tersebut ditulis pada sebuah buku yang wajib dimiliki oleh setiap penyelam yang lazim disebut “Diver Log Book”. e) repetitive dive Catatan tersebut ditulis pada sebuah buku yang wajib dimiliki oleh setiap penyelam yang lazim disebut “Diver Log Book”. 2.3 2.3 Prosedur Atraksi Wisata Olahraga Selam 2.3 Prosedur Atraksi Wisata Olahraga Selam Menjelajahi dunia dasar laut memiliki sensasi yang sama seperti menjelajahi luar angkasa bagi yang menyukai dunia dasar laut dan sangat menarik bagi kebanyakan orang. Olahraga selam menjadi salah satu olahraga bahari yang paling digemari oleh sebagian besar wisatawan yang berkunjung ke Sulawesi Utara (Lagarense, 2002). Pada dasarnya, olahraga ini hanya melibatkan http://www.lpsdimataram.com ___________________________ Volume 10, No. 8,Agustus 2016 ISSN No. 1978-3787 Media Bina Ilmiah71 3. Pengecekan terhadap alat dan persiapannya Di Blue Marlin Dive Centre, proses yang sangat central harus dilakukan pengecekan secara ketat adalah pengecekan alat-alat penyelaman khsusnya pada layak pakai dan dioperasikan. Selain alat yang akan di pakai, juga peserta juga dilakukan pengecekan terhadap pemakaian alat-alat supaya sesuai dengan standard kopetensi serta sesuai dengan manual diving PADI. Adapaun alat-alat yang diapakai adalah tabung masker, snorkel, regulator tahap dua, sumber udara alternative tahap dua, sabuk pemberat dengan penahan pemberat dan gesper sabuk yang dapat dilepas dengan cepat, konsol instrument dengan penunjuk tekanan udara, penunjuk kedalaman, timer selam, kompas, fin scuba, wet suit panjang, tabung oksigen, table selam dan slate, dan BCD dengan implator tekanan rendah. sertifikat dari PADI, harus menjalani kursus professional penyelaman yang di pandu oleh para dive instruktur. Biaya kursus penyelaman sangatlah mahal. Berdasarkan atas hasil penelitian yang telah dilakukan, di Blue Marlin Dive Centre, prosedur meakukan pemanduan dapat di bagi atas beberapa kelompok berdasarkan atas proses yang dialami. Proses katagori tersebut terbagi atas 3 katagori yaitu katagori persiapan, katagori proses saat pelaksanaan diving , dan katagori penutup atau mengakhiri pemanduan wisata selam. a. Katagori Persiapam Pemanduan Wisata Selam. Proses katagori persiapan merupakan proses kegiatan yang sangat menentukan keberhasilan kegiatan pemanduan wisata selam di Blue Marlin Dive Centre. Proses persiapan tersebut dilakukan melalui check list yang baku dan kaku. Adapun yang masuk dalam proses persiapan adalah (1) Menerima Konsumen yang telah memiliki pesanan, (2) Informasi dive site yang akan di tuju, (3) Pengecekan terhadap alat dan persiapannya, (4) Briefing oleh dive master, (5) Persiapan boat menuju area dive site. Sumber: Open Water Diver, 2011 Gambar 1. Peraltan Penyelaman j 1. Menerima Konsumen yang telah memiliki pesanan. Di Blue Marlin Dive Centre dalam melakukan pemanduan dimulai dari menerima wisatawan/peserta dari officer. Penerimaan dilakukan dengan prosedur salam “Good Morning/apternoon/ evening, Welcome in Blue Marlin Dive Centre!” . Selanjutnyadilakukan perkenalan Antara dive master dan peserta sambil mengkoleksi data peserta secara sah, yang datanya didapat dari blangko registrasi. 2. 2. Informasi dive site yang akan di tuju Dive master Blue Marlin Dive Centre, selanjutnya mengajak peserta untuk duduk dan rilek dan diberikan informasi tentang area dive site. Area dive site dijelaskan agar peserta mendapatkan data sementara tentang lokasi dan cara menuju ke lokasi. Informasi lain juga tentang kekuatan dan kelemahan dari kultur laut yang ada di area dive site serta kandungan biomasa yang memperkayanya. Blue Marlin Dive Centre memiliki area wilayah kegiatan penyelaman adalah di wilayah sekitaran Gili Air, sekitaran Gili Meno, dan sekitaran Gili Trawangan. Yang menjadi kekuatan ketiga area dive site ini adalah tumbuh dan berkembangnya blue coral. Di Indonesia sementara hanya baru umbuh di Pulau Lembongan dan sekitaran Gili Air, Meno dan Trawangan. Sumber: Open Water Diver, 2011 Gambar 1. Peraltan Penyelaman 4. Briefing oleh dive master Briefing dilakukan di Blue Marlin Dive centre adalah sebagai media komunikasi untuk saling mengingatkan akan pentingnya mengikuti aturan-aturan professional association dive internasional (PADI). Dalam Briefing di jelaskan aturan-aturan dan prosedur diving dan cara berkomunikasi pada saat berada dibawah laut (under water). Dalam briefing peserta diingatkan lagi akan setiap langkah penyelaman serta simbul atau tanda berkomunikasi di dalam air. Simbul-simbul komunikasi dalam air sangatlah penting bagi keselamatan penyelaman baik secara individu 4. _______________________ Volume 10, No. 8, Agustus 2016 http://www.lpsdimataram.com ISSN No. 1978-3787 72 Media Bina Ilmiah yang dipandu oleh dive mater/dive guide. Data- data penyelaman, mulai dari nama dive master/dive guide,area dive site yang dikunjungi, jumlah team dan namanya, serta pesona alam bawah laut yang dilihat digambarkan dalam logbook yang dibuat wisatawan. Saran dan kritik dalam penyelaman juga di sampaikan dalam logbook tersebut. PENUTUP Dapat ditarik kesimpulan tentang pemanduan wisata selam di Blue Marlin Gili Trawangan Lombok: Prosedur pemanduan wisata selam di blue marlin Gili Trawangan Lombok dilakukan berdasarkan atas tiga katagori tahapan yaitu 1) tahap persiapan penyelaman ((1) Menerima Konsumen yang telah memiliki pesanan, (2) Informasi dive site yang akan di tuju, (3) Pengecekan terhadap alat dan persiapannya, (4) Briefing oleh dive master, (5) Persiapan boat menuju area dive site), 2) tahap pelaksanaan penyelaman, dan 3) tahap penutu/mengakhiri penyelaman. Prosedur Penyelaman yang dilakukan sanagatlah patuh pada aturan dan standar baku PADI, karena mengandung resiko yang patal. Sehingga prosedur penyelaman dan pemanduan harus tepat. Area Dive site yang yang menjadi area penyelaman oleh Blue Marlin Dive Centre adalah daerah sekitaran Pulau Gili Air, Pulau Gili Meno, dan sekitaran Pulau Gili Trawangan. Penyelaman objek wisata bawah laut di ajak pada site tunel dan dome conservation, yang memiliki populasi trumbu karang yang sudah dewasa. Site yang biasa di kunjungi adalah daerah sebelah timur Gili Trawangan, dimana daerah ini pada tahun 2003 – 2009 banyak di tanam tunel dan dome dalam program konservasi trumbu karang di daerah Gili Trawangan dan Sekitarnya oleh Gili Eco Trust. Pada saat didalam air, penyelaman dilakukan berkelompok. Wisatawan mengikuti guide master yang berada didepannya. Penyelaman wisata bawah laut yang dipandu guide master dilakukan anatara 1-1,5 jam penyelaman. Lama penyelaman sangat dipengaruhi oleh konsumsi O2 yang ada dalam tabung. Rata-rata penyelaman yang dilakukan di Blue Marlin adalah satu jam penyelaman. Saran yang dapat disampaikan dalam penerapan prosedur pemanduan wisata selam di bluemarlin Gili Trawangan Lombokadalah: pemanduan wisata selam sangat sarat dengan resiko yang fatal, maka perlu adanya konsistensi pengecekan standard baku PADI pada sebelum, saat dan sesudah penyelaman, serta dibarengi dengan pengecekan kesehatan peserta. Pemanduan dalam satu group seyogyanya di pandu oleh dua master dive atau dive guide, sehingga tingkat keamanan lebih terjamin. Perlu adanya upaya penelitian lanjutan yang mengkaji dengan pendekatan suhu air, kedalaman, dan arus laut, sehingga lebih melengkapi penelitian dimaksud. a. Katagori Persiapam Pemanduan Wisata Selam. Sedangkan Dive master membuat logbook yang berisi tentang tanggal penyelaman, jumlah jam penyelaman, nama wisatawan yang di ajak, site area yang dikunjungi. Logbook tersebut sebagai bukti pengalaman dan jam terbang bagi penyelam, serta berhubungan dengan sertifikasi dan lisensi penyelam. maupun kelompok penyelaman. Persiapan boat menuju area dive site Selesai persiapan briefing oleh master dive, Jika situasi dan peralatan sudah usai maka langsung diajak menuju pada boat yang sudah dipersiapkan oleh karyawan bagian boat. Besar kecilnya boat tergantung jumlah peserta yang ikut dalam group-group. Masing-masing group terdiri atas 4 orang wisatawan dan di pandu oleh satu orang master dive atau dive guide. c. Laporan pemanduan wisata selam Blue Marlin Dive Centre Blue Marlin Dive Centre dalam mengahiri penyelaman, guide master akan memberikan sinyal untuk naik ke permukaan air dan di ajak menuju boat yang telah siap menunggu. Usai penyelaman para wisatawan diajak untuk ke dive centre office. Pengembalian alat-alat, serta penyelesaian transaksi aktifitas penyelaman dilakukan oleh staff office. Wisatawan, dive master dan dive guide sambil bersantai di restaurant, mereka mengisi log book penyelaman. Baik wisatawan maupun dive master/dive guide membuat logbook. Wisatawn membuat logbook tentang aktifitas penyelamannya b. Katagori Pelaksanaan / Aksi pemanduan wisata selam Dalam pelaksanaan penyelaman, para wisatawan diberikan aba-aba untuk melakukan backrool untuk masuk keair dari atas boat yang ditumpangi. Teknik backrool adalah salah satu teknik memasuki air menggunakan peralatan selam dengan selamat. Setelah semua anggota group berada di air, maka master dive/dive guide memberikan aba-aba penyelaman selanjutnya. _______________________ Volume 10, No. 8, Agustus 2016 http://www.lpsdimataram.com DAFTAR PUSTAKA Anonim. (2016), Permenpar No. 7 Tahun 2016: Pedoman Penyelenggaraan Wisata Selam Rekreasi. Kemenpar. Jakarta. Angelin Alow, (2006). Peranan Olahraga Selam dalam Pengembangan Pariwisata Sulawesi Utara. LKTIM 2006. Sulawesi. http://www.lpsdimataram.com ___________________________ Volume 10, No. 8,Agustus 2016 ISSN No. 1978-3787 Media Bina Ilmiah73 Arikunto Suharni. (2010). Metode Penelitian. Jakarta : Rineka Cipta Arikunto Suharni. (2010). Metode Penelitian. Jakarta : Rineka Cipta Narbuko. (2010). Metode Pengolahan Dan Analisa Data. Jakarta : Rineka Cipta Nyoman S. (2010). Dasar – Dasar Pariwisata. Jakarta : PT Gramedia Widiarsan. Richardson, Drew. (2011). Open Water Dive Manual (PADI). Canada. PADI Publish Subagyo. (2014). Metodelogi Penelitian Kualitatif Teori dan Praktis. Jakarta. Bineka Sulastiyono Agus. (2006). Manajement Penyelenggaraan Hotel. Bandung: Alfabeta Suwithi Ni Wayan, Cecil Jr. Boham. (2008). Akomodasi Perhotelan untuk SMK Jilid 1. Jakarta : Direktorat Pembinaan Sekolah Menengah Kejuruan, Direktorat Jenderal Manajemen Pendidikan Dasar dan Menengah, Departemen Pendidikan Nasional Umar, Wanta. (2014). Destinasi Indonesia Panduan Jelajah Indonesia. Auremedia. Denpasar Bali. http://www.lpsdimataram.com _______________________ Volume 10, No. 8, Agustus 2016 http://www.lpsdimataram.com
https://openalex.org/W4285032003
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0271333&type=printable
English
null
COVID-19-associated fungal infections in Iran: A systematic review
PloS one
2,022
cc-by
11,043
PLOS ONE 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. Funding: SM received a grant (grant number: 1400-3-99-22062) from Iran University of Medical Sciences, Tehran, Iran. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Citation: Nazari T, Sadeghi F, Izadi A, Sameni S, Mahmoudi S (2022) COVID-19-associated fungal infections in Iran: A systematic review. PLoS ONE 17(7): e0271333. https://doi.org/10.1371/journal. pone.0271333 Objectives This systematic review aims to summarize the mycological and clinical features of COVID- 19-associated fungal infections (CAFIs) in Iran. Tina NazariID1, Fatemeh Sadeghi2, Alireza Izadi3,4, Setayesh Sameni5, Shahram MahmoudiID2* Tina NazariID1, Fatemeh Sadeghi2, Alireza Izadi3,4, Setayesh Sameni5, Shahram MahmoudiID2* 1 Department of Medical Geriatrics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, 2 Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran, 3 Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, 4 Medical Mycology and Bacteriology Research Center, Kerman University of Medical Sciences, Kerman, Iran, 5 Department of Medical Sciences, Shahrood Branch, Islamic Azad University, Shahrood, Iran * mahmoudi.sh@iums.ac.ir, sh.mahmoudi93@gmail.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Methods PubMed, Web of Science, Scopus, Cochrane Library, SID, Magiran, IranDoc, and Google Scholar were searched for Persian and English articles published from January 1, 2020, to November 5, 2021, using a systematic search strategy. Studies on Iranian patients suffering from CAFIs were included in the review. Editor: Felix Bongomin, Gulu University, UGANDA Received: March 29, 2022 Accepted: June 29, 2022 Published: July 11, 2022 Copyright: © 2022 Nazari et al. This is an open Editor: Felix Bongomin, Gulu University, UGANDA Received: March 29, 2022 Accepted: June 29, 2022 Published: July 11, 2022 Copyright: © 2022 Nazari 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. Editor: Felix Bongomin, Gulu University, UGANDA Results Twenty-two studies comprising 169 patients were retrieved. Reported CAFIs included can- didiasis (85, 50.30%), mucormycosis (35, 20.71%), aspergillosis (29, 17.16%), fusariosis (6, 3.55%), three cases caused by rare pathogens (Rhodotorula mucilaginosa, Diaporthe foeni- culina, and Sarocladium kiliense) and 11 (6.51%) uncharacterized mold infections. The most common underlying diseases were diabetes (67/168, 39.88%), cardiovascular dis- eases (55/168, 32.74%), and hypertension (43/168, 25.59%). The use of antibiotics (111/ 124, 89.52%), corticosteroids (93/132, 70.44%), and mechanical ventilation (66, 51.16%) were the most common predisposing factors. Totally, 72 (50.35%) of 143 patients with CAFIs died (data were not available for 26 patients). Copyright: © 2022 Nazari 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. PLOS ONE PLOS ONE Introduction The coronavirus disease 2019 (COVID-19), which started as a pneumonia epidemic in Wuhan, China, in December 2019, has become a global issue due to its high prevalence and rapid transmission [1]. It has been diagnosed in hundreds of million cases and killed millions of people around the world [2]. In addition to the harmful effects of the virus, such as dysregu- lation of immune response and direct damage to pulmonary and extra-pulmonary tissues, COVID-19 can be accompanied by infections caused by other microorganisms [3, 4]. Fungal, bacterial, and viral co-infections and super-infections have been detected in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), leading to more difficult management and increased morbidity and mortality [5]. Fungal infections have been reported in 3.7% of COVID-19 patients, with a higher occurrence (9.6%) in ICU- admitted cases [6]. They have been shown to increase fatality in COVID-19 patients, which can be reduced with the prescription of antifungal medications [7]. Aspergillosis, candidiasis, and mucormycosis are the most commonly reported CAFIs while other infections such as cryptococcosis, Pneumocystis jirovecii pneumonia, coccidioidomycosis, Paracoccidioidomyco- sis, and histoplasmosis have also been disclosed [8, 9]. COVID-19 weakens the cellular immunity due to decreasing T lymphocytes and alters the respiratory and gastrointestinal microbiota, which can make patients more susceptible to fun- gal infections [10, 11]. Long stays at hospitals, especially ICU, mechanical ventilation, the use of broad-spectrum antibiotics and corticosteroids in COVID-19 patients may also contribute to an increased risk of mycoses [12, 13]. Following the COVID-19 spread in Iran, cases of CAFIs have been reported from different parts of the country [14–16]. To date, there is no comprehensive study of fungal infections in patients with COVID-19 in Iran. In this systematic review, we aim to summarize the studies that have reported CAFIs in Iran. Conclusion Fungal infections are evident to be a complication of COVID-19 in Iran; thus, clinicians should consider them as a differential diagnosis, especially in patients with comorbidities and previous antibiotic or corticosteroid use. Competing interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 Methods This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (S1 File) [17], and the protocol of this review was registered in the International Prospective Register of Systematic Reviews (PROS- PERO registration code: CRD42021287258). We performed a comprehensive search for stud- ies concerning fungal infections diagnosed in COVID-19 patients reported from Iran during 2020 and 2021. Electronic databases, including PubMed, Web of Science, Scopus, Cochrane Library, SID, Magiran, and IranDoc, as well as Google Scholar search engine, were searched on November 5, 2021. The systematic search was conducted with a combination of keywords such as ‘COVID-19’, ‘SARS-CoV-2’, ‘fungal infections’, ‘candidiasis’, ‘mucormycosis’, ‘aspergillosis’, and ‘Iran’. An exhaustive list of keywords and MeSH terms used for PubMed search is pro- vided in S2 File. The search results were filtered for Persian and English languages and publica- tion date from January 1, 2020. There was no limitation on the study design. Moreover, references of selected articles were investigated for additional relevant articles. The inclusion criteria were: (1) the studies concerned patients diagnosed with COVID-19, (2) the subjects became infected with a type of fungi simultaneously or after contracting COVID-19, (3) the studies took place in Iran, (4) the articles were written in English or Per- sian. Reviews, letters, animal studies, and studies that did not contain clinical data of infected patients were excluded. Titles and abstracts of the retrieved articles were screened independently by two reviewers. Next, the full-text articles were reviewed by two independent authors to determine the PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 2 / 19 PLOS ONE COVID-19 associated mycoses in Iran eligibility of reports based on the inclusion criteria. The risk of bias of the studies was assessed by two independent reviewers using the Joanna Briggs Institute critical appraisal tools (https:// jbi.global/critical-appraisal-tools). In case of any disagreement between the two authors in each stage, a third author was consulted. Two researchers independently extracted data, including bibliographic, demographic, and clinical details. Disagreement between data collec- tors was resolved through discussing a third reviewer. The bibliographic data included the first author, year of publication, and study design. Demographic features consisted of the province, sample size, incidence, mortality rate, age, gender, past medical history, and drug history. Candidiasis among Iranian COVID-19 patients Four studies comprising 85 cases of candidiasis were found among Iranian COVID-19 patients. The mean ± SD of age was 61.98 ± 17.10 years, and 44 (51.76%) patients were female. The cases included oropharyngeal candidiasis (53, 62.35%), pulmonary candidiasis (25, 29.41%), candidemia (6, 7.06%), and endocarditis (1, 1.18%). In total, 97 Candida isolates were recovered from the patients (9 cases of mixed infection), and C. albicans (74, 76.29%) was the most common species, followed by C. glabrata (4, 4.71%), C. dubliniensis (3, 3.53%), C. tropica- lis (2, 2.35%), C. parapsilosis (1, 1.18%), and C. krusei (1, 1.18%). Mixed infection cases included C. albicans + C. glabrata (2, 2.35%), C. albicans + C. parapsi- losis (2, 2.35%), C. albicans + C. tropicalis (1, 1.18%), C. albicans + C. dubliniensis (1, 1.18%), C. albicans + C. glabrata + C. dubliniensis (2, 2.35%), and C. albicans + C. glabrata + C. parapsilo- sis (1, 1.18%). Results Database searching resulted in 673 articles, of which 22, including 16 case reports and six cross-sectional studies, were included in the present study (Fig 1) [15, 16, 18–37]. Details of the articles and results of the quality assessment are presented in Table 1 and S3 File, respec- tively. The reviewed studies comprised 169 cases of fungal infections among Iranian COVID- 19 patients with the dominance of candidiasis (85, 50.30%), followed by mucormycosis (35, 20.71%), aspergillosis (29, 17.16%), and fusariosis (6, 3.55%). Three cases of rare infections and 11 (6.51%) cases of uncharacterized mold infections were also found. Details of comorbidities, risk factors, diagnosis, treatment, and outcome are summarized in Tables 2 and 3, separately for the three common fungal infections. Methods Clinical details included signs and symptoms, laboratory and radiological findings, diagnostic methods, and treatment separately for COVID-19 and fungal infections; as well as duration between COVID-19 and fungal infection diagnosis, genera and species of fungi, site of fungal infection, antifungal susceptibility tests, and outcome of treatment. The extracted data were reviewed, and variables with an acceptable proportion of available values were summarized as mean ± SD or frequency (percentage). Mucormycosis among Iranian COVID-19 patients Nine studies composed of 35 cases of mucormycosis, 19 (54.29%) males and 16 (45.71%) females, were retrieved. The mean ± SD of age was 55.97 ± 12.93 years. All patients had rhino- orbital-cerebral manifestations, with orbital involvement in 32 (91.43%) and rhino-sinusitis in three (8.57%) cases. The orbital infections comprised of rhino-sino-orbital (17, 48.57%), rhino-orbital (7, 20%), sino-orbital (5, 14.29%), orbital (2, 5.71%), and rhino-sino-orbito-cere- bral (1, 2.86%) involvements. Definitive identification of the causative agents was made for only two cases of Rhizopus arrhizus (R. oryzae). 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran Fig 1. The PRISMA flow diagram for selection of studies reporting data on COVID-19-associated fungal infections in Iran up to November 5, 2021. https://doi.org/10.1371/journal.pone.0271333.g001 Fig 1. The PRISMA flow diagram for selection of studies reporting data on COVID-19-associated fungal infections in Iran up to November 5, 2021. Fig 1. The PRISMA flow diagram for selection of studies reporting data on COVID-19-associated fungal infections in Iran up to November 5, 2021. https://doi.org/10.1371/journal.pone.0271333.g001 https://doi.org/10.1371/journal.pone.0271333.g001 Fusariosis among Iranian COVID-19 patients Six cases of pulmonary fusariosis, two males (33.33%) and four females (66.67%), were found in PCR-confirmed COVID-19 cases with a mean ± SD age of 67 ± 12.66 years. The causative agents, including Fusarium incranatum (3, 50%), F. fujikuroi (1, 16.67%), F. equiseti (1, 16.67%), and F. solani (1, 16.67%) were detected through bronchoalveolar lavage (BAL) cul- ture; and five patients had positive BAL galactomannan (GM) test. Six cases of pulmonary fusariosis, two males (33.33%) and four females (66.67%), were found in PCR-confirmed COVID-19 cases with a mean ± SD age of 67 ± 12.66 years. The causative agents, including Fusarium incranatum (3, 50%), F. fujikuroi (1, 16.67%), F. equiseti (1, g g f j q 16.67%), and F. solani (1, 16.67%) were detected through bronchoalveolar lavage (BAL) cul- ture; and five patients had positive BAL galactomannan (GM) test. Hypertension (4, 66.67%), cardiovascular diseases (3, 50%), diabetes mellitus (2, 33.33%), and chronic kidney diseases (1, 16.67%) were the reported underlying diseases. All six patients were admitted to ICU, mechanically ventilated, and received antibiotics and corticosteroids. None of the patients received antifungal medications, and all of them regrettably died. PLOS ONE PLOS ONE COVID-19 associated mycoses in Iran Table 1. Details of 22 studies with data of COVID-19-associated fungal infections among Iranian patients published up to November 5, 2021. First author Year of publication Type of study Type of fungal infection (N of patients) Province Ref. Abolghasemi 2021 Case report Aspergillosis (1) Tehran [18] Arastehfar 2021 Cross- sectional Candidemia (6), Rhodotorula mucilaginosa fungemia (1) Khorasan Razavi [19] Davoodi 2021 Case report Candidemia and endocarditis (1) Mazandaran [20] Fazeli 2021 Cross- sectional Mucormycosis (12) Kermanshah [21] Ghazanfari 2021 Cross- sectional Aspergillosis (22), fusariosis (6), unidentified mold infections (11), Diaporthe foeniculina infection (1) Mazandaran [16] Hakimifard 2021 Case report Aspergillosis (1) Isfahan [22] Heydarifard 2021 Case report Mucormycosis (1) Tehran [23] Hosseinikargar 2021 Case report Aspergillosis (1) Khorasan Razavi [24] Karimi-Galougahi 2021 Case report Mucormycosis (1) Tehran [25] Khodavaisy 2021 Case report Aspergillosis (1) Tehran [26] Mehrabi 2021 Case report Mucormycosis (1) Bushehr/Fars [27] Mohammadi 2021 Case report Mucormycosis (1) Qazvin [28] Nasri 2020 Case report Aspergillosis (1) Isfahan [29] Ostovan 2021 Case report Mucormycosis (1) Fars [30] Pakdel 2021 Cross- sectional Mucormycosis (15) Tehran [15] Ranjbar- Mobarake 2021 Case report Sarocladium kiliense infection (1) Isfahan [31] Salehi 2020 Cross- sectional Oropharyngeal candidiasis (53) Tehran [32] Salehi 2021 Case report Aspergillosis (1) Tehran [33] Sharifpour 2021 Case report Aspergillosis (1) Mazandaran [34] Shirvani 2021 Cross- sectional Pulmonary candidiasis (25) Tehran [35] Tabarsi 2021 Case report Mucormycosis (1) Tehran [36] Veisi 2021 Case series Mucormycosis (2) Tehran [37] https://doi org/10 1371/journal pone 0271333 t001 Aspergillosis among Iranian COVID-19 patients Eight studies comprising 29 cases of aspergillosis were reported among patients with COVID- 19 from Iran. The patients were 62.97 ± 12.68 years old, and 15 (51.72%) were male. Pulmo- nary infection (27, 93.1%) was the most common clinical form of the disease; in addition, one patient (3.45%) with disseminated (pulmonary + cerebral) and one patient (3.45%) with rhino- sinusitis were reported. In total, 26 out of 29 isolates were identified to the species level. Asper- gillus flavus (15/26, 57.69%) was the most common etiological agent, and the remaining isolates included A. fumigatus (3, 11.54%), A. japonicus (3, 11.54%), A. niger (2, 7.69%), A. ochraceus (1, 3.85%), A. terreus (1, 3.85%), and A. tubingensis (1, 3.85%). 4 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 Discussion This systematic review showed that the most common fungal infections reported in Iranian COVID-19 patients are candidiasis, mucormycosis, and aspergillosis. Previous global reviews also found members of Candida, Aspergillus, and Mucorales among the most frequent fungi causing secondary infections in people with COVID-19 [6, 8, 9, 38]. Results of the present review are based on a limited number of studies, mainly case reports, which are prone to variation by future reports. Meanwhile, the thorough systematic search and exhaustive data collection provide a comprehensive description of CAFIs to the date of data- base searching across the country, which could be considered a strength of this report. PLOS ONE COVID-19 associated mycoses in Iran Table 2. Frequency of comorbidities and risk factors in COVID-19-associated fungal infection cases reported from Iran up to November 5, 2021. Variables Frequencies based on the type of fungal infection Candidiasis Mucormycosis Aspergillosis Past medical history Diabetes mellitus (21, 24.70%) Diabetes mellitus (27, 77.14%) Diabetes mellitus (10, 35.71%) Immunosuppressive diseases (25, 29.41%) Hypertension (17, 48.57%) Hypertension (13, 46.43%) Cardiovascular diseases (29, 34.11%) Cardiovascular disease (7, 20%) Cardiovascular disease (11, 39.28%) Pulmonary diseases (5, 5.88%) Pulmonary diseases (4, 11.43%) Pulmonary diseases (5, 17.24%) Malignancy (8, 9.41%) Malignancy (2, 5.71%) Malignancy (4, 14.28%) Chronic kidney disease (11, 12.94%) Chronic kidney disease (2, 5.71%) Chronic kidney disease (2, 7.14%) Others (23, 27.05%) Others (9, %) Others (4, 13.79%) Healthy (5, 5.88%) Healthy (4, 11.43%) Healthy (3, 10.71%) Unknown (1) Mechanical ventilation 22/60, 36.7% 3/22, 13.64% 23/27, 85.19% Unknown (25) Unknown (13) Unknown (2) Central venous catheter 11/60, 18.3% Unknown (25) Unknown (35) Unknown (29) Intensive care unit hospitalization 33/60, 55% 9/26, 34.62% 26/27, 96.3% Unknown (25) Unknown (9) Unknown (2) Corticosteroid therapy 25/54, 46.3% 23/32, 71.87% 26/27, 96.3% Unknown (31) Unknown (3) Unknown (2) Antibiotic consumption 56/60, 93.33% 10/16, 62.5% 25/28, 89.29% Unknown (25) Unknown (19) Unknown (1) https://doi.org/10.1371/journal.pone.0271333.t002 https://doi.org/10.1371/journal.pone.0271333.t002 2. Disseminated infection due to Sarocladium kiliense in a 74-year-old diabetic female receiving corticosteroid and antibiotics. She did not survive despite receiving liposomal amphotericin B. 2. Disseminated infection due to Sarocladium kiliense in a 74-year-old diabetic female receiving corticosteroid and antibiotics. She did not survive despite receiving liposomal amphotericin B. 3. Pulmonary infection due to Diaporthe foeniculina in a 67-year-old female with diabetes, hypertension, chronic kidney disease and ischemic heart disease, with a history of ICU admission, mechanical ventilation, corticosteroid and antibiotic therapy. The patient received no antifungal treatment and died. Eleven cases of uncharacterized pulmonary mold infections were also found in ICU-admit- ted patients who underwent mechanical ventilation, corticosteroid and antibiotic therapy. They were detected by high levels of GM (serum GM index > 0.5 or BAL GM index  1). None of the patients were prescribed antifungals, and all except one passed away. Rare or uncharacterized fungal infections among Iranian COVID-19 patients Three cases of CAFI caused by rare fungi were reported: 1. Fungemia due to Rhodotorula mucilaginosa in an ICU-admitted, mechanically ventilated, 68-year-old female without underlying diseases, who was effectively treated with fluconazole. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 5 / 19 PLOS ONE COVID-19 associated mycoses in Iran Table 3. Description of diagnosis, treatment and outcome of COVID-19-associated fungal infection cases reported from Iran up to November 5, 2021. Variables Frequencies based on the type of fungal infection Candidiasis Mucormycosis Aspergillosis COVID-19 diagnostic method (n, %) PCR (29, 48.33%) PCR (18, 51.43%) PCR + CT (28, 96.55%) PCR + CT (1, 1.66%) PCR + CT (14, 40%) CT (1, 3.45%) Clinical (30, 50%) CT (2, 5.71%) Unknown (25) Clinical (1, 2.86%) Days between COVID-19 and fungal infection diagnosis (mean ± SD) 8.97 ± 8.89 17.18 ± 12.44 29 ± 19.3 Unknown (25) Unknown (1) Unknown (23) Fungal infection diagnostic method (n, %) Microscopic + molecular + culture (53, 62.35%) Microscopic + imaging (18, 51.43%) Culture + biomarkera (17, 58.62%) Molecular (25, 29.41%) Microscopic (15, 42.86%) Culture (5, 17.24%) Molecular + culture (6, 7.06%) Microscopic + culture + molecular + imaging (2, 5.71%) Biomarker + imaging (2, 6.89%) Molecular + culture + imaging (1, 1.17%) Microscopic + culture + Molecular + biomarker + imaging (2, 6.89%) Microscopic + culture + Molecular + imaging (2, 6.89%) Microscopic + culture + imaging (1, 3.45%) Fungal infection treatment (n, %) FCZ (22, 36.66%) AMB (1, 2.86%) VCZ (2, 6.9%) FCZ + NYS (20, 33.33%) AMB + SI (25, 71.43%) VCZ + CSP (1, 3.45%) NYS (10, 16.66%) AMB + PSZ+ SI (5, 14.28%) VCZ + AMB (2, 6.9%) FCZ + CSP (5, 8.33%) AMB + PSZ + CSP + SI (1, 2.86%) FCZ + AMB + surgical intervention (1, 3.45%) CSP (1, 1.66%) AMB + CSP + SI (2, 5.71%) AMB (1, 3.45%) AMB (1, 1.66%) Systemic antifungal + SI (1, 2.86%) No treatment (22, 75.86%) No treatment (1, 1.66%) Case fatality rate (%) 16.66% 55.88% 86.2% Unknown (25) Unknown (1) PCR: Polymerase chain reaction; CT: computerized tomography scan; FCZ: fluconazole; AMB: Amphotericin B; VCZ: voriconazole; NYS: nystatin; CSP: caspofungin; PSZ: posaconazole; SI: surgical intervention. aEither galactomannan or β-D-glucan in serum or bronchoalveolar lavage sample. PCR: Polymerase chain reaction; CT: computerized tomography scan; FCZ: fluconazole; AMB: Amphotericin B; VCZ: voriconazole; NYS: nystatin; CSP: caspofungin; PSZ: posaconazole; SI: surgical intervention. aEither galactomannan or β-D-glucan in serum or bronchoalveolar lavage sample. https://doi.org/10.1371/journal.pone.0271333.t003 patients’ registry in an American hospital showed that 12% of patients diagnosed with COVID-19 had candidiasis, and patients with candidiasis were 3.73 times more likely to be diagnosed with COVID-19 [40]. COVID-19-associated candidiasis (CAC) Candida is the most prevalent fungal pathogen causing superinfections and the second most common source of coinfections in COVID-19 patients [39]. A cross-sectional study of the PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 6 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE In this review, around half of the reported CAFI cases were caused by Candida spp., the most common fungal pathogens in Iranian COVID-19 patients. The exact pathogenesis of CAC is not clarified, but several factors have been suggested to explain the predisposition of patients with COVID-19 to Candida infections. The SARS-CoV- 2 infection can cause lymphopenia and consequent impairment of immune defense against fungal agents including Candida spp. [41, 42]. Severe COVID-19 is associated with elevation of blood lactate and acidosis [43, 44], which enables Candida to restructure its cell wall to mask β-glucans and escape the recognition by the host immune system [45]. Moreover, an important virulence factor of Candida that helps it evade phagocytosis is the ability to form biofilms, which can be triggered by COVID-19 through increasing oxidative stress and pH imbalance [41]. p g y p p g COVID-19 had candidiasis, and patients with candidiasis were 3.73 times more likely to be diagnosed with COVID-19 [40]. In this review, around half of the reported CAFI cases were caused by Candida spp., the most common fungal pathogens in Iranian COVID-19 patients. The exact pathogenesis of CAC is not clarified, but several factors have been suggested to explain the predisposition of patients with COVID-19 to Candida infections. The SARS-CoV- 2 infection can cause lymphopenia and consequent impairment of immune defense against fungal agents including Candida spp. [41, 42]. Severe COVID-19 is associated with elevation of blood lactate and acidosis [43, 44], which enables Candida to restructure its cell wall to mask β-glucans and escape the recognition by the host immune system [45]. Moreover, an important virulence factor of Candida that helps it evade phagocytosis is the ability to form biofilms, which can be triggered by COVID-19 through increasing oxidative stress and pH imbalance [41]. On the other hand, COVID-19 patients are at an increased risk of contracting candidiasis because of the treatments they receive. Consumption of corticosteroids, antibiotics, and TNF- α inhibitors, as well as undergoing mechanical ventilation, central venous catheterization, and 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran extracorporeal membrane oxygenation (ECMO) are risk factors for developing candidiasis [41, 46–48]. Concordant to previous studies, a significant proportion of the patients in our review were exposed to antibiotic consumption, corticosteroid use, mechanical ventilation, and central venous catheterization. PLOS ONE However, we did not find any report of patients with CAC who had received a TNF-α inhibitor or went through ECMO, as these are less commonly indi- cated for COVID-19 treatment. Another predictor of developing CAC is admission to ICU, which is often accompanied by interventions such as mechanical ventilation and central venous catheterization and can raise the risk of nosocomial Candida infections [41]. In our study, 55% of the CAC cases (100% of patients with candidemia) stayed in ICU, emphasizing the need for more attention to ICU patients in terms of prompt diagnosis of candidiasis. A global review showed that CAC is more likely to involve male patients with more than 40 years of age [38]. This is probably because of the fact that higher age and male gender are asso- ciated with more severe COVID-19 and the need for ICU admission [49, 50]. Expectedly, the Iranian CAC cases were about 62 years old on average. However, with female patients being reported slightly more than males, no significant gender difference was found in our review. The most common species causing CAC throughout the world is C. albicans, which has also been the most frequent causative agent of candidiasis before the pandemic [38, 51]. Simi- larly, the majority of Candida isolates obtained from Iranian patients were identified as C. albi- cans. The following frequent pathogens in Iran were C. glabrata, C. dubliniensis, C. The most common species causing CAC throughout the world is C. albicans, which has also been the most frequent causative agent of candidiasis before the pandemic [38, 51]. Simi- larly, the majority of Candida isolates obtained from Iranian patients were identified as C. albi- cans. The following frequent pathogens in Iran were C. glabrata, C. dubliniensis, C. parapsilosis, and C. tropicalis; which were also among the most common species in other coun- tries [52–56]. Furthermore, Salehi et al. reported one case of CAC caused by C. krusei [32]. A less prevalent species, this pathogen was detected in two other CAC cases from India and Spain [54, 55]. Whil f C i C l i i C h il i d C i i d cans. The following frequent pathogens in Iran were C. glabrata, C. dubliniensis, C. parapsilosis, and C. tropicalis; which were also among the most common species in other coun- tries [52–56]. Furthermore, Salehi et al. reported one case of CAC caused by C. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE krusei [32]. A less prevalent species, this pathogen was detected in two other CAC cases from India and Spain [54, 55]. While cases of C. auris, C. lusitaniae, C. orthopsilosis, and C. inconspicua were reported globally, they were not found in Iranian patients [55, 57–62]. This is not surprising as we know there are regional differences in the global distribution of Candida species [63]. For example, while C. auris outbreaks have been reported from India, Pakistan, USA, Venezuela, South Africa, Spain and the United Kingdom [64]; only limited sporadic cases of this species were reported from Iran [65–67]. In addition, C. auris and other rare pathogens might have been missed due to the lack of proper diagnostic tools in Iran; thus, the differences might result from the use of culture and PCR for identifying the causative agents [65, 68, 69]. Oropharyngeal candidiasis (OPC) is a common form of Candida infection, which has been reported in people with COVID-19 as well [48, 70]. In the majority of CAC patients described in this review, the infection was localized in the oropharyngeal area. Pulmonary candidiasis rarely happens in critically ill patients with multiple risk factors [71, 72]. Although Shirvani et al. reported 25 cases of COVID-19 patients with Candida spp. detected in their BAL fluid [35], no other pulmonary CAC was reported from other countries. There is no consensus on whether Candida can actually cause pulmonary infection or whether its presence in BAL sam- ples is due to colonization [73, 74]. Candidemia, which is 3–8 times more frequent in COVID-19 patients compared to the non-COVID-19 population [75, 76], was confirmed in six Iranian COVID-19 patients [19]. Moreover, a case of endocarditis caused by C. tropicalis was reported in an Iranian patient with prosthetic heart valves, who died despite receiving liposomal amphotericin B [20]. Another case of endocarditis caused by C. albicans was diagnosed in an Italian COVID-19 patient with a central venous catheter [77]. Fungal endocarditis is a rare and hard-to-treat con- dition leading to a 50% mortality, with C. albicans being the causative agent in 24–46% of cases [78]. In a study conducted in 2009–2011 on Iranian patients with infective endocarditis who did not respond to antibacterial treatment, C. albicans has been documented in two out PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 8 / 19 PLOS ONE COVID-19 associated mycoses in Iran of eleven cases. COVID-19-associated mucormycosis (CAM) Over the last decade, mucormycosis has been more frequently diagnosed, especially in India [80]. A pre-pandemic study showed an increased incidence (about 2.5 times) of mucormycosis during the years 2008–2014 in Iran [81]. Besides, this infection has become 50 times more prevalent in the COVID-19 era globally [82]. CAM has been primarily seen in India, while cases from other countries, including Iran, have also been reported [83]. Two major risk factors of CAM are diabetes mellitus (present in 79–85% of patients) and use of corticosteroids (prescribed in 76.3–85% of cases) [8, 38, 84–86]. Similarly, the most fre- quent risk factor in Iranian CAM patients was diabetes mellitus, present in the majority (77.14%) of patients, and corticosteroid treatment with a 71.87% frequency. It should be noted that corticosteroids are not always prescribed correctly, as it is demonstrated that only 28.8% of cases in India and 52.6% in other countries received corticosteroids based on indication and with accurate dosage [83]. Male gender might also be related to a higher incidence of CAM, as several studies have shown more than 70% of patients were men [38, 85–88]. However, with men accounting for 54% of Iranian patients, the male preponderance was not as significant as in previous studies. Since limited cases have been studied, more extensive research is needed to understand the gender distribution of CAM both in Iran and the world. The average age of CAM patients was 56 years in Iran, roughly similar to a 55 years median in worldwide studies [89]. Making up 57% of culture results, Rhizopus spp. are the most commonly identified fungi in CAM cases worldwide [84]. Mucor spp. and Lichteimia spp. were also diagnosed in a small number of cases [38]. In the studies of this review, the species were not identified except for two cases of R. arrhizus. The rationale for the lack of data about species is that identifying mucormycosis agents to the level of genera and species for clinical management is not strongly supported by evidence, although recommended for epidemiological purposes [90]. Besides, culture has a low sensitivity (50%) to identify the species, and molecular techniques are not yet standardized and yield heterogeneous results [91]. The most common form of the disease, with or without COVID-19, is rhino-orbital-cere- bral mucormycosis (ROCM) [80, 92]. All 35 CAM cases from Iran fell into this category, with a 91.43% orbital and 2.86% cerebral involvement. PLOS ONE These two patients were intravenous drug abusers and responded to treatment with surgical removal and amphotericin B [79]. What this study brings to mind is that surgical removal of the vegetations might have improved the outcome as the C. tropicalis isolate was proved to be sensitive to amphotericin. Based on a review of global studies, CAC has a mortal- ity rate ranging from 11 to 100% based on the infection site and clinical conditions [38]. In this study, all cases with candidemia and endocarditis died; nonetheless, the overall mortality rate was 16.66% because of the low fatality of OPC. Studies with large sample sizes and adjust- ments of underlying factors are warranted to estimate the attributable mortality of different categories of CAC. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 COVID-19-associated aspergillosis (CAA) Observed in 7.7–27.7% of patients with severe COVID-19, aspergillosis is another fungal infec- tion frequently seen along with this viral infection [107]. COVID-19-associated pulmonary aspergillosis (CAPA) affects up to 35% of ICU patients with COVID-19, leading to a 54.9% mortality rate [108, 109]. CAA of the nose and sinuses was also reported from India and Egypt [110–113]. Twenty-nine CAA cases were reported from Iran, of which 27 were diagnosed with CAPA, in addition to two patients with rhinosinusitis and disseminated infection. CAPA is almost exclusive to ICU-admitted patients, while Aspergillus rhinosinusitis was reported in several outpatient COVID-19 cases [108, 111, 114, 115]. All except one of the Ira- nian cases with available data were admitted to ICUs. Corticosteroids (96.3%), antibiotics (89.29%), and mechanical ventilation (85.19%) were used in a large proportion of the patients in our review. A global study revealed a high frequency of mechanical ventilation (94.1%), but corticosteroid use was less frequent (52.7%) [115]. The exact antibiotic use frequency has not been reported in CAPA patients, but a 75% proportion could be presumed based on data from ICU-admitted COVID-19 patients [116]. The differences can be explained by the prescription of dexamethasone, ceftriaxone and azithromycin per institutional protocol in all patients of the Ghazanfari et al. study, which is the reference for the majority of our patients [16]. Hypertension was the most common comorbidity both in our study (46.43%) and a world- wide review (24%) [108]. Another study reported cardiovascular diseases (50.5%) as the most prevalent underlying condition in CAPA patients [115], and we found it in 39.28% of patients in our review. Diabetes mellitus, malignancies and pulmonary diseases are also common among CAPA patients both in Iran and other countries [108, 115, 117]. However, none of these comorbidities are significantly different in CAPA and other ICU-admitted COVID-19 patients [117]. The most prevalent causative agent of CAPA is A. fumigatus, observed in more than 80% of cases globally [108, 114, 116]. However, in Iran, A. fumigatus was the second most common pathogen after A. flavus, identified in more than half of Iranian CAPA cases. This is not unex- pected as previous studies on species distribution of Aspergillus in Iran before the COVID-19 outbreak showed that A. flavus had the highest prevalence, followed by A. fumigatus, A. niger, and A. terreus [118, 119]. The disseminated aspergillosis case was caused by A. COVID-19-associated mucormycosis (CAM) While not found in our review, several cases of lung, bone, gastrointestinal, and skin involvement were reported from other countries [93– 101]. The absence of reported CAM cases of organs other than the nose, sinus, and orbits from Iran might be because of the rarity of these infections, the non-specificity of their presenta- tions, and the lack of awareness among clinicians about the uncommon presentations of mucormycosis. The mortality rate of CAM has been estimated at 30–52% in various review studies [38, 84, 86, 102]. In the present review, 55.88% of patients died despite treatment, com- parable to the 61.9% mortality rate in countries other than India [83]. The mortality rate of CAM in India is lower (14–36.5%), probably because of the higher prevalence of ROCM in PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 9 / 19 PLOS ONE COVID-19 associated mycoses in Iran Indian patients [83, 87], as it is known that disseminated and pulmonary mucormycosis are associated with higher mortality rates than ROCM and cutaneous infection [90]. Nevertheless, since none of the Iranian cases had disseminated or pulmonary infection, this cannot be the reason for the higher fatality rate. However, the higher prevalence of orbital involvement in the Iranian (91.43%) compared to the Indian (72%) CAM cases might be a sign of neglect and delayed diagnosis, as well as the possible source of the poorer prognosis, as orbital involvement with vision loss is associated with a higher mortality rate [87]. The treatment of choice for mucormycosis is amphotericin B, which is more effective when combined with surgical interventions [103]. Isavuconazole and posaconazole, alone or in com- bination with amphotericin B, are recommended as first-line or salvage therapy [104, 105]. Almost all patients in our review received amphotericin B and surgical interventions (Table 3). Although we did not analyze the data statistically, it was noticed that all eight cases that received one or two drugs (caspofungin, posaconazole or both) in addition to Amphotericin B survived while monotherapy with Amphotericin B led to ~73% mortality. This highlights the need for further clinical trials as the efficiency of combination therapy is proposed by limited studies yet not supported by enough evidence [104, 106]. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 COVID-19-associated aspergillosis (CAA) ochraceus, which has not been reported, to our knowledge, in other COVID-19 patients. A. ochraceus is a PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 10 / 19 PLOS ONE COVID-19 associated mycoses in Iran rare pathogen, previously described in two case reports of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis [120, 121]. The fatality rate of CAA in Iran was estimated at 86.2%, which is remarkably higher than CAPA all-cause mortality rates in global studies (42.6–54.1%) [108, 115, 122]. The global rate is even lower when the cause of death is limited to CAPA (17.2%) [115]. Apostolopoulou et al. found an association between age, male sex and pulmonary diseases with mortality of CAPA in a global review [108]. None of these factors can be the reason for the higher mortality in the Iranian patients, who were on average 62.97 years old (vs 67% worldwide), 51.72% male (vs 75.4% worldwide), and 17.85% with pulmonary comorbidities (vs 20% worldwide) [108]. The difference in the mortality rate of Iranian and global cases can be explained by the fact that 22 out of 29 patients, for an unknown reason, did not receive antifungal treatment [16]. Only two out of seven patients who received antifungal treatment survived; both received voriconazole, which is considered the first-line therapy of invasive pulmonary aspergillosis and is the most frequently prescribed drug for CAPA worldwide [108, 114, 116]. COVID-19-associated fusariosis and other CAFIs Scattered cases of fusariosis have been reported in COVID-19 patients across the globe. For instance, two patients with severe COVID-19 infected with F. verticillioides and F. proliferatum were reported from Argentina and France, respectively [123, 124]. In addition, two cases of fusariosis were detected in mechanically-ventilated COVID-19 patients among subjects of observational studies from France and Spain [125, 126]. In one of the studies we reviewed, six COVID-19 patients with acute respiratory distress syndrome (ARDS) had pulmonary fusario- sis [16]. It is noteworthy that none of these patients had the classic risk factors of invasive fusariosis, i.e., hematologic malignancy and history of transplant [127]. However, all of them were mechanically ventilated and received corticosteroids and antibiotics, emphasizing the role of COVID-19 and its treatment in triggering Fusarium invasion. Treatment of fusariosis is diffi- cult due to the resistance to most antifungal drugs, its optimal treatment is yet to be discov- ered, and its mortality rate remains high [128–130]. Thus, it is not unexpected that all Iranian patients died, with this in mind that all were high-risk COVID-19 ICU patients and did not receive any antifungal drugs. COVID-19 patients are prone to infections by uncommon fungal pathogens. Three instances of very rare fungal infections were reported in Iranian COVID-19 patients, namely R. mucilaginosa and S. kiliense, and D. foeniculina [16, 19, 31]. This highlights the importance of considering emerging and rare fungi as a differential diagnosis of COVID-19 complications. To the best of our knowledge, these fungi have not been reported in other patients with COVID-19 at the time of writing this article. Eleven cases described as probable pulmonary mold infections were detected by serum or BAL GM [16]. GM testing in serum and BAL samples is used as a screening and diagnostic tool; however, its positive predictive value ranges between 0 and 100% [131]. Therefore, the diagnosis of mold infection in these cases is undetermined, as the respiratory features and radiological findings can be attributed to SARS-CoV-2 or other organisms. Supporting information S1 File. PRISMA checklist. (DOCX) Supporting information S1 File. PRISMA checklist. (DOCX) S2 File. PubMed search strategy. (DOCX) S3 File. Risk of bias assessment results. (XLSX) S4 File. Extracted data. (XLSX) S2 File. PubMed search strategy. (DOCX) S3 File. Risk of bias assessment results. (XLSX) S3 File. Risk of bias assessment results. Author Contributions Conceptualization: Tina Nazari, Shahram Mahmoudi. Conceptualization: Tina Nazari, Shahram Mahmoudi. Data curation: Tina Nazari, Fatemeh Sadeghi, Setayesh Sameni, Shahram Mahmoudi. , g , y , Investigation: Tina Nazari, Fatemeh Sadeghi, Alireza Izadi, Shahram Mahmoudi. Investigation: Tina Nazari, Fatemeh Sadeghi, Alireza Izadi, Shahram Mahmoudi. Methodology: Tina Nazari, Alireza Izadi, Shahram Mahmoudi. Project administration: Shahram Mahmoudi. Supervision: Shahram Mahmoudi. Supervision: Shahram Mahmoudi. Writing – original draft: Tina Nazari, Fatemeh Sadeghi, Alireza Izadi, Setayesh Sameni, Shah- ram Mahmoudi. Writing – review & editing: Tina Nazari, Fatemeh Sadeghi, Shahram Mahmoudi. Conclusion Fungal infections are evident to be a complication of COVID-19 in Iran, especially in patients with diabetes mellitus, cardiovascular diseases, and those receiving antibiotics, corticosteroids, and mechanical ventilation. Candidiasis, mucormycosis and aspergillosis are the most com- mon CAFIs in Iran. However, less common infections like fusariosis and cases of rare 11 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran infections have also been reported. Accordingly, clinicians should avoid neglecting fungal infections by acknowledging them as differential diagnoses and ordering confirmative labora- tory tests in high-risk patients or those not responding to routine treatments. infections have also been reported. Accordingly, clinicians should avoid neglecting fungal infections by acknowledging them as differential diagnoses and ordering confirmative labora- tory tests in high-risk patients or those not responding to routine treatments. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 References Opportunistic fungal infections in the epidemic area of COVID-19: a clinical and diagnostic perspective from Iran. 2020; 185(4):607–11. 15. Pakdel F, Ahmadikia K, Salehi M, Tabari A, Jafari R, Mehrparvar G, et al. Mucormycosis in patients with COVID-19: A cross-sectional descriptive multicentre study from Iran. Mycoses. 2021; 64 (10):1238–52. https://doi.org/10.1111/myc.13334 PMID: 34096653 16. Ghazanfari M, Arastehfar A, Davoodi L, Charati JY, Moazeni M, Abastabar M, et al. Pervasive but Neglected: A Perspective on COVID-19-Associated Pulmonary Mold Infections Among Mechanically Ventilated COVID-19 Patients. Front Med (Lausanne). 2021; 8:13. https://doi.org/10.3389/fmed.2021. 649675 PMID: 34195207 17. Page MJ, Mckenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021; 372:n71. https://doi.org/ 10.1136/bmj.n71 PMID: 33782057 18. Abolghasemi S, Hakamifard A, Sharifynia S, Toutkaboni MP, Tehrani HA. Fatal invasive pulmonary aspergillosis in an immunocompetent patient with COVID-19 due to Aspergillus terreus: A case study. Clin Case Rep. 2021; 9(4):2414–8. https://doi.org/10.1002/ccr3.4051 PMID: 33936706 19. Arastehfar A, Shaban T, Zarrinfar H, Roudbary M, Ghazanfari M, Hedayati MT, et al. Candidemia among Iranian Patients with Severe COVID-19 Admitted to ICUs. J Fungi (Basel). 2021; 7(4). https:// doi.org/10.3390/jof7040280 PMID: 33917967 20. Davoodi L, Faeli L, Mirzakhani R, Jalalian R, Shokohi T, Kermani FJCMM. Catastrophic Candida pros- thetic valve endocarditis and COVID-19 comorbidity: A rare case. Curr Med Mycol. 2021; 7(2):43–7. https://doi.org/10.18502/cmm.7.2.7157 PMID: 35028485 21. Fazeli MA, Rezaei L, Javadirad E, Iranfar K, Khosravi A, Saman JA, et al. Increased incidence of rhino-orbital mucormycosis in an educational therapeutic hospital during the COVID-19 pandemic in western Iran: An observational study. Mycoses. 2021; 64(11):1366–77. https://doi.org/10.1111/myc. 13351 PMID: 34252988 22. Hakamifard A, Hashemi M, Fakhim H, Aboutalebian S, Hajiahmadi S, Mohammadi R. Fatal dissemi- nated aspergillosis in an immunocompetent patient with COVID-19 due to Aspergillus ochraceus. J Mycol Med. 2021; 31(2):101124. https://doi.org/10.1016/j.mycmed.2021.101124 PMID: 33684835 23. Heydarifard Z, Safaei M, Zadheidar S, Ehsan S, Shafiei-Jandaghi NZ. Mucormycosis infection in severe COVID-19 patient with multiple underlying health conditions. Clin Case Rep. 2021; 9(10). https://doi.org/10.1002/ccr3.5009 PMID: 34745621 24. Hosseinikargar N, Basiri R, Asadzadeh M, Najafzadeh MJ, Zarrinfar H. First report of invasive Asper- gillus rhinosinusitis in a critically ill COVID-19 patient affected by acute myeloid leukemia, northeastern Iran. Clin Case Rep. 2021; 9(10):e04889. https://doi.org/10.1002/ccr3.4889 PMID: 34631073 25. Karimi-Galougahi M, Arastou S, Haseli S. Fulminant mucormycosis complicating coronavirus disease 2019 (COVID-19). Int Forum Allergy Rhinol. 2021; 11(6):1029–30. https://doi.org/10.1002/alr.22785 PMID: 33713565 26. References 1. Naserghandi A, Allameh S, Saffarpour RJNM, Infections N. All about COVID-19 in brief. 2020; 35. 2. Worldometers.Info. Dover, Delaware, U.S.A.2022 [Available from: https://www.worldometers.info/ coronavirus/. 3. Lansbury L, Lim B, Baskaran V, Lim WSJJOI. Co-infections in people with COVID-19: a systematic review and meta-analysis. 2020; 81(2):266–75. 4. Kumar A, Narayan RK, Prasoon P, Kumari C, Kaur G, Kumar S, et al. COVID-19 Mechanisms in the Human Body—What We Know So Far. Frontiers in Immunology. 2021; 12. https://doi.org/10.3389/ fimmu.2021.693938 PMID: 34790191 5. Garcia-Vidal C, Sanjuan G, Moreno-Garcı´a E, Puerta-Alcalde P, Garcia-Pouton N, Chumbita M, et al. Incidence of co-infections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study. 2021; 27(1):83–8. 6. Alhumaid S, Al Mutair A, Al Alawi Z, Alshawi AM, Alomran SA, Almuhanna MS, et al. Coinfections with Bacteria, Fungi, and Respiratory Viruses in Patients with SARS-CoV-2: A Systematic Review and Meta-Analysis. Pathogens. 2021; 10(7). https://doi.org/10.3390/pathogens10070809 7. White PL, Dhillon R, Cordey A, Hughes H, Faggian F, Soni S, et al. A National Strategy to Diagnose Coronavirus Disease 2019-Associated Invasive Fungal Disease in the Intensive Care Unit. Clin Infect Dis. 2021; 73(7):e1634–e44. https://doi.org/10.1093/cid/ciaa1298 PMID: 32860682 8. Abdoli A, Falahi S, Kenarkoohi A. COVID-19-associated opportunistic infections: a snapshot on the current reports. Clin Exp Med. 2021:1–20. https://doi.org/10.1007/s10238-021-00751-7 PMID: 34424451 12 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 9. Amin A, Vartanian A, Poladian N, Voloshko A, Yegiazaryan A, Al-Kassir AL, et al. Root Causes of Fun- gal Coinfections in COVID-19 Infected Patients. Infect Dis Rep. 2021; 13(4):1018–35. https://doi.org/ 10.3390/idr13040093 PMID: 34940403 10. Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet Respiratory medicine. 2020; 8(4):420–2. https:// doi.org/10.1016/S2213-2600(20)30076-X PMID: 32085846 11. Wang B, Zhang L, Wang Y, Dai T, Qin Z, Zhou F, et al. Alterations in microbiota of patients with COVID-19: potential mechanisms and therapeutic interventions. Signal Transduct Target Ther. 2022; 7(1):143. https://doi.org/10.1038/s41392-022-00986-0 PMID: 35487886 12. Song G, Liang G, Liu WJM. Fungal co-infections associated with global COVID-19 pandemic: a clinical and diagnostic perspective from China. 2020:1–8. 13. Gangneux JP, Bougnoux ME, Dannaoui E, Cornet M, Zahar JR. Invasive fungal diseases during COVID-19: We should be prepared. Journal de Mycologie Me´dicale. 2020; 30(2):100971. https://doi. org/10.1016/j.mycmed.2020.100971 PMID: 32307254 14. Salehi M, Ahmadikia K, Badali H, Khodavaisy SJM. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 References Khodavaisy S, Khajavirad N, Izadi A, Dehghan Manshadi SA, Abdollahi A, Aliramezani A, et al. Proven pulmonary aspergillosis in a COVID-19 patient: A case report. Curr Med Mycol. 2021; 7(2):39–42. https://doi.org/10.18502/cmm.7.2.7031 PMID: 35028484 27. Mehrabi Z, Salimi M, Niknam K, Mohammadi F, Mamaghani HJ, Sasani MR, et al. Sinoorbital Mucor- mycosis Associated with Corticosteroid Therapy in COVID-19 Infection. Case Reports in Ophthalmo- logical Medicine. 2021; 2021:9745701. https://doi.org/10.1155/2021/9745701 PMID: 34745674 13 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 28. Mohammadi F, Badri M, Safari S, Hemmat N. A case report of rhino-facial mucormycosis in a non-dia- betic patient with COVID-19: a systematic review of literature and current update. BMC Infect Dis. 2021; 21(1):906. https://doi.org/10.1186/s12879-021-06625-3 PMID: 34479495 29. Nasri E, Shoaei P, Vakili B, Mirhendi H, Sadeghi S, Hajiahmadi S, et al. Fatal Invasive Pulmonary Aspergillosis in COVID-19 Patient with Acute Myeloid Leukemia in Iran. Mycopathologia. 2020; 185 (6):1077–84. https://doi.org/10.1007/s11046-020-00493-2 PMID: 33009966 30. Ostovan VR, Rezapanah S, Behzadi Z, Hosseini L, Jahangiri R, Anbardar MH, et al. Coronavirus dis- ease (COVID-19) complicated by rhino-orbital-cerebral mucormycosis presenting with neurovascular thrombosis: a case report and review of literature. J Neurovirol. 2021; 27(4):644–9. https://doi.org/10. 1007/s13365-021-00996-8 PMID: 34342852 31. Ranjbar-Mobarake M, Nowroozi J, Badiee P, Mostafavi SN, Mohammadi R. Fatal disseminated infec- tion due to Sarocladium kiliense in a diabetic patient with COVID-19. Clin Case Rep. 2021; 9(9):6. https://doi.org/10.1002/ccr3.4596 32. Salehi M, Ahmadikia K, Mahmoudi S, Kalantari S, Jamalimoghadamsiahkali S, Izadi A, et al. Oropha- ryngeal candidiasis in hospitalised COVID-19 patients from Iran: Species identification and antifungal susceptibility pattern. Mycoses. 2020; 63(8):771–8. https://doi.org/10.1111/myc.13137 PMID: 32609906 33. Salehi M, Khajavirad N, Seifi A, Salahshour F, Jahanbin B, Kazemizadeh H, et al. Proven Aspergillus flavus pulmonary aspergillosis in a COVID-19 patient: A case report and review of the literature. Myco- ses. 2021; 64(8):809–16. https://doi.org/10.1111/myc.13255 PMID: 33576014 34. Sharifpour A, Zakariaei Z, Fakhar M, Banimostafavi ES, Nakhaei M, Soleymani M. Post-COVID-19 co-morbidity of emerged Lophomonas infection and invasive pulmonary aspergillosis: First case report. Clin Case Rep. 2021; 9(9):e04822. https://doi.org/10.1002/ccr3.4822 PMID: 34594552 35. Shirvani F, Fattahi A. Pulmonary Candidiasis Associated with COVID-19: Evaluation of Causative Agents and their Antifungal Susceptibility Patterns. Tanaffos. 2021; 20(1):29–35. PMID: 34394367 36. Tabarsi P, Khalili N, Pourabdollah M, Sharifynia S, Naeini AS, Ghorbani J, et al. References COVID-19-Associ- ated Candidiasis (CAC): An Underestimated Complication in the Absence of Immunological Predispo- sitions? J Fungi (Basel). 2020; 6(4). https://doi.org/10.3390/jof6040211 PMID: 33050019 48. Pankhurst CL. Candidiasis (oropharyngeal). BMJ Clin Evid. 2013; 2013:1304. PMID: 24209593 49. Peckham H, De Gruijter NM, Raine C, Radziszewska A, Ciurtin C, Wedderburn LR, et al. Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission. Nat Com- mun. 2020; 11(1):6317. https://doi.org/10.1038/s41467-020-19741-6 PMID: 33298944 50. Romero Starke K, Reissig D, Petereit-Haack G, Schmauder S, Nienhaus A, Seidler A. The isolated effect of age on the risk of COVID-19 severe outcomes: a systematic review with meta-analysis. BMJ Global Health. 2021; 6(12):e006434. https://doi.org/10.1136/bmjgh-2021-006434 PMID: 34916273 51. Turner SA, Butler G. The Candida pathogenic species complex. Cold Spring Harbor perspectives in medicine. 2014; 4(9):a019778–a. https://doi.org/10.1101/cshperspect.a019778 PMID: 25183855 52. Riche CVW, Cassol R, Pasqualotto AC. Is the Frequency of Candidemia Increasing in COVID-19 Patients Receiving Corticosteroids? J Fungi (Basel). 2020; 6(4). https://doi.org/10.3390/jof6040286 PMID: 33203016 53. Nucci M, Barreiros G, Guimarães LF, Deriquehem VaS, Castiñeiras AC, Noue´r SA. Increased inci- dence of candidemia in a tertiary care hospital with the COVID-19 pandemic. Mycoses. 2021; 64 (2):152–6. https://doi.org/10.1111/myc.13225 PMID: 33275821 54. Segrelles-Calvo G, De SaGR, Llopis-Pastor E, Carrillo J, Herna´ndez-Herna´ndez M, Rey L, et al. Can- dida spp. co-infection in COVID-19 patients with severe pneumonia: Prevalence study and associated risk factors. Respir Med. 2021; 188:106619. https://doi.org/10.1016/j.rmed.2021.106619 PMID: 34555702 55. Chowdhary A, Tarai B, Singh A, Sharma A. Multidrug-Resistant Candida auris Infections in Critically Ill Coronavirus Disease Patients, India, April-July 2020. Emerg Infect Dis. 2020; 26(11):2694–6. https:// doi.org/10.3201/eid2611.203504 PMID: 32852265 56. Bishburg E, Okoh A, Nagarakanti SR, Lindner M, Migliore C, Patel P. Fungemia in COVID-19 ICU patients, a single medical center experience. J Med Virol. 2021; 93(5):2810–4. https://doi.org/10.1002/ jmv.26633 PMID: 33107617 57. Villanueva-Lozano H, Treviño-Rangel RJ, Gonza´lez GM, Ramı´rez-Elizondo MT, Lara-Medrano R, Aleman-Bocanegra MC, et al. Outbreak of Candida auris infection in a COVID-19 hospital in Mexico. Clin Microbiol Infect. 2021; 27(5):813–6. https://doi.org/10.1016/j.cmi.2020.12.030 58. Magnasco L, Mikulska M, Giacobbe DR, Taramasso L, Vena A, Dentone C, et al. Spread of Carbape- nem-Resistant Gram-Negatives and Candida auris during the COVID-19 Pandemic in Critically Ill Patients: One Step Back in Antimicrobial Stewardship? Microorganisms. 2021; 9(1). https://doi.org/10. 3390/microorganisms9010095 59. Cataldo MA, Tetaj N, Selleri M, Marchioni L, Capone A, Caraffa E, et al. References Case Report: Corona- virus Disease 2019-associated Rhinosinusitis Mucormycosis Caused by Rhizopus arrhizus: A Rare but Potentially Fatal Infection Occurring After Treatment with Corticosteroids. Am J Trop Med Hyg. 2021; 105(2):449–53. https://doi.org/10.4269/ajtmh.21-0359 PMID: 34237015 37. Veisi A, Bagheri A, Eshaghi M, Rikhtehgar MH, Rezaei Kanavi M, Farjad R. Rhino-orbital mucormyco- sis during steroid therapy in COVID-19 patients: A case report. Eur J Ophthalmol. 2021:11206721211009450. https://doi.org/10.1177/11206721211009450 PMID: 33843287 38. Frı´as-De-Leo´n MG, Pinto-Almaza´n R, Herna´ndez-Castro R, Garcı´a-Salazar E, Meza-Meneses P, Rodrı´guez-Cerdeira C, et al. Epidemiology of Systemic Mycoses in the COVID-19 Pandemic. J Fungi (Basel). 2021; 7(7). https://doi.org/10.3390/jof7070556 PMID: 34356935 39. Musuuza JS, Watson L, Parmasad V, Putman-Buehler N, Christensen L, Safdar N. Prevalence and outcomes of co-infection and superinfection with SARS-CoV-2 and other pathogens: A systematic review and meta-analysis. PLoS One. 2021; 16(5):e0251170. https://doi.org/10.1371/journal.pone. 0251170 PMID: 33956882 40. Katz J. Prevalence of candidiasis and oral candidiasis in COVID-19 patients: a cross-sectional pilot study from the patients’ registry in a large health center. Quintessence Int. 2021; 52(8):714–8. https:// doi.org/10.3290/j.qi.b1491959 PMID: 34076373 41. Ahmed N, Mahmood MS, Ullah MA, Araf Y, Rahaman TI, Moin AT, et al. COVID-19-Associated Candi- diasis: Possible Patho-Mechanism, Predisposing Factors, and Prevention Strategies. Curr Microbiol. 2022; 79(5):127. https://doi.org/10.1007/s00284-022-02824-6 PMID: 35287179 42. Anka AU, Tahir MI, Abubakar SD, Alsabbagh M, Zian Z, Hamedifar H, et al. Coronavirus disease 2019 (COVID-19): An overview of the immunopathology, serological diagnosis and management. Scand J Immunol. 2021; 93(4):e12998. https://doi.org/10.1111/sji.12998 PMID: 33190302 43. Nechipurenko YD, Semyonov DA, Lavrinenko IA, Lagutkin DA, Generalov EA, Zaitceva AY, et al. The Role of Acidosis in the Pathogenesis of Severe Forms of COVID-19. Biology (Basel). 2021; 10(9). https://doi.org/10.3390/biology10090852 PMID: 34571729 44. Carpenè G, Onorato D, Nocini R, Fortunato G, Rizk JG, Henry BM, et al. Blood lactate concentration in COVID-19: a systematic literature review. Clinical Chemistry and Laboratory Medicine (CCLM). 2022; 60(3):332–7. https://doi.org/10.1515/cclm-2021-1115 PMID: 34856090 45. Ballou ER, Avelar GM, Childers DS, Mackie J, Bain JM, Wagener J, et al. Lactate signalling regulates fungal β-glucan masking and immune evasion. Nat Microbiol. 2016; 2:16238. https://doi.org/10.1038/ nmicrobiol.2016.238 PMID: 27941860 46. Concia E, Azzini AM, Conti M. Epidemiology, incidence and risk factors for invasive candidiasis in high-risk patients. Drugs. 2009; 69 Suppl 1:5–14. https://doi.org/10.2165/11315500-000000000- 00000 PMID: 19877728 14 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 47. Arastehfar A, Carvalho A, Nguyen MH, Hedayati MT, Netea MG, Perlin DS, et al. References Incidence of bacterial and fun- gal bloodstream infections in COVID-19 patients in intensive care: An alarming "collateral effect". J Glob Antimicrob Resist. 2020; 23:290–1. https://doi.org/10.1016/j.jgar.2020.10.004 PMID: 33130024 60. Calderaro A, Buttrini M, Montecchini S, Piccolo G, Martinelli M, Dell, et al. Detection of SARS-CoV-2 and Other Infectious Agents in Lower Respiratory Tract Samples Belonging to Patients Admitted to Intensive Care Units of a Tertiary-Care Hospital, Located in an Epidemic Area, during the Italian Lock- down. Microorganisms. 2021; 9(1):185. https://doi.org/10.3390/microorganisms9010185 PMID: 33467079 61. Lv Z, Cheng S, Le J, Huang J, Feng L, Zhang B, et al. Clinical characteristics and co-infections of 354 hospitalized patients with COVID-19 in Wuhan, China: a retrospective cohort study. Microbes Infect. 2020; 22(4–5):195–9. https://doi.org/10.1016/j.micinf.2020.05.007 PMID: 32425649 62. Rodriguez JY, Le Pape P, Lopez O, Esquea K, Labiosa AL, Alvarez-Moreno C. Candida auris: A Latent Threat to Critically Ill Patients With Coronavirus Disease 2019. Clinical Infectious Diseases. 2020; 73(9):e2836–e7. https://doi.org/10.1093/cid/ciaa1595 63. Colombo AL, Perfect J, Dinubile M, Bartizal K, Motyl M, Hicks P, et al. Global Distribution and Out- comes for Candida Species Causing Invasive Candidiasis: Results from an International Randomized Double-Blind Study of Caspofungin Versus Amphotericin B for the Treatment of Invasive Candidiasis. European Journal of Clinical Microbiology and Infectious Diseases. 2003; 22(8):470–4. https://doi.org/ 10.1007/s10096-003-0973-8 PMID: 12884068 64. Lone SA, Ahmad A. Candida auris-the growing menace to global health. Mycoses. 2019; 62(8):620– 37. https://doi.org/10.1111/myc.12904 PMID: 30773703 65. Abastabar M, Haghani I, Ahangarkani F, Rezai MS, Taghizadeh Armaki M, Roodgari S, et al. Candida auris otomycosis in Iran and review of recent literature. Mycoses. 2019; 62(2):101–5. https://doi.org/ 10.1111/myc.12886 PMID: 30585653 15 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 66. Taghizadeh Armaki M, Mahdavi Omran S, Kiakojuri K, Khojasteh S, Jafarzadeh J, Tavakoli M, et al. First fluconazole-resistant Candida auris isolated from fungal otitis in Iran. Curr Med Mycol. 2021; 7 (1):51–4. https://doi.org/10.18502/cmm.7.1.6243 PMID: 34553098 67. Mirhendi H, Charsizadeh A, Aboutalebian S, Mohammadpour M, Nikmanesh B, De Groot T, et al. South Asian (Clade I) Candida auris meningitis in a paediatric patient in Iran with a review of the litera- ture. Mycoses. 2022; 65(2):134–9. https://doi.org/10.1111/myc.13396 PMID: 34780087 68. Mahmoudi S, Agha Kuchak Afshari S, Aghaei Gharehbolagh S, Mirhendi H, Makimura K. Methods for identification of Candida auris, the yeast of global public health concern: A review. Journal de Mycolo- gie Me´dicale. 2019; 29(2):174–9. https://doi.org/10.1016/j.mycmed.2019.04.004 PMID: 31031132 69. References Karimi L, Mirhendi H, Khodadadi H, Mohammadi R. Molecular identification of uncommon clinical yeast species in Iran. Current medical mycology. 2015; 1(2):1–6. https://doi.org/10.18869/acadpub. cmm.1.2.1 PMID: 28680981 70. Riad A, Gomaa E, Hockova B, Klugar M. Oral candidiasis of COVID-19 patients: Case report and review of evidence. J Cosmet Dermatol. 2021; 20(6):1580–4. https://doi.org/10.1111/jocd.14066 PMID: 33713390 71. Masur H, Rosen PP, Armstrong D. Pulmonary disease caused by Candida species. Am J Med. 1977; 63(6):914–25. https://doi.org/10.1016/0002-9343(77)90546-0 PMID: 343588 72. Schnabel RM, Linssen CF, Guion N, Van Mook WN, Bergmans DC. Candida pneumonia in intensive care unit? Open Forum Infect Dis. 2014; 1(1):ofu026. https://doi.org/10.1093/ofid/ofu026 PMID: 25734099 73. Pema´n J, Ruiz-Gaita´n A, Garcı´a-Vidal C, Salavert M, Ramı´rez P, Puchades F, et al. Fungal co-infec- tion in COVID-19 patients: Should we be concerned? Rev Iberoam Micol. 2020; 37(2):41–6. https:// doi.org/10.1016/j.riam.2020.07.001 PMID: 33041191 74. Meena DS, Kumar D. Candida Pneumonia: An Innocent Bystander or a Silent Killer? Med Princ Pract. 2021. https://doi.org/10.1159/000520111 PMID: 34638123 75. Casalini G, Giacomelli A, Ridolfo A, Gervasoni C, Antinori S. Invasive Fungal Infections Complicating COVID-19: A Narrative Review. J Fungi (Basel). 2021; 7(11). https://doi.org/10.3390/jof7110921 PMID: 34829210 76. Baddley JW, Thompson GR, Chen SC 3rd, White PL, Johnson MD, Nguyen MH, et al. Coronavirus Disease 2019-Associated Invasive Fungal Infection. Open Forum Infect Dis. 2021; 8(12):ofab510. https://doi.org/10.1093/ofid/ofab510 PMID: 34877364 77. Antinori S, Bonazzetti C, Gubertini G, Capetti A, Pagani C, Morena V, et al. Tocilizumab for cytokine storm syndrome in COVID-19 pneumonia: an increased risk for candidemia? Autoimmun Rev. 2020; 19(7):102564. https://doi.org/10.1016/j.autrev.2020.102564 PMID: 32376396 78. Yuan SM. Fungal Endocarditis. Braz J Cardiovasc Surg. 2016; 31(3):252–5. https://doi.org/10.5935/ 1678-9741.20160026 PMID: 27737409 79. Badiee P, Amirghofran AA, Ghazi Nour M, Shafa M, Nemati MH. Incidence and outcome of docu- mented fungal endocarditis. Int Cardiovasc Res J. 2014; 8(4):152–5. PMID: 25614858 80. Prakash H, Chakrabarti A. Global Epidemiology of Mucormycosis. J Fungi (Basel). 2019; 5(1):26. https://doi.org/10.3390/jof5010026 PMID: 30901907 81. Dolatabadi S, Ahmadi B, Rezaei-Matehkolaei A, Zarrinfar H, Skiada A, Mirhendi H, et al. Mucormyco- sis in Iran: A six-year retrospective experience. J Mycol Med. 2018; 28(2):269–73. https://doi.org/10. 1016/j.mycmed.2018.02.014 PMID: 29545123 82. Hussain S, Riad A, Singh A, Klugarova´ J, Antony B, Banna H, et al. Global Prevalence of COVID-19- Associated Mucormycosis (CAM): Living Systematic Review and Meta-Analysis. J Fungi (Basel). 2021; 7(11). https://doi.org/10.3390/jof7110985 PMID: 34829271 83. Muthu V, Rudramurthy SM, Chakrabarti A, Agarwal R. Epidemiology and Pathophysiology of COVID- 19-Associated Mucormycosis: India Versus the Rest of the World. References Mycopathologia. 2021; 186(6):739– 54. https://doi.org/10.1007/s11046-021-00584-8 PMID: 34414555 84. Bhattacharyya A, Sarma P, Kaur H, Kumar S, Bhattacharyya J, Prajapat M, et al. COVID-19-associ- ated rhino-orbital-cerebral mucormycosis: A systematic review, meta-analysis, and meta-regression analysis. Indian J Pharmacol. 2021; 53(6):499–510. https://doi.org/10.4103/ijp.ijp_839_21 PMID: 34975140 85. Pal R, Singh B, Bhadada SK, Banerjee M, Bhogal RS, Hage N, et al. COVID-19-associated mucormy- cosis: An updated systematic review of literature. Mycoses. 2021; 64(12):1452–9. https://doi.org/10. 1111/myc.13338 PMID: 34133798 86. Singh AK, Singh R, Joshi SR, Misra A. Mucormycosis in COVID-19: A systematic review of cases reported worldwide and in India. Diabetes Metab Syndr. 2021; 15(4):102146. https://doi.org/10.1016/j. dsx.2021.05.019 PMID: 34192610 16 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 87. Sen M, Honavar SG, Bansal R, Sengupta S, Rao R, Kim U, et al. Epidemiology, clinical profile, man- agement, and outcome of COVID-19-associated rhino-orbital-cerebral mucormycosis in 2826 patients in India—Collaborative OPAI-IJO Study on Mucormycosis in COVID-19 (COSMIC), Report 1. Indian J Ophthalmol. 2021; 69(7):1670–92. https://doi.org/10.4103/ijo.IJO_1565_21 PMID: 34156034 88. Arora R, Goel R, Khanam S, Kumar S, Shah S, Singh S, et al. Rhino-Orbito-Cerebral-Mucormycosis During the COVID-19 Second Wave in 2021—A Preliminary Report from a Single Hospital. Clin Ophthalmol. 2021; 15:3505–14. https://doi.org/10.2147/OPTH.S324977 PMID: 34429582 89. Hoenigl M, Seidel D, Carvalho A, Rudramurthy SM, Arastehfar A, Gangneux J-P, et al. The emer- gence of COVID-19 associated mucormycosis: a review of cases from 18 countries. The Lancet Microbe. 2022. https://doi.org/10.1016/S2666-5247(21)00237-8 PMID: 35098179 90. Cornely OA, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B, et al. Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confeder- ation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis. 2019; 19(12):e405–e21. https://doi.org/10.1016/S1473-3099(19) 30312-3 PMID: 31699664 91. Dannaoui E. Recent Developments in the Diagnosis of Mucormycosis. J Fungi (Basel). 2022; 8(5). https://doi.org/10.3390/jof8050457 92. Ahmadikia K, Hashemi SJ, Khodavaisy S, Getso MI, Alijani N, Badali H, et al. The double-edged sword of systemic corticosteroid therapy in viral pneumonia: A case report and comparative review of influenza-associated mucormycosis versus COVID-19 associated mucormycosis. Mycoses. 2021; 64 (8):798–808. https://doi.org/10.1111/myc.13256 PMID: 33590551 93. Garg D, Muthu V, Sehgal IS, Ramachandran R, Kaur H, Bhalla A, et al. Coronavirus Disease (Covid- 19) Associated Mucormycosis (CAM): Case Report and Systematic Review of Literature. Mycopatho- logia. 2021; 186(2):289–98. https://doi.org/10.1007/s11046-021-00528-2 PMID: 33544266 94. Placik DA, Taylor WL, Wnuk NM. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 References Fungal Biol Rev. 2021; 38:67–91. https://doi.org/10.1016/j.fbr.2021.09.003 PMID: 34548877 106. Sipsas NV, Gamaletsou MN, Anastasopoulou A, Kontoyiannis DP. Therapy of Mucormycosis. Journal of Fungi. 2018; 4(3):90. 107. Rabagliati R, Rodrı´guez N, Nu´ñez C, Huete A, Bravo S, Garcia P. COVID-19–Associated Mold Infec- tion in Critically Ill Patients, Chile. Emerging infectious diseases. 2021; 27(5):1454. https://doi.org/10. 3201/eid2705.204412 PMID: 33760726 108. Apostolopoulou A, Esquer Garrigos Z, Vijayvargiya P, Lerner AH, Farmakiotis D. Invasive pulmonary aspergillosis in patients with SARS-CoV-2 infection: a systematic review of the literature. Diagnostics. 2020; 10(10):807. https://doi.org/10.3390/diagnostics10100807 PMID: 33050499 109. Mitaka H, Kuno T, Takagi H, Patrawalla P. Incidence and mortality of COVID-19-associated pulmo- nary aspergillosis: A systematic review and meta-analysis. Mycoses. 2021; 64(9):993–1001. https:// doi.org/10.1111/myc.13292 PMID: 33896063 110. Sebastian SK, Kumar VB, Gupta M, Sharma Y. Covid Assossiated Invasive Fungal Sinusitis. Indian J Otolaryngol Head Neck Surg. 2021:1–4. https://doi.org/10.1007/s12070-021-02471-6 PMID: 33649716 111. Shetty S, Shilpa C, Kavya S, Sundararaman A, Hegde K, Madhan S. Invasive Aspergillosis of Nose and Paranasal Sinus in COVID-19 Convalescents: Mold Goes Viral? Indian J Otolaryngol Head Neck Surg. 2022:1–6. https://doi.org/10.1007/s12070-022-03073-6 112. Dokania V, Gaikwad NS, Gite V, Mhashal S, Shetty N, Shinde P, et al. Emergence of Invasive Fungal Rhinosinusitis in Recently Recovered COVID-19 Patients. Ann Otol Rhinol Laryngol. 2021:34894211060923. https://doi.org/10.1177/00034894211060923 PMID: 34852669 113. El-Kholy NA, El-Fattah AMA, Khafagy YW. Invasive Fungal Sinusitis in Post COVID-19 Patients: A New Clinical Entity. Laryngoscope. 2021; 131(12):2652–8. https://doi.org/10.1002/lary.29632 PMID: 34009676 114. Chong WH, Neu KP. Incidence, diagnosis and outcomes of COVID-19-associated pulmonary asper- gillosis (CAPA): a systematic review. Journal of Hospital Infection. 2021; 113:115–29. https://doi.org/ 10.1016/j.jhin.2021.04.012 PMID: 33891985 115. Salmanton-Garcı´a J, Sprute R, Stemler J, Bartoletti M, Dupont D, Valerio M, et al. COVID-19-Associ- ated Pulmonary Aspergillosis, March-August 2020. Emerg Infect Dis. 2021; 27(4):1077–86. https:// doi.org/10.3201/eid2704.204895 PMID: 33539721 116. Arastehfar A, Carvalho A, Van De Veerdonk FL, Jenks JD, Koehler P, Krause R, et al. COVID-19 associated pulmonary aspergillosis (CAPA)—from immunology to treatment. Journal of Fungi. 2020; 6 (2):91. https://doi.org/10.3390/jof6020091 PMID: 32599813 117. Prattes J, Wauters J, Giacobbe DR, Salmanton-Garcı´a J, Maertens J, Bourgeois M, et al. Risk factors and outcome of pulmonary aspergillosis in critically ill coronavirus disease 2019 patients-a multina- tional observational study by the European Confederation of Medical Mycology. Clin Microbiol Infect. 2022; 28(4):580–7. https://doi.org/10.1016/j.cmi.2021.08.014 PMID: 34454093 118. Chadeganipour M, Mohammadi R. A 9-Year Experience of Aspergillus Infections from Isfahan, Iran. Infection and Drug Resistance. 2020; 13:2301. https://doi.org/10.2147/IDR.S259162 PMID: 32765006 119. References Bronchopleural fistula development in the setting of novel therapies for acute respiratory distress syndrome in SARS-CoV-2 pneumonia. Radiology Case Reports. 2020; 15(11):2378–81. https://doi.org/10.1016/j.radcr.2020.09.026 PMID: 32983308 95. Johnson AK, Ghazarian Z, Cendrowski KD, Persichino JG. Pulmonary aspergillosis and mucormyco- sis in a patient with COVID-19. Medical Mycology Case Reports. 2021; 32:64–7. https://doi.org/10. 1016/j.mmcr.2021.03.006 PMID: 33842203 96. Kanwar A, Jordan A, Olewiler S, Wehberg K, Cortes M, Jackson BR. A Fatal Case of Rhizopus azy- gosporus Pneumonia Following COVID-19. Journal of Fungi. 2021; 7(3):174. https://doi.org/10.3390/ jof7030174 PMID: 33670842 97. Zurl C, Hoenigl M, Schulz E, Hatzl S, Gorkiewicz G, Krause R, et al. Autopsy Proven Pulmonary Mucormycosis Due to Rhizopus microsporus in a Critically Ill COVID-19 Patient with Underlying Hematological Malignancy. Journal of Fungi. 2021; 7(2):88. https://doi.org/10.3390/jof7020088 PMID: 33513875 98. Mishra N, Mutya VSS, Thomas A, Rai G, Reddy B, Mohanan AA, et al. A case series of invasive mucormycosis in patients with COVID-19 infection. Int J Otorhinolaryngol Head Neck Surg. 2021; 7 (5):867–70. 99. Moorthy A, Gaikwad R, Krishna S, Hegde R, Tripathi KK, Kale PG, et al. SARS-CoV-2, Uncontrolled Diabetes and Corticosteroids—An Unholy Trinity in Invasive Fungal Infections of the Maxillofacial Region? A Retrospective, Multi-centric Analysis. Journal of Maxillofacial and Oral Surgery. 2021; 20 (3):418–25. https://doi.org/10.1007/s12663-021-01532-1 PMID: 33716414 100. Monte Junior ESD, Santos MELD, Ribeiro IB, Luz GDO, Baba ER, Hirsch BS, et al. Rare and Fatal Gastrointestinal Mucormycosis (Zygomycosis) in a COVID-19 Patient: A Case Report. Clin Endosc. 2020; 53(6):746–9. https://doi.org/10.5946/ce.2020.180 PMID: 33207116 101. Khatri A, Chang K-M, Berlinrut I, Wallach F. Mucormycosis after Coronavirus disease 2019 infection in a heart transplant recipient – Case report and review of literature. Journal of Medical Mycology. 2021; 31(2):101125. https://doi.org/10.1016/j.mycmed.2021.101125 PMID: 33857916 102. Afzal S, Nasir M. Aspergillosis and Mucormycosis in COVID-19 Patients; a Systematic Review and Meta-analysis. medRxiv. 2021. 103. Yasmin F, Najeeb H, Naeem A, Dapke K, Phadke R, Asghar MS, et al. COVID-19 Associated Mucor- mycosis: A Systematic Review from Diagnostic Challenges to Management. Diseases. 2021; 9(4). https://doi.org/10.3390/diseases9040065 104. Azhar A, Khan WH, Khan PA, Alhosaini K, Owais M, Ahmad A. Mucormycosis and COVID-19 pan- demic: Clinical and diagnostic approach. J Infect Public Health. 2022; 15(4):466–79. https://doi.org/10. 1016/j.jiph.2022.02.007 PMID: 35216920 17 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 PLOS ONE COVID-19 associated mycoses in Iran 105. Ghosh A, Sarkar A, Paul P, Patel P. The rise in cases of mucormycosis, candidiasis and aspergillosis amidst COVID19. References Tavakoli M, Yazdani Charati J, Hedayati MT, Moosazadeh M, Badiee P, Seyedmousavi S, et al. National trends in incidence, prevalence and disability-adjusted life years of invasive aspergillosis in Iran: a systematic review and meta-analysis. Expert review of respiratory medicine. 2019; 13 (11):1121–34. https://doi.org/10.1080/17476348.2019.1657835 PMID: 31426666 120. Moazam S, Denning DW. Aspergillus nodules in chronic granulomatous disease attributable to Asper- gillus ochraceus. Medical mycology case reports. 2017; 17:31–3. https://doi.org/10.1016/j.mmcr. 2017.06.004 PMID: 28702318 121. Novey HS, Wells ID. Allergic bronchopulmonary aspergillosis caused by Aspergillus ochraceus. Am J Clin Pathol. 1978; 70(5):840–3. https://doi.org/10.1093/ajcp/70.5.840 PMID: 102184 122. Chong WH, Saha BK, Neu KP. Comparing the clinical characteristics and outcomes of COVID-19- associate pulmonary aspergillosis (CAPA): a systematic review and meta-analysis. Infection. 2022; 50 (1):43–56. https://doi.org/10.1007/s15010-021-01701-x PMID: 34570355 123. Barberis F, Benedetti MF, De Abreu MS, Pola SJ, Posse G, Capece P, et al. Invasive fusariosis in a critically ill patient with severe COVID-19 pneumonia: A case report. Medical Mycology Case Reports. 2022; 35:5–8. https://doi.org/10.1016/j.mmcr.2021.12.003 PMID: 34931158 124. Poignon C, Blaize M, Vezinet C, Lampros A, Monsel A, Fekkar A. Invasive pulmonary fusariosis in an immunocompetent critically ill patient with severe COVID-19. Clinical Microbiology and Infection. 2020; 26(11):1582–4. https://doi.org/10.1016/j.cmi.2020.06.026 PMID: 32619736 PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 18 / 19 PLOS ONE COVID-19 associated mycoses in Iran 125. Gangneux J-P, Dannaoui E, Fekkar A, Luyt C-E, Botterel F, De Prost N, et al. Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: The French multicentre MYCOVID study. The Lancet Respiratory Medicine. 2021. https://doi.org/10.1016/S2213-2600(21) 00442-2 PMID: 34843666 126. Meira F, Moreno-Garcı´a E, Linares L, Macaya I, Tome´ A, Herna´ndez-Meneses M, et al. Impact of inflammatory response modifiers on the incidence of hospital-acquired infections in patients with COVID-19. Infectious Diseases and Therapy. 2021; 10(3):1407–18. https://doi.org/10.1007/s40121- 021-00477-9 PMID: 34115314 127. Nucci F, Noue´r SA, Capone D, Anaissie E, Nucci M. Fusariosis. Semin Respir Crit Care Med. 2015; 36(5):706–14. https://doi.org/10.1055/s-0035-1562897 PMID: 26398537 128. Muhammed M, Coleman JJ, Carneiro HA, Mylonakis E. The challenge of managing fusariosis. Viru- lence. 2011; 2(2):91–6. https://doi.org/10.4161/viru.2.2.15015 PMID: 21304267 129. Kim J-Y, Kang C-I, Lee JH, Lee WJ, Huh K, Cho SY, et al. Clinical Features and Outcomes of Invasive Fusariosis: A Case Series in a Single Center with Literature Review. Infect Chemother. 2021; 53(2): e4. 130. Horn DL, Freifeld AG, Schuster MG, Azie NE, Franks B, Kauffman CA. PLOS ONE | https://doi.org/10.1371/journal.pone.0271333 July 11, 2022 References Treatment and outcomes of invasive fusariosis: review of 65 cases from the PATH Alliance(®) registry. Mycoses. 2014; 57 (11):652–8. https://doi.org/10.1111/myc.12212 PMID: 24943384 131. Avcu G, Karapinar DY. A review of a diagnostic tool: galactomannan. Journal of Immunological Sci- ences. 2018; 2(4). 19 / 19
https://openalex.org/W2805357125
http://dea.lib.unideb.hu/bitstreams/b1419427-d71f-462d-b7b4-f996f0771f22/download
English
null
MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis
Scientific reports
2,018
cc-by
11,211
MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis Received: 6 June 2017 Accepted: 19 January 2018 Published: xx xx xxxx Received: 6 June 2017 Accepted: 19 January 2018 Published: xx xx xxxx Barnali Biswas1, Frank Batista1,2, Subha Sundaram1 & Pamela Stanley   1 Mechanisms that regulate spermatogenesis in mice are important to define as they often apply to fertility in man. We previously showed that conditional deletion of the mouse Mgat1 gene (Mgat1 cKO) in spermatogonia causes a germ-cell autonomous defect leading to infertility. MGAT1 is the N-acetylglucosaminyltransferase (GlcNAcT-I) that initiates the synthesis of complex N-glycans. Mechanistic bases of MGAT1 loss were investigated in germ cells from 22- and 23-day males, before any changes in germ cell morphology were apparent. Gene expression changes induced by deletion of Mgat1 were determined using the Affymetrix gene chip Mouse Mogene 2.0 ST array, and relationships were investigated by bioinformatics including Gene Ontology (GO), Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA). The loss of complex N-glycans promoted the premature up-regulation of genes normally expressed later in spermatogenesis and spermiogenesis, and IPA and GSEA implicated ERK signaling. EGFR and PDGFRA transcripts and ERK1/2 signaling were reduced in 22-day Mgat1 cKO germ cells. Basigin, a germ cell target of MGAT1, activated ERK1/2 in CHO cells, but not in a Lec1 CHO mutant that lacks MGAT1 and complex N-glycans. Thus, MGAT1 is required to regulate ERK1/2 signaling during spermatogenesis, potentially via different mechanisms. In mammals, spermatogenesis involves a complicated sequence of cell-cell interactions and signaling pathways1,2. In order to identify roles for glycans in spermatogenesis, we previously generated a number of conditional mutants of protein glycosylation by deleting various glycosyltransferase genes in spermatogonia at 3 days post-partum (dpp) using a Stra8-iCre transgene3. Deletion of C1galt1 that generates core 1 and 2 O-glycans, or deletion of Pofut1 that transfers O-fucose to Notch receptors and is required for Notch signaling, had no major effects on spermatogenesis, but deletion of Mgat1 blocked spermatogenesis. Mgat1 conditional mutant (cKO) males exhibit multinuclear cells (MNC) and produce no sperm3. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (GlcNAcT-I), the transferase that transfers GlcNAc from UDP-GlcNAc to Man5GlcNAc2Asn to generate hybrid and complex N-glycans4,5. In the absence of MGAT1, N-glycans of mature glycoproteins are solely oligomanno- syl, and lack all branch antennae that contain GlcNAc, Gal, Fuc, and sialic acid6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 6 June 2017 Accepted: 19 January 2018 Published: xx xx xxxx 1Department of Cell Biology, Albert Einstein College of Medicine, New York, NY, 10461, USA. 2Present address: Biochemistry and Molecular Biology Department, University of Debrecen, Debrecen, Hungary. Correspondence and requests for materials should be addressed to P.S. (email: pamela.stanley@einstein.yu.edu) MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis Global inactivation of the mouse Mgat1 gene leads to embryonic lethality at approximately E9.57,8.h g g y y pp y The architecture of seminiferous tubules in sections from 7 week Mgat1 cKO mice is disrupted3. All tubules contain MNC or symplasts composed of fused spermatids, and lack sperm. A related phenotype is observed with the inactivation of the alpha-mannosidase IIx gene Man2a2, that encodes a mannosidase that acts immediately after MGAT1 in the generation of complex N-glycans. Man2a2 null mice are infertile and also exhibit MNC in testis tubules9. Interestingly, loss of the glycoprotein basigin, a carrier of complex N-glycans in germ cells gener- ated by MGAT13, also gives rise to MNC and infertility10. In this paper, we determine the earliest time when loss of MGAT1 causes a change in germ cell organization. We show that, at a stage when Sertoli cells, spermatogonia and spermatocyte numbers are not affected in 22 and 23 dpp Mgat1 cKO testes, molecular changes have never- theless occurred that lead to the premature expression of spermiogenic genes, and to reduced ERK1/2 signaling. In addition, we show that basigin, a target of MGAT1 in germ cells3, does not stimulate pERK1/2 levels in Lec1 CHO cells expressing only oligomannosyl N-glycans (a model for Mgat1 cKO germ cells). In contrast, basigin with complex N-glycans stimulates ERK1/2 signaling in wild type CHO cells. 1Department of Cell Biology, Albert Einstein College of Medicine, New York, NY, 10461, USA. 2Present address: Biochemistry and Molecular Biology Department, University of Debrecen, Debrecen, Hungary. Correspondence and requests for materials should be addressed to P.S. (email: pamela.stanley@einstein.yu.edu) Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 1 www.nature.com/scientificreports/ Figure 1. Onset of morphological changes in Mgat1[F/F]:Stra8-iCre testes. (A) Representative control Mgat1[F/F] and Mgat1[F/F]:Stra8-iCre testis sections stained with hematoxylin and eosin at 15–28 dpp (n = 3–4 mice per genotype; 20–30 tubules observed per section). At 22 dpp round spermatids were present in control and Mgat1 cKO tubules (arrows). At 25 dpp, few elongated spermatids were seen in Mgat1 cKO tubules and some MNC were observed in a few tubules (asterisks, inset). At 28 dpp, Mgat1 cKO tubules contained MNC comprised of fused spermatids (asterisks, inset and Supplementary Fig. S1). Images were scanned at 40× in a Perkin Elmer Scanner. (B) Sertoli cells were identified by anti-Sox9 Ab, spermatogonia by anti-PCNA Ab; primary spermatocytes by anti-Sycp3, and acrosomes by PAS. MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis Histograms show numbers of stained cells per 20–30 tubules per section, and numbers of mice examined. Tubules at stage VI and beyond based on morphology with PAS+ acrosomes were counted per 50 tubules. Histograms represent mean ± SEM. Images were photographed at 20×. Controls for antibody specificity are shown in Supplementary Fig. S2. Figure 1. Onset of morphological changes in Mgat1[F/F]:Stra8-iCre testes (A) Representative control Figure 1. Onset of morphological changes in Mgat1[F/F]:Stra8-iCre testes. (A) Representative control Mgat1[F/F] and Mgat1[F/F]:Stra8-iCre testis sections stained with hematoxylin and eosin at 15–28 dpp (n = 3–4 mice per genotype; 20–30 tubules observed per section). At 22 dpp round spermatids were present in control and Mgat1 cKO tubules (arrows). At 25 dpp, few elongated spermatids were seen in Mgat1 cKO tubules and some MNC were observed in a few tubules (asterisks, inset). At 28 dpp, Mgat1 cKO tubules contained MNC comprised of fused spermatids (asterisks, inset and Supplementary Fig. S1). Images were scanned at 40× in a Perkin Elmer Scanner. (B) Sertoli cells were identified by anti-Sox9 Ab, spermatogonia by anti-PCNA Ab; primary spermatocytes by anti-Sycp3, and acrosomes by PAS. Histograms show numbers of stained cells per 20–30 tubules per section, and numbers of mice examined. Tubules at stage VI and beyond based on morphology with PAS+ acrosomes were counted per 50 tubules. Histograms represent mean ± SEM. Images were photographed at 20×. Controls for antibody specificity are shown in Supplementary Fig. S2. Results E l Early testicular changes associated with deletion of Mgat1 in spermatogonia. Our previous study characterized Mgat1[F/F]:Stra8-iCre males at 7 weeks3. To identify the earliest stage at which defective sper- matogenesis is observed, testes from control and Mgat1 cKO males from 15 to 28 dpp were compared by histology (Fig. 1A). At 15 dpp, no apparent differences in seminiferous tubule size or the population of germ cells present in 50 tubules were observed (n = 3 mice/group). At 22 and 23 dpp, round spermatids were present in both control and mutant tubules, and there were still no apparent histological differences (Fig. 1A). At 24 and 25 dpp, fusion of cells adjacent to the lumen was observed in a few tubules (Supplementary Table S1; Fig. 1A). Spermatids were identified based on nuclear size, morphology, location in the tubule or detection of acrosomes by periodic Schiff stain (PAS) at 22–25 dpp (Fig. 1A,B), or the acrosomal protein sp56 at 28 dpp (Supplementary Fig. S1). At 28 dpp, mature spermatozoa were present in control but not Mgat1 cKO mutant testis sections (Fig. 1A). The number of tubules with elongated spermatids was significantly reduced in 28 dpp mutant testes, and MNC were present (Supplementary Table S1). Mgat1 cKO and control testis sections were analyzed at 24–26 dpp to detect Sertoli cells (SOX9), spermatogonia (PCNA), spermatocytes (SYCP3), and spermatids (PAS) (Fig. 1B; Supplementary Fig. S2). The number of Sertoli cells, spermatogonia, spermatocytes and Stage VI and beyond tubules were not significantly reduced in Mgat1 cKO versus control tubules (Fig. 1B histograms). Gene expression in Mgat1 cKO testicular germ cells. To gain insights into molecular mechanisms that occur early, before morphological changes are apparent, and thus give rise to the defective spermatogen- esis of Mgat1 cKO males, microarray analyses were performed on RNA from germ cells from 22 and 23 dpp males (Supplementary Table S2). To investigate the relative purity of germ cells, qRT-PCR for cell-type specific genes was performed on cDNA prepared from 22 dpp germ cells and compared to 22 dpp testis. Transcripts of Rhox5 (Sertoli cells) and Cyp11a1 (Leydig cells) were greatly reduced in germ cell compared to testis preparations Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 2 www.nature.com/scientificreports/ Figure 2. Gene expression changes in Mgat1 cKO germ cells. (A) Western blot analysis for basigin before and after endoglycosidase H treatment of germ cell lysates. Results E l Basigin from Mgat1 cKO germ cells was sensitive to Endo H digestion, consistent with a lack of complex N-glycans. (B) Hierarchical clustering of genes expressed in control versus Mgat1 cKO germ cell cDNA preparations from 22 dpp males, and from 3 replicate cDNA preparations of control versus Mgat1 cKO germ cell RNA pools obtained from 6 mice per genotype at 23 dpp. Red indicates high and green indicates low relative expression. (C) qRT-PCR validation of up-regulated genes in microarray data using cDNA from individual preparations used in microarray experiments. Bars represent mean ± SEM (gray, control; black Mgat1 cKO). Data are from two experiments performed in duplicate (n = 3 mice/group). *p < 0.05, **p < 0.01. (D) qRT-PCR of down-regulated genes identified in microarray data performed on the same RNA samples used in B. Data are from two experiments performed in duplicate (n = 3 mice/group). *p < 0.05, **p < 0.01. Both control and Mgat1 cKO transcripts were determined relative to Actb. Figure 2. Gene expression changes in Mgat1 cKO germ cells. (A) Western blot analysis for basigin befo Figure 2. Gene expression changes in Mgat1 cKO germ cells. (A) Western blot analysis for basigin before and after endoglycosidase H treatment of germ cell lysates. Basigin from Mgat1 cKO germ cells was sensitive to Endo H digestion, consistent with a lack of complex N-glycans. (B) Hierarchical clustering of genes expressed in control versus Mgat1 cKO germ cell cDNA preparations from 22 dpp males, and from 3 replicate cDNA preparations of control versus Mgat1 cKO germ cell RNA pools obtained from 6 mice per genotype at 23 dpp. Red indicates high and green indicates low relative expression. (C) qRT-PCR validation of up-regulated genes in microarray data using cDNA from individual preparations used in microarray experiments. Bars represent mean ± SEM (gray, control; black Mgat1 cKO). Data are from two experiments performed in duplicate (n = 3 mice/group). *p < 0.05, **p < 0.01. (D) qRT-PCR of down-regulated genes identified in microarray data performed on the same RNA samples used in B. Data are from two experiments performed in duplicate (n = 3 mice/group). *p < 0.05, **p < 0.01. Both control and Mgat1 cKO transcripts were determined relative to Actb. (Supplementary Fig. S3A). By contrast, the expression of germ cell-specific genes Sycp3 (spermatocytes) and Dbil5 (round spermatids) were similar in testis and germ cell preparations. Results E l Acrv transcripts (late spermatids) were poorly represented in germ cell preparations. Importantly, the expression of Mgat1 was greatly reduced in Mgat1 cKO germ cells at 22 and 23 dpp (Supplementary Fig. S3B). PCR genotyping of germ cell genomic DNA11 showed that Mgat1 cKO germ cells contained only deleted Mgat1 alleles (520 bp), whereas control germ cells contained only floxed Mgat1 alleles (560 bp; Supplementary Fig. S3C). Therefore, Mgat1 transcripts in germ cell preparations were contributed by a small fraction of non-germ cells.hi (Supplementary Fig. S3A). By contrast, the expression of germ cell-specific genes Sycp3 (spermatocytes) and Dbil5 (round spermatids) were similar in testis and germ cell preparations. Acrv transcripts (late spermatids) were poorly represented in germ cell preparations. Importantly, the expression of Mgat1 was greatly reduced in Mgat1 cKO germ cells at 22 and 23 dpp (Supplementary Fig. S3B). PCR genotyping of germ cell genomic DNA11 showed that Mgat1 cKO germ cells contained only deleted Mgat1 alleles (520 bp), whereas control germ cells contained only floxed Mgat1 alleles (560 bp; Supplementary Fig. S3C). Therefore, Mgat1 transcripts in germ cell preparations were contributed by a small fraction of non-germ cells.hi y g The nature of the N-glycans on basigin (~37 kDa), a germ cell glycoprotein target of MGAT13, confirmed efficient deletion of Mgat1 at 22 and 23 dpp. Thus, control germ cell basigin with complex N-glycans was resistant to digestion by endoglycosidase H (Endo H). By contrast, Mgat1 cKO germ cell basigin was sensitive to Endo H digestion, showing that it carried oligomannosyl N-glycans (Fig. 2A and Supplementary Fig. S3D). Incomplete Endo H digestion probably accounts for traces of undigested basigin in some Mgat1 cKO lysates. Importantly, comparison with ~25 kDa non-glycosylated basigin observed at 22 dpp, showed that levels of basigin were similar in control and Mgat1 cKO germ cells (Fig. 2A). Differentially expressed genes (DEGs) in control versus Mgat1 cKO germ cells. Gene expression profiles of 22 and 23 dpp testicular germ cells from control and Mgat1 cKO males were determined using the Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 3 www.nature.com/scientificreports/ GeneChip™ Mouse Gene 2.0 ST Array (34,472 genes). For the 22 dpp experiment, cDNA from three mice per group was analyzed (Supplementary Table S2). Results E l In 22 dpp germ cells (1,086 genes), the top canonical pathway was connected with sperm motility (Fig. 3A). A comparison of canonical pathways affected in 22 versus 23 dpp germ cells shows them to be similar (Fig. 3A, right panel). The significance value is a measure of the likelihood that genes from the input dataset participate in a pathway or function. Sperm motility was ranked third in the 23 dpp dataset. A comparison of biological functions regulated by Mgat1 cKO at 22 versus 23 dpp is shown in Fig. 3B. The top biological functions in both 22 and 23 dpp Mgat1 cKO germ cells were Cellular Function and Maintenance, Reproductive System Development, and Function and Embryonic Development (Fig. 3B). Top upstream transcriptional regulators. IPA upstream functional analysis was used to predict the top upstream transcriptional regulators in DEGs of Mgat1 cKO germ cells at 22 and 23 dpp based on their gene tar- gets. An overlap p value was calculated on the basis of significant overlap between genes in the test dataset and target genes regulated by the same regulator in the IPA knowledge base. The activation Z score algorithm was used to make predictions. IPA predicted the top transcriptional regulator activated in the 22 and 23 dpp datasets to be TAF7L (Z score 4.96; overlap p value 8.89E-31 for 22 dpp; Z score 4.24, overlap p value 6.16E-24 for 23 dpp; Fig. 3C). FIGLA is predicted to be inhibited in both the 22 dpp (Z score −3.22; overlap p value 1.56E-12) and 23 dpp datasets (Z score −3.28, p 1.17E-11) (Fig. 3C). Target genes of TAF7L were up-regulated in our datasets, and predict activation of this regulator. For FIGLA, the target genes from the input datasets were up-regulated, but the regulator is predicted to inhibit transcription. Comparison analysis for upstream regulators at 22 and 23 dpp showed similar activation Z scores for each of the top regulators at both developmental stages (Fig. 3C, right panel). p We tested whether the expression of TAF7L itself was up-regulated by IHC, qRT-PCR and western blot anal- yses (Supplementary Fig. S6). TAF7L is expressed in the nucleus or cytoplasm of germ cells, depending on their stage of differentiation13. We observed TAF7L in the cytoplasm of spermatocytes at 23 dpp. There was no apparent difference in the intensity of the signal between control and Mgat1 cKO sections (Supplementary Fig. S6A). Results E l For the 23 dpp experiment, RNA (RIN > 9) aliquots from six con- trol and six Mgat1 cKO germ cell preparations were separately pooled, and three aliquots from the control and Mgat1 cKO pool, respectively, were converted to cDNA for analysis. Affymetrix Expression Console was used to process the .CEL files of the array. .CHP files were generated using the RMA sketch workflow after signal sum- marization and data normalization. Gene level analysis was further conducted with Affymetrix Transcriptome Analysis Console v2.0 software (TAC). Hierarchical clustering differentiated control and Mgat1 cKO samples (Fig. 2B). Volcano plots representing gene distributions are shown in Supplementary Fig. S4. Genes that were differentially expressed based on ANOVA p < 0.05 and FDR p < 0.05 were identified. There were 1,643 DEGs in 22 dpp Mgat1 cKO versus control germ cells, with an absolute fold-change (linear) of <−2 to >+2.0. Of those, 1,400 genes were up-regulated and 243 down-regulated at 22 dpp. At 23 dpp, 784 genes were differentially regu- lated at <−2 to >+2.0, 771 genes up-regulated, and 13 genes down-regulated in Mgat1 cKO germ cells. Many of the DEGs in Mgat1 cKO germ cells (Supplementary Table S3) are involved in spermatogenesis and later stages of spermiogenesis12. Validation of microarray experiments by qRT-PCR was performed on cDNA prepared from germ cell RNA obtained from the individual mice used in microarray experiments (Fig. 2C,D). PCR primers are given in Supplementary Table S4. Microarray data are deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO serial accession number GSE99035. Enriched Gene Ontology (GO) analysis. DEGs identified by microarray were subjected to GO analysis, which classified 513 DEGs into three GO categories. The most enriched GO terms that correlated with Mgat1 cKO DEGs were sperm egg recognition (GO:0035036, Biological Process, enrichment score (ES) 9.41, Bonferroni count (BC) 8,371), lysozyme activity (GO:0003796, Molecular Function, ES 24.17, BC 3,018), and sperm fibrous sheath (GO:0035686, Cellular Component (CC), ES 21.76, BC 1,288). The number of genes present in the 22 dpp dataset for each GO function are shown in Supplementary Fig. S5, with fold-enrichment of input genes over expected. Enriched biological pathways by Ingenuity Pathway Analysis (IPA). Using IPA, relationships between significant DEGs of absolute fold-change ±1.5 with adjusted FDR and ANOVA p < 0.05 were examined to determine the most significant canonical pathways and biological networks altered in Mgat1 cKO germ cells. Results E l Right panel shows the heat map for comparison of upstream regulators at 22 and 23 dpp based on activation Z score. (D) IPA downstream effect analysis networks associated with diseases and functions. Z scores for both diseases are negative indicating a reduction in disease. Edges and nodes are color-coded based on the predicted relationship as indicated in the legend. Right panel shows the heat map for comparison of disease conditions at 22 and 23 dpp based on activation Z score. Figure 3. IPA of DEGs in control versus Mgat1 cKO germ cells. (A) Canonical pathways significantly overrepresented in Mgat1 cKO germ cells compared to control according to their -log p value. Colors indicate the activation Z score of processes: activated processes are orange, while inhibited processes are blue. No activity pattern is represented by gray, and no change is denoted by white. Right panel shows comparison analysis of canonical pathways from independent 22 dpp versus 23 dpp mutant germ cells based on p value. (B) Comparison of biological functions based on p value in 22 versus 23 dpp shows Cellular Function and Maintenance and Reproductive System Development and Function as the top hits in both 22 and 23 dpp DEGs. (C) IPA upstream regulators of DEGs in control versus Mgat1 cKO 22 dpp germ cells. Symbols of target proteins or upstream regulators in red indicate a predicted increase or activation. The symbol shapes denote the molecular classes of proteins. Right panel shows the heat map for comparison of upstream regulators at 22 and 23 dpp based on activation Z score. (D) IPA downstream effect analysis networks associated with diseases and functions. Z scores for both diseases are negative indicating a reduction in disease. Edges and nodes are color-coded based on the predicted relationship as indicated in the legend. Right panel shows the heat map for comparison of disease conditions at 22 and 23 dpp based on activation Z score. Genes related to fertility were increased relatively more at 22 than 23 dpp, whereas genes related to cell movement of sperm were relatively increased at 23 compared to 22 dpp (Fig. 3D). Interaction networks of DEGs. IPA was used to map biological relationships of the Mgat1 cKO germ cell DEGs into networks based on published literature. At 22 dpp, a 26-gene network with a score of 44 was the most significant. Results E l By qRT-PCR, TAF7L transcripts in 23 dpp Mgat1 cKO germ cells were slightly reduced (Supplementary Fig. S6B). However, TAF7L protein levels were similar by western blot analyses of germ cell lysates (Supplementary Fig. S6C). Nevertheless, microarray data showed up-regulation of numerous TAF7L target genes (Fig. 3C), and qRT-PCR data validated several of these genes including Spert, Spem1, Prm2, Spata3, Tnp1, and Tnp2 (Figs 2C, 4B). Thus, TAF7L activation leading to up-regulation of target genes appears to be due to factors other than increased expression of TAF7L. Correlation with disease genes. DEGs of Mgat1 cKO germ cells were compared by IPA with genes impli- cated in diseases. Disease categories impacted in 22 and 23 dpp Mgat1 cKO germ cells include Teratozoospermia and Asthenozoospermia diseases of men. Up-regulated DEGs from the 22 dpp dataset are shown in Fig. 3D. The blue dotted lines indicate the predicted suppression of disease in Mgat1 cKO germ cells, as up-regulation of spermatogenic and spermiogenic genes is predicted to reduce development of diseases such as Teratozoospermia and Asthenozoospermia. Comparison of heat maps for disease conditions at 22 and 23 dpp shows that sperm dis- order, Asthenozoopermia, Teratozoospermia, and Oligoszoospermia, as defined in men with fertility disorders, were reduced, whereas sperm capacitation and quantity of male germ cells were increased (Fig. 3D, right panel). Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 4 www.nature.com/scientificreports/ i Figure 3. IPA of DEGs in control versus Mgat1 cKO germ cells. (A) Canonical pathways significantly overrepresented in Mgat1 cKO germ cells compared to control according to their -log p value. Colors indicate the activation Z score of processes: activated processes are orange, while inhibited processes are blue. No activity pattern is represented by gray, and no change is denoted by white. Right panel shows comparison analysis of canonical pathways from independent 22 dpp versus 23 dpp mutant germ cells based on p value. (B) Comparison of biological functions based on p value in 22 versus 23 dpp shows Cellular Function and Maintenance and Reproductive System Development and Function as the top hits in both 22 and 23 dpp DEGs. (C) IPA upstream regulators of DEGs in control versus Mgat1 cKO 22 dpp germ cells. Symbols of target proteins or upstream regulators in red indicate a predicted increase or activation. The symbol shapes denote the molecular classes of proteins. Results E l The functions associated with network 1 are cellular development, reproductive system development and function, and cell morphology. The central molecule in this network is ERK1/2, but it is not indicated as activated or inhibited, and neither ERK1 nor ERK2 are amongst the DEGs in our datasets. PKA, several AKAPs and CAM kinase 4 genes were predicted to be up-regulated. Spermiogenesis-specific genes that include transition protein 2 (Tnp2) and protamine 2 (Prm2) were also predicted to be up-regulated (Fig. 4A). Some extracellular matrix genes such as laminin and collagen, indirectly associated with network 1 and not predicted to change, were removed for clarity. The genes in network 1 overlapped with canonical pathways that show a direct or indi- rect relation with ERK1/2 (Fig. 4A). Five of the up-regulated genes (Akap1, Akap3, Akap4, Tnp1, Camk4) and 1 down-regulated gene (Figf) were validated by qRT-PCR (Fig. 4B). Camk4 is a protein kinase that phosphorylates Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 5 www.nature.com/scientificreports/ Figure 4. The top network identified by IPA in 22 dpp Mgat1 cKO germ cells. (A) Molecules are represented as nodes (see legend). Nodes in red or green represent up-regulated or down-regulated genes, respectively. (B) Validation of Network 1 by qRT-PCR performed in duplicate on germ cell cDNA prepared from 3 mice in each group. Gray bars represent control and black bars represent Mgat1 cKO germ cells (mean ± SEM; *p < 0.05, **p < 0.01). Figure 4. The top network identified by IPA in 22 dpp Mgat1 cKO germ cells. (A) Molecules are represented as nodes (see legend). Nodes in red or green represent up-regulated or down-regulated genes, respectively. (B) Validation of Network 1 by qRT-PCR performed in duplicate on germ cell cDNA prepared from 3 mice in each group. Gray bars represent control and black bars represent Mgat1 cKO germ cells (mean ± SEM; *p < 0.05, **p < 0.01). protamines14. Network 1 from the 23 dpp dataset is associated with activation of TAF7L and YBX2 target genes (not shown). This network had a score of 28 with 16 associated molecules, including genes contributing to cel- lular development, cellular growth, proliferation and embryonic development. The central molecule in network 1 is TAF7L, and PAPOLB, FIGLA and YBX2 all contribute to the up-regulation of DEGs in the 23 dpp dataset. Results E l Additionally, a group of genes involved in metabolism are significantly up-regulated upon loss of Mgat1, and likely reflect both metabolic and spermatogenic functions. Gene set enrichment analysis (GSEA). We utilized GSEA to evaluate Mgat1 cKO DEGs at the level of published, classified gene sets in MSigDB. At 22 dpp, 15 of the 3,658 gene sets in the C2 (curated) gene sets were significantly enriched in Mgat1 cKO, and 1975 were significantly enriched in control germ cells (FDR < 0.25). The most significant C2 gene sets were Matzuk spermatid differentiation and Matzuk spermatozoa formation, and the top C5 (GO) gene set was gamete generation (Fig. 5). Genes involved in spermiogenesis, or expressed in the later stages of spermatogenesis (Tnp1, Tnp2, Prm1, Crem, Cadm1, Ybx2), are among the top up-regulated genes (Fig. 5D‒F; stars). GSEA of 23 dpp Mgat1 cKO germ cell DEGs identified 6 of the C2 gene sets were significantly enriched (FDR < 0.25) in Mgat1 cKO germ cells. Top C2 gene sets were Matzuk spermatozoa (NES 2.14) and Matzuk spermatid differentiation (NES 1.99), and top C5 gene set was gamete generation (NES 2.0), similar to 22 dpp germ cells. Thus, germ cells lacking Mgat1 exhibit premature expression of genes that promote spermat- ogenesis and spermiogenesis.i g p g For 22 dpp control germ cells, the most significant C2 gene set relationships (FDR < 0.1) were Iglesias_E2F_ targets_UP (NES −2.44), Nagashima_EGF Signaling_UP (NES −2.31), and for C5 gene sets were RNA process- ing (NES −2.00), RNA splicing (NES −2.00), mRNA processing (NES −1.92), and RNA binding (NES −1.77). At 23 dpp, the top gene sets enriched for control germ cells in the C2 curated set were PID_Integrin1 pathway (NES −2.19), Hernandez_Mitotic arrest_by_Docetaxel_1_UP (NES −2.08) and PID_Integrin3 pathway (NES −2.06). For C5 gene sets, enrichment in 23 dpp control germ cells was seen for Extracellular Matrix (NES −1.95), Regulation of MAP KINASE ACTIVITY (NES −1.94) and Transmembrane Receptor Protein Kinase Activity (NES −1.77). The 22 and 23 dpp control germ cell gene set relationships determined by GSEA, though differing in certain specifics, were both related to cellular signaling. Signaling through ERK1/2 is reduced in Mgat1 cKO germ cells. GSEA analysis showed that one of the most significant oncogenic signatures (C6) is PDGF ERK (FDR < 0.001), which was enriched in control ver- sus Mgat1 cKO germ cells at 22 dpp (Fig. 6A). Results E l The heat map shows core enrichment genes that affect the pathway that were up-regulated in control germ cells, including PDGFRA, which regulates ERK1/2 signaling (Fig. 6B). Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 6 www.nature.com/scientificreports/ Figure 5. Gene set enrichment analysis (GSEA) of microarray data in 22 dpp Mgat1 cKO germ cells. GSEA enrichment plots of three gene clusters that are enriched in Mgat1 cKO germ cells were (A) Gamete Generation, (B) Matzuk Spermatid Differentiation, and (C) Matzuk Spermatozoa Formation. Heat map of positively- enriched genes in Mgat1 cKO germ cells compared to control for (D) gamete generation (E) Matzuk spermatid differentiation and (F) Matzuk spermatozoa formation. *Genes involved in spermiogenesis that are up- regulated prematurely in Mgat1 cKO males at 22 dpp. Figure 5. Gene set enrichment analysis (GSEA) of microarray data in 22 dpp Mgat1 cKO germ cells. GSEA enrichment plots of three gene clusters that are enriched in Mgat1 cKO germ cells were (A) Gamete Generation, (B) Matzuk Spermatid Differentiation, and (C) Matzuk Spermatozoa Formation. Heat map of positively- enriched genes in Mgat1 cKO germ cells compared to control for (D) gamete generation (E) Matzuk spermatid differentiation and (F) Matzuk spermatozoa formation. *Genes involved in spermiogenesis that are up- regulated prematurely in Mgat1 cKO males at 22 dpp. Microarray data and qRT-PCR identified PDGFRA as well as EGFR as down-regulated genes in Mgat1 cKO germ cells at 22 and 23 dpp (Fig. 2D). Consistent with this, western blot analysis showed that at 22 dpp, pERK1/ERK1 and pERK2/ERK2 levels were markedly reduced in Mgat1 cKO germ cells compared to controls. However, pAKT/ AKT levels were not significantly altered by the loss of Mgat1 (Fig. 6C,D; Supplemental Fig. S7). Basigin signaling is reduced in Mgat1-null Lec1 CHO cells. Basigin is a germ cell glycoprotein with complex N-glycans, and the efficient deletion of Mgat1 in spermatogonia generates basigin with oligomannosyl N-glycans3 (Fig. 2A). Deletion of basigin causes the complete loss of Griffonia simplicifolia II (GSA) lectin binding to germ cells10, suggesting that basigin is a major carrier of germ cell complex N-glycans. Thus, the phenotype of Mgat1 cKO males may arise, in part, from functional defects in signaling by basigin carrying oligomannosyl, rather than complex, N-glycans. To investigate this hypothesis, we examined ERK signaling induced by mouse basigin in Chinese hamster ovary (CHO) cells that express complex N-glycans, compared to Lec1 Mgat1-null CHO mutant cells. Results E l The Lec1 CHO mutant5 is a cell-based model for Mgat1 cKO germ cells that lacks MGAT1 activity and expresses only oligomannosyl N-glycans on glycoproteins6. We also investigated the expression of connexin 43 (Cx43), a component of the blood-testis-barrier (BTB). The BTB is altered in basigin-null testes10. Cx43 immunoreactivity was observed along the basal compartment of seminiferous tubules between sper- matogonia and spermatocytes of 28 dpp control and Mgat1 cKO testis sections, in a pattern typical of BTB pro- teins (Fig. 7). Immunoreactivity was scored blindly as high (score 3), medium (score 2), or low (score 1). There was a significant reduction in score 2 and score 3 staining in Mgat1 cKO sections, suggesting that the BTB may be compromised in Mgat1 cKO testis. Basigin signaling is reduced in Mgat1-null Lec1 CHO cells. Basigin is a germ cell glycoprotein with complex N-glycans, and the efficient deletion of Mgat1 in spermatogonia generates basigin with oligomannosyl N-glycans3 (Fig. 2A). Deletion of basigin causes the complete loss of Griffonia simplicifolia II (GSA) lectin binding to germ cells10, suggesting that basigin is a major carrier of germ cell complex N-glycans. Thus, the phenotype of Mgat1 cKO males may arise, in part, from functional defects in signaling by basigin carrying oligomannosyl, rather than complex, N-glycans. To investigate this hypothesis, we examined ERK signaling induced by mouse basigin in Chinese hamster ovary (CHO) cells that express complex N-glycans, compared to Lec1 Mgat1-null CHO mutant cells. The Lec1 CHO mutant5 is a cell-based model for Mgat1 cKO germ cells that lacks MGAT1 activity and expresses only oligomannosyl N-glycans on glycoproteins6. We also investigated the expression of connexin 43 (Cx43), a component of the blood-testis-barrier (BTB). The BTB is altered in basigin-null testes10. C 43 i i i b d l h b l f i if b l b h Cx43 immunoreactivity was observed along the basal compartment of seminiferous tubules between sper- matogonia and spermatocytes of 28 dpp control and Mgat1 cKO testis sections, in a pattern typical of BTB pro- teins (Fig. 7). Immunoreactivity was scored blindly as high (score 3), medium (score 2), or low (score 1). There was a significant reduction in score 2 and score 3 staining in Mgat1 cKO sections, suggesting that the BTB may be compromised in Mgat1 cKO testis. Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 7 www.nature.com/scientificreports/ Figure 6. Signaling pathways in Mgat1 cKO germ cells at 22 dpp. Results E l (A) GSEA analysis showing enrichment of a PDGF_ERK signature in control germ cells. (B) Heat map shows the cluster of DEGs in the PDGF_ERK signaling pathway positively-enriched in control versus Mgat1 cKO germ cells. Arrow identifies PDGFRA as down-regulated in Mgat1 cKO germ cells. (C) Western blot analysis of phosphorylated and unphosphorylated ERK1, ERK2 and AKT in germ cells of Mgat1 cKO compared to control. The gels from which these data were obtained are shown in Supplementary Fig. S7. (D) Histogram of 2–3 independent experiments analyzed in 4–6 gels is shown (mean ± SEM; ***p < 0.005, ****p < 0.001). Figure 6. Signaling pathways in Mgat1 cKO germ cells at 22 dpp. (A) GSEA analysis showing enrichment of a PDGF_ERK signature in control germ cells. (B) Heat map shows the cluster of DEGs in the PDGF_ERK signaling pathway positively-enriched in control versus Mgat1 cKO germ cells. Arrow identifies PDGFRA as down-regulated in Mgat1 cKO germ cells. (C) Western blot analysis of phosphorylated and unphosphorylated ERK1, ERK2 and AKT in germ cells of Mgat1 cKO compared to control. The gels from which these data were obtained are shown in Supplementary Fig. S7. (D) Histogram of 2–3 independent experiments analyzed in 4–6 gels is shown (mean ± SEM; ***p < 0.005, ****p < 0.001). There are numerous ligands for basigin (including basigin itself and the soluble extracellular domain of basi- gin), that induce ERK1/2 phosphorylation15. To determine if the nature of the N-glycans on basigin could affect basigin signaling, CHO and Lec1 cells were co-transfected with a mouse basigin cDNA, and a plasmid encoding neomycin resistance. G418-resistant transfectants were sorted for high expression of basigin on the cell surface (Fig. 8A,B). Sorted populations of CHO and Lec1 cells expressing equivalent amounts of cell surface basigin, were serum-starved for 24 hr, and medium was replaced with serum-free medium with and without potential ligands, including cyclophilin A (CypA 100, 250 or 500 ng/ml), or 10% fetal calf serum, or 10 μg/ml anti-basigin Ab. After incubation for 15 min at 37 °C, cells were washed and lysates prepared for western blot analysis. Surprisingly, the inclusion of CypA, FCS or anti-basigin Ab in serum-free medium did not consistently stimulate signaling in CHO or Lec1 basigin transfectants (Supplementary Fig. S8 and not shown). Results E l By contrast, activation of ERK1/2 was observed in CHO cells expressing basigin (CHO + Bsg) compared to control CHO cells (CHO) after incubation in serum-free medium alone (Fig. 8C,D; supplemental Fig. S9). The “ligand” in this case may be homomeric interactions between basigin on adjacent cells15. Lec1 cells exhibited somewhat higher pERK1/2 levels compared to the CHO cells from which they were derived5. However, in contrast to CHO + Bsg cells, signaling was not fur- ther stimulated by the presence of basigin in Lec1 + Bsg cells (Fig. 8E,D). A significant difference between CHO and Lec1 cells expressing basigin was apparent when the ratios of CHO + Bsg/CHO and Lec1 + Bsg/Lec1 were compared for ERK1/2 (Fig. 8D). Thus, the combined data are consistent with the hypothesis that basigin carrying oligomannosyl N-glycans signals less well than basigin carrying complex N-glycans. Discussion In this paper we investigated mechanisms that lead to defective spermatogenesis in Mgat1 cKO males following Mgat1 deletion in spermatogonia at 3 dpp. Morphological changes began to appear at 24–25 dpp in a small pro- portion of mutant tubules. About 14% of tubules showed spermatid MNC by 28 dpp. Sertoli cell, spermatogonia and spermatocyte numbers per round tubule were unaffected in Mgat1 cKO testes at the same stage. To obtain insights into early events that might be the basis of defective spermatogenesis in Mgat1 cKO males, we interro- gated gene expression changes in morphologically normal Mgat1 cKO germ cells from 22 and 23 dpp males. Surprisingly, we discovered that many genes involved in later stages of spermatogenesis and spermiogenesis were up-regulated in 22 and 23 dpp Mgat1 cKO germ cells. The majority of up-regulated genes (>85%) encode tran- scripts that promote spermatogenesis. Therefore, the loss of Mgat1 in spermatogonia at 3 dpp leads to mutant germ cells at 22 and 23 dpp in which genes normally turned on much later in spermatogenesis, are prematurely up-regulated. 8 Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 www.nature.com/scientificreports/ Figure 7. CX43 in 28 dpp control and Mgat1 cKO testes. (A) IHC of CX43 expressed in 28 dpp control and Mgat1 cKO testis sections were scored blindly as score 1, score 2, or score 3 to reflect poor, medium or high staining, respectively. (B) Scores from 90 CX43-positive tubules were counted per genotype. Mean ± SEM, **p < 0.01. Figure 7. CX43 in 28 dpp control and Mgat1 cKO testes. (A) IHC of CX43 expressed in 28 dpp control and Mgat1 cKO testis sections were scored blindly as score 1, score 2, or score 3 to reflect poor, medium or high staining, respectively. (B) Scores from 90 CX43-positive tubules were counted per genotype. Mean ± SEM, **p < 0.01. To gain insights into the repertoire of genes connected with functions of MGAT1 in germ cells, we applied molecular network analyses using IPA, GO and GSEA. IPA identified sperm motility as the top canonical path- way with a positive activation Z score, and cAMP signaling as the top canonical pathway with a negative activa- tion Z score. Top biological functions in both 22 and 23 dpp Mgat1 cKO germ cells were cellular function and maintenance, and reproductive system. Top upstream regulators were TAF7L and FIGLA. Discussion Taf7l null males have a post-meiotic block in spermiogenesis and are sterile16. This block occurs beyond the stage at which spermato- genesis is disrupted in Mgat1 cKO males. FIGLA functions in oocytes to suppress male germ cell genes involved in spermatogenesis and spermiogenesis17. YBX2/MSY2 is a germ cell specific RNA and DNA binding protein of the cytoplasm, most highly expressed in round spermatids during spermatogenesis. Deletion of Ybx2/Msy2 leads to incomplete nuclear condensation in spermatids, and a block in spermatogenesis18,19. The poly(A) poly- merase PAPOLB/TRAP polyadenylates a subset of transcription factors and other mRNAs in the cytoplasm. It is expressed in round spermatids and is required for spermatogenesis20. Top disease processes Teratozoospermia and Asthenozoospermia were identified as being inhibited. This suggests that the spermatogenesis-specific genes up-regulated in Mgat1 cKO germ cells normally guard against the development of both these fertility diseases in men. Consistent with this interpretation, GSEA analysis revealed gene enrichment in Mgat1 cKO germ cells over- lapping most significantly with gamete generation, Matzuk spermatid differentiation and Matzuk spermatozoa formation. In summary, the complement of genes up-regulated in 22 and 23 dpp Mgat1 cKO germ cells suggest that germ cells that lack Mgat1 are attempting to differentiate prematurely by up-regulating genes involved in spermatogenesis and spermiogenesis. This means that MGAT1 and complex N-glycans on germ cell glycopro- teins function during normal spermatogenesis to control differentiation by retarding the expression of genes early in spermatogenesis that are required for later stages of spermatogenesis and spermiogenesis.h p g q g p g p g The mechanism(s) by which MGAT1 and complex N-glycans on glycoproteins regulate gene expression must necessarily be indirect. It is well established in cell-based experiments that loss or reduced branching of complex N-glycans leads to reduced cell surface residence time of glycoprotein receptors due to the weakening of their interactions with a galectin lattice21–23. On this basis it would be predicted that growth factor signaling should be reduced in Mgat1 cKO germ cells. In fact, our microarray and qRT-PCR data showed that transcripts of both the EGF and PDGF receptors (Egfr and Pdgfra) were markedly reduced in Mgat1 cKO germ cells, and the top IPA network identified ERK1/2 as regulating numerous genes in Mgat1 cKO germ cells. Thus, on several counts, we predicted that growth factor signaling should be reduced in Mgat1 cKO germ cells. Discussion Western blot analysis showed Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 9 www.nature.com/scientificreports/ Figure 8. Basigin signaling is reduced in Lec1 CHO cells, a model for Mgat1 cKO germ cells. Flow cytometry profiles of CHO wild type (A) and Lec1 mutant (B) CHO cells and basigin transfectants. Also shown is a complex N-glycan typical of CHO cells and the oligomannosyl N-glycan substrate of MGAT1 that accumulates on glycoproteins of Lec1 CHO cells. Blue square, GlcNAc; green circle, Man; yellow circle, Gal; red triangle, fucose; purple diamond, sialic acid. (C) CHO wild type, Lec1 and basigin transfectant lysates following 24 hr serum starvation and a 15 min incubation in serum-free medium were analyzed by western blotting using ERK Abs as shown. The gels from which these data were obtained are shown in Supplementary Fig. S9. (D) The pERK1/ERK1 and pERK2/ERK2 values for CHO, Lec1 and respective basigin transfectants (left panel), and the ratio of these values in basigin expressing versus non-basigin expressing CHO and Lec1 cells (right panel). Data are mean ± SEM from 9–13 gels of lysates run in 6 independent experiments. *p < 0.05 in (D) left panel is based on a one-tailed, unpaired Student’s t test with Welch’s correction; in (E, left panel) significance is based on the non-parametric, two-tailed Wilcoxon matched-pairs signed rank test; in (E) right panel it is based on the unpaired, two-tailed Student’s t test with Welch’s correction. **p < 0.01, ***p < 0.005 and ****p < 0.001. Figure 8. Basigin signaling is reduced in Lec1 CHO cells, a model for Mgat1 cKO germ cells. Flow cytometry profiles of CHO wild type (A) and Lec1 mutant (B) CHO cells and basigin transfectants. Also shown is a complex N-glycan typical of CHO cells and the oligomannosyl N-glycan substrate of MGAT1 that accumulates on glycoproteins of Lec1 CHO cells. Blue square, GlcNAc; green circle, Man; yellow circle, Gal; red triangle, fucose; purple diamond, sialic acid. (C) CHO wild type, Lec1 and basigin transfectant lysates following 24 hr serum starvation and a 15 min incubation in serum-free medium were analyzed by western blotting using ERK Abs as shown. The gels from which these data were obtained are shown in Supplementary Fig. S9. (D) The pERK1/ERK1 and pERK2/ERK2 values for CHO, Lec1 and respective basigin transfectants (left panel), and the ratio of these values in basigin expressing versus non-basigin expressing CHO and Lec1 cells (right panel). Discussion Data are mean ± SEM from 9–13 gels of lysates run in 6 independent experiments. *p < 0.05 in (D) left panel is based on a one-tailed, unpaired Student’s t test with Welch’s correction; in (E, left panel) significance is based on the non-parametric, two-tailed Wilcoxon matched-pairs signed rank test; in (E) right panel it is based on the unpaired, two-tailed Student’s t test with Welch’s correction. **p < 0.01, ***p < 0.005 and ****p < 0.001. that pERK1/2 levels were indeed markedly reduced in Mgat1 cKO germ cells. The MAP kinase pathway that leads to ERK1/2 activation is active in spermatogonia and primary spermatocytes and diminished in pachytene spermatocytes24–26. Interestingly, pAKT levels were not reduced, consistent with the fact that the block in sper- matogenesis in Mgat1 cKO males is distinct from that in males lacking AKT which is essential for survival and proliferation of pre- and post-meiotic cells27. When signaling via basigin was investigated in CHO versus Lec1 cells that lack Mgat1, pERK1/2 levels were enhanced by the presence of basigin in CHO but not Lec1 cells. Basigin is a substrate of MGAT13, and is a major carrier of complex N-glycans of germ cell glycoproteins10. Basigin has potential ligands in testis that might lead to activation of ERK1/2 in germ cells15 including basigin itself and soluble basigin extracellular domain28. Importantly, the spermatogenesis defect in basigin-null males is similar to that in Mgat1 cKO males10. The combined data suggest that defective signaling via ERK1/2 due to the loss of complex N-glycans on basigin leads, along with other factors that reduce ERK1/2 signaling in Mgat1 cKO germ cells, to the block in spermatogenesis in germ cells lacking Mgat1. Testing this hypothesis forms the basis of future experiments in males conditionally lacking basigin in germ cells, and related mouse models. that pERK1/2 levels were indeed markedly reduced in Mgat1 cKO germ cells. The MAP kinase pathway that leads to ERK1/2 activation is active in spermatogonia and primary spermatocytes and diminished in pachytene spermatocytes24–26. Interestingly, pAKT levels were not reduced, consistent with the fact that the block in sper- matogenesis in Mgat1 cKO males is distinct from that in males lacking AKT which is essential for survival and proliferation of pre- and post-meiotic cells27. Methods Animals. Mgat1[F/F]:Stra8-iCre (mutant) and Mgat1[F/F] (control) mice were generated as described3. Mice were genotyped by PCR of tail genomic DNA or germ cell DNA as described3,11. All mice were bred within the Institute for Animal Studies. The Albert Einstein Animal Institute Committee approved experimental protocols. All methods were performed in accordance with the relevant guidelines and regulations. Mice were sacrificed by carbon dioxide asphyxiation and cervical dislocation. Testes were dissected free of surrounding tissue and weighed. Antibodies. Antibodies were rabbit SOX9 pAb (#sc20095, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); rabbit SYCP3 pAb (#NB300–230, Novus Biologicals, Littleton, CO, USA); mouse PCNA mAb PC10 (#P8825, Sigma Aldrich, St. Louis, Mo, USA); rat anti-mouse basigin mAb OX114 (#B3663, LSBio, Inc., Seattle, WA, USA,); mouse connexin 43 mAb CX-1B1 (#13–8300, Invitrogen Corp, Camarillo, CA, USA); rabbit ERK1/2 mAb C33E10, (#3192), mouse pERK1/2 mAb L34F12 (#4696), mouse AKT mAb 40D4 (#2920) and rab- bit pAKT mAb D9E (#4060) were from Cell Signaling Technology, Danvers, MA, USA; horse radish peroxi- dase(HRP)-conjugated goat anti-rabbit IgG (#65–6120, Invitrogen Corp) and goat anti-rat IgG (#112–005–003, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA); LI-COR fluorescent secondary Abs (#P/N 925–32211, goat anti-rabbit IgG IRDye 800CW; #P/N 925–6802, goat anti-mouse IgG IRDye 680LT; LI-COR Biotechnology, Lincoln, NE, USA); and biotinylated goat anti-mouse IgG (#BA-9200). Histology and Immunohistochemistry. Testes from 15–28 dpp males were fixed in Bouin’s fixative (#100503–962, Electron Microscopic Sciences, Radnor, PA, USA) for 48 hr at room temperature (RT) and processed by the Einstein Histotechnology and Comparative Pathology Facility. Testes were paraffin-embedded. Serial sections (5 μm) were collected on positively-charged slides and stained with H&E or the PAS reagent. Unstained sections were used for IHC. Slides were microwaved in 10 mM citrate buffer (pH 6.0), incubated in 3% hydrogen peroxide in methanol (15 min, RT) and blocked in 5% goat serum (#G9023, Sigma-Aldrich) in phosphate-buffered saline (pH 7.2) containing 1 mM CaCl2, 1 mM MgCl2 and 0.05% Tween 20 (PBST) for 60 min at RT. Sections were incubated overnight at 4 °C in blocking buffer containing anti-CX43 (1:200) or 1:500 anti-SOX9, anti-SYCP3 or anti-PCNA Abs in Tris-buffered saline, pH 7.2 (TBS). Secondary antibody alone and peptide inhibition were the negative controls. Methods After washing with PBST (no cations), sections were incubated with biotinylated secondary Ab (60 min, RT; 1:500 in PBST (CX43); 1:1000 in TBS (SOX9, SYCP3 and PCNA), detected using ABC Vectastain (#PK-6100, Vector laboratories, Burlingame, CA, USA) and 3,3′diaminobenzidine (#SK-4100, Vector Laboratories) followed by counterstaining with haematoxylin (#MHS-16, Sigma-Aldrich). Slides were observed by light microscopy (Leica Microsystem, Wetzlar, Germany) and scanned using a Perkin Elmer P250 high capacity slide scanner (3D Histech P250 high capacity slide scanner, Perkin Elmer, Waltham, MA, USA). Germ cell isolation and extraction. Testes were collected in DMEM/F12 (Gibco Life Technologies Corp., Grand Island, NY, USA) on ice, detunicated, and germ cells were isolated as described29. Briefly, testes were minced in 10 ml solution 1 (DMEM/F12 containing 500 μg/ml collagenase 1 A (#CO130–1G) and 200 μg/ml DNase I (#DN25–100MG) for 10 min in a shaking water bath, tubules were allowed to sediment in Hank’s buff- ered saline (HBSS, #55–022–PB, Corning, Manassas, VA, USA) containing 5% Percoll (#17–0891–01, GE Health Care Bio-Sciences, Uppsala, Sweden) in water for 20 min, followed by incubation in 10 ml solution 2 (DMEM/F12 containing 1.25 mg/ml trypsin (#T4799–5G) and 200 μg/ml DNase I), filtered through 40 micron nylon mesh, and washed in PBS. All enzymes were from Sigma-Aldrich. DNA and protein were extracted from germ cells of one testis and RNA from the other. DNA was isolated by the DNeasy Blood & Tissue Kit (#69504, Qiagen, Germantown, MD, USA), protein by homogenization in 200 μl 1.5% Triton X-100 with protease inhibitor (#05 892 791 001; Thermo Fisher Scientific, Springfield Township, NJ, USA) by pellet pestle (#749540–000, Kontes Glass Co, Vineland, NJ, USA) and RNA by the All-Prep RNeasy mini kit (#74104) and RNeasy mini-elute cleanup kit (#74204) from Qiagen. After centrifugation (3000 rpm, 10 min, 4 °C), protein was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). RIN was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and measured using a Nanodrop ND1000 Spectrophotometer. RNA and protein were extracted from 23 dpp germ cells using Trizol Reagent (Sigma Aldrich). Endoglycosidase H digestion. Lysate containing 20–60 μg testis protein was treated with 5 mU Endo H from S. plicatus (#11088726001, Roche Diagnostics, Manheim, Germany) or water in 20 μl manufacturer’s buffer at 37 °C for 2 hr. Reactions were stopped by adding SDS gel loading buffer and heating at 95 °C, 10 min. Microarray analysis. Discussion When signaling via basigin was investigated in CHO versus Lec1 cells that lack Mgat1, pERK1/2 levels were enhanced by the presence of basigin in CHO but not Lec1 cells. Basigin is a substrate of MGAT13, and is a major carrier of complex N-glycans of germ cell glycoproteins10. Basigin has potential ligands in testis that might lead to activation of ERK1/2 in germ cells15 including basigin itself and soluble basigin extracellular domain28. Importantly, the spermatogenesis defect in basigin-null males is similar to that in Mgat1 cKO males10. The combined data suggest that defective signaling via ERK1/2 due to the loss of complex N-glycans on basigin leads, along with other factors that reduce ERK1/2 signaling in Mgat1 cKO germ cells, to the block in spermatogenesis in germ cells lacking Mgat1. Testing this hypothesis forms the basis of future experiments in males conditionally lacking basigin in germ cells, and related mouse models. Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 10 www.nature.com/scientificreports/ Methods Testis RNA (150 ng, RIN ≥ 9) was provided to the Genomics Core Facility of the Albert Einstein College of Medicine for conversion to cDNA, labeling and hybridization to Affymetrix GeneChip™ Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). After gene level normalization, signal summariza- tion and background subtraction, raw intensity data (.CEL files) were transformed to .CHP files using Affymetrix Expression Console software. Genes up- or down-regulated with a fold-change >2.0 or <−2.0 were determined using the Affymetrix Transcriptome Analysis Console. Differences between medians (log2) were determined and transformed to linear fold-change. Statistical significance was assessed using ANOVA and FDR p values. Quantitative PCR. RNA (500 ng) was reverse-transcribed into cDNA using the verso cDNA synthesis kit (#AB1453/B, Thermo Fisher) with an oligo-dT primer according to the manufacturer’s protocol. Real time PCR was performed using Absolute Blue QPCR mix (#AB4162, Thermo Fisher) on a master cycler (ViiA 7, Thermo Fisher). PCR conditions were 95 °C 30 sec, followed by 40 cycles at 95 °C 15 sec, 60 °C 15 sec and 72 °C 20 sec. Gene expression relative to actin (Actb) was calculated as log2 dCT, Mgat1 cKO values were subtracted from control (log2 ddCT) and converted to fold-change. Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 11 www.nature.com/scientificreports/ Gene Ontology. Gene ontology (PANTHER version 11.1)30,31 analysis was used to investigate functions for genes with fold-change ±1.5 and p < 0.05. The analysis classifies functions according to biological process, cellular component and molecular function. Gene Ontology. Gene ontology (PANTHER version 11.1)30,31 analysis was used to investigate functions for genes with fold-change ±1.5 and p < 0.05. The analysis classifies functions according to biological process, cellular component and molecular function. Ingenuity Pathway Analysis. IPA of DEGs was performed using QIAGEN’s Ingenuity Pathway Analysis algorithm (www.qiagen.com/ingenuity, QIAGEN, Redwood City, CA, USA) for genes with fold-change ±1.5 and overlap p < 0.05. Ingenuity Pathway Analysis. IPA of DEGs was performed using QIAGEN’s Ingenuity Pathway Analysis algorithm (www.qiagen.com/ingenuity, QIAGEN, Redwood City, CA, USA) for genes with fold-change ±1.5 and overlap p < 0.05. Gene set enrichment analysis. GSEA was performed to determine DEGs that were enriched in gene lists extracted from MSigDB32,33 to determine enrichment in gene sets from the curated (C2), GO (C5), and oncogenic signatures (C6) collections. Western blot analysis. Methods Protein was extracted from germ cells in 200 μl homogenization buffer (1.5% Triton X-100, protease Inhibitor and PhosSTOP (#04906845001, Roche Diagnostics), centrifuged (3000 rpm, 10 min, 4 °C), and supernatant protein determined. For detection of ERK and AKT, protein (60 μg) was separated on 10% SDS polyacrylamide, transferred to nitrocellulose membrane (#162–0095, Bio-Rad) in 10% methanol, blocked in LI-COR Odyssey blocking buffer (#P/N 927–40100; OBB) 1 hr at RT, and incubated overnight at 4 °C with antibodies in OBB. Membranes washed 3× in PBST and then PBS (both no cations) were probed with LI-COR fluorescent Abs in OBB for 1  hr at RT in the dark. After washing, imaging was performed (LI-COR Odyssey scanner) and band intensity was calculated (LI-COR Odyssey software). For detecting basigin, 40–60 μg protein was separated by SDS-PAGE (10%). After transfer to PVDF membrane (10% methanol) and blocking in 3% fish gelatin (#G7041, Sigma-Aldrich) in TBST, membranes were incubated in anti-basigin mAb (1:1000 in 3% fish gelatin) overnight at 4 °C. The membrane was probed with secondary Ab 1 hr at RT, treated with Super Signal West Pico chemiluminiscent substrate (#34080, Thermo Fisher) and exposed to film. ERK activation by basigin in CHO and Lec1 Cells. CHO wild type (Pro-5) and Mgat1 null Lec1 CHO cells (Pro-5Lec1.3 C)5 were co-transfected with 3 μg mouse basigin plasmid cDNA (MC200670, Origene, Rockville, MD, USA) and 0.3 μg pSV2NEO in 6 well plates with Xtreme Gene HP DNA transfection reagent (#06 366 244 001, Sigma-Aldrich) at 1:3, DNA:reagent. Next day the cells were trypsinized and re-plated in a 10 cm plate in Gibco Alpha + MEM medium (#11900–073, Thermo Fisher) containing 10% fetal calf serum (FCS), pen- icillin, streptomycin and 1.5 mg/ml active G418 (#400–111 P, Gemini, Sunnyvale, CA, USA). Transfectant popu- lations were analyzed by flow cytometry using anti-basigin mAb and anti-rat IgG conjugated to Alexa-Fluor-488. The top 3% of binders were sorted and used in experiments.l h p p To assay ERK activation, cells at ~80% confluency were serum-starved for 24 hr in alpha + MEM medium. To stimulate signaling, medium was replaced with alpha + MEM, or alpha + MEM containing CypA (#3589-CA, R&D Systems), or 10% FCS or 10 μg/ml basigin mAb, for 15 min at 37 °C. After washing, cells were lysed in 100 μl RIPA (#20–188, EMD Millipore, Billerica, MA, USA), 0.1% SDS, protease inhibitor and PhosSTOP (Sigma Aldrich). Methods After centrifugation (3,000 rpm, 5 min, 4 °C), 40 μg protein was electrophoresed on 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose using the Pierce G2 blotter, washed in distilled water, and dried at 4 °C. Membranes were blocked with OBB in TBS (1 hr, RT), incubated in primary mAbs in OBB 0.05% Tween at 4 °C overnight followed, after washing, by LI-COR secondary antibodies for 1 hr at RT in the dark. Blots were washed 4× with TBST, twice with TBS, dried and analyzed as described above. Statistical Analysis. Unless otherwise noted, results are presented as mean ± SEM for independent meas- urements or individual mice. A one-way ANOVA or unpaired, two-tailed Student’s t test with Welch’s correction was used to determine p values in Graph Pad Prism 7.0a (Graph Pad Software Inc., La Jolla, CA, USA), unless otherwise noted. Limited amounts of RNA from 22 and 23 dpp germ cells precluded testing sufficient replicates to establish a Gaussian distribution for qRT-PCR data. However, a Gaussian distribution was established by the D’Agostino-Pearson test in Prism for most data sets in Fig. 7D,E. For other samples a Gaussian distribution was assumed and parametric tests of significance were performed, unless otherwise noted. References 1. O’Bryan, M. K. & de Kretser, D. Mouse models for genes involved in impaired spermatogenesis. Int J Androl 29, 76–89; discussion 105–108, https://doi.org/10.1111/j.1365-2605.2005.00614.x (2006). 1. O’Bryan, M. K. & de Kretser, D. Mouse models for genes involved in impaired spermatogenesis. Int J Androl 29, 76–89; discussion 105–108, https://doi.org/10.1111/j.1365-2605.2005.00614.x (2006). 1. O’Bryan, M. K. & de Kretser, D. Mouse models for genes involved in impaired spermatogenesis. Int J Androl 29, 76–89; discussion 105–108, https://doi.org/10.1111/j.1365-2605.2005.00614.x (2006). p g j 2. Matzuk, M. M. & Burns, K. H. Genetics of mammalian reproduction: modeling the end of the germline. Annu Rev Physiol 74, 503–528, https://doi.org/10.1146/annurev-physiol-020911-153248 (2012). 2. Matzuk, M. M. & Burns, K. H. Genetics of mammalian reproduction: modeling the end of the germline. Annu Rev Physiol 74, 503–528, https://doi.org/10.1146/annurev-physiol-020911-153248 (2012). 3. Batista, F., Lu, L., Williams, S. A. & Stanley, P. Complex N-Glycans Are Essential, but Core 1 and 2 Mucin O-Glycans, O-Fucose Glycans, and NOTCH1 Are Dispensable, for Mammalian Spermatogenesis. Biol Reprod 86(179), 171–112, https://doi.org/10.1095/ biolreprod.111.098103 (2012). 3. Batista, F., Lu, L., Williams, S. A. & Stanley, P. Complex N-Glycans Are Essential, but Core 1 and 2 Mucin O-Glycans, O-Fucose Glycans, and NOTCH1 Are Dispensable, for Mammalian Spermatogenesis. Biol Reprod 86(179), 171–112, https://doi.org/10.1095/ biolreprod.111.098103 (2012). 4. Stanley, P. Taniguchi, N. & Aebi, M. In: Varki, A. et al. editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; Chapter 9, (2017). p g y p 5. Chen, W. & Stanley, P. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology 13, 43–50 (2003).i 5. Chen, W. & Stanley, P. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology 13, 43–50 (2003).i y g y y gy ( ) 6. North, S. J. et al. Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J Biol Chem 285, 5759–5775, https://doi.org/10.1074/jbc.M109.068353 (2010).f y g y y gy 6. North, S. J. et al. Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J Biol Chem 285, 5759–5775, https://doi.org/10.1074/jbc.M109.068353 (2010).f p y p g j 7. Ioffe, E. & Stanley, P. Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc. Natl. Acad. Sci. USA 91, 728–732 (1994). www.nature.com/scientificreports/ Basigin-2 is a cell surface receptor for soluble basigin ligand. J Biol Chem 283, 17805–17814, https://doi.org/10.1074/jbc.M801876200 (2008). p g j 9. Chang, Y. F., Lee-Chang, J. S., Panneerdoss, S., MacLean, J. A. 2nd & Rao, M. K. Isolation of Sertoli, Leydig, and spermatogenic cell from the mouse testis. Biotechniques 51, 341–342, 344, https://doi.org/10.2144/000113764 (2011). p g j 29. Chang, Y. F., Lee-Chang, J. S., Panneerdoss, S., MacLean, J. A. 2nd & Rao, M. K. Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis. Biotechniques 51, 341–342, 344, https://doi.org/10.2144/000113764 (2011). q p g 30. Mi, H. et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res 45, D183–D189, https://doi.org/10.1093/nar/gkw1138 (2017).hi q p g 0. Mi, H. et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis too enhancements. Nucleic Acids Res 45, D183–D189, https://doi.org/10.1093/nar/gkw1138 (2017).hi 1. Mi, H., Muruganujan, A., Casagrande, J. T. & Thomas, P. D. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc 8, 1551–1566, https://doi.org/10.1038/nprot.2013.092 (2013).i y p g p ( ) 2. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles Proc Natl Acad Sci USA 102, 15545–15550, https://doi.org/10.1073/pnas.0506580102 (2005). y p g p 32. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102, 15545–15550, https://doi.org/10.1073/pnas.0506580102 (2005). 33. Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267–273, https://doi.org/10.1038/ng1180 (2003). 3. Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267–273, https://doi.org/10.1038/ng1180 (2003). www.nature.com/scientificreports/ Biol Reprod 76, 48–54, https://doi.org/10.1095/biolreprod.106.055095 ( h b S T S Ok d k b T Ad l b fi l l (A) l g J g p essenger RNA instability leads to spermatogenic arrest. Biol Reprod 76, 48–54, https://doi.org/10.1095/biolreprod.106.055095 (2007). hi b S I T t S Ok d K Y k Y & B b T Ad l ti b t ti ifi t l i l (A) l g y p g p , , p g p ( ) 20. Kashiwabara, S. I., Tsuruta, S., Okada, K., Yamaoka, Y. & Baba, T. Adenylation by testis-specific cytoplasmic poly(A) polymerase, 20. Kashiwabara, S. I., Tsuruta, S., Okada, K., Yamaoka, Y. & Baba, T. Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP is essential for spermatogenesis J Reprod Dev 62 607–614 https://doi org/10 1262/jrd 2016-116 (2016) 20. Kashiwabara, S. I., Tsuruta, S., Okada, K., Yamaoka, Y. & Baba, T. Adenylation by testis specific cytoplasmic poly(A) poly PAPOLB/TPAP, is essential for spermatogenesis. J Reprod Dev 62, 607–614, https://doi.org/10.1262/jrd.2016-116 (2016).f 1. Lau, K. S. et al. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cel 129, 123–134, https://doi.org/10.1016/j.cell.2007.01.049 (2007). p g j 2. Patnaik, S. K. et al. Complex N-glycans are the major ligands for galectin-1, −3, and −8 on Chinese hamster ovary cells. Glycobiology 16, 305–317 (2006).h 23. Song, Y., Aglipay, J. A., Bernstein, J. D., Goswami, S. & Stanley, P. The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression. Cancer Res 70, 3361–3371, https://doi.org/10.1158/0008-5472.CAN-09-2719 (2010). 24. Wong, C. H. & Cheng, C. Y. Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: a review of recent data. Dev Biol 286, 1–15, https://doi.org/10.1016/j.ydbio.2005.08.001 (2005). p g j y 5. Almog, T. & Naor, Z. Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions. Mo Cell Endocrinol 282, 39–44, https://doi.org/10.1016/j.mce.2007.11.011 (2008). p g j 6. Li, M. W., Mruk, D. D. & Cheng, C. Y. Mitogen-activated protein kinases in male reproductive function. Trends Mol Med 15 159–168, https://doi.org/10.1016/j.molmed.2009.02.002 (2009). g j 26. Li, M. W., Mruk, D. D. & Cheng, C. Y. Mitogen-activated pr p g j 7. Ciraolo, E. et al. Essential role of the p110beta subunit of phosphoinositide 3-OH kinase in male fertility. Mol Biol Cell 21, 704–711 https://doi.org/10.1091/mbc.E09-08-0744 (2010). p g 8. Belton, R. J. Jr., Chen, L., Mesquita, F. S. & Nowak, R. A. Acknowledgementsh The authors are grateful to Dr. Boris Bartholdy (Albert Einstein College of Medicine) for help with bioinformatics and for reviewing the early manuscript, and to Dr. Laura Barreyro for help with microarray analysis. This work was supported by NIGMS grant RO1 105399 (P.S.), Lalor Foundation postdoctoral fellowship 342111 (B.B.), equipment grant SIG #1S10OD019961–01 from the NIH, and the Albert Einstein Cancer Center grant NCI PO1 13330 for support with core facilities. Author Contributions B.B. and F.B. performed the experiments. B.B. and P.S. designed experiments and analyzed the data. S.S. did the cell-based assays. B.B. and P.S. wrote the manuscript. All authors read and approved the manuscript. References p y y 8. Metzler, M. et al. Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development. EMBO Journal 13, 2056–2065 (1994). p 9. Akama, T. O. et al. Germ cell survival through carbohydrate-mediated interaction with Sertoli cells. Science 295, 124–127 (2002). 10. Bi, J. et al. Basigin null mutant male mice are sterile and exhibit impaired interactions between germ cells and Sertoli cells. Dev Biol 380, 145–156, https://doi.org/10.1016/j.ydbio.2013.05.023 (2013). Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 12 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1. Shi, S. et al. Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycan develop and implant. Mol Cell Biol 24, 9920–9929 (2004). 11. Shi, S. et al. Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycans develop and implant. Mol Cell Biol 24, 9920–9929 (2004). 12 Jan S Z et al Molecular control of rodent spermatogenesis Biochim Biophys Acta 1822 1838–1850 https://doi org/10 1016/j p p 2. Jan, S. Z. et al. Molecular control of rodent spermatogenesis. Biochim Biophys Acta 1822, 1838–1850, https://doi.org/10.1016/j bbadis.2012.02.008 (2012). ( ) 13. Pointud, J. C. et al. The intracellular localisation of TAF7L, a paralogue of transcription factor TFIID subunit TAF7, is developmentally regulated during male germ-cell differentiation. J Cell Sci 116, 1847–1858 (2003). l d h f b l d l ll l k k f 4. Wu, J. Y. et al. Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4. Nat Genet 25 448–452, https://doi.org/10.1038/78153 (2000). p g 15. Muramatsu, T. Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners. J Biochem 159, 481–490, https://doi.org/10.1093/jb/mvv127. p g j 6. Zhou, H. et al. Taf7l cooperates with Trf2 to regulate spermiogenesis. Proc Natl Acad Sci USA 110, 16886–16891, https://doi org/10.1073/pnas.1317034110 (2013). g p ( ) 7. Joshi, S., Davies, H., Sims, L. P., Levy, S. E. & Dean, J. Ovarian gene expression in the absence of FIGLA, an oocyte-specific transcription factor. BMC Dev Biol 7, 67, https://doi.org/10.1186/1471-213X-7-67 (2007).i p p g 18. Gu, W. et al. Mammalian male and female germ cells express a germ cell-specific Y-Box protein, MSY2. Biol Reprod 59, 1266–1274 (1998). 19 Yang J Morales C R Medvedev S Schultz R M & Hecht N B In the absence of the mouse DNA/RNA binding protein MSY2 18. Gu, W. et al. Mammalian male and female germ cells express a germ cell-specific Y-Box protein, MSY2. Biol Reprod 59, 1266–1274 (1998). g p g pi p p 19. Yang, J., Morales, C. R., Medvedev, S., Schultz, R. M. & Hecht, N. B. In the absence of the mouse DNA/RNA-binding protein M messenger RNA instability leads to spermatogenic arrest. Additional Information upplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-20465-3. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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 Cre- ative 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 per- mitted 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/. © The Author(s) 2018 Scientific Reports | (2018) 8:2022 | DOI:10.1038/s41598-018-20465-3 13
https://openalex.org/W2897678266
http://irep.ntu.ac.uk/id/eprint/35822/1/13358_Sumner.pdf
English
null
Revisiting Models of Concurrent Vowel Identification: The Critical Case of No Pitch Differences
Acta acustica united with Acustica
2,018
cc-by
3,112
1. Introduction the overall improvement with F0 differences, it very poorly accounted for the specific confusions made. Humans demonstrate a significant ability to identify and concentrate on specific speakers within a complex auditory environment. Whilst this clearly relies on a multitude of cues, listeners can still identify both of a pair of steady- state vowels, presented simultaneously [1]. The concurrent vowel identification (CVI) task probes the effect that cues, such as pitch differences, have on this recognition [2]. Even when the F0s of all vowels presented are identical, human CVI performance is greatly above chance [3]. This implies that identification cues beyond pitch differences are utilized that are not well accounted for in existing mod- els. In this identical-F0 scenario, all existing models con- struct predictions of just individual vowels being identified by comparing unseparated representations of concurrent vowel pairs with internal templates of individual vowels. Furthermore, to construct predictions of concurrent vowel pairs being identified, either deterministic algorithms are used (e.g. [4, 5, 7, 8]), or probabilistic decisions are made following assumptions of independence (e.g. [3, 6]). Many models predicting human performance for CVI have been created [3, 4, 5, 6, 7]. The most widely accepted models generate segregated representations of each vowel by segregating information in different frequency regions according to fundamental frequencies (F0s) inferred from the model. The segregated representations are then com- pared to stored templates of individual vowels, to predict the concurrent vowel pair presented. Here we explore the consequences of an alternative recognition process, for the important case where there is no F0 difference between vowel pairs. We hypothesize that predicting the complete internal representation of the presented stimulus would be an optimal solution to the CVI task, and might produce results in line with human behaviour. Therefore, internal representations should de- scribe concurrent vowel pairs (i.e. retaining dependent in- formation), as opposed to individual vowels. Our model simulates different variants of auditory processing, fol- lowed by a naive Bayesian classifier which allows for probabilistic predictions of human decisions and system- atic comparison of different recognition strategies. Meddis and Hewitt’s model [5] is widely cited as it is able to qualitatively predict human improvement in vowel identification when pitch differences are introduced be- tween the vowel-pair. However, when no F0 differences are present, it under-predicts the correct identifications made by humans in their study (human: 57%, model: 37%). © 2018 The Author(s). Published by S. Hirzel Verlag · EAA. This is an open access article under the terms of the CC BY 4.0 license. ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 104 (2018) 922 – 925 ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 104 (2018) 922 – 925 DOI 10.3813/AAA.919244 Summary When presented with two vowels simultaneously, humans are often able to identify the constituent vowels. Com- putational models exist that simulate this ability, however they predict listener confusions poorly, particularly in the case where the two vowels have the same fundamental frequency. Presented here is a model that is uniquely able to predict the combined representation of concurrent vowels. The given model is able to predict listener’s systematic perceptual decisions to a high degree of accuracy. © 2018 The Author(s). Published by S. Hirzel Verlag · EAA. This is an open access article under the terms of the Creative Commons Attribution (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). © 2018 The Author(s). Published by S. Hirzel Verlag · EAA. This is an open access article under the terms of the Creative Commons Attribution (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). PACS no. 4371.An, 43.66Ba, 43.71.Es, 43.71.-k, 43.72.-p, 43.72.Qr, 43.66.-x PACS no. 4371.An, 43.66Ba, 43.71.Es, 43.71.-k, 43.72.-p, 43.72.Qr, 43.66.-x Revisiting Models of Concurrent Vowel Identification: The Critical Case of No Pitch Differences Samuel S. Smith1), Ananthakrishna Chintanpalli2), Michael G. Heinz3), Christian J. Sumner1) 1)Medical Research Council Institute of Hearing Research, University of Nottingham, NG7 2RD, UK. samuel.smith@nottingham.ac.uk Samuel S. Smith1), Ananthakrishna Chintanpalli2), Michael G. Heinz3), Christian J. Sumner1) 1)Medical Research Council Institute of Hearing Research, University of Nottingham, NG7 2RD, UK. samuel.smith@nottingham.ac.uk Samuel S. Smith1), Ananthakrishna Chintanpalli2), Michael G. Heinz3), Christian J. Sumner1) 1)Medical Research Council Institute of Hearing Research, University of Nottingham, NG7 2RD, UK. samuel.smith@nottingham.ac.uk 2) Department of Electrical and Electronics Engineering, Birla Institute of Technology & Science, Pilani-333 031, Rajasthan, India 2) Department of Electrical and Electronics Engineering, Birla Institute of Technology & Science, Pilani-333 031, Rajasthan, India 3) Department of Speech, Language and Hearing Sciences, Purdue University, West Lafayette, Indiana 47907-2028, USA 3) Department of Speech, Language and Hearing Sciences, Purdue University, West Lafayette, Indiana 47907-2028, USA Received 28 February 2018, accepted 14 August 2018. 2.1. Stimuli Synthetic vowels (steady-state harmonic complexes) were created using a Klatt-synthesizer [9]. The formant frequen- cies and bandwidths matched those specified by Chintan- palli and Heinz [8]. The fundamental frequency of all vow- els were 100 Hz, and all vowels were set to 65 dB SPL. All vowels had a duration of 400 ms (including 10ms on- set/offset raised cosine ramps). With a total of 5 individual synthetic vowels (/i/, /a/, /u/, /æ/, /Ç/) there are a total of 15 unique pairwise combina- tions. The waveforms were added to one another to create concurrent vowel pairs. Figure 1. A diagram describing our model of CVI. 3.2. Classification The CVI task and data are detailed in Chintanpalli and Heinz [8]. Five subjects were randomly presented one of the 15 concurrent vowel pairs and were required to iden- tify two vowels from the set of five (different or identical). Each subject responded to 300 trials of concurrent vowels with identical F0s. Participants had considerable training with individual and concurrent vowel stimuli. The task of the listeners, and our classifier, was to deter- mine what stimulus had been presented for all instances of auditory activity (a). We did this using a naive Bayesian classifier, which determined regions of auditory activity (Rk) where a given stimulus class (Ck) was more probable than any other stimulus class to have produced said audi- tory activity (i.e. a ∈Rk if k = arg maxi P (Ci|a)). Given the presentation of a concurrent vowel pair, the probability that our model predicted a certain stimulus class had been presented was 3. Computational Model Our computational model generated ideal-observer based predictions of human decisions. For each concurrent vowel pair a probabilistic distribution of auditory activity was generated from a simulation of the auditory system. This was compared to distributions associated with all se- lectable concurrent vowel pairs, or individual vowels as in previous models. P (Ck|/vi, vj/) =  a∈Rk Pa /vi, vj/ da. (1) (1) These high dimensional integrals were then evaluated nu- merically. We modelled two approaches for classification which differed in the stimulus classes used, each producing a confusion matrix (P (/vx, vy/|/vi, vj/) where vx, vy ∈ {i, a, u, æ, Ç}): 2. Concurrent Vowel Identification Present concurrent vowel waveform /𝑣𝑖,𝑣 𝑗/ Auditory system (spectral/temporal) Add noise 𝝁 𝑖𝑗 Classify auditory activity: Eq. 1 𝑅 𝑘 𝑃𝒂/𝑣𝑖,𝑣 𝑗/ Determine regions of auditory activity most likely to have originated from each class. Figure 1. A diagram describing our model of CVI. 1. Introduction Recently, Chintanpalli and Heinz [8] further high- lighted that although the model qualitatively reproduced 922 Smith et al.: Concurrent vowel identification ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 104 (2018) 4. Results The model predicts the combined auditory response of pre- sented concurrent vowels (Section 3.2: combined classes). Given this assumption it was able to match the mean num- ber of concurrent vowels correctly identified by listeners in the absence of any F0 differences (73%). More impor- tantly, however, the probabilities of individual decisions (i.e. the confusions) predicted by our model are acutely similar to those made by listeners (Figure 2, circles), de- spite the fact that no attempt was made to fit the confusions themselves. Spectral processing models were best at pre- dicting human decision probabilities (r > 0.94, p < 0.01; r was calculated between sets of values, ignoring any ma- trix structure). Decisions predicted using temporal pro- cessing were less accurate (although in all cases r > 0.86, p < 0.01). Figure 4. (Colour online) Average number of concurrent vowels correctly predicted as a function of internal noise, for variants of our model. [11],[12],[13] reference different cochlea filter- shapes. they make the wrong decisions consistently, and despite the probabilistic nature of the models. We also considered a model which compared auditory responses of concurrent vowels to representations of indi- vidual vowels (Section 3.2: individual classes). Like sim- ilar previously published models, it fails to approach the mean number of concurrent vowels correctly identified by listeners for any amount of internal noise, predicting a maximum value of 42% when a temporal pathway was im- plemented. Additionally the probability of individual de- cisions were poorly correlated with human data (max r of 0.42, p <0.01). The combined-class model which predicted human de- cisions best used spectral processing, outperforming the temporal representation. Perhaps surprisingly, neither tem- poral nor spectral processing depended on whether fil- terbanks were based on human or guinea-pig bandwidth estimates (Figure 3b). Further investigation revealed that for spectral processing, filters with narrower bandwidths approached human like performance with more internal noise (Figure 4, solid lines). This was not the case when using a temporal pathway, in which frequency resolution is not such a constraint (Figure 4, dashed lines). In contrast, identification from individual classes (Figure 4, dotted and dash-dotted lines) did not converge on human performance for any amount of internal noise. The predictions from the best fitting of such models (Figure 2, crosses) are clustered close to 0% and 100% correct, suggesting that these errors are much more spe- cific and confident than those of human listeners. 3.1. Auditory System Waveforms of concurrent vowel pairs (/vi, vj/ where vi, vj ∈{i, a, u, æ, Ç}) were bandpass filtered, simulat- ing middle and outer ear effects, and then passed to a lin- ear cochlear filter bank. This comprised 100 gammatone filters centred at logarithmically spaced frequencies from 80 to 4000 Hz. Different filter bandwidths could be imple- mented, determined from masking experiments in humans [10, 11] or guinea-pigs [12]. The outputs of each filter were then half-wave rectified. An auditory representation (µij) followed from one of two processing pathways: • Combined Classes. Each class was a probabilistic tem- plate describing a combination of vowels. These were constructed by passing concurrent vowel pairs through our auditory model. Due to the equivalence of stimuli classes with the stimuli presented, calculating Equation (1) produced a suitable confusion matrix. • Individual Classes. Each class was a probabilistic template describing an individual vowel (calculat- ing Equation (1) resulted in P (/vz/|/vi, vj/) where vz ∈{i, a, u, æ, Ç}). To obtain predictions of con- current vowel pair presentation probabilities, individ- ual vowel presentation probabilities were multiplied to- gether. This approach, assuming individual vowels are identified independently of one another, was initially proposed in [4]. • Spectral processing. The logarithm of the RMS of each channel was calculated and standardised across channels (mean of 0, SD of 1). • Temporal processing. An autocorrelation function was applied to each channel [6]. These were pooled across all channels and then standardised as above. Independent, normal, zero-mean noise with identical vari- ance was then added to each value of this representa- tion. This resulted in a distribution of auditory activity (a ∼N (µij, σ2I)). The variance was the only free pa- rameter in our model. Independent, normal, zero-mean noise with identical vari- ance was then added to each value of this representa- tion. This resulted in a distribution of auditory activity (a ∼N (µij, σ2I)). The variance was the only free pa- rameter in our model. For each model variant, we selected the variance of the in- ternal noise (σ2; single free parameter) to predict the clos- est fit to the overall percent of concurrent vowels correctly identified by listeners. 923 ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 3.1. Auditory System 104 (2018) Smith et al.: Concurrent vowel identification Smith et al.: Concurrent vowel identification Sp Te Sp Te Sp Te 0 0.2 0.4 0.6 0.8 1 Correlation coefficients ( r) a b c [11] [12] [13] Figure 3. Correlation coefficients (r) between predicted confu- sions for model variants, and listener confusions. ‘Sp’: Spec- tral pathway, ‘Te’: Temporal pathway. [11],[12],[13] are refer- ences to different cochlea filter-shapes. a) Individual classes, b) Combined classes, c) Combined classes with non-linear cochlear model [13]. Smith et al.: Concurrent vowel identification Smith et al.: Concurrent vowel identification 0 20 40 60 80 100 Hum anprobabilities(% ) 0 50 100 M odelprobabilities(% ) P(/a,æ/|/a,æ/) P(/a,æ/|/a,æ/) /u,ɝ/ /u,ɝ/ /ɝ,ɝ/ Figure 2. A scatter plot comparing the probabilities with which humans predicted concurrent vowel pairs had been presented, against probabilities predicted from the combined-class (◦; σ2 = 1.03) and individual-class (×; σ2 = 1.20) variants of our spectral model. The probabilities of confusing /Ç,Ç/ for /u,Ç/, and cor- rectly identifying /a,æ/, are indicated. Sp Te Sp Te Sp Te 0 0.2 0.4 0.6 0.8 1 Correlation coefficients ( r) a b c [11] [12] [13] 0 20 40 60 80 100 Hum anprobabilities(% ) 0 50 100 M odelprobabilities(% ) P(/a,æ/|/a,æ/) P(/a,æ/|/a,æ/) /u,ɝ/ /u,ɝ/ /ɝ,ɝ/ Figure 3. Correlation coefficients (r) between predicted confu- sions for model variants, and listener confusions. ‘Sp’: Spec- tral pathway, ‘Te’: Temporal pathway. [11],[12],[13] are refer- ences to different cochlea filter-shapes. a) Individual classes, b) Combined classes, c) Combined classes with non-linear cochlear model [13]. Figure 2. A scatter plot comparing the probabilities with which humans predicted concurrent vowel pairs had been presented, against probabilities predicted from the combined-class (◦; σ2 = 1.03) and individual-class (×; σ2 = 1.20) variants of our spectral model. The probabilities of confusing /Ç,Ç/ for /u,Ç/, and cor- rectly identifying /a,æ/, are indicated. 0 0.5 1 1.5 2 Internal noise (σ2) 0 20 40 60 80 100 Mean % Correct [13] [12] [11] Human data Combined classes (spectral) Combined classes (temporal) Individual classes (spectral) Individual classes (temporal) Figure 4. (Colour online) Average number of concurrent vowels correctly predicted as a function of internal noise, for variants of our model. [11],[12],[13] reference different cochlea filter- shapes. 0 0.5 1 1.5 2 Internal noise (σ2) 0 20 40 60 80 100 Mean % Correct [13] [12] [11] Human data Combined classes (spectral) Combined classes (temporal) Individual classes (spectral) Individual classes (temporal) 5. Discussion The presented model demonstrated how predicting the complete internal representation of concurrent vowels pro- duces decisions in line with listener behaviour, when no F0 differences are presented. However, instead assuming individual vowels are identified independently of one an- other (Section 3.2: individual classes) produced poor esti- mates of listener confusions. In fact fitting a confusion ma- trix in order to optimise the correlation coefficient between predicted and human confusions, under the constraint that individual vowels are identified independently of one an- other, results in a theoretical maximum r of 0.88. ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 104 (2018) BITS Pilani (AKC), and by NIH-NIDCD grants R01- DC009838 (MGH). BITS Pilani (AKC), and by NIH-NIDCD grants R01- DC009838 (MGH). References [1] M. T. M. Scheffers: Sifting vowels: Auditory pitch analysis and sound segregation. Ph.D. thesis, University of Gronin- gen, 1983. [2] C. Micheyl, A. J. Oxenham: Pitch, harmonicity and concur- rent sound segregation: Psychoacoustical and neurophysio- logical findings. Hear Res 266 (2010) 36–51. [3] P. F. Assmann, Q. Summerfield: Modeling the perception of concurrent vowels: Vowels with the same fundamental frequency. J Acoust Soc Am 85 (1989) 327–338. Assmann and Summerfield [3] explored the effect of various transformations to auditory excitation patterns on predictions of listener CVI data, incorporating this as- sumption of independence. They achieved correlations with listener confusions between 0.42 and 0.71, over 0.25 lower than our best prediction. The authors found that em- phasising spectral peaks best matched their listener data. [4] P. F. Assmann, Q. Summerfield: Modeling the perception of concurrent vowels: vowels with different fundamental fre- quencies. J Acoust Soc Am 88 (1990) 680–697. [5] R. Meddis, M. J. Hewitt: Modeling the identification of concurrent vowels with different fundamental frequencies. J Acoust Soc Am 91 (1992) 233–245. [6] J. F. Culling, C. J. Darwin: Perceptual and computational separation of simultaneous vowels: Cues arising from lowfrequency beating. J Acoust Soc Am 95 (1993) 1559– 1569. The work promotes the use of an ideal observer type model as an initial point to investigate cues beyond pitch for the CVI task. The model hints at a process that seeks to optimally predict which concurrent-vowel pair led to a corresponding auditory representation. Considering where listener behaviour deviates most from ‘ideal’ could repre- sent a structured approach to extending, and improving the performance, of this model. [7] A. de Cheveigne: Concurrent vowel identification. III. A neural model of harmonic interference cancellation. J Acoust Soc Am 101 (1997) 2857–2865. [8] A. Chintanpalli, M. G. Heinz: The use of confusion patterns to evaluate the neural basis for concurrent vowel identifica- tion. J Acoust Soc Am 134 (2013) 2988–3000. 6. Conclusion [9] D. H. Klatt: Software for a cascade/parallel formant syn- thesizer. J Acoust Soc Am 67 (1980) 971–995. A novel computational model predicts human CVI be- haviour, when vowels have identical pitches. It is better at predicting listener’s systematic perceptual confusions than existing models, when ideal representations of combined speech were implemented. The model’s simplicity allows potential extension to more complex scenarios with more identification cues (e.g. F0 differences), and to investigate the possible mechanisms underlying CVI. [10] A. J. Oxenham, C. Shera: Estimates of human cochlear tun- ing at low levels using forward and simultaneous masking. J Assoc Res Otolaryngol 4 (2003) 541–554. [11] B. R. Glasberg, B. C. Moore: Derivation of auditory filter shapes from notched-noise data. Hear Res 47 (1990) 103– 138. [12] E. F. Evans: The frequency response and other properties of single fibers in the guinea-pig cochlear nerve. J Physiol 226 (1972) 263–287. 4. Results Consis- tent with this, the entropy of the decision probabilities, corresponding to their randomness, was lower for mod- els of individual-class recognition (<4.86 bits) than either the human data (5.11 bits) or the combined-class recogni- tion model (>5.05 bits). Thus, the models of individual- class recognition make more errors than people because Finally, we tested a more sophisticated model of the guinea-pig cochlea, which incorporated non-linear filter- ing and haircell transduction [13]. This produced the same qualitative relationships aforementioned (Figure 3c). 924 Smith et al.: Concurrent vowel identification ACTA ACUSTICA UNITED WITH ACUSTICA Vol. 104 (2018) Acknowledgement [13] C. J. Sumner, L. P. O’Mard, E. A. Lopez-Poveda, R. Med- dis: A nonlinear filter-bank model of the guinea-pig cochlear nerve: rate responses. J Acoust Soc Am 113 (2003) 3264–3274. Work partially supported by MRC grant #M C_UU_ 00010/1 and U135097127 (SSS,CJS), OPERA and RIG, 925
https://openalex.org/W1979228040
https://www.repo.uni-hannover.de/bitstream/123456789/473/1/sensors-13-00106.pdf
English
null
Influence of Cobalt on the Properties of Load-Sensitive Magnesium Alloys
Sensors
2,012
cc-by
6,760
Sensors 2013, 13, 106-118; doi:10.3390/s130100106 Sensors 2013, 13, 106-118; doi:10.3390/s130100106 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS Article Influence of Cobalt on the Properties of Load-Sensitive Magnesium Alloys Christian Klose *, Christian Demminger, Gregor Mroz, Wilfried Reimche, Friedrich-Wilhelm Bach, Hans Jürgen Maier and Kai Kerber Keywords: physical sensors; magnetic materials; load-sensitive materials; Villari effect; magnesium; cobalt 1. Introduction Magnetic magnesium alloys can be utilized as load-sensitive materials with sensory properties because they enable an online-measurement of the instantaneous mechanical loads to which a structural component is subjected during service. With the aid of measured loading history data, the component’s resulting fatigue life can be derived and subsequent generations of components can be optimized [1]. In mechanical engineering, the use of strain gauges to measure mechanical loads is state of the art. A major limitation of traditional strain sensors, however, is the locally restricted measurement range, as only local strains directly at the measuring position can be detected. Furthermore, the strain gauges and their electrical contacts are typically applied to the component’s surface and thus might be exposed to mechanical damage caused, for example, by external impacts during the use of the component. If the component has material-inherent magnetic properties it can serve as a load sensor itself, because in this case the loading information can be collected with the magnetic material. The application of external mechanical forces to a ferromagnetic material temporarily changes the magnetic susceptibility due to the reversible deformation of the material’s crystal lattice [2]. This magnetoelastic effect (also known as Villari effect) is thermodynamically inverse to magnetostriction and leads to a macroscopic change of the magnetic properties [3], which can be monitored by methods of non-destructive component testing, such as the harmonic analysis of eddy-current signals. The application of magnetoelastic force and torque sensors has been reported in literature, e.g., [4,5]. Common ferromagnetic materials are ferritic steels, cobalt and nickel alloys, whereas light metals are paramagnetic. The overall aim in the development of magnetic magnesium alloys is to obtain a lightweight material with inherent load-sensitive abilities. By alloying pure magnesium or magnesium alloys with ferromagnetic elements, particularly cobalt, and ferromagnetic compounds based on samarium-cobalt, several magnesium alloys were produced which exhibited measurable magnetic properties [6–8]. However, in the magnesium industry the element cobalt in general is considered as an impurity due to its negative impact on the corrosion behavior and therefore avoided in the production of magnesium and standard magnesium alloys such as AZ91 [9–12]. This corrosion-accelerating effect is usually related to the existence of undissolved cobalt particles in the microstructure of the magnesium alloy which result from the low solubility of cobalt in magnesium and hence the formation of active cathodic sites [13]. Christian Klose *, Christian Demminger, Gregor Mroz, Wilfried Reimche, Friedrich-Wilhelm Bach, Hans Jürgen Maier and Kai Kerber Institut für Werkstoffkunde (Institute of Materials Science), Leibniz Universität Hannover, An der Universität 2, D-30823 Garbsen, Germany; E-Mails: demminger@iw.uni-hannover.de (C.D.); mroz@iw.uni-hannover.de (G.M.); reimche@iw.uni-hannover.de (W.R.); bach@iw.uni-hannover.de (F.-W.B.); maier@iw.uni-hannover.de (H.J.M.); kerber@iw.uni-hannover.de (K.K.) * Author to whom correspondence should be addressed; E-Mail: klose@iw.uni-hannover.de; Tel.: +49-511-762-4328; Fax: +49-511-762-5245. * Author to whom correspondence should be addressed; E-Mail: klose@iw.uni-hannover.de; Tel.: +49-511-762-4328; Fax: +49-511-762-5245. Received: 1 November 2012; in revised form: 12 December 2012 / Accepted: 19 December 2012 / Published: 21 December 2012 Received: 1 November 2012; in revised form: 12 December 2012 / Accepted: 19 December 2012 / Published: 21 December 2012 Abstract: In this study, magnesium is alloyed with varying amounts of the ferromagnetic alloying element cobalt in order to obtain lightweight load-sensitive materials with sensory properties which allow an online-monitoring of mechanical forces applied to components made from Mg-Co alloys. An optimized casting process with the use of extruded Mg-Co powder rods is utilized which enables the production of magnetic magnesium alloys with a reproducible Co concentration. The efficiency of the casting process is confirmed by SEM analyses. Microstructures and Co-rich precipitations of various Mg-Co alloys are investigated by means of EDS and XRD analyses. The Mg-Co alloys’ mechanical strengths are determined by tensile tests. Magnetic properties of the Mg-Co sensor alloys depending on the cobalt content and the acting mechanical load are measured utilizing the harmonic analysis of eddy-current signals. Within the scope of this work, the influence of the element cobalt on magnesium is investigated in detail and an optimal cobalt concentration is defined based on the performed examinations. Keywords: physical sensors; magnetic materials; load-sensitive materials; Villari effect; magnesium; cobalt Sensors 2013, 13 Sensors 2013, 13 107 Sensors 2013, 13 Sensors 2013, 13 undissolved cobalt particles in the magnesium matrix. The microstructure and the suitable magnetic properties of a die-cast binary magnesium-cobalt alloy were briefly introduced by the authors in a previous publication. Here, largely eutectic structures of the cobalt-rich precipitations were observed in images recorded with the scanning electron microscope (SEM) [8]. The specific aim of the present work is to broaden the understanding of the influence that varying amounts of cobalt wield on the characteristics of magnesium-cobalt sensor alloys. In order to qualify these alloys as load-sensor materials, the magnetic properties as well as the microstructures depending on the cobalt concentration and the applied mechanical load are in the focus of this work. After a short presentation of the production process, details on the microstructure, the magnetic and the mechanical properties of magnetic magnesium alloys are discussed in the subsequent sections. 1. Introduction Apart from the frequently described inferior corrosion resistance of cobalt-containing magnesium alloys, little is known about the various other effects caused by cobalt as an alloying element. Some authors reported that small additions of cobalt result in an increase of the strength values of magnesium [14] or magnesium-neodymium alloys [15]. Regarding the magnetic properties of magnesium-cobalt materials, the intermetallic compound MgCo2 was found to be ferromagnetic below 321 K [16,17]. One important objective in the development of load-sensitive magnesium alloys is the production of an actual technical alloy in a casting process which is difficult to accomplish due to the high melting point (1,495 °C) and low solubility of cobalt in magnesium. This requirement demands a complete dissolution of the cobalt component in the magnesium melt in order to achieve a homogeneously distributed multiphase microstructure. According to the literature [16,18], a maximum cobalt concentration of 4.5 weight percent (wt.%) is appropriate in order to prevent the formation of 108 Sensors 2013, 13 Sensors 2013, 13 The melt was cast into graphite-coated steel molds (Ø 22 mm × length 250 mm) with horizontal feeders which were preheated to 350 °C. The melt was cast into graphite-coated steel molds (Ø 22 mm × length 250 mm) with horizontal feeders which were preheated to 350 °C. A suitable method for an online-monitoring of the Mg-Co alloys’ load-dependent magnetic properties is the harmonic analysis of eddy current signals which is therefore used in this work. A sinusoidal excitation voltage is generated and fed to the excitation coil integrated into the eddy current sensor which generates an alternating magnetic field via the excitation current. The alternating magnetic field is designated as the primary field and produces the magnetic reversal processes and eddy currents which are dependent on the material’s influencing variables such as, for example, the formation of the microstructure or the stress state. By means of the eddy currents and the magnetic reversal processes, a secondary field is produced. With the aid of the measuring coil, the signal difference between the primary and the secondary field is measured in comparison to the measuring signal in air. Further details are given in [19]. The harmonics’ measured values, computed by a Fast-Fourier-Transformation, provide information about the instantaneous material condition and the lattice distortions as a consequence of the forces acting on the Mg-Co specimens. The described method was used for a preliminary determination of the magnetic properties of machined cylinder specimens (Ø 18 mm × length 20 mm) from the Mg-Co castings. Furthermore, cyclic loading tests using a universal testing machine of the type Z010 (Zwick Roell, Ulm, Germany) were performed by employing stepwise increasing loads from 200 to 1,400 N in order to test whether correlations can be established between the forces applied to the specimens and their magnetic properties measured by means of the eddy current testing. Threaded cylindrical tensile specimens with a nominal diameter of 6 mm (according to DIN 50125) were machined by turning as test pieces for both the cyclic loading experiments using the Villari effect and the determination of the mechanical strength values in standard tensile tests. Three specimens of each alloy were used in the tensile tests. For the examination of the Mg-Co alloys’ microstructure, SEM investigations were carried out employing the compositional contrast mode (RBSD) of a LEO 1455VP SEM (Carl Zeiss Microscopy, Jena, Germany). 2. Experimental Section For the investigations in this study, five binary Mg-Co alloys with a nominal Co concentration of 1 to 5 wt.% were produced by die casting using technically pure Mg as base material (cf. Table 1 for the materials used). Pure Mg specimens were also produced with the same casting parameters for comparison of the magnetic and mechanical properties. In order to facilitate the dispensation of the ferromagnetic powder in the Mg melt and to avoid floating, 40 wt.% of pure Co powder (particle size < 150 µm) were blended with 60 wt.% of pure Mg powder (particle size < 60 µm) by means of a laboratory powder mixer of the type MP-6 (Biomation, Jugenheim, Germany). The device was equipped with a V-shaped mixing container (volume 1.5 L) and was set to reverse the direction of rotation every 120 s throughout the mixing duration of 150 min. For best results, the mixing container was two-thirds full. Subsequently the powder mixture was compacted to billets with an approximate diameter of 30 mm and a length of 280 mm using a cold-isostatic dry-bag pressing process with a maximum pressure of 2,000 bar which was performed by the company Loomis Products (Kaiserslautern, Germany). After machining to a diameter of 29 mm, the powder billets were extruded to rods (Ø 6 mm) at a temperature of 200 °C using a laboratory extrusion press and a maximum force of 500 kN. Table 1. Materials used in this study. Material Supplier Content in wt.% according to Supplier Zn Al Si Mn Co Fe Ca Mg Mg ingots Magnesium Elektron 0.004 0.012 0.012 0.025 - 0.002 0.02 99.92 Mg powder Ecka Granules - - - - - - - 99.8 Co powder Sigma Aldrich - - - - 99.9 - - - Table 1. Materials used in this study. During the subsequent casting experiments the pure Mg melt was inoculated with 1–5 wt.% of Co utilizing the extruded Mg-Co powder rods. The magnetic Mg alloys were manufactured by means of a die casting method using a resistance-heated K4/10 furnace (Nabertherm, Lilienthal, Germany) in a shielding-gas atmosphere (N2 + 0.3% SF6) and by employing a boron nitride coated, unalloyed steel crucible. The basic material was melted and held at a temperature of 730 °C. At the beginning of the stirring process (45 min at 300 min−1), the extruded Mg-Co powder rods were introduced into the melt. 109 Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 110 3. Results and Discussion In the subsequent sections, the Mg-Co alloys’ nomenclature includes the corresponding nominal concentrations of Mg and Co in weight percent. For example, Mg96Co4 means pure Mg alloyed with 4 wt.% of pure Co. The results presented in the following chapters are mainly focused on the alloy Mg96Co4 due to its advantageous properties. Sensors 2013, 13 This method was used by the authors before in order to distinguish the microstructure constituents of binary Mg-Co alloys [8]. Sections of the Mg-Co castings were metallographically prepared by grinding using SiC abrasive papers up to grade P2500. Afterwards, the specimens were polished to 1 µm using diamond paste and rinsed with ethanol. Corresponding to the SEM analyses of the Mg-Co alloys, measurements with energy-dispersive x-ray spectroscopy (EDS) employing a Quantax EDS system (Bruker, Billerica, MA, USA) and a measuring time of 120 s each were performed on the specimens in order to determine the alloys’ overall Co concentrations. The SEM investigations were supplemented with phase analyses by means of XRD measurements using an X’Pert MPD system (PANalytical, Almelo, the Netherlands). The measurement was carried out using CuKα radiation in the range of 5–110° 2Θ with an increment of 0.02° and a measuring time of 16 s per step. An evaluation of the measured diffraction patterns was performed by means of the XRD database PDF-2 ver. 2010. In addition, micro tensile specimens with a rectangular cross section of 3 × 2 mm in the taper and a length of 60 mm were manufactured by continuous-wire electro discharge machining and polished as described above. These samples were examined in-situ with the aid of a SEM-fitted tensile/compression module (Kammrath & Weiss, Dortmund, Germany). At several stages during the tensile test, SEM images of the alloy’s microstructure were recorded. Sensors 2013, 13 obvious dependency exists between the applied mechanical loads and the measured amplitude values which is demonstrated in Figure 2 by means of the alloy Mg96Co4. Following the test sequence from left to right, every dot is the average result of three consecutive measurements at the same test force. While a force of 200 N leads to a slightly greater amplitude than in the initial state, the highest load in this experiment (1,400 N) causes the 3rd harmonic to increase by 30%. These magnetic Mg alloys can therefore be classified as load-sensitive materials. Figure 2. Amplitudes of the 3rd harmonic of Mg96Co4 measured in loading tests; eddy current ring sensor Ø 10 mm, test frequency 1.6 kHz. Figure 2. Amplitudes of the 3rd harmonic of Mg96Co4 measured in loading tests; eddy current ring sensor Ø 10 mm, test frequency 1.6 kHz. current ring sensor Ø 10 mm, test frequency 1.6 kHz. 2.5 3.0 3.5 4.0 50 200 50 600 50 1000 50 1400 50 1000 50 600 50 200 50 3rd Harmonic in V Test Load in N Test Sequence Loaded Relieved of the load Permanent increase of 3rd harmonic 3rd Harmonic in V Permanent increase of 3rd harmonic Test Load in N After each step the specimen was relieved of the load (50 N) with the amplitudes of the 3rd harmonic dropping back to a lower value. There is however a gradual increase of the amplitudes in the unloaded condition. After reaching the maximum test load, the 3rd harmonic’s measured values remain constantly on the higher level. This effect is presumably connected with the low yield strengths of the binary Mg-Co alloys. Concluding from results of the tensile tests which are discussed in Section 3.3 and the sample’s cross-section (~30 mm2), the alloy’s yield strength is exceeded at a load of approx. 1,000 N. The beginning plastic deformation by uniaxial strain affects the microstructure of the magnetic material and leads to a permanent change of the magnetic properties. In a first step in order to examine the reproducibility of this effect, the load test was repeated after a time interval of 6 days utilizing the identical tensile specimen as before. Note that the test rig was disassembled in the meantime and set-up again for the repeated trial. An overview of the results compared with the original measurement is given in Figure 3. 3.1. Magnetic Properties The magnetic properties of the five binary Mg-Co alloys were examined with the aid of a harmonic analysis of eddy current signals. For a material’s magnetic properties the 3rd harmonic is of major significance. It will be close to zero if the tested material is not ferromagnetic; higher values indicate better magnetic properties. In Figure 1 a screening of the produced Mg-Co alloys’ average 3rd harmonics in their initial condition is presented by means of measurements on the cylinder specimens. As one might expect, a higher Co concentration leads to greater amplitudes of the 3rd harmonic and thus to stronger magnetic properties. While the alloy with 1 wt.% Co exhibits very low amplitudes which are on the same level as pure Mg, a significant increase is achieved with alloys containing at least 2 wt.% Co. The alloy Mg96Co4 shows an optimal trade-off between the average amplitude and the scatter of the values. Figure 1. Amplitudes of the 3rd harmonic of the binary Mg-Co alloys’ cylinder specimens; eddy current ring sensor Ø 20 mm, test frequency 1.6 kHz. Figure 1. Amplitudes of the 3rd harmonic of the binary Mg-Co alloys’ cylinder specimens; eddy current ring sensor Ø 20 mm, test frequency 1.6 kHz. In order to qualify the alloys’ material-inherent load sensor abilities, tensile specimens manufactured from all of the Mg-Co castings were subjected to cyclic loading measurements. As the results show, the magnetic properties of alloys containing at least 2 wt.% Co are sufficient to distinguish the values of the 3rd harmonic between the loaded and the relieved condition. Moreover, an 0.0 1.0 2.0 3.0 0 1 2 3 4 5 3rd Harmonic in mV Nominal Cobalt Concentration in wt.% eddy current ring sensor Ø 20 mm, test frequency 1.6 kHz. 0.0 1.0 2.0 3.0 0 1 2 3 4 5 3rd Harmonic in mV Nominal Cobalt Concentration in wt.% 0.0 1.0 2.0 3.0 0 1 2 3 4 5 3rd Harmonic in mV Nominal Cobalt Concentration in wt.% Nominal Cobalt Concentration in wt.% In order to qualify the alloys’ material-inherent load sensor abilities, tensile specimens manufactured from all of the Mg-Co castings were subjected to cyclic loading measurements. As the results show, the magnetic properties of alloys containing at least 2 wt.% Co are sufficient to distinguish the values of the 3rd harmonic between the loaded and the relieved condition. Moreover, an 111 Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 still has to be investigated in greater detail, it suggests its use for the detection of local plastic deformation of structural components made from magnetic Mg alloys. In this case, the chance of quantifying a component’s overloading by eddy current measurements after the end of its lifecycle would be one great advantage in addition to the sensory behavior of Mg-Co alloys. Figure 3. Amplitudes of the 3rd harmonic of Mg96Co4 measured in loading tests which were performed twice on the same tensile specimen with a time interval of 6 days; eddy current ring sensor Ø 10 mm, test frequency 1.6 kHz. 3.2. Microstructure Investigation and Phase Analysis 2.5 3.0 3.5 4.0 50 200 50 600 50 1000 50 600 50 3rd Harmonic in mV Test Load in N Original After 6 days Test Sequence Loaded condition Loaded condition (original trial) 3.2. Microstructure Investigation and Phase Analysis The following SEM images show sections of three of the binary Mg-Co alloys’ microstructures (Figure 4). Due to their higher atomic mass numbers, the Co-rich phases appear as bright structures which are easily distinguished from the Mg matrix. Independent of the global Co concentration, the Co-containing phases exhibit a eutectic structure in all the examined alloys and form a refined, widespread network within the Mg matrix. With the Co content increasing from 3 wt.% (Figure 4(a)) to 4 wt.% (Figure 4(b)), the number of lamellar-arranged precipitations grows and they seem to take up a greater proportion of the material’s volume. Furthermore, the hypereutectic alloy Mg95Co5 (Figure 4(c)) shows an increased quantity of agglomerated particles which are not connected to the eutectic phase structure. Most of the agglomerates are surrounded by areas of darker gray-shades which indicate regions of lower density and thus beginning corrosion. These undissolved particles originate from the ferromagnetic Co powder and, in this work, can be observed mainly in alloys with an overall Co concentration of more than 4 wt.%. These results agree with the data given in literature regarding the limited solubility of Co in the Mg melt [16,18] (see also Section 1: Introduction). 2.5 3.0 3.5 4.0 50 200 50 600 50 1000 50 600 50 3rd Harmonic in mV Test Load in N Original After 6 days Test Sequence Loaded condition Loaded condition (original trial) 3.2. Sensors 2013, 13 During the second loading experiment, the 3rd harmonic’s measured values are constant around 3.45 mV in the relieved state and hence on a similar level like at the end of the original load test (~3.4 mV). Furthermore, the load-sensitive behavior of the alloy Mg96Co4 is still present with an increase of 0.28 mV per 1,000 N. Although the gradual and permanent increase of the 3rd harmonic, which could be observed as a result of high loads, 112 Sensors 2013, 13 Microstructure Investigation and Phase Analysis The following SEM images show sections of three of the binary Mg-Co alloys’ microstructures (Figure 4). Due to their higher atomic mass numbers, the Co-rich phases appear as bright structures which are easily distinguished from the Mg matrix. Independent of the global Co concentration, the Co-containing phases exhibit a eutectic structure in all the examined alloys and form a refined, widespread network within the Mg matrix. With the Co content increasing from 3 wt.% (Figure 4(a)) to 4 wt.% (Figure 4(b)), the number of lamellar-arranged precipitations grows and they seem to take up a greater proportion of the material’s volume. Furthermore, the hypereutectic alloy Mg95Co5 (Figure 4(c)) shows an increased quantity of agglomerated particles which are not connected to the eutectic phase structure. Most of the agglomerates are surrounded by areas of darker gray-shades which indicate regions of lower density and thus beginning corrosion. These undissolved particles originate from the ferromagnetic Co powder and, in this work, can be observed mainly in alloys with an overall Co concentration of more than 4 wt.%. These results agree with the data given in literature regarding the limited solubility of Co in the Mg melt [16,18] (see also Section 1: Introduction). 113 Sensors 2013, 13 Figure 4. SEM images of the microstructures of binary Mg-Co alloys (RBSD mode): (a) Mg97Co3; (b) Mg96Co4; (c) Mg95Co5. In contrast to the authors’ previous publications [7], the results of the EDS measurements (Table 2) which were performed in order to determine the overall amounts of Co in the produced Mg alloys show good compliance to the nominal amounts of the ferromagnetic alloying element the Mg melts were inoculated with during the casting process. This can be credited to the use of the extruded powder rods, as they facilitate the alloying process and obviously increase alloying efficiency. For the hypereutectic alloy Mg95Co5, a low amount of silicon was measured which, in this case, can be attributed to the use of SiC-containing abrasive paper during the sample preparation procedure. Also, the measured carbon and oxygen concentrations are within the usual limits of magnesium alloy samples which were prepared by mechanical grinding and polishing. Sensors 2013, 13 Diffraction pattern of the alloy Mg96Co4 in as-cast state. 3.3. Mechanical Properties Standard tensile tests were performed in order to assess the mechanical strength properties of the produced alloys in the as-cast condition. The results reveal that the influence of the Co addition is reflected mainly in the changes of the ultimate tensile strength (Figure 6). For the alloy containing 1 wt.% Co, an increase of 40% (153 MPa) compared with pure Mg (108 MPa) is observed. With increasing amount of Co, the tensile strength is reduced gradually; the alloy Mg95Co5 has the same tensile strength as pure Mg only. The ultimate elongation tends to correspond with the tensile strength and ascends to approx. 20% higher values in case of the alloy Mg99Co1 (8.9% elongation) but drops to a very low level at the higher Co concentrations (2.4%). In contrast, the yield strength remains almost constant around 30 MPa with the exception of the hypereutectic alloy Mg95Co5 which reaches 40 MPa. Apart from the slightly higher global Co concentration the large number of undissolved Co particle agglomerates is the main difference in the microstructure of Mg95Co5 compared with the other alloys. They result from an oversaturation of the Mg melt with Co and might to some extent have the same effect as a particle reinforcement 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 log Intensity in a.u. 2 Theta in ° Mg Co Co2Mg Mg2-xCo 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 log Intensity in a.u. 2 Theta in ° Mg Co Co2Mg Mg2-xCo 3.3. Mechanical Properties Standard tensile tests were performed in order to assess the mechanical strength properties of the produced alloys in the as-cast condition. The results reveal that the influence of the Co addition is reflected mainly in the changes of the ultimate tensile strength (Figure 6). For the alloy containing 1 wt.% Co, an increase of 40% (153 MPa) compared with pure Mg (108 MPa) is observed. With increasing amount of Co, the tensile strength is reduced gradually; the alloy Mg95Co5 has the same tensile strength as pure Mg only. The ultimate elongation tends to correspond with the tensile strength and ascends to approx. Sensors 2013, 13 In contrast to the authors’ previous publications [7], the results of the EDS measurements (Table 2) which were performed in order to determine the overall amounts of Co in the produced Mg alloys show good compliance to the nominal amounts of the ferromagnetic alloying element the Mg melts were inoculated with during the casting process. This can be credited to the use of the extruded powder rods, as they facilitate the alloying process and obviously increase alloying efficiency. For the hypereutectic alloy Mg95Co5, a low amount of silicon was measured which, in this case, can be attributed to the use of SiC-containing abrasive paper during the sample preparation procedure. Also, the measured carbon and oxygen concentrations are within the usual limits of magnesium alloy samples which were prepared by mechanical grinding and polishing. Table 2. EDS measurements of overall Co concentrations in binary Mg-Co alloy Alloy Nominal Co Content in wt.% Element concentration in wt.% Mg Co C O Si Mg97Co3 3 94.71 2.99 1.93 0.37 - Mg96Co4 4 94.99 3.44 1.57 0.00 - Mg95Co5 5 92.66 4.47 1.79 0.95 0.12 Table 2. EDS measurements of overall Co concentrations in binary Mg-Co alloys. The multiphase structure, as can be seen in the SEM images (Figure 4), suggests the existence of primary Mg and Co crystals as well as intermetallic compounds, though few intermetallic phases are possible in the Mg-Co system [20]. The diffraction pattern resulting from the XRD measurement of the Mg96Co4 specimen is shown in Figure 5. As expected, the peaks with the highest intensities belong to the primary Mg crystals occupying the largest part of the sample’s surface area. Due to the hexagonal crystal structure of both Mg and Co at room temperature, the reflexes of both metals overlap partially, e.g., at 90° 2Θ. However several peaks were found that cannot be fitted to either Mg or Co but, according to the XRD database used in this work, clearly indicate the existence of intermetallic compounds of the types Co2Mg and Mg2−xCo in the cast specimen. The exact stoichiometry of intermetallic phases in the Mg-Co system is discussed controversially in literature [16,20], yet their presence together with the eutectic structure proves the dissolution of the Co powder in the Mg melt. The compound Co2Mg at least contributes to the macroscopic magnetic properties of these alloys because it is ferromagnetic at room temperature [17]. Sensors 2013, 13 114 Figure 5. Sensors 2013, 13 wields a major influence on the strength properties of the binary Mg-Co alloys due to its lamellar morphology and higher hardness than the Mg matrix. Compared with pure Mg (Figure 6), an increasing overall Co content leads to a more brittle behavior which can be attributed to the growing precipitation of Co-rich phases in the microstructure. Figure 6. Mechanical properties of binary Mg-Co alloys in the as-cast state subject to the Co content; tensile tests according to ISO 6892-1, threaded tensile specimens DIN 50125 B 6 × 30 (mean values of 3 specimens for each alloy). 0 2 4 6 8 10 12 14 16 18 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 Strain in % Tensile Strength in MPa Nominal Cobalt Concentration in wt.% Yield Strength (0.2% offset strain) Ultimate Strength Ultimate Elongation Strain in % Figure 7. SEM investigation of the microstructure of the alloy Mg96Co4 during the in-situ tensile test: (a) specimen 1 in unloaded state; (b) specimen 1 loaded with 630 N; (c) fracture in specimen 2 after failure. Figure 7. SEM investigation of the microstructure of the alloy Mg96Co4 during the in-situ tensile test: (a) specimen 1 in unloaded state; (b) specimen 1 loaded with 630 N; (c) fracture in specimen 2 after failure. 4. Conclusions In the present work the production of magnetic Mg alloys based on pure Mg and pure Co was demonstrated by die casting. The aim of this study was to show the influence of Co concentrations Sensors 2013, 13 20% higher values in case of the alloy Mg99Co1 (8.9% elongation) but drops to a very low level at the higher Co concentrations (2.4%). In contrast, the yield strength remains almost constant around 30 MPa with the exception of the hypereutectic alloy Mg95Co5 which reaches 40 MPa. Apart from the slightly higher global Co concentration the large number of undissolved Co particle agglomerates is the main difference in the microstructure of Mg95Co5 compared with the other alloys. They result from an oversaturation of the Mg melt with Co and might to some extent have the same effect as a particle reinforcement. For an explanation of the measured tensile strengths the behavior of the alloy Mg96Co4 during plastic deformation and failure was investigated by means of in-situ tensile tests. In Figure 7 the same area is displayed in its initial state (Figure 7(a)) and under the sample’s highest tolerated force (630 N; Figure 7(b)). The arrows in Figure 7(b) indicate a continuous precipitation which seems to act as a barrier preventing the deformation of the adjacent grain. It is evident that mainly the Mg matrix is influenced while the Co-containing phases partly shield the loads. This may lead to increased stresses in a limited number of grains and finally to early failure of the sample. On a second specimen crack initiation could be observed next to the eutectic phases whereas the cracks propagated along the precipitations (Figure 7(c)). Obviously the Co-rich eutectic phase structure 115 Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 116 magnetic properties. The results contribute towards a better understanding of the challenges in the production and the various properties of Mg sensor alloys. Because the solubility of Co in Mg is negligible [16] and hence the ferromagnetic element Co precipitates in a eutectic structure, it is evident that the Mg-Co alloys’ magnetic properties must be related primarily to the Co-rich eutectic precipitations that could be observed in the SEM images. XRD analyses confirmed the existence of primary Co phases and suggested the formation of intermetallic compounds between Mg and Co in the microstructure. The magnetic properties strongly depend on the overall amount of Co in the alloy. In particular, the higher-alloyed materials Mg96Co4 and Mg95Co5 show better magnetic properties in the sense of greater amplitudes of the 3rd harmonic. As a consequence, this means a higher sensitivity for the online-measurement of the applied mechanical loads because of the larger range of the measured values between the initial, unloaded state and the highest load. However, the large quantity of undissolved Co agglomerates in the hypereutectic alloy Mg95Co5 indicates an oversaturation with Co which can lead to accelerated corrosion due to the particles’ effect as active cathodic sites in the Mg matrix. Hence the authors suggest an optimal Co concentration of 4 wt.% for the production of magnetic Mg alloys. Regarding the mechanical properties in the as-cast state, the low yield strength of the binary Mg-Co alloys is an obvious problem. The tensile tests revealed that the impact of an increasing Co content shows mainly in the decrease of the tensile strength and the elongation at fracture. On the other hand, a connection between the magnetic properties and the yield strength could be observed during the loading experiments which appears as a gradual and permanent increase of the 3rd harmonic of the eddy current signal. This effect occurred usually as a result of high test loads and thus might be of use for the detection of overloaded parts of structural components without the need for an active permanent online-monitoring of the forces acting on the component. 4. Conclusions In the present work the production of magnetic Mg alloys based on pure Mg and pure Co was demonstrated by die casting. The aim of this study was to show the influence of Co concentrations between 1 and 5 wt.% on the microstructure and the phase formation as well as on the mechanical and Sensors 2013, 13 Sensors 2013, 13 Figure (Team e 8. Loa m Horsepow ad-sensitive wer, Leibniz magnesiu Universität um wheel t Hannover) carrier f ). from the RP09 racce car Figure (Team e 8. Loa m Horsepow ad-sensitive wer, Leibniz magnesiu Universität um wheel t Hannover) carrier f ). from the RP09 racce car A “ th “ H in m ( e Acknowled This rese “Magnetic M heir Lifecy “Gentelligen Hübsch for n-situ tensi mechanical t o sepow gments earch is spo Magnesium ycle”. The e nt Part Iden conducting ile tests, An testing. we , e b onsored by Alloys” of eddy curren ntification an g the XRD nja Krabben U ve s the Germa the Collabo nt measurem nd Integrity phase analy nhöft for re ove ) n Research orative Rese ments were y Assessmen ysis, Torste ecording th ). h Foundatio earch Cente e executed nt”. The aut en Heidenb he SEM ima n (DFG) w er 653 “Gen in cooperat thors would lut and Ma ages and H within the su ntelligent Co tion with su d like to than arcus Engelh Harald Kern ubproject E omponents i ubproject S nk Christop hardt for th nbach for th E2 in S3 ph he he Acknowledgments “ th “ H in m This rese “Magnetic M heir Lifecy “Gentelligen Hübsch for n-situ tensi mechanical t earch is spo Magnesium ycle”. The e nt Part Iden conducting ile tests, An testing. onsored by Alloys” of eddy curren ntification an g the XRD nja Krabben the Germa the Collabo nt measurem nd Integrity phase analy nhöft for re n Research orative Rese ments were y Assessmen ysis, Torste ecording th h Foundatio earch Cente e executed nt”. The aut en Heidenb he SEM ima n (DFG) w er 653 “Gen in cooperat thors would lut and Ma ages and H within the su ntelligent Co tion with su d like to than arcus Engelh Harald Kern ubproject E omponents i ubproject S nk Christop hardt for th nbach for th E2 in S3 ph he he 5. Outlook Apart from the high melting points and the poor dissolving behavior of the ferromagnetic alloying elements in Mg the greatest challenge in the development of sensory Mg alloys is to find the optimum between usable magnetic properties and reasonable mechanical properties. The authors already demonstrated the use of Zn as an additional alloying element in low concentrations [7,8] which improves the strength and, to some degree, the castability. In order to examine the interactions of Co and standard alloying elements like Al, Zn and Mn further investigations are currently made which focus on enhancing the mechanical properties in the as-cast state and primarily the yield strength. These results will be published in a future work. Within the scope of this project, the magnetic Mg alloys’ load-sensitive behavior will also be demonstrated on actual structural components with a complex geometry. For these tests, the rear wheel carrier of a Formula Student racing car of Leibniz Universität Hannover was chosen and produced by casting (Figure 8). Sensors 2013, 13 117 References 1. Denken product na, B.; Hen tion enginee nning, H.; ering. Prod. Lorenzen, . Eng. 2010 L.-E. Gen 0, 4, 65–73. netics and intelligence: New appproaches in 2. Bozorth, R.M. Ferromagnetism, 1st ed.; IEEE Press: New York, NY, USAA, 1951. 3. Bieńkow 1980, 1 wski, A.; Ku 19, 120–122 ulikowski, J 2. J. The magneto-elastic VVillari effect in ferritess. J. Magn. M Magn. Mater. 4. Bieńkow sensors wski, A; Sz s. Acta Phys zewczyk, R s. Pol. A 201 .; Salach, J. 10, 118, 100 Industrial a 08–1009. application of magnetooelastic forcce and torque 5. Ekreem magnet 2007, 1 m, N.B.; O tostriction, 191, 96–101 Olabi, A.G. its use and . .; Prescott, methods to T.; Raffe o measure erty, A.; H these prope Hashmi, M erties. J. M M.S.J. An o Mater. Proce overview o ess. Techno of ol. 6. Borman and Ha of Mag Weimar 2010; p nn, D.; Rod ardmagnetic gnesium–8th r, Germany pp. 27–32. dman, M.; K c Magnesium h Internati y, 26–29 Oc Klose, C.; K m Materials ional Confe ctober 2009 Kerber, K.; s as Compo ference on 9; Kainer, K Reimche, W onents of St Magnesium K.U., Ed.; W W.; Wurz, tructural El m Alloys a Wiley-VCH M.C.; Bach lements. In and their A H: Weinheim h, F.-W. So Proceeding Application m, Germany oft gs ns, y, 7. Klose, alloys b C.; Mroz, based on Mg G.; Rodma gZn and Sm an, M.; Kuj mCo with se at, B.; Borm ensory prope mann, D.; B erties. Adv. Bach, F.-W Eng. Mater W. Magnetic r. 2012, 14, c magnesium 28–34. m 118 © 2013 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 license (http://creativecommons.org/licenses/by/3.0/). Sensors 2013, 13 Sensors 2013, 13 8. Klose, C.; Mroz, G.; Angrisani, G.L.; Kerber, K.; Reimche, W.; Bach, F.-W. Casting process and comparison of the properties of adapted load-sensitive magnesium alloys. Prod. Eng. 2012, doi:10.1007/s11740-012-0413-7. 8. Klose, C.; Mroz, G.; Angrisani, G.L.; Kerber, K.; Reimche, W.; Bach, F.-W. Casting process and comparison of the properties of adapted load-sensitive magnesium alloys. Prod. Eng. 2012, doi:10.1007/s11740-012-0413-7. 9. Kammer, C. Aufbau von Magnesiumlegierungen. In Magnesium-Taschenbuch, 1st ed.; Kammer, C., Ed.; Aluminium-Verlag: Düsseldorf, Germany, 2000; Chapter 8, pp. 155–188. 10. Buchmann, W. Festigkeitseigenschaften. In Magnesium und Seine Legierungen, 2nd ed.; Beck, A., Altwicker, H., Eds.; Springer: Berlin, Germany, 2001; Chapter 5, pp. 101–271. 11. Schultze, W. Chemisches Verhalten, Korrosion und Oberflächenschutz. In Magnesium und Seine Legierungen, 2nd ed.; Beck, A., Altwicker, H., Eds.; Springer: Berlin, Germany, 2001; Chapter 6, pp. 272–312. 12. Shaw, B.A. Corrosion Resistance of Magnesium Alloys. In ASM Handbook Volume 13A: Corrosion: Fundamentals, Testing, and Protection, 1st ed.; Cramer, S.D., Covino, B.S., Eds.; ASM International: Novelty, OH, USA, 2003; pp. 692–696. 13. Hawke, D.L. Corrosion Behavior. In ASM Specialty Handbook: Magnesium and Magnesium Alloys, 1st ed.; Avedesian, M., Baker, H., Eds.; ASM International: Novelty, OH, USA, 1999; pp. 194–210. 14. Wetherill, J.P. Magnesium Alloy. U.S. Patent 1,880,614, 4 October 1932. 14. Wetherill, J.P. Magnesium Alloy. U.S. Patent 1,880,614, 4 October 1932. 15. Yang, Z.; Li, J.P.; Zhang, J.X.; Lorimer, G.W.; Robson, J. Review on research and development of magnesium alloys. Acta Metall. Sin. 2008, 21, 313–328. 16. Nayeb-Hashemi, A.A.; Clark, J.B. The Co-Mg (Cobalt-Magnesium) system. Bull. Alloy Phase Diagrams 1987, 8, 352–355. 17. Buschow, K.H.J. Magnetic Properties of MgCo2, MgNi2 and Mg2Ni. Solid State Commun. 1975, 17, 891–893. 18. Polmear, I.J. Grades and Alloys. In ASM Specialty Handbook: Magnesium and Magnesium Alloys, 1st ed.; Avedesian, M., Baker, H., Eds.; ASM International: Novelty, OH, USA, 1999; pp. 12–25. 19. Feiste, K.L. Entwicklung der Harmonischen-Analyse von Wirbelstromsignalen zur Charakterisierung mechanischer Kugelgraphitgusseigenschaften. Ph.D. Thesis, Universität Hannover, Hannover, Germany, 2003. 20. Gennari, F.C.; Castro, F.J. Formation, composition and stability of Mg-Co compounds. J. Alloys Compounds 2005, 396, 182–192. 20. Gennari, F.C.; Castro, F.J. Formation, composition and stability of Mg-Co compounds. J. Alloys Compounds 2005, 396, 182–192. © 2013 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 license (http://creativecommons.org/licenses/by/3.0/). © 2013 by the authors; licensee MDPI, Basel, Switzerland. Sensors 2013, 13 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 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 license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W1968123059
https://www.scielo.br/j/rbh/a/Tm5jPtWrVj7HwSHNSP6DrBN/?lang=pt&format=pdf
Portuguese
null
Escrevendo Cartas: Jesuítas, Escrita e Missão no Século XVI
Revista brasileira de história
2,002
cc-by
19,770
RESUMO E s te arti go examina a produção e troc a de corre s pondência en tre os mission á- rios jesuítas do século XVI e seus su pe- riores, resgatando o texto inaciano cons- truído gradualmente nessa circulação de cartas entre Europa,Ásia e América.Nes- se tex to, rec u perado nos Ex erc í cios Es p i- ri tu a i s, nas Co n s ti tu i ç õ e s e nas cartas do s a n to, e s t a riam as bases da definição de um método mission á rio para a redu ç ã o do infiel à Fé católica. This arti cle deals with the making and exch a n ge of l et ters bet ween the Je su i t m i s s i on a ries and their su peri ors in the s i x teenth cen tu ry. These let ters circ u l a- ting in Eu rope , Asia and Am erica are b a s ed on Saint Ign ac yo’s wri ti n gs ,m a i n ly the Sp i ri tual Ex erci ce s, the Co n s ti tu ti o n s and his own let ters . Th ey are the bases of the mission a ry met h od for the converti on of the unfaithful to the Catholic rel i gi on . Keywords: jesuits; mission; letters. Palavras-chave: jesuítas; missões; cartas. Somente darey conta a V.R. de nossa chegada a esta ter- ra e do que nella fizemos e esperamos fazer em ho Se- n h or No s s o, dei x a n do os fervores de nossa pro s pera viagem aos Irmãos que mais em particular a notaram Serafim Leite Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI Fernando Torres Londoño* PUC-SP Revista Brasileira de História. São Paulo, v. 22, nº 43, pp. 11-32 2002 INTRODUÇÃO Suas cartas foram se ac u mu l a n do em diversas casas de governo e hoje se en con tram nos arqu ivos de Rom a , L i s boa , É vora , Rio de Janeiro e Madrid2. Esse acervo, mesmo espalhado, se con s ti tu iu na referência para a rec u- peração do passado dos jesuítas no Brasil e da con s trução de sua mem ó ri a . Esta rec u peração passou a ser feita depois da re s t a u ra ç ã o, na segunda met ade do XIX. No caso do Bra s i l , o pad re Serafim Lei te , con s c i en te da import â n c i a destas cartas e do imagi n á rio que elas alimen t avam a re s pei to da Com p a n h i a nos pri m ei ros anos da co l onização lusa no Bra s i l , além de utilizá-las para a redação de sua Hi s t ó ria da Companhia de Je sus no Bra s i l , con ti nuou o tra b a- lho já iniciado por histori adores lei gos do IHGB, como Ca p i s trano de Abreu , que de s de 1885 se tinha em pen h ado em localizar e publicar cartas dos pad re s N ó brega e An ch i et a3. Tra b a l h a n do nos arqu ivos da Companhia em Rom a , Portu gal e Espanha e em diversas prov í n c i a s , p ad re Lei te loc a l i zou cartas per- d i d a s , veri f i cou datas, a utores e de s ti n a t á ri o s , tra n s c reveu e po s teri orm en te p u bl i cou as cartas dos pri m ei ros anos da pre s ença jesu í tica no Bra s i l4. INTRODUÇÃO Assim começou o pad re Nóbrega , provavel m en te a 10 de abril de 1549, na Ba h i a , sua pri m ei ra carta como mission á ri o, ao su peri or em Portu ga l ,p a- d re Simão Rod ri g u e s . N ó brega tinha ch egado em 29 de março, na arm ada de Tomé de So u s a , pri m ei ro govern ador geral do Bra s i l . Menos de qu i n ze dias se tinham passado e o superior da pequena missão que se iniciava já escrevia pa- ra seu su peri or con t a n do-lhe como tinha sido recebi do, on de estavam aloja- do s , o que tinham en con trado na terra , o estado de pec ado e abandono mo- ral em que viviam os portu g u e s e s , os pri m ei ros con t a tos com os índios e o Revista Brasileira de História. São Paulo, v. 22, nº 43, pp. 11-32 2002 Fernando Torres Londoño i n teresse que mostravam em adotar a fé, os avanços na comunicação com es- tes e os planos de catequese e de ir a outras regiões como Pern a m bu co (Lei te , 1 9 5 4 , vol I, pp. 1 0 9 - 1 1 5 ) . Como el e , os outros pad res e irmãos também escre- veram para Portu gal e hoje contamos com três grossos vo lumes de cartas do s primeiros jesuítas entre 1549 e 1563. Até a expulsão da Com p a n h i a , no Brasil e no Pa r á - Ma ra n h ã o, su peri o- re s , p ad res e irmãos não dei x a ram de escrever cart a s , i n form e s , rel a t ó rios e crônicas em que se reco l h eu a vida e o co tidiano da Companhia nas co l ô n i a s portuguesas da Am é ri c a1. Revista Brasileira de História, vol. 22, nº 43 INTRODUÇÃO A pu- blicação destas co l etâneas dispon i bi l i zou para estudiosos e histori adores um ri quíssimo material seri ado para a pe s qu i s a , que nos últimos anos tem sido uti l i z ado para a el a boração de IC, m e s trados e do utorados sobre diversos as- pectos, e em particular sobre o processo de cristianização e as missões5. 12 Nos anos seten t a , José Ca rlos Sebe Bom Mei hy advertiu sobre a pre s en ç a de cartas ed i f i c a n tes e de informação en tre a vo lumosa corre s pondência je- su í ti c a6. As s i m , de s de o começo da missão dos inacianos no Bra s i l , uma boa p a rte das cartas teria sido produzida com o prop ó s i to cl a ro de ed i f i c a r, na ex- pressão ascética da époc a , que apon t ava para as ações que serviam para ma- n i festar a pre s ença divi n a , e s ti mular a Fé do próximo e infundir pied ade7. As c a rtas estavam determ i n adas pela sua função, s eus de s ti n a t á rios e obj etivo s p a rti c u l a re s . Em comunicação de 1997 no Con gresso de Am ericanistas em Q u i to, i n s eri a questão da utilização das cartas e das crônicas dos jesuítas na Am é ri c a , den tro da referência básica do carisma inaciano e de sua ge s t a ç ã o no século XV I . Coincidi assim com o apon t ado por Le a n d ro Ka rn a l , Pa u l o Assunção e Julio Quevedo em suas teses e dissertações8. Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI Pelo seu traço de ori entação de tu do a um “princípio e fundamen to”, a es- p i ri tu a l i d ade inaciana co l oc ava todas as ações do indiv í duo a serviço de Deu s . INTRODUÇÃO E ra na ex pect a tiva de Sa n to Ignácio pela proc u ra da von t ade de Deus que se d i ri gia a vida de cada jesu í t a . Pa ra isto, p a rti c u l a rm en te nas Co n s ti tu i ç õ e s d a ordem e nas suas cart a s , o fundador foi de s dobra n do as instruções para seg u i r o método apre s en t ado nos Ex erc í cios Es p i ri tu a i s. Q u a n do os pad res e irm ã o s com e ç a ram a se comunicar por cartas de s de as mais va ri adas partes do gl obo, e s te espíri to inu n d ava sua escrita nas ex pre s s õ e s , nos assu n tos e episódios re- feri do s . Ao escrever sobre sua missão, os jesuítas o faziam uti l i z a n do um regi s- tro ou tom inspirado na su bj etivi d ade de sua vivência do carisma inac i a n o. Como histori ador, ac red i to que não con s i go ouvir esse regi s tro su bj etivo considerando referências e maneiras de escrever só como edificantes.Da mes- ma form a , as informações pre s en tes nas cartas não se deviam unicamen te ao e s p í ri to de con trole ou ao de s ejo de matar curi o s i d ades (As su n ç ã o, 2 0 0 0 , pp. 8 1 - 8 9 ) . Elas seriam recolhidas e envi adas à Eu ropa con s ti tu i n do tex tos dife- ren c i ado s , produ z i dos como parte de um proj eto mission á rio que estava sen- do con s tru í do e para o qual o poder sem pre foi uma referência fundamen t a l . E nessa construção da missão, a escrita cumpriu um papel estratégico. INTRODUÇÃO Form a n do parte de um proj eto em andamen to, a pre s en to aqui alguns elementos referentes ao contexto jesuítico e do próprio Ignácio de Loyola, que explicam a importância estra t é gica da corre s pondência e o privi l é gio da es- c rita en tre os jesuítas como meio de comu n i c a ç ã o. A partir daí e com p u l s a n- do doc u m en tos da ordem do século XV I , a pon to para a importância que esta correspondência teve para construir um imaginário sobre as Missões da Com- panhia que as fizesse pre s en tes na Eu rop a . Faço isto saben do que estou tri- l h a n do terreno pantanoso para um histori ador, como o da espiri tu a l i d ade , mas ac red i to que explicitar pon derações de método sobre o tra t a m en to da doc u m entação rel i giosa pode ser útil para o futu ro da pe s quisa do cato l i c i s- mo entre nós. 13 Julho de 2002 1. AS CARTAS E A VISIBILIDADE DA “UNIVERSAL COMPANHIA” 5 6 1 - 5 6 3 ) . Im port ava as- s i m , n e s te mom en to, e s t a bel ecer os canais e formas de comunicação da Com p a n h i a , das corri qu ei ras às mais com p l ex a s , como a que passava pel a convocação da Con gregação Gera l . In tere s s ava , em parti c u l a r, cuidar da cir- culação de informações pessoalmente ou por “letras”(número 679). No que diz re s pei to às “l etras missiva s”, determ i n a ram-se obri gações em dois sentidos: entre súditos e superiores e entre casas e províncias. No primei- ro sen ti do, o pad re geral e os provinciais deveriam saber e “en ten der las nu e- vas e inform ac i ones que de unas y otras partes vi en en” ( n ú m ero 673). Pa ra ga ra n tir que as cartas fo s s em re a l m en te envi ad a s , os su peri ores deveriam es- c rever para os provinciais cada semana e estes re s pon deriam e escreveri a m também ao padre geral a cada mês. (número 674). Ainda para 14 (...) mas inform ación de todos se imbíe cada cuatros meses al provi n c i a l , de ca- da casa y co l egio una lista breve du p l i c ada de todos los que hay en la tal casa, y los que faltan por muerte o por otra causa” (número 676). (...) mas inform ación de todos se imbíe cada cuatros meses al provi n c i a l , de ca- da casa y co l egio una lista breve du p l i c ada de todos los que hay en la tal casa, y los que faltan por muerte o por otra causa” (número 676). No seg u n do sen ti do, o governo cen tral ou provincial deveria ordenar “co- mo em cada parte se pueda saber de las otras lo que es para con s o l ación y ed i- f i c ación mutua em el Se ñ or ”( n ú m ero 673). 1. AS CARTAS E A VISIBILIDADE DA “UNIVERSAL COMPANHIA” A Companhia de Je sus nasceu e se esten deu no século XVI a qu a tro con- ti n en tes sob o domínio da escri t a . No mom en to em que a pri m ei ra dúzia de “com p a n h ei ro s” se co l ocou a serviço do papa, com preen deu-se que a disper- são poderia ameaçar sua união e para se manterem unidos em Je sus Cri s to n a s ceu o In s ti tuto. Nas Co n s ti tu i ç õ e s c u i dou Sa n to Inácio de dedicar uma das dez parte s , a oi t ava , a “ De lo que ayuda para unir los rep a rti dos com su cabe- za en tre sí”9. No pri m ei ro número dessa parte co l ocou que pela dificuldade Julho de 2002 Julho de 2002 Fernando Torres Londoño de união dos mem bros com a cabeça e por estarem espalhados en tre fiéis e infiéis deveriam contar com diversas ajudas, (...) pues no con s erva rse puede ni regi rs e , ni por con s i g u i en te con s eguir el fin que preti en de la Compañia a mayor gl oria divina sin estar en tre si y com su ca- beza unidos los miembros della (Loyola, 1963, p. 556, número 655). (...) pues no con s erva rse puede ni regi rs e , ni por con s i g u i en te con s eguir el fin que preti en de la Compañia a mayor gl oria divina sin estar en tre si y com su ca- beza unidos los miembros della (Loyola, 1963, p. 556, número 655). O vínculo entre súditos e superiores através da obediência (número 659), o incentivo do“espírito de corpo”, a uniformidade de vida e doutrina e o com- bate às divisões (números 663-665 e 671-672),a chamada “união dos ânimos” e a comunicação perm a n en te através de cartas (números 662 e 673-676), fo- ram pre s c ri tos por Loyola como re s postas ao desafio da dispersão e da “d i- vers i d ad que no dañe a la unión” ( Loyo l a , 1 9 6 3 , pp. Revista Brasileira de História, vol. 22, nº 43 1. AS CARTAS E A VISIBILIDADE DA “UNIVERSAL COMPANHIA” Pa ra isto os su peri ores escreveri a m ao provincial a cada “qu a tro meses uma letra que con ten ga solamen te las co s a s de ed i f i c ación em la lengua vu l gar de la província y outra em latín del mismo ten or ”( n ú m ero 675). Ambas deveriam ir du p l i c adas para que uma fosse man- d ada ao geral e da outra se fize s s em cópias “tantas vece s , que baste para dar no- ticia a los otros de su Provi n c i a” ( n ú m ero 675). Pa ra ganhar tem po e ga ra n ti r que as notícias circ u l a ri a m , os su peri ores poderiam escrever diret a m en te ao ge- n era l ,m a n d a n do cópia ao provincial e ainda avi s a n do “a los demas de su pro- v í n c i a ,i m bi á n doles copias de las que escri ben al provi n c i a l ”( n ú m ero 675). Foi tra ç ado assim, nas Con s ti tu i ç õ e s , um con ju n to de operações de co- Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI municação que compunha um sistema de inform a ç õ e s . Ne s te sistema se esta- bel eciam re s pon s a bi l i d ades para a geração das informações e de s ti n a t á ri o s de s t a s . Foram fixados pra zo s , determ i n ada a produção de cópias, definida a c i rculação de s t a s , con s i deradas as línguas e apon t ados os temas a serem tra- t ados nas cart a s . Coeren te com o en ten d i m en to hier á rqu i co de Loyo l a , a co- municação deveria ex i s tir de forma vertical para o governo e hori zontal para a união dos ânimos. 1. AS CARTAS E A VISIBILIDADE DA “UNIVERSAL COMPANHIA” Para conseguir este último, nas condições do século XVI, se recorria à duplicação, assim (...) dara orden el general que de las letras que se imbián de las províncias,se ha- gan tantas cop i a s , que basten para proveer a todos los otros provi n c i a l e s” ( n ú- mero 675). (...) dara orden el general que de las letras que se imbián de las províncias,se ha- gan tantas cop i a s , que basten para proveer a todos los otros provi n c i a l e s” ( n ú- mero 675). Sen do a divers i d ade lingüística um ob s t á c u l o, se uti l i z ava o lati m , m a n- tendo as línguas ditas “vulgares”na articulação jesuítica do universal e do par- ti c u l a r. Tu do isto para ga ra n tir a função das cart a s : consolar e ed i f i c a r, d a n do a con h ecer as obras feitas em nome de Deu s ,“ Pa ra que lo de uma prov í n c i a se sepa em outra” ( 6 7 5 ) . E s c rever para que outros lessem , cop i a s s em , d i f u n- d i s s em e guard a s s em . As s i m , na parte tercei ra das Con s ti tuições sobre a for- mação dos jovens jesu í t a s , ao tratar das refei ç õ e s , se manda ler algum livro p i edoso ou “cosas sem eja n tes son como leer letras de ed i f i c ac i ó n” ( Loyo l a , 1963, 470). 15 Ac red i to que este sistema de informações atu ava como su porte para um s i s tema de decisões niti d a m en te inac i a n o : h i er á rqu i co e verti c a l . In formar a p a rtir da base nas cartas peri ó d i c a s . 1. AS CARTAS E A VISIBILIDADE DA “UNIVERSAL COMPANHIA” Reunir regi s tros e interc a m biar op i n i õ e s à proc u ra de uma dec i s ã o. Comunicar por escri to a decisão a partir do gover- no gera l . Acatar e exec utar a decisão nas instâncias. E m bora fosse po s s í vel re- pre s en t a r, em várias oc a s i õ e s , a obed i ê n c i a , p a rti c u l a rm en te o en ten d i m en to desta se deveria impor1 0. Tal sistema de informações perm i tiu pelo menos a proc u ra de alguma uniform i d ade das po l í ticas numa infinidade de ações às ve zes discord a n te s . E s te sistema foi cen tral na ordem e se ge s tou a partir do próprio percurso letrado do fundador e do relevo concedido às letras na Com- panhia de Jesus como se verá em seguida. Julho de 2002 2. ESCRITA E AÇÃO EM INÁCIO DE LOYOLA Esta capac i d ade de cre s cer rápido teria sim que ver com o en ten d i m en to dado pelos pri m ei ros jesuítas ao regi s tro e s c ri to como ex pressão de uma práxis co l oc ada ao serviço da proc u ra da von- t ade divi n a . Aqui como em outras partes realço a figura de Ignácio de Loyo l a . A mem ó ria jesu í tica fixou a imagem de Sa n to Ignácio de Loyola como o h om em da ação. Apre s enta-se aqui outro traço que con s i dero essen c i a l : Lo- yola foi também um hom em da escri t a . E m bora isso reduza sua rica pers o- nalidade,é difícil não ser levado em consideração para quem entre 1524-1556 e s c reveu seis mil oi tocentas e qu i n ze cart a s1 2. De s de sua conva l e s cença na ci- d ade de Loyo l a , pelo feri m en to recebi do em Pa m p l on a , se sabe pelo pr ó pri o santo que a escrita o acompanhou nas suas primeiras experiências espirituais, qu a n do escrevia num caderno palavras de Cri s to e Nossa Sen h ora uti l i z a n do tintas de diferen tes cores para cada um e com “bu ena letra porque era muy bu en escri b a n o” ( Loyo l a , 1 9 6 3 , p. 9 3 ) , como lem brou ao pad re Gonzalez de Ca m a ra sem nen huma mod é s ti a . Na sua autobi ografia ele fala também qu e em Ma n re s a , on de começou propri a m en te sua ex periência espiri tu a l , co l o- cou seus pec ados por escri to, e seg u n do o pad re Ip a rra g u i rre , de s c revia pr á ti- cas espiri tuais num caderno de notas. ( Loyo l a , 1 9 6 3 ,1 8 1 ) . 2. ESCRITA E AÇÃO EM INÁCIO DE LOYOLA A Companhia de Je sus começou a ser ge s t ada em Pa ri s , a partir de 1531, e seu núcl eo inicial foi um gru po de estu d a n tes do Co l é gio de Santa Bárb a ra que se reunia em torno de um basco que tinha fama de homem santo. Incluin- do o pr ó prio Loyo l a , os pri m ei ros jesuítas eram todos mestres em letra s . Na s Julho de 2002 Julho de 2002 Fernando Torres Londoño gerações seg u i n tes já estariam pre s en tes também os do utore s . Os ch a m ado s pri m ei ros com p a n h ei ros va l ori z a ram de s de o início os aspectos rel ac i on ado s com as letra s , o que com preendia escrever e ler em vernáculo e em lati m , ter con h ec i m en to de outras línguas e de tex tos ex i s ten tes em grego e lati m . As- s i m , obri gações comuns aos pad res de maior gra u , os ch a m ados profe s s o s , como o de viver de esmolas, não se aplicava aos jovens que deveriam ded i c a r- se ao estu do. Pa ra isto, se bu s cou doações e pro teções que foram con s ti tu i n- do um patrimônio destinado a ser investido na formação dos jovens. Uma or- dem de s cen tra l i z ada mas ao mesmo tem po hier á rquica como a Com p a n h i a de Jesus só podia ser letrada no século XVI11. p Porém o fato de se tratar de uma ordem de letrados não explica su f i c i en- tem en te uma força de expansão tão gra n de . Revista Brasileira de História, vol. 22, nº 43 2. ESCRITA E AÇÃO EM INÁCIO DE LOYOLA Aprox i m ad a m en te dez anos depoi s ,e s tes pri m ei ros regi s tros eram já o livro dos Exercícios Espi- ri tuais (Loyo l a , 1 9 6 3 , p. 1 9 6 ) . Em matéria de ascética e místi c a , Loyola ainda produ z iu o Di á rio Espiri tual (Loyo l a , 1 9 6 3 , p. 3 1 8 ) , uma minuciosa de s c ri ç ã o dos estados de sua alma du ra n te um processo de oração en tre 1544 e 1545, p a ra tomar uma decisão a re s pei to da pobreza nas casas professas da nascen- te Companhia. De forma mais aprimorada foram registrados aqui estados es- p i ri tuais e sensações viven c i adas e lidas por Loyola na proc u ra de sinais da von t ade divi n a . E s te vínculo en tre escrita e devoção o acompanhou até o fim de sua vi d a , qu a n do escreven do as Con s ti tuições “c ada dia escri bia lo que pa- saba por su alma” (Loyola, 1963, p. 159). A é é d f d ã d C hi d J Iñi à 16 Até as vésperas da fundação da Companhia de Je su s ,I ñ i go ac re s cen tou à sua cultu ra letrada os estu dos de artes e teo l ogia em Pa ri s , e pri n c i p a l m en te Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI e s c reveu cartas às mais va ri adas pe s s oas def i n i n do assim um traço de sua ati- vi d ade ep i s to l a r. 2. ESCRITA E AÇÃO EM INÁCIO DE LOYOLA Pri m ei ro como Iñigo e depois como o pad re Ign á c i o, Loyo l a e s c reveu a diversas pe s s oas preem i n en tes en tre elas o papa, o imperador Ca r- los V, reis, rainhas,nobres, doutores,e também pessoas de condição mais sim- ples como rel i gi o s o s , cl é ri gos e as ch a m adas pe s s oas espiri tu a i s1 3. E s te traço é i m port a n te porque Loyola tra n s i t ava en tre tra t a m en to s , eti qu etas e esti l o s bem diferen tes de forma flu i d a . A todas essas pe s s oas Loyola se diri giu com um intu i to, mesmo que fosse tão simplório como informá-las de que re z ava por el a s1 4. A consciência das ex pect a tivas do interl oc utor e dos obj etivos do red a tor, o su peri or geral quis passar para seus com p a n h ei ros que não ti n h a m sua mesma ex periência e históri a . As s i m , e s c reven do ao pad re Ber ze , em 24/2/1554, recomendava que quando se escrevesse para pessoas de muita qua- l i d ade e inteligência de fora da Companhia "se deten ga menos el que escri be en las cosas que particolarmente tocan a personas de la Compañia, extendien- dose mas en las generales". (Loyola, 1963, p.854). E s c rever era para Loyola um ato com a n d ado por um sen ti do. Ele escre- veu os Ex erc í cios Es p i ri tu a i s p a ra ensinar e acom p a n h a r, as Co n s ti tuições p a ra regulamentar, as Instruções aos membros da Companhia para manter a união, s eus diários para en ten der sua pr ó pria espiri tu a l i d ade , e as cartas como for- ma de agir e comunicar sobre os mais va ri ados assu n tos e situ a ç õ e s . 2. ESCRITA E AÇÃO EM INÁCIO DE LOYOLA Loyo l a ac red i t ava na comunicação como forma privi l egi ada de ação, e se seg u i rm o s a Ba rthes no seu ensaio sobre os Ex erc í cios Es p i ri tu a i s on de iden ti f i cou qu a- tro tex to s , podemos dizer que nas cartas do santo coex i s tiam vários tex tos e v á rios de s ti n a t á ri o s1 5. Em tem pos de con f l i to ou de pers eg u i ç õ e s , como as acon tecidas qu a n do estu d a n te , recorreu à escrita para se defen der, a r g ü i n do, ref ut a n do. Q u a n do já havia se torn ado influ en te escrevia para conven cer, de- finir, decidir, reclamar, dissuadir, agradecer. Posteriormente, quando em exer- cício como pri m ei ro geral da Com p a n h i a , e s c revia ainda para influ i r, i n for- m a r, d i s cordar e ped i r1 6. Como assinalou Ba rt h e s , i den ti f i c a n do na el eição a função dos Exercícios Espirituais: a escrita em Loyola era uma práxis17. 17 17 3. LETRAS, INFORMAÇÃO E GOVERNO Sob a influência do padre Ignácio a Companhia, desde os primeiros anos, uti l i zou a escrita como forma predom i n a n te de comu n i c a ç ã o, ação e regi s tro. Já foi men c i on ado que no início os jesuítas estavam dispersos pela It á l i a , Ir- l a n d a , Portu ga l , E s p a n h a , França e depois Ásia. As s i m , o obj etivo fundamen- tal de qu a l qu er carta era a união dos ânimos em torno da proc u ra da von t a- de de Deus. Ignácio de Loyola, como primeiro superior geral, teve muito claro que havia de produzir uma imagem da Companhia através das letra s .Q u a l- Julho de 2002 Fernando Torres Londoño qu er notícia deveria pri m ei ro ed i f i c a r, e para con s eguir a consolação nad a m el h or que mostrar os avanços da gl ó ria divina nas obras e ações apo s t ó l i c a s dos pad res e irm ã o s . Sen do este o obj etivo, a missiva não poderia ser dei x ad a ao acaso das impertinências co tidianas dos pad res ou à inten s i d ade de seu s s en ti m en tos espiri tu a i s . E s c reven do para serem lidos por mu i tos outro s , o s p ad res deveriam ter a consciência de que estavam produ z i n do um tex to para ser interpretado e lembrado. Porém su peri ore s , provinciais e o governo gera l , prec i s avam de inform a- ções e notícias para tomar decisões rel a tivas ao envio de pad re s , à abertu ra de re s i d ê n c i a s ,n omeação de su peri ore s , proc u ra de auxílio de nobres e podero- s o s , e em mu i tos casos, correção de de s vios e abu s o s . 3. LETRAS, INFORMAÇÃO E GOVERNO Sobre isto era urgen te e s c rever mas de forma sep a rad a , e s t a bel ecen do a diferença en tre o produ z i do p a ra mostrar e edificar e as novi d ades do com p l exo co tidiano das casas, eiva- do de sen ti m en tos que deveriam perm a n ecer re s ervados aos su peri ores e in- tere s s ado s . Di ferença de tex to pre s c rita por qu em fez da iden tificação de dis- tinção de estados e meios um dos el em en tos fundamentais do método do s Exercícios Espirituais. E n tre várias manifestações do santo con cern en tes a esse en ten d i m en to da mu l ti p l i c i d ade de tex tos pre s en tes na corre s pon d ê n c i a , ex i s te uma cart a de dezembro de 1542,escrita para o padre Pedro Fabro, que estava por ordem do papa na Al em a n h a , na corte de Ca rlos V. O pri m ei ro com p a n h ei ro de Iñi- go de s de os tem pos de Pa ris se qu ei x ava de ter que escrever com freq ü ê n c i a , dei x a n do o santo impac i en te pela falta de com preensão da importância das cartas para a Companhia e de suas diversas formas, conteúdos e sentidos.San- to Inácio escreve ao companheiro de forma direta e repetitiva na ênfase de es- c rever a Roma a cada qu i n ze dias. O pad re geral distingue aqui o que seria a “carta principal” das “hijuelas”. 18 A “carta principal” deveria ser escrita de tal forma que ( . . . ) se pudiese mostrar a cualqu i er pers on a ; porque a mu chos que nos son bi en a f i c i on ados y desan ver nu e s tras cart a s ,no las osamos mostrar por no traer ni guar- dar orden alguna y halando de cosas imperti n en tes en ellas (Loyo l a ,1 9 6 3 , p. Revista Brasileira de História, vol. 22, nº 43 3. LETRAS, INFORMAÇÃO E GOVERNO Ainda para não deixar dúvi d a ,m en c i on a- va como refazia a cópia das cartas que recebia de diversas partes antes de en- viá-las a outros jesu í t a s , reti ra n do “lo que es ed i f i c ación y pon er po s tpon er las mismas palabra s , cort a n do e qu i t a n do las imperti n en te s , por daros a to- dos placer en el sen or nu e s tro y ed i f i c ación de los que las oyeren de nu evo “(Loyola, 1963, p.650)18. Ap a rece aqui a consciência cl a ra de que nas cartas se produzia a imagem da Companhia para provocar edificação e apoi o. Re s pon s a bi l i d ade de todo s os jesu í t a s , mas pri m ei ro do pad re gera l , a qu em corre s pondia distri buir a in- formação e acertá-la de acordo com o modelo de ed i f i c a ç ã o. Esta proc u ra era o que con duzia o cri t é rio para a definição do perti n en te . Gestação da escri t a en c a rada como uma mon t a gem definida pelos seus fins e de s ti n a t á ri o s . F i c a- va assim em evidência tanto o con h ec i m en to do universo letrado de sua épo- c a , á vi do que era o santo pelo saber, qu a n to sua consciência da carta com o tex to que não podia ser dilu í do para não ter que “m o s trar em parte y en c u- brir em parte” (Loyola, 1963, p.649). 19 As “h iju el a s”, por sua ve z , não eram meros anexo s , em bora o santo não as privilegiasse neste texto. 3. LETRAS, INFORMAÇÃO E GOVERNO 6 4 9 ) . ( . . . ) se pudiese mostrar a cualqu i er pers on a ; porque a mu chos que nos son bi en a f i c i on ados y desan ver nu e s tras cart a s ,no las osamos mostrar por no traer ni guar- dar orden alguna y halando de cosas imperti n en tes en ellas (Loyo l a ,1 9 6 3 , p. 6 4 9 ) . Nessa carta principal escrita para provocar edificação em “oi dores o lec- tore s”, se deveria co l ocar o que se fazia em serm õ e s , con f i s s õ e s , Exerc í c i o s , obras pias. Pa ra que ficasse mais cl a ra a forma de produzir esta carta pri n c i- pal, o santo dizia como ele fazia as suas, narrando as coisas que edificavam: (...) y despues mira n do y corri g u i en do, h ac i en do cuenta que todos la han de ver, torno a escri bir o hacer escri bir otra ve z , porque lo que se escri be es aún mu ch o mas de mirar que lo que se habl a ; porque la escri tu ra qu ed a , y da siem pre te s ti- Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI m on i o, y no se puede así bi en soldar ni glosar tan fac i l m en te como qu a n do ha- blamos (Loyola, 1963, p.649). Desta forma,segundo o santo, escrevendo duas vezes ele se persuadia que as letras fo s s em “más con cert adas y más disti n t a s”. Produção do tex to com a cl a reza de seu caráter doc u m en t a l . 3. LETRAS, INFORMAÇÃO E GOVERNO Ali seriam colocadas as “otras particularidades im- perti n en tes para la carta pri n c i p a l ” como doen ç a s ,n ova s ,n eg ó c i o s , poden do “d i l a t a rse em palabras ex h ort a n do”. Desta forma “en las hiju elas puede cad a uno escri bir a priesa de la abundancia del corazón con cert ado o sin con c i er- to ; mas en la principal no se su f re , si no va con algun estudio disti n to e ed i f i- c a tivo para poderse mostrar e ed i f i c a r ”( Loyo l a , 1 9 6 3 , p. 6 5 0 ) . Ca beria aqu i tu do o que pudesse não ed i f i c a r, o em oc i on a l , o pri m á ri o, o espon t â n eo ou sem elaboração e por isso não deveria ser mostrado ou dado a público. F i n a l m en te o santo, i nvoc a n do a obed i ê n c i a , dizia que seg u i ria insisti n do nesse pon to e assim rogava e pedia para que “os em en deis em vu e s tro escri- bi r, prec i á n doos dello y de s e a n do edificar a vu e s tros hermanos y a los proj i- mos com vu e s tras letra s” ( Loyo l a ,1 9 6 3 , p. 6 5 0 ) .A edificação ganha assim, n e s- te con tex to, um traço de modern i d ade on de através da escrita se co l oc ava em evidência o real além de uma rel ei tu ra à luz da ex periência de cri s ti a n i z a ç ã o. Não precisou Sa n to Ignácio de todos aqu eles rogo s , no que dizia re s pei- to a escrever, p a ra com outro com p a n h ei ro e gra n de amigo Fra n c i s co Xavi er. Julho de 2002 3. LETRAS, INFORMAÇÃO E GOVERNO Q u eri a s a ber, que não era só curi o s i d ade ou fervor mission á ri o, mas também inte- resse em estar inform ado para poder participar e intervir com recom en d a- ções e com en t á ri o s . Fra n c i s co Xavi er se fazia assim pre s en te de s de a Ásia com suas cartas e não dem orou para que mu i tos de s eja s s em lê-las e con h ec ê - l a s na Europa. 20 Porém o que a Companhia qu eri a , ou mel h or, prec i s ava saber? A esta pergunta feita pelo reitor da casa de formação de Coimbra,padre Urbano Fer- n a n de s , re s pon deu o pad re Po l a n co mandado por pad re In á c i o, em pri m ei ro de junho de 1551. 3. LETRAS, INFORMAÇÃO E GOVERNO Si n ton i z ados em mu i tos aspecto s , os dois santos se iden ti f i c a ram também na sua utilização da escrita na Com p a n h i a . Fra n c i s co Xavi er fez também das le- tras o seu principal meio de comunicação em relação aos com p a n h ei ros qu e Fernando Torres Londoño f i c a ram na Eu ropa e com os outros mission á rios que estavam se espalhando pela Ásia. As s i m , qu a n do saiu de Goa , em 14 de abril de 1552, dei xou instru- ções ao padre Barzeo, de escrever todos os anos ao padre Ignácio, sobre as no- vas dos irmãos (Xavi er, 1953 , p. 489) “y esto de escri bir y rec i bir cartas harei s com mucha diligência como esto se cumpla”(Xavier, 1953, p. 492). Recomen- d ava , a i n d a , que se fize s s em várias cópias das cart a s . Também insistia para que lhe escrevessem a Malaca “todos los años no faltareis”(Xavier,1953,p.492), acrescentando que (...) y sea la carta que vos me escri bi reis muy cumplida, en que me hagais saber muchas cosas así de las nuevas del estado de la Índia,como del fruto que los otros religiosos hacen a gloria de Dios y el fruto de las almas (Xavier, 1953, p. 492). (...) y sea la carta que vos me escri bi reis muy cumplida, en que me hagais saber muchas cosas así de las nuevas del estado de la Índia,como del fruto que los otros religiosos hacen a gloria de Dios y el fruto de las almas (Xavier, 1953, p. 492). Pa rti n do para tão lon ge , Fra n c i s co Xavi er qu eria se manter a par do qu e acon tecia em Goa tanto no plano do tem poral como no avanço da gl ó ria de Deu s . Q u eria saber “as nova s” e em que condições andava a missão. Revista Brasileira de História, vol. 22, nº 43 3. LETRAS, INFORMAÇÃO E GOVERNO O sec ret á rio do su peri or gera l , diz inicialmen te , que isto de escrever aos su peri ores uma vez por mês não só com preen de as novas de edificação e o fruto espiri tual em confissões e pregações porqu e , p a ra isto, basta escrever cada qu a tro meses, mas o que “nu e s tro pad re desea saber es to- do aqu ello (en cuanto se podrá) que convi ene sepa para mas ayudar y mej or satisfazer el cargo que Dios Nuestro Señor le ha dado”.“Como existen inúme- ras coisas das que podem cuidar os su peri ores loc a i s”, con ti nua pad re Po l a n- co, “h o l ga ria nu e s tro pad re se le diese inform ación de las cosas que mas im- portan y de las que tienen mas dificultad”(Loyola,1963, p.766).Como se trata de uma casa de form a ç ã o, o santo está intere s s ado no modo de proceder do s e s tu d a n te s , nos seus estu dos e na vida espiri tu a l , qu em aproveita mais qu e o utros na do utrina e na graça de prega r,“quiénes estan en disposición de ser i m bi ados por unas partes e outras ya ac a b ado el curso ord i n á rio de sus estú- dios”(Loyola, 1963, p.766). Nas cartas mensais ao su peri or, o pad re geral de s eja saber inform a ç õ e s con c ret a s , tais como o número dos que estão na casa ou “qu i enes en tran y se s a l en o se de s p i den”, como é o co tidiano da form a ç ã o, qu em mostra já sinais Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI de poder ser aprovei t ado em algum apo s to l ado. 3. LETRAS, INFORMAÇÃO E GOVERNO F i c a n do o tex to ed i f i c a n te , com os números de confissões e pregações e atos notáveis por conta das car- tas principais de cada qu a tro meses, i n teressa aqui o import a n te , o que inci- dia na vida dos jesuítas e na sua missão, a quilo no qual havia dúvidas ou difi- c u l d ade s . O que supunha sel ec i onar as informações que deveriam ser a pre s en t adas às diversas instâncias da obed i ê n c i a , pon dera n do sem pre às hie- ra rquias su peri ore s . O que não foi fácil, a ju l gar pelas histórias das prov í n c i a s e pelas ve zes em que este tem a , pre s en te também nas Con s ti tu i ç õ e s , vo l tou a a p a recer nas cartas do fundador, como na célebre “ In s trução sobre el modo de tratar o negociar com cualqu i er su peri or ” de maio de 1555, já com en t ad a aqui (Loyola, 1963, p.923) Com a importância con ferida a estas cartas de mês nas Con s ti tu i ç õ e s , como já foi vi s to, e com o em penho do gen eral em recebê-las e con te s t á - l a s através de seu secretário e copistas,se foi constituindo a partir do governo ge- ra l , a base de um sistema de inform a ç õ e s . O que não é de estranhar numa or- dem inserida num tem po em que a corre s pon d ê n c i a , a crônica, o informe se- c reto, o mapa, os vi s i t adore s , faziam parte dos instru m en tos de con trole e das formas de pre s ença de Estados co l onialistas como os da pen í n sula ibéri c a , aliás Estados que compunham a base regi onal da Companhia no século XV I . 3. LETRAS, INFORMAÇÃO E GOVERNO Ainda que com a prec a ri ed ade das comunicações da época e com a re s i s t ê n- cia de não po u cos su peri ore s , esse sistema envo lvia toda uma estrutu ra de re- gi s tro s , cop i s t a s , envi o s , a rqu ivo s , em função de asseg u rar a comunicação e forn ecer ao su peri or geral e outros su peri ores os el em en tos para suas dec i- sões naqu ele del i c ado equ i l í brio en tre o cen tralismo e a auton om i a , o alto e o b a i xo que con s ti tu iu a Companhia de Je su s . Em cartas ou rel a t ó rios o jesu í t a se comu n i c ava com os su peri ores distantes inform a n do, con su l t a n do, op i- n a n do, d i s cord a n do, a s s i n a l a n do sua disposição a obedecer. E ra também por c a rtas que o governo gera l , os provinciais e os rei tores de co l é gio tra n s m i ti a m suas decisões, envios e destinos aos súditos que se encontravam longe. 21 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES Nesse processo de ida e volta na proc u ra da von t ade divi n a , ac red i to qu e se foi con s tru i n do e def i n i n do nas suas caracter í s ticas a missão jesu í ti c a . A pre s ença da Companhia na Eu ropa e em outros con ti n en tes envo lveu de s de o começo uma teia de relações e interesses tanto fora como den tro da ordem , que foram determ i n a n tes e ex i gi ram gra n de habi l i d ade para seu geren c i a- m en to a partir do governo cen tra l : o serviço ao papa na defesa e expansão da F é ,a s s ociação com Estados católico s , com os reis de Portu ga l , com o impera- dor Ca rlos V e com os reis da Espanha na cri s tianização das colônias e terri- Julho de 2002 Fernando Torres Londoño t ó rios de ultra m a r. E s t a bel eceu ainda vínculos com nobres e gra n des famílias da Itália e da Espanha de on de provinham voc a ç õ e s , auxílios econ ô m i co s , doa ç õ e s , pro te ç ã o. Com relação à expansão da Companhia fora de Espanha, Portu ga l , Itália e Fra n ç a , era feita fundamen t a l m en te a partir da pre s ença de padres e irmãos,às vezes os mais qualificados membros destas províncias. De- veriam também ser arbi trados ciúmes e qu eixas numa mediação en tre a obe- diência e o apelo pela dispon i bi l i d ade que deveria caracterizar o jesu í t a . 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES O papado, os Estados,os nobres, os padres da Companhia criaram assim expec- t a tivas que eram maiore s , na medida em qu e , com obras como os co l é gios ou com a presença fora da Europa, os jesuítas ficavam em evidência.Estas expec- t a tivas deveriam ser sati s feitas tanto na con s trução e ad m i n i s tração de uma i m a gem , como na direção e caracter í s ticas da ex p a n s ã o. No intu i to de alcan- çar estes obj etivo s , a corre s pondência foi fart a m en te uti l i z ada e a produ ç ã o de textos privilegiada. Nessa teia de ex pect a tivas com relação às missões fora da Eu rop a , os je- suítas re s pon deram produ z i n do um imagi n á rio mission á rio e cri a n do um m é todo de atuação en tre infiéis, a m bos se con f i g u ra n do na corre s pon d ê n c i a entre os padres e irmãos. Í O envio de jesuítas à Índia em 1541, pre s i d i dos por Fra n c i s co Xavi er, con s ti tui o pri m ei ro gra n de referen te na produção desses dois instru m en to s . A partir de sua atuação na Índia, em diversas partes da Ásia até ch egar ao Ja- pão em 1549 e morrer em 1552, Fra n c i s co Xavi er passou a en c a rnar o mis- sionário, o apóstolo jesuíta por excelência.Essa imagem de Francisco que ser- vi ria de modelo a todos os outros mission á rios da ordem , começou a ser con s truída ainda em Lisboa com a distinção que ele logo alcançou na corte , recolhida na corre s pondência dos pri m ei ros jesuítas portugueses e nas su a s pr ó prias cart a s1 9. Revista Brasileira de História, vol. 22, nº 43 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES As s i m , de Goa , Xavi er escrevia a santo Ign á c i o, em outu bro de 1542, do sen ti m en to causado en tre as crianças que catequ i z ava dizen do que “no me dejaban los mu ch achos ni rezar mi of i c i o, ni com er ni dorm i r, s i- no que les enseñase algunas orac i on e s” ( Xavi er, 1 9 5 3 , p. 1 0 8 ) . A figura do de- d i c ado e incansável mission á rio e dos frutos que produzia no anúncio da Fé católica foi se cri s t a l i z a n do no modelo de edificação que se podia ex trair das missões en tre infiéis, p a ra ser espalhado nas cortes de Eu ropa e en tre os jesu í- t a s . Ao mesmo tem po Xavi er se con s ti tu iu no pri m ei ro referencial para a de- finição de um método de atuação da companhia en tre infiéis. Con s c i en te de s er o pri m ei ro a estar en tre infiéis, qu a n do ainda em Lisboa , a n tes de parti r p a ra a Índia, Xavi er pedia a santo Inácio que lhe escrevesse “muy largo”s obre o modo de proceder en tre eles e a mel h or manei ra de servir a Deus “que all á os pareciere que debemos hacer” (Xavier, 1953, p. 76). 22 A edificação que se podia extrair da atuação missionária e que se foi cons- Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI truindo nas cartas de Xavier ficou definida pelo componente da conversão no en ten d i m en to inac i a n o. Pa ra a produção da ed i f i c a ç ã o, os reinos da África e da Ásia onde chegavam os missionários, traziam novos cenários definidos pe- la pre s ença do fiel na fren te do infiel . 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES Uma po l a ri d ade com traços pr ó pri o s que tinha como paradigma mais distante o tempo dos apóstolos Pedro e Pau- lo en tre ju deu s , gregos e rom a n o s . Po l a ri d ade que só podia ser su primida pe- la produção da conversão e da incorporação à Igreja dos novos cristãos. Como nas com posições de tem po e lu gar das meditações dos Exerc í c i o s Espirituais,os cenários das missões presentes nas cartas de Xavier e seus com- p a n h ei ros são def i n i dos pelos pers on a gens que neles se localizam e pelas re- lações assim gerad a s . No cen tro o mission á rio caracteri z ado pelo seu zelo na s a lvação das almas pre s en te nas inúmeras prega ç õ e s , b a ti s m o s , confissões e pelos perigos que corre por amor a Jesus Cristo. Junto a ele os europeus, prin- c i p a l m en te as autori d ades co l oniais que patrocinam a cri s tianização e os co- l onos en tre os que se produ zem as pri m ei ras reformas de vida movidas pel a pregação con s o l adora e a reconciliação da con f i s s ã o. À sua fren te os infiéis. 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES Os govern a n te s , os interl oc utores por excel ê n c i a , os pri m ei ros a serem con- tem p l ado s , que são tra t ados com con s i deração e su avi d ade na esperança qu e tra gam também seus súdito s , os hom ens sábios aos quais se prega para mo- ver suas inteligências em direção à sabedoria divi n a , as crianças que são fac i l- mente atraídas e para quem se fundam escolas.Finalmente os homens e mulhe- res adultos,tanto aqueles que se convertem e passam a ser cristãos exemplares, como os que relutam em aceitar o Eva n gelho por se pren der a seus erros an- ti go s ,“pues los gra n des ni a malas ni a bu enas qu i eren ir al para í s o”, como es- c reve Xavi er, de Goa , a Ign á c i o, em 1544 (Xavi er, 1 9 5 3 , p. 1 3 7 ) . Não falta fi- nalmente neste cenário insinuada mais clara,a presença do demônio,o inimigo do gênero humano na linguagem inaciana. 23 A cristalização de um modelo de edificação mission á rio cen trado nas n a rra tivas da conversão dos infiéis e sua circulação en tre os jesuítas e não-je- suítas foi percebida cl a ra m en te por santo Inácio nos anos cinqüen t a , qu a n do escrevendo para o Brasil e para a Índia por intermédio do padre Polanco, aler- t ava sobre o peri go de só escrever cartas de ed i f i c a ç ã o. Como já o tinha fei to com os com p a n h ei ros que atu avam na Eu rop a , o santo dizia a Nóbrega , em julho de 1553, que a ele intere s s ava saber “no solamen te de cosas de ed i f i c a- c i ó n” ( Lei te , 1 9 5 4 , I , p. 5 1 2 ) . 4. FRANCISCO XAVIER E O MODELO DA EDIFICAÇÃO NAS MISSÕES Também pad re Ber ze , m i s s i on á rio da Índia, l h e e s c reve em feverei ro de 1554 dizen do que “Es verd ad que para la ed i f i c ac i ó n de las pers onas de la Compañia lo que toca a los parti c u l a res de ella es muy al prop ó s i to ; pero pod ria venir de por si” ( Loyo l a , 1 9 6 3 , p. 8 5 5 ) . As s i m , a ed i f i c a- ção tão bu s c ada no início dos anos qu a ren t a , já nos cinqüenta estava pre s en- te num modelo cristalizado podendo “Venir de por si”. Julho de 2002 Fernando Torres Londoño 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL 24 (...) y algunos parti c u l a res de más autori d ad y dep u é s , sin tu mu l to, s i en do esto s d i p u e s to s , se mire se pod ra fazer ay u n t a m i en to de los que más esti m ados son en do trina en aqu ellos rei n o s ; y sin que se les quitase interesse ninguno ni cosa qu e ellos mucho estimen,hacerles capaces de las verdades católicas y de lo que se de- be tener em la Iglesia. (Loyola, 1963, p.911) (...) y algunos parti c u l a res de más autori d ad y dep u é s , sin tu mu l to, s i en do esto s d i p u e s to s , se mire se pod ra fazer ay u n t a m i en to de los que más esti m ados son en do trina en aqu ellos rei n o s ; y sin que se les quitase interesse ninguno ni cosa qu e ellos mucho estimen,hacerles capaces de las verdades católicas y de lo que se de- be tener em la Iglesia. (Loyola, 1963, p.911) Tratava-se pois, de “reducirlos a uniformidad con la Iglesia católica”, mas s em violência con tra os ânimos habi tu ados a outra forma de viver. Recom en- d ava que olhassem os abusos e de s ordens que podiam ser reform ados su ave- m en te “y em modo que los de la ti erra vean cl a ra m en te que la reform ac i ó n era nece s á ri a , y de aqu ellos se com i en ce , por que sera gran autori d ad para la reformación de otros” (Loyola, 1963, p.913). 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL Como na Eu rop a , a principal preocupação do su peri or geral na corre s- pondência com seus súditos nos outros continentes foi a conservação da Com- panhia e a qu a l i d ade e permanência dos frutos produ z i dos pelos mission á- rios,o que para o padre Ignácio e seus sucessores dependia do aperfeiçoamento do método para atuar en tre infiéis. Na instrução ao pad re Nuñes envi ado em 1555 como patriarca de uma missão à Etiópia pedida por dom João III de Por- tugal,aparece da própria pena de santo Ignácio alguns dos principais elemen- tos do entendimento da missão entre infiéis.A Etiópia era governada pelo ne- gus Cl á u d i o, que para os portugueses de s cendia do len d á rio Pre s te Jo ã o, de qu em se ac red i t ava seria soberano de um reino cristão na Áfri c a . Na instru- ção santo Ignácio realça o que chama “ganar el ánimo del Pre s te” e de “a l g u- nas pers onas gra n de s”. Recom enda que o patri a rca deverá utilizar todos os m eios hon e s tos para se fazer qu erer bem do Pre s te , comu n i c a n do sobre a sal- vação na Igreja católica rom a n a , ac red i t a n do o santo que “en este gen era l , s i se le puede persu ad i r, se ganan mu chos parti c u l a re s , que dél depen den y po- co a poco se pueden dedu c i r ”( Loyo l a , 1 9 6 3 , p. 9 1 0 ) . Como se tra t ava de um reino cristão que teria caído em erro s , deveriam os mission á rios fazer tom a r ciência destes ao Preste. 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL (Loyola, 1963, p.912) (...) ayudaria mucho para la reducción entre de aquellos reinos,así para los prin- cipios como para todo ti em po, que allá en Eti opia hiciesen mu chas escuelas de l eer y escri bi r, y otras letras y co l egios para insti tuir la juven tu d , y tambi en los demas que lo habran men e s ter en la leguna lati n a , y co s tu m bres y doctrina cri s- ti a n a , que esto seria la salud de aqu ella nac i ó n ; porque estos crec i en do ten d ri a n afición a lo que al principio hibi e s en apren d i do, y en lo que le parecería exceder a sus mayores,y en breve caerian y se extinguirián los errores y abusos de los vie- jos. (Loyola, 1963, p.912) Tod avi a , Inácio tra t ava dos mais va ri ados aspecto s , como a formação de i n t é rprete s , os ti pos de orn a m en to s , a pre s ença de livro s , a vinda de rel í qu i a s de santos, a distribuição de dioceses e o governo do patriarca. Contando já com os resultados das várias missões fundadas por São Fran- c i s co Xavi er na Ásia, de posse das informações que ch egavam do Bra s i l , com as Con s ti tuições en tra n do na sua fase final de redação e servi n do-se da ex pe- riência ac u mu l ada no com b a te dos jesuítas à “h ere s i a” na Al em a n h a , Sa n to Ignácio estava em condições de aplicar a um proj eto mission á rio tão pec u l i a r como o da Eti ó p i a , os meios e pr á ticas ado t ados num perc u rso espiri tual qu e começava nos Exercícios Espirituais. Cen tral na espiri tu a l i d ade inaciana o princípio de que “o bem qu a n to mais universal mais divi n o”, teve na missão um de seus principais campos de a p l i c a ç ã o. 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL Para esta reforma (...) en sus abusos deveria ayudar ademas de los sac ra m en tos la introdu cción de algunas fiestas sen s i bl e s , como serían proce s i ones del Cu erpo de Cri s to Nu e s tro Se ñ or, o otras usadas en la Iglesia católica en lu gar de sus bapti s m o s , etc ; porqu e aun nuestro vulgo,que es menos grosero,se ayuda con esto”(Loyola,1963,p.911). (...) en sus abusos deveria ayudar ademas de los sac ra m en tos la introdu cción de algunas fiestas sen s i bl e s , como serían proce s i ones del Cu erpo de Cri s to Nu e s tro Se ñ or, o otras usadas en la Iglesia católica en lu gar de sus bapti s m o s , etc ; porqu e aun nuestro vulgo,que es menos grosero,se ayuda con esto”(Loyola,1963,p.911). Preten den do ainda a con s ervação católica da Eti ó p i a ,ac red i t ava o santo que Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI (...) ayudaria mucho para la reducción entre de aquellos reinos,así para los prin- cipios como para todo ti em po, que allá en Eti opia hiciesen mu chas escuelas de l eer y escri bi r, y otras letras y co l egios para insti tuir la juven tu d , y tambi en los demas que lo habran men e s ter en la leguna lati n a , y co s tu m bres y doctrina cri s- ti a n a , que esto seria la salud de aqu ella nac i ó n ; porque estos crec i en do ten d ri a n afición a lo que al principio hibi e s en apren d i do, y en lo que le parecería exceder a sus mayores,y en breve caerian y se extinguirián los errores y abusos de los vie- jos. 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL As s i m , na parte sétima das Co n s ti tu i ç õ e s, no número 612, com rel a- ção ao envio a missões por parte do papa, se deveria esco l h er os que fo s s em “conven i en tes o mas pr ó prios para ello mira n do el mayor bi en univers a l ”( Lo- yo l a ,1 9 6 3 , p. 5 4 5 ) . No tem po do envio no número 615 também se deveria con- templar “ el mayor o men or fru cto espiri tual que se sinti ere hacerse o en otra p a rte se espera , o segun pare s c i ere mas conven i en te par algun bi en univers a l ” ( Loyo l a ,1 9 6 3 , p. 5 4 5 ) .E s te en ten d i m en to do bem universal como mais divi n o ganhou nas Con s ti tuições formulação cl a ra no número 622, ao con s i derar pa- ra o envio lu ga res e pe s s oas qu e , por sua con d i ç ã o, o bem re a l i z ado se esten- deria a mu i to s ,t a n to no caso daqu eles con s i derados gra n de s , tais como pr í n- c i pe s , prel ado s ,s en h ore s ,m a gi s trado s , como da mesma forma para com 25 (...) a pers onas señaladas em letras y auctori d ad , debe ten erse por más de im- port â n c i a , por la mesma ra zon del bi en ser mas univers a l , pó lo cual también la ayuda que se hiciese a gen tes gra n des como a lãs Índias, o a pru eblos pri n c i p a l e s o a Un iveri d ade s , don de su el en con c u rrir mas pers on a s , que ay u d adas pod ra n ser operários para ayudar a otros, deben preferirse. 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL (Loyola, 1963, p.548) V á rios cri t é rios de escolha para os lu ga res da pre s ença da Companhia se Julho de 2002 Julho de 2002 Fernando Torres Londoño de s prendiam desta aplicação do universal à missão: on de houvesse mais ne- ce s s i d ade , on de a porta estivesse mais abert a , on de houvesse maior devoção e de s ej o, on de houvesse mais dívida ou maior peri go “que el en em i go de Cri s to nuestro Señor há sembrado cizaña” (Loyola, 1963, p.548). ( y , , p ) É nos Ex erc í cios Es p i ri tu a i s que se en con tra uma das referências básicas p a ra a ju s ti f i c a tiva da missão en tre infiéis: sua redução à Fé católica para as- sim salvar sua alma. É também lá que en con tramos os principais el em en to s da forma como os jesuítas deveriam tratar com os infiéis, como era recom en- d ado nas instruções ao patri a rca de Eti ó p i a . Na pri m ei ra anotação dos Ex er- c í cios Es p i ri tu a i s, Sa n to Inácio os def i n iu com p a ra n do-os com os exerc í c i o s corporais de caminhar, passear ou correr, de sorte que todo “m odo de prep a- rar o dispon er el ánima, p a ra quitar de sí todas las afecc i ones de s orden adas y despues de qu i t adas para buscar y hallar la vo lu n t ad divina se llaman ej erc i- cios espirituales”(Loyola,1963, p.196).A espiritualidade de Ignácio e sua apli- cação na salvação das almas estava marc ad a , poi s , pelo reorden a m en to da von t ade , ao en ten d i m en to e aos sen ti dos do ser humano em função da von- t ade divi n a . 5. INÁCIO DE LOYOLA E A REDUÇÃO DO INFIEL Os Ex erc í cios Es p i ri tu a i s deveri a m , poi s , d i s por a pe s s oa para ou- vir e seguir o ch a m a m en to do “rei etern a l ”( Loyo l a ,1 9 6 3 , p. 2 1 8 ) . Tra t ava - s e , em termos ign ac i a n o s , de “recon duzir aos fins”, i s to é, po s tular ou diri gir os fins almejados pelo hom em , de acordo com o que Ignácio de Loyola ch a m o u de princípio ou fundamen to ou ainda finalidade pri m ei ra de todo ser hu m a- no: amar, louvar e servir a Deus e assim salvar sua alma (Loyola,1963, p.203). E s t a ria na nota 22 que precedia os Ex erc í cios Es p i ri tu a i s a matriz da redu ç ã o que se pretendia en tre infiéis, qu a n do se recom en d ava para a relação en tre o diretor dos exercícios e o exercitante 26 (...) pre su pon er que todo bu en Cri s tiano há de ser mas pron to a salvar la propo s i- ción del próximo que a con den a rl a ; y si no la puede salva r, i n qu i ra como la en- ti en de , y si mal la en ti en de , corr í jale com amor, y si no basta, bu s que todos los mé- dios conven i en tes para qu e , bi en en ten d i é n do l a , se salve .( Loyo l a ,1 9 6 3 , p. 2 0 2 ) Revista Brasileira de História, vol. 22, nº 43 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. Buscar todos os meios para que o outro en ten desse era o gra n de de s a f i o que apresentava a missão entre infiéis. Porém na prática missionária dos anos c i n q ü enta e sessenta do século XVI os re su l t ados e o avanço da Fé estava m mu i to lon ge dos rel a tos ed i f i c a n te s . São Fra n c i s co Xavi er tinha en con trado i n ú m eros obstáculos na Ásia, pri n c i p a l m en te no que ele sonhou como su a gra n de missão: a introdução do cri s tianismo no Ja p ã o. Em Satzuma teve qu e Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI a m a r g u rar a pre s ervação do mon opólio rel i gioso por parte dos “bonsos shin- gon”. Em Miya ko, capital do impéri o, a Ki o to modern a , Xavi er su portou in- j ú rias e pers eguições e a decepção da indiferença e falta de apoio dos gra n de s s en h ore s , os maiores interl oc utores no seu método de reduzir à Fé2 0. No Bra- s i l , os índios adu l to s , pre s erva n do os co s tumes de seus pais,“c i erran las ore- jas para no oir la palabra de salud y converterse al verd adero culto de Di o s”, o que levou então o irmão An ch i et a , em junho de 1560, a dizer ao pad re gera l Di ego Laynes que há “tan pocas cosas dignas de se escrivir que no sé que es- c riva , porque si espera V. P. que hay mu chos de los bra s i lles converti dos en ga- ñ a rse a sua espera n z a” ( Lei te , 1 9 5 4 , I I I , p. 2 4 9 ) . 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. Também nas Índias espanho- las, onde os jesuítas iniciaram em 1566,uma missão entre os índios da Flórida, se começou mal,com o assassinato de um dos dois padres.Insistindo em 1568, os trabalhos não produ z i ram os frutos esperados e em 1571 foram assassina- dos mais missionários e a missão foi abandonada21. As s i m , em bora o teor ed i f i c a n te não tenha de s a p a rec i do efetiva m en te das cartas para “não mostra r ”, c ada vez mais foi preva l ecen do na escrita qu e vinha das missões o envio de con sultas dos mais va ri ados temas e a que vi- nha de Rom a ,L i s boa ou Espanha a adoção de instruções e determinações do p ad re geral e dos provinciais sobre as formas da pre s ença jesu í tica e o méto- do mission á rio na Ásia e na Am é ri c a . Du ra n te o mandato do pad re Lay n e s , ele e o sec ret á rio Po l a n co escreveram cartas on de , além de manifestar aos je- suítas do Brasil e da Índia que “ten emos puestos los ojos en vo s o tro s” ( Lei te , 1 9 5 4 , I I I , p.9) instruíam sobre várias questões del i c adas e urgen te s , a maiori a delas formu l adas a partir de perguntas feitas nas cartas do pad re Nóbrega ( Lei te ,1 9 5 4 , I I I , p. 5 4 1 ) . Pad re Lay n e s , em de zem bro de 1562, re s pondia di- zen do que pelas cartas recebidas en ten deu “la va ri ed ad que en el modo de proceder en ciertas co s a s , se há ten i do en esa prov í n c i a” ( Lei te , 1 9 5 4 , I I I , p. 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. 5 1 3 ) , e sen do a informação recebida su f i c i en te até o envio de um vi s i t ador, re s o lveu determinar algumas coisas escreven do “lo que me oc u rre a algunos p u n ctos principales de vu e s tra letra” ( Lei te ,1 9 5 4 , I I I , p. 5 1 3 ) . Aprovou assim, en tre outro s , o estabel ec i m en to de casas para meninos e meninas do gen ti o, a s eleção de alguns da terra para serem mandados para Portu ga l , a seleção do s que estavam sen do ad m i ti dos à Companhia e ter escravos para tratar das fa- zen d a s , do gado e pescar para manter as casas que “no lo ten go por inconve- n i en te com que sean ju s t a m i en te po s s e í do s , lo qual digo porque he en ten d i- do que algunos se hazen esclavos injustamente” (Leite, 1954, III, p. 514). 27 Si tuações como estas de po u co ou nen hum con s enso en tre os mission á- ri o s , ou polêmicas como os “re sga tes de índios”, as doações de terra , a ad m i- n i s tração exclu s iva dos sac ra m en to s , o de s tino das esmolas do rei , traziam pa- ra a missão nas colônias uma com p l ex i d ade que pedia re s postas não imagi n ad a s Julho de 2002 Julho de 2002 Fernando Torres Londoño nas Con s ti tu i ç õ e s . Perguntas qu e , como dizia Po l a n co,“porque aca no se sabe lo particular no se puede re s pon der ”( Lei te , 1 9 5 4 , I I I , p. 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. 5 4 2 ) , dei x a n do para o d i s cern i m en to e a prudência dos mission á rios as re s po s t a s .E m bora como diz Lei te ,c i t a n do ao pad re Bor ja em carta de 1567, em questões de missões todo s os pad res gerais qu eriam ajudar (Lei te ,1 9 5 4 ,I , p. 5 6 ) . Essa ajuda do governo cen tral e das províncias da Eu rop a , na medida em que se divers i f i c avam as missões, s i gn i f i cou na pr á tica uma maior interven- ç ã o, vi s a n do ao estabel ec i m en to de um pad r ã o. Esta intervenção se re a l i zo u recorren do ao uso de instruções escri t a s , uti l i z adas por santo Ignácio de s de os pri m ei ros envi o s ,e s t a bel ecen do cri t é ri o s , proced i m en to s ,n ormas e mei o s p a ra a atuação dos jesu í t a s , que term i n a ram sen do def i n i tivas para o método m i s s i on á ri o2 2. Uma amostra de s te espíri to que se impunha no clima de difi- c u l d ades mission á rias nos anos sessenta do século XVI é a Ca rta de Bor ja , em 1 5 6 7 , ao provincial do Peru ,p ad re Porti ll o, p a ra o aprovei t a m en to do envi o de oi to jesuítas ao Peru , como re s posta aos vi n te solicitados por Fel i pe II. 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. O p ad re geral dispunha nas suas instruções ir a poucas parte s ,a ten der pri m ei ro aos já fei tos cri s t ã o s ,c u i d a n do em con s ervá-los e depois converter os outro s “y así, no ten gan por cosa ex ped i en te discurrir de una en otras partes para convertir gen te , com las cuales despues no pueden ten er cuen t a” ( As tra i n , 1 9 1 4 , vol II, p. 3 0 5 ) ,a p l i c a n do assim o cri t é rio bem inaciano de ganhar po u- co a po u co, forti f i c a n do o que se ga n h o u . Também advertia para não se co l o- car em peri go de vida en tre gen te não con qu i s t ad a ,“pues não seria útil para el bi en com ú n , por la mu cha falta que hay de obreros para aqu ella viña y la d i f i c u l t ad que tenía la Compañía de enviar otros en su lu ga r ”( As tra i n , 1 9 1 4 , vol II, p. 3 0 6 ) . A essa altu ra já havia sido assassinado o pad re Ped ro Ma rti n e z na Flóri d a , e ga n h ava força uma visão mais realista do que se podia espera r nas Índias. Na verd ade , nesta pon derada con s i deração de situações se esta- vam aplicando as Constituições na Parte Sétima número 22: 28 Asi mismo entre las obras pías de igual importância y priesa y necesidad,habien- do algunas mas seg u ras para qu i en las trata y otras mas pel i grosas y algunas qu e más fácil y brevem en te y otras com mas dificultad y com mas largo ti em po se concluirán, las primeras asi mesmo deberán preferirse. (Loyola, 1963, p. Revista Brasileira de História, vol. 22, nº 43 CONCLUSÃO Ao con s i derar a produção de cartas no âmbi to da con s trução da missão na Companhia de Je sus no século XV I , se trabalhou aqui numa lei tu ra de s s a doc u m entação por parte do histori ador. No con tex to da espiri tu a l i d ade do s j e suítas no século XV I , de alguma forma aqui tra t ad a ,t a n to a missão como o que dela se escrevia estava con du z i do pela busca da von t ade divi n a . Pri n c í p i o e fundamen to do ser humano e da criação que se re a l i z ava na iden ti f i c a ç ã o do bem universal como o mais divi n o. Na proposta de uma ordem gerada em gra n de parte por ibéri cos para servir à Igreja no século XV I , o bem univers a l foi iden ti f i c ado, en tre outro s , na convocação para trabalhar no anúncio da boa nova aos infiéis da Ásia e da América. 29 Re sga tou-se poi s , mesmo que tem era ri a m en te , a evidência do tex to ina- ciano nas cartas dos mission á rios e se lhe co l ocou numa abrangência que su- pera o ed i f i c a n te , a pon t a n do para uma mu l ti p l i c i d ade de sen ti do s . Is to den- tro da proposta qu e , esse regi s tro, por espec í f i co que seja , tem que ser ouvi do e interpretado pela análise histórica. Fu n d ada por alguém que escreveu quase sete mil cartas e com posta por l etrado s , a Companhia de Je sus fez da corre s pondência o lu gar on de ficava vi s í vel a “u n iversal Com p a n h i a”. 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. 549) Pad re Bor ja determ i n ava também, que em regiões de índios ainda não con qu i s t ad a s , os pad res fixariam sua residência em lu gar seg u ro como on de residia o govern ador, proi bi n do as “salidas pel i gro s a s” mesmo com ordem do superior. Para a entrada nestas regiões se deveria procurar saber que gente ) es aqu ella em que han de aprovech a r, que errores y sectas de gen ti l i d ad si- i li i í i ti i h d t d édit t (...) es aqu ella em que han de aprovech a r, que errores y sectas de gen ti l i d ad si- g u en , que incl i n ac i ones y vícios ti en en , si hay doctos o pers onas de crédito en tre ( ) q q p , q y g g u en , que incl i n ac i ones y vícios ti en en , si hay doctos o pers onas de crédito en tre Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI ell o s , p a ra que estos se proc u ren ganar como cabezas de los otros y qué rem e- d i o s , con formes a estas cosas se les puedan y deban aplicar y com los de más en- ten d i m i en to proc u re antes com su avi d ad de palabras y ej emplo de vida aficio- narlos al verdadero camino, que por otros rigores. (Atrain, 1914, vol II, p. 305) De novo a pre s ença nas instruções mission á rias do tercei ro pad re gera l ,o s traços da espiri tu a l i d ade inaciana na con s i deração real dos fins a alcançar, a s condições do lu ga r, os mei o s . 6. AS INSTRUÇÕES E O MÉTODO MISSIONÁRIO. Também aqui o estabel ec i m en to das diferenças e h i era rquias en tre as gen tes e o discern i m en to sobre as ações mais pr ó prias a s erem diri gi d a s , bu s c a n do sem pre o maior bem univers a l . Ainda a redução en- tendida como um processo de s ti n ado a persu ad i r, a mover o en ten d i m en to pa- ra se abrir a Deu s . Bases da con s ti tuição de um método mission á rio que espe- rava por novos intérpretes como José de Acosta e Ma t teo Ri cc i . Julho de 2002 CONCLUSÃO G erador de múltiplos tex tos qu a n do escre- vi a , Inácio de Loyola tra n s feriu com paciência a seus súditos a consciência da d i s tinção de diversos interl oc utores e a el a boração cri teriosa da escri t a . Na s c a rt a s , os mission á rios apareciam como os com p a n h ei ros de Je sus que qu e- ri a m ,s eg u n do os Exercícios Espiri tu a i s ,s er co l oc ados ao lado do Cri s to po- bre e padecer com el e . Mas para esse seg u i m en to, os pad res prec i s avam saber o que fazer para alcançar seus fins. As cartas con s ti tuíam assim um espaço de Fernando Torres Londoño ten s ã o, de negoc i a ç ã o, de recuos e pri n c i p a l m en te de ação. Ac red i t a n do que a edificação deveria vir por si, se preoc u pou Loyola pri n c i p a l m en te por estabe- l ecer a base do que aqui foi ch a m ado de um sistema de informações de s ti n a- do a ajudar na tomada de decisões e na realização de ações. No caso das missões, na cópia e envio de cartas com diversos de s ti n o s , foi con s tru í do e def i n i do o proj eto jesu í ti co mission á rio numa troca de in- formações que se re a l i z ava no ei xo Rom a ,L i s boa ,Í n d i a , Bra s i l . Tal proj eto foi ex a m i n ado aqui a partir da proc u ra de um método para levar a boa nova en- tre infiéis. NOTAS * Este trabalho faz parte de uma pe s quisa maior em andamen to sobre Índios e Mi s s i on á- rios nos séculos XVI e XV I I , que tem con t ado com a participação de bolsistas do CNPQ. Sou grato ao professor Ênio Brito e Lícia Masagão por comentários e correções. * Este trabalho faz parte de uma pe s quisa maior em andamen to sobre Índios e Mi s s i on á- rios nos séculos XVI e XV I I , que tem con t ado com a participação de bolsistas do CNPQ. Sou grato ao professor Ênio Brito e Lícia Masagão por comentários e correções. 30 1 Com re s pei to às cartas da Companhia e suas co l e ç õ e s , ver para o Bra s i l ,L E I T E , Sera f i m . C a rtas dos Pri m ei ros Je suítas do Bra s i l. São Pa u l o : Comissão do IV Cen ten á rio da Ci d ade de São Paulo, 1954, vol I, p.61 e seguintes. 1 Com re s pei to às cartas da Companhia e suas co l e ç õ e s , ver para o Bra s i l ,L E I T E , Sera f i m . C a rtas dos Pri m ei ros Je suítas do Bra s i l. São Pa u l o : Comissão do IV Cen ten á rio da Ci d ade de São Paulo, 1954, vol I, p.61 e seguintes. 2Pa ra as cartas de Sa n to Inácio e a Assistência da Espanha, ver a introdução bi bl i ogr á f i c a de A S T RA I N , An ton i o. Hi s to ria de la Co m pañia de Je sus en la As i s ten cia de Es pa ñ a. Ma- drid: Razon y Fé, 1912, vol I p. XIII. 2Pa ra as cartas de Sa n to Inácio e a Assistência da Espanha, ver a introdução bi bl i ogr á f i c a de A S T RA I N , An ton i o. CONCLUSÃO Nessa proc u ra por um método no tem po de Loyo l a , L aynes e Bor- ja , teria havi do um rec u rso con t í nuo à matriz inaciana no seu en ten d i m en to do bem universal e da redução espiri tual e o reorden a m en to de fins e mei o s , que se materi a l i zou na produção de instruções mission á rias que term i n a ra m definindo os rumos da atuação da Companhia fora da Europa. NOTAS Hi s to ria de la Co m pañia de Je sus en la As i s ten cia de Es pa ñ a. Ma- drid: Razon y Fé, 1912, vol I p. XIII. 3 Sobre este trabalho pion ei ro de Ca p i s trano de Abreu , ver as notas de rodapé de José Ho- n ó rio Rod rigues em tex to sobre a Hi s tori ografia rel i gi o s a , que forma parte de sua Hi s t ó ri a da História do Brasil. Historiografia colonial. São Paulo:Companhia Editora Nacional,1979, p.256. 4 O pad re Lei te fez isto no marco do que seria a Mo nu m enta Brasiliae S.I. se inscreven do e s eg u i n do os cri t é rios da gi ga n tesca coleção Mo nu m enta Hi s t ó rica S.I. , ver LEITE, Sera f i m , op. ci t ., vol I, p. 6 9 . Ainda LEITE, Serafim tinha ed i t ado Novas Cartas Je su í ti c a s. São Pa u l o : Companhia Editora Nac i onal 1940, p u bl i c a n do depois C a rtas do Brasil e mais escri to s. Coimbra: O, 1955. 5E n tre outros podemos men c i onar GADELHA, Regina Ma ria F. As missões je su í ticas do It a ti m . Es tru tu ras so ci oe conômicas do Pa raguai col o n i a l .S é c u l o s , XVI e XV I I. Rio de Ja n ei- ro : Paz e Terra ,1 9 8 0 ; BA E TA NEV E S , Luis Fel i pe . O co m ba te do soldados de Cri s to na Terra dos Pa pa ga i o s . Colonialismo e represão cultu ra l. Rio de Ja n ei ro : Foren s e ,1 9 7 8 ;G A M B I N I , Roberto. O espelho do Índio, revi s ado e reed i t ado. Revista Brasileira de História, vol. 22, nº 43 NOTAS Las misiones jesuíticas en la América colonial, Quito/Lima, Abya-Yala-PUC Peru, 1999. 31 31 9As Constituições foram redigidas por santo Inácio num processo que foi de 1541 até 1556. A partir de 49, pon tos def i n i dos por seis dos pri m ei ros mem bro s , o santo pri m ei ro só e depois com ajuda de seu sec ret á rio P. Po l a n co, foi ava n ç a n do via con sultas a diversos ra s- cunhos e versões em 1547, 1 5 5 0 , até ch egar às dez partes de 1556. A re s pei to ver a intro- dução ao tex to das Co n s ti tu i ç õ e s do P. Ip a rra g u i rre em San Ign á cio de Loyol a , Ob ras Co m- pl et a s. Mad ri d : BAC ,1 9 6 3 , p. 4 0 0 . Uti l i zo esta edição das obras com p l etas do santo pel a sua fidelidade, trasladando as citações no original. 1 0 Pa ra fixar as formas de repre s entação e a obed i ê n c i a , além da ch a m ada carta da obe- diência aos pad res e irmãos de Portu gal em março de 1553, Sa n to Ignácio via pad re Po l a n co, s eu sec ret á ri o, e s c reveu a “ In s trução sobre el modo de tratar negociar com c u a l qu i er su peri or ” em maio de 1555, on de o pon to qu a rto diz “ Si a la determ i n ac i ó n del su peri or, o lo que a el toc a re , rep l i c a re alguna cosa que bi en le pare z c a , torn a n do el su peri or a determ i n a r, no haya replica ni ra zones algunas por en ton ce s” ( Loyo l a ,1 9 6 3 , p. 9 2 4 ) . NOTAS c i m en to e poder no Brasil co l on i a l :e s tu dos históri cos psico l ó gi co s”, tese de Livre Doc ê n- c i a , Fac u l d ade de Filosof i a , Ciências e Letra s ,U S P, Ri beirão Preto 1995; RA M I N E L L I , Ro- n a l d . Im a gens da col o n i z a ç ã o. A repre sentação do Índio de Caminha a Vi ei ra. Rio de Ja n ei- ro : Jor ge Zahar Editor, 1 9 9 6 ;A S S U N Ç Ã O, Pa u l o. A terra dos brasis: a natu reza da Am é ri c a po rtuguesa vista pelos pri m ei ros je suítas (1549-1596). São Pa u l o : An n a blu m e ,2 0 0 0 . Pa ra o sul do Bra s i l , QU E B E DO, Ju l i o. Gu errei ros e Je suítas na Utopia do Pra t a. Ba u ru :E D U S C , 2000; ainda os doutorados de FLECK, Eliane Cristina D. e MARTINS, Maria Bohn. 6 M E I H Y, José Ca rlos Sebe Bom .“A pre s ença no Brasil da Companhia de Je sus 1549-1649”, tese de Do utorado apre s en t ada ao Dep a rt a m en to de Hi s t ó ria da USP em 1975, a pon to u p a ra a importância de levar em con s i deração a diferença en tre cartas ed i f i c a n tes e cart a s informativas. 7A S S U N Ç Ã O, Pa u l o. Op ci t ., p. 8 2 , diz que com relação às terras americanas era o verbo que melhor definia a ação dos missionários,já que nas colônias nada era digno de ser man- ti do. Nas cartas de Loyola aqu ela ex pressão aparece rel ac i on ada a sen ti m en tos de con s o l a- ção gerados em terceiros por nossas ações em oposição à censura ou rejeição. NOTAS 7A S S U N Ç Ã O, Pa u l o. Op ci t ., p. 8 2 , diz que com relação às terras americanas era o verbo que melhor definia a ação dos missionários,já que nas colônias nada era digno de ser man- ti do. Nas cartas de Loyola aqu ela ex pressão aparece rel ac i on ada a sen ti m en tos de con s o l a- ção gerados em terceiros por nossas ações em oposição à censura ou rejeição. 8Os trabalhos de s tes autores vi ra ram livro s . Ver KA R NA L , Le a n d ro. Te a tro da Fé. São Pa u- l o : Hu c i tec , 1 9 9 8 , pp. 4 8 - 6 1 ;A S S U N Ç Ã O, Pa u l o. Op. ci t. , pp. 5 7 - 9 1 ; QU E B E DO, Ju l i o. Op. ci t ., pp. 2 1 - 4 8 . Minha con tri buição é “La ex peri encia rel i giosa jesu í tica y la crônica de Pa r á y Ma ranhão em el siglo XV I I ”. In Sa n d ra Negro y Ma nu el Ma r z a l , Un reino en la fro n tera . Las misiones jesuíticas en la América colonial, Quito/Lima, Abya-Yala-PUC Peru, 1999. 8Os trabalhos de s tes autores vi ra ram livro s . Ver KA R NA L , Le a n d ro. Te a tro da Fé. São Pa u- l o : Hu c i tec , 1 9 9 8 , pp. 4 8 - 6 1 ;A S S U N Ç Ã O, Pa u l o. Op. ci t. , pp. 5 7 - 9 1 ; QU E B E DO, Ju l i o. Op. ci t ., pp. 2 1 - 4 8 . Minha con tri buição é “La ex peri encia rel i giosa jesu í tica y la crônica de Pa r á y Ma ranhão em el siglo XV I I ”. In Sa n d ra Negro y Ma nu el Ma r z a l , Un reino en la fro n tera . NOTAS São Pa u l o : Axis Mu n d i / Tercei ro Nom e , 2 0 0 0 ;C H A M BO U L EY RO N , Ra f ael . “Os lavradores de almas “, d i s s ertação de mestrado, Dep a rt a m en to de Hi s t ó ri a ,U S P, 1 9 9 4 ;M A S S I M I , Ma ri n a .“ De s cobri m en to, a ç ã o, con h e- Revista Brasileira de História, vol. 22, nº 43 Escrevendo Cartas. Jesuítas, Escrita e Missão no Século XVI c i m en to e poder no Brasil co l on i a l :e s tu dos históri cos psico l ó gi co s”, tese de Livre Doc ê n- c i a , Fac u l d ade de Filosof i a , Ciências e Letra s ,U S P, Ri beirão Preto 1995; RA M I N E L L I , Ro- n a l d . Im a gens da col o n i z a ç ã o. A repre sentação do Índio de Caminha a Vi ei ra. Rio de Ja n ei- ro : Jor ge Zahar Editor, 1 9 9 6 ;A S S U N Ç Ã O, Pa u l o. A terra dos brasis: a natu reza da Am é ri c a po rtuguesa vista pelos pri m ei ros je suítas (1549-1596). São Pa u l o : An n a blu m e ,2 0 0 0 . Pa ra o sul do Bra s i l , QU E B E DO, Ju l i o. Gu errei ros e Je suítas na Utopia do Pra t a. Ba u ru :E D U S C , 2000; ainda os doutorados de FLECK, Eliane Cristina D. e MARTINS, Maria Bohn. NOTAS 1 4Um bom exemplo é a carta a Fel i pe , pr í n c i pe da Espanha, de feverei ro de 1549, on de o santo manifesta o apreço pelo príncipe herdeiro da Espanha,sem dizer ou pedir nada,mas que tinha sido escrita no con tex to de uma visita do con t ador- m or do pr í n c i pe e em b a i x a- dor ex tra ord i n á rio a Rom a , qu a n do se tra tou de questões referen tes à reforma dos mostei- ros em Catalunha, ver, San Ignácio de Loyola, Obras Completas, op. cit., p. 715. 5BARTHES, Roland. Sade, Fourier, Loyola.. Paris: Éditions Du Seuil, 1971, p. 47. 1 6Dom i n i que Bertra n d , na La pol i tique de Saint Ign a ce de Loyol a, op. ci t ., el a borou uma sé- rie de tabelas das categorias das cartas p. 7 3 , dos gra n des temas p. 9 1 , e dos meios soc i a i s aos quais se dirigiu santo Inácio, p.115. 17BARTHES, Roland. Op cit., p.54. 1 8E s te ref a zer a carta e mexer no seu tex to pra ti c ado por santo In á c i o, foi apren d i do e se- g u i do por seu sec ret á rio pad re Po l a n co, p a ra de s e s pero dos histori adore s , como men c i on a LEITE, Serafim. Op. cit., vol. I, p.58. 32 19Ver, Cartas e escritos de San Francisco Xavier. Madrid: BAC, 1953. 2 0Pa ra as missões de São Fra n c i s co Xavi er no Ja p ã o, ver LAC O U T U R E , Je a n . Je suítas I Lo s conquistadores. Paidos: Barcelona 1991, pp. 206-211. 2 1Pa ra a missão da Florida e o assassinato do pad re Ped ro Ma rti n e z , ver A S T RA I N , An to- nio. Op. cit., vol. II, p. 288. NOTAS 2 2Nos leva n t a m en tos de Dom i n i que Bertra n d ,s a n to Inácio teria escri to 90 cartas sobre as- suntos de missões.Ver La politique de Saint Ignace de Loyola, op. cit., p.73.Em 24 de setem- bro de 1549, s a n to Inácio red i giu uma serie de instruções para os jesuítas que iriam lec i o- nar teo l ogia na univers i d ade de In go l s t adt na Al emanha . Dessas instruções con s t avam o que podia ajudar para alcançar o fim pri m á ri o, a pureza na Fé e a obediência à Igreja , e o fim sec u n d á rio de prom over a Companhia na Al emanha e os meios comuns para alcançar a ambos. Ver, Obras completas de San Ignácio de Loyola, op. cit., p. 740. NOTAS Ai n d a , o santo abria po s s i bi l i d ades de repre s entar com o tem po “porque la ex- peri encia com el ti em po de s c u bre mu chas co s a s” ;i s to sem óbi ce de obedecer. 1 1Há nas Co n s ti tu i ç õ e s d iversas disposições rel ac i on adas ao con h ec i m en to e estu do de le- tra s . Aqui é import a n te men c i onar os “ Im ped i m en tos para en trar dos que preten dem ser coad jutores espiri tu a i s”, on de se men c i ona “ Falta de letras o apti tud de ingenio o mem ó- ria para apren dellas o lengua para ex p l i c a ll a s”, Co n s ti tu i ç õ e s, Pa rte I, c a p. 3 , nº 1 8 3 , em Obras Completas de Santo Ignácio de Loyola,. Op. cit.,p.454. 1 2Si go aqui o cri terioso estu do de DO M I N I QUE BERT RA N D, S . J. , La pol i tique de Saint Ign a ce de Loyol a. Pa ri s : Lês Editi ons du Cerf ,1 9 8 5 , on de com p a ra nu m eri c a m en te a cor- Julho de 2002 Fernando Torres Londoño respondência do santo com a de outros contemporâneos,Erasmo 1.980, Lutero 3.141, Cal- vino .1247, Catarina de Médecis 6.381, entre outros, p.39. 13Pelo levantamento feito por DOMINIQUE BERTRAND, op. cit., Loyola escreveu mil qui- n h entas e qu a tor ze cartas a não-mem bros da Com p a n h i a , duas cartas ao papa e duas ao i m perador, n oventa e duas a reis e pe s s oas da família re a l , oi tenta e duas a gra n des nobre s eu ropeu s , s etenta e uma a carde a i s , cen to e qu a renta a altos funcion á rios e suas famílias, cento e quarenta a bispos e 301 a nobres, etc., p.42. Artigo recebido em 01/2002. Aprovado em 05/2002. Revista Brasileira de História, vol. 22, nº 43
https://openalex.org/W2050559379
https://europepmc.org/articles/pmc4140099?pdf=render
English
null
Analysis of Asymmetry of the Forces Applied on the Lower Limb in Subjects with Nonspecific Chronic Low Back Pain
BioMed research international
2,014
cc-by
4,249
Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 289491, 6 pages http://dx.doi.org/10.1155/2014/289491 Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 289491, 6 pages http://dx.doi.org/10.1155/2014/289491 Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 289491, 6 pages http://dx.doi.org/10.1155/2014/289491 1 Musculoskeletal Research Center, Isfahan University of Medical Sciences, P.O. Box 81745-164, Isfahan, Iran 2 Department of Health Professions, Manchester Metropolitan University, UK 1 Musculoskeletal Research Center, Isfahan University of Medical Sciences, P.O. Box 81745-164, Isfahan, Iran 2 Department of Health Professions, Manchester Metropolitan University, UK Correspondence should be addressed to Mohammad Taghi Karimi; mohammad.karimi.bioengineering@gmail.com Received 20 December 2013; Revised 5 April 2014; Accepted 6 May 2014; Published 1 July 2014 Copyright © 2014 Maryam Hassan Zahraee 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. Several studies have investigated asymmetry and loading patterns in different spine pathologies, motor disorders, and other conditions; there is a lack of knowledge on these aspects in chronic low back pain (CLBP). The aim of this study was to analyse asymmetry and loading patterns in patients with nonspecific chronic low back pain (NCLBP) compared to normal individuals, during walking. Method. Forty participants (20 healthy subjects and 20 patients with NCLBP) participated in the study. Asymmetry of the force was measured based on the Asymmetry Index (ASI). The difference in the mean values of all data between the two groups was examined using the independent t-test. Results. The mean value of the first peak of ground reaction force of normal subjects was 1.02 ± 0.0354 N/BW compared to 1.038 ± 0.099 N/BW in NCLBP patients (𝑃= 0.25) and 0.1004 ± 0.036 N/BW mediolateral force applied on the leg in normal subjects compared to 0.089±0.022 N/BW in NCLBP patients (𝑃= 0.214). The Asymmetry Index (ASI) of the first peak of vertical force was 2.59% ± 1.89% and 3.88% ± 2.94% for NCLBP and normal subjects, respectively, P = 0.2. Conclusion. Therefore, it can be concluded that NCLBP subjects follow avoidance-endurance model without any limitation during walking. 1. Introduction 11]. Clinicians commonly recommend gait reeducation pro- gramme for people with LBP as part of their rehabilitation. Therefore, it is important to have a good understanding of how LBP affects walking so as to develop more appropriate walking programs for this population. However, individu- als with LBP generally have walking problems, which, in turn, may reduce their willingness to participate in exercise programs [12]. Low back pain (LBP) remains a prevalent and persistent prob- lem that is associated with functional limitations and par- ticipation restrictions [1–3]. Evidence suggests that increased activity level such as walking is an effective way of managing patients with LBP; hence, therapeutic exercise programmes have been recommended for individuals with this condition [4–8]. While some people with LBP remain active, others have difficulty doing so due to physical, psychological, and social reasons, and this can contribute to distress and disabil- ity, increasing the economic cost of chronic LBP. As a valuable tool for the understanding of motion disorders and treatment outcomes, clinical gait analysis is based on the quantification and evaluation of deviations from normal values [12–14]. Kinetic, as a gait parameter, reflects the cause of movement, and therefore the forces, power, and energy that affect the manner in which an individual moves. Also, GRFs measured with force plates imbedded in the ground or treadmill refer to the forces that act on the body throughout the stance phase. The symmetry of motions and also the forces applied on the legs have being used to Walking is a complex task that involves the coordination of the muscular system to simultaneously produce and sustain a variety of multidirectional forces around each joint and with the ground, that is, ground reaction forces (GRFs) and maintaining balance in an upright posture [9]. It is evident that LBP has a profound impact on gait [10, BioMed Research International 2 Table 1: The characteristics of NCLBP and control subjects who participated in the study. Table 1: The characteristics of NCLBP and control subjects who participated in the study. cut-off frequency of 10 Hz. The peaks of the vertical force, anteroposterior force, and mediolateral force applied on the right and left legs were used for final analysis. Moreover, the symmetry of the force applied on legs in normal and NCLBP patients was determined, according to Asymmetry Index measure. The asymmetry was measured based on the Asymmetry Index (ASI) described by Herzog et al. 3. Results Table 3 shows the mean values of spatiotemporal gait parameters of normal subjects and those with NCLBP. The mean values of walking speed in healthy participants and patients with NCLBP were 9.53 ± 0.99 and 9.2 ± 1.3 cm/s, respectively, 𝑃= 0.245. There was no significant differ- ence in the mean values of spatiotemporal gait parame- ters between health participants and patients with NCLBP (𝑃> 0.05). These findings may help to provide a good understand- ing of the possible effects of CLBP on gait parameters. Ultimately, they can be used for identifying any abnormal- ity and to inform appropriate treatment plan for patients with this condition. The present study, therefore, aimed at analyzing asymmetry and loading patterns of legs in patients with CLBP compared with their healthy counter- parts. The main hypothesis associated with this study was that there was no asymmetry of applied forces in subjects with CLBP. Table 4 summarizes the mean values of forces applied on the leg in three directions. The mean value of the first peak of ground reaction force of normal subjects was 1.02 ± 0.0354 N/BW compared to 1.038 ± 0.099 N/BW in patients with NCLBP (𝑃= 0.25). There was a significant difference in the vertical force applied on the leg during push-off phase (third peak) between healthy subjects and patients with NCLBP groups (𝑃= 0.038). The mediolateral force applied on the leg in healthy subjects was 0.1004 ± 0.036 N/BW compared to 0.089 ± 0.022 N/BW in patients with NCLBP (𝑃= 0.214). Table 5 shows the ASI in healthy subject and patients with NCLBP groups during walking on a level surface. The ASI of walking speed in healthy subjects was 5.03 ± 4.6% compared with 3.8 ± 3.4% in patients with NCLBP (𝑃= 0.3). The ASI of the first peak of vertical force was 2.59 ± 1.89% and 3.88±2.94% in patients with NCLBP and healthy participants, respectively, 𝑃= 0.2. The ASI of the second and third peaks of vertical ground reaction force varied between 2.4 to 2.48 in healthy subjects compared to 2.4 to 17.12 in patients with NCLBP. 1. Introduction [22], based on the following equation: Characteristics Groups NCLBP∗(𝑛= 20) Control (𝑛= 20) Age (y) 41.56 ± 9.57 40.18 ± 8.55 Height (m) 158.81 ± 5.56 158.18 ± 5.74 Weight (kg) 61.68 ± 8.88 60.25 ± 6.38 Sex Female Female Values are mean ± SD. ∗Nonspecific low back pain. ASI = 𝑋right −𝑋left (𝑋right + 𝑋left) /2, (1) (1) in which 𝑋is the value of the parameter. An ASI value of zero indicates that there is perfect symmetry for the particular gait variable. Shapiro-Wilk test revealed that the data was normally distributed; hence, the independent t-test was used to compare the two groups. determine the severity of disease and also the effectiveness of treatment interventions [15–18]. In pathological gait a notice- able asymmetry has been recorded between the affected and unaffected lower limbs [18, 19]. Hence, asymmetry may be a significant factor of pathology. Therefore, it is important that this parameter should also be evaluated as part of a gait analysis. Although there are several studies on asymmetry and loading patterns in different spine pathologies such as scoliosis [19], motor disorders, cerebral palsy [20, 21], and other conditions such as lower limb amputations [17], there is a lack of knowledge on the asymmetry of gait parameters in patients with CLBP. 2. Materials Forty participants (20 healthy subjects and 20 patients with NCLBP) participated in the study. Healthy subjects were selected from the staff members of Rehabilitation Faculty of Isfahan University of Medical Sciences and were matched with patients based on age and height. Table 1 shows the characteristics of both groups. Patients were recruited from the subjects referred to Phys- ical Therapy clinic of the faculty, based on inclusion/exclusion criteria shown in Table 2. Ethical approval was obtained from the Ethics Committee of Isfahan University of Medical Sciences. Informed written consent was obtained from all the participants. The participants were asked to walk along the gait lab path with a comfortable speed, from which 5 successful trials were selected. A Kistler force plate was used to record the forces applied on leg during walking. The data of the force plate was collected with a frequency of 120 Hz and was filtered with a Butterworth low pass filter with a 4. Discussion Approximately 80% of adult population experience LBP at any time during their life [3]. It has been shown that nearly 90% of all patients with LBP suffer from a pain with an unknown origin, which is defined as nonspecific low back pain (NCLBP) [23, 24]. To date, there is no information regarding the loads applied on the leg in patients with NCLBP. Moreover, the symmetry of loads applied on limb in patients 3 BioMed Research International 3 0 0.01 0.02 0.03 0.04 0.05 0.06 Force (N/BW) 0 50 100 The gait cycle (%) NCLBP subject Normal subject −0.02 −0.03 −0.04 −0.01 (a) 0 0.05 0.1 0.15 0.2 0.25 0.3 Force (N/BW) 0 50 100 The gait cycle (%) NCLBP subject Normal subject −0.15 −0.05 −0.1 (b) 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 Force (N/BW) The gait cycle (%) NCLBP subject Normal subject −0.2 (c) Figure 1: The mediolateral (a), anteroposterior (b), and vertical forces (c) applied on the leg of a normal and a NCLBP su h NCLBP is currently unknown. Therefore, the aim of this earch was to evaluate the loads and symmetry of loading plied on the leg in patients with NCLBP., Figure 1 show The diolateral anteroposterior and ertical forces applied on The mean values of the loads applied on the side of normal subjects and those with NCLBP are Table 4. As it can be seen, there was no differenc the peaks of most of the forces applied on th 0 0.01 0.02 0.03 0.04 0.05 0.06 Force (N/BW) 0 50 100 The gait cycle (%) −0.02 −0.03 −0.04 −0.01 0 0.05 0.1 0.15 0.2 0.25 0.3 Force (N/BW) 0 50 100 The gait cycle (%) −0.15 −0.05 −0.1 0 0.01 0.02 0.03 0.04 0.05 Force (N/BW) 0 50 100 The gait cycle (%) NCLBP subject Normal subject −0.02 −0.03 −0.04 −0.01 (a) 0 0.05 0.1 0.15 0.2 0.25 Force (N/BW) 0 50 100 The gait cycle (%) NCLBP subject Normal subject −0.15 −0.05 −0.1 (b) 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 Force (N/BW) The gait cycle (%) NCLBP subject Normal subject −0.2 (c) The mediolateral (a), anteroposterior (b), and vertical forces (c) applied on the leg of a normal and a NCLBP subject. 4. Discussion Inclusion criteria Exclusion criteria Female Age: 25–55 years Pain group: (i) History of NSCLBP ≥6-month duration (ii) Without peripheral pain referral (iii) Pain in the area from T12 to gluteal folds (iv) Moderate ongoing LBP (a) Average daily pain level—VAS > 2/10 (b) Experienced most days of the week (v) Mechanically induced localized LBP Control group: No history of spinal pain (i) Specific diagnosis associated with LBP such as spondylolisthesis, disc prolapse, and inflammatory disorders (ii) Presence of other conditions affecting the spine including neurological or metastatic disease (iii) Any neurological deficit (iv) Any surgery involving the lumbar spine (v) Any diagnosed pelvic or abdominal pain disorder in the last 12 months (vi) Pregnancy or being less than six months after partum (vii) Any lower limb surgery in the last 2 years (viii) Current lower limb injury (ix) An inability to understand written or spoken Persian Table 2: Inclusion and exclusion criteria for patients who participated in the study. Table 3: Spatiotemporal gait parameters. Table 3: Spatiotemporal gait parameters. Parameter Velocity (cm/s) Cadence (steps/min) Stride length (cm) CLBP 9.2 ± 1.3 97.7 ± 9 1.13 ± 0.093 Normal 9.53 ± 0.99 98.3 ± 7.1 1162.62 ± 0.77 𝑃value 0.245 0.4 0.17 patients with CLBP may be due to the fear of exerting a push- off.h f The asymmetry indices of loads applied on legs are shown in Table 5. There was no asymmetry of loads between legs in NCLBP. Therefore, it can be concluded that the loads transmitted through the legs cannot be a source of this pathology. Actually, there are two theories to explain the source of the pain and its relation with physical activity in subjects with NCLBP [26]. The first theory is known as fear avoidance model (FAM) and emphasizes the fact that NCLBP patients decrease their physical activities to reduce their pain. In contrast, the second theory, avoidance-endurance model (AEM), emphasizes that NCLBP patients ignore their pain and persist on moving around despite their pain [26]. As there was no difference between the loads applied on the legs and also symmetry of the loads between health subjects and patients with NCLBP, it can be concluded that these subjects try to not change their physical performance. 4. Discussion It should be emphasized that asymmetry of loads on the right and left sides has been studied in some disorders including scoliosis and cerebral palsy, which also correlates with the severity of pathology. phase. This suggests that NCLBP patients walked nearly the same as normal subjects, as there was no difference between the spatiotemporal gait parameters of both groups. This finding contradicts that reported by Khodadadeh et al., 1998, and Keefer and Hill, 1985, who observed that patients with LBP tend to walk slower than normal sub- jects as observed in previous studies. The reason for our findings is not obvious, as clinical gait assessments such as functional gait assessment or Tenetti were not carried out to verify if gait was actually affected by back pain or not in these patients. Finally, there are limitations in this study. First, only the force applied on the legs was evaluated. Therefore, it is recommended that in the future studies the moment applied on the leg will also be evaluated. Secondly, no information regarding the site of pain in patients with LBP was collected in our study. Hence, it is difficult to say whether the activities of the lumbar muscles during the stance phase were associated with the site of pain. Lastly, a cross-sectional design was used in this study. The results from such design may not reflect the population studied because different observations may be obtained if the intervention was carried out in another time frame. Therefore, the clinicians are to interpret the findings of this study with caution. It is hoped that these limitations could be addressed in future studies. p However, the main reason for decrease in vertical force applied on the leg during push-off phase may be related to the angle of the joints in this stage of gait cycle. If the flexion angle of knee or hip joint decreases, then the loads applied on the leg will decrease as well (due to decrease in displacement of center of gravity in downward direction) [9, 25]. Therefore, it can be concluded that the decrease in loads of leg in this stage may be related to the kinematic of the joints. However, it should be emphasized that there was no asymmetry in the peaks of the vertical force applied on the leg during push-off between normal subjects and those with NCLBP. 4. Discussion It means that the reduction of this force occurred in both legs simultaneously. In addition, the back muscles are usually active during the stance phase to counteract forward momentum produced by the lower limb (Nelson et al., 1995). The higher the push-off force is, the more the activation of back muscles required sufficient antagonistic moments to stabilize the upper body and or trunk. Hence, the reduction in vertical push-off could be a compensatory mechanism to alleviate back pain. As a result, the reduced vertical GRF in 4. Discussion Force (N/BW) NCLBP subject Normal subject (a) NCLBP subject Normal subject (b) 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 Force (N/BW) The gait cycle (%) NCLBP subject Normal subject −0.2 (c) Figure 1: The mediolateral (a), anteroposterior (b), and vertical forces (c) applied on the leg of a normal and a NCLBP subject. (b) 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 Force (N/BW) The gait cycle (%) NCLBP subject Normal subject −0.2 (c) 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 Force (N/BW) The gait cycle (%) NCLBP subject Normal subject −0.2 (c) ediolateral (a), anteroposterior (b), and vertical forces (c) applied on the leg of a normal and a NCLBP sub Figure 1: The mediolateral (a), anteroposterior (b), and vertical forces (c) applied on the leg of a normal and a NCLBP subject. The mean values of the loads applied on the dominant side of normal subjects and those with NCLBP are shown in Table 4. As it can be seen, there was no difference between the peaks of most of the forces applied on the leg. The only difference was related to the vertical force of push-off with NCLBP is currently unknown. Therefore, the aim of this research was to evaluate the loads and symmetry of loading applied on the leg in patients with NCLBP., Figure 1 show The mediolateral, anteroposterior, and vertical forces applied on the leg of a normal and a NCLBP subject. BioMed Research International 4 Table 2: Inclusion and exclusion criteria for patients who participated in the study. References [1] C. Cato, D. K. Olson, and M. Studer, “Incidence, prevalence, and variables associated with low back pain in staff nurses,” AAOHN Journal, vol. 37, no. 8, pp. 321–327, 1989. [14] J. Crosbie, R. de Faria Negr˜ao Filho, D. P. Nascimento, and P. Ferreira, “Coordination of spinal motion in the transverse and frontal planes during walking in people with and without recurrent low back pain,” Spine, vol. 38, no. 5, pp. E286–E292, 2013. [2] M. Laslett, C. Crothers, P. Beattie, L. Cregten, and A. Moses, “The frequency and incidence of low back pain/sciatica in an urban population,” New Zealand Medical Journal, vol. 104, no. 921, pp. 424–426, 1991. [15] H. Sadeghi, P. Allard, F. Prince, and H. Labelle, “Symmetry and limb dominance in able-bodied gait: a review,” Gait and Posture, vol. 12, no. 1, pp. 34–45, 2000. [3] D. Hoy, P. Brooks, F. Blyth, and R. Buchbinder, “The Epidemi- ology of low back pain,” Best Practice and Research: Clinical Rheumatology, vol. 24, no. 6, pp. 769–781, 2010. [16] K. R. Archer, R. C. Castillo, E. J. MacKenzie et al., “Gait sym- metry and walking speed analysis following lower-extremity trauma,” Physical Therapy, vol. 86, no. 12, pp. 1630–1640, 2006. [4] J. N. Morris and A. E. Hardman, “Walking to health,” Sports Medicine, vol. 23, no. 5, pp. 306–332, 1997. [17] A. Tura, M. Raggi, L. Rocchi, A. G. Cutti, and L. Chiari, “Gait symmetry and regularity in transfemoral amputees assessed by trunk accelerations,” Journal of NeuroEngineering and Rehabili- tation, vol. 7, no. 1, article 4, 2010. [5] J. Rainville, C. Hartigan, E. Martinez, J. Limke, C. Jouve, and M. Finno, “Exercise as a treatment for chronic low back pain,” Spine Journal, vol. 4, no. 1, pp. 106–115, 2004. [6] S. D. Liddle, G. D. Baxter, and J. H. Gracey, “Exercise and chronic low back pain: what works?” Pain, vol. 107, no. 1-2, pp. 176–190, 2004. [18] K. K. Patterson, W. H. Gage, D. Brooks, S. E. Black, and W. E. McIlroy, “Evaluation of gait symmetry after stroke: a comparison of current methods and recommendations for standardization,” Gait and Posture, vol. 31, no. 2, pp. 241–246, 2010.i [7] D. Joffe, M. Watkins, L. Steiner, and B. A. Pfeifer, “Treadmill ambulation with partial body weight support for the treatment of low back and leg pain,” Journal of Orthopaedic and Sports Physical Therapy, vol. 32, no. 5, pp. 202–213, 2002. [19] N. Chockalingam, P. 5. Conclusion 19, no. 10, pp. 1613–1620, 2010. [12] M. J. Simmonds, C. E. Lee, B. R. Etnyre, and G. S. Morris, “The influence of pain distribution on walking velocity and horizontal ground reaction forces in patients with low back pain,” Pain Research and Treatment, vol. 2012, Article ID 214980, 10 pages, 2012. Conflict of Interests The authors declare that there is no conflict of interests regarding the publishing of this paper. [13] P. Y. M. Chung and G. Y. F. Ng, “Comparison between an accelerometer and a three-dimensional motion analysis system for the detection of movement,” Physiotherapy, vol. 98, no. 3, pp. 256–259, 2012. 5. Conclusion There were neither any differences between the forces applied on the leg between normal and NCLBP subjects nor any differences between asymmetry indices. Therefore, it can be BioMed Research International 5 Table 4: The peaks of the forces applied on the leg (dominant side) in normal and NCLBP subjects. Parameter Fz1 (N/BW) Fz2 (N/BW) Fz3 (N/BW) Fy (N/BW) Fx1 (N/BW) Fx2 (N/BW) NCLBP 1.038 ± 0.099 0.86 ± 0.0398 1.106 ± 0.0356 0.089 ± 0.022 0.178 ± 0.03 0.055 ± 0.011 Normal 1.02 ± 0.0354 0.0857 ± 0.0389 1.13 ± 0.045 0.1004 ± 0.036 0.19 ± 0.0218 0.053 ± 0.016 𝑃value 0.25 0.42 0.038 0.214 0.138 0.353 Values are mean ± SD. N: newton, BW: body weight, Fz: vertical force, Fy: mediolateral force, and Fx: anteroposterior force. Table 4: The peaks of the forces applied on the leg (dominant side) in normal and NCLBP subjects. Table 5: The results of asymmetry analysis of spatiotemporal and force parameters of normal and NCLBP subjects. Parameter WS (%) Cadence (%) SL (%) Fz1 (%) Fz2 (%) Fz3 (%) Fy (%) Fx1 (%) Fx2 (%) CLBP 3.8 ± 3.4 3.23 ± 2.62 1.54 ± 1.33 2.59 ± 1.89 17.12 ± 48.46 2.4 ± 2 16.73 ± 12.9 7.21 ± 6.3 27.74 ± 26.1 Normal 5.03 ± 4.6 5.88 ± 4.84 2.42 ± 1.5 3.88 ± 2.94 2.4 ± 2.3 2.48 ± 2.2 16.3 ± 8.2 5.59 ± 8.8 22.56 ± 28.9 𝑃value 0.3 0.15 0.152 0.2 0.169 0.48 0.46 0.36 0.37 WS: walking speed, SL: stride length, Fz: vertical force, Fy: mediolateral force, and Fx: anteroposterior force. le 5: The results of asymmetry analysis of spatiotemporal and force parameters of normal and NCLBP subjects. Table 5: The results of asymmetry analysis of spatiotemporal and force parameters of nor concluded that NCLBP subjects follow avoidance-endurance model without any limitation during walking. It also can be concluded that the asymmetry of force cannot be a reason of low back pain. [11] P. Hendrick, A. M. Te Wake, A. S. Tikkisetty, L. Wulff, C. Yap, and S. Milosavljevic, “The effectiveness of walking as an intervention for low back pain: a systematic review,” European Spine Journal, vol. 19, no. 10, pp. 1613–1620, 2010. [11] P. Hendrick, A. M. Te Wake, A. S. Tikkisetty, L. Wulff, C. Yap, and S. Milosavljevic, “The effectiveness of walking as an intervention for low back pain: a systematic review,” European Spine Journal, vol. References H. Dangerfield, A. Rahmatalla, E.-N. Ahmed, and T. Cochrane, “Assessment of ground reaction force during scoliotic gait,” European Spine Journal, vol. 13, no. 8, pp. 750–754, 2004. h [8] N. F. Taylor, O. M. Evans, and P. A. Goldie, “The effect of walking faster on people with acute low back pain,” European Spine Journal, vol. 12, no. 2, pp. 166–172, 2003. [20] H. B¨ohm and L. D¨oderlein, “Gait asymmetries in children with cerebral palsy: do they deteriorate with running?” Gait and Posture, vol. 35, no. 2, pp. 322–327, 2012. [9] J. Rose and J. G. Gamble, Eds., Human Walking, Lippincott Williams & Wilkins, Philadelphia, Pa, USA, 3rd edition, 2006.h [21] V. Femery, P. Moretto, H. Renaut, G. Lensel, and A. Thevenon, “Asymmetries in dynamic plantar pressure distribution mea- surement in able-bodied gait: application to the study of the gait asymmetries in children with hemiplegic cerebral palsy,” [10] P. Hendrick, S. Milosavljevic, L. Hale et al., “The relationship between physical activity and low back pain outcomes: a systematic review of observational studies,” European Spine Journal, vol. 20, no. 3, pp. 464–474, 2011. BioMed Research International 6 Annales de Readaptation et de Medecine Physique, vol. 45, no. 3, pp. 114–122, 2002. [22] W. Herzog, B. M. Nigg, L. J. Read, and E. Olsson, “Asymmetries in ground reaction force patterns in normal human gait,” Medicine and Science in Sports and Exercise, vol. 21, no. 1, pp. 110–114, 1989. [23] B. M. Wand and N. E. O’Connell, “Chronic non-specific low back pain—Sub-groups or a single mechanism?” BMC Musculoskeletal Disorders, vol. 9, article 11, 2008. [24] B. W. Koes, M. W. van Tulder, and S. Thomas, “Diagnosis and treatment of low back pain,” The British Medical Journal, vol. 332, no. 7555, pp. 1430–1434, 2006. [25] M. Atarod, C. B. Frank, and N. G. Shrive, “Kinematic and kinetic interactions during normal and ACL-Deficient gait: a longitudinal in vivo study,” Annals of Biomedical Engineering, vol. 42, no. 3, pp. 566–578, 2013. [26] D. W. Griffin, D. C. Harmon, and N. M. Kennedy, “Do patients with chronic low back pain have an altered level and/or pattern of physical activity compared to healthy individuals? A systematic review of the literature,” Physiotherapy, vol. 98, no. 1, pp. 13–23, 2012.
https://openalex.org/W4281986837
https://discovery.ucl.ac.uk/id/eprint/10150656/1/Schrag_2920255.pdf
English
null
Abbreviated MDS-UPDRS for Remote Monitoring in PD Identified Using Exhaustive Computational Search
Parkinson's disease
2,022
cc-by
5,653
Gareth Morinan ,1 Robert A. Hauser ,2 Anette Schrag ,3 Jingxuan Tang,1 Jonathan O’Keefe ,1 and MDS-NMS Scale Development Study Group4 1Machine Medicine Technologies Ltd., Te Leather Market Unit 1.1.1, 11/13 Weston Street, London SE1 3ER, UK 2Parkinson’s Disease and Movement Disorders Center, Department of Neurology, Parkinson Foundation Center of Excellence, University of South Florida, 4001 E Fletcher Ave, Tampa, FL 33613, USA 1Machine Medicine Technologies Ltd., Te Leather Market Unit 1.1.1, 11/13 Weston Street, London SE1 3ER, UK 2Parkinson’s Disease and Movement Disorders Center, Department of Neurology, Parkinson Foundation Center of Excellence, University of South Florida, 4001 E Fletcher Ave, Tampa, FL 33613, USA y f p Department of Clinical and Movement Neurosciences, Institute of Neurology, University College London, Queen London WC1N 3AR, UK y f p 3Department of Clinical and Movement Neurosciences, Institute of Neurology, University College London, Queen Square, London WC1N 3AR, UK 4Movement Disorder Society, 555 East Wells Street, Suite 1100 Milwaukee, WI 53202-3823, USA 4Movement Disorder Society, 555 East Wells Street, Suite 1100 Milwaukee, WI 53202-3823, USA Correspondence should be addressed to Jonathan O’Keefe; jonathan@machinemedicine.com Received 23 March 2022; Accepted 11 May 2022; Published 6 June 2022 Received 23 March 2022; Accepted 11 May 2022; Published 6 June 2022 Academic Editor: Antonio Pisani Copyright © 2022 Gareth Morinan et al. Tis 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. Background. Te Movement Disorder Society Unifed Parkinson’s Disease Rating Scale (MDS-UPDRS) comprises 50 items, consisting of historical questions and motor ratings, typically taking around 30 minutes to complete. We sought to identify an abbreviated version that could facilitate use in clinical practice or used remotely via telemedicine. Methods. To create an 8-item version we conducted an “exhaustive search” of all possible subsets. We measured explained variance in comparison to the 50- item version using linear regression, with the “optimal” subset maximising this while also meeting remote assessment practicality constraints. Te subset was identifed using a dataset collected by the Parkinson’s Progression Markers Initiative and validated using an MDS Non-Motor Symptoms Scale validation study dataset. Results. Te optimal remote version comprised items from all parts of the MDS-UPDRS and was found to act as an unbiased estimator of the total 50-item score. Tis version had an explained variance score of 0.844 and was highly correlated with the total MDS-UPDRS score (Pearson’s r  0.919, p-value <0.0001). Gareth Morinan ,1 Robert A. Hauser ,2 Anette Schrag ,3 Jingxuan Tang,1 Jonathan O’Keefe ,1 and MDS-NMS Scale Development Study Group4 Another subset that maximised explained variance score without adhering to remote assessment practicality constraints provided similar results. Conclusion. Tis result demonstrates that the total scores of an abbreviated form identifed by computational statistics had high agreement with the MDS-UPDRS total score. Whilst it cannot capture the richness of information of the full MDS-UPDRS, it can be used to create a total score where practicality limits the application of the full MDS-UPDRS, such as remote monitoring. Further validation will be required, including in specifc subgroups and advanced disease stages, and full validation of clinimetric properties. Previous work has suggested the duration of as- sessment required for a PD rating scale can restrict adoption in clinical practice [2–4]. Creation of a short version would reduce patient and clinician burden. It is also of increasing interest given information technology advances and rising demand for remote assessment, with shorter and potentially higher frequency assessments. Te MDS-UPDRS has not yet been validated for remote use. Hindawi Parkinson’s Disease Volume 2022, Article ID 2920255, 7 pages https://doi.org/10.1155/2022/2920255 2. Methods 2.1.Data. We utilised two datasets for this work: a “training” dataset, which was used to determine the optimal 8-item subset, and a “validation” dataset, which was used to validate the effectiveness of the optimal remote subset on a patient group that was independent of the training dataset. 1. Introduction Severity of Parkinson’s disease (PD) is commonly assessed using the Movement Disorder Society Unifed PD Rating Scale (MDS-UPDRS), consisting of historical questions and motor ratings, with an average completion time of around 30 minutes [1]. Te MDS-UPDRS consists of four parts, requiring a total of 65 ratings made on a 5- point (0–4) scale (see Supplement Table 1). 2 Parkinson’s Disease 2.3. Identification of MDS-UPDRS Subsets. While our ulti- mate aim was to identify an “optimal” subset for remote assessment, and certain items were not be included due to practicality constraints, we began by carrying out an ex- haustive search of every possible 8-item combination of the 50 MDS-UPDRS items. Tis required evaluating 536,878,650 (“50 choose 8”) possible subsets and was done in order to measure, using our evaluation metrics, the impact of applying remote practicality constraints. Te optimal subset across all combinations was termed the “any-item” subset, and the subset that was optimal while meeting practicality constraints was termed the “remote” subset. Even in most developed countries, public access to healthcare is still restricted [5]. Telemedicine can benefit pa- tients by saving cost and time spent attending appointments and reducing travel [6]. Preliminary research suggested that telemedicine for PD is feasible [7, 8], and most of the PD motor examinations can be performed remotely [7, 9]. Previous work proposed an 8-item shortened version of the original UPDRS based upon clinical relevance and ex- pertise [4]. We aimed to develop an 8-item version of the MDS-UPDRS which minimises information loss, measured using explained variance, compared to the MDS-UPDRS total score, while meeting remote assessment practicality constraints. Tis was achieved by conducting an “exhaustive search” (testing every possible 8-item combination). 2.4. Evaluation Metrics. Our criterion for model selection was the model that maximised the primary metric, the explained variance score (EVS) [14] of a linear regression. To verify this procedure, we used two secondary metrics: Spearman’s Rank Correlation Coefficient (SRC) [15] and Pearson’s Correlation Coefficient (PCC) [16]. Supplement Section 2.4 provides more detail on metric computation. 2.4. Evaluation Metrics. Our criterion for model selection was the model that maximised the primary metric, the explained variance score (EVS) [14] of a linear regression. To verify this procedure, we used two secondary metrics: Spearman’s Rank Correlation Coefficient (SRC) [15] and Pearson’s Correlation Coefficient (PCC) [16]. Supplement Section 2.4 provides more detail on metric computation. 3. Results For training we used the Parkinson’s Progression Markers Initiative (PPMI) dataset [10] (accessed 12 July 2021), while for validation we used the MDS-UPDRS scores collected in the MDS-Non-Motor Symptoms Scale (MDS- NMS) validation study [11]. Both datasets contain MDS- UPDRS examinations conducted by trained assessors, per- formed across multiple sites. 3.1. Exhaustive Search. An exhaustive search was conducted of the 536,878,650 possible 8-item combinations. Te dis- tribution of the three metrics, computed using the training dataset, can be seen in the Supplement Figure 2 and Sup- plement Table 4. Examining the top 100,000 ranked subsets found the rate of occurrence of items varied considerably (see Supplement Figure 3 for occurrences of each item); however, item 1.13 Fatigue appeared in more than 50% of these subsets, indicating it is relatively informative, whereas 18 different items appeared in less than 5% of these subsets, such as item 3.3 Rigidity, which was the motor examination item with the lowest rate of occurrence. For an assessment to be included in this analysis, we re- quired it to have a rating for every MDS-UPDRS item. Of the 15,986 assessments in the training dataset, 7,594 satisfied this requirement, while of the 402 assessments in the validation dataset, 377 did. Tis selection process did not introduce a bias with respect to age or gender (see Supplement Table 2). Te MDS-UPDRS rating distributions for these two datasets were visually similar; however, the median total MDS-UPDRS score of the validation dataset was signifi- cantly higher (Mann–Whitney’s U ˆ 1153785, p-value <0.0001) [12]. Te median Hoehn and Yahr stage [13] was the same. For a full comparison between datasets, see Supplement Figure 1 and Supplement Table 3. 3.2. Highest Ranked 8-Item Subsets. Ranking all subsets by EVS, we arrived at the optimal “remote” and “any-item” subsets (see Table 1). Te two subsets included similar clinical features although half of the individual items differed between them. Items 1.13 Fatigue, 2.5 Dressing, 2.10 Tremor, and 4.3 Time Spent in the OffState were included in both, whereas 3.2 Facial Movement, 3.4 Finger Tapping, 2.12 Walking and Balance, and 3.9 Arising From Chair appeared in the remote subset and 2.1 Speech, 3.5 Hand Movement, 3.7 Toe Tapping, and 3.13 Posture appeared in the any-item subset. 2.2. Remote Assessment Practicality Constraints. 4. Discussion We demonstrated that our results are robust by testing this remote subset, selected using the training dataset, on a separate validation dataset. Te explained variance on this validation dataset was found to be slightly lower (EVS ˆ 0.805); however, the 95% credible intervals of explained variance of the two datasets overlapped. Te statistical metrics were similar between the training and validation datasets (training, validation; EVS ˆ 0.844, 0.805; PCC ˆ 0.919, 0.897; SRC ˆ 0.900, 0.888, see Supple- ment Table 7). Te 95% credible intervals of the two datasets overlapped for each metric (see Supplement Figure 6). Finally, we show that the linear regression model con- structed using this remote subset acts as an unbiased esti- mator of the total 50-item rating, as seen from the 95% credible interval of the mean residual of this model being centered on zero for both training and validation datasets. 3.4. Subset Explanatory Power. Te linear regression model (described in Section 2.4) employing the remote 8-item subset was used to estimate the total 50-item MDS-UPDRS ratings. Comparing these estimations to the 50-item rating (upper row Figure 2) showed a highly significant correlation for both the training dataset (Pearson’s r ˆ 0.919, p-value <0.0001) and the validation dataset (Pearson’s r ˆ 0.897, p-value <0.0001). Te equation of the model trained on the entire training dataset was y ˆ 4.87x + 5.59, where x is the sum of the subset ratings. While this equation could be used to convert between the 8-item and 50-item scales, the resulting 50-item rating would only be an approximation (as seen in Figure 1, there was a range of 50-item ratings for each possible 8-item rating) rather than an equivalent score. 4.2. Comparison to Previous Work. Previous work proposed an 8-item subset of the original UPDRS based upon clinical experience [4]. Whilst this is not directly comparable as it was a subset of the original UPDRS [17], approximately mapping these 8 items from the original UPDRS to items from the MDS-UPDRS, we find this previous subset has lower explained variance (EVS ˆ 0.681) than the ones identified by our analyses. However, this is unsurprising as the methodology and motivation behind the previous work was not statistical optimisation of explained variance with respect to the total score, but selection of items based on the individual item clinical relevance. 4. Discussion Remote subset Any-item subset (1.13) fatigue (1.13) fatigue (2.5) dressing (2.1) speech (2.10) tremor (2.5) dressing (2.12) walking and balance (2.10) tremor (3.2) facial movement (3.5) hand movement (3.4) finger tapping (3.7) toe tapping (3.9) arising from chair (3.13) posture (4.3) time spent in the offstate (4.3) time spent in the offstate Remote subset Any-item subset (1.13) fatigue (1.13) fatigue (2.5) dressing (2.1) speech (2.10) tremor (2.5) dressing (2.12) walking and balance (2.10) tremor (3.2) facial movement (3.5) hand movement (3.4) finger tapping (3.7) toe tapping (3.9) arising from chair (3.13) posture (4.3) time spent in the offstate (4.3) time spent in the offstate 4.1. Summary ofResult. We aimed to provide an abbreviated 8-item version of the 50-item MDS-UPDRS that was suitable for remote assessment through identifying a subset of items that minimised information loss, as measured by explained variance with respect to the MDS-UPDRS total score, while adhering to practicality constraints. We conducted an ex- haustive search of all possible 8-item subsets of the 50-items (n ˆ 536,878,650). Tis computationally intensive approach resulted in finding subsets for which the sum ratings were highly correlated (Pearson’s r > 0.9) with the full 50-item UPDRS. comparison of, and constituent items within, each of the top 40 subsets). Tis suggests the amount of information con- tained within each of these 40 subsets is similar, and so the application of remote assessment practicality constraints (i.e., selecting the 20th ranked by EVS, instead of the 1st) has minimal impact upon our evaluation metric. Te subset that maximised explained variance, while adhering to the remote assessment practicality constraints, contained three motor examination items (3.2 Facial Movement, 3.4 Finger Tapping, 3.9 Arising From Chair) along with five question items (1.13 Fatigue, 2.5 Dressing, 2.10 Tremor, 2.12 Walking and Balance, 4.3 Time Spent in the OffState). 3.3. Validation Dataset. We tested the remote subset on the validation dataset, to examine whether the degree of explained variance was similar when applied to a different patient group. For each possible 8-item rating, the range of possible total 50-item ratings was similar in both the training and validation sets (see Figure 1). We found the explained variance for this “remote” subset (EVS ˆ 0.844) and the “any-item” subset (EVS ˆ 0.847) were similar (their 95% credible intervals overlapped), and these subsets shared four of the same items. Parkinson’s Disease scatter-plot (upper left Figure 2) where it can be seen that the model tends to under-estimate when the actual MDS- UPDRS rating is above 100. Tis means that, while the estimator is unbiased on average, there is a conditional bias for patients with very high MDS-UPDRS ratings, and therefore care should be taken in interpreting results in such cases. Table 1: Items contained within the subsets that rank highest, by explained variance score on the training dataset, while adhering (“remote”) or not adhering (“any-item”) to the remote assessment constraints defined in Section 2.3. Tese subsets share four of the same items, and both correspond to the same core aspects of Parkinson’s disease; movement of upper limbs (3.4 or 3.5) and lower limbs (3.9 or 3.7), tremor (2.10), axial (2.12 or 3.13), speech/ facial muscles (3.2 or 2.1), activities of daily living (2.5), fatigue (1.13), and time in Off(4.3). 3. Results We set out to find a subset of 8 MDS-UPDRS items that would be practical for high-frequency remote assessment, which meant excluding certain items from consideration. We excluded items that require face-to-face assessments, which are 3.3 Rigidity and 3.12 Postural Stability [1]. Also, as patients generally carry out remote assessments using an electronic device placed on a desk or table in front of them and assessment of lower body movements and observation of gait requires repositioning of the device or patient, we excluded six items that require the lower limbs of the patient to be observed (items 3.7 Toe Tapping, 3.8 Leg Agility, 3.10 Gait, 3.11 Freezing of Gait, 3.13 Posture, and 3.14 Body Bradykinesia [1]). 2.2. Remote Assessment Practicality Constraints. We set out to find a subset of 8 MDS-UPDRS items that would be practical for high-frequency remote assessment, which meant excluding certain items from consideration. We excluded items that require face-to-face assessments, which are 3.3 Rigidity and 3.12 Postural Stability [1]. Also, as patients generally carry out remote assessments using an electronic device placed on a desk or table in front of them and assessment of lower body movements and observation of gait requires repositioning of the device or patient, we excluded six items that require the lower limbs of the patient to be observed (items 3.7 Toe Tapping, 3.8 Leg Agility, 3.10 Gait, 3.11 Freezing of Gait, 3.13 Posture, and 3.14 Body Bradykinesia [1]). Te statistical metrics for these two subsets were similar (remote, any-item; EVS ˆ 0.844, 0.847; PCC ˆ 0.919, 0.920; SRC ˆ 0.900, 0.905, see Supplement Table 5). Te correlation between the sum of the 8-item ratings for these two subsets was highly significant by both PCC (Pearson’s r ˆ 0.898, p-value <0.0001) and SRC (Spearman’s r ˆ 0.880, p-value <0.0001), see Supplement Figure 4. Te remote subset was ranked 20th among all subsets evaluated. Furthermore, we found a large overlap in the 95% credible intervals for each of the 40 highest ranked subsets (see Supplement Figure 5 and Supplement Table 6 for 3 4. Discussion For example, item 1.3 Depressed Mood, which was included in the previous work’s 8-item scale is a recognised important clinical feature in that Use of the remote 8-item subset to estimate the 50-item rating provided an unbiased estimator, as can be seen from the credible interval of the mean residuals of this linear model estimator being centered on zero (lower row Fig- ure 2). We note there is a slight right skew in the residuals of the training dataset, which can also be observed in the Parkinson’s Disease 4 2 0 20 40 60 Total MDS-UPDRS rating 80 100 120 Training data Validation data 3 4 5 6 7 8 9 Remote subset 8-item rating 10 11 12 13 14 15 16 17 18 Figure 1: Boxplots showing the distribution of the sum rating of the total 50-item MDS-UPDRS rating for each value of the remote subset rating (see Table 1) for both training and validation datasets, with the interquartile range indicated by the boxes and the range from the 2.5th to 97.5th percentile indicated by the whiskers. Note this figure only displays boxplots of 8-item ratings for which n ≥5, e.g., 8-item rating of zero is not displayed because n ≥5 in the validation dataset (Supplement Table 8 shows the (n) for each rating). For each of these ratings, there is a large overlap between the range from 2.5th to 97.5th percentile of the training and validation datasets. Total MDS-UPDRS rating Figure 1: Boxplots showing the distribution of the sum rating of the total 50-item MDS-UPDRS rating for each value of the remote subset rating (see Table 1) for both training and validation datasets, with the interquartile range indicated by the boxes and the range from the 2.5th to 97.5th percentile indicated by the whiskers. Note this figure only displays boxplots of 8-item ratings for which n ≥5, e.g., 8-item rating of zero is not displayed because n ≥5 in the validation dataset (Supplement Table 8 shows the (n) for each rating). For each of these ratings, there is a large overlap between the range from 2.5th to 97.5th percentile of the training and validation datasets. if a patient is depressed he may benefit from antidepressant treatment. However, our work found it to be relatively uninformative for estimating the total score. 4. Discussion Similarly, item 4.1 Time Spent with Dyskinesias was uninformative for estimation of the total score, most likely because dyskinesias affect a relatively small proportion of patients. Our proposed 8-item subset is therefore intended to provide information on the overall disease severity equivalent to the total MDS- UPDRS score. Our proposal does not aim to provide the same comprehensive clinical information regarding indi- vidual items of the MDS-UPDRS or individual aspects of PD that might require treatment. Rather, similar to the total MDS-UPDRS score, it can be considered an index of overall disease severity. intrapatient comparison, comparing a patient over time, rather than interpatient comparison. We note that the nature of our statistical approach means we inevitably arrived at a subset that was optimised for the “average” PD patient. Other approaches, such as the previous work discussed above, could result in a subset that may be more useful to specific patients and patient groups but slightly less so for the majority. Future work could examine ways of segmenting patient populations and the intent of assessments in order to arrive at differently opti- mised subsets for different patient groups. For instance, one subset might be more useful for patients in a research setting vs clinical care or be better suited for patients who are being considered for deep brain stimulation. Our method quantifies information loss with respect to the total MDS-UPDRS score, but for certain monitoring purposes only the motor examination (part III) may be relevant, and for others only the aspects of daily living (parts I & II). We note the 8-item subset resulting from our ap- proach consists mostly of motor items (parts II & III) and includes only a single part I nonmotor item, meaning there is greater information loss with respect to nonmotor symp- toms as compared to motor symptoms. Future work could identify item subsets that maximise different parts of the MDS-UPDRS, such as a 5-item nonmotor assessment. 4.3. Limitations and Future Work. Te selection of our proposed 8-item subset relied upon the training dataset, which primarily represents patients in earlier stages of PD. Patients included in the validation dataset had greater later stage representation, but the results were similar, indicating the results are robust. Nevertheless, future work should examine whether other 8-item subsets may be more ap- propriate for later disease stages. 4.3. Limitations and Future Work. 4. Discussion Te selection of our proposed 8-item subset relied upon the training dataset, which primarily represents patients in earlier stages of PD. Patients included in the validation dataset had greater later stage representation, but the results were similar, indicating the results are robust. Nevertheless, future work should examine whether other 8-item subsets may be more ap- propriate for later disease stages. Te use of an 8-item scale will lead to measurements that have a greater level of noise compared to 50-items, par- ticularly for later stage PD. Tis means that comparisons between patients, particularly those with later stage PD, using the 8-item rating should be done with caution. We believe the scale will have more practical benefit for Te MDS-UPDRS has not yet been validated for remote use. If and when this validation occurs, future work could validate our abbreviated scale by a similar method. Lastly, our proposed 8-item subset is not suitable for use as a diagnostic tool, just as the full MDS-UPDRS is not a 5 Parkinson’s Disease Training data (PPMI, n=7594) 175 150 125 100 Actual total score 75 50 25 0 0 25 50 75 100 Estimated total score 125 150 175 Validation data (NMS, n=377) 175 0.84 95% credible interval 0.82 Explained variance score 0.80 0.78 150 125 100 Actual total score 75 50 25 0 0 25 50 75 100 Estimated total score 125 150 175 –40 0 100 200 300 400 Frequency 500 600 700 –30 –20 –10 0 Residuals 10 20 30 40 –40 0 5 10 15 20 Frequency 25 30 35 –30 –20 –10 0 Residuals 10 20 30 40 –1.5 –1.0 –0.5 Mean residual 0.0 0.5 1.0 1.5 25 –30 –20 –10 0 10 Residuals 20 30 40 50 75 100 Mean of actual and estimate 125 150 –1.96 S.D +1.96 S.D Mean 175 25 –30 –20 –10 0 10 Residuals 20 30 40 50 75 100 Mean of actual and estimate 125 150 –1.96 S.D +1.96 S.D Mean 175 Figure 2: A linear regression model was used to estimate the total 50-item MDS-UPDRS rating (see Section 2.4), using the 8-item rating of the “remote” subset (see Table 1). (Upper left) Te correspondence between ground truth and estimations for the training dataset, and (upper center) the validation dataset. Note: the X ˆ Y line is marked in grey. 4. Discussion For both datasets, the correlations between ground truth and estimations were highly significant (p-value <0.0001), and (upper right) the 95% credible intervals of these correlations overlapped. Tis suggests the ability of this 8-item subset to estimate the total 50-item MDS-UPDRS rating generalises across different datasets. (Mid left) Te residuals of the estimator for the training dataset, and (mid center) the validation dataset. Te mean residuals for both datasets were close to zero, and (mid right) the 95% credible intervals of the mean residual crossed zero for both datasets. Tis suggests the linear regression model is an unbiased estimator of the total 50-item MDS-UPDRS rating. (Lower left) Te Bland–Altman plot of agreement between the actual and estimated total score for training (lower center) and validation, both of which show that residuals are approximately equally distributed either side of zero across the range of possible values of total score, indicating a lack of proportional bias. 4. Discussion Validation data (NMS, n=377) 175 150 125 100 Actual total score 75 50 25 0 0 25 50 75 100 Estimated total score 125 150 175 Training data (PPMI, n=7594) 175 150 125 100 Actual total score 75 50 25 0 0 25 50 75 100 Estimated total score 125 150 175 Validation data (NMS, n=377) Estimated total score –40 0 100 200 300 400 Frequency 500 600 700 –30 –20 –10 0 Residuals 10 20 30 40 25 –30 –20 –10 0 10 Residuals 20 30 40 50 75 100 Mean of actual and estimate 125 150 –1.96 S.D +1.96 S.D Mean 175 –1.5 –1.0 –0.5 Mean residual 0.0 0.5 1.0 1.5 –40 0 100 200 300 400 Frequency 500 600 700 –30 –20 –10 0 Residuals 10 20 30 40 –40 0 5 10 15 20 Frequency 25 30 35 –30 –20 –10 0 Residuals 10 20 30 40 Mean residual 25 –30 –20 –10 0 10 Residuals 20 30 40 50 75 100 Mean of actual and estimate 125 150 –1.96 S.D +1.96 S.D Mean 175 Residuals Residuals Mean of actual and estimate Mean of actual and estimate Figure 2: A linear regression model was used to estimate the total 50-item MDS-UPDRS rating (see Section 2 on model was used to estimate the total 50-item MDS-UPDRS rating (see Section 2.4), using the 8-item rating of Figure 2: A linear regression model was used to estimate the total 50-item MDS-UPDRS rating (see Section 2.4), using the 8-item rating of the “remote” subset (see Table 1). (Upper left) Te correspondence between ground truth and estimations for the training dataset, and (upper center) the validation dataset. Note: the X ˆ Y line is marked in grey. For both datasets, the correlations between ground truth and estimations were highly significant (p-value <0.0001), and (upper right) the 95% credible intervals of these correlations overlapped. Tis suggests the ability of this 8-item subset to estimate the total 50-item MDS-UPDRS rating generalises across different datasets. (Mid left) Te residuals of the estimator for the training dataset, and (mid center) the validation dataset. Te mean residuals for both datasets were close to zero, and (mid right) the 95% credible intervals of the mean residual crossed zero for both datasets. Tis suggests the linear regression model is an unbiased estimator of the total 50-item MDS-UPDRS rating. Data Availability Te supplementary materials report included with this paper contains additional tables and figures, as well as a more detailed explanation of the statistical methodology. (Sup- plementary Materials) Te Parkinson’s disease assessment data used to support the findings of this study were supplied by the Parkinson’s Progression Markers Initiative (PPMI). Qualified re- searchers may obtain access by direct request to the PPMI (https://www.ppmi-info.org/). Additional Parkinson’s disease assessment data used to support the findings of this study were supplied by the Movement Disorder Society (MDS) Non-Motor Symptoms Scale Develop- ment Study Group. Requests for access to these data should be made to the MDS (https://www. movementdisorders.org/). Consent Te authors state that ethics agreements and informed consent have been obtained for studies on patients. [3] L. Henderson, C. Kennard, T. J. Crawford et al., “Scales for rating motor impairment in Parkinson’s disease: studies of reliability and convergent validity,” Journal of Neurology Neurosurgery and Psychiatry, vol. 54, no. 1, pp. 18–24, 1991. Conflicts of Interest [4] R. A. Hauser, K. E. Lyons, and R. Pahwa, “Te updrs-8: A brief clinical assessment scale for parkinson’s disease,” Interna- tional Journal of Neuroscience, vol. 122, no. 7, pp. 333–337, 2012. Te authors declare that there are no conflicts of interest. Authors’ Contributions [5] R. B. Schneider and K. M. Biglan, “Te promise of tele- medicine for chronic neurological disorders: the example of Parkinson’s disease,” Te Lancet Neurology, vol. 16, no. 7, pp. 541–551, 2017. Gareth Morinan performed conceptualization, formal analysis, investigation, methodology, project adminis- tration, software, supervision, validation, visualization, and writing—original draft, review, and editing. Robert A. Hauser contributed to conceptualization and wri- ting—review and editing. Anette Schrag performed data curation and writing—review and editing. Jingxuan Tang performed formal analysis, investigation, software, and validation. Jonathan O’Keeffe contributed to conceptu- alization, funding acquisition, methodology, project administration, resources, supervision, and wri- ting—review and editing. MDS-NMS Scale Development Study Group (Anette Schrag, Ray Chaudhuri, Daniel Weintraub, and Pablo Martinez-Martin) performed data curation. All authors have read and approved the final manuscript. [6] D. Alajmi, S. Almansour, and M. S. Househ, “Recommen- dations for implementing telemedicine in the developing world,” Studies in Health Technology and Informatics, vol. 190, pp. 118–120, 2013. [7] E. R. Dorsey, L. M. Deuel, T. S. Voss et al., “Increasing access to specialty care: a pilot, randomized controlled trial of telemedicine for Parkinson’s disease,” Movement Disorders: Official Journal of the Movement Disorder Society, vol. 25, no. 11, pp. 1652–1659, 2010. [8] V. Venkataraman, S. J. Donohue, K. M. Biglan, P. Wicks, and E. R. Dorsey, “Virtual visits for Parkinson disease: a case series,” Neurology: Clinical Practice, vol. 4, no. 2, pp. 146–152, 2014. [9] E. Cubo, J. M. T. Gabriel-Gal´an, J. S. Mart´ınez et al., “Comparison of officebased versus home web-based clinical assessments for Parkinson’s disease,” Movement Disorders: Official Journal of the Movement Disorder Society, vol. 27, no. 2, pp. 308–311, 2012. 4. Discussion National Institute for Health Research University College London Hospitals Biomedical Research Centre. Tis study was funded by Innovate UK, Capital Enterprise, and Machine Medicine Technologies. References [1] C. G. Goetz, B. C. Tilley, S. R. Shaftman et al., “Movement disorder society-sponsored revision of the unified parkinson’s disease rating scale (MDS-UPDRS): scale presentation and clinimetric testing results,” Movement disorders: official journal of the Movement Disorder Society, vol. 23, no. 15, pp. 2129–2170, 2008. [2] P. Martinez-Martin, A. Gil-Nagel, L. M. Gracia et al., “Unified Parkinson’s disease rating scale characteristics and structure,” Movement Disorders, vol. 9, no. 1, pp. 76–83, 1994. 4. Discussion (Lower left) Te Bland–Altman plot of agreement between the actual and estimated total score for training (lower center) and validation, both of which show that residuals are approximately equally distributed either side of zero across the range of possible values of total score, indicating a lack of proportional bias. due to the time required, which is estimated to be about 30 minutes. In addition, many PD clinical visits are being performed via telehealth, and we suspect even fewer MDS-UPDRS ratings are performed under these cir- cumstances due to additional barriers, including diffi- culty visualizing various body parts and performance tasks. We have therefore derived brief, 8-item versions of the MDS-UPDRS that correlate well with the overall total MDS-UPDRS score. Tese scales, an any-item scale that can be used in person and a remote scale that can be used for telehealth visits, can be used as an overall index of disease status and only add an additional 5–10 minutes. We believe this may be appealing to busy clinicians as an adjunct to their usual history and exam. diagnostic tool. We note in particular that our 8-item subset does not include rigidity, which the PD diagnosis criteria mention as a key feature of the disease. Tus, our 8-item subset should only be administered in patients who have received a PD diagnosis. Additionally, the 8-item scale is not directly comparable to, and therefore should not replace, the full MDS-UPDRS scale. 4.4. Practical Application. We estimate that for the vast ma- jority of PD patients the “remote” 8-item assessments could be completed in 5–10 minutes. Tis estimate is based upon each motor examination item typically taking 30–60 seconds to complete, along with an assumption that each question would take 15–30 seconds, and 2-3 minutes being required to initially set up the assessment and transition between items. Our result demonstrates that an 8-item subset iden- tified by computational statistics appears to be a robust abbreviated version of the total MDS-UPDRS, which could be used to create a total score where practicality Outside of clinical trials and academic centers, MDS- UPDRS ratings are rarely performed. Tis appears to be 6 Parkinson’s Disease limits the application of the full MDS-UPDRS. Addi- tionally, given this subset was constrained to not include items that may be impractical for home assessment, this version is well suited for remote monitoring. Acknowledgments [10] K. Marek, D. Jennings, S. Lasch et al., “Te Parkinson pro- gression marker initiative (PPMI),” Progress in Neurobiology, vol. 95, no. 4, pp. 629–635, 2011. Te data was collected as part of the Parkinson’s Pro- gression Marker’s Initiative. Te authors thank the PPMI Steering Committee and all staffat the PPMI clinical sites. Te data was collected as part of the MDS-NMS vali- dation study [11]. Te authors thank the MDS-NMS Scale Development Study Group (Anette Schrag, Pablo Mar- tinez-Martin, Daniel Weintraub, and Kallol Ray Chaudhuri). Anette Schrag was supported by the [11] K. R. Chaudhuri, A. Schrag, D. Weintraub et al., “Te movement disorder society nonmotor rating scale: initial validation study,” Movement Disorders: Official Journal of the Movement Disorder Society, vol. 35, no. 1, pp. 116–133, 2020. [12] H. B. Mann and D. R. Whitney, “On a test of whether one of two random variables is stochastically larger than the other,” Te Annals of Mathematical Statistics, vol.18, pp. 50–60, 1947. 7 Parkinson’s Disease [13] M. M. Hoehn and M. D. Yahr, “Parkinsonism: onset, pro- gression, and mortality,” Neurology, vol. 50, no. 2, p. 318, 1998. [14] C. H. Achen, “What does “explained variance” explain?” Reply, Political Analysis, vol. 2, pp. 173–184, 1990. [15] C. Spearman, “Reprinted: the proof and measurement of association between two things (2010),” International Journal of Epidemiology, vol. 39, pp. 1137–1150, 1904. f p gy [16] K. Pearson, “Notes on regression and inheritance in the case of two parents,” Proceedings of the Royal Society of London, vol. 58, pp. 240–242, 1895. [17] S. Fahn, “Members of the updrs development committee. unified Parkinson’s disease rating scale,” Recent developments in Parkinson’s disease, vol. 2, pp. 293–304, 1987.
https://openalex.org/W2606827187
https://europepmc.org/articles/pmc5468145?pdf=render
English
null
The combined effects of reactant kinetics and enzyme stability explain the temperature dependence of metabolic rates
Ecology and evolution
2,017
cc-by
9,243
J. P. DeLong  | J. P. Gibert | T. M. Luhring | G. Bachman | B. Reed |  A. Neyer | K. L. Montooth School of Biological Sciences, University of Nebraska – Lincoln, Lincoln, NE, USA School of Biological Sciences, University of Nebraska – Lincoln, Lincoln, NE, USA Received: 12 December 2016  |  Revised: 28 February 2017  |  Accepted: 7 March 2017 Received: 12 December 2016  |  Revised: 28 February 2017  |  Accepted: 7 March 2017 Received: 12 December 2016  |  Revised: 28 February 2017  |  Accepted: 7 March 2017 DOI: 10.1002/ece3.2955 DOI: 10.1002/ece3.2955 Abstract A mechanistic understanding of the response of metabolic rate to temperature is essential for understanding thermal ecology and metabolic adaptation. Although the Arrhenius equation has been used to describe the effects of temperature on reaction rates and metabolic traits, it does not adequately describe two aspects of the thermal performance curve (TPC) for metabolic rate—that metabolic rate is a unimodal function of temperature often with maximal values in the biologically relevant temperature range and that activation energies are temperature dependent. We show that the temperature dependence of metabolic rate in ectotherms is well described by an enzyme-­assisted Arrhenius (EAAR) model that accounts for the temperature-­ dependent contribution of enzymes to decreasing the activation energy required for reactions to occur. The model is mechanistically derived using the thermodynamic rules that govern protein stability. We contrast our model with other unimodal functions that also can be used to describe the temperature dependence of metabolic rate to show how the EAAR model provides an important advance over previous work. We fit the EAAR model to metabolic rate data for a variety of taxa to demonstrate the model’s utility in describing metabolic rate TPCs while revealing significant differences in thermodynamic properties across species and acclimation temperatures. Our model advances our ability to understand the metabolic and ecological consequences of increases in the mean and variance of temperature associated with global climate change. In addition, the model suggests avenues by which organisms can acclimate and adapt to changing thermal environments. Furthermore, the parameters in the EAAR model generate links between organismal level performance and underlying molecular processes that can be tested for in future work. Correspondence J. P. DeLong, School of Biological Sciences, University of Nebraska – Lincoln, Lincoln, NE, USA. Email: jpdelong@unl.edu Present Address J. P. Gibert, The University of California, Merced, Merced, CA, USA Funding information United States-Israel Binational Science Foundation, Grant/Award Number: 2014295; Directorate for Biological Sciences, Grant/ Award Number: 1501668 and 1505247 Correspondence J. P. DeLong, School of Biological Sciences, University of Nebraska – Lincoln, Lincoln, NE, USA. Email: jpdelong@unl edu 3940  |   Ecology and Evolution. 2017;7:3940–3950 www.ecolevol.org 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. Ecology and Evolution published by John Wiley & Sons Ltd. Abstract 1 | INTRODUCTION Temperature plays a major role in setting biological rates across all lev- els of organization, from biochemical reactions within cells to nutrient turnover in ecosystems (Brown, Gillooly, Allen, Savage, & West, 2004 Hochachka & Somero, 2002; Kleiber, 1961; Schulte, 2015; Yvon- Durocher et al., 2012). The temperature dependence of metabolic rate is among the most fundamental of thermal relationships, playing a Ecology and Evolution. 2017;7:3940–3950. The combined effects of reactant kinetics and enzyme stability explain the temperature dependence of metabolic rates J. P. DeLong  | J. P. Gibert | T. M. Luhring | G. Bachman | B. Reed |  A. Neyer | K. L. Montooth significant role in setting the temperature dependence of many other biological processes (Brown et al., 2004; Dell, Pawar, & Savage, 2011; Gillooly, Brown, West, Savage, & Charnov, 2001). As such, an under- standing of the response of metabolic rate to temperature is essential for understanding thermal ecology. to describe the temperature dependence of metabolic rate. First, the Arrhenius factor is a monotonically increasing function of tem- perature, whereas metabolic rate and many other biological rates are unimodal functions of temperature, generally known as thermal per- formance curves (TPC; Huey & Stevenson, 1979; Huey & Kingsolver, 1989; Angilletta, Niewiarowski, & Navas, 2002). It is often argued that the Arrhenius equation is sufficient because it applies to the range of temperatures sometimes referred to as the “biologically relevant temperature range” (usually 0–40°C; Figure S1) or, alternatively, the temperature range between the minimum temperature and the tem- perature at which maximal metabolic rates are observed (Topt) (Gillooly et al., 2001). The justification for focusing on restricted temperature ranges is that these are the temperatures at which organisms spend most of their time. Although this may be true in some cases, many organisms experience temperatures above their Topt, where metabolic rates decrease and thus are not expected to be well described by the Arrhenius equation, as the Arrhenius factor increases monotonically with temperature. This problem is currently becoming more import- ant, as future climate scenarios predict warmer and more variable temperatures (Schulte, 2015), causing organisms to spend more time at the upper extremes of their viable temperature ranges where the Arrhenius equation does not apply, or, if used, would overestimate metabolic rates. The metabolic rate of ectotherms typically increases rapidly as temperature increases from lower temperatures, and this increase is often described using an Arrhenius function (Dell et al., 2011; Gillooly et al., 2001; Robinson, Peters, & Zimmermann, 1983). The Arrhenius equation models the effect of temperature on the rate (V) of a reaction by scaling the potential reaction rate A0 (set by the availability of ap- propriately conformed reactants) by the Arrhenius factor, e−Ea∕kT, where k is Boltzmann’s constant (8.61 × 10−5 eV K−1), T is absolute temperature (K), and Ea is the activation energy—the minimum energy that must be available for the chemical reaction to occur (eV) (Figure 1a; Laidler, 1984): (1) V=A0e−Ea∕kT, (1) The Arrhenius factor varies between 0 and 1 (Figure S1), giving the proportion of the potential reaction rate A0 that can occur given the kinetic state of the reactants. The product kT is the average kinetic energy of the reactants, such that as temperature increases, the energy of the reactants increases, reducing the value of the exponent and raising e−Ea∕kT toward 1. Second, the activation energy is a constant in the standard Arrhenius model, yet activation energy can vary across biologically relevant temperatures (Gibert, Chelini, Rosenthal, & DeLong, 2016; Knies & Kingsolver, 2010; Pawar, Dell, Savage, & Knies, 2016; Schulte, 2015). Such empirical observations suggest that even over tem- perature ranges where the Arrhenius equation is thought to apply, it may not be sufficiently nuanced to enable prediction of metabolic responses to temperature under warmer and more variable climates (Gibert et al., 2016; Pawar et al., 2016). The Arrhenius equation was originally applied to describe the temperature dependence of chemical reaction rates in controlled settings, but it has also been applied to describe the thermal depen- dence of biological rates, including enzyme-­catalyzed reactions and organismal metabolic rate, an application widely promoted in the met- abolic theory of ecology (MTE; Brown et al., 2004). Despite the good fit of the Arrhenius equation to many data sets and its widespread use by ecologists (Allen, Brown, & Gillooly, 2002; Anderson-­Teixeira, DeLong, Fox, Brese, & Litvak, 2010; Dell et al., 2011; Ernest et al., 2003; López-­Urrutia, San Martin, Harris, & Irigoien, 2006; O’Connor, Piehler, Leech, Anton, & Bruno, 2009; Yvon-­Durocher et al., 2012), there are two unresolved problems with using the Arrhenius equation There have been several efforts to modify the Arrhenius equa- tion and generate models that describe the unimodal response of reaction rates to temperature (Box 1). 1 | INTRODUCTION Ecology and Evolution. 2017;7:3940–3950. DELONG et  al 3941 Box 1 A history of models describing the unimodal temperature dependence of enzyme-­catalyzed reaction rates A range of models have been developed to describe the dependence of a reaction rate (V, for reaction velocity) on temperature (T). Most of these models were originally developed to describe enzyme-­catalyzed reactions, rather than metabolic rate per se, but model 4 was developed to describe population growth rate and model 3 originally dealt with development rate. Nonetheless, they all have potential as descriptions of the temperature dependence of metabolic rate. All of the models begin with a monotonically increasing function of tem- perature, either the Eyring or the Arrhenius equation, the difference being that the Eyring model explicitly includes temperature in the constant. All of the models invoke a reduction in enzyme performance at low and/or high temperature due to the decreased probability of enzymes being in an active state. A key difference between the EAAR model and models 1-­5 is that in the EAAR model, enzymes increase reaction rates over some baseline rate, while in models 1-­5, reduced enzyme performance lowers the reaction rate from a maximal rate (Figure B1). The models invoke different assumptions, some of which are shared across models and others which are unique to specific models. The key assumptions invoked by each model are indicated with a check-­mark in the Table and listed here: (1) enzymes are inactive at high temperature, (2) enzymes are inactive at low temperature, (3) active state is given by a three-­state transition process, (4) active state is given by a protein stability process, (5) substrate supply is unlimited, (6) the proportion of enzymes that are in an active state is at equilib- rium, (7) enzymes denature through time, (8) the activation energy of the catalyzed reaction is equal to the free energy of the catalyzing enzymes, (9) heat capacity is negative, (10) activation energy corresponds to maximal enzyme activity level, (11) there is no activation en- ergy in the absence of enzymes, (12) the activation energy of the reaction is lowered as a function of the free energy of the catalyzing enzymes. Box 1 A history of models describing the unimodal temperature dependence of enzyme-­catalyzed reaction rates The common parameters in these models are ΔH, enthalpy change of folding the enzymes, relative to a reference or melting temperature, subscripted A for active state and L for lower temperature inactive state; ΔS, entropy change of an enzyme with temperature, relative to the melting temperature, subscripted A for active state and L for lower temperature inactive state; ΔCp, the difference in heat capacity between the folded and unfolded state of the enzymes, relative to the melting temperature; Tm, melting temperature. The Schoolfield model includes three summary parameters: ρ(25°C), rate at 25°C assuming no inactivation; T1/2, the temperature at which the enzyme is half active and half inactive, subscripted L for low-­temperature inactive and H for high-­temperature inactive. The equilibrium model includes four parameters: Teq, the temperature at which the concentration of active and inactive enzymes is equal; ΔHeq, the change in enthalpy associated with the equilibrium; ΔGcat, activation energy for the reaction; and ΔGinact, activation energy for enzyme inactivation. The EAAR model introduces Eb, the baseline activation energy, the change in activation energy associated with the change in enthalpy of the cata- lysts (EΔCp), and the change in activation energy associated with the change in heat capacity of the catalysts (EΔH). The physical constants are k, Boltzmann’s constant, R, the gas constant, and h, Planck’s constant. Beginning with Johnson and Lewin (1946), at least five models have modified an Arrhenius (or the FIGURE 1 (a) Reactions proceed as the reactants gain enough energy to clear the hurdle of the activation energy (Ea) to form products. (b) Organisms contribute some energy to reactions occurring within their bodies with enzymes. The contributed energy lowers the kinetic hurdle that reactants must clear, such that the net activation energy is Eb − Ec(T), the latter of which is temperature dependent via effects of temperature on protein stability. (c) Metabolic reactions have to clear their particular activation energy (Eb, gray bar), but enzymes provide a temperature-­dependent contribution to the starting energetic state of the reactants through a temperature-­dependent increase in stability (ΔH, orange arrow). Below the melting temperature, however, temperature lowers the energetic state through its effect on heat capacity (ΔCp, red arrow) FIGURE 1 (a) Reactions proceed as the reactants gain enough energy to clear the hurdle of the activation energy (Ea) to form products. (b) Organisms contribute some energy to reactions occurring within their bodies with enzymes. The contributed energy lowers the kinetic hurdle that reactants must clear, such that the net activation energy is Eb − Ec(T), the latter of which is temperature dependent via effects of temperature on protein stability. (c) Metabolic reactions have to clear their particular activation energy (Eb, gray bar), but enzymes provide a temperature-­dependent contribution to the starting energetic state of the reactants through a temperature-­dependent increase in stability (ΔH, orange arrow). Below the melting temperature, however, temperature lowers the energetic state through its effect on heat capacity (ΔCp, red arrow) DELONG et  al 3942 Box 1 A history of models describing the unimodal temperature dependence of enzyme-­catalyzed reaction rates These energies are not the same, as recognized by the previous models that separate out an activation energy for a reaction from the free energy of the catalysts (Box 1). Second, the model requires the heat capacity of the catalysts to be negative (Box 1, Assumption 9), when this value must be positive, as indicated in previous work (Becktel & Schellman, 1987; Feller, 2010; Ratkowsky et al., 2005). Third, the model reduces to a linear function of temperature in the absence of enzymes (Box 1, Assumption 11) with a universal slope of the Boltzmann constant divided by Plank’s constant, rather than an Arrhenius type function. For these and other reasons (see key assumptions in Box 1), the current unimodal modifications of the Arrhenius function are not sufficient. A model that describes and predicts metabolic rate TPCs based on more realistic biological mechanisms is therefore urgently needed (Schulte, 2015). Here, we derive a general model for the temperature dependence of metabolic rate with biologically meaningful parameters that captures the unimodal shape of a metabolic rate TPC. We do this by incorporating a mechanistically derived temperature-­dependent protein stability curve, which specifies the extent to which enzymes can catalyze metabolic reactions, into the Arrhenius equation to cap- ture the temperature dependence in the ability of enzymes to lower activation energies, yielding the enzyme-­assisted Arrhenius (EAAR) model. Box 1 A history of models describing the unimodal temperature dependence of enzyme-­catalyzed reaction rates Note that model 1 has a lowered performance region only at high temperature. (b) The EAAR model begins with the assumption that there is a physical baseline reaction rate (black line) that may occur outside of an organism and that has a relatively high activation energy. Inside an organism, enzymes assist the process by lowering the activation energy, boosting the reaction rate above what would otherwise be expected, to the blue line FIGURE B1 How the EAAR model and other models bend Arrhenius type functions to create a unimodal function. (a) Most models assume a maximal reaction rate (red line) that is discounted by lowered probability of enzyme performance, reducing the reaction rate to the blue line. Note that model 1 has a lowered performance region only at high temperature. (b) The EAAR model begins with the assumption that there is a physical baseline reaction rate (black line) that may occur outside of an organism and that has a relatively high activation energy. Inside an organism, enzymes assist the process by lowering the activation energy, boosting the reaction rate above what would otherwise be expected, to the blue line similar Eyring) function by discounting the rates at low and/or high temperatures (Daniel & Danson, 2010; Ratkowsky, Olley, & Ross, 2005; Schoolfield, Sharpe, & Magnuson, 1981; Sharpe & DeMichele, 1977). These models use different functions to reduce the probabil- ity of enzymes being in an active state at low and/or high tempera- tures, but are consistent in the assumption that the activation energy in the Arrhenius type function corresponds to the state of maximal enzyme activity (Box 1, Assumption 10, Figure B 1). This assumption is problematic because it requires that in the absence of enzymes, the reaction would still occur with a low activation energy corre- sponding to fully active enzymes. Furthermore, this assumption con- tradicts what enzymes are actually thought to do, which is to lower the activation energy below a baseline level (Box 1, Assumption 12). The more recent macromolecular rates model (Hobbs et al., 2013) uses a different approach, altering the activation of a reaction di- rectly through a protein stability curve. Although this approach shows promise, there are three problematic assumptions with this model. First, the activation energy of the reaction is made equal to the free energy of the enzymes themselves (Box 1, Assumption 8). Box 1 A history of models describing the unimodal temperature dependence of enzyme-­catalyzed reaction rates Model # Model/source Model Assumptions 1 2 3 4 5 6 7 8 9 10 11 12 1 Johnson-­Lewin (Johnson & Lewin, 1946) V= cTeΔHA∕RT 1+eΔS∕ReΔHA∕RT ✔ ✔ ✔ 2 Sharpe-­DeMichele (Sharpe & DeMichele, 1977) V= kT h e(ΔSA−ΔHA∕T)∕R 1+e(ΔSL−ΔHL∕T)∕R+e(ΔSH−ΔHL∕T)∕R ✔ ✔ ✔ ✔ ✔ 3 Schoolfield (Schoolfield et al., 1981) V= 휌( 25 ◦C ) T 298e [ ΔHA R ( 1 298 −1 T )] 1+e [ ΔHL R ( 1 T1∕2L −1 T )] +e [ ΔHH R ( 1 T1∕2H −1 T )] ✔ ✔ ✔ ✔ ✔ 4 Ratkowsky (Ratkowsky et al., 2005) V= cTe(ΔHA∕RT) 1+e(−n[ΔH−TΔS+ΔCp[(T−Tm)−T ln (T∕Tm)])]∕RT) ✔ ✔ ✔ ✔ ✔ 5 Equilibrium model (Daniel & Danson, 2010) V= kT h e−(ΔGcat∕RT)E0e ⎡ ⎢ ⎢ ⎢ ⎢⎣ kTe−(ΔGinact∕RT)e  ΔHeq  1 Teq −1 T  ∕R  t 1+e  ΔHeq  1 Teq −1 T  ∕R  ⎤ ⎥ ⎥ ⎥ ⎥⎦ 1+e  ΔHeq  1 Teq −1 T  ∕R  ✔ ✔ ✔ ✔ ✔ 6 Macromolecular rates (Hobbs et al., 2013) V= kT h e − ( ΔH−TΔS+ΔCp ( T−Tm−T ln T Tm )) RT ✔ ✔ ✔ ✔ ✔ ✔ 7 Enzyme-­assisted Arrhenius (This study) V=A0e − ( Eb− ( EΔH ( 1−T Tm ) +EΔCp ( T−Tm−T ln T Tm ))) kT ✔ ✔ ✔ ✔ DELONG et  al 3943 |  394 DELONG et  al FIGURE B1 How the EAAR model and other models bend Arrhenius type functions to create a unimodal function. (a) Most models assume a maximal reaction rate (red line) that is discounted by lowered probability of enzyme performance, reducing the reaction rate to the blue line. Note that model 1 has a lowered performance region only at high temperature. (b) The EAAR model begins with the assumption that there is a physical baseline reaction rate (black line) that may occur outside of an organism and that has a relatively high activation energy. Inside an organism, enzymes assist the process by lowering the activation energy, boosting the reaction rate above what would otherwise be expected, to the blue line Box 1 (Continued) Box 1 (Continued) Box 1 (Continued) FIGURE B1 How the EAAR model and other models bend Arrhenius type functions to create a unimodal function. (a) Most models assume a maximal reaction rate (red line) that is discounted by lowered probability of enzyme performance, reducing the reaction rate to the blue line. What is missing from the Arrhenius model is a recognition that the activity level of enzymes follows a hump-­shaped relation with tem- perature (Feller, 2010; Peterson, Daniel, Danson, & Eisenthal, 2007), as recognized in previous unimodal models (Box 1). At low and/or high temperatures, enzymes may occur in inactive states, either through reversible unfolding or denaturation. As a consequence, enzymes are less effective at reducing the Eb of metabolic rate at low and high temperatures. Thus, increasing enzymatic contributions with increas- ing temperature helps to increase the metabolic rate up to the Topt, while decreasing enzymatic contributions as temperature continues to increase generates the decreasing slope observed for metabolic rate TPCs. activation by an amount EL, such that Ec =EL ΔG ΔGmax. Thus, we replace each parameter in Equation 3 to account for this transformation (i.e., EΔH =EL ΔH ΔGmax, EΔCp =EL ΔCp ΔGmax) and rewrite Equation 3 as follows: (4) Ec =EΔH ( 1−T Tm ) +EΔCp ( T−Tm −T ln T Tm ) (4) Substituting Equation 4 into Equation 2, we get (5) V=A0e − ( Eb− ( EΔH ( 1−T Tm ) +EΔCp ( T−Tm−T ln T Tm ))) kT , (5) which now provides a mechanistic description of the temperature de- pendence of metabolic rate that is generated by both reactant ­kinetics and temperature-­dependent enzyme stability. Here, we incorporate a model for protein stability/free energy into the Arrhenius equation to provide a mechanistic basis for the tem- perature dependence of metabolic rate that accounts for the contri- butions of both reactant kinetics and the temperature dependence of enzyme activity. This is an important conceptual advancement over our current description of the rising portion of metabolic rate TPCs as a function of the energetic state of reactants. This change in viewpoint clarifies how and why activation energies should change during the rising portion of the TPC, and why metabolic rate should decline again above an optimal temperature. The thermodynamic parameters in Equation 5 provide a nonphe- nomenological depiction of how enzyme stability alters reaction rates. ΔH is by definition zero at the melting point and increases below the melting point, meaning that the colder it gets, the more stable the enzyme is and the more effectively it can contribute to a reaction (Figure 1c). ΔCp scales the loss of free energy as temperature goes below the melting point, meaning the colder it gets below the melting temperature, the more energy the enzyme can retain without chang- ing temperature, in effect scrubbing free energy from the system. The expression ( T−Tm −T ln T Tm ) thus represents the loss of function as- sociated with being unfolded and is zero at the melting temperature. The parameters in our model reflect the change in activation energy associated with the change in enthalpy of the catalysts (EΔCp) and the change in activation energy associated with the change in heat capac- ity of the catalysts (EΔH). 3 | THE ENZYME-­ASSISTED ARRHENIUS MODEL (EAAR) Biochemical reactions within organisms require sufficient kinetic acti- vation and the catalytic contribution of enzymes (Segel, 1975). Protein stability curves depict the ΔG (change in Gibbs free energy, kcal/mol or equivalently in eV) between the folded and unfolded states as a function of temperature, or the amount of work that must be done to induce a transition in a protein from the folded to the unfolded state at each temperature (Haynie, 2008). Mechanistic derivations of protein stability curves indicate that the temperature dependence of ΔG follows a hump-­shaped function of temperature (Becktel & Schellman, 1987; Feller, 2010): Taking the derivative of Equation 5 with respect to temperature and then rearranging terms provides an explicit expression for the op- timal temperature (Topt), where the top of the unimodal TPC is reached: (6) Topt = Eb −EΔH +EΔCpTm EΔCp . (6) This expression shows that modifying Topt through acclimation or adaptation potentially involves changes in the activation energy of the reaction as well as the thermodynamic properties of enzymes, EΔH, EΔCp, and Tm. The EAAR model generates clear predictions about the acclimation and adaptation of metabolic rate TPCs to match environmental condi- tions (Figure 2). For example, ΔCp—the difference in heat capacity be- tween the folded and unfolded state of enzymes—directly affects the spread or breadth of the TPC, so one clear route to becoming a thermal generalist is to lower enzymatic ΔCp. The model predicts that lowering ΔCp not only broadens the curve, but also elevates the curve, suggest- ing that we might not predict a specialist—generalist trade-­off for all TPCs. Enzymes with higher ΔH are predicted to elevate the TPC due to increases in the enzyme contribution to Eb and to shift Topt to lower temperatures, while increases in Tm will lower the curve and move the Topt to the right. Any potential genetic correlations among parameters, however, could constrain the options for acclimation or evolution of the curves with changing thermal environments (Ratkowsky et al., 2005). (3) ΔG=ΔH ( 1−T Tm ) +ΔCp ( T−Tm −T ln T Tm ) , (3) where ΔH is the enthalpy of folding the enzymes used in the meta- bolic reaction, relative to the melting temperature, Tm, and ΔCp is the difference in heat capacity between the folded and unfolded state of the enzymes, again relative to the melting temperature. 2 | WHAT IS THE ARRHENIUS EQUATION MISSING? Within an organism, biological reactions are assisted by enzymes that connect reactants in a spatially appropriate way and lower the kinetic energy needed for the reaction to proceed. It is generally appreciated that the Ea in the Arrhenius equation, as it is used to describe biologi- cal rates, will be set by enzymes. However, it is more precise to say that the realized Ea represents the difference between the kinetic re- quirements of a reaction as it would occur outside of an organism (i.e., without catalysts, the baseline energy, Eb) and the enzymatic contribu- tion (Ec) of the organism to the reaction (Figure 1b). We can therefore rewrite the Arrhenius equation to explicitly include both the baseline energy and the energetic contribution of enzymes to the process: (2) V=A0e −(Eb−Ec) kT , (2) The observed activation energy (Ea), then, is the kinetic hurdle that remains after enzymes have done their job (Eb − Ec). 3944 DELONG et  al 3 | THE ENZYME-­ASSISTED ARRHENIUS MODEL (EAAR) ΔG reflects the stability of the enzyme, and critically, the probability of an enzyme being in an active state and thus its ability to lower the activation energy of the reaction (Feller, 2010; Hobbs et al., 2013; Ratkowsky et al., 2005). As indicated above, the free energy of the catalyst is not equal to the reduction in the activation energy. Rather, the probabil- ity that enzymes are in an active state approaches 1 at the maximum ΔG. We therefore divide Equation 3 by ΔGmax to transform it into a probability. Given that it is in an active state, the catalyst lowers the |  3945 HE EAAR MODEL METABOLIC RATE in the shape of TPCs. The TPC data were plotted as means with stand- ard errors in the original sources. We extracted the data from the fig- ures converted the units to whole-organism metabolic rate in watts s a es e c all DELONG et  al 3945 |  3945 DELONG et  al 4 | HOW WELL DOES THE EAAR MODEL DO IN DESCRIBING REAL METABOLIC RATE DATA? As an initial assessment of the model’s ability to describe real data, we fit the model to metabolic rate TPCs for three species of amphi- pod (Issartel Hervant Voituron Renault & Vernon 2005) a stonefly in the shape of TPCs. The TPC data were plotted as means with stand- ard errors in the original sources. We extracted the data from the fig- ures, converted the units to whole-­organism metabolic rate in watts (W) from oxygen consumption, and modeled the full data set by ran- domly drawing data for each temperature given the reported sample size and the mean and standard deviation of metabolic rate for that temperature. We then log-­transformed the metabolic rate data and FIGURE 2 Effect of parameter variation on metabolic rate TPCs. Black is a representative TPC with parameter values similar to those derived from data on the amphipod Niphargus verei (Issartel et al., 2005): (Table S1). Other lines represent decreases (green and blue) or increases (magenta and red) in parameter values. With the exception of A0, all parameters modify the Topt (open circle) of metabolic rate, although the effect of Eb is very small |  3945 FIGURE 2 Effect of parameter variation on metabolic rate TPCs. 3 | THE ENZYME-­ASSISTED ARRHENIUS MODEL (EAAR) Black is a representative TPC with parameter values similar to those derived from data on the amphipod Niphargus verei (Issartel et al., 2005): (Table S1). Other lines represent decreases (green and blue) or increases (magenta and red) in parameter values. With the exception of A0, all parameters modify the Topt (open circle) of metabolic rate, although the effect of Eb is very small in the shape of TPCs. The TPC data were plotted as means with stand- ard errors in the original sources. We extracted the data from the fig- ures, converted the units to whole-­organism metabolic rate in watts (W) from oxygen consumption, and modeled the full data set by ran- domly drawing data for each temperature given the reported sample size and the mean and standard deviation of metabolic rate for that temperature. We then log-­transformed the metabolic rate data and fit the log-­transformed EAAR model to each data set using nonlinear regression in MATLAB. We conducted the fitting in two steps. In step (1), we identified the melting temperature (Tm) using a fit of a quadratic function to the right side of the data. This step is essential because the model is defined with respect to Tm, and otherwise attempting to fit Tm in the overall fitting process can provide poor estimates of both Tm and in the shape of TPCs. The TPC data were plotted as means with stand- ard errors in the original sources. We extracted the data from the fig- ures, converted the units to whole-­organism metabolic rate in watts (W) from oxygen consumption, and modeled the full data set by ran- domly drawing data for each temperature given the reported sample size and the mean and standard deviation of metabolic rate for that temperature. We then log-­transformed the metabolic rate data and fit the log-­transformed EAAR model to each data set using nonlinear regression in MATLAB. We conducted the fitting in two steps. In step (1), we identified the melting temperature (Tm) using a fit of a quadratic function to the right side of the data. This step is essential because the model is defined with respect to Tm, and otherwise attempting to fit Tm in the overall fitting process can provide poor estimates of both Tm and 4 | HOW WELL DOES THE EAAR MODEL DO IN DESCRIBING REAL METABOLIC RATE DATA? As an initial assessment of the model’s ability to describe real data, we fit the model to metabolic rate TPCs for three species of amphi- pod (Issartel, Hervant, Voituron, Renault, & Vernon, 2005), a stonefly (Heiman & Knight, 1975), and zebra mussels (Dreissena polymorpha) acclimated to two different temperatures (5 and 25°C; Alexander & McMahon, 2004). We emphasize that this is not a test of the model but an illustration of its utility for describing metabolic rate TPCs cor- rectly and understanding how underlying mechanisms lead to changes DELONG et  al 3946 DELONG et  al 3946  |     DELONG et  al FIGURE 3 Left column. Thermal performance curves for metabolic rate with fits of the EAAR model for three amphipods: Niphargus rhenorhodanensis, N. virei, and Gammarus fossarum data from (Issartel et al., 2005), the stonefly Acroneuria californica (Heiman & Knight, 1975), and for zebra mussels (Dreissena polymorpha) acclimated to 5 and 25°C (Alexander & McMahon, 2004). Blue dots are means for each temperature as reported in the original source, and the gray dots are a sample of a simulated data set we used for fitting that has the same sample size as the original data set and is generated by randomly sampling from a normal distribution set by the reported mean and standard deviation. The orange bar is the 95% confidence intervals of Topt, calculated using equation 6, and the gray shaded region is the 95% confidence interval of the fit, from each of 1000 modeled data sets. Middle column. Model components and their effect on the kinetic hurdle of metabolic reactions (i.e., the y-­axis in Figure 1). The black dot is the melting temperature, Tm. Right column. The proportion of the potential reaction that can occur given the temperature. The Arrhenius factor is the standard model, while the EAAR factor is the Arrhenius factor that considers the temperature dependence of enzyme stability. Fitted curves suggest that enzymatic properties are altered by acclimation in the zebra mussels so that both the melting (Tm) and optimal temperatures (Topt) are higher when acclimated to higher temperatures FIGURE 3 Left column. Thermal performance curves for metabolic rate with fits of the EAAR model for three amphipods: Niphargus rhenorhodanensis, N. virei, and Gammarus fossarum data from (Issartel et al., 2005), the stonefly Acroneuria californica (Heiman & Knight, 1975), and for zebra mussels (Dreissena polymorpha) acclimated to 5 and 25°C (Alexander & McMahon, 2004). 5 There is a long history of work attempting to understand the tem- perature dependence of metabolic rate. The issues became somewhat controversial in the 2000s with the advent of MTE and opposing views (Allen & Gillooly, 2007; Brown et al., 2004; Clarke, 2004, 2006; Gillooly et al., 2006; Knies & Kingsolver, 2010; O’Connor et al., 2007). Important problems with the use of the Arrhenius equation included the model itself, because it neither shows a unimodal response to temperature nor allows for variation in activation energy across tem- perature. The Arrhenius equation also has been criticized as being not mechanistic, because it ascribes all of the temperature dependence of metabolism to kinetics and bypasses a wide range of physiologi- cal processes (Clarke, 2006). Despite the controversy, the Arrhenius equation has enabled considerable insights into thermal ecology, even as an understanding of the full response to temperature has remained unresolved. The EAAR model captures the shape of the metabolic TPCs ex- ceedingly well across all these organisms, including the rising and fall- ing portions of the curves as well as the optimal temperature (Figure 3, first column, Table S1). Most of the parameters varied widely, but EΔH seemed somewhat conserved across TPCs (Figure 4, Table S1). The second column in Figure 3 shows how the components of the model interact to set the modified Arrhenius factor in the EAAR model (i.e., the EAAR factor). Heat capacity and enthalpy of formation interact to set the temperature-­dependent energetic contribution to the re- action, generating an upward opening Ec. The model fits to the data also reveal that the baseline activation energy, Eb, is in line with previ- ous expectations of the observed activation energy, Ea, of near .65 eV (confidence intervals overlap .65 in all but one case), although there is some variation among taxa (Table S1). The third column of Figure 3 shows the Arrhenius factor given only the reactant kinetic response. The enzymatic contributions are large compared to the kinetic contri- butions, driving the reaction up and over the ­thermal optima. Several attempts have been made to create models with a uni- modal response of enzyme-­catalyzed reactions to temperature (Box 1). These models make some implausible assumptions, particularly that enzymes do not lower the reaction’s activation energy (Assumption #12). 4 | HOW WELL DOES THE EAAR MODEL DO IN DESCRIBING REAL METABOLIC RATE DATA? Blue dots are means for each temperature as reported in the original source, and the gray dots are a sample of a simulated data set we used for fitting that has the same sample size as the original data set and is generated by randomly sampling from a normal distribution set by the reported mean and standard deviation. The orange bar is the 95% confidence intervals of Topt, calculated using equation 6, and the gray shaded region is the 95% confidence interval of the fit, from each of 1000 modeled data sets. Middle column. Model components and their effect on the kinetic hurdle of metabolic reactions (i.e., the y-­axis in Figure 1). The black dot is the melting temperature, Tm. Right column. The proportion of the potential reaction that can occur given the temperature. The Arrhenius factor is the standard model, while the EAAR factor is the Arrhenius factor that considers the temperature dependence of enzyme stability. Fitted curves suggest that enzymatic properties are altered by acclimation in the zebra mussels so that both the melting (Tm) and optimal temperatures (Topt) are higher when acclimated to higher temperatures |  3947 DELONG et  al FIGURE 4 Parameter estimates and curve properties with 95% confidence intervals from fits of the EAAR model to TPC data from the three amphipods, the stonefly, and the zebra mussels shown in Figure 3. Dashed line shows 0.65 ev DELONG et  al 3947 FIGURE 4 Parameter estimates and curve properties with 95% confidence intervals from fits of the EAAR model to TPC data from the three amphipods, the stonefly, and the zebra mussels shown in Figure 3. Dashed line shows 0.65 ev the remaining parameters. In step (2), we fit the EAAR model to the rest of the data with the Tm set at the value identified in step (1). For each modeled data set, we also calculated the Topt using Equation 6. We repeated this process 1,000 times per data set and used these distributions to identify mean and 95% confidence ­intervals for the model parameters, Tm, and Topt. shapes TPCs for aerobic metabolic rate (Dahlhoff & Somero, 1993; Gibbs & Somero, 1990; Weinstein & Somero, 1998). The molecular evolution underlying divergence in intrinsic protein stability across the range of temperatures inhabited by life appears to be shaped by a common set of thermodynamic rules that govern pro- tein folding (Feller, 2010; Hochachka & Somero, 2002). Nonetheless, adaptive molecular changes in protein conformational thermostability involve diverse amino acid substitutions that can affect the strength of noncovalent interactions, the binding of stabilizing ions, the sur- face charges of the molecule, or modify conformational entropy (Feller, 2010; Hochachka & Somero, 2002). The EAAR model can therefore help link specific pathways of molecular evolution to whole-­organism function via the parameters that reflect thermostability, and as such generates new opportunity to provide insight into adaptation to different thermal environments. The right column of Figure 3 suggests a surprising response of met- abolic rate to temperature. Our data and model suggest that the rise and fall of the EAAR factor comes mostly from an increase and decrease in the enzymatic contributions to the reaction, as the activation hurdle drops and then rises again. The right side of this curve is anchored at the melting temperature, Tm, where by definition the enzymes are no longer contributing to the reaction and the organism is near death. Above this point, however, the reactant kinetic effect of temperature is still increasing, which suggests that the remaining reactants present in an organism that is pushed above this temperature should react at a faster rate, even if the organism has died, at least until the reactants decline in availability. Surprisingly, this outcome has been observed in what is known as thermolimit respirometry, where the metabolic rate of an organism is measured as the temperature is ramped up, and an increase in the rate of metabolism is observed after the organism dies (Lighton, 2007). Although we do not claim that the good fits to data we obtained above should be treated as tests of the EAAR model, the mod- el’s novel prediction of an uptake in metabolic rate above the melting temperature was made independent of data, providing an unintended qualitative test. No other metabolic rate TPC model that we know of predicts this aspect of thermolimit respirometry (Box 1). The EAAR model is built on a mechanistic derivation of protein stability in which all the parameters have thermodynamic meaning. However, we do not know whether this reflects the thermal depen- dence of a single key protein (e.g., a rate-­limiting step), the sharing of similar thermal dependencies of the many enzymes that underlie metabolic rate within an organism, or whether it is the average of many enzymes which share control of metabolism, each of which may differ somewhat in their thermal optima (Darveau, Suarez, Andrews, & Hochachka, 2002). The standard interpretation in MTE is that the observed activation energy (Ea) for metabolic rates rep- resents an average activation energy for the rate-­limiting enzyme-­ catalyzed biochemical reactions that govern metabolism (Gillooly et al., 2001, 2006). We recognize that our model obscures some of the underlying physiological mechanisms driving metabolic rate by focusing on the net outcome of many individual reactions operating within a complex system of biochemical networks and structures. For example, while the downward slope of the TPC in multicellular organisms may be explained by the thermal dependence of enzyme ΔG, it may also be the result of failure at higher levels of biological organization (e.g., neural processes, membrane-­associated func- tions, mitochondrial failure or any number of processes that affect oxygen and energy supply and demand). Protein stability may be modified by extrinsic changes in pH, thermoprotectant osmolytes, protein concentration, and, in the case of membrane-­localized pro- teins, the membrane architecture. Additionally, a variety of stressors including temperatures near and above thermal optima activate the heat-­shock protein response (Verghese, Abrams, Wang, & Morano, 2012) or the signals that promote the production of heat-­shock proteins (Kaspari et al., 2016). Finally, although we did not observe this in the data sets compared here, regulation of substrate availabil- ity (A0) via regulation of flux in response to temperature may be a critical component of thermal responses of metabolic rate (Schulte, 2015; Suarez & Moyes, 2012). These mechanisms could modify the TPC beyond what could be expected from the EARR model (or any of the other models shown in Box 1). Thus, we do not argue that the EAAR model is a complete depiction of the processes that drive metabolic rate but a useful tool for understanding thermal ecology and predicting the consequences of changes in temperature on organism performance. 5 Like the other models (Box 1), the EAAR model maintains the importance of reactant kinetics in driving metabolic rates, but it in- corporates the more complex reality of how enzymes facilitate the reaction. Metabolism does not run by itself even in the biologically relevant temperature range—it requires enzymes to lower the kinetic hurdle. Enzymes modify the activation energy, which is why the ob- served activation energy should be understood as the kinetic hurdle that remains after enzymes have done their job (Eb − Ec). The EAAR model uses the thermodynamics of protein stability to describe how enzymes increase and then decrease in effectively lowering the acti- vation energy as temperatures rise, and the model describes well the dependence of metabolic rate on temperature for diverse organisms (Figures 3). Because the EAAR model has a mechanistic derivation, parameter differences across data sets or conditions have biological meaning, al- though because these parameters are compound we should interpret them with some caution. Nonetheless, some parameter differences were evident among the data sets shown in Figure 3. For example, the amphipod N. rhenorhodanensis has a lower EΔCp than the other amphipods, consistent with the higher thermal optimum and broader TPC for this species. In contrast, the stonefly has a much higher EΔCp, generating its narrow TPC. The zebra mussels acclimated to 5 and 25°C showed no shifts in the stability properties of the enzymes, but the Tm and Topt were higher at the higher acclimation temperature (Figures 3; Table S1). 3948 DELONG et  al ACKNOWLEDGMENTS TML thanks the University of Nebraska—Lincoln Program of Excellence in population ecology and the School of Biological Sciences. Support for this work also came from NSF CAREER Award 1505247 (KLM), NSF DDIG Award 1501668 (JPG), and BSF Award 2014295 (JPD). We appreciate the input of Mary O’Connor, Matt Osmond, and Brent Lockwood on this manuscript. Dell, A. I., Pawar, S., & Savage, V. M. (2011). Systematic variation in the tem- perature dependence of physiological and ecological traits. Proceedings of the National Academy of Sciences, 108, 10591–10596. Deutsch, C. A., Tewksbury, J. J., Huey, R. B., Sheldon, K. S., Ghalambor, C. K., Haak, D. C., & Martin, P. R. (2008). Impacts of climate warming on ter- restrial ectotherms across latitude. Proceedings of the National Academy of Sciences, 105, 6668–6672. Ernest, S. K. M., Enquist, B. J., Brown, J. H., Charnov, E. L., Gillooly, J. F., Savage, V. M., … Tiffney, B. (2003). Thermodynamic and metabolic ef- fects on the scaling of production and population energy use. Ecology Letters, 6, 990–995. The authors declare no conflict of interest. Feller, G. (2010). Protein stability and enzyme activity at extreme biolog- ical temperatures. Journal of Physics Condensed Matter: An Institute of Physics Journal, 22, 323101. AUTHOR CONTRIBUTIONS Gillooly, J. F., Brown, J. H., West, G. B., Savage, V. M., & Charnov, E. L. (2001). Effects of size and temperature on metabolic rate. Science, 293, 2248–2251. All authors were involved in the conception of the paper and contrib- uted to the collection of data. JPD and JPG developed the model. JPD and TML analyzed the data. All authors contributed critically to the drafts and figures and gave final approval for publication. and TML analyzed the data. All authors contributed critically to the drafts and figures and gave final approval for publication. Haynie, D.T. (2008) Biological thermodynamics. Cambridge, UK: Cambridge University Press. Heiman, D. R., & Knight, A. W. (1975). The influence of temperature on the bioenergetics of the carnivorous Stonefly Nymph, Acroneuria califor- nica Banks (Plecoptera: Perlidae). Ecology, 56, 105–116. DATA ACCESSIBILITY Gibbs, A., & Somero, G. N. (1990). Pressure adaptation of teleost gill Na+/ K+-­adenosine triphosphatase: Role of the lipid and protein moieties. Journal of Comparative Physiology B, 160, 431–439. The data used in this article are previously published and available in the original sources. Our digitization of the data will be made available on Data Dryad upon acceptance of the manuscript. Journal of Comparative Physiology B, 160, 431–439. Gibert, J. P., Chelini, M.-C., Rosenthal, M. F., & DeLong, J. P. (2016). Crossing regimes of temperature dependence in animal movement. Global Change Biology, 22, 1722–1736. Gillooly, J. F., Allen, A. P., Savage, V. M., Charnov, E. L., West, G. B., & Brown, J. H. (2006). Response to Clarke and Fraser: Effects of temperature on metabolic rate. Functional Ecology, 20, 400–404. et al., 2016). Similarly, the role of metabolism in driving production and population growth can incorporate the EAAR model to capture and un- derstand the responses of population processes to a wider range of tem- peratures. This will be increasingly important, as climate variation across unimodal responses to temperature are critical to predicting future organism performance (Deutsch et al., 2008; Vasseur et al., 2014), and TPCs can change quickly as ecological conditions change (Kingsolver, Massie, Ragland, & Smith, 2007; Luhring & DeLong, 2016), indicating an immediate need for understanding and predicting the shape of TPCs. Brown, J., Gillooly, J., Allen, A., Savage, V., & West, G. (2004). Toward a met- abolic theory of ecology. Ecology, 85, 1771–1789. Clarke, A. (2004). Is there a Universal Temperature Dependence of metab- olism? Functional Ecology, 18, 252–256. Clarke, A. (2006). Temperature and the metabolic theory of ecology. Functional Ecology, 20, 405–412. Dahlhoff, E. A., & Somero, G. N. (1993). Effects of temperature on mito- chondria from abalone (genus Haliotis): Adaptive plasticity and its lim- its. Journal of Experimental Biology, 185, 151–168. Daniel, R. M., & Danson, M. J. (2010). A new understanding of how tem- perature affects the catalytic activity of enzymes. Trends in Biochemical Sciences, 35, 584–591. Darveau, C.-A., Suarez, R. K., Andrews, R. D., & Hochachka, P. W. (2002). Allometric cascade as a unifying principle of body mass effects on me- tabolism. Nature, 417, 166–170. In summary, the EAAR model captures the empirical patterns and in- corporates the minimum necessary processes shaping the temperature dependence of metabolic rate in a relatively simple and useful manner. It resolves long-­term problems with the Arrhenius equation and other reaction models and provides a way to move forward with temperature in metabolic ecology. In particular, the EAAR model will facilitate more comparative analyses to elucidate mechanisms involved in acclimation and adaptation to different thermal environments. Furthermore, the many biological processes that depend on metabolic rate can be un- derstood as extensions of the EAAR process. For example, the EAAR model should apply equally well to photosynthesis, which also shows a unimodal response to temperature (Padfield, Yvon-­Durocher, Buckling, Jennings, & Yvon-­Durocher, 2015). In addition, the role of metabolic rate in driving other processes such as locomotion can be captured by in- corporating the EAAR model into biomechanical models of movement to understand the unimodal temperature dependence of animal move- ment and the consequences of warming on these processes (Gibert In addition, the model makes useful connections between whole-­ organism rates and underlying mechanisms by building on fundamen- tal, thermodynamic aspects of all protein stability curves (Feller, 2010). Thus, our model reveals potential mechanistic links between individual reactions and whole-­organism rates which can serve as hypotheses about climate adaptation and point toward additional research. In the case of the zebra mussels (Figures 3, 4), acclimation to warmer temperatures involved a change in metabolic processes to be more stable at high temperatures (increased Tm and Topt), but other parame- ters, such as substrate levels (A0), EΔH, and EΔCp were unchanged. One possibility for such changes is that acclimation of mitochondrial mem- branes to temperature may be a factor determining the thermal sta- bility of the membrane-­embedded protein oxidative phosphorylation complexes, which may play a critical role in how thermal acclimation DELONG et  al 3949 Schoolfield, R. M., Sharpe, P. J. H., & Magnuson, C. E. (1981). Non-­linear regression of biological temperature-­dependent rate models based on absolute reaction-­rate theory. Journal of Theoretical Biology, 88, 719–731. Kaspari, M., Clay, N.A., Lucas, J., Revzen, S., Kay, A., & Yanoviak, S.P. (2016) Thermal adaptation and phosphorus shape thermal performance in an assemblage of rainforest ants. Ecology, 97, 1038–1047. Kingsolver, J. G., Massie, K. R., Ragland, G. J., & Smith, M. H. (2007). Rapid population divergence in thermal reaction norms for an invading spe- cies: Breaking the temperature-­size rule. Journal of Evolutionary Biology, 20, 892–900. Schulte, P. M. (2015). The effects of temperature on aerobic metabolism: Towards a mechanistic understanding of the responses of ectotherms to a changing environment. Journal of Experimental Biology, 218, 1856–1866. Kleiber, M. (1961) The fire of life : An introduction to animal energetics/max kleiber. New York: Wiley Segel, I.H. (1975) Enzyme kinetics: Behavior and analysis of rapid equilibrium and steady state enzyme systems. New York, NY: Wiley. Knies, J. L., & Kingsolver, J. G. (2010). Erroneous arrhenius: Modified Arrhenius model best explains the temperature dependence of ecto- therm fitness. American Naturalist, 176, 227–233. Sharpe, P. J. H., & DeMichele, D. W. (1977). Reaction kinetics of poikilo- therm development. Journal of Theoretical Biology, 64, 649–670. Suarez, R. K., & Moyes, C. D. (2012). Metabolism in the age of ‘omes’. Journal of Experimental Biology, 215, 2351–2357. Laidler, K. J. (1984). The development of the Arrhenius equation. Journal of Chemical Education, 61, 494–498. Vasseur, D. A., DeLong, J. P., Gilbert, B., Greig, H. S., Harley, C. D. G., McCann, K. S., … O’Connor, M. I. (2014). Increased temperature varia- tion poses a greater risk to species than climate warming. Proceedings of the Royal Society B: Biological Sciences, 281, 20132612. Lighton, J. R. B. (2007). Hot hypoxic flies: Whole-­organism interactions between hypoxic and thermal stressors in Drosophila melanogaster. Journal of Thermal Biology, 32, 134–143. López-Urrutia, Á., San Martin, E., Harris, R. P., & Irigoien, X. (2006). Scaling the metabolic balance of the oceans. Proceedings of the National Academy of Sciences of the United States of America, 103, 8739–8744. Verghese, J., Abrams, J., Wang, Y., & Morano, K. A. (2012). Biology of the heat shock response and protein chaperones: Budding yeast (Saccharomyces cerevisiae) as a model system. Microbiology and Molecular Biology Reviews: MMBR, 76, 115–158. Luhring, T.M., & DeLong, J.P. (2016) Predation changes the shape of ther- mal performance curves for population growth rate. Current Zoology, 62, 501–505. Weinstein, R. B., & Somero, G. N. (1998). Effects of temperature on mito- chondrial function in the Antarctic fish Trematomus bernacchii. Journal of Comparative Physiology B, 168, 190–196. O’Connor, M. P., Kemp, S. J., Agosta, S. J., Hansen, F., Sieg, A. E., Wallace, B. P., … Dunham, A. E. (2007). Reconsidering the mechanistic basis of the metabolic theory of ecology. Oikos, 116, 1058–1072. Yvon-Durocher, G., Caffrey, J. M., Cescatti, A., Dossena, M., Giorgio, P. D., Gasol, J. M., … Allen, A. P. (2012). Reconciling the temperature depen- dence of respiration across timescales and ecosystem types. Nature, 487, 472–476. O’Connor, M. I., Piehler, M. F., Leech, D. M., Anton, A., & Bruno, J. F. (2009). Warming and resource availability shift food web structure and metab- olism. PLoS Biology, 7, e1000178. Padfield, D., Yvon-Durocher, G., Buckling, A., Jennings, S., & Yvon-Durocher, G.(2015) Rapid evolution of metabolic traits explains thermal adapta- tion in phytoplankton. Ecology Letters, 19, 133–142. REFERENCES Hobbs, J. K., Jiao, W., Easter, A. D., Parker, E. J., Schipper, L. A., & Arcus, V. L. (2013). Change in heat capacity for enzyme catalysis determines tem- perature dependence of enzyme catalyzed rates. ACS Chemical Biology, 8, 2388–2393. Alexander, J. E. Jr, & McMahon, R. F. (2004). Respiratory response to temperature and hypoxia in the zebra mussel Dreissena polymor- pha. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology, 137, 425–434. Hochachka, P. W., & Somero, G. N. (2002) Biochemical adaptation: Mechanism and process in physiological evolution. Oxford, UK: Oxford University Press Allen, A. P., Brown, J. H., & Gillooly, J. F. (2002). Global biodiversity, bio- chemical kinetics, and the energetic-­equivalence rule. Science, 297, 1545–1548. Huey, R. B., & Kingsolver, J. G. (1989). Evolution of thermal sensitivity of ectotherm performance. Trends in Ecology and Evolution, 4, 131–135. Allen, A. P., & Gillooly, J. F. (2007). The mechanistic basis of the metabolic theory of ecology. Oikos, 116, 1073–1077. Huey, R. B., & Stevenson, R. D. (1979). Integrating thermal physiology and ecology of ectotherms: A discussion of approaches. American Zoologist, 19, 357–366. Anderson-Teixeira, K., DeLong, J., Fox, A., Brese, D., & Litvak, M. (2010). Differential responses of production and respiration to temperature and moisture drive the carbon balance across a climatic gradient in New Mexico. Global Change Biology, 17, 410–424. Issartel, J., Hervant, F., Voituron, Y., Renault, D., & Vernon, P. (2005). Behavioural, ventilatory and respiratory responses of epigean and hypo- gean crustaceans to different temperatures. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology, 141, 1–7. Angilletta, M. J. Jr, Niewiarowski, P. H., & Navas, C. A. (2002). The evolu- tion of thermal physiology in ectotherms. Journal of Thermal Biology, 27, 249–268. Johnson, F. H., & Lewin, I. (1946). The growth rate of E. coli in relation to temperature, quinine and coenzyme. Journal of Cellular and Comparative Physiology, 28, 47–75. Becktel, W. J., & Schellman, J. A. (1987). Protein stability curves. Biopolymers, 26, 1859–1877. 3950 DELONG et  al SUPPORTING INFORMATION Additional Supporting Information may be found online in the sup- porting information tab for this article. Pawar, S., Dell, A. I., Savage, V. M., & Knies, J. L. (2016). Real versus artifi- cial variation in the thermal sensitivity of biological traits. The American Naturalist, 187, E41–52. Peterson, M. E., Daniel, R. M., Danson, M. J., & Eisenthal, R. (2007). The dependence of enzyme activity on temperature: Determination and validation of parameters. Biochemical Journal, 402, 331–337. How to cite this article: DeLong JP, Gibert JP, Luhring TM, et al. The combined effects of reactant kinetics and enzyme stability explain the temperature dependence of metabolic rates. Ecol Evol. 2017;7:3940–3950. https://doi.org/10.1002/ ece3.2955 Ratkowsky, D. A., Olley, J., & Ross, T. (2005). Unifying temperature effects on the growth rate of bacteria and the stability of globular proteins. Journal of Theoretical Biology, 233, 351–362. Robinson, W. R., Peters, R. H., & Zimmermann, J. (1983). The effects of body size and temperature on metabolic rate of organisms. Canadian Journal of Zoology, 61, 281–288.
https://openalex.org/W2806071374
https://link.springer.com/content/pdf/10.1007/s00535-018-1482-y.pdf
English
null
Crohn’s disease-specific mortality: a 30-year cohort study at a tertiary referral center in Japan
Journal of gastroenterology
2,018
cc-by
7,613
J Gastroenterol (2019) 54:42–52 https://doi.org/10.1007/s00535-018-1482-y ORIGINAL ARTICLE—ALIMENTARY TRACT Crohn’s disease-specific mortality: a 30-year cohort study at a tertiary referral center in Japan Shigeyoshi Yasukawa1 • Toshiyuki Matsui1 • Yutaka Yano1 • Yuho Sato1 • Yasumichi Takada1 • Masahiro Kishi1 • Yoichiro Ono1 • Noritaka Takatsu1 • Takashi Nagahama1 • Takashi Hisabe1 • Fumihito Hirai2 • Kenshi Yao3 • Toshiharu Ueki1 • Daijiro Higashi4 • Kitaro Futami4 • Suketo Sou5 • Toshihiro Sakurai6 • Tsuneyoshi Yao7 • Hiroshi Tanabe8 • Akinori Iwashita8 • Masakazu Washio9 Received: 18 February 2018 / Accepted: 31 May 2018 / Published online: 9 June 2018  The Author(s) 2018 the life table method and compared with that of a sex- and age-matched population model from the year 2000. the life table method and compared with that of a sex- and age-matched population model from the year 2000. Results The study included 1108 patients whose mean age at diagnosis was 25.6 ± 10.8 years. The mean duration of follow-up was 14.6 ± 9.4 years, and there were 52 deaths. The cumulative survival rate was significantly lower 25 years after the diagnosis of CD (91.7%) than in the standard population model (95.7%). SMRs for both all causes [3.5; 95% confidence interval (CI): 2.7–4.6] and CD-specific causes (36.7; 95% CI 26.1–51.6) were high. Among the CD-specific causes, SMRs were especially high for small intestine and colorectal cancers, gastrointestinal diseases including intestinal failure (IF), perioperative complications, and amyloidosis. Assessment of mortality Date and cause of death were determined based on medical records, and for the patients whose deaths were identified through the follow-up questionnaire survey were deter- mined through an inquiry to the facility where they passed away. As in previous reports, the causes of death were classified in accordance with the International Classifica- tion of Diseases 10th revision (ICD-10) [13, 14, 19]. Gastrointestinal disease (excluding non-alcoholic liver disease, ICD codes K00–K70, 77–93) included intestinal complications of CD such as intestinal obstruction and perforation, perioperative complications, state of undernu- trition due to short-bowel syndrome, and sudden death of unknown cause that occurred during home parenteral nutrition (HPN) or the active stage of the disease. Non- alcoholic liver disease (ICD code: K71–76) included chronic and progressive liver diseases that were not con- sidered associated with CD. In the present study, (1) the cumulative mortality of CD patients was compared with that of the standard population, (2) the causes of death in CD and the standardized mor- tality ratios (SMRs) by cause of death were investigated, and (3) background characteristics that affect mortality in CD patients were investigated. There are no previous reports of the SMRs for deaths related to CD. Therefore, with the aim to calculate CD- specific mortality, the total mortality for small intestinal cancer (ICD code: C17), colorectal cancer (CRC) (ICD code: C18–21), and amyloidosis (ICD code: E85), as well as gastrointestinal diseases, as CD-specific causes was determined, and the numbers of expected cases of death Abstract Mary’s College, Kurume, Japan 3 J Gastroenterol (2019) 54:42–52 43 Study design and populations This was a single-center, retrospective, cohort study con- ducted at Fukuoka University Chikushi Hospital. Among the patients with a definitive diagnosis of CD who had a history of visiting our hospital between May 1967 and December 2015, patients with a history of treatment for at least 6 months since diagnosis were selected, and a database based on their medical records was subsequently created [3]. Patients who were followed up for less than 6 months after diagnosis were excluded. First, sex, age at diagnosis, date of diagnosis, smoking history, disease type at diagnosis, pres- ence/absence of perianal disease, survival to the last observed day, surgical history, and use of medications (aminosalicylates, corticosteroids, thiopurines, and anti-tu- mor necrosis factor [TNF] agents) were determined from the medical records. Subsequently, a questionnaire survey of the patients who had not visited our hospital for at least 3 years was conducted between May 2015 and May 2016. Diagnosis and treatment of CD CD was diagnosed based on clinical symptoms, endoscopic findings, X-ray findings, and histological findings in accordance with the diagnostic criteria for CD proposed by the Japanese Ministry of Health, Labour and Welfare [15–17]. Furthermore, using the Montreal classification, disease location at diagnosis was classified into ileal type, colonic type, or ileocolonic type, and disease behavior at diagnosis was classified into inflammatory, stricturing, or penetrating type [18]. Introduction was determined based on medical records at our hospital, and the last observed day was the date of the final visit to our hospital according to the medical records, or the date of survey response or date of death for patients whose infor- mation could be collected through the follow-up ques- tionnaire survey. Crohn’s disease (CD) is a chronic inflammatory bowel dis- ease (IBD) of unknown etiology. It often develops at an early age and can affect the entire gastrointestinal tract from the oral cavity to the anus. It is also known to frequently require intestinal resection due to the development of intestinal complications such as stenosis and fistula through repeated relapses or recurrences [1–4]. Although there are some reports of the long-term course of patients with this disease, there are very few reports related to their survival and cause of death; moreover, most previous reports are derived from population-based cohort studies, and there are very few, long-term, hospital-based, cohort studies with a long dura- tion of follow-up and a large sample size [5–14]. In the present study, a database based on the medical records of CD patients at our hospital with the addition of information obtained from a questionnaire survey was created, and the long-term prognosis of CD patients was investigated. Ever-use of medicine The use of medications was reviewed for the duration of follow-up in all patients, and the use of aminosalicylates, corticosteroids, thiopurines, and anti-TNF agents was examined. Duration of medication use, dose, and side effects were not taken into consideration. Abstract Background In this study, survival and cause of death were investigated in patients with Crohn’s disease (CD) at a tertiary referral center. Methods A database was created based on the medical records of 1108 CD patients who had a history of visiting our hospital to investigate background characteristics, cumulative survival rates from diagnosis, causes of death, and the standardized mortality ratio (SMR) for each cause of death. A follow-up questionnaire survey of patients followed up inadequately was also conducted. The cumu- lative survival rate from diagnosis was determined using & Toshiyuki Matsui matsui@fukuoka-u.ac.jp 1 Department of Gastroenterology, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-0067, Japan 2 Inflammatory Bowel Disease Center, Fukuoka University Chikushi Hospital, Fukuoka, Japan 3 Department of Endoscopy, Fukuoka University Chikushi Hospital, Fukuoka, Japan 4 Department of Surgery, Fukuoka University Chikushi Hospital, Fukuoka, Japan 5 Department of Gastroenterology, Tobata Kyoritsu Hospital, Kitakyushu, Japan 6 Department of Gastroenterology, Ashiya Central Hospital, Kitakyushu, Japan 7 Department of Gastroenterology, Sada Hospital, Fukuoka, Japan 8 Department of Pathology, Fukuoka University Chikushi Hospital, Fukuoka, Japan 9 Department of Community Health and Clinical Epidemiology, St. Mary’s College, Kurume, Japan & Toshiyuki Matsui matsui@fukuoka-u.ac.jp Conclusion The SMRs for both all causes and CD-specific causes were high in CD patients. CD-specific causes including intestinal cancer, IF, perioperative complications, and amyloidosis showed especially high SMRs. 1 Department of Gastroenterology, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-0067, Japan 1 Department of Gastroenterology, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-0067, Japan Keywords Crohn’s disease-specific mortality  Standardized mortality ratios  Cohort study  Intestinal cancer  Amyloidosis 2 Inflammatory Bowel Disease Center, Fukuoka University Chikushi Hospital, Fukuoka, Japan 3 Department of Endoscopy, Fukuoka University Chikushi Hospital, Fukuoka, Japan Abbreviations CD Crohn’s disease CI Confidence interval CRC Colorectal cancer HPN Home parenteral nutrition HR Hazard ratio IBD Inflammatory bowel disease ICD International Classification of Diseases IF Intestinal failure SMR Standardized mortality ratio SIR Standardized incidence ratio TNF Tumor necrosis factor 4 Department of Surgery, Fukuoka University Chikushi Hospital, Fukuoka, Japan 5 Department of Gastroenterology, Tobata Kyoritsu Hospital, Kitakyushu, Japan 6 Department of Gastroenterology, Ashiya Central Hospital, Kitakyushu, Japan 7 Department of Gastroenterology, Sada Hospital, Fukuoka, Japan 8 Department of Pathology, Fukuoka University Chikushi Hospital, Fukuoka, Japan 8 Department of Pathology, Fukuoka University Chikushi Hospital, Fukuoka, Japan 9 Department of Community Health and Clinical Epidemiology, St. Patient populations and background characteristics This study included 1108 patients whose duration of fol- low-up was at least 6 months from among the 1165 CD patients who were diagnosed after May 1967 and had a history of visiting our hospital to December 2015. A questionnaire survey was distributed to 303 CD patients who did not have a history of visiting our hospital for at least 3 years, and information on their present condition (alive, dead), cause of death, last observed day, smoking history, surgical history, and previously used medications was added from the 106 responses. It was, therefore, pos- sible to obtain information on background characteristics and survival to the last observed day for all patients. The patients’ clinical characteristics are shown in Table 1. There were 758 men and 350 women who were 7–76 years old at the time of diagnosis. The mean age at diagnosis was 25.6 ± 10.8 years, and the mean duration of follow-up was 14.6 ± 9.4 years (range 0.5–47.2 years). The study inclu- ded 16,199 person-years, and 583 patients (52.6%) were diagnosed before 2000. Disease location at diagnosis was ileal type in 386 patients (34.8%), colonic type in 197 patients (17.8%), and ileocolonic type in 525 patients (47.4%). Disease behavior was inflammatory in 531 patients (47.9%), stricturing in 365 patients (32.9%), pen- etrating in 208 patients (18.8%), and unknown in 4 patients (0.4%). A perianal fistula was present at diagnosis in 594 patients (53.6%). There were 303 current smokers (27.3%), 610 never-smokers (55.1%), and 117 ex-smokers (10.6%). There were 662 patients (59.7%) who had undergone intestinal surgery by the last observed day The mean 2. 2. SMRs of patients with CD 2. SMRs of patients with CD The SMR is the ratio of the observed number of deaths among the patients with CD in this cohort study to the expected number of deaths in the study population under the assumption that the age by sex-specific mortality rates for the study population are the same as those for a reference population. The expected number of deaths was calculated by multiplying the person- years of the study population by the age by sex-specific death rates of the Japanese population in 2000. The SMRs were calculated for 5-year age bands of both sexes in this study. 3. Factors associated with mortality of patients with CD Hazard ratios of death and their 95% confidence intervals were estimated with a Cox proportional hazards model. Duration of follow-up Duration of follow-up was defined as from the date of diagnosis to the last observed day. The date of diagnosis 12 J Gastroenterol (2019) 54:42–52 44 package. A two-sided P less than 0.05 was considered significant. and SMRs were calculated from the above mortality and person-years in the sex- and age-matched population model. and SMRs were calculated from the above mortality and person-years in the sex- and age-matched population model. Statistical analysis This study was conducted in accordance with the 1964 Helsinki Declaration and its subsequent amendments. Personal information listed in the medical records or obtained through questionnaire surveys, including informed consent, was managed on a database such that individuals were anonymized. This study was approved by the institutional review board of Fukuoka University Chi- kushi Hospital (R14-011, 2014.6.4–2017.2.28). 1. Expected and observed cumulative survival rates from diagnosis The age by sex-specific death rates of the Japanese population in 2000 were applied to the distribution of the age groups of the study population by sex to esti- mate the yearly expected survival rate of the general population during the follow-up period. The death rates for 5-year age bands of both sexes were used in this study. According to the past reports, the age by sex-specific mortality of the Japanese population in 2000 [20], which was almost the median of the year of diagnosis, was used [13]. The life table method was used to estimate the cumulative survival rate and 95% confidence intervals of the study subjects. p diagnosis The age by sex-specific death rates of the Japanese population in 2000 were applied to the distribution of the age groups of the study population by sex to esti- mate the yearly expected survival rate of the general population during the follow-up period. The death rates for 5-year age bands of both sexes were used in this study. According to the past reports, the age by sex-specific mortality of the Japanese population in 2000 [20], which was almost the median of the year of diagnosis, was used [13]. The life table method was used to estimate the cumulative survival rate and 95% confidence intervals of the study subjects. Overall mortality (23.1%). There were 31 patients (59.6%) presenting with perianal disease, 2 current smokers (3.9%), and 45 patients (86.5%) who had undergone surgery during the follow-up period. The mean number of surgeries per patient was 2.3 ± 1.8. Previously used medications were aminosali- cylates in 29 patients (55.8%), corticosteroids in 26 patients (50.0%), thiopurines in 12 patients (23.1%), and anti-TNF agents in 21 patients (40.4%). Fifty-two deaths occurred among the 1108 patients with CD. Among the 52 deaths, 9 were reported through the additional questionnaire survey. The background charac- teristics of the deceased patients are shown in Table 2. Of the 52 patients who died, 32 were men (61.5%). The mean age at diagnosis was 29.1 ± 14.0 years, the mean age at death was 48.1 ± 14.6 years, and the mean duration of follow-up from diagnosis to death was 18.3 ± 8.8 years. Disease location at diagnosis was ileal type in 15 patients (28.9%), colonic type in 6 patients (11.5%), and ileo- colonic type in 31 patients (59.6%). Disease behavior at diagnosis was inflammatory in 21 patients (40.4%), stric- turing in 19 patients (36.5%), and penetrating in 12 patients Patient populations and background characteristics Age was treated as a continuous variable, while other factors were treated as indicator variables. 4. Number of intestinal resections and mortality in patients with CD. Number of intestinal resection was classified into three categories: 0, 1–4, 5 or more. A Chi-squared test was used to evaluate if the number of intestinal resections was related to the death rate. The dose-dependent trend was tested by evaluating the regression coefficient when the categories were treated as equally spaced numerical variables in Cox’s model There were 662 patients (59.7%) who had undergone intestinal surgery by the last observed day. The mean number of surgeries per patient was 1.2 ± 1.4. Previously used medications included aminosalicylates in 864 patients (77.9%), corticosteroids in 346 patients (31.2%), thiopuri- nes in 423 patients (38.1%), and anti-TNF agents in 570 patients (51.4%). Person-years at risk were computed from the date of CD diagnosis until the date of death, loss to follow-up, or end of follow-up. Person-years at risk were computed from the date of CD diagnosis until the date of death, loss to follow-up, or end of follow-up. All statistical analyses were conducted using the Sta- tistical Analysis System (SAS Institute, Cary, NC, USA) 123 123 45 J Gastroenterol (2019) 54:42–52 Sex (male/female) 758/350 Age at diagnosis (mean ± SD, years) 25.6 ± 10.8 (7–76) Follow-up period (mean ± SD, years) 14.6 ± 9.4 (0.5–47.2) Calendar year at diagnosis \ 2000 583 (52.6%) C 2000 525 (47.4%) Disease location at diagnosisa Ileal 386 (34.8%) Colonic 197 (17.8%) Ileocolonic 525 (47.4%) Disease behavior at diagnosisa Inflammatory 531 (47.9%) Stricturing 365 (32.9%) Penetrating 208 (18.8%) Unknown 4 (0.4%) Perianal disease at diagnosis Absent 514 (46.4%) Present 594 (53.6%) Smoking at last observed day Never-smoker 610 (55.1%) Ex-smoker 117 (10.6%) Current smoker 303 (27.3%) Unknown 78 (7.0%) Intestinal resection at last observed day Absent 446 (40.3%) Present 662 (59.7%) Number of intestinal resections (mean ± SD) 1.2 ± 1.4 (0–7) Ever-use of medication Aminosalicylates 864 (77.9%) Corticosteroids 346 (31.2%) Thiopurines 423 (38.1%) Anti-TNF agents 570 (51.4%) SD standard deviation, TNF tumor necrosis factor aDisease location and behavior at diagnosis are according to the Montreal classification Table 1 Characteristics of patients with Crohn’s disease (n = 1108) Present Aminosalicylates Corticosteroids Thiopurines Anti-TNF agents aDisease location and behavior at diagnosis are according to the Montreal classification SMRs Fifty-two all-cause deaths were observed in this study, while the expected value calculated from the mortality in the sex- and age-matched general population was 14.8. Thus, the SMR for all-cause mortality was 3.5 (95% CI 2.7–4.6). Analysis by disease revealed a high SMR of 5.4 (95% CI 3.5–8.5) for malignant tumors. SMRs were especially high for small intestinal cancer at 200 (95% CI 500.1–7997.1), CRC at 29.7 (95% CI 16.5–53.7), and brain tumor at 12.5 (95% CI 1.8–88.7). In addition, SMRs were also high for gastrointestinal disease at 48.4 (95% CI 29.2–80.3), non- alcoholic liver disease at 14.3 (95% CI 4.6–44.3), and amyloidosis at 1000 (95% CI 416.2–2402.6). However, high SMRs were not observed for many conditions, such as respiratory disease (1.4; 95% CI 0.4–5.7), cardiovascular disease (1.3; 95% CI 0.4–3.9), suicide (1.2; 95% CI Expected and observed cumulative survival rates from diagnosis Gastrointestinal disease (n = 15) consisted of intestinal failure (IF) (n = 7), severe (fistulous) disease including perioperative complications (n = 6), and intesti- nal perforation (n = 2). Amyloidosis (n = 5) consisted of kidney failure (n = 3) and massive bleeding (n = 2). (n = 19; small intestinal cancer, n = 2; CRC, n = 11; lung cancer, n = 2; lymphoma, n = 2; brain tumor, n = 1; and cancer of unknown primary, n = 1), gastrointestinal disease (n = 15), liver disease (n = 3), respiratory disease (n = 2), cardiovascular disease (n = 3), suicide (n = 3), metabolic disease (n = 5), and traffic injuries (n = 1). Information about the cause of death was not provided on the ques- tionnaire survey in one case. There were a total of 33 deaths related to CD, including small intestinal cancer, CRC, gastrointestinal disease, and amyloidosis (patholog- ically secondary amyloidosis as shown by immunostaining AA amyloid). Gastrointestinal disease (n = 15) consisted of intestinal failure (IF) (n = 7), severe (fistulous) disease including perioperative complications (n = 6), and intesti- nal perforation (n = 2). Amyloidosis (n = 5) consisted of kidney failure (n = 3) and massive bleeding (n = 2). 10 years (expected cumulative survival rate: 98.3%, 95% CI 97.5–99.0%), 95.1% (95% CI 93.3–96.9%) after 20 years (expected cumulative survival rate: 96.5%, 95% CI 95.5–97.6%), and 87.1% (95% CI 82.8–91.4%) after 30 years (expected cumulative survival rate: 94.9%, 95% CI 93.6–96.1%). The cumulative survival rate of CD patients in the first 24 years since diagnosis was not dif- ferent from that of the standard population model. How- ever, it was significantly lower in CD patients (91.7%; 95% CI 89.0–94.4%) at 25 years after diagnosis compared to that in the standard population model (95.7%; 95% CI 94.5–96.9%), and this pattern continued thereafter. Expected and observed cumulative survival rates from diagnosis The cumulative survival rates from diagnosis in CD patients are shown in Fig. 1. The cumulative survival rates among CD patients were 99.3% (95% CI 98.8–99.9) after 123 12 3 3 J Gastroenterol (2019) 54:42–52 46 Fig. 1 Expected and observed cumulative survival rates from diagnosis in 1108 patients with CD. Although the cumulative survival rate at 25 years from diagnosis is not different compared to that of the standard population model, it is significantly lower C 25 years after diagnosis Table 2 Background characteristics of deceased patients (n = 52) Sex (male) 32 (61.5%) Age at diagnosis (mean ± SD, years) 29.1 ± 14.0 Age at death (mean ± SD, years) 48.1 ± 14.6 Duration of follow-up (mean ± SD, years) 18.3 ± 8.8 Disease location at diagnosisa Ileal 15 (28.9%) Colonic 6 (11.5%) Ileocolonic 31 (59.6%) Disease behavior at diagnosisa Inflammatory 21 (40.4%) Stricturing 19 (36.5%) Penetrating 12 (23.1%) Perianal disease at diagnosis 31 (59.6%) Smoking at last observed day Never-smoker 38 (73.1%) Ex-smoker 11 (21.1%) Current smoker 2 (3.9%) Unknown 1 (1.9%) Intestinal resection at last observed day Present 45 (86.5%) Number of intestinal resections (mean ± SD) 2.3 ± 1.8 (0-7) Ever-use of medication Aminosalicylates 29 (55.8%) Corticosteroids 26 (50.0%) Thiopurines 12 (23.1%) Anti-TNF agents 21 (40.4%) SD standard deviation, TNF tumor necrosis factor aDisease location and behavior at diagnosis are according to the Montreal classification Fig. 1 Expected and observed cumulative survival rates from diagnosis in 1108 patients with CD. Although the cumulative survival rate at 25 years from diagnosis is not different compared to that of the standard population model, it is significantly lower C 25 years after diagnosis (n = 19; small intestinal cancer, n = 2; CRC, n = 11; lung cancer, n = 2; lymphoma, n = 2; brain tumor, n = 1; and cancer of unknown primary, n = 1), gastrointestinal disease (n = 15), liver disease (n = 3), respiratory disease (n = 2), cardiovascular disease (n = 3), suicide (n = 3), metabolic disease (n = 5), and traffic injuries (n = 1). Information about the cause of death was not provided on the ques- tionnaire survey in one case. There were a total of 33 deaths related to CD, including small intestinal cancer, CRC, gastrointestinal disease, and amyloidosis (patholog- ically secondary amyloidosis as shown by immunostaining AA amyloid). Discussion The results of long-term follow-up of CD patients in the present study showed that the cumulative survival rate decreased and SMR increased C 25 years after diagnosis. Among individual complications, CD-specific causes, such as intestinal cancer, IF, severe (fistulous) diseases including perioperative complications, and amyloidosis, devel- oped C 20 years after diagnosis, increasing mortality. Causes of death in CD and the SMRs by cause of death The numbers of deaths and SMRs by cause of death in CD patients are shown in Table 3. Fifty-two of the 1108 CD patients died. The causes of death were: malignant tumor 123 J Gastroenterol (2019) 54:42–52 47 Table 3 All causes of death in Crohn’s disease patients and standardized mortality ratios by cause of death Cause ICD-10 code Observed cases Expected cases SMR 95% CI All causes 52 14.8 3.5 2.7–4.6 Malignant tumor C00–D48 19 3.5 5.4 3.5–8.5 Small intestinal cancera C17 2 0.01 200 500.1–7997.1 Colorectal cancera C18–C21 11 0.37 29.7 16.5–53.7 Lung cancer C33–C34 2 0.45 4.4 1.1–17.8 Lymphatic cancer C81–C96 2 0.72 2.8 0.7–11.1 Brain tumor C70–C72 1 0.08 12.5 1.8–88.7 Cancer of unknown primary – 1 – – – Gastrointestinal diseasea (excluding K71–76) K00–70,77–93 15 0.31 48.4 29.2–80.3 Non-alcoholic liver disease K71–76 3 0.21 14.3 4.6–44.3 Respiratory disease J00–J99 2 1.4 1.4 0.4–5.7 Cardiovascular disease I00–I99 3 2.4 1.3 0.4–3.9 Suicide X60–X84 3 2.6 1.2 0.4–3.6 Metabolic disease (amyloidosis)a E85 5 0.005 1000 416.2–2402.6 Traffic injuries V00–V89 1 1.4 0.7 0.1–5.1 Unknown – 1 – – – CD-specific causesa 33 0.9 36.7 26.1–51.6 ICD-10 International Classification of Diseases-10, SMR standardized mortality ratio, CI confidence interval aCD-specific causes are small bowel and colorectal cancer, gastrointestinal disease, and amyloidosis CD-10 International Classification of Diseases-10, SMR standardized mortality ratio, CI confidence interval CD-specific causes are small bowel and colorectal cancer, gastrointestinal disease, and amyloidosis squared test, p for trend \ 0.01), as well as in patients who were followed up for 25 years and more (Table 5, p = 0.03 by Chi-squared test, p for trend = 0.02). 0.4–3.6), and traffic injuries (0.7; 95% CI 500.1–7997.1). The SMR for CD-specific causes of death, which included small intestinal cancer, CRC, gastrointestinal disease, and metabolic disease, was high at 36.7 (95% CI 26.1–51.6). Thus, the results showed that many deaths of CD patients were caused by intestinal cancer, IF, severe (fis- tulous) disease including perioperative complications, and amyloidosis. Factors associated with mortality in patients with CD Factors associated with mortality are shown in Table 4. Sex, calendar year, disease location and behavior at diag- nosis, smoking habits, perianal fistula, thiopurines, and use of anti-TNF agents were not significant factors. However, old age at diagnosis (C 60 years) [hazard ratio (HR) 19.93; 95% CI 5.56–71.54] and use of corticosteroids (HR 1.94; 95% CI 1.12–3.25) had high HRs. p y g g y There are several reports primarily from Western countries concerning the survival and causes of death of CD patients. It has recently been reported that mortality is similar or slightly higher in CD patients than in the general population [5–14]. However, most data are derived from population-based studies, which are considered not con- ducive to patient follow-up or detailed investigation of the causes of deaths, and there are very few, long-term, hos- pital-based studies with a large sample size [5, 6, 14, 21–25]. Therefore, this hospital-based cohort study was conducted at a tertiary referral center in Japan to investigate survival, cause of death, and factors that affect mortality of CD patients in detail. Number of intestinal resections and the death rate The number of intestinal resections and the death rate in all subjects and in patients followed up more than 25 years were examined (Table 5). A higher number of surgeries was significantly associated with higher mortality during the entire follow-up period (Table 5, p \ 0.01 by Chi- 12 3 3 J Gastroenterol (2019) 54:42–52 48 Table 4 Factors associated with mortality of Crohn’s disease patients Data at the baseline survey Person-years No. of cases Age- and sex-adjusted HR (95% CI) Data at the baseline survey Person-years No. of cases Age- and sex-adjusted HR (95% CI) Data at the baseline survey Person-years No. of cases Age- and sex-adjusted HR (95% CI) Sex Female 5438 20 1.00 (reference) Male 10,759 32 0.97 (0.55–1.07) Age (years) \ 20 5549 14 0.64 (0.34–1.19) 20–59 10,505 35 1.00 (reference) C 60 142 3 19.93 (5.56–71.54) Calendar year \ 1999 12,126 47 1.00 (reference) C 2000 4071 5 0.61 (0.21–1.74) Disease location at diagnosis Ileal 5859 15 0.57 (0.31–1.05) Colonic?ileocolonic 10,338 37 1.00 (reference) Disease behavior at diagnosis Inflammatory 6795 15 1.03 (0.59–1.80) Stricturing ? penetrating 9335 37 1.00 (reference) Perianal disease Absent 6558 21 1.00 (reference) Present 9639 31 1.07 (0.60–1.90) Smoking habits Never-smoker ? ex-smoker 10,619 40 1.00 (reference) Current smoker 4649 11 0.77 (0.38–1.52) Intestinal resection Absent 4273 7 1.00 (reference) Present 11,925 45 1.47 (0.65–3.35) Ever-use of medicine corticosteroids Without 10,502 26 1.00 (reference) With 5509 26 1.94 (1.12–3.25) Thiopurines Without 9868 40 1.00 (reference) With 6184 12 0.59 (0.30–1.13) Anti-TNF agents Without 7945 31 1.00 (reference) With 8253 21 0.87 (0.49–1.55) HR, hazard ratio; CI, confidence interval; TNF, tumor necrosis factor The cumulative survival rates of CD patients from diagnosis were 98.9% at 10 years, 94.0% at 20 years, and 86.7% at 30 years according to Lee et al. from Korea [14], and the rates were generally consistent with the results of the present study. Some reports compared the cumulative survival rate from diagnosis with that of the standard population model and found no significant differences between the two groups [5, 8, 11]. Consistent with previous reports, the present study results also did not show signif- icant differences for 25 years from diagnosis. However, longer follow-up demonstrated that the cumulative survival rate decreased significantly C 25 years from diagnosis. These findings suggest that long-term follow-up of 20 years or longer may show a decrease in cumulative survival among CD patients. J Gastroenterol (2019) 54:42–52 49 Table 5 Number of intestinal resections and death rates by follow-up period Present condition Number of intestinal resections p for trend** 0 (n = 446) 1–4 (n = 620) C 5 (n = 42) (a) Death rate and number of intestinal resections in all subjects (total observation period) Dead (n = 52) 7 (1.6%) 36 (5.8%)* 9 (21.4%)* \0.01 Present condition Number of intestinal resections p for trend** 0 (n = 24) 1–4 (n = 137) C 5 (n = 23) (b) Death rate and number of intestinal resections in patients observed more than 25 years Dead (n = 12) 0 (0.0%) 8 (5.8%) 4 (17.4%)* 0.02 Data were presented as n (%) *p \ 0.03 vs no intestinal resection * Chi-squared test **Regression coefficient using Cox’s model J Gastroenterol (2019) 54:42–52 49 Present condition Number of intestinal resections p for trend** 0 (n = 446) 1–4 (n = 620) C 5 (n = 42) (a) Death rate and number of intestinal resections in all subjects (total observation period) Dead (n = 52) 7 (1.6%) 36 (5.8%)* 9 (21.4%)* \0.01 Present condition Number of intestinal resections p for trend** 0 (n = 24) 1–4 (n = 137) C 5 (n = 23) (b) Death rate and number of intestinal resections in patients observed more than 25 years Dead (n = 12) 0 (0.0%) 8 (5.8%) 4 (17.4%)* 0.02 Data were presented as n (%) *p \ 0.03 vs no intestinal resection * Chi-squared test **Regression coefficient using Cox’s model Table 5 Number of intestinal resections and death rates by follow-up period the long course of the disease, requiring repeated surgeries. Frequent surgeries and intestinal complications can result in short-bowel syndrome and IF. According to Watanabe et al., IF was observed relatively frequently at a specialized hospital, with an incidence of 3.6% at 10 years and 8.5% at 20 years after initial surgery for CD, and the mortality after diagnosis of IF was 3.7% at 5 years and 8.9% at 10 years [28]. HPN-related mortality according to reports from overseas ranges from 2 to 28% at 5 years, which is by no means low [29]. Loly et al. collected data from numerous cases and stated that the mortality of CD patients who received HPN was 10% at 5 years, and Limketkai et al. aPerson-years at risk were estimated by the author (cases 9 follow-up periods) N.D: no data in the publication SMR standardized mortality ratio, CI confidence interval SMR standardized mortality ratio, CI confidence interval bN.D: no data in the publication bN.D: no data in the publication Number of intestinal resections and the death rate Table 6 shows SMRs for all causes from previous hos- pital-based studies [5, 6, 14, 21–25]. Prior et al. [21] and Weterman et al. [22] reported high SMRs, but recent reports did not find increased SMRs. Except for the study by Lee et al., previous reports encompassed a low number of person-years (range 2712–7424), which takes into account the number of patients and the duration of follow- up. Furthermore, compared to the report by Lee at al., the present study had a longer mean duration of follow-up (14.6 years) with 16,197 person-years. It is evident from Fig. 1 that the cumulative survival rate [ 25 years after 123 123 J Gastroenterol (2019) 54:42–52 J Gastroenterol (2019) 54:42–52 J Gastroenterol (2019) 54:42–52 stated that mortality caused by IF was 6% at 1 year and up to 20% at 4 years [30, 31]. It could be expected that mortality due to intestinal complications, as well as short- bowel syndrome and IF caused by such complications, would be high, and the SMR for gastrointestinal disease is considered to be high, similar to previous reports. diagnosis was decreased, indicating that the present study might be informative, since there are no other studies with both high person-years at risk and a long duration of fol- low-up. Regarding SMRs by disease, previous reports showed that SMRs were high for neoplasms, especially small intestinal cancer and CRC, as well as gastrointestinal dis- eases [13, 14, 26, 27]. As in previous studies, the present study also found high SMRs for malignant tumors, espe- cially small intestinal cancer and CRC, as well as gas- trointestinal disease and amyloidosis, and not very high SMRs for respiratory and cardiovascular disease. The SMR of all causes was high in the present study, likely because of the high SMR for CD-specific causes. In the present study, the SMR for gastrointestinal dis- ease was high, because the data showed that the number of intestinal resections was significantly related to the death rate (Table 5). CD induces intestinal complications over Table 6 Hospital-based studies examining mortality in Crohn’s disease Author Year Country Years of diagnosis Follow-up period (years) Cases Deaths SMR 95% CI %Death Person-years at risk Prior 1981 UK 1941–1976 14.5 513 102 2.0 1.85–2.47 19.9 7424 Weterman 1990 Netherlands 1934–1984 9.9 659 64 2.2 1.75–2.85 9.7 6590a Cottone 1996 Italy 1973–1993 7.8 531 9 1.0 0.4–1.8 1.7 2712 Farrokhyar 2001 UK 1978–1986 15.0 196 23 0.9 0.59–1.4 11.7 3623 Uno 2003 Japan 1967–1997 8.4 544 6 1.4 0.53–3.12 1.1 4570a Oriuchi 2003 Japan 1965–1998 9.9 276 11 N.Db N.Db 4.0 2732a Kennedy 2012 Scotland 1998–2000 3.0 1595 144 3.3 2.8–3.89 9.0 4785a Lee 2017 Korea 1981–2013 8.6 2414 35 1.4 0.97–1.94 1.4 20,712 Yasukawa Japan 1968–2015 14.6 1108 52 3.5 2.7–4.6 4.7 16,199 SMR standardized mortality ratio, CI confidence interval Table 6 Hospital-based studies examining mortality in Crohn’s disease 12 123 50 J Gastroenterol (2019) 54:42–52 Malignant tumors had the second highest SMR in the present study. Recently, Caini et al. from Italy conducted long-term follow-up of 231 CD patients and compared SMRs by causes of death. J Gastroenterol (2019) 54:42–52 Their results showed that the SMR was 1.79 for all causes (95% CI 1.39–2.27), and it was very high for cancer, at 2.57 (95% CI 1.28–5.13) [32]. Furthermore, most reports have demonstrated that the SMR is higher for lower gastrointestinal cancer among malignant tumors. Reports from Western countries have shown the standardized incidence ratio (SIR) to be 2.5 (95% CI 1.3–4.7) for CRC and 33.2 (95% CI 15.9–60.9) for small-bowel cancer [33]. It has also been reported in Japan that the SIR for CRC is 2.79–5.8 [34, 35].Higashietal.reportedthat the frequency ofcomorbid CRC in CD from multiple facilities specialized for IBD was 3.5% (122/3454 patients), and that the 5-year survival rate of CRC was 88% in Stage I, 68% in Stage II, 71% in Stage IIIa, 25% in Stage IIIb, and 0% in Stage IV [36]. Of these cases, 91% of the diagnosed gastrointestinal cancers were advanced cancers. In addition, they claimed that comorbid CRC has increased markedly in Japan since 2008. Sugita et al. analyzed 20 CD patients with rectal and anal cancers, and they reported a mortality of 33% after 36 months [37]. As described here, the incidences of intestinal cancer and anal cancer have also increased in Japan, and mortality associated with these dis- eases is not low. affiliated hospital network and added a questionnaire sur- vey, we succeeded in gathering sufficient data related to mortality and cause of death over a long term. Because the present study was a retrospective cohort study, disease location and behavior observed at initial examination were not significant factors associated with mortality. Furthermore, while the usefulness of anti-TNF-a agents and a decrease in the intestinal resections performed for refractory CD have been suggested recently, there was no association with the use of medications [44–47]. Nutritional therapy was not considered in the present study; it was considered difficult to elucidate the contribution of nutritional therapy to mortality since the data were old and retrospective, and the dose and duration of the therapy were not known in many of the patients. Further investi- gations will be necessary to clarify the associations with treatment and changes in disease type. Compliance with ethical standards Conflict of interest Toshiyuki Matsui received fees for lectures from Eisai Co. Ltd. and AbbVie GK. Fumihito Hirai received fees for lectures from EA Pharm Co. Ltd., Eisai Co Ltd., AbbVie GK, and Mitsubishi Tanabe Pharm Co. Ltd. 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. The limitations of this study are as follows. It is very difficult to obtain long-term outcomes including mortality rates in a cohort group, because CD patients have younger disease onset, and follow-up for more than 20 years is extremely difficult due to their higher social activity and frequent changes in residence. Because we have an Conclusion The long-term follow-up of CD patients in the present study showed that the cumulative survival rate decreases and SMR increases C 25 years after diagnosis. Among individual complications, CD-specific causes such as intestinal cancer, IF, severe (fistulous) diseases including perioperative complications, and amyloidosis devel- oped C 20 years after diagnosis, increasing the mortality. In the future, it may be possible to improve the prognosis by predicting these serious complications or by diagnosing them at an early stage. One of the major differences between this study and previous reports is that the SMR for amyloidosis was very high. The prevalence of comorbid secondary amyloidosis in patients with CD has been reported to be 2.5–5.3% in Japan and 0.9–5.6% in Western countries [38–42]. It is considered that, when amyloidosis develops in conjunction with CD, serious complications including kidney failure ensue, ultimately increasing mortality [42]. A recent study from the USA showed that mortality increases in the presence of secondary amyloidosis in hospitalized CD patients [41]. A report by Miyaoka from Japan stated that secondary amyloidosis does not occur frequently in CD (2.5%), but that 40% of these patients died, indicating that the prognosis is poor with secondary amyloidosis [40]. In addition, a long-term outcome report by Weterman et al. showed that 4 of 64 deaths (6.3%) were due to amyloidosis [22], indicating that this is not rare. Patients are diagnosed with amyloidosis between 12.0 and 25.0 years after the diagnosis of CD [38, 40, 43], and they die of complications such as kidney failure. Thus, death caused by amyloidosis may only be captured by performing long-term follow-up, as demonstrated in the present study. Acknowledgements This work was supported by the Health and Labour Sciences Research Grants for Research on Intractable Disease from the Ministry of Health, Labour and Welfare of Japan. 1. Peyrin-Biroulet L, Loftus EV Jr, Colombel JF, et al. The nature history of adult Crohn’s disease in population based cohorts. Am J Gastroenterol. 2010;105:289–97. 123 References Survival and cause specific mortality in patients with inflammatory bowel disease: a long term outcome study in Olmsted Country, Minnesota, 1940–2004. Gut. 2006;55:1248–54. 28. Watanabe K, Sasaki I, Fukushima K, et al. Long-term incidence and characteristics of intestinal failure in Crohn’s disease: a multicenter study. J Gastroenterol. 2014;49:231–8. 9. Canavan C, Abrams KR, Mayberry JF. Meta-analysis: mortality in Crohn’s disease. Aliment Pharmacol Ther. 2007;25:861–70. 29. Howard L, Malone M. Current status of home parenteral nutrition in the United States. Transplant Proc. 1996;28:2691–5. 30. Loly C, Belaiche J, Louis E. Predictors of severe Crohn’s disease. Scand J Gastroenterol. 2008;43:948–54. 10. Jess T, Riis L, Vind I, et al. Changes in clinical characteristics, course, and prognosis of inflammatory bowel disease during the last 5 decades: a population-based study from Copenhagen, Denmark. Inflamm Bowel Dis. 2007;13:481–9. 31. Limketkai BN, Parian AM, Shah ND, et al. Short bowel syn- drome and intestinal failure in Crohn’s disease. Inflamm Bowel Dis. 2016;22:1209–18. 11. Selinger CP, Andrews J, Dent OF, et al. Cause-specific mortality and 30-year relative survival of Crohn’s disease and ulcerative colitis. Inflamm Bowel Dis. 2013;19:1880–8. 32. Caini S, Bagnoli S, Palli D, et al. Total and cancer mortality in a cohort of ulcerative colitis and Crohn’s disease patients: the Florence inflammatory bowel disease study, 1978–2010. Dig Liver Dis. 2016;48:1162–7. 12. Hovde Ø, Kempski-Monstad I, Sma˚stuen MC, et al. Mortality and causes of death in Crohn’s disease: results from 20 years of follow-up in the IBSEN study. Gut. 2014;63:771–5. 33. Canavan C, Abrams KR, Mayberry J. Meta-analysis: colorectal and small bowel cancer risk in patients with Crohn’s disease. Aliment Pharmacol Ther. 2006;23:1097–104. 13. Bitton A, Vutcovici M, Sewitch M, et al. Mortality trends in crohn’s disease and ulcerative colitis: a population-based study in Que´bec, Canada. Inflamm Bowel Dis. 2016;22:416–23. 34. Yano Y, Matsui T, Hirai F, et al. Cancer risk in Japanese Crohn’s disease patients: investigation of the standardized incidence ratio. J Gastroenterol Hepatol. 2013;28:1300–5. 14. Lee HS, Choe J, Kim SO, et al. Overall and cause-specific mor- tality in Korean patients with inflammatory bowel disease: a hos- pital-based cohort study. J Gastroenterol Hepatol. 2017;32:782–8. p 35. Mizushima T, Ohno Y, Nakajima K, et al. Malignancy in Crohn’s disease: incidence and clinical characteristics in Japan. Digestion. 2010;81:265–70. 15. Matsui T, Hirai F, Hisabe T. Proposed diagnostic criteria for Crohn’s disease. References 1. Peyrin-Biroulet L, Loftus EV Jr, Colombel JF, et al. The nature history of adult Crohn’s disease in population based cohorts. Am J Gastroenterol. 2010;105:289–97. 123 J Gastroenterol (2019) 54:42–52 51 tclass2=000001053061&tclass3=000001053065&result_back=1& result_page=1&second2=1. Accessed 1 May 2018. 2. Lakatos PL, Golovics PA, David G, et al. Has there been a change in the natural history of Crohn’s disease? Surgical rates and medical management in a population-based inception cohort from Western Hungary between 1977–2009. Am J Gastroenterol. 2012;107:579–88. 21. Prior P, Gyde S, Cooke WT, et al. Mortality in Crohn’s disease. Gastroenterology. 1981;80:307–12. 22. Weterman IT, Biemond I, Pen˜a AS. Mortality and cause of death in Crohn’s disease: review of 50 years’ experience in Leiden University Hospital. Gut. 1990;31:1387–90. 3. Sato Y, Matsui T, Yano Y, et al. Long-term course of Crohn’s disease in Japan: incidence of complications, cumulative rate of initial surgery, and risk factors at diagnosis for initial surgery. J Gastroenterol Hepatol. 2015;30:1713–9. 23. Cottone M, Magliocco A, Rosselli M, et al. Mortality in patients with Crohn’s disease. Scand J Gastroenterol. 1996;31:372–5. 4. Yano Y, Matsui T, Matsushima Y, et al. Time trend and risk factors of initial surgery for Crohn’s disease in Japan. J Colitis Diverticulitis. 2016;1:107. https://doi.org/10.4172/jcdc.1000107. 24. Farrokhyar F, Swarbrick ET, Grace RH, et al. Low mortality in ulcerative colitis and Crohn’s disease in three regional centers in England. Am J Gastroenterol. 2001;96:501–7. 5. Uno H, Yao T, Matsui T, et al. Mortality and cause of death in Japanese patients with Crohn’s disease. Dis Colon Rectum. 2003;46:S15–21. 25. Kennedy NA, Clark DN, Bauer J, et al. Nationwide linkage analysis in Scotland to assess mortality following hospital admission for Crohn’s disease: 1998–2000. Aliment Pharmacol Ther. 2012;35:142–53. 6. Oriuchi T, Hiwatashi N, Kinouchi Y, et al. Clinical course and longterm prognosis of Japanese patients with Crohn’s disease: predictive factors, rates of operation, and mortality. J Gastroen- terol. 2003;38:942–53. 26. Hutfless SM, Weng X, Liu L, et al. Mortality by medication use among patients with inflammatory bowel disease, 1996–2003. Gastroenterology. 2007;133:1779–86. 7. Wolters FL, Russel MG, Sijbrandij j, et al. Crohn’s disease: increased mortality 10 years after diagnosis in a Europe-wide population based cohort. Gut. 2006;55:510–8. gy 27. Masala G, Bagnoli S, Ceroti M, et al. Divergent patterns of total and cancer mortality in ulcerative colitis and Crohn’s disease patients: the Florence IBD study 1978–2001. Gut. 2004;53:1309–13. 8. Jess T, Loftus EV Jr, Harmsen WS, et al. References In: Annual of Research Group of Intractable Inflammatory Bowel Disease subsidized by the Min- istry of Health, Labour, and Welfare of Japan. 2011, pp. 52–54 (in Japanese). 36. Higashi D, Katsuno H, Kimura H, et al. Current state of and problems related to cancer of the intestinal tract associated with Crohn’s disease in Japan. Anticancer Res. 2016;36:3761–6. 16. Ueno F, Matsui T, Matsumoto T, et al. Evidence-based clinical practice guidelines for Crohn’s disease, integrated with formal consensus of experts in Japan. J Gastroenterol. 2013;48:31–72. 37. Sugita A, Koganei K, Tatsumi K, et al. Management of rectal cancer including cancer in the anal fistula with Crohn’s disease. Nihon Shokakibyo Gakkai Zasshi. 2013;110:396–402. 17. Hisabe T, Hirai F, Matsui T, et al. Evaluation of diagnostic cri- teria for Crohn’s disease in Japan. J Gastoenterol. 2014;49:93–9. 38. Yamamoto J, Uno H, Hirai F, et al. Clinical study of 11 cases of amyloidosis secondary to Crohn’s disease (in Japanese). Stomach Intestine. 1999;34:1255–66. 18. Silverberg MS, Satsangi J, Ahmad T, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol. 2005;19:5–36. 39. Tanimura S, Nozaki R, Ohwan T, et al. Clinical study of sec- ondary amyloidosis in surgically treated cases of Crohn’s disease (in Japanese). J Jpn Soc Coloproctol. 2006;59:441–7. 40. Miyaoka M, Matsui T, Hisabe T, et al. Clinical and endoscopic features of amyloidosis secondary to Crohn’s disease: diagnostic value of duodenal observation and biopsy. Dig Endosc. 2011;23:157–65. 19. Kim HJ, Hann HJ, Hong SN, et al. Incidence and natural course of inflammatory bowel disease in Korea, 2006-2012: a nationwide population-based study. Inflamm Bowel Dis. 2015;21:623–30. 41. Sharma P, Aguilar R, Siddiqui OA, et al. Secondary systemic amyloidosis in inflammatory bowel disease: a nationwide anal- ysis. Ann Gastroenterol. 2017;30:504–11. 20. Statistics bureau of the ministry of internal affairs and commu- nications. https://www.e-stat.go.jp/stat-search/files?page=1&lay out=datalist&toukei=00450011&kikan=00450&tstat=000001028 897&cycle=7&year=20000&month=0&tclass1=000001053058& 12 3 23 52 J Gastroenterol (2019) 54:42–52 45. Hanauer SB, Feagan BG, Lichtenstein GR, et al. Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet. 2002;359:1541–9. 42. Tosca Cuquerella J, Bosca-Watts MM, Anton Ausejo R, et al. Amyloidosis in inflammatory bowel disease: a systematic review of epidemiology, clinical features, and treatment. J Crohns Colitis. 2016;10:1245–53. 46. Sands BE, Anderson FH, Bernstein CN, et al. Infliximab main- tenance therapy for fistulizing Crohn’s disease. N Engl J Med. 2004;350:876–85. References 43. Lowdell CP, Shousha S, Parkins RA. The incidence of amyloi- dosis complicating inflammatory bowel disease. A prospective survey of 177 patients. Dis Colon Rectum. 1986;29:351–4. 47. Rungoe C, Langholz E, Andersson M, et al. Changes in medical treatment and surgery rates in inflammatory bowel disease: a nationwide cohort study 1979–2011. Gut. 2014;63:1607–16. 44. Rutgeerts P, D’Haens G, Targan S, et al. Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn’s disease. Gastroenterology. 1999;117:761–9. 123
https://openalex.org/W4377242330
https://repository.ubn.ru.nl//bitstream/handle/2066/295889/295889.pdf
English
null
The neurophysiology of continuous action monitoring
iScience
2,023
cc-by
19,234
The neurophysiology of continuous action monitoring Wilken, S.; Böttcher, A.; Adelhöfer, N.; Raab, M.; Hoffmann, S.; Beste, C. 2023, Article / Letter to editor (iScience, 26, 7, (2023), article 106939) Doi link to publisher: https://doi.org/10.1016/j.isci.2023.106939 Version of the following full text: Publisher’s version Downloaded from: https://repository.ubn.ru.nl/handle/2066/295889 Download date: 2024-10-24 Download date: 2024-10-24 Note: To cite this publication please use the final published version (if applicable). iScience iScience ll OPEN ACCESS ll OPEN ACCESS Article The neurophysiology of continuous action monitoring Saskia Wilken, Adriana Bo¨ ttcher, Nico Adelho¨ fer, Markus Raab, Sven Hoffmann, Christian Beste christian.beste@ uniklinikum-dresden.de Highlights Continuous action monitoring is crucial for everyday goal-directed behavior We investigate the underlying neurophysiology with a new EEG paradigm Modulation of alpha, beta, and theta band activity in widespread cortical regions Saskia Wilken, Adriana Bo¨ ttcher, Nico Adelho¨ fer, Markus Raab, Sven Hoffmann, Christian Beste christian.beste@ uniklinikum-dresden.de Highlights Continuous action monitoring is crucial for everyday goal-directed behavior We investigate the underlying neurophysiology with a new EEG paradigm Modulation of alpha, beta, and theta band activity in widespread cortical regions iScience iScience ll OPEN ACCESS SUMMARY Monitoring actions is essential for goal-directed behavior. However, as opposed to short-lasting, and regularly reinstating monitoring functions, the neural pro- cesses underlying continuous action monitoring are poorly understood. We inves- tigate this using a pursuit-tracking paradigm. We show that beta band activity likely maintains the sensorimotor program, while theta and alpha bands probably support attentional sampling and information gating, respectively. Alpha and beta band activity are most relevant during the initial tracking period, when sensorimotor calibrations are most intense. Theta band shifts from parietal to frontal cortices throughout tracking, likely reflecting a shift in the functional rele- vance from attentional sampling to action monitoring. This study shows that resource allocation mechanisms in prefrontal areas and stimulus-response map- ping processes in the parietal cortex are crucial for adapting sensorimotor pro- cesses. It fills a knowledge gap in understanding the neural processes underlying action monitoring and suggests new directions for examining sensorimotor inte- gration in more naturalistic experiments. 1General Psychology: Judgment, Decision Making, and Action, Institute of Psychology, University of Hagen, Hagen, Germany 2Cognitive Neurophysiology, Department of Child and Adolescent Psychiatry, Faculty of Medicine, TU Dresden, Dresden, Germany 3University Neuropsychology Center, Faculty of Medicine, TU Dresden, Dresden, Germany 4Donders Institute of Cognition and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands 5Performance Psychology, Institute of Psychology, German Sport University Cologne, Cologne, Germany 6School of Applied Sciences, London South Bank University, London, UK 7These authors contributed equally 8Senior author 9Lead contact *Correspondence: christian.beste@ uniklinikum-dresden.de https://doi.org/10.1016/j.isci. 2023.106939 Article The neurophysiology of continuous action monitoring Saskia Wilken,1,7 Adriana Bo¨ ttcher,2,3,7 Nico Adelho¨ fer,2,4 Markus Raab,5,6 Sven Hoffmann,1,8 and Christian Beste2,3,8,9,* Saskia Wilken,1,7 Adriana Bo¨ ttcher,2,3,7 Nico Adelho¨ fer,2,4 Markus Raab,5,6 Sven Hoffma and Christian Beste2,3,8,9,* iScience 26, 106939, July 21, 2023 ª 2023 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 1General Psychology: Judgment, Decision Making, and Action, Institute of Psychology, University of Hagen, Hagen, Germany iScience 26, 106939, July 21, 2023 ª 2023 The Author(s). C BY license (http://creativecommons.org/licenses/by/4.0/). 1 iScience 26, 106939, July 21, 2023 ª 2023 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 1General Psychology: Judgment, Decision Making, and Action, Institute of Psychology, University of Hagen, Hagen, Germany 2Cognitive Neurophysiology, Department of Child and Adolescent Psychiatry, Faculty of Medicine, TU Dresden, Dresden, Germany 3University Neuropsychology Center, Faculty of Medicine, TU Dresden, Dresden, Germany 4Donders Institute of Cognition and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands 5Performance Psychology, Institute of Psychology, German Sport University Cologne, Cologne, Germany 6School of Applied Sciences, London South Bank University, London, UK 7These authors contributed equally 8Senior author 9Lead contact *Correspondence: christian.beste@ uniklinikum-dresden.de https://doi.org/10.1016/j.isci. 2023.106939 INTRODUCTION The latter is likely because the intraparietal sulcus and the dorsal premotor cortex probably constitute the major cortical network to control complex movement kinematics.26 Despite oscillatory activity reflecting a fundamental information processing principle in the human brain,7 the limited temporal resolution of the time-frequency analytical approach is a drawback. Performance in pursuit tracking varies considerably28 depending on acquired implicit knowledge and the predictability of the target trajectory.3 Especially after trajectory direction changes, adaptive processes have to be triggered to reduce spatial tracking errors. Thus, there are discrete points where adaptive processes are required. The analysis of time-domain EEG data is necessary to capture the neurophysiological substrates of these temporal dy- namics. An ERP component capturing these processes may be the error-related negativity (ERN)29–33 origi- nating from medial frontal cortices.34 The ERN reflects a comparison process between the desired and the achieved action outcome,35 which is also required during continuous sensorimotor processing.26 It is commonly accepted that such aspects of performance monitoring are necessary when adjusting action-to-ac- tion outcomes.34 In addition, it is also mandatory to increase processing capacities to adapt complex move- ments when sensorimotor processes have to be adapted (because they deviate too much from the desired state). Such resource allocation processes are likely reflected by the P2 ERP component,36–38 while adaptations in the mapping of the stimulus on the appropriate response are reflected by the P3 ERP component.39,40 Since monitoring and adaptation are highly dynamic,41,42 we also relate tracking and EEG dynamics43 by relating single-trial EEG dynamics of the ERP parameters mentioned previously and tracking performance. To experimentally disentangle demands on continuous action monitoring processes and to examine their un- derlying neurophysiology in detail, participants performed two consecutive tasks employing a pursuit-tracking paradigm requiring continuous tracking of a moving target with a cursor using a joystick: In the first task, we examined the execution of complex movements requiring online sensorimotor adaptation. For that, one trajec- tory segment was kept constant across trials, andtwo randomly generated trajectory segments were appended at the end and beginning of this constant trajectory, respectively. Comparisons were performed between the constant and random trajectory segments. The second task tested the prediction of visual trajectories in prep- aration for actions using segments without visual feedback about the cursor location. Like in the first task, the target trajectory was a concatenation of two random parts and constant part in the middle. INTRODUCTION The monitoring of one’s actions is a central requirement for goal-directed behavior. Despite its ubiquitous na- ture, the underlying neural mechanisms are only incompletely understood. Existing conceptions on action monitoring, and especially considerations on the functional relevance of neurophysiological correlates of ac- tion monitoring, are based on analyses/experiments during relatively short-lasting monitoring or demands that reinstate regularly.1,2 However, in the rarest cases, real-time action monitoring is short-lasting and regu- larly reinstates in discretized occasions.1 Instead, action monitoring is performed continuously over several seconds or even longer with little prior knowledge about the intensity with which action monitoring processes are demanded, such as in driving.3–5 Moreover, actions do not consist solely of sequential action outcome evaluation. Instead, they require online adjustment of movements and continuous prediction of motor pro- gram outcomes (e.g., playing soccer and controlling the ball while passing opponents). Thus, it is tentative whether knowledge from classical experiments and the involved neurophysiological functions6 can be gener- alized to other more naturalistic settings. There is thus an urgent need to clarify the neural mechanisms of continuous action monitoring, which is done in the current study through pursuit-tracking tasks and an in- depth analysis of neurophysiological processes applying various electroencephalography (EEG) analysis methods. We combine the analysis of oscillatory dynamics, as a fundamental communication mechanism of the human brain,7 with high-resolution time-domain event-related potential (ERP) analysis to achieve a detailed overview of the neurophysiological correlates of continuous action monitoring. INTRODUCTION Previous studies examining action monitoring during reinstating and short-lasting demands have consis- tently found that theta band activity plays a role,8–10 such that it likely serves as a ‘‘surprise signal’’ necessary to adapt actions.8 The theta-related surprise signal is used to alter learning processes to adjust behavioral strategy and the need to increase cognitive control.8 The importance of medial frontal theta band activity during action monitoring relates to biophysical principles of high-amplitude/low-frequency oscillations.11 These types of oscillations are optimal for synchronizing activities between multiple remote functional neuroanatomical regions.8 Of note, theta band activity and other EEG correlates associated with the medial frontal and superior parietal cortex are central for integrating sensory and motor processes,12–14 which well-reflects overarching conceptualizations of the medial frontal cortex.15 However, theta band iScience Article ll OPEN ACCESS activity also plays a role during attentional selection processes16,17 and especially so in parietal cortices, which are known to be involved in binding sensory and motor information into a sensorimotor representa- tion (program) necessary for goal-directed action.18 Accordingly, these brain structures should also reveal theta band activity modulation during continuous action monitoring. Crucially, when considering the role of theta band activity, beta band activity needs to be considered as well since both are jointly involved in motor control.19 Moreover, beta band activity has been associated with top-down controlled processing,20 including attention to upcoming motor tasks.21,22 It may thus reflect premotor mechanisms guiding motor actions.23 Therefore, beta band activity may reflect the computation of the ‘‘status quo,’’24 meaning that beta band activity reflects the continuous maintenance of a sensorimotor program depending on the pre- diction of what is likely to happen soon, to adapt the sensorimotor program.25,26 All these aspects make it likely that beta band activity plays an essential role during the continuous monitoring of actions. However, a conceptually related role has been ascribed to alpha band activity.27 Alpha band activity likely reflects the gating and controlled access of incoming information to a domain-general ‘‘knowledge system.’’27 Sup- posedly, alpha band activity plays a role in anticipatory attentional processes needed to maintain target information.27 Based on these considerations, we examine the theta, alpha, and beta band activity dy- namics during continuous action monitoring combining time-frequency and EEG-beamforming methods in a pursuit-tracking task in the current study. We hypothesize that these activities are modulated during continuous action monitoring and that frontoparietal cortices reflect these dynamics. INTRODUCTION At regular intervals, the cursor was occluded. Theseparadigms are standard in the field of pursuit tracking.3–5 In this study, we inves- tigate the association of alpha, beta, and theta band activity with the execution of complex movements with different sensorimotor demands, i.e. predictability of the target trajectory and visibility of the cursor. Further- more, we investigate ERPs related to target direction changes with different sensorimotor demands. Finally, we will examine the predictability of the ERP amplitudes by the behavioral tracking performance. 2 iScience 26, 106939, July 21, 2023 Figure 1. Flow diagram overview of analyses g g y After general preprocessing of the EEG data, we split EEG analyses in two analysis strands: the time-frequency domain and the time domain analyses which both culminate respectively in a suitable source localization. The analysis of the behavioral data was used in conjunction with the time-domain EEG data. Additionally, exploring the observed differences in tracking behavior during the first 500 ms of a trial as compared to the proceeding trial led to the introduction of an additional contrast in the time-frequency analyses. Analyses are shown in green shaded rectangles, data are depicted in white diamonds. RESULTS To gain an overview of the order of data analysis steps for the current study, please refer to Figure 1. The analyses were performed for both tasks. After summarizing explorative findings in the behavioral data that shaped our analysis of task 1, we present the results grouped by the two tasks. 2 iScience 26, 106939, July 21, 2023 ll OPEN ACCESS iScience Article igure 1 Flow diagram overview of analyses General behavioral data In both tasks, the participants were asked to continuously track a steadily moving target with a cursor along Figure 1. Flow diagram overview of analyses After general preprocessing of the EEG data, we split EEG analyses in two analysis strands: the time-frequency domain and the time domain analyses which both culminate respectively in a suitable source localization. The analysis of the behavioral data was used in conjunction with the time-domain EEG data. Additionally, exploring the observed differences in tracking behavior during the first 500 ms of a trial as compared to the proceeding trial led to the introduction of an additional contrast in the time-frequency analyses. Analyses are shown in green shaded rectangles, data are depicted in white diamonds. General behavioral data g g g g Figure (A) shows the distribution of all subjects’ tracking error magnitudes (y axis) over time (x axis) during the first 2000 ms of all trials, with the color scale denoting the frequency (number of trials) of tracking error magnitudes per frame. A more peaked and more predictable frequency distribution can be observed during the first 500 ms of the trial compared to the rest. Figure (B) depicts the quantification of this observation using sample entropy as a measure of time-series predictability. It shows the frequency (y axis) of sample entropy values (x axis) of all subjects and all trials for the first 500 ms (blue) compared to the following 1500 ms (red). The median of the sample entropy values in the first 500 ms interval (blue line) is fi l ll h h d f h 501 2000 l l l ( d l ) Figure (B) depicts the quantification of this observation using sample entropy as a measure of time-series predictability. It shows the frequency (y axis) of sample entropy values (x axis) of all subjects and all trials for the first 500 ms (blue) compared to the following 1500 ms (red). The median of the sample entropy values in the first 500 ms interval (blue line) is significantly smaller than the median of the 501–2000 ms interval sample entropy values (red line). To quantify this difference, we calculated the sample entropy44 of the z-transformed tracking error of the first 500 ms interval of each trial of each participant respectively and the z-transformed tracking error of the following 1500 ms interval of each trial (see Figure 2B). Sample entropy quantifies the predictability of a time series without requiring assumptions about its distribution. We used a Wilcoxon rank-sum test to compare sample entropy values (one for each subject) from the first 500 ms interval to the sample entropy values of the following 1500 ms interval. We found that entropy is significantly lower in the first (median = 0.104) compared to the second (median = 0.122) interval (Z = 3.65, p < 0.001, r = 0.67). Upon visually inspecting tracking and pursuit of the individual trials, we noticed that participants frequently did not actively move the cursor from the middle line during the first 500 ms interval of a trial (Figure 3). Task 1: Online sensorimotor adaptation In the first task, participants tracked the target with a cursor along a trajectory concatenated from two random trajectory segments (at the trial start and end) and one constant, i.e., repeated segment in between (see trial overview in Figure 4A). 4 iScience 26, 106939, July 21, 2023 General behavioral data At the start of the trial, the difference between the cursor position and target position was more determined by the target path than by the rest of the trial. Nevertheless, the discrepancy was evident for the participants due to the visual input. Due to the observed differences between the first 500 ms interval and the remaining part of the trial, the analysis of the neurophysiological (EEG) data was performed separately for the first 500 ms and the remaining time of the trial. General behavioral data In both tasks, the participants were asked to continuously track a steadily moving target with a cursor along a trajectory using a joystick. First, we investigated whether the tracking data revealed changes in tracking behavior patterns across the trial. We calculated the tracking error for each frame by subtracting the y-po- sition of the cursor on the screen from the y-position of the target on the screen. Then, we inspected the distribution of the absolute tracking error for each frame over one trial, across all trials of all participants. As shown in Figure 2A, the pattern of tracking errors differed in the first 500 ms interval of the trial compared to the later trial interval. iScience 26, 106939, July 21, 2023 3 iScience Article ll OPEN ACCESS Figure 2. Visualization of change in tracking error between first 500 ms and following 1500 ms Figure (A) shows the distribution of all subjects’ tracking error magnitudes (y axis) over time (x axis) during the first 2000 ms of all trials, with the color scale denoting the frequency (number of trials) of tracking error magnitudes per frame. A more peaked and more predictable frequency distribution can be observed during the first 500 ms of the trial compared to the rest. Figure (B) depicts the quantification of this observation using sample entropy as a measure of time-series predictability. It shows the frequency (y axis) of sample entropy values (x axis) of all subjects and all trials for the first 500 ms (blue) compared to the following 1500 ms (red). The median of the sample entropy values in the first 500 ms interval (blue line) is significantly smaller than the median of the 501–2000 ms interval sample entropy values (red line). ure 2. Visualization of change in tracking error between first 500 ms and following 1500 ms g g g g Figure (A) shows the distribution of all subjects’ tracking error magnitudes (y axis) over time (x axis) during the first 2000 ms of all trials, with the color scale denoting the frequency (number of trials) of tracking error magnitudes per frame. A more peaked and more predictable frequency distribution can be observed during the first 500 ms of the trial compared o the rest. Behavioral data the follow 1500 ms Th fi h d l k d l (6 fi A F) f b A b b d Figure 3. Example trials illustrating the difference in tracking behavior during the first 500 ms vs. the following This figure shows randomly picked trials (6, figure parts A–F) from various subjects. As can be seen, subjects do not immediately move the joystick in pursuit of the target after the target started moving, which is why the tracking error during this time is mainly due to the course of the random trajectory. The dashed line indicates the moment the participant started moving the joystick in pursuit of the target. Note that the apparently linear sections of the trajectory are due to omitting x-coordinate information in this plot so that time can be plotted with equidistant tick marks on the x axis. The trajectory on screen had no linear sections. vincentiles of pursuit latency to obtain a measure of association between the two tracking performance measures. We found a slightly positive association between the epoch error and pursuit latency as can be seen in Figure 5. This means that lower accuracy is associated with slower reaction times to target di- rection changes. The size of this effect ranges from d = 0.07 to d = 0.61. Behavioral data Two measures were used to quantify the accuracy of pursuit tracking: First, the mean of the vertical differ- ence between cursor and target was calculated, thus measuring the tracking error (accuracy) for the respec- tive epoch (epoch error). Second, for each target direction change, the latency of the following matching pursuit direction change was calculated (pursuit latency). This reflects the time needed to adjust the cursor movement to a change in the target trajectory. First, we were curious to know how differences between conditions change with increasing magnitude of tracking error and pursuit latency. To this end, we ordered the tracking error and pursuit latency into ten vincentiles per subject. For data analysis, we took 5,000 boot- strap samples from each vincentile and performed paired t-tests on the respective vincentiles, comparing the error measure magnitudes between the first random and the constant trajectory segments. Both, tracking error and pursuit latency were significantly larger in the constant condition in almost all vincentiles (except for the first three epoch error vincentiles) (see Figure 5), indicating slower reaction times to changes in the target direction and lower tracking accuracy in the constant segments compared to the random seg- ments. The effect sizes remain relatively stable across vincentiles, ranging from d = 0.36 to d = 0.30 for epoch error and from d = 0.19 to d = 0.31 for pursuit latency. Finally, we sorted the epoch error by the 4 ll OPEN ACCESS Figure 3. Example trials illustrating the difference in tracking behavior during the first 500 ms vs. the following 1500 ms This figure shows randomly picked trials (6, figure parts A–F) from various subjects. As can be seen, subjects do not immediately move the joystick in pursuit of the target after the target started moving, which is why the tracking error during this time is mainly due to the course of the random trajectory. The dashed line indicates the moment the participant started moving the joystick in pursuit of the target. Note that the apparently linear sections of the trajectory are due to omitting x-coordinate information in this plot so that time can be plotted with equidistant tick marks on the x axis. The trajectory on screen had no linear sections. Figure 3. Example trials illustrating the difference in tracking behavior during the first 500 ms vs. Time-frequency decomposition and EEG beamforming analyses The first and last segments were randomly generated for each trial, whereas the middle segment was repeated in every trial. (B) Since we found significant differences in the tracking behavior pattern during the first 500 ms of each trial compared to the rest of the trial, we segmented the EEG into two intervals for the random and constant trajectory, respectively. The first interval consisted of the first 500 ms of the random and constant trajectory segment (depicted in blue). The second interval compared 500–3000 ms for the random trajectory segment to 0–2500 ms for the constant segment (depicted in green). Figure 4. One trial of our pursuit-tracking paradigm measuring online sensorimotor adaptation and the segmentation for EEG data analysis (A) Participants were asked to follow a continuously moving target (a red square) along an invisible trajectory, depicted as a white dashed line, using a cursor (a white cross). Horizontal cursor movements were locked to the target. Following a start vector at the beginning of each trial, the target trajectory consisted of three segments connected via cubic splines The first and last segments were randomly generated for each trial, whereas the middle segment was repeated in every trial Figure 4. One trial of our pursuit-tracking paradigm measuring online sensorimotor adaptation and the segmentation for EEG data analysis g y (A) Participants were asked to follow a continuously moving target (a red square) along an invisible trajectory, depicted as a white dashed line, using a cursor (a white cross). Horizontal cursor movements were locked to the target. Following a start vector at the beginning of each trial, the target trajectory consisted of three segments connected via cubic splines. The first and last segments were randomly generated for each trial, whereas the middle segment was repeated in every trial. (B) Since we found significant differences in the tracking behavior pattern during the first 500 ms of each trial compared to the rest of the trial, we segmented the EEG into two intervals for the random and constant trajectory, respectively. The first interval consisted of the first 500 ms of the random and constant trajectory segment (depicted in blue). The second interval compared 500–3000 ms for the random trajectory segment to 0–2500 ms for the constant segment (depicted in green). proceeding trial) at the sensor level using cluster-based permutation tests. This way, we identified which contrasts revealed significant differences at the sensor level. 6 iScience 26, 106939, July 21, 2023 Time-frequency decomposition and EEG beamforming analyses Based on the behavioral data analysis (cf. General behavioral data section), the EEG data recorded during the random and constant trajectory segments were analyzed separately for the first 500 ms interval and the rest of the trial. This resulted in two segments for the random trajectory: 0–500 ms as the interval for the trial start (from now on referred to as ‘‘first interval’’), and 500–3000 ms for the proceeding trial (from now on named ‘‘second interval’’). The EEG data for the constant trajectory segment were also split into two parts of the same lengths as the first and second intervals of the random trajectory, respectively. The first interval of the constant trajectory ranged from 0 to 500 ms, whereas the second interval ranged from 0 to 2500 ms (see Figure 2A). We chose to use the first 500 ms for both intervals twice to underline that we do not assume that there is any difference in between the EEG activity within the first 500 ms of the constant trajectory (since it is the central part of the trial) compared to the following 2500 ms (see Figure 4). Then, in prepara- tion of time-frequency decomposition, we analyzed differences in alpha, beta, and theta band power be- tween constant and random trajectory segments for the first (i.e., trial start) and second time interval (i.e., iScience 26, 106939, July 21, 2023 5 iScience Article ll OPEN ACCESS proceeding trial) at the sensor level using cluster-based permutation tests. This way, we identified which contrasts revealed significant differences at the sensor level. We then reconstructed the source activity for each significant contrast using dynamic imaging of coherent sources (DICS) beamforming.45 We calcu- lated the normalized difference of source activity and extracted demarcated brain areas with a clustering algorithm (DBSCAN), as done in previous studies.46,47 The results are shown in Figure 6. Figure 4. One trial of our pursuit-tracking paradigm measuring online sensorimotor adaptation and the segmentation for EEG data analysis (A) Participants were asked to follow a continuously moving target (a red square) along an invisible trajectory, depicted as a white dashed line, using a cursor (a white cross). Horizontal cursor movements were locked to the target. Following a start vector at the beginning of each trial, the target trajectory consisted of three segments connected via cubic splines. Time-frequency decomposition and EEG beamforming analyses We then reconstructed the source activity for each significant contrast using dynamic imaging of coherent sources (DICS) beamforming.45 We calcu- lated the normalized difference of source activity and extracted demarcated brain areas with a clustering algorithm (DBSCAN), as done in previous studies.46,47 The results are shown in Figure 6. proceeding trial) at the sensor level using cluster-based permutation tests. This way, we identified which contrasts revealed significant differences at the sensor level. We then reconstructed the source activity for each significant contrast using dynamic imaging of coherent sources (DICS) beamforming.45 We calcu- lated the normalized difference of source activity and extracted demarcated brain areas with a clustering algorithm (DBSCAN), as done in previous studies.46,47 The results are shown in Figure 6. Figure 5. Vincentiles of tracking performance measures by condition constant vs. random The two left plots visualize which magnitudes the error measures assume (y axis), per vincentile (dots) and separated by condition (blue resp. black line). Additionally, at the bottom, there is a bar plot indicating whether there are significant differences between conditions and how large that difference is. The smallest, still significant t-value is t(29) = -1.79, p = 0.03. Note that the effect size values are absolute values (not considering direction of effect) for easier visualization. The rightmost plot shows the epoch error in pixels sorted by the vincentiles of pursuit latency per condition. It visualizes a slightly positive association between the two variables in task 1 for constant as well as random trajectory segments. Figure 5. Vincentiles of tracking performance measures by condition constant vs. random performance measures by condition constant vs. random The two left plots visualize which magnitudes the error measures assume (y axis), per vincentile (dots) and separated by condition (blue resp. black line). Additionally, at the bottom, there is a bar plot indicating whether there are significant differences between conditions and how large that difference is. The smallest, still significant t-value is t(29) = -1.79, p = 0.03. Note that the effect size values are absolute values (not considering direction of effect) for easier visualization. The rightmost plot shows the epoch error in pixels sorted by the vincentiles of pursuit latency per condition. It visualizes a slightly positive association between the two variables in task 1 for constant as well as random trajectory segments. Time-frequency decomposition and EEG beamforming analyses 6 iScience 26, 106939, July 21, 2023 6 iScience 26, 106939, July 21, 2023 ll OPEN ACCESS or the first 500 ms (first interval), the cluster-based permutation tests at the sensor level revealed signifi- t diff b t th t t d d t j t i th ti fi t 500 i b t igure 6. Results of the time-frequency and beamforming analysis for the contrast of constant and random trajectory segment (constant-random) n the first interval of the pursuit-tracking paradigm requency power differences for significant electrodes in cluster-based permutation test on the left. The topography for the respective power difference is hown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant egative differences (random > constant) were found for the alpha, beta, and theta band. The dashed lines approximately enclose the significant time terval and frequency range. On the right, source activity differences for the respective frequency band are depicted in the clusters identified by the BSCAN algorithm. A) Theta (4–7 Hz) power differences, including the results of the beamforming analysis. Significant negative power differences were found for 68–500 ms. B) Alpha (8–12 Hz) power differences, including the results of the beamforming analysis. Significant negative differences were found for the whole time terval. C) Beta (13–30 Hz) power differences, including the results of the beamforming analysis. Significant negative differences were found for 0–436 ms. OPEN ACCESS Article Figure 6. Results of the time-frequency and beamforming analysis for the contrast of constant and random trajectory segment (constant-random) in the first interval of the pursuit-tracking paradigm in the first interval of the pursuit tracking paradigm Frequency power differences for significant electrodes in cluster-based permutation test on the left. The topography for the respective power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant negative differences (random > constant) were found for the alpha, beta, and theta band. The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the respective frequency band are depicted in the clusters identified by the DBSCAN algorithm. (A) Theta (4–7 Hz) power differences, including the results of the beamforming analysis. Significant negative power differences were found for 68–500 ms. iScience 26, 106939, July 21, 2023 7 Time-frequency decomposition and EEG beamforming analyses Results of the time-frequency and beamforming analysis for the contrast of constant and random trajectory (constant-random) in the second interval (i.e., proceeding trial) of the pursuit-tracking paradigm Theta band (4–7 Hz) power differences for significant electrodes in cluster-based permutation test on the left. Note that the time information on the x axis applies to the constant trajectory segment, whereas for the random trajectory segment, the time window would be 500–3000 ms (see Figure 4). The topography for the theta power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant positive power differences (constant > random) were found for 928–1944 ms. The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the theta frequency band are depicted in the clusters identified by the DBSCAN algorithm. ll OPEN ACCESS iScience Article iScience Article ll OPEN ACCESS Figure 7. Results of the time-frequency and beamforming analysis for the contrast of constant and random trajectory (constant-random) in the second interval (i.e., proceeding trial) of the pursuit-tracking paradigm Theta band (4–7 Hz) power differences for significant electrodes in cluster-based permutation test on the left. Note that the time information on the x axis applies to the constant trajectory segment, whereas for the random trajectory segment, the time window would be 500–3000 ms (see Figure 4). The topography for the theta power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant positive power differences (constant > random) were found for 928–1944 ms. The dashed lines approximately d beamforming analysis for the contrast of constant and random trajectory (constant-random) in the e pursuit-tracking paradigm Figure 7. Results of the time-frequency and beamforming analysis for the contrast of constant and random trajectory (constant-random) in the second interval (i.e., proceeding trial) of the pursuit-tracking paradigm Theta band (4–7 Hz) power differences for significant electrodes in cluster-based permutation test on the left. Note that the time information on the x axis applies to the constant trajectory segment, whereas for the random trajectory segment, the time window would be 500–3000 ms (see Figure 4). The topography for the theta power difference is shown in the top right corner. Time-frequency decomposition and EEG beamforming analyses (B) Alpha (8–12 Hz) power differences, including the results of the beamforming analysis. Significant negative differences were found for the whole time interval. (C) Beta (13–30 Hz) power differences, including the results of the beamforming analysis. Significant negative differences were found for 0–436 ms. p g p g Frequency power differences for significant electrodes in cluster-based permutation test on the left. The topography for the respective power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant negative differences (random > constant) were found for the alpha, beta, and theta band. The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the respective frequency band are depicted in the clusters identified by the g g Frequency power differences for significant electrodes in cluster-based permutation test on the left. The topography for the respective power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant negative differences (random > constant) were found for the alpha, beta, and theta band. The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the respective frequency band are depicted in the clusters identified by the DBSCAN algorithm. (A) Theta (4–7 Hz) power differences, including the results of the beamforming analysis. Significant negative power differences were found for 68–500 ms. For the first 500 ms (first interval), the cluster-based permutation tests at the sensor level revealed signifi- cant differences between the constant and random trajectory in the respective first 500 ms, i.e., between the trial onset and the middle of the trial, in the alpha, beta, and theta frequency band. The test revealed one significant negative cluster in the alpha frequency band (p < 0.001) on sensor level during the whole time interval. Since the test is based on the difference between frequency power during constant and random trajectory segments (constant-random), a negative cluster indicates higher power in the random compared to the constant trajectory. On source level, DICS beamforming for constant-random revealed activity modulations in the left superior and medial parietal lobe (BA7). For the beta band, N = 7, significant iScience 26, 106939, July 21, 2023 7 Figure 7. Time-frequency decomposition and EEG beamforming analyses Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant positive power differences (constant > random) were found for 928–1944 ms. The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the theta frequency band are depicted in the clusters identified by the DBSCAN algorithm. negative clusters for constant-random were detected on the sensor level (all p < 0.001) from 0 to 436 ms. The DICS beamforming for this contrast revealed activity modulations in the left-hemispheric parietal cor- tex (BA5, BA7) and superior frontal areas of the left hemisphere (BA4, BA6). For the theta frequency band, the cluster-based permutation test also revealed three significant negative clusters (random > constant) at the sensor level (all p < 0.001) from 68 to 500 ms. For this contrast, the source reconstruction revealed ac- tivity modulations in the left and right precuneus, extending to superior parietal areas (BA7). In the second interval, ranging from 500 to 3000 ms in the random trajectory and 0 to 2500 ms in the constant trajectory segment, we found significant differences on the sensor level only in the theta band (see Figure 7). For the theta band, two significant positive clusters (both p < 0.001) were identified on sensor level from 928 to 1944 ms for the random trajectory (or 428 to 1444 ms for the constant trajec- tory, due to the different time windows). Positive clusters indicate higher frequency power in the constant compared to the random segment. Reconstructing source-level activity for this contrast, activity modu- lations in the right frontoparietal cortex (BA4, BA5, and BA6) and the left frontoparietal cortex (BA4 and BA5) were obtained. Moreover, activity modulations were evident in the right supplementary motor area (BA4). iScience 26, 106939, July 21, 2023 Time domain analyses In addition to testing the differences between conditions in the frequency domain, we also investigated whether there are significant differences in ERPs following target direction changes. To that end, we epoched the EEG data into 0–750 ms sections after each target direction change. Then, we performed a cluster-based permutation test over the whole epoch duration, comparing the ERPs of the random and constant condition epochs on sensor level. The test detected four significant negative clusters and eight significant positive clusters. Positive clusters indicate that the ERP in the constant trajectory segment was significantly more negative than the ERP in the random trajectory segment during the respective time interval and for the respective electrodes. Yet, none of the significant clusters could be interpreted because none surpassed our criteria for cluster selection (see STAR Methods section for details). iScience 26, 106939, July 21, 2023 8 ll OPEN ACCESS iScience Article igure 8. One trial of our pursuit-occlusion paradigm measuring the prediction of sensorimotor trajectories and he segmentation for EEG data analysis A) Participants were asked to follow a steadily moving target along the concatenation of random and constant trajectory egments. The cursor was occluded for 2 s every 2 s (depicted as gray shaded areas). During the occlusion periods, articipants had to predict the cursor position. B) The EEG data were segmented according to the occlusion periods, with occluded segments depicted in blue and sible in green. Figure 8. One trial of our pursuit-occlusion paradigm measuring the prediction of sensorimotor trajectories and the segmentation for EEG data analysis (A) Participants were asked to follow a steadily moving target along the concatenation of random and constant trajectory segments. The cursor was occluded for 2 s every 2 s (depicted as gray shaded areas). During the occlusion periods, participants had to predict the cursor position. Figure 8. One trial of our pursuit-occlusion paradigm measuring the prediction of sensorimotor trajectories and the segmentation for EEG data analysis (A) Participants were asked to follow a steadily moving target along the concatenation of random and constant trajectory segments. The cursor was occluded for 2 s every 2 s (depicted as gray shaded areas). During the occlusion periods, participants had to predict the cursor position. (A) Participants were asked to follow a steadily moving target along the concatenation of random and constant trajectory segments. Task 2: Prediction of sensorimotor trajectories To investigate the prediction of sensorimotor trajectories, we modified our pursuit-tracking paradigm (Task 1). Again, participants were asked to follow a steadily moving target along the concatenation of trajectory segments. Importantly, the cursor moved by the participants was occluded for 2 s every 4 s (see Figure 8). During the occlusion period, participants had to predict the cursor’s current position and continue tracking the target. Therefore, we focused on these segments in the data analysis (see Figure 8). Time domain analyses The cursor was occluded for 2 s every 2 s (depicted as gray shaded areas). During the occlusion periods, participants had to predict the cursor position. (B) The EEG data were segmented according to the occlusion periods, with occluded segments depicted in blue and visible in green. participants had to predict the cursor position. (B) The EEG data were segmented according to the occlusion periods, with occluded segments depicted in blue and visible in green. p p p p (B) The EEG data were segmented according to the occlusion periods, with occluded segments depicted in blue and visible in green. iScience 26, 106939, July 21, 2023 9 Behavioral data Here, we applied the same procedure as in the first task, using the epoch error and pursuit latency to quan- tify the accuracy of pursuit tracking. We ordered pursuit latencies and epoch errors into ten vincentiles per subject and drew 5,000 bootstrap samples per vincentile. We then performed t-tests to compare the occluded and non-occluded trajectory segments. Analogously to task one, the epoch error and pursuit la- tency differed significantly in almost all vincentiles (see Figure 9). For the epoch error vincentiles, effect sizes monotonously increased from the first to the eight vincentile (min d = 0.77, max d = 1.69). In each vin- centile, the epoch error was larger when the cursor was occluded compared to the non-occluded condi- tion. So, tracking accuracy is reduced when the cursor is invisible. For the pursuit latency vincentiles, we observed a monotonous reduction in effect sizes from large effects in low vincentiles, i.e., short pursuit la- tency, to non-significant medium effects in the highest vincentile, i.e. long pursuit latency (min d = 1.02, max d = 0.15). The pursuit latencies of every vincentile were larger in the visible condition compared to the occluded condition. In other words, seeing the cursor while tracking is associated with a slower reaction time to changes in the target movement direction. These findings likely reflect an interference of processes: tracking the cursor might interfere with the ability to respond to changes in the trajectory quickly. Finally, we also investigated how the epoch error and pursuit latency are related by sorting the epoch error by the pursuit latency vincentiles. We found that there is no clear association between epoch error and pursuit iScience 26, 106939, July 21, 2023 9 Figure 9. Vincentiles of tracking performance measures by condition occluded vs. visible The two left plots visualize which values the error measures assume per vincentile and separated by condition. Additionally, it is plotted whether the conditions are significantly different and how large the effect is. The smallest, still significant t-value is t(29) = -1.87, p = 0.02. Note that the effect size values are absolute values (not considering direction of effect) for a simpler visualization. The right plot shows the epoch error in pixels sorted into the vincentiles of pursuit latency per condition. It visualizes that there is no clear association between epoch error and pursuit latency in task two neither in the occluded nor the visible condition. Behavioral data ll OPEN ACCESS iScience Article Figure 9. Vincentiles of tracking performance measures by condition occluded vs. visible Th t l ft l t i li hi h l th i til d t d b diti Additi ll it i l tt d h th th ll OPEN ACCESS iScience Article Figure 9. Vincentiles of tracking performance measures by condition occluded vs. visible The two left plots visualize which values the error measures assume per vincentile and separated by condition. Additionally, it is plotted whether the conditions are significantly different and how large the effect is. The smallest, still significant t-value is t(29) = -1.87, p = 0.02. Note that the effect size values are absolute values (not considering direction of effect) for a simpler visualization. The right plot shows the epoch error in pixels sorted into the vincentiles of pursuit latency per condition. It visualizes that there is no clear association between epoch error and pursuit latency in task two neither in the occluded nor the visible condition. The two left plots visualize which values the error measures assume per vincentile and separated by condition. Additionally, it is plotted whether the conditions are significantly different and how large the effect is. The smallest, still significant t-value is t(29) = -1.87, p = 0.02. Note that the effect size values are absolute values (not considering direction of effect) for a simpler visualization. The right plot shows the epoch error in pixels sorted into the vincentiles of pursuit latency per condition. It visualizes that there is no clear association between epoch error and pursuit latency in task two neither in the occluded nor the visible condition. latency. However, the difference in epoch error between the conditions when sorted by pursuit latency is overall large, ranging from d = 0.84 to d = 1.72. Time-frequency decomposition and EEG beamforming analyses To analyze neurophysiological processes associated with predicting sensorimotor trajectories, we analyzed differences in alpha, beta, and theta power between occluded and non-occluded trajectory seg- ments. We reconstructed the source activity using the DICS beamformer for significant effects at the sensor level and analyzed the contrast by calculating the normalized difference between both conditions. Then, we identified source-level clusters using the DBSCAN algorithm. The cluster-based permutation test for the contrast occluded vs. visible in the theta band on the sensor level revealed four significant negative clusters for the entire time interval (all p < 0.001). The contrast was calculated using the difference between the occluded and visible conditions (occluded- visible). Hence, a positive cluster indicates higher power in the occluded compared to the visible condition (Figure 10). Reconstructing the source-level activity in the theta band for this contrast, three neuroanatom- ical regions revealed activity modulations. Activity modulations were evident in the right-hemispheric infe- rior occipital and inferior temporal areas (BA19, BA37) and the inferior occipitotemporal cortex of the left hemisphere (BA19, BA37). Moreover, activity modulations were evident in the right hemisphere’s middle temporal and occipital cortex (BA19, BA21, BA37) (Figure 10). No significant clusters were identified for the alpha or beta band. 10 iScience 26, 106939, July 21, 2023 Time domain analyses Results of the time-frequency and beamforming analysis for the contrast of occluded and visible occlusion paradigm requency and beamforming analysis for the contrast of occluded and visible cursor (occluded-visible) in the pursuit- Figure 10. Results of the time-frequency and beamforming analysis for the contrast of occluded and visible cursor (occluded-visible) in the pursuit- occlusion paradigm 0. Results of the time-frequency and beamforming analysis for the contrast of occluded and visible cursor (occluded-vi p g Theta band power differences (occluded-visible) for significant electrodes in cluster-based permutation test on the left. The topography for the theta power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant negative power differences (visible > occluded) were found for the entire time interval (0–2 s). The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the theta frequency band are depicted in the clusters identified by the DBSCAN algorithm. As shown in Figure 12, the earliest positive clusters (Figure 12A) extended from 72 to 164 ms and over the central parietal electrodes. For the visible condition, sLORETA revealed significant activation in the precentral gyrus (BA6; MNI(x,y,z) = 50, 0, 30; p < 0.05) (Figure 13A left column). During occlusion, the most prominent activation was detected in the anterior cingulate (ACC, BA24; MNI(x,y,z) = 0, 30, 15; p < 0.05) (Figure 13A, right column). Contrasting visible-occluded in sLORETA revealed for this time range the most prominent activation in the cuneus (BA18; MNI(x,y,z) = 15, 85, 10; p < 0.05) (Figure 12A). The second positive cluster (Figure 12B) was found at 172–212 ms at the right hemisphere’s parietal and occipital electrodes. Here, the sLORETA revealed that for the visible condition, activation differences in the precentral gyrus (BA4; MNI(x,y,z) = 30, 30, 50; p < 0.05) and during occlusion in the cingulate gyrus were evident (BA24; MNI(x,y,z) = 15, 20, 40; p < 0.05) (see Figure 13). Contrasting both conditions, the sLORETA revealed the most prominent activation in the precentral gyrus (BA4; MNI(x,y,z) = 40, 20, 50; p <. 05) (see Figure 12B). Figure 11. Scree plots of t-value sums per cluster Figure (A) shows the absolute t-value sums of each significant positive (left) and negative cluster (right), respectively, of the condition, occluded-visible. Time domain analyses As for task one, we analyzed whether there is a significant difference between ERPs following target direc- tion changes. Therefore, we epoched the data 0 to 750 ms after target direction change. We contrasted trial segments during which the cursor was occluded with trial segments during which the cursor was visible. A cluster-based permutation test was performed over the whole epoch duration with a minimum cluster size of four electrodes. The test yielded N = 20 significant negative clusters and N = 14 significant positive clusters. Positive clusters indicate that the ERP resulting from the occluded condition was significantly more negative than the ERP of the visible condition in the respective time window and for the respective electrodes. To decide which clus- ters are relevant to analyze in more detail, we inspected a scree plot of each cluster’s sum of t-values (see Figure 11). The cluster sum of t-values is the respective summed t-values of all the t-tests for each combi- nation of spatial and temporal points that belong to a cluster. Using an adjusted elbow criterion, we decided to only further investigate the three largest clusters in each direction based on their respective t-value sums. Figure 12 shows the ERPs, the result of applying an sLORETA algorithm to the occluded-visible contrast and the results of an analysis that reveals the association between tracking performance and EEG data (see section Single-trial time domain analysis and inter-relation with behavioral data). Figure 13 reveals the activity sources in occluded and visible conditions contrasted against zero. 10 iScience 26, 106939, July 21, 2023 10 Figure 10. Results of the time-frequency and beamforming analysis for the contrast of occluded and visible cursor (occluded-visible) in the pursuit- occlusion paradigm Theta band power differences (occluded-visible) for significant electrodes in cluster-based permutation test on the left. The topography for the theta power difference is shown in the top right corner. Significant electrodes are highlighted with red crosses for negative and black crosses for positive differences. Significant negative power differences (visible > occluded) were found for the entire time interval (0–2 s). The dashed lines approximately enclose the significant time interval and frequency range. On the right, source activity differences for the theta frequency band are depicted in the clusters identified by the DBSCAN algorithm. ll OPEN ACCESS iScience Article iScience Article ll OPEN ACCESS Figure 10. gure 12. Relevant clusters in the occluded-visible contras g From Figure (A) to Figure (E), this figure visualizes the clusters detected by the cluster-based permutation test in the condition occluded-visible that are relevant for interpretation. In the left column are ERP plots contrasting the grand average ERPs after CSD transformation and weighting of all epochs. Time point 0 denotes the time point of a trajectory peak. The dashed line marks the time window of significant differences in the ERPs, as indicated by the cluster test. The topography next to the ERP plot marks the electrodes which were identified by the cluster test and whose signal is included in the ERP. The colors on the topography are the differences between the two conditions per electrode averaged across the whole time interval of the cluster. The central column shows the results of the sLORETA analysis of the time window of the cluster including all electrodes. The contrast between conditions occluded and visible is shown. Red voxels indicate that the activation in the occluded condition is significantly larger than in the visible condition; blue voxels indicate a significant effect in the opposite direction (note that the color scale range changes between Figures A–E). In the right column, the tracking performance vincentiles (x axis) are associated with the EEG data time bins (x axis). The colors of the cells indicate size and direction of the difference between conditions occluded- visible is for that vincentile-EEG bin combination, expressed in cohen’s d. Overall, the plots show no clear association between magnitude of condition differences and tracking performance. Cells with gray color denote t-tests between conditions that did not indicate significant differences. The third positive cluster (Figure 12C) lasted from 172 to 304 ms and included bilateral parietal and central electrodes with sources in the precuneus (visible; BA7; MNI(x,y,z) = 10, 60, 40; p < 0.05) and the cingu- late gyrus (occlusion; BA31; MNI(x,y,z) = 15, 25, 40; p < 0.05) (see Figure 13B). The contrast of both was in the medial frontal gyrus (BA6; MNI(x,y,z) = 15, 15, 55; n.s.) (Figure 12C). The earliest negative cluster (Figure 12D) started at 192 ms and ended at 240 ms and extended over frontal medial electrodes with sources in the parietal lobe (visible; BA7; MNI(x,y,z) = 50, 60, 55; p < 0.05) and precuneus (occlusion; BA7; MNI(x,y,z) = 15, 50, 60; p < 0.05) (Figure 13). gure 12. Relevant clusters in the occluded-visible contras The contrast for this time range was located in the precuneus (BA7; MNI(x,y,z) = 20, 70, 35; p < 0.05) (Figure 12D). Of note is that clusters C and D show very similar ERP waveforms, only in opposite orientations (D is nega- tive while C is positive). Also, the locations of the electrodes that form these clusters are essentially mirrored across the midline of the head, indicating that the same neuronal source might be producing both activation patterns. The final and second negative cluster (Figure 12E) was detected between 304 and 444 ms and included medial central, medial frontal, and medial parietal electrodes. Its sources were located in the anterior cingulate cortex (visible; BA32; MNI(x,y,z) = 0, 40, 10; p < 0.05) and inferior parietal lobule (occlusion; BA40; MNI(x,y,z) = 35, 35, 45; p < 0.05) (see Figure 13). sLORETA revealed the most prominent activation for the contrast of both conditions in the inferior parietal lobule (BA 39; MNI(x,y,z) = 40, –70, 40; n.s.) (Fig- ure 12E). Cluster E encompasses almost the same electrodes as cluster A and therefore results in very similar ERP waveforms, revealing significant condition differences in two discrete time windows. Investi- gating cluster E more closely, there was a positive peak in the ERP in the occluded condition, which showed almost the same amplitude as the visible condition but at an earlier latency. In an exploratory analysis, a paired two-tailed t-test comparing the occluded and visible ERP peak latencies showed that the peak in the occluded condition occurred significantly earlier (mean = 290 ms) than the peak in the visible condition (mean = 345 ms; t(35,99) = 3.69, d = 1.17). iScience 26, 106939, July 21, 2023 13 iScience Article iScience Article Time domain analyses Figure (B) shows the same for the clusters of the contrast constant – random (positive left, negative right). The dashed line indicates our adjusted elbow criterion for cluster selection (see STAR Methods section for details). Figure 11. Scree plots of t-value sums per cluster Figure 11. Scree plots of t value sums per cluster Figure (A) shows the absolute t-value sums of each significant positive (left) and negative cluster (right), respectively, of the condition, occluded-visible. Figure (B) shows the same for the clusters of the contrast constant – random (positive left, negative right). The dashed line indicates our adjusted elbow criterion for cluster selection (see STAR Methods section for details). g p p Figure (A) shows the absolute t-value sums of each significant positive (left) and negative cluster (right), respectively, of the condition, occluded-visible. Figure (B) shows the same for the clusters of the contrast constant – random (positive left, negative right). The dashed line indicates our adjusted elbow criterion for cluster selection (see STAR Methods section for details). iScience 26, 106939, July 21, 2023 11 iScience 26, 106939, July 21, 2023 11 iScience Article OPEN ACCESS Arti 12 iScience 26, 106939, July 21, 2023 12 ll OPEN ACCESS Time-frequency results Due to the observed behavioral adjustments during the first 500 ms of each trial, the time-frequency data analysis during pursuit tracking was constrained by the different time intervals for the random and constant trajectory segments (see Figure 4). Within the first 500 ms, theta, alpha, and beta band activity significantly differed between the constant trajectory at the middle of the trial and random trajectories at trial onset, and power in the examined frequency bands was significantly larger while tracking random trajectory compared to constant trajectory. For all examined frequency bands, activity modulations were sustained over the entire 500 ms period, and the beamforming analysis revealed activity modulations in superior parietal areas and the precuneus (BA7). These brain regions are involved in processes contributing to the selection of mo- tor responses.48–51 Superior and posterior parietal areas are known to integrate perception and action by binding sensory and motor information into a sensorimotor representation (program) necessary for goal- directed action.18 When faced with a random trajectory at trial start and when it is thus not possible to effi- ciently plan the specification of the sensorimotor program (incl. its execution) ahead of time, it is evident that activity in these regions has to be increased, compared to a situation where sensorimotor processes can be planned ahead of time (i.e., a constant trajectory during ongoing tracking). Crucially, three distinct frequency bands are involved in these mechanisms (i.e., theta, alpha, and beta band activity), each poten- tially playing specific roles in information processing as discussed in the following section. Out of these three frequency bands, the beta band activity is likely to play a unique role since beta band activity is not only associated with the superior parietal cortex and the precuneus (BA7) but also with superior frontal regions, including the supplementary motor area and the premotor cortex (BA4, BA6). Both the parietal regions and the observed modulations in superior frontal regions have been attributed to be central in the specification of complex sensorimotor programs and their monitoring.25,26 The obtained findings specify the associated oscillatory architecture of these processes and the functional relevance of these brain structures in continuous action monitoring. DISCUSSION The presented experiments provide detailed insights into the neurophysiological mechanisms underlying continuous action monitoring and fill a critical gap in knowledge of how the brain accomplishes action monitoring in ecologically more realistic situations that are not characterized by relatively short-lasting and regularly reinstating monitoring demands.1 Tracking performance results Sensorimotor demands on pursuit tracking increase when visual feedback on the cursor position is missing, which is indicated by the higher tracking error during cursor occlusion. However, participants reacted faster to target direction changes during cursor occlusion. This pattern of faster reaction times and lower tracking accuracy points to a potential speed-accuracy trade-off. Moreover, we found significant differences in the tracking behavior during the first 500 ms of the first random trajectory segment compared to the first 500 ms of the middle, i.e. constant trajectory segment. Adjustments of sensorimotor processes seemed to be particularly necessary during the first 500 ms of each trial. This finding led us to compare the first 500 ms of the random and constant trajectory separately from the rest of the trial. iScience Article g O g From Figure (A) to Figure (E), this figure visualizes significant sLORETA estimates of the conditions ‘‘occluded’’ and ‘‘visible’’ of the reported clusters A–E in g g From Figure (A) to Figure (E), this figure visualizes significant sLORETA estimates of the conditions ‘‘occluded’’ and ‘‘visible’’ of the reported clusters A–E in Figure 12 against zero. Figure 12B) were also evident in this time window. However, a scaling between the effect between the occluded and the visible condition (expressed using Cohen’s d) and the degree of behavioral tracking error (measures using epoch error or pursuit latency) was not evident. For cluster E (Figure 12E), the cluster- based permutation tests revealed differences between the visible and the occluded condition between 304 and 444 ms. Significant differences in the vincentiles were also evident during this time window. Since all other clusters did not reveal a consistent pattern of results between cluster-based permutation tests and vincentile analysis (also including the behavioral data), we consider the processes reflected in clusters B and E to be the most important. iScience 26, 106939, July 21, 2023 15 Single-trial time domain analysis and inter-relation with behavioral data As mentioned in the previous section, we examined whether the ERPs of the occluded and visible condi- tions in the significant clusters were related to the tracking performance. To this end, we used the ten pur- suit-latency and epoch-error vincentiles for the occluded and visible conditions. The results are shown in Figure 12 (the rightmost plots in each line). We grouped the ERPs of the epochs associated with the respec- tive vincentiles and averaged them across the electrodes of the clusters. We then split this average ERP into 50 ms time bins and averaged the ERP across the respective bins. The first bin started at 50 ms after a target direction change, and the last (10th bin) ended at 550 ms. This resulted in a 10x10 vincentile-bin matrix of combinations per cluster and error measure, each containing one average value per subject and condition. We then performed a t-test on each matrix cell and compared the occluded with the visible condition. Thus, we examined whether there were significant differences in the ERP related to the tracking task performance and established an association between tracking performance and neuronal processes. Figure 12 shows that results from the vincentile analysis and the cluster-based permutation ERP analyses only converge for clusters B and E. More precisely, only for these clusters, the vincentile analysis revealed effects in the same time windows that were also obtained for the cluster-based permutation tests. For cluster B, the clus- ter-based permutation tests revealed differences between the visible and the occluded condition between 172 and 212 ms, and significant differences in the vincentiles (see color shading in the rightmost plot in iScience 26, 106939, July 21, 2023 13 iScience Article ll OPEN ACCESS ll OPEN ACCESS iScienc Artic 14 iScience 26, 106939, July 21, 2023 ll OPEN ACCESS ll OPEN ACCESS iScience Article Time-frequency results This supporting role of alpha band activity is likely because alpha band activity likely reflects processes of gating and the controlled access of incoming information to a domain-general ‘‘knowledge system’’ (i.e., not restricted to perception, attention, working memory, and long-term memory) containing integrated infor- mation.27 Beta band activity may use the information gated through parietal alpha band activity to adapt the sensorimotor program during pursuit tracking. Crucially, this necessitates that incoming information is sampled and provided for the alpha band activity-controlled gating. This may be the role of the observed su- perior parietal theta band activity modulations. Aside from cognitive control and surprise signaling in medial/ superior frontal cortices,8 posterior theta band activity plays a role in attentional control16 and especially the parietal source of theta band activity fits conceptions of non-spatial attentional processes enabled by superior parietal and precuneus regions.18 Moreover, several lines of evidence suggest that information sampling fol- lows a theta rhythm.52,53 The theta band activity was stronger during the random trajectories at trial onset than constant trajectories in the ongoing trial. The finding substantiates the increased theta band activity-related information sampling during the initial period of pursuit tracking that in experiment 2, in which the visibility of the to-be-tracked trajectory was the central experimental manipulation, only theta band activity in the ventral stream was modulated in areas long-known to be central in the processing of visual motion informa- tion,54 as induced by visible tracking cursor movements. At this point, it should be critically noted that the observed beta band activity difference occurs at the boundary between the alpha and beta band. However, we assume that the observed beta activity does not represent an extended alpha band activity, since we find different neuroanatomical sources for both activity differences and our experimental manipulation is likely to induce beta band activity differences. Of note, after the initial 500 ms, only modulations in theta band activity were evident, suggesting that processes reflected by alpha and beta band activity in the initial period of pursuit tracking became less important. This can be attributed to sensorimotor learning and adaptation processes known to occur dur- ing pursuit tracking.5 This is not to say that processes reflected by alpha and beta band activity are no longer involved—they are just not differentially modulated between occasions imposing higher or lower demands on sensorimotor processes during pursuit tracking. Time-frequency results Crucially, however, the quality of theta band activity modulation changed considerably compared to the initial period of pursuit tracking (i.e., the first 500 ms). Opposed to superior parietal theta band activity in the initial period of tracking, theta band activity modulations were stronger during constant than random trajectories and medial superior frontal structures (BA6) (instead of superior parietal ones) were associated with the modulations. The source in the medial superior frontal gyrus reflects a pattern observed in commonly used experiments to examine action monitoring,8,9 that is where relatively short-lasting and regularly reinstating monitoring demands are evident.1 Based on data from such approaches, it has been coined that theta band activity likely reflects a ‘‘surprise signal’’ necessary to adapt actions.8 For the pursuit of trajectories, tracking performance varies,28 depending on acquired implicit knowledge and the predictability of the target tra- jectory.3 Therefore, the finding that theta band activity is higher during constant than random trajectories can be explained well. Only when it is possible to build an expectancy of how events unfold, it can emit a surprise signal. Such expectancies cannot reliably be built when the trajectory is random. 16 iScience 26, 106939, July 21, 2023 Time-frequency results As mentioned, beta band activity supports attention to upcoming motor tasks,21,22 premotor mechanisms guiding motor actions and likely the computation of the ‘‘status quo.’’24 According to the status quo interpre- tation, the continuous maintenance of a sensorimotor program depends on predicting what is likely to happen iScience 26, 106939, July 21, 2023 15 iScience Article ll OPEN ACCESS soon to adapt the sensorimotor program. These processes must be intensified when faced with situations where the sensorimotor program needs almost permanent adjustments (i.e., in random trajectories). The observed modulations in theta and alpha band activity possibly support these beta band activity-related pro- cesses. This supporting role of alpha band activity is likely because alpha band activity likely reflects processes of gating and the controlled access of incoming information to a domain-general ‘‘knowledge system’’ (i.e., not restricted to perception, attention, working memory, and long-term memory) containing integrated infor- mation.27 Beta band activity may use the information gated through parietal alpha band activity to adapt the sensorimotor program during pursuit tracking. Crucially, this necessitates that incoming information is sampled and provided for the alpha band activity-controlled gating. This may be the role of the observed su- perior parietal theta band activity modulations. Aside from cognitive control and surprise signaling in medial/ superior frontal cortices,8 posterior theta band activity plays a role in attentional control16 and especially the parietal source of theta band activity fits conceptions of non-spatial attentional processes enabled by superior parietal and precuneus regions.18 Moreover, several lines of evidence suggest that information sampling fol- lows a theta rhythm.52,53 The theta band activity was stronger during the random trajectories at trial onset than constant trajectories in the ongoing trial. The finding substantiates the increased theta band activity-related information sampling during the initial period of pursuit tracking that in experiment 2, in which the visibility of the to-be-tracked trajectory was the central experimental manipulation, only theta band activity in the ventral stream was modulated in areas long-known to be central in the processing of visual motion informa- tion,54 as induced by visible tracking cursor movements. soon to adapt the sensorimotor program. These processes must be intensified when faced with situations where the sensorimotor program needs almost permanent adjustments (i.e., in random trajectories). The observed modulations in theta and alpha band activity possibly support these beta band activity-related pro- cesses. iScience Article iScience Article sensorimotor integration processes become complicated through the lack of visual information otherwise useful to inform motor program formation. Resource allocation processes may be predominantly needed to enable motor aspects of pursuit tracking in the absence of sensory information but are not decisive for the performance during tracking. This interpretation is corroborated by two other findings of this study: First, the analysis of tracking performance vincentiles and ERP data revealed that there was neither an in- ter-relation between the P2 amplitudes (locked at the trajectory peak) and the magnitude of the tracking error nor a relationship between the P2 amplitude and the latency of the pursuit peaks following the trajectory turning points. Second, there were no modulations of the P2 amplitude (and other ERP compo- nents) depending on whether there was a constant or a random trajectory to be tracked—in spite of mod- ulations of behavioral tracking performance. Possibly, resource allocation processes are not volatile enough to be modulated during tracking and only provide a defined amount of processing capacity, which is then used by other processes that we find reflected in the theta, alpha, and beta band activity. Besides cluster B, effects were also evident in cluster E (see Figure 12E) between 304 and 444 ms, suggesting that the P3 ERP component is modulated as well. The P3 in response control paradigms likely reflects the map- ping of the stimulus input onto the appropriate motor output.40,55 In line with that, P3 amplitudes are usu- ally found to be smaller when demands on the mapping processes are increased12,40 and the herein found source of the difference in the inferior parietal lobule (BA39) substantiates that the P3 is modulated. The source localization analyses revealed that the cortical source of the P3 cluster E in the occluded trials was associated with activity modulations in the inferior parietal lobule (BA40). In the visible trials, the source was located in the ACC (BA32). The occlusion effect (i.e., the difference between occluded and non- occluded trials) was also reflected in theta band activity modulations during pursuit tracking (see discussion above). Previous findings show that parietal areas contribute to the selection of motor responses,18,48 and evidence that superior and posterior parietal areas serve to integrate perception and action by binding sensory and motor information into a sensorimotor representation (program) necessary for goal-directed action18 are also well in line with the current findings. Limitations of the study While the current study provides details and in-depth insights into the role of specific neural oscillatory ac- tivity, it does not reveal insights into the causal relevance of the oscillatory activity and the associated func- tional neuroanatomical structures of the processes focused in this study. This should be focus of future studies using non-invasive brain stimulation approaches. iScience Article In conclusion, our study provides new insights into the neural processes underlying continuous action monitoring and the regular reinstatement of monitoring functions, which are critical for goal-directed behavior. The superior parietal and frontal cortices play a crucial role in this process, with beta band activity maintaining the sensorimotor program, and theta and alpha bands supporting attentional sampling and information gating, respectively. We also found that modulating sensorimotor demands through cursor vis- ibility led to changes in the theta band and P2- and P3-like components. Our findings suggest that resource allocation mechanisms in prefrontal areas and stimulus-response mapping processes in the parietal cortex are crucial for adapting sensorimotor processes. This study fills a significant gap in our understanding of action monitoring by shedding light on the neural processes involved in continuous action monitoring, and thereby offers an alternative to the predominant reliance on experimental procedures imposing short-lasting and regularly reinstating monitoring demands. iScience 26, 106939, July 21, 2023 17 Time domain results Especially after changes in the direction of the target trajectory, adaptive processes must be triggered to reduce tracking error. Avoiding the reduced temporal resolution of the time-frequency analyses, the ERP analysis provides insights into processes that happen on these occasions. The results were only reliable for task 2. Here, we consider the processes reflected in clusters B and E (Figures 12B and 12E) to be most important because only these clusters revealed a consistent pattern of results between cluster-based per- mutation tests and vincentile analysis (also including the behavioral data). For none of the clusters did the effects at the neurophysiological level scale with the degree of behavioral performance. Cluster B (Figure 12B) resembles a P2/N2-like ERP complex that was larger for occluded and visible trials. Previous studies suggest that the P2 likely reflects resource allocation processes.36–38 Thus, resource allo- cation processes are intensified in the occluded condition and seem to take place in premotor cortices (BA4) as suggested by the sLORETA analysis. This is reasonable since, in the occluded condition, 16 iScience 26, 106939, July 21, 2023 ll OPEN ACCESS ll OPEN ACCESS d QUANTIFICATION AND STATISTICAL ANALYSIS B Calculation of tracking performance B Behavioral data significance tests B Association of tracking performance and ERPs B Time-domain source localization analysis AUTHOR CONTRIBUTIONS Conceptualization, A.B., S.W., C.B., and S.H.; Methodology, A.B., S.W., N.A., C.B., and S.H.; Software, N.A., S.W., and A.B.; Validation, S.W. and A.B.; Formal Analysis, S.W. and A.B.; Investigation, A.B.; Resources, C.B. and A.B.; Data Curation, A.B. and S.W.; Writing – Original Draft, A.B., S.W., and C.B.; Writing – Re- view & Editing, N.A., M.R., S.H., C.B., A.B., and S.W.; Visualization, A.B. and S.W., Supervision, S.H. and C.B., Project Administration, S.H. and C.B.; Funding Acquisition, M.R., S.H., and C.B. ACKNOWLEDGMENTS A grant from the Deutsche Forschungsgemeinschaft to HO 5054/8-1, BE4045/44-1, FOR 2790 and RA 940/ 25-1 supported this work. SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.106939 DECLARATION OF INTERESTS The authors declare no competing interests. Received: March 1, 2023 Revised: April 28, 2023 Accepted: May 18, 2023 Published: May 22, 2023 Received: March 1, 2023 Revised: April 28, 2023 Accepted: May 18, 2023 Published: May 22, 2023 REFERENCES task. Front. Psychol. 8, 2241. https://doi.org/ 10.3389/fpsyg.2017.02241. 11. Buzsa´ki, G., and Draguhn, A. (2004). Neuronal oscillations in cortical networks. Science 304, 1926–1929. https://doi.org/10.1126/science. 1099745. task. Front. Psychol. 8, 2241. https://doi.org/ 10.3389/fpsyg.2017.02241. 1. Frings, C., Hommel, B., Koch, I., Rothermund, K., Dignath, D., Giesen, C., Kiesel, A., Kunde, W., Mayr, S., Moeller, B., et al. (2020). Binding and retrieval in action control (BRAC). Trends Cognit. Sci. 24, 375–387. https://doi.org/10. 1016/j.tics.2020.02.004. 6. Beste, C., Mu¨ nchau, A., and Frings, C. (2023). Towards a systematization of brain oscillatory activity in actions. Commun. Biol. 6, 137. https://doi.org/10.1038/s42003-023-04531-9. 12. Opitz, A., Beste, C., and Stock, A.-K. (2020). Using temporal EEG signal decomposition to identify specific neurophysiological correlates of distractor-response bindings proposed by the theory of event coding. Neuroimage 209, 116524. https://doi.org/10. 1016/j.neuroimage.2020.116524. 2. Keye, D., Wilhelm, O., Oberauer, K., and Stu¨ rmer, B. (2013). Individual differences in response conflict adaptations. Front. Psychol. 4, 947. https://doi.org/10.3389/fpsyg.2013. 00947. 7. Ward, L.M. (2003). Synchronous neural oscillations and cognitive processes. Trends Cognit. Sci. 7, 553–559. https://doi.org/10. 1016/j.tics.2003.10.012. 13. Prochnow, A., Eggert, E., Mu¨ nchau, A., Mu¨ ckschel, M., and Beste, C. (2022). Alpha and theta bands dynamics serve distinct functions during perception–action integration in response inhibition. J. Cognit. Neurosci. 34, 1053–1069. https://doi.org/10. 1162/jocn_a_01844. 3. Broeker, L., Ewolds, H., de Oliveira, R.F., Ku¨ nzell, S., and Raab, M. (2021). The impact of predictability on dual-task performance and implications for resource-sharing accounts. Cogn. Res. Princ. Implic. 6, 1. https://doi.org/ 10.1186/s41235-020-00267-w. 8. Cavanagh, J.F., and Frank, M.J. (2014). Frontal theta as a mechanism for cognitive control. Trends Cognit. Sci. 18, 414–421. https://doi.org/10.1016/j.tics.2014.04.012. 9. Cohen, M.X. (2014). A neural microcircuit for cognitive conflict detection and signaling. Trends Neurosci. 37, 480–490. https://doi. org/10.1016/j.tins.2014.06.004. 4. Broeker, L., Haeger, M., Bock, O., Kretschmann, B., Ewolds, H., Ku¨ nzell, S., and Raab, M. (2020). How visual information influences dual-task driving and tracking. Exp. Brain Res. 238, 675–687. https://doi.org/ 10.1007/s00221-020-05744-8. 14. Takacs, A., Zink, N., Wolff, N., Mu¨ nchau, A., Mu¨ ckschel, M., and Beste, C. (2020). Connecting EEG signal decomposition and response selection processes using the theory of event coding framework. Hum. Brain Mapp. 41, 2862–2877. https://doi.org/ 10.1002/hbm.24983. 10. Pscherer, C., Mu¨ ckschel, M., Summerer, L., Bluschke, A., and Beste, C. (2019). On the relevance of EEG resting theta activity for the neurophysiological dynamics underlying motor inhibitory control. Hum. Brain Mapp. 40, 4253–4265. https://doi.org/10.1002/hbm. 24699. 5. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Lead contact B Materials availability B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Participants d METHOD DETAILS B Task B EEG recording and preprocessing iScience 26, 106939, July 21, 2023 17 iScience Article ll OPEN ACCESS ll OPEN ACCESS d QUANTIFICATION AND STATISTICAL ANALYSIS B Calculation of tracking performance B Behavioral data significance tests B Time-frequency decomposition B Beamforming analysis B Time-domain analysis B Association of tracking performance and ERPs B Time domain source localization analysis iScience Article 40. Verleger, R., Jaskowski, P., and Wascher, E. (2005). Evidence for an integrative role of P3b in linking reaction to perception. J. Psychophysiol. 19, 165–181. https://doi. org/10.1027/0269-8803.19.3.165. integration. Neurosci. Biobehav. Rev. 46, 3–29. https://doi.org/10.1016/j.neubiorev. 2014.05.008. 29. Beste, C., Saft, C., Konrad, C., Andrich, J., Habbel, A., Schepers, I., Jansen, A., Pfleiderer, B., and Falkenstein, M. (2008). Levels of error processing in Huntington’s disease: a combined study using event- related potentials and voxel-based morphometry. Hum. Brain Mapp. 29, 121–130. https://doi.org/10.1002/hbm. 20374. integration. Neurosci. Biobehav. Rev. 46, 3–29. https://doi.org/10.1016/j.neubiorev. 2014.05.008. 16. Spyropoulos, G., Bosman, C.A., and Fries, P. (2018). A theta rhythm in macaque visual cortex and its attentional modulation. Proc. Natl. Acad. Sci. USA 115, E5614–E5623. https://doi.org/10.1073/pnas.1719433115. 41. Debener, S., Ullsperger, M., Siegel, M., Fiehler, K., von Cramon, D.Y., and Engel, A.K. (2005). Trial-by-Trial coupling of concurrent electroencephalogram and functional magnetic resonance imaging identifies the dynamics of performance monitoring. J. Neurosci. 25, 11730–11737. https://doi. org/10.1523/JNEUROSCI.3286-05.2005. 30. Beste, C., Saft, C., Yordanova, J., Andrich, J., Gold, R., Falkenstein, M., and Kolev, V. (2007). Functional compensation or pathology in cortico-subcortical interactions in preclinical Huntington’s disease? Neuropsychologia 45, 2922–2930. https://doi.org/10.1016/j. neuropsychologia.2007.06.004. 17. Wendiggensen, P., Adelho¨ fer, N., Jamous, R., Mu¨ ckschel, M., Takacs, A., Frings, C., Mu¨ nchau, A., and Beste, C. (2022). Processing of embedded response plans is modulated by an interplay of fronto-parietal theta and beta activity. J. Neurophysiol. https://doi. org/10.1152/jn.00537.2021. 42. Hoffmann, S., and Beste, C. (2015). A perspective on neural and cognitive mechanisms of error commission. Front. Behav. Neurosci. 9, 50. https://doi.org/10. 3389/fnbeh.2015.00050. 31. Falkenstein, M., Hohnsbein, J., Hoormann, J., and Blanke, L. (1991). Effects of crossmodal divided attention on late ERP components. II. Error processing in choice reaction tasks. Electroencephalogr. Clin. Neurophysiol. 78, 447–455. 18. Gottlieb, J., and Snyder, L.H. (2010). Spatial and non-spatial functions of the parietal cortex. Curr. Opin. Neurobiol. 20, 731–740. https://doi.org/10.1016/j.conb.2010.09.015. 43. Hoffmann, S., Borges, U., Bro¨ ker, L., Laborde, S., Liepelt, R., Lobinger, B.H., Lo¨ ffler, J., Musculus, L., and Raab, M. (2018). The psychophysiology of action: a multidisciplinary endeavor for integrating action and cognition. Front. Psychol. 9, 1423. https://doi.org/10.3389/fpsyg.2018.01423. 19. Herrmann, C.S., Stru¨ ber, D., Helfrich, R.F., and Engel, A.K. (2016). EEG oscillations: from correlation to causality. Int. J. Psychophysiol. 103, 12–21. https://doi.org/10.1016/j. ijpsycho.2015.02.003. 32. Gehring, W.J., Goss, B., Coles, M.G.H., Meyer, D.E., and Donchin, E. (1993). A neural system for error detection and compensation. Psychol. Sci. 4, 385–390. https://doi.org/10.1111/j.1467-9280.1993. tb00586.x. 20. iScience Article Fries, P. (2015). Rhythms for cognition: communication through coherence. Neuron 88, 220–235. https://doi.org/10.1016/j. neuron.2015.09.034. 44. Richman, J.S., and Moorman, J.R. (2000). Physiological time-series analysis using approximate entropy and sample entropy. Am. J. Physiol. Heart Circ. Physiol. 278, H2039–H2049. https://doi.org/10.1152/ ajpheart.2000.278.6.H2039. 33. Ullsperger, M., Fischer, A.G., Nigbur, R., and Endrass, T. (2014). Neural mechanisms and temporal dynamics of performance monitoring. Trends Cognit. Sci. 18, 259–267. https://doi.org/10.1016/j.tics.2014.02.009. 21. Fetz, E.E. (2013). Volitional control of cortical oscillations and synchrony. Neuron 77, 216–218. https://doi.org/10.1016/j.neuron. 2013.01.003. 45. Gross, J., Kujala, J., Hamalainen, M., Timmermann, L., Schnitzler, A., and Salmelin, R. (2001). Dynamic imaging of coherent sources: studying neural interactions in the human brain. Proc. Natl. Acad. Sci. USA 98, 694–699. https://doi.org/10.1073/pnas.98. 2.694. 34. Ridderinkhof, K.R., Ullsperger, M., Crone, E.A., and Nieuwenhuis, S. (2004). The role of the medial frontal cortex in cognitive control. Science 306, 443–447. https://doi.org/10. 1126/science.1100301. 22. Seki, K., and Fetz, E.E. (2012). Gating of sensory input at spinal and cortical levels during preparation and execution of voluntary movement. J. Neurosci. 32, 890–902. https://doi.org/10.1523/ JNEUROSCI.4958-11.2012. 35. Holroyd, C.B., Nieuwenhuis, S., Yeung, N., Nystrom, L., Mars, R.B., Coles, M.G.H., and Cohen, J.D. (2004). Dorsal anterior cingulate cortex shows fMRI response to internal and external error signals. Nat. Neurosci. 7, 497–498. https://doi.org/10.1038/nn1238. 46. Adelho¨ fer, N., Schreiter, M.L., and Beste, C. (2020). Cardiac cycle gated cognitive- emotional control in superior frontal cortices. Neuroimage 222, 117275. https://doi.org/10. 1016/j.neuroimage.2020.117275. 23. MacKay, W.A. (1997). Synchronized neuronal oscillations and their role in motor processes. Trends Cognit. Sci. 1, 176–183. https://doi. org/10.1016/S1364-6613(97)01059-0. 47. Adelho¨ fer, N., and Beste, C. (2020). Pre-trial theta band activity in the ventromedial prefrontal cortex correlates with inhibition- related theta band activity in the right inferior frontal cortex. Neuroimage 219, 117052. https://doi.org/10.1016/j.neuroimage.2020. 117052. 36. Ghin, F., Stock, A.-K., and Beste, C. (2022). The importance of resource allocation for the interplay between automatic and cognitive control in response inhibition – an EEG source localization study. Cortex 155, 202–217. https://doi.org/10.1016/j.cortex. 2022.07.004. 24. Engel, A.K., and Fries, P. (2010). Beta-band oscillations — signalling the status quo? Curr. Opin. Neurobiol. 20, 156–165. https://doi. org/10.1016/j.conb.2010.02.015. 25. Ptak, R. (2012). The frontoparietal attention network of the human brain: action, saliency, and a priority map of the environment. Neuroscientist 18, 502–515. https://doi.org/ 10.1177/1073858411409051. 48. Bernier, P.-M., Cieslak, M., and Grafton, S.T. (2012). Effector selection precedes reach planning in the dorsal parietofrontal cortex. J. Neurophysiol. 108, 57–68. https://doi.org/ 10.1152/jn.00011.2012. 37. 39. Mu¨ ckschel, M., Stock, A.-K., and Beste, C. (2014). Psychophysiological mechanisms of interindividual differences in goal activation modes during action cascading. Cereb. Cortex N. Y. N 24, 2120–2129. https://doi. org/10.1093/cercor/bht066. 50. Jaffard, M., Longcamp, M., Velay, J.-L., Anton, J.-L., Roth, M., Nazarian, B., and Boulinguez, P. (2008). Proactive inhibitory control of movement assessed by event- related fMRI. Neuroimage 42, 1196–1206. https://doi.org/10.1016/j.neuroimage.2008. 05.041. REFERENCES Ewolds, H.E., Bro¨ ker, L., de Oliveira, R.F., Raab, M., and Ku¨ nzell, S. (2017). Implicit and explicit knowledge both improve dual task performance in a continuous pursuit tracking 15. Ridderinkhof, K.R. (2014). Neurocognitive mechanisms of perception–action coordination: a review and theoretical iScience 26, 106939, July 21, 2023 18 ll OPEN ACCESS ll OPEN ACCESS ll OPEN ACCESS 56. Delorme, A., and Makeig, S. (2004). EEGLAB: an open source toolbox for analysis of single- trial EEG dynamics including independent component analysis. J. Neurosci. Methods 134, 9–21. https://doi.org/10.1016/j. jneumeth.2003.10.009. 62. Kayser, J., and Tenke, C.E. (2006). Principal components analysis of Laplacian waveforms as a generic method for identifying ERP generator patterns: I. Evaluation with auditory oddball tasks. Clin. Neurophysiol. 117, 348–368. https://doi.org/10.1016/j. clinph.2005.08.034. 51. Sulpizio, V., Lucci, G., Berchicci, M., Galati, G., Pitzalis, S., and Di Russo, F. (2017). Hemispheric asymmetries in the transition from action preparation to execution. Neuroimage 148, 390–402. https://doi.org/ 10.1016/j.neuroimage.2017.01.009. 57. Ratcliff, R. (1979). Group reaction time distributions and an analysis of distribution statistics. Psychol. Bull. 86, 446–461. https:// doi.org/10.1037/0033-2909.86.3.446. 52. Fiebelkorn, I.C., Saalmann, Y.B., and Kastner, S. (2013). Rhythmic sampling within and between objects despite sustained attention at a cued location. Curr. Biol. 23, 2553–2558. https://doi.org/10.1016/j.cub.2013.10.063. 63. Pascual-Marqui, R.D. (2002). Standardized low-resolution brain electromagnetic tomography (sLORETA): technical details. Methods Find. Exp. Clin. Pharmacol. 24 (Suppl D), 5–12. 58. Vincent, S.B. (1912). The function of the vibrissae in the behavior of the white rat. Anim. Behav. Monogr. 1, 84. 53. Helfrich, R.F., Fiebelkorn, I.C., Szczepanski, S.M., Lin, J.J., Parvizi, J., Knight, R.T., and Kastner, S. (2018). Neural mechanisms of sustained attention are rhythmic. Neuron 99, 854–865. https://doi.org/10.1016/j.neuron. 2018.07.032. 64. Ocklenburg, S., Friedrich, P., Fraenz, C., Schlu¨ ter, C., Beste, C., Gu¨ ntu¨ rku¨ n, O., and Genc¸, E. (2018). Neurite architecture of the planum temporale predicts neurophysiological processing of auditory speech. Sci. Adv. 4, eaar6830. https://doi. org/10.1126/sciadv.aar6830. 59. Oostenveld, R., Fries, P., Maris, E., and Schoffelen, J.-M. (2011). FieldTrip: open source software for advanced analysis of MEG, EEG, and invasive electrophysiological data. Comput. Intell. Neurosci. 2011, 156869. https://doi.org/10.1155/2011/156869. 54. Goodale, M.A., Kro´ liczak, G., and Westwood, D.A. (2005). Dual routes to action: contributions of the dorsal and ventral streams to adaptive behavior. Prog. Brain Res. 149, 269–283. https://doi.org/10.1016/ S0079-6123(05)49019-6. 65. Sekihara, K., Sahani, M., and Nagarajan, S.S. (2005). Localization bias and spatial resolution of adaptive and non-adaptive spatial filters for MEG source reconstruction. Neuroimage 25, 1056–1067. https://doi.org/10.1016/j. neuroimage.2004.11.051. 60. Mu¨ ckschel, M., Stock, A.-K., Dippel, G., Chmielewski, W., and Beste, C. (2016). Interacting sources of interference during sensorimotor integration processes. Neuroimage 125, 342–349. https://doi.org/ 10.1016/j.neuroimage.2015.09.075. 55. Verleger, R., Metzner, M.F., Ouyang, G., Smigasiewicz, K., and Zhou, C. (2014). Testing the stimulus-to-response bridging function of the oddball-P3 by delayed response signals and residue iteration decomposition (RIDE). 66. Fuchs, M., Kastner, J., Wagner, M., Hawes, S., and Ebersole, J.S. (2002). A standardized boundary element method volume conductor model. Clin. Neurophysiol. 113, 702–712. 20 iScience 26, 106939, July 21, 2023 iScience Article Stock, A.-K., Friedrich, J., and Beste, C. (2016). Subliminally and consciously induced cognitive conflicts interact at several processing levels. Cortex 85, 75–89. https:// doi.org/10.1016/j.cortex.2016.09.027. 26. Ptak, R., Schnider, A., and Fellrath, J. (2017). The dorsal frontoparietal network: a core 26. Ptak, R., Schnider, A., and Fellrath, J. (2017). The dorsal frontoparietal network: a core system for emulated action. Trends Cognit. Sci. 21, 589–599. https://doi.org/10.1016/j. tics.2017.05.002. 49. Cisek, P., and Kalaska, J.F. (2005). Neural correlates of reaching decisions in dorsal premotor cortex: specification of multiple direction choices and final selection of action. Neuron 45, 801–814. https://doi.org/10.1016/ j.neuron.2005.01.027. 38. Sugimoto, F., and Katayama, J. (2013). Somatosensory P2 reflects resource allocation in a game task: assessment with an irrelevant probe technique using electrical probe stimuli to shoulders. Int. J. Psychophysiol. 87, 200–204. https://doi.org/ 10.1016/j.ijpsycho.2013.01.007. system for emulated action. Trends Cognit. Sci. 21, 589–599. https://doi.org/10.1016/j. tics.2017.05.002. 27. Klimesch, W. (2012). Alpha-band oscillations, attention, and controlled access to stored information. Trends Cognit. Sci. 16, 606–617. https://doi.org/10.1016/j.tics.2012.10.007. 50. Jaffard, M., Longcamp, M., Velay, J.-L., Anton, J.-L., Roth, M., Nazarian, B., and Boulinguez, P. (2008). Proactive inhibitory control of movement assessed by event- related fMRI. Neuroimage 42, 1196–1206. https://doi.org/10.1016/j.neuroimage.2008. 05.041. 39. Mu¨ ckschel, M., Stock, A.-K., and Beste, C. (2014). Psychophysiological mechanisms of interindividual differences in goal activation modes during action cascading. Cereb. Cortex N. Y. N 24, 2120–2129. https://doi. org/10.1093/cercor/bht066. 28. Wulf, G., and Schmidt, R.A. (1997). Variability of practice and implicit motor learning. J. Exp. Psychol. Learn. Mem. Cogn. 23, 987– 1006. https://doi.org/10.1037/0278-7393.23. 4.987. iScience 26, 106939, July 21, 2023 19 62. Kayser, J., and Tenke, C.E. (2006). Principal components analysis of Laplacian waveforms as a generic method for identifying ERP generator patterns: I. Evaluation with auditory oddball tasks. Clin. Neurophysiol. 117, 348–368. https://doi.org/10.1016/j. clinph.2005.08.034. iScience Article ll OPEN ACCESS Materials availability There are no newly generated materials. Lead contact Further information and request for resources should be directed and will be fulfilled by the lead contact, Christian Beste (Christian.Beste@uniklinikum-dresden.de). ll OPEN ACCESS Neuroimage 100, 271–280. https://doi.org/ 10.1016/j.neuroimage.2014.06.036. 66. Fuchs, M., Kastner, J., Wagner, M., Hawes, S., and Ebersole, J.S. (2002). A standardized boundary element method volume conductor model. Clin. Neurophysiol. 113, 702–712. 61. Ester, M., Kriegel, H.-P., and Xu, X. (1996). A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise6. 20 iScience 26, 106939, July 21, 2023 20 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Software and algorithms MATLAB 2021b https://de.mathworks.com/products/matlab. html RRID:SCR_001622 BrainVision Recorder https://www.brainproducts.com/productdetails.php?id=21 RRID:SCR_016331 PsychoPy http://www.psychopy.org RRID:SCR_006571 EEGLAB http://sccn.ucsd.edu/eeglab/index.html RRID:SCR_007292 Fieldtrip https://www.fieldtriptoolbox.org RRID:SCR_004849 Deposited data Raw data behavior This paper https://osf.io/pv49m/ Raw data EEG This paper https://osf.io/pv49m/ ll OPEN ACCESS iScience Article ll OPEN ACCESS REAGENT or RESOURCE SOURCE IDENTIFIER Software and algorithms MATLAB 2021b https://de.mathworks.com/products/matlab. html RRID:SCR_001622 BrainVision Recorder https://www.brainproducts.com/productdetails.php?id=21 RRID:SCR_016331 PsychoPy http://www.psychopy.org RRID:SCR_006571 EEGLAB http://sccn.ucsd.edu/eeglab/index.html RRID:SCR_007292 Fieldtrip https://www.fieldtriptoolbox.org RRID:SCR_004849 Deposited data Raw data behavior This paper https://osf.io/pv49m/ Raw data EEG This paper https://osf.io/pv49m/ Data and code availability De-identified raw data for the behavioral analysis as well as raw EEG data have been deposited at Open Science Forum: https://osf.io/pv49m/. EEG data at different processing steps are available on reasonable request. Participants The initial study sample consisted of N = 34 healthy adults. Three participants were excluded due to incom- plete data recording. One participant was excluded since the behavioral data indicated that this partici- pant was not following the instructions, resulting in a final sample of N = 30 subjects aged from 20 to 30 years (on average 25.42 G 3.04 years). This sample comprised N = 11 male and N = 19 female participants who were right-handed. All participants had normal or corrected-to-normal vision and reported no neuro- logical or psychiatric illness history in the past six months. The study was conducted in accordance with the Declaration of Helsinki and approved by the IRB of the TU Dresden (EK 390082019). The participants were compensated for their participation and provided written informed consent. EEG recording and preprocessing For the EEG recording, we used EasyCaps with 60 passive Ag-AgCl electrodes and BrainAmp amplifiers from Brain Products Inc. (Brain Products GmbH, Gilching, Germany). The electrode placement was an equi- distant setup based on the 10%-system with a reference electrode at position Fpz. The EEG recording was carried out with a sampling rate of 500 Hz. Later, offline down-sampling to 250 Hz was applied. The impedances of all electrodes did not exceed 10 kU. EEG data were pre-processed using EEGlab56 on MATLAB 2021b (IBM Mathworks Corp.). Firstly, the data were high-pass filtered at 1 Hz using a Hamming windowed FIR filter. A line-noise filter at 50 Hz was applied using the pop_cleanline plugin for EEGlab. Then, flat channels and those with minimum channel correlation were removed. A low-pass filter (Hamming windowed FIR filter) for 40 Hz was applied. Subsequently, the removed channels were interpo- lated using a spherical method. This was done to ensure all participants have the same number of channels to facilitate further analyses. Next, the data were re-referenced to the common average. Before conducting the ICA, the appended data was detrended, and artifacts in this dataset were removed using a joint prob- ability method (as implemented in pop_jointprob.56 An independent component analysis (using an extended infomax algorithm) was conducted for the appended trials of both the pursuit-tracking and the pursuit-occlusion task. To identify all components not labeled brain activity, we applied the ‘‘IClabel algorithm.’’ All components not labeled brain activity with at least 50% probability were removed automat- ically. To further check for artifactual components, we inspected the topography of the first five remaining components visually. Detected artifactual components were excluded and all remaining components were applied to the data of each task respectively. Subsequently, the EEG data were parallelized with the behav- ioral data of each paradigm, thereby excluding all EEG data for trials that were excluded based on the behavioral data. Task In this study, the participants executed two pursuit-tracking tasks, with the order of the tasks randomized across participants. In both tasks, participants were asked to track a moving red square (the target) using an equally sized crosshair (the cursor) controlled by a joystick (Thrustmaster T16000M) during each trial. The objective was to maintain the cursor on the target as accurately as possible (see Table S1 for precise instruc- tions). Participants could only control the vertical movement of the cursor, with the joystick’s deflection pro- portionally affecting the cursor’s position on the screen: maximal joystick deflection moved the cursor to the edge of the screen, while no deflection centered the cursor. The target, a 32 x 32-pixel red square (0.79 visual angle), moved across the screen along a three-segment trajectory, while the cursor and target maintained the same horizontal position. Each segment was created by concatenating three sine and cosine terms28: iScience 26, 106939, July 21, 2023 21 iScience Article ll OPEN ACCESS f ðxÞ = X 3 i = 1 ai$sinði $ xÞ + bi$cosði $ xÞ The first and last segments had randomly generated coefficients between 40 and 40 pixels which ensured that the trajectory did not reach the top or bottom of the screen. The central segment remained constant across all trials, with fixed coefficients: a1 = 37, a2 = 3, a3 = 26, b1 = 23, b2 = 15, and b3 = 9. Participants were not informed of the constant central segment. Smooth transitions between the segments were achieved by placing a 30-pixel gap between them and using cubic splines for interpolation. The target began at the center of the screen and moved either left or right at one of three possible velocities, which remained constant within a trial. The direction and velocities of the target’s movement were counterbal- anced, with each combination repeated twelve times, resulting in a total of 72 trials. Each trial was separated by a white fixation square of the same size as the target, which appeared for a random duration averaging 1.5 s. After every ten trials, participants received a 30-s break. The average trial lasted 12 s, mak- ing the total task duration approximately 20 min. In task 1, participants completed the basic pursuit-tracking paradigm as described above. Task In task 2, the paradigm was modified by occluding the cursor for 2 s every 4 s during each trial, starting with a visible cursor. This alteration aimed to investigate the effect of intermittent cursor visibility on participants’ tracking performance. The task was programmed using PsychoPy2. iScience 26, 106939, July 21, 2023 iScience Article For two samples to be regarded as ‘‘the same,’’ they need to be less than the similarity criterion ‘‘r’’ apart, which is traditionally set to 0.2 times the standard deviation. Notably, sample entropy does not depend on the length of the investigated time series. For this analysis, we z-transformed each segment to be investigated for each trial individually. This made the trial segments by different participants comparable and also allowed us to know the standard deviation, which allowed us to use the same similarity criterion across all trials, avoiding introducing bias in the analysis based on a var- iable similarity criterion. Subsequently, we compared the sample entropy values of the first 500 ms to the sample entropy values of the following 1500 ms. Since the sample entropy values of the first 500 ms are not normally distributed (see Figure 2), we used a Wilcoxon rank-sum test to compare the two conditions. The significant difference between the conditions can be easily explained by investigating images of single trial pursuits plotted against trajectory courses. The tracking error in this initial interval, in contrast to the interval after the initial 500 ms, mainly depends on the target’s movements. It is either increasing (by moving away from the cursor) or decreasing (by crossing the cursor) the tracking error. We analyzed the respective EEG data from the first 500 ms separately since this time interval likely reflects sensorimotor calibration pro- cesses needed to engage in continuous action monitoring required during pursuit-tracking. Behavioral data significance tests To test whether there are differences in tracking performance between conditions, we ordered the behav- ioral data of each epoch into ten vincentiles per subject and condition, which means that we first ordered the error measures (epoch error and pursuit latency) by size and then split the ordered data into ten equal- populated bins. The vincentile procedure57,58 is preferred above analyzing quantiles because it assures the independence of sample size within the analysis bins. We then averaged the data inside each bin so that every subject was represented by one value per condition vincentile. For each vincentile, we performed a paired two-tailed t-test with an alpha level of 0.001 by taking 5,000 bootstrap samples from the subject sample data, comparing the conditions occluded and visible as well as constant and random. Finally, we performed an FDR correction with q = 0.05 on the resulting ten p values per comparison to reduce the risk of false positives. iScience Article Since the sample entropy values of the first 500 ms are not normally distributed (see Figure 2), we used a Wilcoxon rank-sum test to compare the two conditions. The significant difference between the conditions can be easily explained by investigating images of single trial pursuits plotted against trajectory courses. The tracking error in this initial interval, in contrast to the interval after the initial 500 ms, mainly depends on the target’s movements. It is either increasing (by moving away from the cursor) or decreasing (by crossing the cursor) the tracking error. We analyzed the respective EEG data from the first 500 ms separately since this time interval likely reflects sensorimotor calibration pro- cesses needed to engage in continuous action monitoring required during pursuit-tracking. as the threshold above which a change in joystick direction implies an intentional direction change. To calculate the pursuit latency, we matched the trajectory peaks with the pursuit peaks that followed them. We considered a pursuit peak valid if it occurred between 80 ms and 300 ms after a trajectory peak (since the trajectory peaks are, on average, 300 ms apart) and was in the same direction as the trajec- tory peak (both local minima resp. maxima). The time between the trajectory peak and a valid pursuit peak reflects the pursuit latency. Of 50,488 epochs, 29,593 have a valid pursuit latency (58,61%). Subsequently, we used the error measures to exclude trials during which participants did not follow the experiment in- structions adequately: the tracking performance in all the trials with a maximum tracking error more than three standard deviations above the mean maximum tracking error per trial was inspected visually. The trial was excluded if the pursuit did not follow the target trajectory. Finally, upon inspection of the dis- tribution of the frequency of tracking errors over the trial, we noticed that the distribution peaked more during the first 500 ms as compared to the rest of the trial (see Figure 2). Subsequently, we quantified this difference in the randomness of the error distribution by calculating sample entropy44 using the MATLAB function. Sample entropy quantifies how complex a time series is. It is the negative natural log- arithm of the probability that a pattern of samples of length m (standard: m = 2) found later in the time se- ries will also be the same for the next element. iScience Article iScience Article as the threshold above which a change in joystick direction implies an intentional direction change. To calculate the pursuit latency, we matched the trajectory peaks with the pursuit peaks that followed them. We considered a pursuit peak valid if it occurred between 80 ms and 300 ms after a trajectory peak (since the trajectory peaks are, on average, 300 ms apart) and was in the same direction as the trajec- tory peak (both local minima resp. maxima). The time between the trajectory peak and a valid pursuit peak reflects the pursuit latency. Of 50,488 epochs, 29,593 have a valid pursuit latency (58,61%). Subsequently, we used the error measures to exclude trials during which participants did not follow the experiment in- structions adequately: the tracking performance in all the trials with a maximum tracking error more than three standard deviations above the mean maximum tracking error per trial was inspected visually. The trial was excluded if the pursuit did not follow the target trajectory. Finally, upon inspection of the dis- tribution of the frequency of tracking errors over the trial, we noticed that the distribution peaked more during the first 500 ms as compared to the rest of the trial (see Figure 2). Subsequently, we quantified this difference in the randomness of the error distribution by calculating sample entropy44 using the MATLAB function. Sample entropy quantifies how complex a time series is. It is the negative natural log- arithm of the probability that a pattern of samples of length m (standard: m = 2) found later in the time se- ries will also be the same for the next element. For two samples to be regarded as ‘‘the same,’’ they need to be less than the similarity criterion ‘‘r’’ apart, which is traditionally set to 0.2 times the standard deviation. Notably, sample entropy does not depend on the length of the investigated time series. For this analysis, we z-transformed each segment to be investigated for each trial individually. This made the trial segments by different participants comparable and also allowed us to know the standard deviation, which allowed us to use the same similarity criterion across all trials, avoiding introducing bias in the analysis based on a var- iable similarity criterion. Subsequently, we compared the sample entropy values of the first 500 ms to the sample entropy values of the following 1500 ms. QUANTIFICATION AND STATISTICAL ANALYSIS Calculation of tracking performance Since the cursor and target coordinates were recorded at 60 Hz and the EEG data was processed with a sampling rate of 250 Hz, we up-sampled the coordinates to 250 Hz using cubic spline interpolation. Then, two error measures describing behavioral performance were calculated: tracking error and pursuit latency. The tracking error quantifies how accurately participants were tracking throughout a trial/epoch. It is defined as the root mean squared error (RMSE) between the target y axis position and the cursor y axis position for each sample. The pursuit latency is the time that passes between a target direction change and the following direction change of the cursor. To obtain this, peaks in the target trajectory and pursuit tracking coordinates were identified first. For the target trajectory, all peaks that exceeded a prominence (how much the peak’s height stands out compared to surrounding peaks) of 1% of the screen size were defined as peaks that the participants likely perceived and could respond to. For the pursuit, which natu- rally contains more jitter due to motor noise, a prominence threshold of 5% of the screen size was defined iScience 26, 106939, July 21, 2023 22 ll OPEN ACCESS ll OPEN ACCESS iScience 26, 106939, July 21, 2023 Beamforming analysis Using dynamic imaging of coherent sources (DICS) beamformer,45 we reconstructed source-level activity for the frequency bands and contrasts that showed significant power differences in the cluster-based per- mutation tests. For that, we extracted the cross-spectral density matrix for each condition using a Fast Four- ier Transformation (FFT) on the alpha (8–12 Hz), beta (13–30 Hz) and theta (4–8 Hz) frequency bands. Based on the appended data for each contrast, we calculated a common spatial filter with the DICS beamformer using a 5 mm grid based on the template-based forward model implemented in the Fieldtrip toolbox. The common spatial filter was applied to the respective conditions in a second step. The contrast was calcu- lated as the source power difference divided by the sum of both conditions, thereby normalizing the dif- ference using the following ratio: r = powercondition1  powercondition2 powercondition1+powercondition2 r = powercondition1+powercondition2 Based on the assumption that there is an equal noise distribution in both conditions, this ratio mitigates potential bias due to noise and diminishes the effect of outliers.60 We then used the ‘‘Density-Based Spatial Clustering of Applications with Noise’’ (DBSCAN) algorithm61 to identify clusters of alpha-, beta- and theta- band for the significant contrasts, using the implementation in MATLAB. The clusters were identified with the respective ratio of both conditions for each contrast. This approach limits the source activity to demar- cated functional neuroanatomical regions.17,46 Depending on the direction of the difference, the most pos- itive or negative 1% of the power distribution of all voxels within labeled neuroanatomical regions on the Automatic Anatomical Labeling (AAL) atlas was used for the DBSCAN. As the epsilon parameter, denoting the neighborhood search radius, we used 1.5 times the edge length of each voxel. The clusters identified by the algorithm were inspected visually and defined based on the number of voxels and anatomical labels. Time-frequency decomposition To achieve comparable trial segments of equal length for random and constant trajectory, we also segmented the constant trajectory interval in two parts, with the first interval from 0 to 500 ms of the constant trajectory and the second interval from 0 to 2500 ms of the constant trajectory. Here, the start of the segment was also included for the second interval since the behavioral difference was only observed at the trial start and the constant trajectory segment always ap- peared in the middle of a trial. For a detailed overview of the segmentation, see Figures 3 and 7). To investigate significant differences in the power of the three frequency bands, we conducted cluster- based permutation tests with 1000 Monte-Carlo iterations and a cluster alpha level of 0.001. For task 1, we compared the constant and random trajectory segments for the first and second time interval (i.e. first 500 ms and 500 ms onwards) respectively. For task 2, we compared the occluded and non-occluded segments. Time-frequency decomposition For the time-frequency analysis, a baseline correction was conducted for all trials, using the interval before the trial start (750 ms to trial start), i.e., the time interval in which only the fixation cross was presented, as a baseline. Then, the data were epoched considering the entire trial length (i.e., 13 s from trial start trigger) and bad trials were rejected using a joint probability method (as implemented in pop_jointprob.56 The data were then segmented according to critical experimental manipulations: for task 1, we divided the data into random and constant trajectory segments, each 3 s long. For task 2, we segmented the data into occluded and non-occluded trajectory parts, each 2 s long. After conducting the preprocessing in EEGlab, the data was imported to the Fieldtrip Toolbox.59 A time-frequency analysis was performed using Morlet wavelets with a width of 5 Gaussians for the theta (4–7 Hz), alpha (8–12 Hz) and beta (13–30 Hz) frequency band. Since the behavioral tracking data indicated different tracking behavior in the first 500 ms of each trial compared to the proceeding trial (see Results section), we separated the trajectory segments for the trial start and the proceeding trial, respectively. Since every trial starts with the random trajectory segment, we iScience 26, 106939, July 21, 2023 23 iScience Article ll OPEN ACCESS separated the random segment into a trial start interval (0–500 ms, ’first interval’) and an interval for the pro- ceeding random trajectory (500–3000 ms, ’second interval’). To achieve comparable trial segments of equal length for random and constant trajectory, we also segmented the constant trajectory interval in two parts, with the first interval from 0 to 500 ms of the constant trajectory and the second interval from 0 to 2500 ms of the constant trajectory. Here, the start of the segment was also included for the second interval since the behavioral difference was only observed at the trial start and the constant trajectory segment always ap- peared in the middle of a trial. For a detailed overview of the segmentation, see Figures 3 and 7). separated the random segment into a trial start interval (0–500 ms, ’first interval’) and an interval for the pro- ceeding random trajectory (500–3000 ms, ’second interval’). Association of tracking performance and ERPs We further analyzed whether the ERP time course after a target direction change was associated with the behavioral tracking performance. To this end, we used the behavioral tracking performance vincentiles and grouped the EEG data in 10 time bins, starting from 50 ms after a target direction change until 550 ms. For the EEG bins, we only used data from electrodes in significant clusters and from epochs with a valid pursuit latency. We chose a duration of 50 ms per EEG data time bin as a reasonable compromise between signal to noise ratio and time resolution, with 12–13 samples contributing to the average of a bin. We ended up with a matrix of EEG data for each subject and condition where each row represented a vincentile and each column a 50 ms time bin. Which results in a total of 100 vincentile-time cells. We calculated the average ERP amplitude for each vincentile-time cell. Thus, we obtained a representation of the association between the ERP time course following a target direction change and the different degrees of tracking performance. Akin to the test of the behavioral data, we subsequently performed a paired two-tailed t-test with 5000 bootstrap samples for each vincentile-time cell, which resulted in 100 tests per condition using an alpha level of 0.001. To reduce the risk of false positive findings, we performed an FDR correction with a q-value of 0.05. To quantify the degree of difference between the vincentile-time bins, we calculated cohen’s d for each cell. iScience Article Since the ‘‘elbow’’ of the scree plot of cluster t-value sums was at the second or the third cluster in all scree plots, to establish con- sistency across contrasts, we chose to analyze the three largest clusters per condition further. Of these clus- ters, we investigated topo plots of the averaged ERP across the cluster time intervals alongside ERP plots of the averaged signal of the electrodes on one cluster. This led us to exclude all constant-random clusters from further analyses and one of the occluded-visible clusters for the following reasons, respectively. For one, we excluded all clusters that showed significant condition differences across a very short time (less than five samples), likely not reflecting meaningful physiological activity. Second, we rejected clusters that appeared more than 300 ms after the target direction change. The reason is that on average, after 300 ms, the following target direction change already took place, which means that activity after 300 ms is to more than 50% a mixture of activity related to the target direction change at time point 0 as well as activity related to the following target direction change. iScience Article iScience Article activation in less than four neighboring electrodes were ignored. The algorithm detected many clusters, which we further narrowed down by applying a multi-step selection process. First, we investigated a scree plot of the cluster t-value sums for both contrasts (constant - random and occluded - visible) and directions (positive and negative) (see Figure 11). The cluster t-value sums are the summed-up t-values of all t-tests that are organized in a cluster, so one test per electrode-sample combination. It is a measure of cluster magnitude in the dimension’s spatial extent, temporal duration and effect size. Since the ‘‘elbow’’ of the scree plot of cluster t-value sums was at the second or the third cluster in all scree plots, to establish con- sistency across contrasts, we chose to analyze the three largest clusters per condition further. Of these clus- ters, we investigated topo plots of the averaged ERP across the cluster time intervals alongside ERP plots of the averaged signal of the electrodes on one cluster. This led us to exclude all constant-random clusters from further analyses and one of the occluded-visible clusters for the following reasons, respectively. For one, we excluded all clusters that showed significant condition differences across a very short time (less than five samples), likely not reflecting meaningful physiological activity. Second, we rejected clusters that appeared more than 300 ms after the target direction change. The reason is that on average, after 300 ms, the following target direction change already took place, which means that activity after 300 ms is to more than 50% a mixture of activity related to the target direction change at time point 0 as well as activity related to the following target direction change. activation in less than four neighboring electrodes were ignored. The algorithm detected many clusters, which we further narrowed down by applying a multi-step selection process. First, we investigated a scree plot of the cluster t-value sums for both contrasts (constant - random and occluded - visible) and directions (positive and negative) (see Figure 11). The cluster t-value sums are the summed-up t-values of all t-tests that are organized in a cluster, so one test per electrode-sample combination. It is a measure of cluster magnitude in the dimension’s spatial extent, temporal duration and effect size. iScience 26, 106939, July 21, 2023 25 Time-domain analysis For the time domain analysis, the EEG data were epoched around the trajectory peaks, with epochs ex- tending from 500 ms to 750 ms. A baseline correction was applied to the first 500 ms of each epoch. As with the time-frequency data, bad trials were rejected using a combination of the joint probability method and the same criteria used for the time-frequency analysis. The average tracking error and the pursuit latency were calculated for each epoch. To improve the topographical localization of scalp surface potentials, we used a spherical spline algorithm to compute the current source density (CSD), as imple- mented in the CSD Toolbox.62 We used the default parameters of this toolbox. Epoching was done for each experimental version of the tracking task separately. For task 1, epochs were created with time point zero denoting the trajectory peak in the first random segment of each trial or the constant segment in each trial. For task 2, epochs around peaks in the trajectory were created and sorted depending on whether the cursor was occluded or non-occluded at the time of the peak. The data were weighted depending on the number of epochs per participant to account for an unequal number of epochs between participants. The weight per subject was determined by calculating the median of the number of trials per subject for each condition and dividing it by the number of trials that subject had. This weight was then multiplied with the EEG data of the respective condition and subject. The condition-specific grand average of all epochs was calculated. Cluster-based permutation tests across the epoch interval from 0 to 750 ms with 3,000 Monte Carlo iterations were performed to identify significant differences between conditions. A conservative alpha and cluster alpha level of 0.001, respectively, was chosen. Also, all clusters that found significant 24 ll OPEN ACCESS ll OPEN ACCESS Time-domain source localization analysis The source localization of the time-domain data was carried out using the sLORETA software (standardized low-resolution tomography (sLORETA).63 sLORETA has been validated in numerous studies using brain stimulation-EEG and fMRI/EEG or structural imaging and yields reliable source estimations.64,65 sLORETA, as used herein, uses the single-subject averaged data (i.e., averaged across single trials in a specific con- dition) as input. The method is based on a realistic head model66 and partitions the intracerebral volume into 6239 voxels with 5 mm spatial resolution. Based on the MNI152 template, the standardized current density at each voxel is calculated to localize the sources of scalp electrical activities. In the current study, sLORETA was used to compare conditions in significant cluster time windows revealed by the cluster-based permutation tests in the time-domain analysis to locate brain regions activated between the contrasted conditions. For all comparisons, sLORETA’s built-in randomization tests, based on nonparametric statisti- cal mapping (SnPM), were utilized to correct for multiple comparisons. For this, 5000 permutations were used. The significance level was set to 0.05. iScience 26, 106939, July 21, 2023 25
https://openalex.org/W4386724484
https://academicareview.com/index.php/jh/article/download/64/47
Indonesian
null
ANALISIS PROSES PEMBELAJARAN JARAK JAUH MENGGUNAKAN MEDIA BERBASIS TEKNOLOGI INFORMASI PADA MASA PANDEMI COVID-19
Jurnal Hurriah
2,022
cc-by
4,046
E-ISSN : 2774-8472 P-ISSN: 2774-8480 E-ISSN : 2774-8472 P-ISSN: 2774-8480 Jurnal Hurriah:Jurnal Evaluasi Pendidikandan Penelitian Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Hurriah:Jurnal Evaluasi Pendidikandan Penelitian Vol. 3, No. 1, Mar 2022, hal. 48-58 Abstrak Penelitian ini bertujuan untuk mengetahui dan mengukur jumlah penggunaan media pembelajaran yang digunakan dalam proses pembelajaran jarak jauh secara daring serta untuk mengetahui implementasi pembelajaran daring menurut pelajar dan mahasiswa. Penelitian ini merupakan penelitian kuantitatif dengan populasi mahasiswa dari akun Bot Twitter @collegemenfess dengan sampel 16 pelajar dan 47 mahasiswa melalui angket terbuka pada Google Form. Hasil dari penelitian ini: (1) Para pelajar dan mahasiswa lebih dominan menggunakan Android dengan jumlah 55 (90,2%) pengguna sebagai teknologi informasi pada pembelajaran jarak jauh secara daring. (2) Media yang paling sering digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring yaitu adalah aplikasi Whatsapp dengan 50 (82%), Zoom dengan 47 (77%) dan Google Classroom 39 (63,9%). (2) Pelajar dan mahasiswa yang tidak nyaman, tidak memahami materi dan kesulitan melakukan proses pembelajaran secara daring. Kemudian, pelajar dan mahasiswa lainnya berpendapat bahwa pembelajaran daring tidak sama dengan pembelajaran tatap muka di sekolah atau di kampus dan sangat tidak efektif untuk diterapkan di tahun-tahun berikutnya. Dan yang terakhir, pelajar dan mahasiswa banyak yang berpendapat bahwa guru/dosen mereka dalam pembelajaran secara daring ini tidak hanya memberikan tugas saja tetapi juga menjelaskan materi pembelajaran. Kata Kunci: Media pembelajaran; Masalah Pembelajaran; Pengguna Twitter; Covid-19 ANALISIS PROSES PEMBELAJARAN JARAK JAUH MENGGUNAKAN MEDIA BERBASIS TEKNOLOGI INFORMASI PADA MASA PANDEMI COVID-19 Fanesa Maura Aprillia Program Studi Tadris Bahasa Inggris, Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi Corresponding Author: fanesamaura@gmail.com Fanesa Maura Aprillia Program Studi Tadris Bahasa Inggris, Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi Corresponding Author: fanesamaura@gmail.com Abstract This study aims to determine and measure the amount of use of learning media used in the distance learning process boldly and to determine the implementation of bold learning according to students and college students. This research is a quantitative study with a student population from the Twitter Bot account @collegemenfess with a sample of 16 students and 47 college students through an open questionnaire on Google Form. The results of this study: (1) Students and college students are more dominant in using Android with a number of 55 (90.2%) users as information technology in distance learning boldly. (2) The media most often used by students and college students in the distance learning process is the Whatsapp application with 50 (82%), Zoom with 47 (77%) and Google Classroom 39 (63.9%). (2) Students and college students who are uncomfortable, do not understand the material and have difficulty carrying out the learning process boldly. Then, students and college students argue that learning is not the same as face-to-face learning at school or on campus and is very ineffective to be applied in the following years. And lastly, many students and college students think that their teachers/lecturers in this bold learning not only give assignments but also explain learning materials. Keywords: Instructional Media; Learning Problems; Twitter users; Covid-19 A. Latar Belakang 1, Mar 2022, hal. 48-58 50 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 A. Latar Belakang A. Latar Belakang Pendidikan adalah bagian yang paling penting untuk memajukan suatu bangsa karena suatu bangsa tersebut akan mengalami kemajuan dan perkembangan yang bagus khususnya dalam bidang ilmu pengetahuan yang telah diberikan maka akan menjadikan warga Negara bisa berkembang kearah 48 Analisis Proses Pembelajaran ... Fanesa Maura Aprillia 49 kemajuan. Namun, pada masa pandemi Covid-19 ini para pelajar dan mahasiswa diharuskan untuk melakukan proses pembelajaran jarak jauh secara daring. Menurut KH. Lalu Gede Muhammad Zainuddin Atsani 2020, kita seolah dipaksa bertransformasi untuk beradaptasi secara tima-tiba untuk melakukan proses pembelajaran jarak jauh secara daring dari rumah. Ini bukanlah hal yang mudah karena belum adanya persiapan sepenuhnya bagi masyarakat khususnya para pelajar dan mahasiswa. Dengan adanya Pandemi Covid-19 membuat perubahan pada semua bidang tidak terkecuali dalam dunia pendidikan. Perubahan tersebut telah membuat perubahan pada bidang pendidikan dengan melakukan proses pembelajaran jarak jauh secara daring dengan menggunakan teknologi informasi dan juga media pembelajaran. Walaupun demikian sampai saat ini pembelajaran secara daring tetap dilakukan mengingat peningkatan Virus Corona yang semakin meningkat. Adapun beberapa teknologi yang digunakan dalam proses pembelajaran secara daring yaitu menggunakan Komputer, Laptop, Tablet, Smartphone berbasis android, Iphone dan sebagai. Hal ini telah dimanfaatkan oleh pelajar dan mahasiswa serta pendidik dalam melakukan proses pembelajaran jarak jauh yang efektif secara daring. Namun, sejauh ini beberapa orangtua dari pelajar dan mahasiswa mengeluhkan proses pembelajaran yang dinilai kurang efektif dari pandangan mereka. B. Rumusan masalah 1. Bagaimana teknologi informasi dan media pembelajaran yang digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring? 2. Bagaimana implementasi pembelajaran daring menurut pelajar dan mahasiswa? C. Tujuan Penulisan 1. Untuk mengetahui dan mengukur media pembelajaran yang digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring 2. Untuk mengetahui implementasi pembelajaran daring menurut pelajar dan mahasiswa D. Manfaat Penulisan Penelitian dari Artikel Ilmiah ini diharapkan dapat memberikan manfaat sebagai berikut: 1. Manfaat Teoritis 1) Untuk meningkatkan mutu pendidikan serta memanfaatkan teknologi informasi secara maksimal. 2) Untuk meningkatkan wawasan keilmuan dan sumber bacaan tentang implementasi media pembelajaran daring. 2. Manfaat Praktis 1. Penelitian ini diharapkan untuk menumbuhkan minat belajar siswa dan mendorong siswa agar lebih mandiri dalam belajar dirumah. 2. Penelitian ini diharapkan untuk meningkatkan kreativitas guru dalam mengajar serta memanfaatkan teknologi informasi sebaik mungkin. 3. Penelitian ini diharapkan bermanfaat untuk kedepan nya dalam memanfaatkan teknologi informasi sebagai alat pembelajaran untuk meningkatkan mutu pendidikan. Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 A. Landasan Teori Webex Meeting m. Moodle n. Quizizz o. E-learning Kampus Berdasarkan pemaparan diatas, pendidik harus cerdas dalam memilih media pembelajaran yang harus digunakan dalam proses pembelajaran jarak jauh secara daring pada masa Pandemi Covid- 19 supaya tidak ketinggalan materi. Maka dari itu, pendidik seharusnya menguasai banyak media pembelajaran (Nasution, 2008) A. Landasan Teori A. Landasan Teori Pendidikan merupakan salah satu wadah untuk membentuk karakter anak bangsa (Ulyan Nasri, 2018). Kerena itu, pendidik harus menguasai berbagai media pembelajaran dan dituntut untuk menyesuaikan dengan keadaankarena kondisi selalu berubah-berubah mengingat Pandemi Covid-19 yang melanda dunia saat ini. Media merupakan hal yang terpenting saat ini yang menentukan berhasil atau tidaknya proses pembelajaran jarak jauh secara daring. Media adalah suatu hal yang dapat dimanfaatkan untuk mengirim pesan dari pengirim ke penerima pesan sehingga dapat merangsang perasaan, perhatian, pikiran dan minat pelajar dan mahasiswa sehingga terbentuklah proses belajar (Ahmad Arsyad, 2005). Dabbagh dan Ritland berpendapat bahwa pembelajaran daring merupakan sistem belajar yang terbuka dan tersebar yang memproses pembelajaran dan pengetahuan melalui aksi dan interaksi melalui perangkat Teknologi Informasi dan Jaringan Internet (Dabbagh, 2009; Mayer, 2009). Dari uraian tersebut dapat disimpulkan bahwa proses pembelajaran daring adalah suatu proses pembelajaran guru dan siswa yang memanfaatkan Teknologi Informasi sebagai perangkat dengan menghubungkannya dengan jaringan internet dan juga menggunakan beberapa media pembelajaran. Media pembelajaran jarak jauh secara daring adalah sebagai media yang dioperasikan oleh pengguna yang disebut user sehingga dapat mengendalikan dan mengakses kebutuhan pembelajaran (DeVito. Joseph A., 2011). Adapun keuntungan yang didapat adalah bersifat mandiri dan interaktif, meningkatkan ingatan, memberikan pengalaman belajar (Melalui Teks, audio,video dan animasi) yang digunakan untuk menyampaikan informasi dan juga memberikan kemudahan kepada penggunanya dalam menyampaikan, memperbaharui isi, mengunduh, mengirim komentar pada forum diskusi, memakai ruang chat, hingga berbagi link videoconference untuk berkomunikasi langsung (Dryden, 2001). Dabbagh dan Ritland juga bependapat ada tiga bagian yang ada pada pembelajaran jarak jauh secara daring yaitu : (1) Model pembelajaran, (2) Strategi instruksional dan pembelajaran, (3) Media pembelajaran daring (Dabbagh, 2009). Dengan adanya Pandemi Covid-19 yang terjadi saat ini telah membuat dunia pendidikan juga merasakan dampaknya yang diharuskan melakukan proses pembelajaran jarak jauh secara daring karena pemerintah melarang adanya perkumpulan yang dapat menyebabkan terjadinya penularan virus. Maka dari itu, ada beberapa media pembelajaran daring yang bisa digunakan oleh pelajar dan mahasiswa serta pendidik dalam melakukan proses pembelajaran jarak jauh. Beberapa diantaranya yaitu : a. Whatsapp Group b. Email c. Telegram d. Instagram e. Youtube a. Whatsapp Group d. Instagram e. Youtube 51 51 Analisis Proses Pembelajaran ... Fanesa Maura Aprillia f. Zoom g. Google Meeting h. Google Classroom i. Edmodo j. Ms. Teams k. Panopto l. Webex Meeting m. Moodle n. Quizizz o E-learning Kampus f. Zoom g. Google Meeting h. Google Classroom i. Edmodo j. Ms. Teams k. Panopto l. B. Kajian Literatur B. Kajian Literatur Dalam penulisan penelitian ini, peneliti mengukur informasi dari penelitian sebelumnya sebagai bahan perbandingan, baik mengenai kekuatan maupun kelemahan yang ada. Selain itu, peneliti juga menggali informasi dari buku-buku dan skripsi guna memperoleh informasi yang sudah ada sebelumnya tentang teori yang berkaitan dengan judul yang digunakan untuk memperoleh landasan teori ilmiah. 1. Jurnal Agus Ramdani, A. Wahab Jufri, Jamaluddin 2020 dengan judul “Pengembangan Media Pembelajaran Berbasis Android pada Masa Pandemi Covid-19 untuk Meningkatkan Literasi Sains Peserta Didik” Program Studi Magister Pendidikan IPA Penelitian ini merupakan penelitian pengembangan (Research & Development) yang menggunakan prosedur model Four D . Instrumen dari penelitian ini adalah angket kevalidan silabus, kevalidan RPP, dan kevalidan instrumen literasi sains. Hasil penelitian ini menunjukan bahwa validitas 84% dengan kriteria sangat valid. Hasil studi kelayakan media memperoleh nilai rata-rata sebesar 88%, silabus memperoleh nilai rata-rata sebesar 83%, RPP memperoleh nilai rata-rata sebesar 82%, dan instrumen literasi sains memperoleh skor rata-rata 83%. Kesimpulan dari penelitian ini adalah produk media berbasis android pada materi IPA layak dapat diimplementasikan dalam pembelajaran khususnya pada saat pandemi covid-19 (Agus Ramdani, A. Wahab Jufri, 2020) 2. Jurnal Baroroh Indiani dengan judul “Mengoptimalkan Proses Pembelajaran Dengan Media Daring Pada Masa Pandemi COVID-19” SMKN 1 Wonogiri Kabupaten Wonogiri, Indonesia. ISSN: 2721-5407 (Online) Volume 1 Nomor 3 Tahun 2020. 2. Jurnal Baroroh Indiani dengan judul “Mengoptimalkan Proses Pembelajaran Dengan Media Daring Pada Masa Pandemi COVID-19” SMKN 1 Wonogiri Kabupaten Wonogiri, Indonesia. ISSN: 2721-5407 (Online) Volume 1 Nomor 3 Tahun 2020. 52 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 52 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Penelitian ini menggunakan metode penelitian kualitatif deskriftif. Hasil dari penelitian ini adalah gambaran dari kegiatan dari guru BK yang melayani pelajar dengan beberapa layanan BK melalui proses pembelajaran secara daring dengan media pembelajaran daring dan kemudian kegiatan dari peserta didik yaitu mengunduh materi yang diberikan melalui media pembelajaran daring dan melaksanakan dan mengumpulkan serta mengupload tugas melalui media pembelajaran daring yang selanjutnya akan dievaluasi oleh Guru BK. Kesimpulan dari penelitian ini adalah Banyak media daring yang bisa digunakan saat ini baik melalui whatsaap group, goggle classroom, kelas maya, email, telegram, google form, zoom dll. Media yang baik belum tentu menghasilkan output yang maksimal. B. Kajian Literatur Faktor kesiapan pendidik dalam menggunakan media dan pemilihan media daring yang tepat adalah faktor yang menentukan proses pembelajaran berjalan optimal. Semua media daring memiliki kelebihan dan kekurangan dalam penggunaannya, maka pemilihan media yang sesuai dengan peruntukannya, menarik perhatian peserta didik, bahkan kolaborasi penggunaan media daring menjadi sangat penting bagi proses pembelajaran agar berjalan optimal (Indiani, 2020) METODE METODE Jenis penelitian ini adalah penelitian kuantitatif. Penelitian ini dilaksanakan secara daring melalui media sosial Twitter yang dapat diakses oleh pelajar dan mahasiswa seluruh Indonesia pada tanggal 17 Agustus 2021 sampai dengan 24 Agustus 2021. Sumber data diperoleh dari hasil pengisian Angket pada Google Form yang dapat diakses melalui https://forms.gle/hBvA5sNcQaSg3HCdA yang disebar ke akun Twitter melalui Akun Bot : @collegemenfess. Dalam penelitian ini peneliti mendapatkan sampel sebanyak 16 pelajar dan 47 mahasiswa. Adapun Prosedur pengumpulan data yang digunakan yaitu : a) Observasi Dengan melakukan observasi, peneliti dapat mendokumentasikan dan merefleksikan secara sistematis terhadap kegiatan dan interaksi subjek peneliti. b) Survey Survey yang dilakukan oleh peneliti menggunakan angket yang ada pada media Google Form yang dapat diakses melalui https://forms.gle/hBvA5sNcQaSg3HCdA Survey yang dilakukan oleh peneliti menggunakan angket yang ada pada media Google Form yang dapat diakses melalui https://forms.gle/hBvA5sNcQaSg3HCdA c) Dokumentasi Berdasarkan data yang diperoleh dari analisis dokumen dapat digunakan sebagai data pendukung dan data pelengkap yang diperoleh melalui obeservasi dan survey. Dokumen adalah data dari peristiwa yang suddah berlalu. Dokumen bisa berbentuk tulisan, gambar atau sebagainya. c) Dokumentasi Berdasarkan data yang diperoleh dari analisis dokumen dapat digunakan sebagai data pendukung dan data pelengkap yang diperoleh melalui obeservasi dan survey. Dokumen adalah data dari peristiwa yang suddah berlalu. Dokumen bisa berbentuk tulisan, gambar atau sebagainya. Analisis data adalah proses mencari dan menyusun secara sistematis data yang diperoleh dari hasil survey melalui Google Form dan dokumentasi dengan cara mengelompokkan data kedalam kategori, menjabarkan ke dalam unit-unit, menyusun pola, melakukan sintesa, dan membuat kesimpulan yang mudah dipahami oleh diri sendiri maupun orang lain. Dalam penelitian kuantitatif Analisis Proses Pembelajaran ... Fanesa Maura Aprillia Analisis Proses Pembelajaran ... Fanesa Maura Aprillia 53 Fanesa Maura Aprillia ini, peneliti menggunakan teknik analisis data deskriptif dalam menganalisis data penelitiannya yang diperoleh dari proses mencari dan menyusun secara sistematis data yang diperoleh dari hasil pengumpulan data dengan menggunakan survey yang dibuat dari Google Form yang di sebarkan melalui akun Bot Twitter @collegemenfess, yaitu : 1) Pengumpulan data ) g p Data pada pertemuan pertama sampai terakhir telah dicatat dan disusun dan dikelompokkan secara intensif kemudian diberi kode agar mudah dalam analisis data. Data pada pertemuan pertama sampai terakhir telah dicatat dan disusun dan dikelompokkan secara intensif kemudian diberi kode agar mudah dalam analisis data. 2) Redukasi data Meredukasi artinya merangkum dan memilih hal-hal pokok, memfokuskan pada hal-hal yang penting, dicari tema dan polanya. 3) Penyajian data Dalam penelitian Kuantitatif ini, penyajian data dilakukan dalam bentuk uraian singkat bagan, hubungan antar aktegore, flowchart dan sebagainya. Selain teks naratif, penyajian data dapat berupa grafik, matrik, network dan chart. 3) Penyajian data Dalam penelitian Kuantitatif ini, penyajian data dilakukan dalam bentuk uraian singkat bagan, hubungan antar aktegore, flowchart dan sebagainya. Selain teks naratif, penyajian data dapat berupa grafik, matrik, network dan chart. 4) Kesimpulan dan verifikasi Kesimpulan dapat berupa temuan baru yang sebelumnya belum pernah ada. Temuan dapat berupa deskripsi atau gambaran suatu obyek yang sebelumnya masih remang-remang sehingga setelah diteliti menjadi jelas, dapat berupa hubungan kausal atau interaktif, hipotesis atau teori. 4) Kesimpulan dan verifikasi Kesimpulan dapat berupa temuan baru yang sebelumnya belum pernah ada. Temuan dapat berupa deskripsi atau gambaran suatu obyek yang sebelumnya masih remang-remang sehingga setelah diteliti menjadi jelas, dapat berupa hubungan kausal atau interaktif, hipotesis atau teori. HASIL Berdasarkan hasil penelitian yang dilakukan peneliti pada Akun Bot Twitter @collegemenfess mengenai Media Pembelajaran Jarak Jauh Secara Daring adalah sebagai berikut : Berdasarkan hasil penelitian yang dilakukan peneliti pada Akun Bot Twitter @collegemenfess mengenai Media Pembelajaran Jarak Jauh Secara Daring adalah sebagai berikut : a) Media pembelajaran yang digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring a) Media pembelajaran yang digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring Pada masa Pandemi Covid-19, para pelajar dan mahasiswa diwajibkan untuk belajar dari rumah secara daring dengan beberapa media pembelajaran daring. Adapun beberapa teknologi yang lebih sering digunakan pelajar dan mahasiswa adalah : Tabel 1. Teknologi Digital NO TEK. INFORMASI JUMLAH/Org JUMLAH/% 1 Komputer 7 11,5 % 2 Tablet 7 11,5 % 3 Laptop 44 72,1 % 4 Android 55 90,2 % 5 Iphone 3 4,9 % 6 Hp 1 1,6 % NO TEK. INFORMASI JUMLAH/Org JUMLAH/% 54 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 54 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Berdasarkan hal tersebut, para pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut lebih dominan menggunakan Android dengan jumlah 55 pengguna atau 90,2 % sebagai teknologi informasi pada pembelajaran jarak jauh secara daring. Kemudian, para pendidik harus menggunakan media pembelajaran yang digunakan dalam pembelajaran jarak jauh secara daring. Adapun media pembelajaran yang sering digunakan adalah : Tabel 2. Media Pembelajaran Daring Tabel 2. Media Pembelajaran Daring Tabel 2. Media Pembelajaran Daring Tabel 2. Media Pembelajaran Daring NO TEK. HASIL INFORMASI JUMLAH/Org JUMLAH/% 1 Whatsapp 50 82 % 2 Telegram 16 26,2 % 3 Instagram 8 13,1 % 4 Youtube 30 49,2 % 5 Zoom 47 77 % 6 GoogleMet 37 60,7 % 7 Google Classroom 39 63,9 % 8 Edmodo 4 6,6 % 9 Email 9 14,8 % 10 Microsoft Teams 6 9,7 % 11 Panopto 1 1,6 % 12 Webex 1 1,6 % 13 Moodle 1 1,6 % 14 Quizizz 1 1,6 % 15 E-Learning Kampus 2 3,2 % 16 Schoology 1 1,6 % Berdasarkan data yang diperoleh tersebut, dapat dipastikan bahwa jumlah pengguna Whatsapp yaitu sebanyak 50 pengguna atau 82 %. Pengguna Telegram sebanyak 16 atau 26,2 %. Pengguna Instagram sebanyak 8 pengguna atau 13,1 %. Pengguna Youtube sebanyak 30 pengguna atau 49,2 %. Pengguna Zoom sebanyak 47 pengguna atau 77 %. Pengguna GoogleMet sebanyak 37 penguna atau 60,7 %. Pengguna Google Classroom sebanyak 39 pengguna atau 63,9 %. Pengguna Edmodosebanyak 4 pengguna atau 6,6 %. Pengguna Email sebanyak 9 pengguna atau 14,8%. Pengguna Microsoft Teams sebanyak 6 pengguna atau 9,7 %. Pengguna Panopto sebanyak 1 pengguna atau 1,6 %. Sama hal-nya dengan Media Webex, Moodle, Quizizz, dan Schoology. Kemudian yang terakhir pengguna E-Learning Kampus ada sebanyak 2 pengguna atau 3,2 %. Berdasarkan hal tersebut, dapat dipastikan bahwa media yang sering digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring yaitu adalah Analisis Proses Pembelajaran ... Fanesa Maura Aprillia 55 Fanesa Maura Aprillia aplikasi Whatsapp dengan 50 pengguna atau 82 %, Zoom dengan 47 pengguna atau 77 % dan Google Classroom 39 pengguna atau 63,9 %. mplementasi pembelajaran daring menurut pelajar dan mahasiswa b) Implementasi pembelajaran daring menurut pelajar dan mahasiswa Selama Pandemi Covid-19 berlangsung dari awal tahun 2020, para pelajar dan mahasiswa merasakan proses pembelajaran jarak jauh secara daring. Adapun beberapa pertanyaan yang diajukan pada survey untuk menemuka pendapat para pelajar dan mahasiswa dari akun Twitter @collegemenfess mengenai implementasi pembelajaran daring, yaitu : Gambar 1. Persentase Kenyamanan Belajar Berdasarkan hal tersebut, ada 25 pelajar daring. Ada 36 pelajar dan mahasiswa atau 59 % yang tidak nyaman belajar secara daring. Berdasarkan hal tersebut, ada 24 Gambar 1. Persentase Kenyamanan Belajar Gambar 2. Persentase Pemahaman Materi Gambar 3. Perentase Pengerjaan Tugas pelajar dan mahasiswa atau 39,3 % yang dapat memahami materi pembelajaran jarak jauh yang dilakukan secara daring. Ada 37 pelajar dan mahasiswa atau 60,7 % pelajar dan mahasiswa tidak dapat memahami materi pembelajaran jarak jauh yang dilakukan secara daring. pelajar dan mahasiswa atau 39,3 % yang dapat memahami materi pembelajaran jarak jauh yang dilakukan secara daring. Ada 37 pelajar dan mahasiswa atau 60,7 % pelajar dan mahasiswa tidak dapat memahami materi pembelajaran jarak jauh yang dilakukan secara daring. Hal ini membuktikan bahwa 23 pelajar dan mahasiswa atau 37,7 % yang dapat mengerjakan tugas tanpa kendala dalam pembelajaran daring. Ada 38 pelajar dan mahasiswa atau 62,3 % yang mengalami kesulitan dalam mengerjakan tugas. Gambar 3. Perentase Pengerjaan Tugas Gambar 3. Perentase Pengerjaan Tugas Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 56 Gambar 4. Persentase Perasaan Siswa Gambar 5. Persentase Efektifitas Pembelajaran Gambar 4. Persentase Perasaan Siswa Gambar 4. Persentase Perasaan Siswa Hal ini membuktikan bahwa 10 pelajar dan mahasiswa atau 16,4 % yang merasa bahwa pembelajaran daring sama dengan pembelajaran tatap muka. Ada 51 pelajar dan mahasiswa atau 83,6 % yang tidak menyamakan hal tersebut. Gambar 4. Persentase Perasaan Siswa Gambar 4. Persentase Perasaan Siswa Gambar 4. Persentase Perasaan Siswa Hal ini membuktikan 11 orang atau 18 % yang merasa pembelajaran daring sangat efektif untuk diterapkan ditahun-tahun berikutnya. Ada 50 pelajar dan mahasiswa atau 82 % yang tidak menyamakan hal tersebut. Gambar 5. Persentase Efektifitas Pembelajaran Gambar 5. Persentase Efektifitas Pembelajaran Gambar 6. Pesentase Persiapan Materi Hal ini membuktikan bahwa 38 pelajar dan mahasiswa atau 62,3 % berpendapat bahwa dosen/ guru tidak hanya memberikan tugas tetapi juga menjelaskan materi pembelajaran daring. Ada 50 pelajar dan mahasiswa atau 82 % yang tidak menyamakan hal tersebut. Gambar 6. Pesentase Persiapan Materi Berdasarkan hasil survey tersebut, dapat dipastikan bahwa banyak pelajar dan mahasiswa dari akun Bot Twitter @collegemenfes yang tidak nyaman melakukan proses pembelajaran secara daring, tidak memahami materi secara daring, dan juga merasa kesulitan dalam mengerjakan tugas secara daring. KESIMPULAN Berdasarkan hasil penelitian ini, dapat disimpulkan bahwa para pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut lebih dominan menggunakan Android dengan jumlah 55 pengguna atau 90,2 % sebagai teknologi informasi pada pembelajaran jarak jauh secara daring. Dan kemudian untuk media yang paling sering digunakan oleh pelajar dan mahasiswa dalam proses pembelajaran jarak jauh secara daring yaitu adalah aplikasi Whatsapp dengan 50 pengguna atau 82 %, Zoom dengan 47 pengguna atau 77 % dan Google Classroom 39 pengguna atau 63,9 %. Untuk implementasi pembelajaran jarak jauh yang dilakukan secara daring tersebut, dapat dipastikan bahwa banyak pelajar dan mahasiswa dari akun Bot Twitter @collegemenfes yang tidak nyaman melakukan proses pembelajaran secara daring, tidak memahami materi secara daring, dan juga merasa kesulitan dalam mengerjakan tugas secara daring. Kemudian, pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut banyak yang berpendapat bahwa pembelajaran daring tidak sama dengan pembelajaran tatap muka di sekolah atau di kampus dan sangat tidak efektif untuk diterapkan di tahun-tahun berikutnya. Namun, selama adanya Pandemi yang semakin parah dan tidak ada yang mengetahui kapan akan berakhir, maka terpaksa selama itu pelajar dan mahasiswa akan melaksanakan proses pembelajaran secara daring. Dan yang terakhir, pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut banyak yang berpendapat bahwa guru/dosen mereka dalam pembelajaran secara daring ini tidak hanya memberikan tugas saja tetapi juga menjelaskan materi pembelajaran. Berdasarkan hal tersebut, ada 24 Kemudian, pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut banyak yang berpendapat bahwa pembelajaran daring tidak sama dengan pembelajaran tatap muka di sekolah atau di kampus dan sangat tidak efektif untuk diterapkan di tahun-tahun berikutnya. Dan yang terakhir, pelajar dan mahasiswa dari akun Bot @collegemenfess tersebut banyak yang berpendapat bahwa guru/dosen mereka dalam pembelajaran secara daring ini tidak hanya memberikan tugas saja tetapi juga menjelaskan materi pembelajaran. Analisis Proses Pembelajaran ... Fanesa Maura Aprillia Analisis Proses Pembelajaran ... Fanesa Maura Aprillia Analisis Proses Pembelajaran ... 57 Fanesa Maura Aprillia Jurnal Huriah: Jurnal Evaluasi dan Penelitian Pendidikan, Vol. 3, No. 1, Mar 2022, hal. 48-58 Mayer, R. E. (2009). Multi Media Learning Prinsip-Prinsip Dan Aplikasi (2009 Yogyakarta: Pustaka Pelajar (ed.)). Ulyan Nasri. (2018). Bersahabat dengan Ilmu: Sebuah Pengantar Filsafat Ilmu (Cet. k-lim). Mataram: CV. Haramain Lombok, 2018. Nasution, B. (2008). Berbagai Pendekatan Dalam Proses Belajar dan Mengajar. Jakarta: Bumi Aksara, 2008. DAFTAR PUSTAKA Agus Ramdani, A. Wahab Jufri, J. (2020). Pengembangan Media Pembelajaran Berbasis Android pada Masa Pandemi Covid-19 untuk Meningkatkan Literasi Sains Peserta Didik. Program Studi Magister Pendidikan IPA Pascasarjana Universitas Mataram Jurnal Kependidikan November 2020, 6(3). Ahmad Arsyad. (2005). Media Pembelajaran. Jakarta: Raja Grafindo Persada, 2005. Dabbagh, N. and R. B. B. (2009). Online Learning, Concepts, Strategies And Application. Ohio: Pearson, 2005. DeVito. Joseph A. (2011). Komunikasi Antarmanusia (Agus (ed.); Edisi Keli). Tangerang Selatan : Karisma, 2011. Dryden, G. and V. J. (2001). Revolusi Cara Belajar. Bandung: Kaifa, 2001. Indiani, B. (2020). Mengoptimalkan Proses Pembelajaran Dengan Media Daring Pada Masa Pandemi COVID-19. SMKN 1 Wonogiri Kabupaten Wonogiri, Indonesia. Jurnal Sipatokkong BPSDM Sulawesi Selatan., 1(3), 227–232. Istiqomah, N. A. I., & Akhmad, F. . (2021). PROBLEMATIKA PEMBELAJARAN DARING PAI SERTA UPAYA KEPALA SEKOLAH DALAM MENGATASINYA. JURNAL HURRIAH: Jurnal Evaluasi Pendidikan Dan Penelitian, 2(4), 1-9. Retrieved from https://www.academicareview.com/index.php/jh/article/view/32 58 Mayer, R. E. (2009). Multi Media Learning Prinsip-Prinsip Dan Aplikasi (2009 Yogyakarta: Pustaka Pelajar (ed.)). Mayer, R. E. (2009). Multi Media Learning Prinsip-Prinsip Dan Aplikasi (2009 Yogyakarta: Pustaka Pelajar (ed.)). Nasution, B. (2008). Berbagai Pendekatan Dalam Proses Belajar dan Mengajar. Jakarta: Bumi Aksara, 2008. Nasution, B. (2008). Berbagai Pendekatan Dalam Proses Belajar dan Mengajar. Jakarta: Bumi Aksara, 2008. Ulyan Nasri. (2018). Bersahabat dengan Ilmu: Sebuah Pengantar Filsafat Ilmu (Cet. k-lim). Mataram: CV. Haramain Lombok, 2018. Ulyan Nasri. (2018). Bersahabat dengan Ilmu: Sebuah Pengantar Filsafat Ilmu (Cet. k-lim). Mataram: CV. Haramain Lombok, 2018.
https://openalex.org/W4367626984
https://link.springer.com/content/pdf/10.1007/s42113-023-00168-3.pdf
English
null
Similarity-Based Interference in Sentence Comprehension in Aphasia: a Computational Evaluation of Two Models of Cue-Based Retrieval
Computational brain & behavior/Computational Brain & Behavior
2,023
cc-by
21,920
1In formal analyses, it is usually assumed that the syntactic dependency within the relative clause is between the verb and the relative pronoun who, which enters into a semantic dependency with the noun phrase the boy in the matrix clause, to which it refers back. https://doi.org/10.1007/s42113-023-00168-3 Computational Brain & Behavior (2023) 6:473–502 https://doi.org/10.1007/s42113-023-00168-3 Computational Brain & Behavior (2023) 6:473–502 ORIGINAL PAPER Abstract Sentence comprehension requires the listener to link incoming words with short-term memory representations in order to build linguistic dependencies. The cue-based retrieval theory of sentence processing predicts that the retrieval of these memory representations is affected by similarity-based interference. We present the first large- scale computational evaluation of interference effects in two models of sentence processing — the activation- based model and a modification of the direct-access model — in individuals with aphasia (IWA) and control participants in German. The parameters of the models are linked to prominent theories of processing deficits in aphasia, and the models are tested against two linguistic constructions in German: pronoun resolution and relative clauses. The data come from a visual-world eye-tracking experiment combined with a sentence-picture matching task. The results show that both control participants and IWA are susceptible to retrieval interference, and that a combination of theoretical explanations (intermittent deficiencies, slow syntax, and resource reduction) can explain IWA’s deficits in sentence processing. Model comparisons reveal that both models have a similar predictive performance in pronoun resolution, but the activation-based model outperforms the direct-access model in relative clauses. Keywords Computational modeling · Cue-based retrieval · Sentence comprehension · Aphasia · Cognitive modeling Keywords Computational modeling · Cue-based retrieval · Sentence comprehension · Aphasia · Cognitive modeling Similarity-Based Interference in Sentence Comprehension in Aphasia: a Computational Evaluation of Two Models of Cue-Based Retrieval ´on1 · Dario Paape1 · Dorothea Pregla1 · Frank Burchert1 · Nicole Stadie1 · Shravan Vasishth Accepted: 5 February 2023 © The Author(s) 2023 / Published online: 1 May 2023 Accepted: 5 February 2023 © The Author(s) 2023 / Published online: 1 May 2023 1 Department of Linguistics, University of Potsdam, Potsdam, Germany  Dario Paape paape@uni-potsdam.de (2) The girl who the boy greeted plays with the dog. (2) The girl who the boy greeted plays with the dog. In this case, when the comprehender reaches the verb greeted, there is one item in memory, boy, that matches all the retrieval cues set by the verb, but there is also another item, girl, that matches some of the retrieval cues ([+animate, −RC subject]). Following J¨ager, Engelmann, & Vasishth (2017), we will refer to the fully matching item (boy) as the retrieval target, and to items with partial feature match (girl) as distractors. In the activation-based model implemented in Lewis and Vasishth (2005), each memory item has a fluctuating activation value that determines both the probability and the latency of retrieval. When a retrieval is triggered, the retrieval cues spread activation to all matching items available in memory. Items with more matches accrue more activation, making them more likely to be retrieved, and decreasing retrieval latency. However, if the cued feature is present on multiple items in memory, the cue’s activation is shared across all items, so that each item receives comparatively less activation. The reduced activation of the target item in memory and the increased activation of competing items are the source of similarity-based interference. A core assumption of the cue-based retrieval theory is that memory retrieval is subject to interference: When a retrieval is triggered, processing difficulty is predicted if multiple items in memory match the same retrieval cues. Therefore, the verb greeted should be more difficult to process in (2) than in (1). This effect is known as similarity-based interference and is indexed by a slowdown at the retrieval site and/or by occasional misretrievals of a distractor item, which results in misinterpretation, such as the girl being interpreted as the agent in (2). Similarity- based interference has been attested in multiple linguistic constructions across different languages (e.g., Van Dyke and Lewis, 2003; Van Dyke & McElree, 2006, 2011; Van Dyke, 2007; Vasishth, Br¨ussow, Lewis, & Drenhaus, 2008; Van Dyke & McElree, 2011; Martin, Nieuwland, & Carreiras, 2012; J¨ager et al., 2017; Engelmann, J¨ager, & Vasishth, 2019; J¨ager, Mertzen, Van Dyke, & Vasishth, 2020; Vasishth & Engelmann, 2022). The direct-access model, developed by McElree and colleagues (McElree, 2000, 2006; McElree et al., 2003; Martin & McElree, 2011), also predicts similarity-based interference. Introduction from memory in order to understand who greeted whom.1 (e.g., Chomsky, 1977). However, for the sake of simplicity, and assuming that both the noun phrase and the relative pronoun inherit the relevant retrieval features from the head noun boy, we will refer directly to the retrieval of boy. In linguistics, resolving who did what to whom is known as thematic role assignment. The doer of the action expressed by the relative clause verb greeted is the agent (boy) and the recipient of the action is the theme (girl). When hearing a sentence, the listener has to link incoming words together and build up syntactic and semantic relations in real time. For instance, verbs have to be linked with their dependent arguments, which is commonly assumed to require retrieval from memory (Lewis, 1999; Van Dyke & Lewis, 2003; McElree, 2006). In a sentence like (1), focusing on the relative clause (RC) The boy [who greeted the girl], when the verb greeted inside the relative clause is reached, the comprehender must retrieve the subject boy (1) The boy who greeted the girl plays with the dog. Cue-based retrieval theory (McElree, 2000; McElree, Foraker, & Dyer, 2003; Lewis & Vasishth, 2005; Lewis, Vasishth, & Van Dyke, 2006; Vasishth, Nicenboim, Engel- mann, & Burchert, 2019) posits that items are retrieved from Paula Liss´on and Dario Paape contributed equally to this work. 474 Computational Brain & Behavior (2023) 6:473–502 regarding the underlying latent processes that unfold in memory retrieval. memory based on their syntactic and semantic features,2 we refer to all the different types of cue-based retrieval accounts as “the” cue-based retrieval theory, even though there are important differences in the underlying latent process assumed and the quantitative predictions of the different variants (e.g., Nicenboim and Vasishth, 2018; Liss´on et al., 2021a). According to cue-based retrieval as implemented computationally in Lewis and Vasishth (2005), items are stored in memory as a bundle of feature-value pairs. In a subject relative clause such as in (1), the embedded verb greeted triggers the retrieval of an item in memory whose features match the retrieval cues [+animate] and [+RC sub- ject], which identify the agent of the relative clause. When the relative clause verb greeted is read, the only item in memory that matches these retrieval cues is boy. Consider now sentence (2), which is an object relative clause. 3A Shiny app provides easy access to this simplified model’s quantitative predictions under parametric variation: https://vasishth. github.io/RetrievalModels/. 2Following Parker et al. (2017). Introduction The activation-based model was originally implemented in the cognitive architecture ACT-R (Anderson et al., 2004). Because the full ACT-R based model is implemented in the programming language Lisp, it is not easily accessible to the wider community in psycholinguistics. Partly in response to this problem, Engelmann et al. (2019) developed an implementation in R (R Core Team, 2020) that represents a simplified version of the full model.3 The match between simulated data from the Lewis and Vasishth (2005) model and human experimental data has been studied in subject-verb and reflexive-antecedent dependencies, negative polarity items, and other linguistic constructions (Vasishth & Lewis, 2006; Wagers, Lau, & Phillips, 2009; J¨ager et al., 2020; Dillon, Mishler, Sloggett, & Phillips, 2013; J¨ager, Engelmann, & Vasishth, 2015; Nicenboim & Vasishth, 2016; Patil, Vasishth, & Lewis, 2016; Parker & Phillips, 2017; Vasishth et al., 2019). (2) The girl who the boy greeted plays with the dog. It assumes that the availability of items in memory — that is, the probability of successful retrieval — decreases as a function of interference, but that retrieval times remain unaffected. However, low availability can lead to misretrievals and/or parsing failure: if a retrieval fails completely, that is, if no appropriate chunk can be retrieved to perform a syntactic attachment, words will be left “stranded,” that is, fail to be integrated into the syntactic structure (Lewis & Vasishth, 2005; Bartek, Lewis, Vasishth, & Smith, 2011), and the parse will crash. If retrieval fails or if an incorrect chunk is retrieved, in a certain proportion of trials, a backtracking process is initiated that requires some extra processing time (Martin Within the cue-based retrieval framework, two different models of retrieval processes have been proposed: the activation-based model (Lewis & Vasishth, 2005) and the direct-access model (McElree, 2000). The two models share the assumption that retrieval cues mediate access to items in memory. However, they make different assumptions Computational Brain & Behavior (2023) 6:473–502 475 & McElree, 2008). Backtracking, also known as reanalysis, is implicitly assumed to lead to the retrieval of the target (McElree, 1993). the modified direct-access model and the original direct- access model against self-paced listening data from IWA and control participants in German (Pregla et al., 2021). The models were compared using Bayes factors, and the result was inconclusive. In the present study, we compare the modified direct-access model against the activation-based model using a larger dataset. Nicenboim and Vasishth (2018) compared the activation- based model and the direct-access model using self-paced reading data from unimpaired adult readers (Nicenboim, Vasishth, Engelmann, & Suckow, 2018). Nicenboim and Vasishth (2018) showed that the predictive performance of the default activation-based model implementation was worse compared to that of the direct-access model. However, the models had similar quantitative performance when the activation-based model was implemented with different variances for target and distractors. Liss´on et al. (2021a) tested the two competing models against self- paced listening data from individuals with aphasia and control participants (Caplan, Michaud, & Hufford, 2015). The authors modeled the comprehension of English subject and object relative clauses as in (1) and (2), in self- paced listening and a sentence-picture matching task. Model comparisons showed similar quantitative performance, but major qualitative differences. Liss´on et al. (2) The girl who the boy greeted plays with the dog. concluded that in order to disentangle the differences between the models, more studies using different linguistic constructions and different experimental paradigms were needed. This is the empirical gap that the current study aims to fill. We model interference effects in IWA and control par- ticipants using a subset of the data from Pregla, Vasishth, Liss´on, Stadie, & Burchert (2022). We focus on two lin- guistic constructions in German: pronoun resolution and relative clauses. These constructions are well-suited for our modeling goals because IWA have difficulty pro- cessing them (Burchert, de Bleser, & Sonntag, 2003; Choy & Thompson, 2010; Caplan et al., 2015; Adelt et al., 2017; Pregla et al., 2021). Furthermore, given that cue-based retrieval is intended as a general model of sen- tence processing, it is necessary to investigate different con- structions and test whether the proposed implementations are able to account for the entire range of data. We also aim to establish links between model parameters and prominent theories of processing deficits in aphasia. Linking these verbally stated theories to model parameters is crucially important, because it enables us to derive constrained, testable predictions for each theory and to evaluate them against the data. Finally, our study is, to our knowledge, the first to compare two different models of cue-based retrieval using online eye-tracking data from the visual-world paradigm in combination with an offline sentence-picture matching task.4 In the context of aphasic sentence processing, one assumption of the direct-access model is potentially overly constraining, namely the assumption that backtracking, if initiated, always leads to correct retrieval of the target. Because of this assumption, due to the added backtracking time in some of the correct trials, the direct-access model assumes that correct retrievals are, on average, slower than misretrievals. However, it is known from different cognitive tasks that “slow errors” can occur in addition to “fast errors” (e.g., Van Maanen, Katsimpokis, & Van Campen, 2019). The direct-access model’s assumption that correct retrievals are on average slower than incorrect retrievals leads to incorrect predictions when modeling data from individuals with aphasia with the direct-access model (Liss´on et al., 2021a): Individuals with aphasia (IWA) often have slower latencies in incorrect trials relative to correct trials (see Hanne, Burchert, de Bleser, & Vasishth, 2015; Adelt, Stadie, Lassotta, Adani, & Burchert, 2017, Pregla, Liss´on, Vasishth, Burchert, & Stadie, 2021). We seek to answer the following questions: 1. 4Patil, Hanne, Burchert, de Bleser, & Vasishth (2016) modeled visual- world eye-tracking data from 7 IWA and 8 controls with different versions of the Lewis and Vasishth (2005) model, but the (modified) direct-access model has never been tested against visual-world eye- tracking data, and never with such a relatively large-scale dataset from IWA and controls. (2) The girl who the boy greeted plays with the dog. Which model of cue-based retrieval offers a better account of interference effects in IWA and control participants across pronoun resolution and relative clauses? 2. How do the parameters of each model map onto theories of processing deficits in IWA? The paper is structured as follows: We begin by summarizing the theories of processing deficits in aphasia that we will evaluate, as well as their proposed connection to the parameters of the activation-based and modified direct-access models. We then introduce the two linguistic constructions of interest, namely pronoun resolution and relative clauses. Next, on the basis of these constructions, we discuss the implementation of the competing models Based on the high prevalence of “slow errors” in IWA, Liss´on et al. (2021b) implemented a modified version of the direct-access model. In this model, there is not only a distinction between trials in which backtracking is initiated and trials in which it is not, but also a distinction between trials in which backtracking is successful and trials in which it fails. Trials with failed backtracking are slower than trials without backtracking, but the parser is stuck with the result of the original misretrieval. Liss´on et al. (2021b) tested Computational Brain & Behavior (2023) 6:473–502 476 in greater detail, and link the assumed parameters to influential theories from the aphasia literature. Finally, we fit the models to the data and evaluate whether the theoretical predictions are borne out. We also assess each model’s predictive fit by repeatedly fitting the models to subsets of the data, simulating new data and comparing the simulated data to a different subset of the original data. To anticipate our results, across IWA and control participants, and across both linguistic constructions, neither of the two implementations of cue-based retrieval performs decisively better than the other in terms of predictive fit. However, the parameter estimates from each of the two models are informative with regard to the underlying deficits in IWA: There is support in the data for slow syntax, intermittent deficiencies, and resource reduction (see discussion below), but less support for delayed lexical access. lexical access — have been tested using the cross-modal lexical decision paradigm. However, studies using the visual-world paradigm in aphasia do not support a delay in lexical access or in syntactic structure building as the main source of comprehension deficits in IWA (e.g., Dickey, Choy, & Thompson, 2007). Theories of Processing Deficits in Aphasia Individuals with aphasia have difficulties processing non- canonical sentences (e.g., Caramazza & Zurif, 1976; McAl- lister et al., 2009; Schumacher et al., 2015) such as object relative clauses and passives, especially when the thematic roles are semantically reversible. That is, IWA experience difficulties identifying the agent and theme of the verb (who did what to whom) based on morpho-syntactic cues alone. Similarly, IWA also experience difficulties compre- hending binding relations, that is, pronouns and reflexives (e.g., Justin told [Thomasi to shave himselfi]; Edwards & Varlokosta, 2007; Choy & Thompson, 2010). All of these theoretical proposals can be incorporated into computational models of retrieval. For instance, Patil, Hanne, Burchert, de Bleser, & Vasishth (2016) modeled the comprehension of active vs. passive sentences in a visual-world eye-tracking experiment in German (Hanne, Sekerina, Vasishth, Burchert, & de Bleser, 2011), using different implementations of the Lewis and Vasishth (2005) model. The best-fitting model for IWA was one that assumed generally slowed processing (understood as a combination of delayed lexical access and slow syntax), as well as intermittent deficiencies. In another modeling study, M¨atzig, Vasishth Engelmann, Caplan, and Burchert mapped parameters of the Lewis and Vasishth (2005) model to slowed processing, intermittent deficiencies, and resource reduction. The authors modeled accuracies in English sub- ject and object relative clauses using the data from Caplan et al. (2015). They concluded that IWA’s performance can be explained by a combination of these three deficits, and that slowed processing, intermittent deficiencies, and resource reduction may affect each individual with aphasia to a different degree. Similarly, in English relative clauses, using self-paced listening data from Caplan et al. (2015), Liss´on et al. (2021a) also found that intermittent deficien- cies, delayed lexical access, and slow syntax can explain the main processing deficits in IWA. Caplan et al. (2015) discuss the different theories that aim to explain why these constructions are challenging for IWA5. For instance, Burkhardt, Pi˜nango, & Wong (2003) and Burkhardt, Avrutin, Piango, & Ruigendijk (2008) argue that IWA compute syntactic dependencies at a slower-than- normal pace, which can lead to comprehension failure. According to this theory, known as slow syntax, the process- ing deficit in IWA is specific to syntactic structure building. By contrast, Ferrill, Love, Walenski, & Shapiro (2012), and Love, Swinney, Walenski, & Zurif (2008) posit that delayed lexical access causes the slowdown in the formation of syntactic dependencies. 5Although the slow syntax and the delayed lexical access theories were originally proposed for Broca’s aphasia, the studies by Caplan and colleagues (Caplan, Michaud, & Hufford, 2013, 2015) show that these deficits could also be playing an important role in impaired sentence comprehension in patients with other types of aphasia. (2) The girl who the boy greeted plays with the dog. The data modeled in the present work is well-suited to test the predictions of these two theo- ries, because we consider visual-world data in combination with reaction times and accuracies from a picture-selection task, which require similar motor responses as in the cross- modal lexical decision task. Another theory, proposed by Caplan and colleagues, argues that IWA may have an impairment in the resources needed for parsing, such as memory capacity (Caplan, Waters, DeDe, Michaud, & Reddy, 2007; Caplan, 2012). Complex sentences create greater processing demands, and therefore IWA have more difficulties in complex sentences. This account is known as resource reduction. In addition, Caplan et al. (2013) claim that IWA may also exhibit intermittent deficiencies in the parsing system that block access to parsing operations such as relating the surface and base positions of words in the structure. The intermittent nature of these breakdowns would explain why IWA are able to understand complex sentences on some but not all trials. (3) a. Mismatch. Peter+subj +masc verspricht nun Lisa−subj −masc, dass er{subj masc} das kleine Lamm streichelt und krault. Peter+subj +masc promises now Lisa−subj −masc, that he{subj masc} the small lamb pets ‘Peter now promises Lisa that he will pet and ruffle the little lamb.’ b. Match. Peter+subj +masc verspricht nun Thomas−subj +masc, dass er{subj masc} das kleine Lamm streichelt und krault. Peter+subj +masc promises now Thomas−subj +masc, that he{subj masc} the small lamb pets and ruffl Peter+subj +masc promises now Thomas−subj +masc, that he{subj masc} the small lamb pets a ‘Peter now promises Thomas that he will pet and ruffle the little lamb.’ Across the two sentences, the main clause object nouns, Lisa in (3a) and Thomas in (3b), partially match the retrieval cues from the pronoun. Both mismatch the [+subj] cue that the pronoun inherits from the verb, but Thomas matches the gender cue from the pronoun, which should lead to increased similarity-based interference. We will refer to sentences like (3a) as mismatch conditions, because the target noun (Peter) and the distractor noun (Lisa) do not share the same gender, and sentences like (3b), as match conditions. had difficulties in pronoun resolution, but these studies did not target the gender mismatch configurations that Pregla et al. (2022) tested, and that we model in the present work. had difficulties in pronoun resolution, but these studies did not target the gender mismatch configurations that Pregla et al. (2022) tested, and that we model in the present work. In Experiment 1, we model interference as a function of the gender cue at the pronoun in the Pregla et al. (2022) data. Based on cue-based retrieval theory, we predict a processing advantage in gender mismatch conditions relative to the gender match conditions. We aim to (a) compare how the activation-based and the modified direct-access model fit these data, and (b) evaluate the theoretical accounts of processing deficits in aphasia by mapping them onto model parameters. In Experiment 1, we model interference as a function of the gender cue at the pronoun in the Pregla et al. (2022) data. Based on cue-based retrieval theory, we predict a processing advantage in gender mismatch conditions relative to the gender match conditions. We aim to (a) compare how the activation-based and the modified direct-access model fit these data, and (b) evaluate the theoretical accounts of processing deficits in aphasia by mapping them onto model parameters. Experiment 1: Pronoun Resolution In our modeling, we follow Liss´on et al. (2021a) and Liss´on et al. (2021b) and implement intermittent deficiencies as increased stochastic noise in memory activations/availabilities. A higher noise value in IWA would mean more mistretrievals and presumably more parsing failures due to failed retrievals compared to unimpaired individuals. Delayed lexical access or slow syntax is assumed to delay the retrieval of the target item from memory, leading to a slowdown at the retrieval site in the activation-based model, and/or to misretrieval in both the activation-based and the direct-access model. In the DA model, which assumes backtracking as a key resource in parsing, resource reductions could disrupt this mechanism and lead to comprehension deficits. In our modeling, we follow Liss´on et al. (2021a) and Liss´on et al. (2021b) and implement intermittent deficiencies as increased stochastic noise in memory activations/availabilities. A higher noise value in IWA would mean more mistretrievals and presumably more parsing failures due to failed retrievals compared to unimpaired individuals. Delayed lexical access or slow syntax is assumed to delay the retrieval of the target item from memory, leading to a slowdown at the retrieval site in the activation-based model, and/or to misretrieval in both the activation-based and the direct-access model. In the DA model, which assumes backtracking as a key resource in parsing, resource reductions could disrupt this mechanism and lead to comprehension deficits. Consider sentence (3a). When the pronoun er (“he”) is encountered, cue-based retrieval predicts that a search for its antecedent is triggered, using the cues [+animate, +masculine, +singular].6 The experiment makes use of the fact that for some verbs, the implicit subject of a sentential complement is coreferential with the main clause subject (subject control) while for others it is coreferential with the main clause object (object control, e.g., Chomsky, 1981; Comrie, 1985). The verb versprechen (“promise”) is lexically specified as a subject control verb (M¨uller, 2002). Even though we do not investigate control structures, we assume that the additional retrieval cue [+subj] is set at the pronoun, and that Peter is the retrieval target: Because versprechen (“promise”) enforces subject control, replacing er (“he”) with sie (“she”) to refer to Lisa in (3a) results in unacceptability, and there is no alternative antecedent in the discourse to which the pronoun could refer. 6The parser presumably also honors structural constraints during retrieval, such as adherence to Binding Principle B (Chomsky, 1981), as opposed to first retrieving and then eliminating structurally illicit antecedents (e.g., Chow, Lewis, & Phillips, 2014). In the current context, however, both possible antecedents are structurally available, so that we abstract away from structural retrieval cues. Experiment 1: Pronoun Resolution We fit the models to data from a picture-selection task and a visual-world experiment that tested the comprehen- sion of pronouns and relative clauses in German (Pregla et al., 2022). We now introduce each linguistic construction in turn. Theories of Processing Deficits in Aphasia Love, Swinney, Walenski, & Zurif (2008) claim that when the sentence requires the reactiva- tion of a lexical item in order to complete a dependency, the lexical reactivation is too slow, and an extragrammatical heuristic may be used instead, which can lead to comprehen- sion errors. These two theories — slow syntax and delayed In the current work, we focus on the role of the proposed processing deficits in the context of the activation- based and the direct-access models of cue-based retrieval. Computational Brain & Behavior (2023) 6:473–502 477 (3) a. Mismatch. In unimpaired populations, a processing advantage in gen- der mismatch conditions has been observed in English by Badecker and Straub (2002) and Runner and Head (2014), but not by Chow et al. (2014). Laurinavichyute, J¨ager, Akinina, Roß, & Dragoy (2017) reported mixed results for German. In the aphasia literature, Choy and Thompson (2010) and Engel, Shapiro, & Love (2018) found that IWA 6The parser presumably also honors structural constraints during retrieval, such as adherence to Binding Principle B (Chomsky, 1981), as opposed to first retrieving and then eliminating structurally illicit antecedents (e.g., Chow, Lewis, & Phillips, 2014). In the current context, however, both possible antecedents are structurally available, so that we abstract away from structural retrieval cues. 478 Computational Brain & Behavior (2023) 6:473–502 Experiment 2: Relative Clauses Our study mainly focuses on the effect of number interference within subject and object relative clauses rather than on the well-studied SR/OR asymmetry. This is because similarity-based interference alone cannot account for the asymmetry in German relative clauses, as the retrieval point for both SRs and ORs is the clause-final verb (see discussion below). However, it is still informative to check if the asymmetry can be captured by assuming changes in the parameters of the activation-based model and the modified direct-access model. Relative clauses have been extensively studied in psycholin- guistics. Subject relatives (SR) have been found to be easier to process than object relatives (OR) in multiple languages for both unimpaired controls (e.g., Grodner and Gibson, 2005; Fedorenko, Gibson, & Rohde, 2006; Gordon, Hen- drick, Johnson, & Lee, 2006; Staub, 2010, Staub, Dillon, & Clifton Jr, 2017) and for IWA (e.g., Caramazza & Zurif, 1976; Burchert et al., 2003; Caplan et al., 2007; Dickey & Thompson, 2009; Caplan et al., 2013, 2015; Pregla et al., 2021). The subject-object asymmetry in IWA and controls has been computationally modeled in the cue-based retrieval framework (M¨atzig et al., 2018; Vasishth et al., 2019; Liss´on et al., 2021a) using self-paced listening data and offline measures in English. The present study is the first to model number interference in relative clauses in German, in IWA and unimpaired controls. Consider the sentences in (4). When the verb badet/baden (bathes/bathe) is encountered at the end of the sentence, two retrievals are triggered, because the agent and the theme of the action expressed by the verb need to be identified. Our modeling focuses on the retrieval of the agent because that is the theoretically interesting event (this is explained below). (4) a. SR, match (4) a. SR, match b. OR, match c. SR, mismatch d. OR, mismatch d. OR, mismatch 479 Computational Brain & Behavior (2023) 6:473–502 In (4a) and (4b), both noun phrases in the sentence are singular (der Esel, der/den Tiger). This is expected to cause similarity-based interference during the retrieval of the subject. By contrast, in (4c) and (4d), the second noun phrase is plural (die Tiger), which should result in easier identification of the subject, based on the retrieval cue from the verb ([singular] or [plural]). We will refer to sentences like (4c) and (4d) as mismatch conditions, because target and distractor do not share the same number, and sentences like (4a) and (4b), as match conditions. Both types of relative clauses should be easier to process in mismatch configurations compared to match conditions. Thus, (4c) and (4d) should be easier to process than (4a) and (4b), respectively. education = 18.1 years, SD = 4.0), all native speakers of German, took part in an auditory sentence-picture matching task combined with visual-world eye-tracking. Individuals with aphasia were in the chronic phase (at least 1 year after onset of the aphasia). More information about the patients is given in Appendix A. The procedure was as follows: At the beginning of the trial, a preview phase of 4000 ms was used to introduce two pictures to the participants. One of the pictures (target) corresponded to the correct meaning of the sentence, whereas the other picture (foil) depicted the opposite thematic interpretation. After the preview phase, an auditory recording of the sentence started playing. Sentences were presented at a normal speech rate, and participants were instructed to select the picture that matched the meaning of the sentence. The pictures were displayed until participants made a choice, or for a maximum time of 30 s. Once the participant pressed the choice button, the trial ended. During the trial, eye movements were recorded using a SensoMotoric Instruments eye-tracker (SMI RED250mobile; binocular tracking, Experiment Center version 3.7, sampling rate 250 Hz). The proportion of looks to the target picture against looks to the foil (or no picture) was calculated. The response time and the accuracy of the picture selection were also recorded. (4) a. SR, match Each participant completed the experiments twice, in two sessions (test and retest), with a gap of approximately 2 months.7 Participants also performed a battery of tests in order to assess auditory and visual comprehension, morphological discrimination, and lexical decision latency. In German, when the head noun is masculine (such as in our items), the morphological form of the relativizer (der for nominative, den for accusative) provides disambiguating information. Therefore, in our items, by the time the comprehender reaches the relativizer, they should be able to identify the agent or the theme of the relative clause due to the overt case marking. Retrieval should occur at the verb, and the number cue should facilitate processing in (4c) vs. (4a) and (4d) vs. (4b), because in (4c) and (4d) only the subject noun phrase matches the number cue at the verb. Studies addressing the comprehension of subject vs. object relatives in German with case-unambiguous relativiz- ers (such as our items) are scarce and have mainly addressed the SR/OR asymmetry (Friederici, Steinhauer, Mecklinger, & Meyer, 1998; Burchert et al., 2003; Adelt et al., 2017). By contrast, the main goals in our Experiment 2 are to compare the performance of the activation-based and the modified direct-access model by modeling number interference in both types of relative clauses, and to evaluate the different theories of processing deficits in aphasia based on the model estimates. Materials In Experiment 1, two conditions (mach and mismatch, as in example 3), with 10 items each were included. Example pictures accompanying the pronoun sentences are shown in Fig. 1. The pronoun items always used subject-control verbs, so that the target of the retrieval was always the first noun phrase.8 Control verbs were selected from the ZAS Database of Clause-Embedding Predicates (Stiebels et al., 2018). 7The test vs. retest main effect is not included in our modeling (see the section on dependent variables) because the model structure had to be simplified due to convergence issues. The test-retest reliability for these data has been investigated in Pregla et al. (2021). 8The pronoun items that we use here were extracted from a larger experiment that also contained object-control verbs, and fillers. 8The pronoun items that we use here were extracted from a larger experiment that also contained object-control verbs, and fillers. 7The test vs. retest main effect is not included in our modeling (see the section on dependent variables) because the model structure had to be simplified due to convergence issues. The test-retest reliability for these data has been investigated in Pregla et al. (2021). Methods The data that we model come from the experiments carried out by Pregla et al. (2022). The participants, procedure, and materials described here summarize the methods in Pregla et al. (2022). By contrast, the dependent variables and contrast coding described here are specific to the present paper. Pregla et al. (2022) analyze the visual-world eye- tracking data, whereas we model the reaction times from the picture-selection task that followed the visual-world paradigm task. In experiment 2, 20 items per relative clause type (subject/object relative) were included. The noun phrase of the matrix clause (henceforth NP1) was always masculine and singular. Out of the 20 items, 10 had a singular embedded noun phrase (henceforth NP2), and 10 had a plural embedded noun phrase. The items were constructed using 10 bisyllabic transitive action verbs, and the noun Participants and Procedure Twenty-one IWA (9 females, mean age = 60.2 years, SD = 11.4; mean education = 15.2 years, SD = 3.2) and fifty control participants (32 females, mean age = 47.7 years, SD = 19.6; mean Participants and Procedure Twenty-one IWA (9 females, mean age = 60.2 years, SD = 11.4; mean education = 15.2 years, SD = 3.2) and fifty control participants (32 females, mean age = 47.7 years, SD = 19.6; mean Computational Brain & Behavior (2023) 6:473–502 480 Fig. 1 Example pictures used in the picture-selection task in Experiment 1. For the sentences in example (3) the left picture is the target and the right picture is the foil Fig. 1 Example pictures used in the picture-selection task in Experiment 1. For the sentences in example (3) the left picture is the target and the right picture is the foil phrases were always bisyllabic animal names. Example pictures for the relative clause conditions are given in Fig. 2. IWA. For instance, Caplan et al. (2015) correlated the lexical decision times for correct responses with the accuracy and reading times in a self-paced listening task. We centered and scaled the LDT predictor within groups. An LDT × group interaction would thus tell us whether an increase in LDT leads to a larger increase in RT for IWA compared to controls. Dependent Variables and Contrast Coding To assess partic- ipants’ lexical access speed, which is important for evalu- ating the delayed lexical access hypothesis of Love et al. (2008) and Ferrill et al. Methods This is the convention used to report summaries of parameter values for which there is support in the data. When interpreting the estimates, the width of the CrI should be taken into account, as it shows the range of plausible parameter values that lie with 95% probability given our model and data. The second retrieval event happens at the verb region, that is, at the end of the sentence. In both experiments, the retrieval target must be re-accessed at this point, as a dependency with the verb needs to be established. We assume that the second retrieval is linked to the first, so that more looks to the target at the critical region (the pronoun/relativizer) go along with higher activation/availability, resulting in faster and/or more accurate retrieval at the verb region. As the picture-selection task takes place immediately after hearing the verb region, we assume that accuracies and RT in this task should show the interference effects predicted by the cue-based retrieval theory. For both the activation-based model and the modified direct-access model, the data from the pronoun experiment and the relative clause experiment were fitted separately.9 The four models were implemented in Stan (Carpenter et al., 2017) and fitted in R (R Core Team, 2020) via the rstan package (Stan Development Team, 2020). The packages brms (B¨urkner, 2017) and bayesplot (Gabry, Simpson, Vehtari, Betancourt, & Gelman, 2019) were used for examining and plotting the posterior distributions of the parameters. For each model, three chains each, with at least 6000 iterations each were run. Each chain included at least 3000 warm-up iterations. Convergence was assessed by checking that  R was below 1.01 and by visually inspecting the convergence of the chains (Gelman et al., 2013). We also verified that the models could recover simulated parameter values. For both models, mildly informative priors were used (Sorensen, Hohenstein, & Vasishth, 2016; Nicenboim et al., 2021; Schad, Betancourt, & Vasishth, 2021). Details about the implementation and the priors are available in Appendix C. We do not model retrieval failures, that is, trials in which neither the target nor the distractor can be retrieved. This simplification is necessary because participants in the Pregla et al. (2021) study had to select one of the two pictures and were not given the option to respond “I don’t know,” which could be interpreted to index retrieval failure (Nicenboim & Vasishth, 2018). 9Initially, we intended to model the pooled data from both experiments. However, the complex structure of fixed and random effects lead to convergence issues in the modified direct-access model. Therefore, we ended up implementing two separate versions of each model, one for each experiment. Methods (2012), we computed their average reaction times in a lexical decision task, based on LEMO 2.0 (Stadie, Cholewa, & de Bleser, 2013). In this task, par- ticipants have to decide whether an auditorily presented item is a word or a non-word. Participants responded by pressing one of two buttons on a computer keyboard, and the accuracy and response times were recorded. We com- puted the average reaction times in correct trials, which yielded a single measure (lexical decision time, LDT) for each participant that we use as a continuous predictor in the models. Another predictor in the models is the proportion of fixations on the target picture (centered and scaled within groups) at the critical sentence region, where retrieval is assumed to take place. We use the proportions of looks to the target at the critical region as a proxy for retrieval (see the next section for more details). The remaining predictors used in both models were sum-coded, with the following contrasts: group was coded with IWA as +1 and controls as −1; the high interference conditions (gender match in pronouns, number match in relative clauses) were coded as +1, and the low interference conditions (gender mismatch, number mismatch) as −1. In the relative clauses Response times in lexical decision tasks have been previously used as a measure of lexical access speed in Fig. 2 Example pictures used in the picture-selection task in Experiment 2. The pictures correspond to the correct interpretations of the example sentences in (4). Within both the match and the mismatch conditions, participants had to select the correct interpretation (SR interpretation vs. OR interpretation) Fig. 2 Example pictures used in the picture-selection task in Experiment 2. The pictures correspond to the correct interpretations of the example sentences in (4). Within both the match and the mismatch conditions, participants had to select the correct interpretation (SR interpretation vs. OR interpretation) Computational Brain & Behavior (2023) 6:473–502 481 sub-experiment, the relative clauses were coded as OR +1, and SR −1. target. Therefore, we predict that more looks to the target picture at the retrieval site correspond to a higher probability that the target has been retrieved at this point. We base our statistical inferences on the posterior distribution of the parameters, which we summarize with the mean and 95% credible interval (CrI). Methods Furthermore, even in the presence of such an option, participants may resort to guessing rather than responding “I don’t know,” although it has been suggested that IWA only use such compensatory processes rarely (Hanne et al., 2011; Burchert, Hanne, & Vasishth, 2013; Arantzeta, Webster, Laka, Mart´ınez-Zabaleta, & Howard, 2018). Parameters related to guessing can, in principle, be implemented in probabilistic cognitive models (e.g., Oberauer, 2006; Logaˇcev & Vasishth, 2016), including the direct-access model, but we do not attempt such an implementation here, given that our models are already relatively complex. Modeling Assumptions Neither the activation-based model nor the direct-acccess model have a linking function that maps proportions of looks to a picture to retrieval times and/or retrieval probabilities of memory chunks. Therefore, we need to specify linking assumptions between fixations on the target picture, the assumed retrieval processes, and the reaction times and accuracies in the picture-selection task. In what follows, we will present the implementation and the fits of the activation-based model and the modified direct-access model to the data in turn. We also present quantitative model comparisons, which allow us to assess the relative goodness of fit of each model to the data. For the two sentence types, we assume two retrieval events. The first retrieval takes place in the middle of the sentence, at the critical region. In pronoun resolution, the critical region is the pronoun, and in relative clauses, it is the relativizer. Our linking assumption is that proportions of looks to the target at the critical region can be used as a proxy for retrieval. This assumption is based on the fact that the critical region provides the necessary cues to retrieve the Modified Direct-Access Model We implement the modified direct-access model as a hierarchical mixture model in the Bayesian framework, following Liss´on et al. (2021b). Mixture models integrate multiple generative processes in one model (see Nicenboim et al., 2021 chapter 20, for a tutorial on these models in Stan). The implementation of the modified direct-access model as a mixture model allows us to take into account the probability and cost of backtracking as a latent variable. We assume that both correct and incorrect responses are generated from one of two distributions: Responses without backtracking follow a distribution with parameters μ and σ, while responses with backtracking follow another distribution with parameters μ⋆= μ + δ and σ, where δ is the time needed for backtracking. The direct-access model assumes that the availability of items in memory determines their probability of retrieval. In our implementation, we map availability to the parameter θ, which is the probability of retrieval of the target. Given that interference is expected to affect availability, we add a main effect of condition to θ. Because we expect IWA to have lower base availability compared to control participants, we also add a main effect of group to θ. We also add a main effect of fixations, following the same logic as for the activation-based model: More fixations on the target at the critical region should lead to a higher probability of retrieval of the target at the verb. In order to evaluate the delayed lexical access theory, we include LDT as a fixed effect to θ, and the interaction LDT × group. This interaction tests the delayed lexical access theory in IWA: If longer LDT leads to a larger decrease in θ for IWA, this would suggest that delayed lexical access lowers the probability of retrieval of the target, causing difficulties in the retrieval process. 1. An increase in fixations to the target picture at the critical region should lead to a decrease in RT for the target accumulator in the picture-selection task, as we assume that the first retrieval influences the second retrieval. If participants retrieved the target at the critical region, re-accessing it at the verb should be easier, meaning that there should be more correct retrievals as well as faster retrieval times. 2. The mean finishing time of the target accumulator should be faster for the mismatch conditions relative to the match conditions, as similarity-based interference slows retrieval. Activation-Based Model The activation-based model can be implemented as a lognormal race of evidence accumulators (Nicenboim & Vasishth, 2018): For the two experiments, we assume that there are two accumulators of noisy evidence that correspond to the retrieval candidates in memory, namely the first and the second noun phrase (NP1 or NP2, target or distractor). For each trial i, the finishing times FT for NP1 and NP2 are each sampled from a lognormal distribution with location μNP 1 or μNP 2 respectively, and scale σ. The accumulator with the faster FTi determines both the Computational Brain & Behavior (2023) 6:473–502 482 difficulty in retrieving the target. This would be in line with delayed lexical access. difficulty in retrieving the target. This would be in line with delayed lexical access. selected picture (target or foil) and the reaction time for trial i. This implementation maps straightforwardly onto the notion of memory chunks with fluctuating activation values, with the chunk with the highest activation being retrieved on a particular trial. In addition, given earlier results (Burchert et al., 2003; Adelt et al., 2017), in Experiment 2, IWA should have longer mean finishing times for the target accumulator in OR compared to SR. The hierarchical structure of the models is implemented in the μ of both accumulators, which include fixed and random effects. The fixed effects added to μNP 1 and μNP 2 in the model for pronoun resolution are as follows: group (IWA vs. control), condition (match vs. mismatch), and the group × condition interaction. We also added the average reaction time from the lexical decision task (LDT), and the group × LDT interaction. Furthermore, we added the proportion of looks to the target at the critical region (fixations), the fixations × group interaction, and the three- way interaction fixations × condition × group. In addition, both μ included by-subject and by-item varying intercepts; the fixed effect of group included an adjustment by item, and the fixed effect of condition included an adjustment by subject. The parameter σ included a fixed effect of group. In addition, the model for relative clause conditions also included a fixed effect for RC type, an RC type × group and an RC × condition interaction, and the RC × group × condition three-way interaction. The predictions of the activation-based model for the two experiments are as follows: Modified Direct-Access Model The probability of successful retrieval θ should be higher for non-interference conditions relative to interference conditions, that is, higher in mismatch vs. match conditions. 3. IWA should have slower RTs relative to controls, so IWA’s μ should be higher. This would be in line with the slow syntax theory. Similarly, IWA should have a higher σ, which would be in line with intermittent deficiencies. 4. If IWA’s slower access to items from memory leads to difficulties in the retrieval, we would expect LDT to lead to a bigger decrease in θ for IWA relative to controls. This would be in line with delayed lexical access. The mixture process for a given trial i works as follows: (a) if the retrieval of the target succeeds, with probability θ, RTi is drawn from LogNormal(μ, σ). (b) if the retrieval of the target fails (1 −θ), backtracking is initiated with probability Pb. RTi is sampled from LogNormal(μ + δ, σ). After backtracking, the target is retrieved with probability θb, and the distractor with probability 1 −θb. 5. We expect IWA to have a lower probability of backtracking, Pb, and a lower probability of retrieval of the target after backtracking, θb. This would be in line with the resource reduction theory, assuming that backtracking is a parsing resource that is impaired in IWA. 5. We expect IWA to have a lower probability of backtracking, Pb, and a lower probability of retrieval of the target after backtracking, θb. This would be in line with the resource reduction theory, assuming that backtracking is a parsing resource that is impaired in IWA. (c) if the retrieval of the target fails and there is no backtracking, a misretrieval is predicted with probability (1−θ)·(1−Pb), and RTi is sampled from LogNormal(μ, σ). 6. Similarly, we also expect IWA to have a higher cost of backtracking, δ, which would be in line with slow syntax. 6. Similarly, we also expect IWA to have a higher cost of backtracking, δ, which would be in line with slow syntax. Notice that the probability of successful retrieval of the target, θ, and the probability of backtracking, Pb are assumed to be independent. Interference can only indirectly affect response times through lower θ and the added cost of backtracking δ. Modified Direct-Access Model The mean of the distractor accumulator should be similar or slower in mismatch conditions relative to match conditions. 3. IWA should have slower RT relative to controls, so IWA’s μ should be higher. This would be in line with the slow syntax theory. Similarly, IWA should have a higher σ, that is, more noisy accrual of evidence corresponding to more variable activation values, which would be in line with intermittent deficiencies. The original direct-access model assumes that when the initial retrieval fails, a costly process of backtracking (or reanalysis) can be triggered, which leads to correct retrieval of the target (McElree et al., 2003; Martin & McElree, 2008). Our modified direct-access model adds the assumption that backtracking can fail. This is reflected in the added parameter θb, which represents the probability of correct retrieval after backtracking. The additional parameter makes the modified direct-access model more 4. If a delay in lexical access is causing processing difficulties in IWA, we would expect the effect of LDT to lead to a bigger increase in RTs for the target accumulator for IWA relative to controls, as higher RT for the target accumulator would indicate more 4. If a delay in lexical access is causing processing difficulties in IWA, we would expect the effect of LDT to lead to a bigger increase in RTs for the target accumulator for IWA relative to controls, as higher RT for the target accumulator would indicate more Computational Brain & Behavior (2023) 6:473–502 483 of the main effect of fixations to the target on the probability of successful retrieval θ should be positive. suitable for modeling data from individuals with aphasia, as it allows for slow, incorrect responses. If IWA show a lower θb, relative to controls, this could point towards a disruption in the process of backtracking as a main source of comprehension difficulties in IWA. The parameter Pb estimates the proportion of trials for which backtracking is performed after an initial misretrieval. The parameter δ estimates the amount of time (in log ms) that backtracking takes. Main effects of group are added to the parameters θb, Pb and δ. Pb and θb additionally have by-subject random intercepts.10 of the main effect of fixations to the target on the probability of successful retrieval θ should be positive. 2. 10Ideally, δ should also have a by-subject adjustment. However, this is a complex hierarchical model, and a by-subject intercept on δ led to convergence issues. Modified Direct-Access Model Therefore, in the μ parameter, which estimates the mean average RT, we do not include an adjustment by condition, but we do include an adjustment by group, since retrieval may generally be slower in IWA compared to controls. The noise parameter, σ, also has an adjustment by group, as IWA may have more variable retrieval times. The priors used, as well as the full hierarchical model, are shown in Appendix C. In addition, given earlier results showing that OR are more difficult to process than SR (Burchert et al., 2003; Adelt et al., 2017), for the relative clause construction, θ should be lower in OR compared to SR. We now move on to the modeling results, which will be presented separately for pronoun resolution and relative clauses. Modified Direct-Access Model We begin by assessing the posterior distribution of θ, which is the probability of retrieving the target during the first retrieval attempt. Figure 4 shows that the probability of retrieval of the target is very high for controls: The mean of the distribution lies above 95% in both conditions (CrI mismatch: [98, 99]%, CrI match: [96, 98]%). By contrast, IWA show lower retrieval probabilities overall. This can be also seen in Fig. 4, where IWA’s mean estimate for mismatch is 72% CrI: [66, 77]%, whereas the estimate for match is 55% CrI: [47, 62]%. The group × condition interaction is inconclusive (2% CrI: [−1, 5]%). With regard to the fixed effects on μNP 1 and μNP 2, due to space limitations, we will only comment on the estimates that are relevant to the processing theories of aphasia that we are evaluating. The estimates for all parameters in this model and their credible intervals are shown in Appendix D. The NP1 accumulator showed an indication of an LDT × group interaction (836 ms, CrI: [539, 1152] ms), but no indication of such an interaction was observed for the NP2 accumulator. This suggests that additional time needed for lexical access leads to a larger slowdown in IWA in the target accumulator, as predicted by the delayed lexical access theory. The estimates for fixations and the fixations × group interaction do not point in the predicted direction: Rather than facilitating correct retrieval of the target, an increase in fixations on the target picture leads to an increase in RTs in both accumulators (NP1: 41 ms, CrI: [−77, 160] ms; NP2: 518 ms, CrI: [−141, 1212] ms). However, due to large uncertainty around the estimates, the results are also compatible with no effect of fixations. With regard to the fixed effects on μNP 1 and μNP 2, due to space limitations, we will only comment on the estimates that are relevant to the processing theories of aphasia that we are evaluating. The estimates for all parameters in this model and their credible intervals are shown in Appendix D. A unit increase in LDT leads to −5% CrI: [−8, −1]% in θ, and a negative LDT × group interaction (−9% CrI: [−12, −7]%) is consistent with the assumption that IWA are more affected by increased LDT. Experiment 1 — Pronoun Resolution Due to the cost of backtracking δ, correct and incor- rect responses following backtracking (b) are expected to be slower, on average, than correct retrievals (a) and misre- trievals without backtracking (c). The RTs corresponding to an initial successful retrieval of the target (a) and to misre- trievals without backtracking (c) are sampled from the same distribution. The predictions of the modified direct-access model for the two experiments are explained below. In the pronoun resolution items, NP1 is always the target of the dependency. Therefore, μNP 1 accumulates evidence for the retrieval of the target, and μNP 2 for the retrieval of the distractor. Figure 3 shows the distribution of estimated finishing times for the two accumulators (NP1 and NP2) across conditions and groups. The results confirm our predictions: IWA have longer finishing times relative to controls in both conditions. In controls, the means of the NP1 accumulator in the mismatch and match conditions are quite similar (1391 ms vs. 1465 ms), although responses are faster on average in the mismatch condition, as expected. IWA show a larger effect of interference: The mean of the NP1 accumulator in the mismatch condition is 4532 ms, compared to 5735 ms in the match condition. The interference effect can also be seen in the overlap of the 1. Fixations to the target picture at the critical region should lead to an increase in the probability of retrieval of the target, as we assume that the first retrieval influences the second retrieval. Therefore, the estimate Computational Brain & Behavior (2023) 6:473–502 484 Controls IWA Match Mismatch 1000 4000 7000 10000 15000 1000 4000 7000 10000 15000 Finishing times (ms) Accumulator NP1 NP2 Pronoun resolution ot. Whereas the distributions er in controls, in IWA, the log ms, CrI: [0.62,0.69] log ms, σcontrols: 0.28 log ms, CrI: [0.27,0.29] log ms). Fig. 3 Distribution of the accumulators of evidence across groups and conditions for the Experiment 1 (pronoun resolution). The dashed lines represent the means of the distributions log ms, CrI: [0.62,0.69] log ms, σcontrols: 0.28 log ms, CrI: [0.27,0.29] log ms). distributions within each plot. Whereas the distributions lie far apart from each other in controls, in IWA, the distributions overlap. This indicates that IWA are more likely to retrieve the distractor than controls, especially in the interference condition (match). In general, the plots show that IWA experience a bigger interference effect. Experiment 1 — Pronoun Resolution This is in line with the estimates for the group × condition interaction (μNP 1: 199 ms, CrI: [81, 322] ms and μNP 2: 1424 ms, CrI: [400, 2720] ms). Modified Direct-Access Model There was neither an indication of an effect of fixations (2% [−1, 5]%), nor of a fixation × group interaction (−2% CrI: [−5, 2]%). This means that for both groups, there is no indication that an increase in fixations to the target picture led to an increase in the probability of successful retrieval of the target. The estimated probability of backtracking for IWA is 22% CrI: [13, 31]% compared to 66% CrI: [51, 79]% for controls. The distribution of the cost of backtracking, δ, is centered around 5592 ms, CrI: [3924, 7738] ms for IWA, and around 2827 ms, CrI: [2277, 3551] ms for controls. The Finally, as predicted under the intermittent deficiencies theory, IWA have higher noise than controls (σIWA: 0.65 485 Computational Brain & Behavior (2023) 6:473–502 Fig. 4 Posterior distribution of the probability of initial retrieval of the target, θ for the two groups, in the pronoun conditions. The vertical lines stand for the means of the distributions, and the shaded areas indicate the 95% credible interval Match Mismatch 40 60 80 100 [%] Controls Match Mismatch 40 60 80 100 [%] IWA Pronoun conditions, posterior distribution of θ probability of retrieval of the target after backtracking θb CrI: [0 23 0 25] log ms) as predicted under the slow syntax Mismatch [%] CrI: [0.23,0.25] log ms), as predicted under the slow syntax and intermittent deficiencies theories. probability of retrieval of the target after backtracking, θb, is shown in Fig. 5. Backtracking leads to the retrieval of the target around 84% of the time for controls (CrI: [70, 94]%), and 58% of the time for IWA (CrI: [42, 73%]). Slower and less successful backtracking is consistent with slow syntax and resource reduction in IWA. Activation-Based Model Finally, IWA’s μ (2376 ms, CrI: [2079, 2701] ms) is higher than controls’ μ (1320 ms, CrI: [1202, 1447] ms); and IWA also have a higher noise estimate (0.46 log ms, CrI: [0.43,0.5] log ms) relative to controls (0.24 log ms, In the relative-clause items, the accumulator mean μNP 1 stands for the retrieval of NP1 as the agent of the action, whereas μNP 2 stands for the retrieval of NP2 as the agent. Depending on the trial, NP1 (in subject relatives) or NP2 (in object relatives) will be the target of the retrieval, as we model the retrieval of the agent. 40 60 80 100 [%] Group Controls IWA Posterior distribution of θb Fig. 5 Posterior distribution of the probability of retrieval of the target after backtracking, for the two groups, in the pronoun conditions. The dashed lines stand for the means of the distributions 40 60 80 100 [%] Group Cont IWA Posterior distribution of θb Figure 6 shows the distribution of finishing times of the two accumulators in subject relative clauses. As expected, IWA have higher finishing times than controls across conditions. The mean of the NP1 accumulator (target) is roughly the same across conditions, whereas the mean of the NP2 accumulator (distractor) is higher in the mismatch condition than in the match condition. In general, controls show almost no overlap between the distributions, which indicates that controls retrieve the target (NP1) most of the time. By contrast, in IWA, the two distributions partially overlap, meaning that IWA often retrieve the distractor (NP2). Figure 7 shows the distribution of finishing times of the two accumulators in object relative clauses. IWA have higher finishing times than controls across conditions, and Fig. 5 Posterior distribution of the probability of retrieval of the target after backtracking, for the two groups, in the pronoun conditions. The dashed lines stand for the means of the distributions Computational Brain & Behavior (2023) 6:473–502 486 Fig. 6 Distribution of the accumulators of evidence across groups and conditions for subject relative clauses. The dashed lines indicate the means of the distributions Controls IWA Match Mismatch 2000 6000 10000 15000 2000 6000 10000 15000 Finishing times (ms) Accumulator NP1 NP2 Subject relatives Finishing times (ms) both groups have slightly lower finishing times in the NP2 accumulator (target) in mismatch vs. match conditions. Crucially, in the match condition, for IWA (right upper panel in Fig. Fig. 7 Distribution of the accumulators of evidence across groups and conditions for object relative clauses. The dashed lines indicate the means of the distributions. Note that in the mismatch condition, for IWA, the means of the two distributions overlap Activation-Based Model 7, light dashed line), the mean of the NP1 accumulator is lower than the mean of the NP2 accumulator. Since NP2 is the retrieval target in object relatives, the pattern indicates that in the match condition, IWA retrieve the distractor more often than the target. That is, in the match condition, IWA are more likely to misinterpret the sentence than to interpret it correctly. However, in the mismatch condition, the mean of the two accumulators overlap, which indicates that IWA are equally likely to retrieve NP1 or NP2 on average. Comparisons between Figs. 6 and 7 show that con- trols perform similarly in subject and object relatives, whereas IWA display a subject-object asymmetry: IWA are estimated to correctly interpret subject relatives most of the time. By contrast, IWA are estimated to misinter- pret object relatives more often, especially in the match condition. Fig. 7 Distribution of the accumulators of evidence across groups and conditions for object relative clauses. The dashed lines indicate the means of the distributions. Note that in the mismatch condition, for IWA, the means of the two distributions overlap Controls IWA Match Mismatch 2000 6000 10000 15000 2000 6000 10000 15000 Finishing times (ms) Accumulator NP1 NP2 Object relatives ss ct Controls IWA Match Mismatch 2000 6000 10000 15000 2000 6000 10000 15000 Finishing times (ms) Accumulator NP1 NP2 Object relatives Computational Brain & Behavior (2023) 6:473–502 487 The model estimates for the fixed effects and interactions on μNP1 and μNP2 are shown in Appendix D. No indication of an effect was found for condition or the condition × group interaction, but there was a RC type × condition interaction on μNP2 (631 ms, CrI: [385, 884] ms): Interference (match) in OR lead to higher finishing times for μNP2 relative to no-interference (mismatch). The three- way interaction RC type × group × condition for μNP2 (−249 ms, CrI: [−491, −10] ms) indicates that the effect of condition within RC is different for the two groups in the μNP2 accumulator: In the SR trials, the difference between match and mismatch conditions is bigger for controls. By contrast, in OR trials, the difference between match and mismatch conditions is bigger for IWA. in Fig. 8. While controls have a slightly lower θ in OR relative to SR in the match conditions, SR and OR have a similar θ in mismatch conditions, around 95%. Activation-Based Model This indicates that, in line with the model predictions, mismatch facilitates the retrieval of the target, especially in OR. The number mismatch also benefits IWA on average, but IWA exhibit a stronger subject-object asymmetry, irrespective of the number manipulation, with higher θ in SR relative to OR for both match and mismatch conditions. The estimates of the model confirm the patterns shown in Fig. 8. The effect of condition (−10% CrI: [−13, −6]%) suggests that match conditions elicit a lower θ across the board, but a condition × group interaction (7% CrI: [3, 10]%) suggests that the effect of condition is stronger for controls than for IWA. There was no indication of a RC type × condition interaction (−1% CrI: [−5, 2]%), nor of a RC type × condition × group interaction (1% CrI: [−3, 4]%). The effect of RC type (−13%, CrI: [−18, −9]%) and the RC type × group interaction (−7%, CrI: [−12, −3]%) indicate that object relatives are more difficult to process than subject relatives, and more so for IWA than for controls. There was no indication of an LDT × group interaction, a LDT × condition interaction, or a LDT × condition × group interaction. There was an effect of fixations on μNP2 (289 ms, CrI: [61, 521] ms). This main effect is uninformative, given that NP2 was the retrieval target in OR but not in SR. There was no indication of a fixations × condition interaction or a fixations × group × condition interaction, so that the role of fixations remains inconclusive. Finally, as predicted, IWA have higher noise than controls (σIWA 0.55 log ms, CrI: [0.53, 0.57 log ms], σcontrols 0.31 log ms, CrI: [0.3, 0.32 log ms]). There was no indication of an effect of LDT on θ (−3%, CrI: [−8, 2]%), nor of a group × LDT interaction (3%, CrI: [−2, 8]%). A unit increase in fixations led to −2%, CrI: [−5, 1]% on θ, and the interaction group × fixations 4%, CrI: [1, 7]% suggests that the effect of fixations is different for IWA and control participants: In IWA, an increase in fixations of looks to the target leads to a higher θ. Modified Direct-Access Model The posterior distributions of θ, the probability of initial retrieval of the target, by group and condition are displayed The estimated probability of backtracking given an initial incorrect retrieval is 35% CrI: [20, 50]% for IWA, whereas Fig. 8 Posterior distribution of the probability of retrieval of the target (θ) across groups and conditions in relative clauses. The vertical lines stand for the means of the distributions, and the shaded areas represent the 95% credible interval OR, mismatch OR, match SR, mismatch SR, match 20 40 60 80 100 [%] Controls OR, mismatch OR, match SR, mismatch SR, match 20 40 60 80 100 [%] IWA Relative clauses, posterior distribution of θ tion of al of the and uses. or the ns, and nt the OR, mismatch OR, match SR, mismatch SR, match 20 40 60 80 100 [%] Controls OR, mismatch OR, match SR, mismatch SR, match 20 40 60 80 100 [%] IWA Relative clauses, posterior distribution of θ IWA Relative clauses, posterior distribution of θ SR, mismatch Computational Brain & Behavior (2023) 6:473–502 488 20 40 60 80 [%] Group Controls IWA Posterior distribution of θb Fig. 9 Posterior distribution of the probability of retrieval of the target after backtracking (θb) across groups in the relative clauses experiment 20 40 60 80 [%] Posterior distribution of θb more difficulties processing object relatives, in line with previous findings. Posterior distribution of θb The conclusions for the rest of our predictions are more complex, since the results differ across the two experiments. For instance, a group × LDT interaction was found for the target accumulator in pronoun resolution. This interaction indicates that slower lexical access leads to increased processing difficulty for IWA, as predicted by the delayed lexical access theory. However, there was no indication of such an interaction in relative clauses. We therefore conclude that more research is needed in order to establish the role of delayed lexical access in the activation-based model. Fig. 9 Posterior distribution of the probability of retrieval of the target after backtracking (θb) across groups in the relative clauses experiment The effect of looks to the target at the critical region also remains inconclusive. No effect of fixations was found in pronoun resolution. In relative clauses, an effect of fixations was found for the NP2 accumulator, but no indication of an interaction between fixations and RC type was found. Modified Direct-Access Model Given that NP2 was the retrieval target in OR but not in SR, the main effect of fixations is uninformative. for controls, it is 87% CrI: [83, 91]%. The posterior distribution of θb is shown in Fig. 9: After backtracking, IWA retrieve the target about half of the time. By contrast, controls retrieve the target more than 80% of the time. In addition, IWA are estimated to need 3457 ms, CrI: [2425, 4659] ms for backtracking, whereas controls need 1829 ms, CrI: [1637, 2032] ms. Modified Direct-Access Model We expected similarity-based interference to result in a lower probability θ of successful retrieval for the target in the match conditions compared to the mismatch conditions. The data from both experiments are in line with this prediction. In addition, in Experiment 2, IWA show a large effect of relative clause type, irrespective of the condition: IWA have more difficulties understanding object relatives compared to subject relatives. This subject-object asymmetry is broadly in line with the accuracies in Adelt et al. (2017), although Adelt et al. (2017) found this pattern in both IWA and controls. We expected similarity-based interference to result in a lower probability θ of successful retrieval for the target in the match conditions compared to the mismatch conditions. Finally, as predicted, μ is higher for IWA (3744 ms, CrI: [3304, 4234] ms) than for controls (1613 ms, CrI: [1492, 1736] ms), and σ is also higher for IWA (0.41 log ms, CrI: [0.38 log ms, 0.44 log ms]) than for controls (0.24 log ms, CrI: [0.23 log ms, 0.24 log ms]). Discussion In the two models, the location and scale parameters (μ and σ) of the RT distribution were consistently higher for IWA than for controls. We linked these parameters to the slow syntax and intermittent deficiencies theories, respectively. Both models thus seem to be generally in line with these two theories of processing deficits in aphasia. We will now discuss the implications for the remaining theories within each model. The probability of backtracking is consistently lower for IWA than for controls, as is the probability of retrieval of the target after backtracking (θb). This pattern is expected under the resource reduction theory, assuming that backtracking makes use of parsing resources. In addition, the average cost of backtracking, δ, is twice as high for IWA compared to controls in both experiments. This adds support for the slow syntax theory. 11Given that our dataset contains data from two experimental sessions (test and retest), an alternative way to perform cross-validation would be to train the models on the test data and to use the retest data to compute predictive accuracy. However, we chose to use the pooled data from each of the two experiments, in order to maximize the amount of data, especially for IWA. General Discussion The procedure for 10-fold cross-validation is as follows: The data are partitioned into 10 balanced subsets containing about the same amount of data per subject.11 One of the 10 subsets is held out, and the model is fit to the remaining subsets. The posterior distributions from the resulting model are used to compute predictive accuracy on the held-out subset. This is repeated 10 times, so that all subsets are covered. The expected log pointwise predictive density,  elpd, is then calculated as a measure of predictive accuracy.  elpd is the summed log-likelihood of all observed, held- out data points under each model. Models are compared by computing the difference in  elpd, (  elpd), with higher  elpd indicating better predictive fit. Because  elpd is an estimate, the difference in  elpd between two models has an associated standard error, which has the standard frequentist interpretation:  elpd ± 2 × SE gives a 95% confidence interval. If the difference in  elpd between the models is greater than 2 × SE, we conclude that there are grounds to assume that the model with the higher  elpd provides the better fit for the given data. This is the first-ever computational investigation of com- peting models of similarity-based interference in German language comprehension in IWA and unimpaired controls. We investigated interference in two linguistic constructions, namely pronoun resolution and relative clauses. Two mod- els of cue-based retrieval were implemented in a Bayesian framework: The activation-based model of Lewis and Vasishth (2005) and a modified version of the direct-access model of McElree (2000) as implemented by Nicenboim and Vasishth (2018). The activation-based model assumes a direct connection between retrieval latency and retrieval probability for memory items, whereas the modified direct- access model assumes a constant retrieval latency, along with a costly backtracking mechanism that triggers when retrieval fails. In the original direct-access model, back- tracking leads to the correct retrieval of the target item from memory (McElree, 1993). In our modified direct-access model, backtracking can fail, leading to a costly misre- trieval. We argue that this is a more suitable model for individuals with aphasia, as it can account for slow incorrect responses, a pattern that is frequently found in the apha- sia literature (Hanne et al., 2015; Adelt et al., 2017; Liss´on et al., 2021b; Pregla et al., 2021). Activation-Based Model According to the delayed lexical access theory, IWA should be more affected by delays in lexical access, as measured by a lexical decision task. The observed group × LDT interaction lends some support to this theory in the pronoun resolution sub-experiment, but not in relative clauses. Therefore, the effect of delayed lexical access in the modified direct-access model remains inconclusive, as for the activation-based model. The effect of fixations is also inconclusive: Although in relative clauses there is some indication that fixations at the critical region may lead to a increase in the probability of retrieving the target We hypothesized that the accumulators in the activation- based model should reflect the interference effect predicted by cue-based retrieval theory, namely, lower mean finishing times for the target accumulator, and similar or higher mean finishing time for the distractor accumulator in the mismatch conditions compared to the match conditions. The accumulators show this pattern across the two experiments. In addition, in Experiment 2, the distribution of the accumulators across relative clause types shows that IWA experience a subject-object asymmetry, that is, IWA have Computational Brain & Behavior (2023) 6:473–502 489 Table 1 Differences in  elpd between the two models and their corresponding SE Model  elpd SE Pronoun resolution −109 133 Relative clauses 403 167 Positive differences indicate an advantage for the activation-based model, whereas negative differences indicate an advantage for the modified direct-access model Table 1 Differences in  elpd between the two models and their corresponding SE for IWA, no effect of fixations was found in pronoun resolution. for IWA, no effect of fixations was found in pronoun resolution. Model Comparisons The activation-based model and the modified direct-access model make different assumptions about the retrieval mechanism, and thus the generative process behind the observed data. Within the framework of each model’s assumptions, conclusions can be drawn about plausible underlying deficits. However, one crucial question remains open: Which model captures the generative process better? In order to answer this question, we performed 10-fold cross-validation (Vehtari, Gelman, & Gabry, 2017; see also Nicenboim et al., 2021, chapter 17, for a tutorial on carrying-out cross-validation for Bayesian models such as the ones discussed here). This is a standard procedure in machine learning for quantifying the relative predictive fit of two or more models. Importantly, cross-validation can also be applied when the models assume different generative processes, as is the case with the activation-based and the modified direct-access models. Positive differences indicate an advantage for the activation-based model, whereas negative differences indicate an advantage for the modified direct-access model the activation-based model has a clear advantage over the modified direct-access model, but the advantage is mostly driven by the control participants, as shown in Appendix E. the activation-based model has a clear advantage over the modified direct-access model, but the advantage is mostly driven by the control participants, as shown in Appendix E. We also evaluated the predictive performance of the original direct-access model, that is, a model in which backtracking can only lead to the retrieval of the target. The results are shown in Appendix F. In relative clauses, the activation-based model outperforms both the original and the modified-direct access model, while the result for pronoun resolution is inconclusive. We also evaluated the predictive performance of the original direct-access model, that is, a model in which backtracking can only lead to the retrieval of the target. The results are shown in Appendix F. In relative clauses, the activation-based model outperforms both the original and the modified-direct access model, while the result for pronoun resolution is inconclusive. Number Mismatch Versus Subject-Object Asymmetry assumed to occur, were used as a predictor, along with the mean reaction times from a lexical decision task. We linked the parameters of each computational model to prominent theories of processing deficits in aphasia, aiming to answer two main questions: (a) Which model is better able to fit the data from IWA and control participants across the two experiments? and (b) What do the parameters in each model tell about the processing deficits and about interference in IWA? We will now discuss the answers to these questions, as well as the relation of our results to prior work in computational modeling of processing deficits in aphasia. The results of the models show that for IWA, the presence of two candidate NPs with distinctive number features is of limited use in both subject and object relatives with regard to successful comprehension (see also the descriptive statistics for relative clauses, split by condition, in Appendix B). The modified direct-access model estimates no effect of number mismatch for IWA, while pronouns did show some indication of a gender mismatch effect. The activation-based model estimates that number mismatch between the NPs in object relative clauses slightly increases the probability of retrieving the target; however, the target is only retrieved about half the time in these conditions. For IWA, the main difference is between subject and object relative clauses, not between high- and low-interference conditions, as shown in Fig. 10. The subject-object asymmetry in relative clauses in IWA is in line with the canonicity effects reported in several German studies with IWA (e.g., Burchert et al., 2003; Burchert & de Bleser, 2004; Hanne et al., 2011; Adelt et al., 2017; Pregla et al., 2021). Canonicity effects refer to the fact that sentences with non-canonical word order (e.g., object-subject-verb in German) are more difficult to process than sentences with canonical word order. First, both models of retrieval perform well across the two linguistic constructions tested, in the sense that the relevant parameters are affected in the expected direction by group differences and by similarity-based interference. The activation-based model outperforms the modified direct- access model in the relative clauses experiment, mainly because it provides a better predictive fit for the data from control participants. However, both models perform similarly at fitting data from IWA. In pronoun resolution, the two models show similar predictive fit across groups and across conditions. Number Mismatch Versus Subject-Object Asymmetry Second, with regard to the underlying processing deficits in aphasia, both models are in line with slow syntax (Burkhardt et al., 2003; Burkhardt et al., 2008) and intermit- tent deficiencies (Caplan et al., 2013). Resource reduction (Caplan et al., 2007; Caplan, 2012), as implemented here, can only be evaluated with respect to the modified direct- access model, and the results show that the model is in line with this deficit. There was no strong indication in our data, across the two experiments and for both models, that delayed lexical access (Love et al., 2008; Ferrill et al., 2012) is a source of processing deficits in IWA: The predicted relationship between individual lexical decision latency and participant group was only found in some conditions. More experiments are needed in order to explore the role of this deficit. In contrast to English, where subject and object relative clauses are distinguished by the subject NP intervening or not intervening in the object-verb dependency, cue-based retrieval cannot explain the subject-object asymmetry in German: Both in subject and in object relative clauses, the verb is clause-final and two NPs have to be retrieved, one that is adjacent to the verb and one that is not. Consequently, cue-based retrieval would predict no processing difference between subject and and object relatives in German. One possible explanation for the differential effects of number marking and RC type is that German relative clauses feature case marking, and that IWA may pay more attention to case than to number cues. Case marking may be more difficult to process in German object relative clauses compared to subject relative clauses, given that there is a case mismatch between the target noun phrase and the rel- ative pronoun in the former. One influential proposal is that of case attraction. Case attraction is analogous to the well-studied phenomenon of number attraction, which has been explained in terms of feature percolation (e.g., Schle- sewsky, 1996; Eberhard, 1997; Nicol, Forster, & Veres, 1997; Bader & Meng, 1999; Fanselow, Schlesewsky, Cavar, & Kliegl, 1999; Logaˇcev & Vasishth, 2012; Czypionka, D¨orre, & Bayer, 2018). If the head noun and the relative pronoun mismatch in case, as in object relatives, the [+nom- Regarding the effect of similarity-based interference, based on the results for the activation-based model, we can conclude that in pronoun resolution, IWA are more sensitive to gender interference than control participants. General Discussion The results of the cross-validation are shown in Table 1. In pronoun resolution, the modified direct-access model has a predictive advantage, but since the SE of  elpd is large, the result is not conclusive. In relative clauses, The predictive performance of the two models was compared against data from a visual-world experiment (Pregla et al., 2021), using the reaction time and accuracy in the picture selection task as dependent variables. Looks to the target at the critical sentence region, where retrieval is Computational Brain & Behavior (2023) 6:473–502 490 Number Mismatch Versus Subject-Object Asymmetry The interaction group × condition was inconclusive in the modified direct-access model. Both models estimate that in relative clauses, the effect of number interference is rather small for both groups. The models suggest that IWA experience a subject-object asymmetry, whereas control participants do not. Below, we discuss the comparatively small effect of number mismatch in relative clauses and some possible explanations of the subject-object asymmetry in IWA. Computational Brain & Behavior (2023) 6:473–502 491 Fig. 10 Descriptive statistics for the relative clauses experiment. The dots stand for the means, and the error bars show the standard error of the means 40 60 80 100 SR OR Accuracy [%] Accuracy 2000 3000 4000 5000 SR OR Reaction times [ms] Reaction times Group Controls IWA 40 60 80 100 SR OR Accuracy [%] Accuracy 2000 3000 4000 5000 SR OR Reaction times [ms] Reaction times authors found that IWA have a general processing advantage in the case-unambiguous conditions. The study of Adelt et al. (2020) supports the idea that IWA may rely more on case cues than on number cues in relative clauses. However, neither Adelt et al. (2020) nor Hanne et al. (2015) included both case and number cues within the same items. Our modeling shows that when both case and number cues are included in a sentence, IWA do not benefit from the extra number cue, suggesting that cue weighting may be a factor. inative] case feature of the head noun could percolate down to the relative pronoun, overriding its original [+accusative] case feature. Because the case feature of the relative pro- noun signals the syntactic role of the relativized noun phrase, the object relative could be misinterpreted as a sub- ject relative. The case attraction theory thus predicts that subject relatives are easier to process than object relatives, because the head noun and the relativizer have an identical [+nominative] feature in subject relatives. The proposal that IWA pay more attention to case than to number cues can be seen a differential weighting of retrieval cues. The cue-weighting proposal, implemented by Engelmann (2016) in the framework of the Lewis and Vasishth (2005) model, claims that depending on the linguistic structure, some retrieval cues may receive more weight, and therefore contribute more strongly to memory activation, than others (Dillon et al., 2013; Cunnings & Sturt, 2014; Parker & Phillips, 2017; Engelmann et al., 2019; Vasishth et al., 2019). Number Mismatch Versus Subject-Object Asymmetry Differences in weighting between case and number cues could be integrated in both the activation-based and the direct-access model, possibly at the individual participant level, as recently proposed by Yadav, Paape, Smith, Dillon, & Vasishth (2021). Limitations and Future Directions The data that we modeled in this paper (Pregla et al., 2021) is the largest-ever compilation of online measures for IWA in German. Nevertheless, the size of the IWA group (21 subjects) remains relatively small when compared to the number of subjects tested in typical eye-tracking experiments with unimpaired participants. Collecting online data from impaired populations is difficult, which is why most studies in the aphasia literature have a smaller number of participants. Usually, online experiments have 3 to 12 IWA and 10 to 20 control participants (e.g., Burkhardt et al., 2003; Love et al., 2008; Dickey & Thompson, 2009; Choy & Thompson, 2010; Hanne et al., 2011; Mack, Ji, & Thompson, 2013; Hanne et al., 2015; Mack et al., 2016; Adelt et al., 2017; Engel et al., 2018). An exception is the data presented in Caplan et al. (2013, 2015), with more than 50 IWA, although it does not include eye-tracking data. With respect to model comparisons, Liss´on et al. (2021a) found that the activation-based model had a predictive advantage over the original direct-access model, although the difference was not decisive, given the large standard error of  elpd. In the present study, we show that in relative clauses in German, the activation-based model provides a better fit than both the original and the modified direct- access model. Our results contrast with Nicenboim and Vasishth (2018), who compared the predictive performance of the activation-based model and the original direct-access model using self-paced reading data from unimpaired controls in German (Nicenboim et al., 2018). Nicenboim and Vasishth (2018) found that the original direct-access model provided a better fit to their data. However, the data modeled in Nicenboim et al. (2018) differ from our data in one important aspect: In correct trials (that is, trials with correct responses to the comprehension question), RTs were on average higher than the RTs in incorrect trials. This pattern in Nicenboim et al.’s data is crucial, because higher RTs for correct responses is what the original direct-access model assumes. Consequently, the advantage in predictive performance of the original direct- access model vs. the activation-based model came from the slow, correct responses. By contrast, our data show the opposite pattern. Correct responses are, on average, faster than incorrect responses, especially for IWA. Comparison with Previous Work In the present study, controls did not have a lower θ relative to IWA, and controls’ θ was always above 90% for all conditions. One possibility is that in our modeling, the RT at the end of the sentence do not reflect the agent-first bias, given that the initial interpretation has already been revised at this point. in the estimates for the direct-access model. Controls had a lower probability of initial correct retrieval than IWA in object relatives (controls 40%, IWA 50%). That is, controls were estimated to initially retrieve the distractor more often than the target in object relatives. The model could still account for the higher accuracy of controls by assuming a high probability of backtracking for controls (80%) relative to IWA (20%). This pattern supported the notion that controls initially processed the first noun phrase in object relatives as the agent, until they revised and corrected their interpretation by backtracking. Surprisingly, in the present modeling of the German data, we do not see such an agent- first bias. In the present study, controls did not have a lower θ relative to IWA, and controls’ θ was always above 90% for all conditions. One possibility is that in our modeling, the RT at the end of the sentence do not reflect the agent-first bias, given that the initial interpretation has already been revised at this point. activation-based model (Patil et al., 2016; M¨atzig et al., 2018; Liss´on et al., 2021a). If we assume that sentence comprehension is mediated by an activation-based model of cue-based retrieval, the performance of IWA can be explained by a combination of processing deficits, namely slow syntax and intermittent deficiencies. Comparison with Previous Work Liss´on et al. (2021a) investigated English relative clause processing in IWA vs. controls using a large-scale dataset from Caplan et al. (2015). Liss´on et al. found an agent-first bias for control participants in English: In non-canonical clauses, such as object relatives, unimpaired controls tend to initially assign the agent role to the first noun phrase in the sentence, which is incorrect in object relatives. By contrast, IWA do not show an agent-first bias. The agent- first bias in unimpaired controls has been attested in visual- world studies in both English (Mack, Wei, Gutierrez, & Thompson, 2016, passives) and German (Hanne et al., 2015, OVS sentences; Hanne et al., 2015, object relatives). In these studies, control participants initially show increased looks to the foil picture in non-canonical sentences. The foil picture in non-canonical sentences depicts the canonical interpretation of the sentence. As soon as they hear the relevant morphological cues (e.g., the relativizer in unambiguous German relative clauses), control participants start looking at the target picture. Studies that have investigated processing of number and case in IWA in German provide mixed evidence with regard to differential cue weighting. For instance, Hanne et al. (2015) investigated IWA’s use of case and number cues to interpret semantically reversible SVO vs. OVS sentences in German. Their data indicate that processing of case marking may be more impaired than processing of number marking. This contrasts with the results in Adelt, Burchert, Adani, & Stadie (2020), who tested case-unambiguous vs. case- ambiguous, number-disambiguated object relatives. The In the study of Liss´on et al. (2021a), which modeled self-paced listening data, this processing bias was reflected Computational Brain & Behavior (2023) 6:473–502 492 in the estimates for the direct-access model. Controls had a lower probability of initial correct retrieval than IWA in object relatives (controls 40%, IWA 50%). That is, controls were estimated to initially retrieve the distractor more often than the target in object relatives. The model could still account for the higher accuracy of controls by assuming a high probability of backtracking for controls (80%) relative to IWA (20%). This pattern supported the notion that controls initially processed the first noun phrase in object relatives as the agent, until they revised and corrected their interpretation by backtracking. Surprisingly, in the present modeling of the German data, we do not see such an agent- first bias. Limitations and Future Directions Although in the present paper we have implemented a modified direct- access model that can account for slow incorrect responses, the cross-validation shows that the activation-based model outperforms both the original and the modified-direct access model in relative clauses in German. The activation-based model outperforms the modified direct-access model in relative clauses, but both models perform similarly in pronoun resolution. We believe that the inability to find a clear answer in pronoun resolution has to do with the inherent limitations of the sample size, both in terms of subjects and items. Crucially, the relative clause experiment tested 40 items per subject (10 items per condition, 4 conditions), whereas the pronoun resolution tested 20 items per subject (10 items per condition, 2 conditions). Future work comparing these models will require much more data in order to distinguish between the models. We have reported the most complex hierarchical structure that yielded converging fits, but in order to account for individual differences, an even more complex hierarchical structure would be necessary. This is especially true for the modified direct-access model, where an individual adjustment for the effect of δ would help in understanding the variability in the process of backtracking. The same holds for both models regarding the effect of eye fixations in the visual world paradigm. In addition, this work focuses on average, group-level effects. While the comparisons between groups yield an estimate of the average performance, it has been shown that IWA have large within and between-subject variability (Patil et al., 2016; M¨atzig et al., 2018; Pregla et al., 2021). Overall, our results are in line with previous modeling work in sentence comprehension in aphasia using the Computational Brain & Behavior (2023) 6:473–502 493 A future direction is to develop an individual-level modeling approach, in which parameters are estimated for each subject, in a similar vein to the modeling work in M¨atzig et al. (2018) for offline data. Recent modeling work shows that under the cue-based retrieval, even among unimpaired participants, individual differences can modulate interference effects (Yadav et al., 2021). Therefore, obtaining individual parameter estimates for IWA would be more informative regarding both interference effects, and the extent to which each processing deficit plays a role for each IWA. Furthermore, the possible role of feature percolation, that is, of target features being overwritten by features of distractor items, has been recently investigated by Yadav, Smith, & Vasishth (2021). Conclusion We conducted the first large-scale evaluation of two computational models of sentence processing in individuals with aphasia (IWA) in German. Our study tested two competing models of cue-based retrieval — the activation- based model and a modified version of the direct-access model — against online and offline data from IWA and control participants. The data came from a visual- world eye-tracking experiment with a picture selection task. Similarity-based interference was manipulated in two linguistic constructions, namely pronoun resolution and relative clauses. Reaction times from the picture selection task were modeled as a function of interference, group (IWA vs. control), lexical access speed, and fixations to the target picture at the critical region of the sentence. The results show that in pronoun resolution, IWA experience greater gender interference effects relative to control participants. In relative clauses, the data suggest that IWA exhibit a larger subject-object asymmetry than controls. In IWA, the subject-object asymmetry is much stronger than the effect of number interference, suggesting that in relative clauses, IWA may rely more strongly on case cues compared to number cues. The parameter estimates from both implemented models are in line with the slow syntax and the intermittent deficiencies accounts. In addition, the parameters of the modified direct-access model are also in line with the resource reduction theory. The cross- validation results show that while both models have similar quantitative performance for the pronoun structures, the activation-based model outperforms the modified direct- access model in relative clauses. A second limitation of this work concerns the implemen- tation of the models. In order to compare the two competing models in a common architecture, we implemented simpli- fied versions that focus on a single retrieval event. However, in order to account for sentence processing deficits in IWA across the whole trial, the models should include a parser. The original Lisp implementation of the activation-based model in ACT-R (Anderson et al., 2004; Lewis & Vasishth, 2005) includes a left-corner parsing algorithm that could conceivably be added to our model. However, as far as we are aware, there exists no computational implementation of a parser in the (modified) direct-access model. A future direction would be to incorporate a parser into the (mod- ified) direct-access model, and to fit Bayesian versions of both models that account for individual parsing steps. Conclusion This is especially challenging because it requires finding a com- mon architecture that supports a parsing algorithm for both models, so that model comparisons can be performed. Finally, another limitation concerns the use of looks to the target at the critical region as an index of retrieval. An analysis of eye fixation patterns across the entire trial would possibly be more informative regarding the time- course of interference during sentence processing. This is especially true for relative clauses, where our results show that higher fixations to the target at the critical region do not lead to faster reaction times in the picture selection task. Specific modeling techniques for visual-world-data could be considered, such as growth curve analysis (Mirman, 2017) or divergence point analysis (Stone, Lago, & Schad, Limitations and Future Directions Their results support the view that in unimpaired populations, feature percolation feeds retrieval processes, suggesting that a hybrid model may better explain similarity-based interference. 2020). Integrating these analyses with our computational modeling approach may be possible, and may yield important insights into the differences between IWA and controls over the course of the trial. Note: Age and education are given in years. T3 and T11 are part of the LEMO 2.0 (Stadie et al., 2013) battery of tests and the scores given in the table are raw scores; T3 (n=80) = auditory lexical decision, T11 (n=20) = auditory word-picture matching. IWA, individual with aphasia; P.O., post-onset; F, female; M, male; L, left; R, right; IMI, ischemic arteria cerebri media infarct; I, infarct; MA, arteria cerebri media aneurysm; VH, vertebrobasilar hemorrhage Appendix A: Details about the individuals with aphasia Table 2, adapted from Pregla et al. (2021), shows a summary of the demographics and neurological information about the individuals with aphasia that took part in the experiments. Computational Brain & Behavior (2023) 6:473–502 494 Table 2 Demographic and neurological data of the individuals with aphasia that took part in the experiment, adapted from Pregla et al. (2021) IWA Gender Age Education Years P.O. Etiology Localization T3 T11 Aphasia type 2 F 72 8 7 IMI L 77 19 Anomic 3 M 76 20 17 IMI L/R 61 20 Not-classifiable 4 F 47 13 21 IMI L 78 20 Anomic 6 M 55 14 10 IMI L 67 20 Anomic 8 F 51 19 7 MA L 74 20 Anomic 9 M 64 15 2 IMI L 73 20 Anomic 10 M 58 18 1 IMI L 52 20 Broca 11 F 63 12 1 IMI L 73 20 Broca 12 F 46 12 13 IMI L 65 20 Broca 13 M 74 13 8 IMI L 57 20 Broca 14 M 66 13 17 IMI L 75 20 Anomic 15 F 59 21 4 I L 77 20 Broca 16 M 67 17 26 VH R 72 19 Broca 17 F 43 14 10 IMI L 65 20 Broca 18 M 57 13 1 I L 67 18 Wernicke 19 F 52 19 8 IMI L 76 20 Broca 20 M 38 13 3 IMI L 73 19 Broca 21 M 57 18 2 IMI L 66 18 Broca 22 F 67 16 5 IMI L 76 20 Anomic 23 M 74 15 7 IMI L 67 20 Anomic Note: Age and education are given in years. T3 and T11 are part of the LEMO 2.0 (Stadie et al., 2013) battery of tests and the scores given in the table are raw scores; T3 (n=80) = auditory lexical decision, T11 (n=20) = auditory word-picture matching. IWA, individual with aphasia; P.O., post-onset; F, female; M, male; L, left; R, right; IMI, ischemic arteria cerebri media infarct; I, infarct; MA, arteria cerebri media aneurysm; VH, vertebrobasilar hemorrhage Note: Age and education are given in years. T3 and T11 are part of the LEMO 2.0 (Stadie et al., 2013) battery of tests and the scores given in the table are raw scores; T3 (n=80) = auditory lexical decision, T11 (n=20) = auditory word-picture matching. Appendix A: Details about the individuals with aphasia IWA, individual with aphasia; P.O., post-onset; F, female; M, male; L, left; R, right; IMI, ischemic arteria cerebri media infarct; I, infarct; MA, arteria cerebri media aneurysm; VH, vertebrobasilar hemorrhage Computational Brain & Behavior (2023) 6:473–502 495 Appendix B: Descriptive statistics Figure 11 shows descriptive statistics for the pronoun sub-experiment by group and condition. Figure 12 shows descriptive statistics for the relative clause sub-experiment by group and condition. 40 60 80 100 Match Mismatch Accuracy [%] Group Controls IWA Accuracy 2000 3000 4000 Match Mismatch Reaction times [ms] Group Controls IWA Reaction times −0.10 −0.05 0.00 0.05 0.10 Match Mismatch Proportions Group Controls IWA Fixations to the target picture at the critical region Pronoun Fig. 11 Descriptive statistics for the pronoun sub-experiment. The dots stand for the means, and the error bars show the standard error of the means. In the plot showing the fixations to the target picture at the critical region, the proportions are centered and scaled within groups 40 60 80 100 Match Mismatch Accuracy [%] Group Controls IWA Accuracy Pronoun Pronoun 2000 3000 4000 Match Mismatch Reaction times [ms] Group Controls IWA Reaction times Reaction times Reaction times Group −0.10 −0.05 0.00 0.05 0.10 Match Mismatch Proportions Group Controls IWA Fixations to the target picture at the critical region Fixations to the target picture at the critical region Fixations to the target picture at the critical region Fig. 11 Descriptive statistics for the pronoun sub-experiment. The dots stand for the means, and the error bars show the standard error of the means. In the plot showing the fixations to the target picture at the critical region, the proportions are centered and scaled within groups Computational Brain & Behavior (2023) 6:473–502 496 Fig. 12 Descriptive statistics for the relative clauses sub-experiment. The dots stand for the means, and the error bars show the standard error of the means. Appendix B: Descriptive statistics In the plot showing the fixations to the target picture at the critical region, the proportions are centered and scaled within groups SR OR Match Mismatch Match Mismatch 40 60 80 100 Accuracy [%] Group Controls IWA Accuracy SR OR Match Mismatch Match Mismatch 2000 3000 4000 5000 Reaction times [ms] Group Controls IWA Reaction times SR OR Match Mismatch Match Mismatch −0.1 0.0 0.1 0.2 Proportions Group Controls IWA Fixations to the target picture at the critical region Relative clauses SR OR Match Mismatch Match Mismatch 40 60 80 100 Accuracy [%] Group Controls IWA Accuracy SR OR Match Mismatch Match Mismatch 2000 3000 4000 5000 Reaction times [ms] Group Controls IWA Reaction times SR OR Match Mismatch Match Mismatch −0.1 0.0 0.1 0.2 Proportions Group Controls IWA Fixations to the target picture at the critical region Relative clauses SR OR Match Mismatch Match Mismatch 40 60 80 100 Accuracy [%] Group Controls IWA Accuracy Relative clauses Relative clauses SR Match Mismatch 40 60 80 100 Accuracy [%] Accuracy Rela SR OR Match Mismatch Match Mismatch 2000 3000 4000 5000 Reaction times [ms] Group Controls IWA Reaction times Mismatch Fixations to the target picture at the critical region SR OR Match Mismatch Match Mismatch −0.1 0.0 0.1 0.2 Proportions Group Controls IWA Fixations to the target picture at the critical region OR Match Mismatch Group Controls IWA 497 Computational Brain & Behavior (2023) 6:473–502 RTi = min(FTNP1i, FTNP2i) (3) (3) Equation (7) shows the priors used in the direct-access model. The priors for α, αb, and γ are in logit space, and the rest of priors are in log space. The hierarchical structure in the location parameter of the lognormal distribution for each accumulator (μ1 and μ2) and in the standard deviation (σ) is shown in Eq. (4). α ∼normal(1, 0.5) αb ∼normal(0, 1) β1,...,12 ∼normal(0, 0.5) μ0 ∼normal(7.5, 0.6) γ ∼normal(−1, 0.5) δ0 ∼normal(0, 1) σ0 ∼normal(0, 0.5) (7) Accumulator NP1 μNP1 = α1 + uα1 + wα1 + (β1 + wβ1) · group+ (β3 + uβ3) · condition + β5 · group · condition+ β7 · LDT + β9 · group · LDT + β11 · fixations + β13 · fixations · group Accumulator NP2 μNP2 = α2 + uα2 + wα2 + (β2 + wβ2) · group+ (β4 + uβ4) · condition + β6 · group · condition+ β8 · LDT + β10 · group · LDT + β12 · fixations + β14 · fixations · group Noise parameter σ = σ0 + β15 · group (4) Accumulator NP1 μNP1 = α1 + uα1 + wα1 + (β1 + wβ1) · group+ (β3 + uβ3) · condition + β5 · group · condition+ β7 · LDT + β9 · group · LDT + β11 · fixations + β13 · fixations · group Accumulator NP2 μNP2 = α2 + uα2 + wα2 + (β2 + wβ2) · group+ (β4 + uβ4) · condition + β6 · group · condition+ β8 · LDT + β10 · group · LDT + β12 · fixations + β14 · fixations · group Noise parameter σ = σ0 + β15 · group (4) Accumulator NP1 (7) Accumulator NP2 Appendix D: Model estimates (activation-based model) (4) Appendix C: Details about the computational implementations μ = μ0 + uμ0 + wμ0 + β1 · group θ = α + uα + wα + β2 · LDT + β3 · LDT · group (β4 + wβ4) · group + (β5 + uβ5) · condition+ β6 · group · condition + β7 · fixations+ Accumulator NP1 β4 β5 β6 · group · condition + β7 · fixations+ FTNP1i ∼lognormal(μ1, σ) Accumulator NP2 FTNP2i ∼lognormal(μ2, σ) (2) (6) g p β8 · group · fixations g p β8 · group · fixations β8 · group · fixations θb = αb + uαb + β9 · group Pb = γ + uγ + β10 · group δ = δ0 + β11 · group σ = σ0 + β12 · group (6) β8 · group · fixations θb = αb + uαb + β9 · group Pb = γ + uγ + β10 · group δ = δ0 + β11 · group σ = σ0 + β12 · group (2) RTi = min(FTNP1i, FTNP2i) (3) RTi = min(FTNP1i, FTNP2i) (3) Appendix C: Details about the computational implementations condition, fixations × RC type, fixations × group × RC type. The slope for the effect of RC type also had a by- subject adjustment. Equation (5) shows the priors used in the activation-based model, all in log space. For both the activation-based and the direct-access model, the varying intercepts and slopes for subject, u, come from a multivariate normal distribution with x dimensions (where x = number of by-subject adjustments), abbreviated as MV Nx. The varying intercepts and slopes for items, w, also come from a multivariate normal distribution with y dimensions, MV Ny (where y = number of by- item adjustments). In the equations below, 0 is a column vector of zeros with the x or y dimensions. The σ are the variance-covariance matrices of the multivariate normal distributions. α1,2 ∼normal(7.5, 0.6) β1,...,15 ∼normal(0, 0.5) σ0 ∼normal+(0, 0.5) (5) (5) σ0 ∼normal+(0, 0.5) Direct-Access Model In Eq. (6), the parameter θ stands for the probability of retrieval of the target; μ is the location of the distribution from which the reaction times are sampled. Pb is the probability of backtracking, δ is the cost of backtracking (in log ms), and σ is the noise parameter. In addition to the adjustments shown in (6), in the model for relative clauses, the following fixed effects were added to θ: RC type, RC type × condition, RC type × group, and RC type × group × condition. The slope for the effect of RC type also had a by-subject adjustment. Direct-Access Model In Eq. (6), the parameter θ stands for the probability of retrieval of the target; μ is the location of the distribution from which the reaction times are sampled. u = MV Nx(0, u)w = MV Ny(0, w) (1) (1) Activation-Based Model For each trial i, values from two independent distributions are sampled, as shown in Eq. (2). The lowest value becomes the estimated reaction time for trial i (see Eq. (3)). Pronouns In addition to the adjustments shown in Eq. (4), in the model for relative clauses, the following fixed effects were also added to both accumulators: RC type, RC type × condition, RC type × group, RC type group × group × Table 3 shows model estimates for the activation-based model for the pronoun sub-experiment. Computational Brain & Behavior (2023) 6:473–502 498 Table 3 Model estimates for the fixed effects on μNP1 and μNP2 and corresponding credible intervals, backtransformed to ms Parameter Estimate 95% CrI Accumulator (μ) Group 2006 ms CrI: [1604, 2453] ms NP1 Group −797 ms CrI: [−2459, 629] ms NP2 Condition 310 ms CrI: [193, 433] ms NP1 Condition −1478 ms CrI: [−2820, -431] ms NP2 Condition × group 199 ms CrI: [81, 322] ms NP1 Condition × group 1424 ms CrI: [400, 2720] ms NP2 LDT 836 ms [539, 1152] ms NP1 LDT 588 ms [−621, 1811] ms NP2 LDT × group 671 ms [377, 973] ms NP1 LDT × group 51 ms [−1147, 1243] ms NP2 Fixations 41 ms [−77, 160] ms NP1 Fixations 518 ms [−141, 1212] ms NP2 Fixations × group 96 ms [−23, 219] ms NP1 Fixations × group 251 ms [−430, 949] ms NP2 Appendix F: Comparisons with original direct-access model Adelt, A., Burchert, F., Adani, F., & Stadie, N. (2020). What matters in processing German object relative clauses in aphasia–timing or morpho-syntactic cues? Aphasiology, 34(8), 970–998. The  elpd between the original direct-access model and the modified direct-access model is −41, SE 134 for pronoun resolution; and −175, SE 166 for relative clauses. The negative  elpd estimates indicate that the modified direct- access model may have a better predictive performance, but given the large SE, the  elpd are inconclusive. When comparing the activation-based model with the original direct-access model, the difference is not conclusive in the pronoun experiment (  elpd −68, SE 140), but in relative clauses, the activation-based model performs better (  elpd 578, SE 173). Thus, in relative clauses, the activation-based model outperforms both the original and the modified-direct access model. Adelt, A., Stadie, N., Lassotta, R., Adani, F., & Burchert, F. (2017). Feature dissimilarities in the processing of German relative clauses in aphasia. Journal of Neurolinguistics, 44, 17–37. Anderson, J. R., Bothell, D., Byrne, M. D., Douglass, S., Lebiere, C., & Qin, Y. (2004). An integrated theory of the mind. Psychological Review, 111(4), 1036–1060. Arantzeta, M., Webster, J., Laka, I., Mart´ınez-Zabaleta, M., & Howard, D. (2018). What happens when they think they are right? Error awareness analysis of sentence comprehension deficits in aphasia. Aphasiology, 32(12), 1418–1444. Badecker, W., & Straub, K. (2002). The processing role of structural constraints on interpretation of pronouns and anaphors. Journal of Experimental Psychology: Learning, Memory, and Cognition, 28(4), 748. Bader, M., & Meng, M. (1999). Case attraction phenomena in German. Unpublished Manuscript. University of Jena. Author Contribution The first draft of the manuscript was written by Paula Liss´on and all authors commented on previous versions of the manuscript. Dario Paape carried out revisions after the first round of review. All authors read and approved the final manuscript. Data analysis and modeling were carried out by Paula Liss´on and supervised by Shravan Vasishth and Dario Paape. Nicole Stadie, Frank Burchert, and Dorothea Pregla contributed to the study conception and design. Material preparation and data collection were performed by Dorothea Pregla. Bartek, B., Lewis, R. L., Vasishth, S., & Smith, M. R. (2011). In search of on-line locality effects in sentence comprehension. Journal of Experimental Psychology: Learning, Memory, and Cognition, 37(5), 1178–1198. Burchert, F., & de Bleser, R. (2004). Passives in agrammatic sentence comprehension: A German study. Aphasiology, 18(1), 29–45. Appendix E: Cross-validation results by group and construction Appendix E: Cross-validation results by group and construction Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by the Deutsche Forschungsgemein- schaft (DFG) - Project-ID 317633480 - SFB1287, project B02 (PIs: Shravan Vasishth, Frank Burchert, and Nicole Stadie). Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by the Deutsche Forschungsgemein- schaft (DFG) - Project-ID 317633480 - SFB1287, project B02 (PIs: Shravan Vasishth, Frank Burchert, and Nicole Stadie). Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by the Deutsche Forschungsgemein- schaft (DFG) - Project-ID 317633480 - SFB1287, project B02 (PIs: Shravan Vasishth, Frank Burchert, and Nicole Stadie). Figure 13 shows cross-validation results by group and construction. Code Availability The data generated and/or analyzed during the current study are available in the OSF repository, https://tinyurl.com/ yz9acste. Controls, OR, match Controls, OR, mismatch Controls, SR, match Controls, SR, mismatch IWA, OR, match IWA, OR, mismatch IWA, SR, match IWA, SR, mismatch −100 0 100 200 300 Δelpd Activation−based model vs. MDA Fig. 13 Graphical representation of the  elpd between the activation- based and the modified direct-access model across groups and conditions. The dot stands for the  elpd and the bars indicate to the 95% confidence interval. Positive values indicate an advantage for the activation-based model, and negative values indicate an advantage for the modified direct-access model (MDA) Controls, OR, match Controls, OR, mismatch Controls, SR, match Controls, SR, mismatch IWA, OR, match IWA, OR, mismatch IWA, SR, match IWA, SR, mismatch −100 0 100 200 300 Δelpd Activation−based model vs. MDA Conflict of Interest The authors declare no 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 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/. Fig. 13 Graphical representation of the  elpd between the activation- based and the modified direct-access model across groups and conditions. The dot stands for the  elpd and the bars indicate to the 95% confidence interval. Positive values indicate an advantage for the activation-based model, and negative values indicate an advantage for the modified direct-access model (MDA) Consent to Participate Not applicable. Consent for Publication Not applicable. Consent for Publication Not applicable. Conflict of Interest The authors declare no competing interests. Relative Clauses Table 4 shows model estimates for the activation-based model for the relative clauses sub-experiment. Table 4 Model estimates for the fixed effects on μNP2 and μNP2 and corresponding credible intervals, backtransformed to ms Parameter Estimate 95% CrI Accumulator (μ) Group 2348 ms [1687, 3055] ms NP1 Group 4057 ms [3279, 4916] ms NP2 RC type 2417 ms [2076, 2807] ms NP1 RC type −1897 ms [−2372, -1459] ms NP2 Condition −68 ms [−262, 122] ms NP1 Condition −228 ms [−506, 40] ms NP2 Condition × group −37 ms [−224, 153] ms NP1 Condition × group 86 ms [−180, 355] ms NP2 RC type × group −1670 ms [−2019, -1366] ms NP1 RC type × group 1847 ms [1443, 2293] ms NP2 Condition × RC type −150 ms [−323, 19] ms NP1 Condition × RC type 631 ms [385, 884] ms NP2 Condition × RC type × group 103 ms [−68, 276] ms NP1 Condition × RC type × group −249 ms [−491, -10] ms NP2 LDT 779 ms [243, 1337] ms NP1 LDT 812 ms [252, 1398] ms NP2 LDT × group −50 ms [−594, 496] ms NP1 LDT × group −460 ms [−1045, 121] ms NP2 LDT × RC type −5 ms [−258, 255] ms NP1 LDT × RC type 19 ms [−347, 385] ms NP2 LDT × RC type × group 5 ms [−251, 262] ms NP1 LDT × RC type × group 291 ms [−72, 677] ms NP2 Fixations −37 ms [−205, 130] ms NP1 Fixations 289 ms [61, 521] ms NP2 Fixations × group −46 ms [−211, 118] ms NP1 Fixations × group 57 ms [−173, 285] ms NP2 Fixations × RC type 69 ms [−97, 235] ms NP1 Fixations × RC type −27 ms [−248, 204] ms NP2 Fixations × RC type × group 75 ms [−96, 244] ms NP1 Fixations × RC type × group −111 ms [−341, 112] ms NP2 Table 4 shows model estimates for the activation-based model for the relative clauses sub-experiment. ates for the activation-based model for the relative clauses sub-experiment. le 4 shows model estimates for the activation-based model for the relative clauses sub-experiment. Table 4 shows model estimates for the activation-based model for the relative clauses sub-experiment. 499 Computational Brain & Behavior (2023) 6:473–502 Declarations Ethics Approval Not applicable. Consent to Participate Not applicable. Appendix F: Comparisons with original direct-access model Caplan, D., Waters, G., DeDe, G., Michaud, J., & Reddy, A. (2007). A study of syntactic processing in aphasia I: Behavioral (psycholinguistic) aspects. Brain and Language, 101(2), 103–150. Gabry, J., Simpson, D., Vehtari, A., Betancourt, M., & Gelman, A. (2019). Visualization in Bayesian workflow. Journal of the Royal Statistical Society, 182, 389–402. Caramazza, A., & Zurif, E. (1976). Dissociation of algorithmic and heuristic processes in language comprehension: Evidence from aphasia. Brain and Language, 3(4), 572–582. Gelman, A., Carlin, J. B., Stern, H. S., Dunson, D. B., Vehtari, A., & Rubin, D. B. (2013). Bayesian data analysis. Boca Raton, FL: Chapman & Hall/CRC. Carpenter, B., Gelman, A., Hoffman, M. D., Lee, D., Goodrich, B., Betancourt, M. ,..., & Riddell, A. (2017). Stan: A probabilistic programming language. Journal of Statistical Software, 76(1), 1–32. Gordon, P. C., Hendrick, R., Johnson, M., & Lee, Y. (2006). Similarity- based interference during language comprehension: Evidence from eye tracking during reading. Journal of Experimental Psychology: Learning, Memory, and Cognition, 32(6), 1304– 1321. Chomsky, N. (1977). On Wh-Movement. In P. Culicover, T. Wasow, & A. Akmajian (Eds.) Formal syntax. New York: Academic Press. Grodner, D., & Gibson, E. (2005). Consequences of the serial nature of linguistic input. Cognitive Science, 29, 261–290. Chomsky, N. (1981). Lectures on government and binding. Dordrecht: Foris. Hanne, S., Burchert, F., de Bleser, R., & Vasishth, S. (2015). Sentence comprehension and morphological cues in aphasia: What eye- tracking reveals about integration and prediction. Journal of Neurolinguistics, 34, 83–111. Chow, W.-Y., Lewis, S., & Phillips, C. (2014). Immediate sensitivity to structural constraints in pronoun resolution. Frontiers in Psychology, 5, 630. Choy, J. J., & Thompson, C. K. (2010). Binding in agrammatic aphasia: Processing to comprehension. Aphasiology, 24(5), 551– 579. Hanne, S., Sekerina, I. A., Vasishth, S., Burchert, F., & de Bleser, R. (2011). Chance in agrammatic sentence comprehension: What does it really mean? Evidence from eye movements of German agrammatic aphasic patients. Aphasiology, 25(2), 221–244. Comrie, B. (1985). Reflections on subject and object control. Journal of Semantics, 4(1), 47–65. J¨ager, L. A., Engelmann, F., & Vasishth, S. (2015). Retrieval interference in reflexive processing: Experimental evidence from Mandarin, and computational modeling. Frontiers in Psychology, 6, 617. Cunnings, I., & Sturt, P. (2014). Coargumenthood and the processing of reflexives. Journal of Memory and Language, 75, 117–139. Czypionka, A., D¨orre, L., & Bayer, J. (2018). Inverse Case attraction: Experimental evidence for a syntactically guided process. Appendix F: Comparisons with original direct-access model Burchert, F., de Bleser, R., & Sonntag, K. (2003). Does morphology make the difference? Agrammatic sentence comprehension in German. Brain and Language, 87(2), 323–342. 500 Computational Brain & Behavior (2023) 6:473–502 Engel, S., Shapiro, L. P., & Love, T. (2018). Proform-antecedent linking in individuals with agrammatic aphasia: A test of the Intervener Hypothesis. Journal of Neurolinguistics, 45, 79–94. Burchert, F., Hanne, S., & Vasishth, S. (2013). Sentence comprehen- sion disorders in aphasia: The concept of chance performance revisited. Aphasiology, 27(1), 112–125. Burkhardt, P., Avrutin, S., Piango, M. M., & Ruigendijk, E. (2008). Slower-than-normal syntactic processing in agrammatic Broca’s aphasia: Evidence from Dutch. Journal of Neurolinguistics, 21(2), 120–137. Engelmann, F. (2016). Toward an integrated model of sentence processing in reading. (Doctoral dissertation, University of Potsdam). Engelmann, F., J¨ager, L. A., & Vasishth, S. (2019). The effect of prominence and cue association in retrieval processes: A computational account. Cognitive Science, 43(12), e12800. Burkhardt, P., Pi˜nango, M. M., & Wong, K. (2003). The role of the anterior left hemisphere in real-time sentence comprehension: Evidence from split intransitivity. Brain and Language, 86(1), 9–22. Fanselow, G., Schlesewsky, M., Cavar, D., & Kliegl, R. (1999). Optimal parsing: Syntactic parsing preferences and optimality theory, vol. 367. Rutgers State University of New Jersey. B¨urkner, P.-C. (2017). brms: An R Package for Bayesian multilevel models using stan. Journal of Statistical Software, 80(1), 1–28. Fedorenko, E., Gibson, E., & Rohde, D. (2006). The nature of working memory capacity in sentence comprehension: Evidence against domain-specific working memory resources. Journal of Memory and Language, 54(4), 541–553. Caplan, D. (2012). Resource reduction accounts of syntactically based comprehension disorders. In C. K. Thompson, & R. Bastiannse (Eds.) Perspectives on Agrammatism, pp. 34–48. Caplan, D., Michaud, J., & Hufford, R. (2013). Dissociations and associations of performance in syntactic comprehension in aphasia and their implications for the nature of aphasic deficits. Brain and Language, 127(1), 21–33. Ferrill, M., Love, T., Walenski, M., & Shapiro, L. P. (2012). The time-course of lexical activation during sentence comprehension in people with aphasia. American Journal of Speech-Language Pathology, 21(2), S179. g g Caplan, D., Michaud, J., & Hufford, R. (2015). Mechanisms under- lying syntactic comprehension deficits in vascular aphasia: New evidence from self-paced listening. Cognitive Neuropsychology, 32(5), 283–313. Friederici, A. D., Steinhauer, K., Mecklinger, A., & Meyer, M. (1998). Working memory constraints on syntactic ambiguity resolution as revealed by electrical brain responses. Biological Psychology, 47(3), 193–221. Appendix F: Comparisons with original direct-access model The Journal of Comparative Germanic Linguistics, 21(2), 135–188. J¨ager, L. A., Engelmann, F., & Vasishth, S. (2017). Similarity-based interference in sentence comprehension: Literature review and Bayesian meta-analysis. Journal of Memory and Language, 94, 316–339. Dickey, M. W., Choy, J. J., & Thompson, C. K. (2007). Real- time comprehension of wh-movement in aphasia: Evidence from eyetracking while listening. Brain and Language, 100(1), 1–22. J¨ager, L. A., Mertzen, D., Van Dyke, J. A., & Vasishth, S. (2020). Interference patterns in subject-verb agreement and reflexives revisited: A large-sample study. Journal of Memory and Language, 111, 104063. Dickey, M. W., & Thompson, C. K. (2009). Automatic processing of wh- and NP-movement in agrammatic aphasia: Evidence from eyetracking. Journal of Neurolinguistics, 22(6), 563–583. Laurinavichyute, A., J¨ager, L. A., Akinina, Y., Roß, J., & Dragoy, O. (2017). Retrieval and encoding interference: Cross-linguistic evidence from anaphor processing. Frontiers in Psychology, 8, 965. Dillon, B., Mishler, A., Sloggett, S., & Phillips, C. (2013). Contrasting intrusion profiles for agreement and anaphora: Experimental and modeling evidence. Journal of Memory and Language, 69(2), 85–103. Eberhard, K. M. (1997). The marked effect of number on subject–verb agreement. Journal of Memory and Language, 36(2), 147–164. Lewis, R. L. (1999). Specifying architectures for language processing: Process, control, and memory in parsing and interpretation. In M. W. Crocker, M. Pickering, & C. Clifton (Eds.) Architectures and mechanisms for language processing, pp. 56–89. Edwards, S., & Varlokosta, S. (2007). Pronominal and anaphoric refe- rence in agrammatism. Journal of Neurolinguistics, 20(6), 423–444. Computational Brain & Behavior (2023) 6:473–502 501 Chapman and Hall/CRC statistics in the social and behavioral sciences series. Lewis, R. L., & Vasishth, S. (2005). An activation-based model of sentence processing as skilled memory retrieval. Cognitive Science, 29(3), 375–419. Nicenboim, B., & Vasishth, S. (2016). Statistical methods for linguistic research: Foundational ideas - Part II. Language and Linguistics Compass, 10(11), 591–613. Lewis, R. L., Vasishth, S., & Van Dyke, J. A. (2006). Computational principles of working memory in sentence comprehension. Trends in Cognitive Sciences, 10(10), 447–454. Nicenboim, B., & Vasishth, S. (2018). Models of retrieval in sentence comprehension: A computational evaluation using Bayesian hierarchical modeling. Journal of Memory and Language, 99, 1–34. Liss´on, P., Pregla, D., Nicenboim, B., Paape, D., Van Het Nederend, M. L., Burchert, F. ,..., & Vasishth, S. (2021a). A computational evaluation of two models of retrieval processes in sentence processing in aphasia. Cognitive Science, 45(4), e12956. Appendix F: Comparisons with original direct-access model Nicenboim, B., Vasishth, S., Engelmann, F., & Suckow, K. (2018). Exploratory and confirmatory analyses in sentence processing: A case study of number interference in German. Cognitive Science, 42, 1075–1100. Liss´on, P., Pregla, D., Paape, D., Burchert, F., Stadie, N., & Vasishth, S. (2021b). Modeling sentence comprehension deficits in aphasia: A computational evaluation of the direct-access model of retrieval. In Proceedings of the Workshop on Cognitive Modeling and Computational Linguistics, NAACL, pp. 177–185. Nicol, J. L., Forster, K. I., & Veres, C. (1997). Subject–verb agreement processes in comprehension. Journal of Memory and Language, 36(4), 569–587. Logaˇcev, P., & Vasishth, S. (2012). Case matching and conflicting bindings interference. In M. Lamers, & P. de Swart (Eds.) Case, word order and prominence, vol. 40, pp. 187–216. Oberauer, K. (2006). Reasoning with conditionals: A test of formal models of four theories. Cognitive Psychology, 53(3), 238–283. Logaˇcev, P., & Vasishth, S. (2016). Understanding underspecification: a comparison of two computational implementations. Quarterly Journal of Experimental Psychology, 69(5), 996–1012. Parker, D., & Phillips, C. (2017). Reflexive attraction in comprehen- sion is selective. Journal of Memory and Language, 94, 272–290. Parker, D., Shvartsman, M., & Van Dyke, J. A. (2017). The cue-based retrieval theory of sentence comprehension: New findings and new challenges. In L. Escobar, V. Torres, & T. Parodi (Eds.) Language processing and disorders, pp. 121–144. Newcastle: Cambridge Scholars Publishing. Love, T., Swinney, D., Walenski, M., & Zurif, E. (2008). How left inferior frontal cortex participates in syntactic processing: Evidence from aphasia. Brain and Language, 107(3), 203–219. Mack, J. E., Ji, W., & Thompson, C. K. (2013). Effects of verb meaning on lexical integration in agrammatic aphasia: Evidence from eyetracking. Journal of Neurolinguistics, 26(6), 619–636. Patil, U., Hanne, S., Burchert, F., de Bleser, R., & Vasishth, S. (2016). A computational evaluation of sentence processing deficits in aphasia. Cognitive Science, 40(1), 5–50. Mack, J. E., Wei, A. Z.-S., Gutierrez, S., & Thompson, C. K. (2016). Tracking sentence comprehension: Test-retest reliability in people with aphasia and unimpaired adults. Journal of Neurolinguistics, 40, 98–111. Patil, U., Vasishth, S., & Lewis, R. L. (2016). Retrieval interference in syntactic processing: The case of reflexive binding in English. Frontiers in Psychology, 7, 329. Martin, A. E., & McElree, B. (2008). A content-addressable pointer mechanism underlies comprehension of verb-phrase ellipsis. Journal of Memory and Language, 58(3), 879–906. Pregla, D., Liss´on, P., Vasishth, S., Burchert, F., & Stadie, N. (2021). Appendix F: Comparisons with original direct-access model Variability in sentence comprehension in aphasia in German. Brain and Language, 222, 105008. Martin, A. E., & McElree, B. (2011). Direct-access retrieval during sentence comprehension: Evidence from sluicing. Journal of Memory and Language, 64(4), 327–343. Pregla, D., Vasishth, S., Liss´on, P., Stadie, N., & Burchert, F. (2022). A visual world study of sentence processing in aphasia in German: The resource reduction hypothesis revisited. PsyArXiv preprint: psyarxiv.com/c456b. Martin, A. E., Nieuwland, M. S., & Carreiras, M. (2012). Event- related brain potentials index cue-based retrieval interference during sentence comprehension. Neuroimage, 59(2), 1859– 1869. R Core Team, R. (2020). R: A language and environment for statistical computing (Version 4.0.2). Vienna, Austria. Runner, J. T., & Head, K. D. (2014). What can visual world eye- tracking tell us about the binding theory? Empirical Issues in Syntax and Semantics, 10, 269–286. M¨atzig, P., Vasishth, S., Engelmann, F., Caplan, D., & Burchert, F. (2018). A computational investigation of sources of variability in sentence comprehension difficulty in aphasia. Topics in Cognitive Science, 10(1), 161–174. Schad, D. J., Betancourt, M., & Vasishth, S. (2021). Toward a principled Bayesian workflow in cognitive science. Psychological Methods, 26(1), 103–126. McAllister, T., Bachrach, A., Waters, G., Michaud, J., & Caplan, D. (2009). Production and comprehension of unaccusatives in aphasia. Aphasiology, 23, 989–1004. Schlesewsky, M. (1996). Kasusph¨anomene in der Sprachverarbeitung (Doctoral dissertation, University of Potsdam). McElree, B. (1993). The locus of lexical preference effects in sentence comprehension: A time-course analysis. Journal of Memory and Language, 32(4), 536–571. Schumacher, R., Cazzoli, D., Eggenberger, N., Preisig, B., Nef, T., Nyffeler, T. ,..., & M¨uri, R. M. (2015). Cue recognition and integration–eye tracking evidence of processing differences in sentence comprehension in aphasia. Plos One, 10(11), e0142853. McElree, B. (2000). Sentence comprehension is mediated by content- addressable memory structures. Journal of Psycholinguistic Research, 29(2), 111–123. Sorensen, T., Hohenstein, S., & Vasishth, S. (2016). Bayesian linear mixed models using stan: A tutorial for psychologists, linguists, and cognitive scientists. The Quantitative Methods for Psychology, 12(3), 175–200. McElree, B. (2006). Accessing recent events. Psychology of Learning and Motivation, 46, 155–200. McElree, B., Foraker, S., & Dyer, L. (2003). Memory structures that subserve sentence comprehension. Journal of Memory and Language, 48(1), 67–91. Stadie, N., Cholewa, J., & de Bleser, R. (2013). LEMO 2.0: Lexikon modellorientiert: Diagnostik f¨ur Aphasie, Dyslexie und Dysgraphie. Hofheim: NAT. Mirman, D. (2017). Growth curve analysis and visualization using R. Boca Raton, FL: Chapman & Hall/CRC. Appendix F: Comparisons with original direct-access model Stan Development Team, S. (2020). RStan: The R interface to Stan. Staub, A. (2010). Eye movements and processing difficulty in object relative clauses. Cognition, 116(1), 71–86. M¨uller, S. (2002). Complex predicates: verbal complexes, resultative constructions, and particle verbs in German. Stanford: center for the study of language and information. Staub, A., Dillon, B., & Clifton Jr, C. (2017). The matrix verb as a source of comprehension difficulty in object relative sentences. Cognitive Science, 41, 1353–1376. Nicenboim, B., Schad, D. J., & Vasishth, S. (2021). Introduction to Bayesian data analysis for cognitive science. Under contract with 502 Computational Brain & Behavior (2023) 6:473–502 Vasishth, S., & Engelmann, F. (2022). Sentence comprehension as a cognitive process: A computational approach. Cambridge, UK: Cambridge University Press. Stiebels, B., McFadden, T., Schwabe, K., Solstad, T., Kellner, E., Sommer, L., & Stoltmann, K. (2018). ZAS database of clause- embedding predicates, release 1.0. In OWID Plus. Stone, K., Lago, S., & Schad, D. J. (2020). Divergence point analyses of visual world data: Applications to bilingual research. Bilingualism: Language and Cognition, pp. 1–9. Vasishth, S., & Lewis, R. L. (2006). Argument-head distance and processing complexity: Explaining both locality and antilocality effects. Language, 82(4), 767–794. Vasishth, S., Nicenboim, B., Engelmann, F., & Burchert, F. (2019). Computational models of retrieval processes in sentence processing. Trends in Cognitive Sciences, 23, 968–982. Van Dyke, J. A. (2007). Interference effects from grammatically unavailable constituents during sentence processing. Journal of Experimental Psychology: Learning, Memory, and Cognition, 33(2), 407–430. Vehtari, A., Gelman, A., & Gabry, J. (2017). Practical Bayesian model evaluation using leave-one-out cross-validation and WAIC. Statistics and Computing, 27(5), 1413–1432. Van Dyke, J. A., & Lewis, R. L. (2003). Distinguishing effects of structure and decay on attachment and repair: A cue-based parsing account of recovery from misanalyzed ambiguities. Journal of Memory and Language, 49(3), 285–316. Wagers, M. W., Lau, E. F., & Phillips, C. (2009). Agreement attraction in comprehension: Representations and processes. Journal of Memory and Language, 61(2), 206–237. Van Dyke, J. A., & McElree, B. (2006). Retrieval interference in sentence comprehension. Journal of Memory and Language, 55(2), 157–166. Yadav, H., Paape, D., Smith, G., Dillon, B., & Vasishth, S. (2021). Individual differences in cue-weighting in sentence comprehension: An evaluation using approximate Bayesian computation. Van Dyke, J. A., & McElree, B. (2011). Cue-dependent interference in comprehension. Journal of Memory and Language, 65(3), 247–263. Yadav, H., Smith, G., & Vasishth, S. (2021). Appendix F: Comparisons with original direct-access model Is similarity-based interference caused by lossy compression or cue-based retrieval? A computational evaluation. In Proceedings of the International Conference on Cognitive Modeling. Van Maanen, L., Katsimpokis, D., & Van Campen, A. D. (2019). Fast and slow errors: Logistic regression to identify patterns in accuracy–response time relationships. Behavior Research Methods, 51(5), 2378–2389. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Vasishth, S., Br¨ussow, S., Lewis, R. L., & Drenhaus, H. (2008). Processing polarity: How the ungrammatical intrudes on the grammatical. Cognitive Science, 32(4), 685–712.
https://openalex.org/W4307877358
https://www.nauka-dialog.ru/jour/article/download/4083/2042
Russian
null
Literary Prototypes of Linguistic and Cultural Types of Natives of Tula
Naučnyĭ dialog/Naučnyj dialog
2,022
cc-by
8,617
Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Токарев Г. В. Литературные прототипы лингвокультурных типажей туляка / Г. В. Тока- рев // Научный диалог. — 2022. — Т. 11. — № 8. — С. 75—91. — DOI: 10.24224/2227-1295- 2022-11-8-75-91. Tokarev, G. V. (2022). Literary Prototypes of Linguistic and Cultural Types of Natives of Tula. Nauchnyi dialog, 11(8): 75-91. DOI: 10.24224/2227-1295-2022-11-8-75-91. (In Russ.). Жу р н а л в к л ю ч е н в П е р е ч е н ь ВА К DOI: 10.24224/2227-1295-2022-11-8-75-91 Литературные прототипы лингвокультурных типажей туляка Токарев Григорий Валериевич orcid.org/0000-0002-2362-0902 доктор филологических наук, профессор grig72@mail.ru Тульский государственный педагогический университет им. Л. Н. Толстого (Тула, Россия) Благодарности: Исследование «Симболарий региональной идентичности» выполнено за счет средств гранта Российского научного фонда № 22-28-20342 и Правительства Тульской области (соглашение № 6 от 19 апреля 2022 г.). Literary Prototypes of Linguistic and Cultural Types of Natives of Tula Grigoriy V. Tokarev orcid.org/0000-0002-2362-0902 Doctor of Philology, Professor grig72@mail.ru Tula State Lev Tolstoy Pedagogical University (Tula, Russia) Acknowledgments: The study “Symbolary of Regional Identity” was funded by a grant from the Russian Science Foundation № 22-28-20342 and the Government of the Tula Region (agreement No. 6 dated April 19, 2022). Literary Prototypes of Linguistic and Cultural Types of Natives of Tula Аннотация: Stereotyped ideas about the natives of Tula are revealed. The literary images of Abram Buly- gin (“History of the city of Tula by the trades- man Abram Bulygin, his wonderful adven- tures in the world, gaiety and his works”), Lefty (“Lefty” by N. S. Leskov) and Tychka (“Legends of Master Tychka” by I. F. Pan- kina). Based on the analysis of the textual features of the characters, the interpretation of selected fragments of three works of art, variants of the linguacultural type of Tula are reconstructed. It has been established that in the “History of the city of Tula ...” by Abram Bulygin, the features of self-identifi cation of a native of Tula in the 18th century are repre- sented. It was revealed that one of the main character traits of a native of Tula is sentimen- tality. Native of Tula has a practical mind, ra- tional and economic. In the tale of N. S. Les- kov “Lefty”, such qualities as a practical mind, ingenuity, the ability to conduct business, pas- sion for one’s occupation, religiosity, devotion to business, Motherland, disinterestedness are typifi ed. The most full and complete image of the Tula master was created by I. F. Pan- kin in “Legends of Master Poke”. Native of Tula is a craftsman, businesslike, silent, hardworking, courageous, willing to share his knowledge, transfer skills to his students, ap- preciating practical knowledge, having a sense of humor. The study revealed that the Tula cul- ture is of the masculine type. The inhabitant of the city in the cultural consciousness is rep- resented by the image of a skilled craftsman, gunsmith, blacksmith. Выявляются стереотипные представления о жителе Тулы. Исследуются литератур- ные образы Абрама Булыгина («История города Тулы мещанина Абрама Булыгина, о чудных его на свете похождениях, об охотах, весёлостях, и об работах»), Левши («Левша» Н. С. Лескова) и Тычки («Леген- ды о мастере Тычке» И. Ф. Панькина). На основе анализа текстовых характеристик персонажей, интерпретации избранных фрагментов трех художественных произве- дений реконструируются варианты лингво- культурного типажа туляка. Установлено, что в «Истории города Тулы…» Абрама Булыгина репрезентированы особенности самоидентификации туляка в XVIII веке. Выявлено, что одной из главных черт ха- рактера туляка является сентименталь- ность. Туляк обладает практическим умом, рационален и хозяйствен. В сказе Н. С. Ле- скова «Левша» типизированы такие ка- чества, как практический ум, смекалка, умение вести дела, увлечённость своим за- нятием, религиозность, преданность делу, Родине, бескорыстность. Литературные прототипы лингвокультурных типажей туляка Токарев Григорий Валериевич orcid.org/0000-0002-2362-0902 доктор филологических наук, профессор grig72@mail.ru Tula State Lev Tolstoy Pedagogical University (Tula, Russia) Тульский государственный педагогический университет им. Л. Н. Толстого (Тула, Россия) Acknowledgments: The study “Symbolary of Regional Identity” was funded by a grant from the Russian Science Foundation № 22-28-20342 and the Government of the Tula Region (agreement No. 6 dated April 19, 2022). Acknowledgments: The study “Symbolary of Regional Identity” was funded by a grant from the Russian Science Foundation № 22-28-20342 and the Government of the Tula Region (agreement No. 6 dated April 19, 2022). © Токарев Г. В., 2022 75 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Аннотация: Наиболее полный и завершённый образ тульского мастера создан И. Ф. Панькиным в «Легендах о ма- стере Тычке». Туляк — человек мастеро- вой, деловой, молчаливый, трудолюбивый, смелый, желающий делиться своими зна- ниями, передать умения своим ученикам, ценящий практические знания, обладаю- щий чувством юмора. В ходе исследования выявлено, что тульская культура маскулин- ного типа. Житель города в культурном сознании представлен образом искусного мастера, оружейника, кузнеца. Ключевые слова: региональная идентификация; симбола- рий; квазиэталон; квазимера; лингвокуль- турный типаж; стереотип. Key words: regional identifi cation; symbolary; quasi standard; quasi-measure; linguacultural type; stereotype. 76 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 УДК 811.161.1’42+821.161.1(470.312) Литературные прототипы лингвокультурных типажей туляка © Токарев Г. В., 2022 Литературные прототипы лингвокультурных типажей туляка © Токарев Г. В., 2022 1. Введение = Introduction д Процесс идентификации заключается в сортировке знаний и знаков культуры на основании культурного таксона «свой / чужой», а его резуль- тат состоит в формировании стереотипов, то есть устойчивых представле- ний, культурных норм [Краткий словарь …, 1996, с. 177], конфигурации ценностей, отражённых в культурном симболарии [Телия, 1999, с. 21]. Региональная идентификация особенно ярко воплощается в стереотипных представлениях о жителях той или иной локации. Кристаллизации устой- чивых представлений способствуют литературные произведения, которые типизируют образы, способные стать эталонами регионального характера. Эти образы, с одной стороны, создаются на основе сложившихся представ- лений, с другой — развивают, стабилизируют их. Типизированные лите- ратурные образы можно отнести к лингвокультурным типажам. «Лингво- культурный типаж может конкретизироваться как персонаж художествен- ного произведения. Вместе с тем типаж представляет собой обобщение реально существующих исторических лиц либо вымышленных персона- жей» [Карасик и др., 2005, с. 17]. Понятие лингвокультурного типажа пред- ложено В. И. Карасиком: это «… лингвокультурные типажи, узнаваемые образы представителей определённой культуры, совокупность которых и составляет культуру того или иного общества…» [Там же, с. 8]. В отличие от квазиэталона, который объективирует стереотипное представление об одном из свойств или качеств человека, лингвокультурный типаж вопло- щает целый комплекс признаков, проявляющихся в поведенческих практи- ках. В. И. Карасик и О. А. Дмитриева отмечают: «Лингвокультурный ти- паж строит своё поведение в соответствии с ролевыми предписаниями…» [Там же, с. 12]. Тем самым лингвокультурный типаж призван выразить ту или иную социокультурную роль. О. А. Дмитриева отмечает: «Лингво- культурный типаж является особым типом лингвокультурных концептов, важнейшие характеристики которого состоят в типизируемости опреде- ленной личности, значимости этой личности для лингвокультуры, возмож- ности ее как фактического, так и фикционального существования, возмож- ности ее упрощенной и карикатурной репрезентации» [Дмитриева, 2009, с. 55]. Лингвокультурный типаж находит разнообразные средства репре- 77 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 зентации: в лексиконе и речевых тактиках, поведении, образе жизни, ма- нере одеваться и т. д. В когнитивной парадигме лингвокультурный типаж можно охарактеризовать как прототип — типичный представитель той или иной категории [Краткий словарь …, 1996, с. 140—145]. 2. Материал, методы, обзор = Material, Methods, Review Целью данной статьи является рассмотрение трёх литературных об- разов туляка: Абрама Булыгина, Левши, Тычки. К задачам исследования относятся: 1) экспликация специфических черт туляка в каждом из про- изведений; 2) моделирование регионального характера на основе стерео- типных представлений о туляке, воплощённых в художественных произ- ведениях. 1. Введение = Introduction Достижение поставленной цели осуществляется следующими метода- ми и приёмами: сплошной выборки фраз, характеризующих литературные персонажи, компонентного анализа значений, интерпретации избранных фрагментов текста. Моделирование черт характера туляка опирается на процесс типизации, под которым мы, вслед за И. А. Мурзиновой, по- нимаем «вычленение в единицах, накопленных в памяти, постоянных и переменных признаков, то есть становление в сознании модели, образца» [Мурзинова, 2014, с. 4]. Материалом исследования стали три произведения, в которых освеща- ется характер туляка: «История города Тулы мещанина Абрама Булыгина, о чудных его на свете похождениях, об охотах, весёлостях, и об работах», «Левша» Н. С. Лескова, «Легендах о мастере Тычке» И. Ф. Панькина. Из- бранные тексты созданы в разные эпохи: в XVIII, XIX, XX веках соот- ветственно и соотносятся с разными жанрами. «История…» А. Булыгиина представляет собой мемуарное, лирическое повествование автора о соб- ственной жизни, «Левша» Н. С. Лескова — сказ, истории мастера Тыч- ки — легенды. Выбор данных текстов объясняется, во-первых, их темати- ческими особенностями: все они освещают быт и судьбы жителей Тулы; во-вторых, они никогда не рассматривались в лингвокультурологическом аспекте в целях моделирования регионального типажа, который является результатом процесса идентификации (работы И. Н. Юркина, А. М. Ран- чина, А. Д. Макаровой и др. посвящены культурно-историческому и лите- ратуроведческому анализу данных текстов [Юркин, 1994; Макарова, 2014; Ранчин, 2018]). Данная проблема является новой для лингвистики. Опи- сание в языковедеческих работах особенностей идентификации носит на сегодняшний день фрагментарный характер. Так, П. А. Гончаровым [Гон- чаров, 2015] предпринято исследование типажа сибиряка в произведениях А. Вампилова. 78 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Абрам рассказывает о сво- их занятиях: …сапоги, вершки и хомутины шивал…; …замки и приборы обтирал. К новокорпусным ножам крыжи, бляхи и наконечники обтирал. И всякие разные поделки наждаком чищал, притом на штыки и ножны шивал [Там же, с. 74—75]. Работа, по мнению героя, делает жизнь спо- койной и беззаботной, но однообразной и скучной. Особая веха в жизни Абрама связана с его приходом в Оружейную слободу. Изготовление де- талей оружия описано как сложная, тяжёлая и даже опасная для жизни работа: «…бывало точилом до смерти…» [Там же, с. 75]. Затем в жизни героя наступают перемены: его увольняют с завода, сажают в тюрьму, хо- тят отдать в солдаты. Мытарства рассказчика заканчиваются тем, что он бросает свою работу на заводе и уходит служить целовальником в кабак. Целовальник должен был следить за выручкой заведения, которая направ- лялась в царскую казну. На этом поиски работы не закончились. Торговое дело герой оценивает как грешное: … торговые дела во грехе замечал… [Там же, с. 79], — что говорит о его честности и бескорыстности. Прора- ботав у купца два года и не получив жалованья, рассказчик думает снова вернуться на казённую работу, но потом решает остаться в лавке у хозяина. Отношение к труду как к чему-то второстепенному подтверждается тем, что за время работы в лавке Абрам даже не научился считать на счётах. Представления о труде вербализованы номинациями, которые используют- ся как синонимичные: работа / забота. Им противопоставлены светские весёлости. Именно они являются отдушиной для героя. Он любит петь, музицировать, плясать, слушать сказки, ловить птиц, участвовать в кулач- ных боях, устраивать фейерверки. В этих занятиях отражена повседнев- ность жителя Тулы, его любимое времяпрепровождение [Шариков, 2016]. Туляки любили разводить голубей. Эта птица стала символом любви и вер- ности: по наблюдениям, голубиная пара создается на всю птичью жизнь. Символизация этой птицы отражает черты регионального характера: сен- тиментальность, верность чувствам. Кулачный бой был важным событием в жизни города. Как известно, он завершал масленичную неделю и носил, говоря современным языком, компенсаторный характер: помогал человеку избавиться от накопившихся отрицательных эмоций. Абрам хвастает сво- ей удалью, говорит о том, что был предводителем и участвовал в неравных боях. Характерно, что история заканчивается описанием кулачного боя как своего рода финала жизни. Герой расстаётся со своим любимым заняти- ем — песенной артелью — и отказывается принимать участие в кулачных боях. Положительная интерпретация этих состязаний также отражает осо- бенности регионального характера: умение долго терпеть, сдержанность, составляющей жизни. Повествование начинается именно с описания ра- боты. Трудиться герою пришлось с десяти лет. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Автостереотипы как результаты процесса самоидентификации объ- ективируются в текстах с перволичностным повествованием. Нарратор непосредственно воплощает индивидуальное мировосприятие, выражает своё «я». В данном аспекте большой интерес представляет повествование Абрама Булыгина, воспроизводящее особенности самоидентификации ту- ляка в XVIII веке. Этот текст является своего рода автобиографией, на- писанной в вольном, отчасти лирическом стиле. Как следует из повество- вания, Абрам Антонович Булыгин происходил из купеческого рода, он обучился оружейному делу и затем работал на тульском казённом заво- де. Статус казюка (так называли рабочих этого завода) позволил ему из- бежать призыва в армию. Позднее ему удалось организовать свою артель, чтобы заниматься проведением увеселительных мероприятий. Хотя Абрам Булыгин в детстве был обучен грамоте, навыками письма и чтения он не обладал, поскольку из-за отсутствия необходимости разучился читать и писать: При отце и матери в малолетстве обучен был я грамоте, когда отец и мать земли предался, а я с учёной грамотою навек расстался. … Пятьдесят лет в руки я учёную грамоту не брал, да и забвению предал… [История …, 1994, с. 128]. История жизни, рассказанная шестидесятилетним Булыгиным, чу- десным образом была записана. Как отмечает И. Н. Юркин, «при участии какого-то неизвестного нам лица рассказы тульского мещанина превра- тились в письменный текст…» [Юркин, 1994, с. 9]. В жизнеописании за- печатлены представления жителя Тулы восемнадцатого столетия о мире. Абрама Булыгина можно считать типичным представителем своего горо- да и времени, поэтому справедливой будет оценка его произведения как воплощения коллективных ценностей. Правдивость своих слов Булыгин подтверждает предварительными замечаниями: Когда эвти книжки кто будет читать — друзья, приятели и незнакомые люди, не сумневайтесь. Ни одного тут слова ложного нет: так написано, как в книге напечата- но [История …, 1994, с. 74]. Полное название текста отражает ценности рассказчика: «История города Тулы мещанина Абрама Булыгина, о чудных его на свете похождениях, об охотах, весёлостях, и об работах». Обратим внимание, что первое место отведено именно развлечениям, а не работе. Они представляют для героя большую ценность. Рассказчик так хроноло- гизирует свою жизнь: … десять лет разные работы работал, сорок лет во одной лавке хлебом торговал… [Там же, с. 105]. Эта фраза эксплицирует представления об обязательности труда, понимание его как непременной 79 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 составляющей жизни. Повествование начинается именно с описания ра- боты. Трудиться герою пришлось с десяти лет. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Положительная интерпретация этих состязаний также отражает осо- бенности регионального характера: умение долго терпеть, сдержанность, 80 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 готовность постоять за своих. Главными ценностями для героя остаются веселье, забава: А я на свете большего богатства не имел, а больше в ве- сёлостях разумел [История …, 1994, с. 84]. Абрам пишет о своей любви к путешествиям: он ездит в Москву и Петербург смотреть «чудности» и «отличности», встречается с разными людьми: … с сапожниками, с вер- шечниками, с ямщиками и с старинными деньщиками. С оружейниками: с русскими, с прусскими, с шведскими и немецкими, со азиатскими… [Там же, с. 87]. Рассказчик скрупулёзно перечисляет все места, в которых он побывал: Кронштадт, Царское Село, Петергоф, Каменный остров, Кун- сткамера и др. Всё это свидетельствует об открытости ко всему новому, любознательности, дружелюбности. готовность постоять за своих. Главными ценностями для героя остаются веселье, забава: А я на свете большего богатства не имел, а больше в ве- сёлостях разумел [История …, 1994, с. 84]. Абрам пишет о своей любви к путешествиям: он ездит в Москву и Петербург смотреть «чудности» и «отличности», встречается с разными людьми: … с сапожниками, с вер- шечниками, с ямщиками и с старинными деньщиками. С оружейниками: с русскими, с прусскими, с шведскими и немецкими, со азиатскими… [Там же, с. 87]. Рассказчик скрупулёзно перечисляет все места, в которых он побывал: Кронштадт, Царское Село, Петергоф, Каменный остров, Кун- сткамера и др. Всё это свидетельствует об открытости ко всему новому, любознательности, дружелюбности. Особое место в повествовании составляет «Толкование о чижиной охо- те». Как было сказано ранее, туляки были любителями и ценителями птиц. Птица стала для них символом райской жизни, беззаботности и забавы, до- статка, семьи и верности. Туляки с любовью относились к певчим птицам, к голубям. С птицей связаны представления о чужом пространстве, иной, экзотической заморской стороне: И станут они лететь на полуденную сторону опять к морю на своё место [Там же, с. 94]. Далее следует де- тальное, пошаговое описание охоты, расстановки сетей, в чём отражается практическое, точное знание туляка: И выбираем на эвтих полянах дре- ва пристойные. Чтоб были двух сажен, а понужнее трёх, и чтоб стояли круговиною сажени на три, чтоб порожнее место промеж их было. И чи- стим на этом месте точок, и прибиваем на этом точку сеточные понцы по две сажени длины, по пяти четвертей ширины. И под эвтими кры- льями вырыты ямки по два аршина длины, по полуаршину ширины [Там же, с. 94—95]. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Абрам рассказывает о сво- их занятиях: …сапоги, вершки и хомутины шивал…; …замки и приборы обтирал. К новокорпусным ножам крыжи, бляхи и наконечники обтирал. И всякие разные поделки наждаком чищал, притом на штыки и ножны шивал [Там же, с. 74—75]. Работа, по мнению героя, делает жизнь спо- койной и беззаботной, но однообразной и скучной. Особая веха в жизни Абрама связана с его приходом в Оружейную слободу. Изготовление де- талей оружия описано как сложная, тяжёлая и даже опасная для жизни работа: «…бывало точилом до смерти…» [Там же, с. 75]. Затем в жизни героя наступают перемены: его увольняют с завода, сажают в тюрьму, хо- тят отдать в солдаты. Мытарства рассказчика заканчиваются тем, что он бросает свою работу на заводе и уходит служить целовальником в кабак. Целовальник должен был следить за выручкой заведения, которая направ- лялась в царскую казну. На этом поиски работы не закончились. Торговое дело герой оценивает как грешное: … торговые дела во грехе замечал… [Там же, с. 79], — что говорит о его честности и бескорыстности. Прора- ботав у купца два года и не получив жалованья, рассказчик думает снова вернуться на казённую работу, но потом решает остаться в лавке у хозяина. Отношение к труду как к чему-то второстепенному подтверждается тем, что за время работы в лавке Абрам даже не научился считать на счётах. Представления о труде вербализованы номинациями, которые используют- ся как синонимичные: работа / забота. Им противопоставлены светские весёлости. Именно они являются отдушиной для героя. Он любит петь, музицировать, плясать, слушать сказки, ловить птиц, участвовать в кулач- ных боях, устраивать фейерверки. В этих занятиях отражена повседнев- ность жителя Тулы, его любимое времяпрепровождение [Шариков, 2016]. Туляки любили разводить голубей. Эта птица стала символом любви и вер- ности: по наблюдениям, голубиная пара создается на всю птичью жизнь. Символизация этой птицы отражает черты регионального характера: сен- тиментальность, верность чувствам. Кулачный бой был важным событием в жизни города. Как известно, он завершал масленичную неделю и носил, говоря современным языком, компенсаторный характер: помогал человеку избавиться от накопившихся отрицательных эмоций. Абрам хвастает сво- ей удалью, говорит о том, что был предводителем и участвовал в неравных боях. Характерно, что история заканчивается описанием кулачного боя как своего рода финала жизни. Герой расстаётся со своим любимым заняти- ем — песенной артелью — и отказывается принимать участие в кулачных боях. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Главным жизненным приоритетом для рассказчика является веселье: Не думал о доме, о жене, о детях, а желал весело жить на све- те… [Там же, с. 105]. В понимании Абрама, веселье — это любимые его занятия: концерты, спектакли, фейерверки, пение, музицирование. Всё это помогает герою вырваться из монотонных будней тяжёлого и однообразно- го физического труда, рутинных обязанностей обывателя. Трогательно по- вествование о роспуске песенной артели: прощальном концерте, сжигании музыкальных инструментов и артистических нарядов. На память Булыгин оставляет свою скрипку: … Оная скрыпка будет на стене висеть, а я буду на неё глядеть… [Там же, с. 117], — но предводитель разбивает её. Друзья Абрама склеивают скрипку, однако тот решает её сжечь. Этот поступок также подтверждает сентиментальность характера туляка. Таким образом, в повествовании представлен образ тульского обыва- теля, в сознании которого противопоставлены работа и отдых. Булыгин не бездельник. Он пытается найти себя в творчестве. Ему присущи качества 81 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 лидера: он возглавляет кулачные бои, руководит музыкальной артелью. Абрама Булыгина нельзя отнести к искусным мастерам. Он с неприязнью относится к тяжёлому физическому труду. Разнообразные развлечения ка- жутся ему отдушиной. Жизнь туляка в восемнадцатом веке — это серые трудовые будни, скрашиваемые редкими праздниками или любимыми за- нятиями. Абрам Булыгин — сплав типичного и индивидуального. С одной стороны, он живёт жизнью обычного чернорабочего, подмастерья, с дру- гой — скрыто не принимает её, пытается найти себя в творчестве, люби- мых занятиях. Пожалуй, одной из главных черт характера туляка является сентиментальность, душевность. Для жителя Тулы важно иметь занятие, которое ему будет доставлять удовольствие, отвлекать от тяжёлой рабо- ты. Он обладает практическим умом, рационален. В сравнении с образами Левши и Тычки имя Абрама Булыгина не стало прецедентным, эмблемой тульского культурного текста, однако интерес представляет обыденное мировоззрение героя, которое, без сомнения, было типичным. Следует от- метить, что, сопоставляя представления о туляке, отражённые в «Истории города Тулы…» Абрама Булыгина, с более поздними, можно сделать вывод о том, что земляк-современник Булыгина не осознавал себя умельцем. По всей вероятности, данный автостереотип сформировался позже. 3.2. Объективация характера туляка в сказе Н. С. Лескова «Левша» Наибольшую известность получил образ Левши. Именно он стал эта- лоном тульского мастера, оружейника, металлиста, гармонщика. Наибольшую известность получил образ Левши. Именно он стал эта- лоном тульского мастера, оружейника, металлиста, гармонщика. Н. С. Лесков воссоздал образ тульского мастерового в сказе «Левша». Как отмечает Т. А. Павлишак, «Левша — один из символов русского на- рода» [Павлишак, 2016, с. 134]. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Определим типичные черты туляка в этом тексте. Как заключает О. А. Дмитриева, «…лингвокультурный типаж яв- ляется сгустком ценностных предпочтений языковой личности…» [Дми- триева, 2009, с. 55]. Высшее мастерство Левши подчёркивается тем, что он всё делает левой рукой. По словам И. В. Столяровой, Левша воспринима- ется как «апофеоз талантливости русского человека, который “все может”» [Столярова, 1996, с. 61]. Мастер имеет неказистый, чудаковатый вид, кото- рый выступает контрастным фоном внутреннему миру Левши и его умени- ям: … косой левша, на щеке пятно родимое, а на висках волосья при ученье выдраны… [Лесков, 1979, с. 466]. Получая задание, Левша вместе с други- ми мастерами отправляется в Мценск поклониться иконе Святого Николая Чудотворца. Тульские оружейники — люди набожные, религиозные: Ту- ляки, люди умные и сведущие в металлическом деле, известны также как первые знатоки в религии. Их славою в этом отношении полна и родная земля, и даже святой Афон: они не только мастера петь с вавилонами, но они знают, как пишется картина «вечерний звон»… Туляк полон церков- 82 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 ного благочестия… [Там же, с. 467]. Левша предан своей вере, считает её наиболее правильной. ного благочестия… [Там же, с. 467]. Левша предан своей вере, считает её наиболее правильной. Туляки — люди хозяйственные: «…если кто из них посвятит себя большему служению и пойдет в монашество, то таковые слывут лучши- ми монастырскими экономами, и из них выходят самые способные сбор- щики…» [Там же, с. 467]. Несмотря на свою мастеровитость, Левша живёт небогато: «Мы люди бедные и по бедности своей мелкоскопа не имеем…» [Там же, с. 475]. Хотя у него золотые руки, Левша не скопил состояния. Он относится к своему делу как к искусству, а не как к ремеслу и способу зарабатывания денег. В сказе о Левше акцент делается на умении тульских мастеров, позво- ляющем им создать более совершенные изобретения, чем иностранные. А. М. Ранчин отмечает: «Левша — настоящий герой, не чуждый, как и дру- гие тульские мастера, чувства собственного достоинства» [Ранчин, 2018, с. 136]. Т. Ю. Березина и Е. И. Вырвич указывают на погруженность Левши в свое искусство, на то, что во время работы он отдалялся от окружающе- го мира: «Мужицкая сила мастеровых сконцентрирована в образе потной спирали. Трудясь, они были полностью отстранены от мира» [Березина и др., 2018, с. 30]. С образом Левши связана квазимера [Токарев, 2021, с. 15] мастерства блоху подковать, указывающая на уровень искусности. Квазимера подчёр- кивает природную выучку мастера. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Как видим, в сказе эталонизируются иные качества туляка, нежели в повествовании Абрама Булыгина: религи- озность, преданность делу и Родине, бескорыстность. Сходными эталони- зируемыми чертами являются практический ум, смекалка, умение вести дела. Туляк всегда увлечён своим занятием. В целом же характер туляка Н. С. Лесковым прорисован схематично. 3.3. Отражение характера туляка в «Легендах о мастере Тычке» И. Ф. Панькина 3.3. Отражение характера туляка в «Легендах о мастере Тычке» И. Ф. Панькина Заданный Н. С. Лесковым эталон тульского умельца получил развитие в повести о мастере Тычке, написанной И. Ф. Панькиным. В одной из ле- генд рождение Тычки мифологизируется, а начало признания его как масте- ра связывается с посещением Тулы Петром I: … Когда родился Тычка, про то никто толком не знает. Одни говорят — с первым ударом кузнечного молота о наковальню, другие — позже. Но когда бы он ни появился на свет, а приметили его при Петре I... [Панькин, 1989, с. 9]. В легендах о мастере Тычке Тула сравнивается с колчаном со стрелами, в основу образа положены представления об искусности тульских мастеров-оружейников. Тычка — это собирательный образ тульского мастера: …Я тоже не учился у мастера Тычки и никогда не видел его. Я полагаю, такого ма- 83 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 стера не было и нет. Тычка — не что иное, как плод фантазии тульских мастеров — личность, хитро и умело нарисованная воображением людей многих поколений, начиная со времен Петра I. Но с детских лет мы так сроднились с легендами о веселом и остроумном мастере Тычке, с торже- ством переходящего из века в век, что он нам стал казаться живым. Мно- гие даже в зрелом возрасте не перестают утверждать, что они учились у самого настоящего Тычки, хотя и учились у разных людей и их мастеров звали Макарами, Захарами и прочими именами. Дело в том, что в Туле всех знатных умельцев-виртуозов за глаза называли Тычками [Там же, с. 128]. Имя Тычки представлено в тексте как образованное от нарицательного существительного, обозначающего измерительный инструмент и мотивиро- ванного глаголом тыкать: В давние времена на ружейном заводе тычкой называли керн — инструмент для разметки деталей. А наш веселый мастер мог не только на железе, а даже на самой плоской стороне человеческой жизни «откернить» такую деталь, которая людям запоминалась на века [Там же, с. 60]. Тем самым подчёркиваются профессиональные качества ма- стера. В отличие от Левши Тычка не изготавливает изделия на глазок. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 стера не было и нет. Тычка — не что иное, как плод фантазии тульских мастеров — личность, хитро и умело нарисованная воображением людей многих поколений, начиная со времен Петра I. Но с детских лет мы так сроднились с легендами о веселом и остроумном мастере Тычке, с торже- ством переходящего из века в век, что он нам стал казаться живым. Мно- гие даже в зрелом возрасте не перестают утверждать, что они учились у самого настоящего Тычки, хотя и учились у разных людей и их мастеров звали Макарами, Захарами и прочими именами. Дело в том, что в Туле всех знатных умельцев-виртуозов за глаза называли Тычками [Там же, с. 128]. Имя Тычки представлено в тексте как образованное от нарицательного существительного, обозначающего измерительный инструмент и мотивиро- ванного глаголом тыкать: В давние времена на ружейном заводе тычкой называли керн — инструмент для разметки деталей. А наш веселый мастер мог не только на железе, а даже на самой плоской стороне человеческой жизни «откернить» такую деталь, которая людям запоминалась на века [Там же, с. 60]. Тем самым подчёркиваются профессиональные качества ма- стера. В отличие от Левши Тычка не изготавливает изделия на глазок. И. Ф. Панькин так рисует портрет мастера: У Тычки был облик самого обыкновенного человека. Бородка у него, как у Емельки Пугачева — лопа- точкой. Волосы подстрижены скобочкой, ремешком опоясаны. Глаза карие, озорные, и в них всегда светились веселые звездочки. Ничем он не отличался от других мастеров, а если и отличался, может быть, только тем, что, кроме своих ног на ложевой стороне, имел еще четыре табуреточные нож- ки, которые его с утра до поздней ночи держали у верстака. Да еще на пле- чах-коромыслах он носил для солидности, как рудничные бадьи, два кулака [Там же, с. 66]. Как видим, образы Левши и Тычки написаны в одинаковой семантической тональности: внешностью мастера обладали заурядной. Сло- жение Тычки отражает его профессиональные занятия. И. Ф. Панькин так рисует портрет мастера: У Тычки был облик самого обыкновенного человека. Бородка у него, как у Емельки Пугачева — лопа- точкой. Волосы подстрижены скобочкой, ремешком опоясаны. Глаза карие, озорные, и в них всегда светились веселые звездочки. Ничем он не отличался от других мастеров, а если и отличался, может быть, только тем, что, кроме своих ног на ложевой стороне, имел еще четыре табуреточные нож- ки, которые его с утра до поздней ночи держали у верстака. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Да еще на пле- чах-коромыслах он носил для солидности, как рудничные бадьи, два кулака [Там же, с. 66]. Как видим, образы Левши и Тычки написаны в одинаковой семантической тональности: внешностью мастера обладали заурядной. Сло- жение Тычки отражает его профессиональные занятия. Обобщим стереотипные представления о туляке, воплощённые в «Леген- дах…». Туляки — люди деловые, молчаливые. Длинные разговоры воспри- нимаются ими как безделье, пустая трата времени. Для того чтобы отметить увлечённость туляка делом, автор с иронией говорит о коротких прозвищах, которые мастера давали друг другу, чтобы якобы сэкономить время: Мы, ту- ляки, народ занятой, и зря тарабарить нам недосуг. Мы даже не позволяем себе такой роскоши, чтобы в занятое время называть друг друга длинны- ми и величальными именами. По отчеству наши деды назывались только в престольные праздники и в воскресные дни. Туляки всегда любили короткие имена, чтобы они свободно проходили через горло, не застревали в зубах и пролетали через цех, как пуля: «Чиж-ж! Левша-а! Тычка!» Да и меня на ра- боте зовут именем короче ружейного залпа — Ив, хотя мать по наивности 84 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 думала: как только я овладею грамотой, меня будут звать не только пол- ным именем — Иваном, но и по батюшке — Федоровым [Там же, с. 9]. думала: как только я овладею грамотой, меня будут звать не только пол- ным именем — Иваном, но и по батюшке — Федоровым [Там же, с. 9]. Туляк скромен: А кто любит хвалу и чтоб о нем в медные трубы дули, тому нечего делать в Туле. Так говорили наши деды, так говорим и мы [Там же]. Туляк трудолюбив, он не может представить себе жизнь без дела, в ко- торое он вкладывает свою душу. Его качества оцениваются по желанию чему-то научиться, что-то сделать: … издавна у нас повелось: когда перед молодым человеком впервые открывают заводские ворота, его не спра- шивают, чего доброго принес он в своей душе. Его спрашивают, к какой работе хочет он приложить руки [Там же, с. 13]. Своего рода символом мастерства туляка являются его руки. Оно про- является в придумывании чего-то необычного, в выполнении ювелирной работы: …А вот мой мастер Тычка всегда свои руки держал на виду. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина К нему на учение тянулось столько мальцов, что если бы он взялся всех выучить, то ему не хватило бы трех жизней. А руки у него были веселые. Он ими мог рассмешить целый город... [Там же, с. 15]. Чтобы изделие стало уникальным, в него нужно вложить душу: Могуч тот, кто душой чувствует свое ремесло и владеет многими знаниями. По-моему, таким могучим является мастер Тычка [Там же, с. 32]. В подтверждение автор рассказывает историю о том, как один фабри- кант заказал ко дню рождения своей жены медальон. Тычка выполнил за- каз, в центре медальона расположив фабриканта, сидящего в позе царя. На носу изображённого жена рассмотрела царапины. В увеличительное стек- ло в них гости увидели ещё одно изображение — губернатора, сидящего в конуре в позе собаки. Эта история эксплицирует такие черты в характере туляка, как смелость, независимость, обострённое чувство достоинства, презрение к богатым. Для туляка один авторитет — мастерство. Смелость выражается не только в том, что туляк не боится дать отпор обидчику, но и в планах, замыслах, в намерении создать что-то невероятное. Так, в туль- ском ментальном пространстве сформировалась ещё одна наивная мера мастерства: … подковать комара на лету: кроме задумки, каждому творя- щему человеку нужна та смелость, которой, кажется, ни у кого из нас до сих пор не было. В этом, наверно, и есть секрет мастерства и, наверное, поэтому нам не удается до сих пор подковать комара на лету... [Там же]. Не только профессиональные, но и человеческие качества выделяют Тычку. Он добр, желает быть полезным людям, передать им свой опыт и знания. Фигурально Тычка представлен образом дерева, на котором птицы устраивают свои гнёзда: … Мастер Тычка был как то дерево, на котором птицы вьют гнезда. К нему всегда тянулись люди... [Панькин, 1989, с. 44]. 85 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Тычка открыт для других: … Мастер Тычка жил широко и раздольно. А го- ворят, кто широко живет, тот не запирает ворот… [Там же, с. 66]. Ещё одним качеством характера мастера Тычки является остроумие и юмор. Тычка всегда найдёт ответ и сможет посмеяться, в том числе и над собой: Большим шутником был Тычка. Как-то мастера ружейного завода встре- тились в засечном лесу и расхвастались. Один мастер сказал: — Прошлым летом я сделал такое ружье, которым у6ил птицу вели- чиной с годовалого бычка. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Положили ее на крыло, а другое крыло покрыло весь стол. Тычка выслушал его и проговорил: — Сколько я ни делал ружей, никогда не приходилось убивать таких птиц, но однажды мне тоже повезло. Иду по лесу, гляжу — у елки лежит яйцо. Разбил его. Хотел размешать ложкой желток, но вдруг уронил ее вовнутрь. Попробовал достать пальцем — не достал. Просунул руку по локоть — не достал. Пришлось нырять за ложкой. — Откуда же в нашем лесу взялось такое яйцо? — спросил мастер. — От той птицы, которую ты убил прошлым летом, — ответил Тычка [Там же, с. 55]. Тычка смелый, стоит горой за правду: …Когда я пришел на завод, то с первого же дня стал поступать так, как советовал он — о хорошем го- ворить хорошо, о плохом — плохо… [Там же, с. 93]. Смелость проявляется не только в нежелании склонять голову перед сильными, но и в мастерстве, творчестве, в умении действовать не по шаблону, делать то, что не при- нято: … Хорошим мог бы стать мастером, но нет в тебе смелости. А художник без смелости — не художник. Иди-ка лучше делать замки, пока люди не научились жить без замков... [Там же, с. 108]. Залогом успеха мастера является желание творить не для собственной выгоды, а для людей: …Тульские мастеровые чем отличались от других, они словно жили не для себя, а для памяти людей. Они жили с постоян- ной надеждой сделать то, что еще никто не делал и никогда не сумеет сделать. Поэтому работали много, зная, что без большого труда себя выразить нельзя [Там же, с. 110]. Тульские мастера ценят знания, но проверенные на практике: Ради сохранения святости высочайшего Тульского мастерства, обретенного многими поколениями работных людей, они даже установили свой особый закон, в котором говорилось: «Какое бы учебное заведение человек ни за- кончил, а без проверки его знаний ни один завод к себе на работу не должен брать…ʼ» [Там же, с. 115]. 86 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Автор так обобщает символические значения образа Тычки: Тычка ни- когда не видел себя, а только других. Недаром его имя стало символом чести, доброты и справедливости рабочего люда [Там же, с. 132]. Рассказ о Тычке сопровождается авторскими комментариями как го- ворили наши деды, издавна повелось и подобными, подчёркивающими типичность и стабильность описываемых ситуаций, устойчивость стерео- типных представлений о туляке. Таким образом, И. Ф. 3. Результаты и обсуждение = Results and Discussion 3.1. Отражение характера туляка в «Истории города Тулы…» А. Булыгина Панькин создал наиболее полный и завершён- ный образ тульского мастера, обладающий следующими чертами харак- тера: искусный мастер, деловой, молчаливый, трудолюбивый, смелый, стремящийся передать умения ученикам, ценящий практические знания, обладающий чувством юмора. Источники 1. История города Тулы мещанина Абрама Булыгина, о чудных его на свете похож- дениях, об охотах, весёлостях, и об работах. — В книге : Юркин И. Н. Абрам Булыгин : чудности, веселости, “непонятная философия” / И. Н. Юркин. — Тула : Рарус, 1994. — С. 74—128. 1. История города Тулы мещанина Абрама Булыгина, о чудных его на свете похож- дениях, об охотах, весёлостях, и об работах. — В книге : Юркин И. Н. Абрам Булыгин : чудности, веселости, “непонятная философия” / И. Н. Юркин. — Тула : Рарус, 1994. — С. 74—128. 2. Краткий словарь когнитивных терминов / Е. С. Кубрякова, В. З. Демьянков, Ю. Г. Панкрац, Л. Г. Лузина // Краткий словарь когнитивных терминов. — Москва : МГУ им. М. В. Ломоносова, 1996. — 245 с. — ISBN 5-89042-018-1. 2. Краткий словарь когнитивных терминов / Е. С. Кубрякова, В. З. Демьянков, Ю. Г. Панкрац, Л. Г. Лузина // Краткий словарь когнитивных терминов. — Москва : МГУ им. М. В. Ломоносова, 1996. — 245 с. — ISBN 5-89042-018-1. МГУ им. М. В. Ломоносова, 1996. — 245 с. — ISBN 5-89042-018-1. 3. Лесков Н. С. Избранное / Н. С. Лесков. — Кишинев : Литература артистикэ, 1979. — 672 с. 3. Лесков Н. С. Избранное / Н. С. Лесков. — Кишинев : Литература артистикэ, 1979. — 672 с. 4. Панькин И. Легенды о мастере Тычке / И. Панькин. — Москва : Детская литера- тура, 1989. — 139 с. 4. Панькин И. Легенды о мастере Тычке / И. Панькин. — Москва : Детская литера- тура, 1989. — 139 с. 4. Заключение = Conclusions Итак, на основе анализа литературно-художественных региональных текстов мы можем заключить, что тульская культура маскулинного типа, поскольку эталонными образами, обладающими прецедентным характе- ром, в произведениях выступают персонажи-мужчины, коллективное со- знание фокусирует внимание на мужском занятии, связанном с обработкой металла. Проанализированные лингвокультурные типажи, сформирован- ные на основе трех литературных образов, различаются. Они воплощают неодинаковые стереотипные черты. Абрам Булыгин относится к работе как к безысходности. Она необходима, чтобы не умереть с голоду. Булыгин не- гативно оценивает физический труд и находит для себя отдушиину в раз- влечениях: ловле и разведении птиц, путешествиях, участии в спектаклях, музицировании и пении. Булыгин честен, ответствен, трудолюбив, испол- нителен. Он человек тонкой душевной организации, хочет наслаждаться жизнью. В типаже Абрама Булыгина показано мироощущение обычного человека из рабочего сословия, то есть в произведении жизнь этого пер- сонажа описана через своего рода приемы саморефлексии. Типажи Лев- ши и Тычки воплощают представления о тульском мастере, сложившиеся у людей, живущих рядом с таким человеком, то есть раскрыто восприятие его со стороны, это своего рода «региональный гетеростереотип». Житель города в культурном сознании представлен образом мастера, оружейника, кузнеца, умельца на все руки. Он самовыражается в труде. В отличие от Булыгина с его особым мироощущением и страстным желанием преодо- леть безысходность обыденности развлечениями Левша и Тычка относятся к своему делу как искусству. Типаж Тычки конкретизирует типаж Левши. Ведущим стереотипом выступает туляк как человек мастеровой. Главные его ценности — работа, знание, опыт. Тульский мастер религиозен. К до- 87 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 минирующим культурным установкам относятся следующие: цени время, будь предан своей вере и Родине, сочетай труд и отдых. Три рассмотрен- ных типажа обладают и общей чертой: скрытый бунт против обыденности. Но если Булыгин пытается освободиться от гнёта повседневности с помо- щью весёлостей, то Левша и Тычка — посредством создания уникальных предметов, в которые они вкладывают душу. В центре симболария регио- нальной культуры располагаются эталоны мастера, представленные номи- нациями Левша, Тычка, и квазимеры мастерства: блоху подковать, комара на лету подковать. Литература 1. Абашин И. Политико-философские воззрения Н. С. Лескова : национальная идентичность / И. Абашин // Современные проблемы науки и образования. — 2013. — № 2. — С. 495. 2. Беглова Е. И. Лексика в произведении Н. С. Лескова «Левша» в аспекте народ- ной русской речи в оппозиции «своё»-«чужое» / Е. И. Беглова // Мир Н. С. Лескова : поэтика, прагматика, стилистика. Сборник докладов участников Национальной научно- практической конференции, посвящённой 190-летию со дня рождения Н. С. Лескова. — Брянск : Издательство БГУ им. И. Г. Петровского, 2021. — С. 81—91. 3. Березина Т. Ю. Судьба талантливого человека в России в сказе Н. С. Лескова «Левша» / Т. Ю. Березина, Е. И. Вырвич // Седьмые Сильвестровские педагогические чтения. Духовность и нравственность в образовательном пространстве — фундамент будущего России. Материалы Всероссийской научной конференции. — Омск : Амфора, 2018. — С. 30—33. 4. Васантада В. Д. Лингвокультурный типаж как отражение языкового созна- ния / В. Д. Васантада // Актуальные проблемы гуманитарных и естественных наук. — 2013. — № 5. — С. 230—232. 4. Васантада В. Д. Лингвокультурный типаж как отражение языкового созна- ния / В. Д. Васантада // Актуальные проблемы гуманитарных и естественных наук. — 2013. — № 5. — С. 230—232. 5. Гончаров П. А. Сибиряк в персонажной сфере А. Вампилова / П. А. Гонча- ров, О. В. Чербаева // Современные научные исследования и инновации. — 2015. — № 12 (56). — С. 1009—1020. 5. Гончаров П. А. Сибиряк в персонажной сфере А. Вампилова / П. А. Гонча- ров, О. В. Чербаева // Современные научные исследования и инновации. — 2015. — № 12 (56). — С. 1009—1020. 6. Дмитриева О. А. Ценностные характеристики в структуре лингвокультурного типажа / О. А. Дмитриева // Система ценностей современного общества. — 2009. — № 9. — С. 55—59. 6. Дмитриева О. А. Ценностные характеристики в структуре лингвокультурного типажа / О. А. Дмитриева // Система ценностей современного общества. — 2009. — № 9. — С. 55—59. 88 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 7. Ибатуллина Г. М. Историософский миф в рассказе Н. Лескова «Левша» / Г. М. Ибатуллина // Печать и слово Санкт-Петербурга. Петербургские чтения-2012. Сборник научных трудов : в 2 частях. — Санкт-Петербург : Санкт-Петербургский го- сударственный университет технологии и дизайна, Северо-Западный институт печати, 2013. — С. Литература 122—131. 7. Ибатуллина Г. М. Историософский миф в рассказе Н. Лескова «Левша» / Г. М. Ибатуллина // Печать и слово Санкт-Петербурга. Петербургские чтения-2012. Сборник научных трудов : в 2 частях. — Санкт-Петербург : Санкт-Петербургский го- сударственный университет технологии и дизайна, Северо-Западный институт печати, 2013. — С. 122—131. 8. Ибатуллина Г. М. Национально-исторический миф и пути его интерпретации в рассказе Н. С. Лескова «Левша» / Г. М. Ибатуллина // Современные исследования со- циальных проблем (электронный научный журнал). — 2014. — № 4. — С. 2. 9. Карасик В. И. Лингвокультурный типаж : к определению понятия / В. И. Кара- сик, О. А. Дмитриева // Аксиологическая лингвистика : лингвокультурные типажи. — Волгоград : Парадигма, 2005. — С. 5—25. 10. Лаптева О. А. Путь к познанию русского национального характера (по произ- ведению Н. С. Лескова «Левша») / О. А. Лаптева // Педагогический поиск. — 2021. — № 4. — С. 14—16. 11. Макарова А. Д. Особенности жанра сказа на примере произведения Ивана Пань- кина “Легенды о мастере Тычке” / А. Д. Макарова // Мировая словесность для детей и о детях : XIX Всероссийская научно-практическая конференция, Москва, 03—04 февра- ля 2014 года. — Москва : Литера, 2014. — С. 97—101. р 12. Мурзинова И. А. Фиксированный линговокультурный типаж : интегральные и дифференциальный признаки / И. А. Мурзинова // Научный руководитель. — 2014. — № 2 (3). — С. 28—37. 12. Мурзинова И. А. Фиксированный линговокультурный типаж : интегральные и дифференциальный признаки / И. А. Мурзинова // Научный руководитель. — 2014. — № 2 (3). — С. 28—37. 13. Ощепкова Н. А. Методы исследования лингвокультурных типажей / Н. А. Ощеп- кова // Заметки учёного. — 2021. — № 8. — С. 85—88. 14. Павлишак Т. А. Лингвокультуроллогическая интепретация метафор сказа Н. С. Лескова «Левша» / Т. А. Павлишак // Язык и текст. — 2016. — Т. 3. — № 4. — С. 130—137. 15. Ранчин А. М. «Левша» Н. С. Лескова и русская национальная мифология / А. М. Ранчин // Россия XXI. — 2018. — № 3. — С. 114—141. 16. Столярова И. В. Лесков и Россия / И. В. Столярова // Лесков Н. С. Полное со- брание сочинений : В 30 т. — Москва : Терра, 1996. — Т. 1 : Сочинения. 1859—1862. — C. 7—100. — ISBN 5-85255-715-3. 17. Тамуркин С. А. Из жизни тульских оружейников : голубиная охота / С. А. Та- муркин // История. Историки. Источники : электронный научный журнал. Литература — 2016. — № 3. — С. 109—116. 18. Телия В. Н. Первоочередные задачи и методологические проблемы исследова- ния фразеологического состава языка в контексте культуры / В. Н. Телия // Фразеология в контексте культуры. — Москва : Языки русской культуры, 1999. — С. 13—24. — ISBN 5-88766-061-9. 19. Телия В. Н. Русская фразеология : семантико-прагматический и лингвокультуро- логический аспекты / В. Н. Телия. — Москва : Яз. рус. культуры, 1996. — 284 с. — ISBN 5-88766-061-9. 20. Токарев Г. В. Лингвокультурный симболарий : квазисимволы / Г. В. Токарев. — Тула : ТППО, 2021. — 112 с. 21. Цинк А. Деконструкция национального дискурса в творчестве Н. С. Леско- ва / А. Цинк // Филология и культура. — 2017. — № 4 (50). — С. 191—196. 22. Шариков В. Е. Повседневная жизнь туляков во второй половине XIX — начале XX в. — Тула : Борус-Принт, 2016. — 268 с. — ISBN 978-5-905154-26-3. 89 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 23. Шелковникова Л. Ф. Тернарность как основа мироощущения и миропонимания Н. С. Лескова-художника / Л. Ф. Шелковникова // Культура и текст. — 1997. — № 1. — С. 63—65. 24. Юркин И. Н. Абрам Булыгин : чудности, веселости, “непонятная философия” / И. Н. Юркин. — Тула : Рарус, 1994. — 186 с. — ISBN 5-86967-004-7. Material resources History of Tula’s philistine Abram Bulygin, about his wonderful adventures in the world, about hunting, merriment, and about work. (1994). In: Yurkin, I. N. Abram Bulygin: mira- cles, gaiety, “incomprehensible philosophy”. Tula: Rarus. Pp. 74—128. (In Russ.). Kubryakova, E. S., Demyankov, V. X., Pankrats, Yu. G., Luzina, L. G. (1996). A concise dictionary of cognitive terms. In: A concise dictionary of cognitive terms. Moscow: Lomonosov Moscow State University. 245 p. ISBN 5-89042-018-1. (In Russ.). Leskov, N. S. (1979). Favorites. Chisinau: Literatura artistique. 672 p. (In Russ.). Pankin, I. (1989). Legends about the master Poke. Moscow: Children’s Literature. 139 p. (In Russ.). References Ab hi ( Abashin, I. (2013). Political and philosophical views of N. S. Leskov: national identity. Mod- ern problems of science and education, 2: P. 495. (In Russ.). Beglova, E. I. (2021). Vocabulary in the work of N. S. Leskov “Lefty” in the aspect of Rus- sian folk speech in opposition “own”-“alien”. In: The world of N. S. Leskov: po- etics, pragmatics, stylistics. Collection of reports of participants of the National Scientifi c and Practical Conference dedicated to the 190th anniversary of the birth of N. S. Leskov. Bryansk: Publishing House of the I. G. Petrovsky BSU. 81—91. (In Russ.). Berezina, T. Yu., Vyrvich, E. I. (2018). The fate of a talented person in Russia in the tale of N. S. Leskov “Lefty”. In: Seventh Sylvester pedagogical readings. Spiritual- ity and morality in the educational space are the foundation of the future of Rus- sia. Materials of the All-Russian Scientifi c Conference. Omsk: Amphora. 30—33. (In Russ.). ( ) Dmitrieva, O. A. (2009). Value characteristics in the structure of linguistic and cultural type. The value system of modern society, 9: 55—59. (In Russ.). Goncharov, P. A., Cherbaeva, O. V. (2015). Sibiryak in the character sphere of A. Vampilov. Modern scientifi c research and innovation, 12 (56): 1009—1020. (In Russ.). Ibatullina, G. M. (2013). Historiosophical myth in N. Leskov’s story “Lefty”. In: Print and Word of St. Petersburg. St. Petersburg Readings-2012. Collection of scientifi c pa- pers: in 2 parts. St. Petersburg: St. Petersburg State University of Technology and Design, Northwestern Institute of Printing. 122—131. (In Russ.). Ibatullina, G. M. (2014). National historical myth and ways of its interpretation in the story of N. S. Leskov “Lefty”. Modern studies of social problems (electronic scientifi c journal), 4: P. 2. (In Russ.). Karasik, V. I., Dmitrieva, O. A. (2005). Linguocultural type: to the defi nition of the concept. In: Axiological linguistics: linguistic and cultural types. Volgograd: Paradigm. 5—25. (In Russ.). Lapteva, O. A. (2021). The path to the knowledge of the Russian national character (based on the work of N. S. Leskov “Lefty”). Pedagogical search, 4: 14—16. (In Russ.). , O. A. (2021). The path to the knowledge of the Russian national character (based on the work of N. S. Leskov “Lefty”). Pedagogical search, 4: 14—16. (In Russ.). 90 Научный диалог = Nauchnyi dialog = Nauchnyy dialog ISSN 2225-756X, eISSN 2227-1295 Makarova, A. D. (2014). References Ab hi ( Features of the genre of a fairy tale on the example of the work of Ivan Pankin “Legends of the master Poke”. In: World literature for children and about children: XIX All-Russian Scientifi c and Practical Conference, Moscow, Feb- ruary 03-04, 2014. Moscow: Litera. 97—101. (In Russ.). Murzinova, I. A. (2014). Fixed lingovocultural type: integral and differential signs. Scientifi c supervisor, 2 (3): 28—37. (In Russ.). Oshchepkova, N. A. (2021). Methods of research of linguocultural types. Notes of the scien- tist, 8: 85—88. (In Russ.). Pavlishak, T. A. (2016). Linguoculturological interpretation of metaphors of N. S. Leskov’s tale “Lefty”. Language and text, 3 (4): 130—137. (In Russ.). Ranchin, A. M. (2018). “Lefty” N. S. Leskova and Russian national mythology. Russia XXI, 3: 114—141. (In Russ.). ( ) Sharikov, V. E. (2016). The daily life of Tulyaks in the second half of the XIX — early XX cen- tury. Tula: Borus-Print LLC. 268 p. ISBN 978-5-905154-26-3. (In Russ.). Shelkovnikova, L. F. (1997). Ternarity as the basis of N. S. Leskov’s attitude and understand- ing of the world. Culture and text, 1: 63—65. (In Russ.). g ( ) Stolyarova, I. V. (1996). Leskov and Russia. In: Leskov N. S. Complete works: In 30 vols, 1: Essays. 1859—1862. Moscow: Terra. 7—100. ISBN 5-85255-715-3. (In Russ.). Tamurkin, S. A. (2016). From the life of Tula gunsmiths: pigeon hunting. History. Historians. Sources: electronic scientifi c journal, 3: 109—116. (In Russ.). Teliya, V. N. (1996). Russian phraseology: semantic-pragmatic and linguoculturological as- pects. Moscow: Yaz. rus. Kultury. 284 p. ISBN 5-88766-061-9. (In Russ.). Teliya, V. N. (1999). Priority tasks and methodological problems of the study of the phra- seological composition of language in the context of culture. In: Phraseology in the context of culture. Moscow: Languages of Russian Culture. 13—24. ISBN 5-88766-061-9. (In Russ.). Tokarev, G. V. (2021). Linguocultural symbolarium: quasi-symbols. Tula: TPPO. 112 p. (In Russ.). Vasantada, V. D. (2013). Linguocultural type as a refl ection of linguistic consciousness. Actual problems of humanities and natural sciences, 5: 230—232. (In Russ.). problems of humanities and natural sciences, 5: 230—232. (In Russ.). Yurkin, I. N. (1994). Abram Bulygin: miracles, gaiety, “incomprehensible philosophy”. Tula: Rarus. 186 p. ISBN 5-86967-004-7. (In Russ.). I. N. (1994). Abram Bulygin: miracles, gaiety, “incomprehensible philosophy”. Tula: Rarus. 186 p. ISBN 5-86967-004-7. (In Russ.). Zink, A. (2017). Deconstruction of national discourse in the works of N. S. Leskov. Philology and culture, 4 (50): 191—196. References Ab hi ( (In Russ.). Статья поступила в редакцию 10.08.2022, одобрена после рецензирования 04.10.2022, подготовлена к публикации 17.10.2022. 91 91
https://openalex.org/W2163270681
https://coughjournal.biomedcentral.com/track/pdf/10.1186/1745-9974-8-7
English
null
Antitussive effects of the peripherally restricted GABAB receptor agonist lesogaberan in guinea pigs: Comparison to baclofen and other GABAB receptor-selective agonists
Cough
2,012
cc-by
6,276
RESEARCH Open Access * Correspondence: bjc@jhmi.edu 1Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224, USA Full list of author information is available at the end of the article © 2012 Canning 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. Abstract Background: Gastroesophageal reflux disease (GERD) is a common cause of chronic cough. Both acid and nonacid reflux is thought to play a role in the initiation of coughing and cough hypersensitivity. The GABAB receptor agonist lesogaberan was developed as a peripherally restricted anti-reflux therapy that reduces the frequency of transient lower esophageal sphincter relaxations (TLESR; the major cause of reflux) in animals and in patients with GERD. GABAB receptor agonists have also been shown to possess antitussive effects in patients and in animals independent of their effects on TLESR, suggesting that lesogaberan may be a promising treatment for chronic cough. Methods: We have assessed the direct antitussive effects of lesogaberan (AZD3355). The effects of other GABAB receptor agonists were also determined. Coughing was evoked in awake guinea pigs using aerosol challenges with citric acid. Results: Lesogaberan dose-dependently inhibited citric acid evoked coughing in guinea pigs. Comparable effects of the GABAB receptor agonists baclofen and 3-aminopropylphosphinic acid (3-APPiA) on cough were also observed. Baclofen produced obvious signs of sedation and respiratory depression. By contrast, both lesogaberan and 3-APPiA (both inactivated centrally by GABA transporters) were devoid of sedative effects and did not alter respiratory rate. Conclusions: Together, the data suggest that lesogaberan and related GABAB receptor agonists may hold promise as safe and effective antitussive agents largely devoid of CNS side effects. Keywords: Gastroesophageal reflux, Esophagus, LES relaxation, C-fiber, TRPV1, Lesogaberan Cough is one of the most commonly reported symptoms amongst patients seeking medical advice. Acute cough is triggered primarily by viral infections, while the most common causes of chronic cough are asthma, upper air- way inflammatory disorders, and gastroesophageal reflux disease (GERD). Therapeutics used specifically for the treatment of cough are either minimally effective or have unwanted side effects that limit their utility. In patients with chronic cough, treatment of their underlying disease can improve patient quality of life and reduce coughing. But for many patients with chronic, troublesome cough, even after aggressive medical treatment of their underlying illnesses, cough can remain a significant health problem that adversely impacts quality of life. New and more ef- fective and selective treatments for cough thus represent an unmet need in respiratory medicine [1,2]. Antitussive effects of the peripherally restricted GABAB receptor agonist lesogaberan in guinea pigs: comparison to baclofen and other GABAB receptor-selective agonists Brendan J Canning1*, Nanako Mori1 and Anders Lehmann2 Brendan J Canning1*, Nanako Mori1 and Anders Lehmann2 Cough Cough Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Methods The institutional animal care and use committee approved all of the studies described below. Male Hartley guinea pigs (200-400 g, Charles River) were placed in a flow through chamber filled with air by an air pump. A pressure transducer was connected to the outflow of the chamber to monitor respiratory efforts and coughing in response to citric acid challenge. Data was recorded digi- tally using a Biopac data acquisition system. Key to the side effect profile of systemically adminis- tered GABAB receptor agonists is their CNS penetrance and susceptibility to inactivation by uptake [23,24]. GABA and analogs of GABA are subject to uptake in the central nervous system by the 4 identified GABA transporters (GAT1-GAT4). Baclofen is not a substrate for uptake and can act centrally when administered peripherally [25]. By contrast, 3-APPiA is a GABAB receptor agonist that is inactivated centrally by trans- port [24-26]. There are reports of antitussive effects of 3-APPiA in guinea pigs and cats [27,28]. It is thus possible that GABAB receptor agonists work periphe- rally to prevent vagal reflexes. Guinea pigs were pretreated 30 minutes prior to citric acid challenge with vehicle, lesogaberan (0.3-10 mg/ kg), baclofen (0.3 and 3 mg/ kg), 3-APPiA (0.3 and 3 mg/ kg) or SKF97541 (0.1 and 0.3 mg/ kg), administered by sub- cutaneous injection. After a 10 minute equilibration period in the exposure chamber when basal respiratory rate was monitored, guinea pigs were then challenged with increasing concentrations of citric acid (0.01, 0.1, 0.3 and 1 M), delivered by nebulizer (particle size: <5 μm) connected in series with the air pump. Each dose was delivered for 5 minutes, with a 5 minute interval in between doses. The total number of coughs evoked by each concentration of citric acid both during the 5 minute challenge and during the 5 minutes following the chal- lenge was determined. The results of these studies are pre- sented as the cumulative number of coughs evoked. Lesogaberan (AZD3355) is a GABAB receptor agonist with limited CNS side effects that was developed for the treatment of GERD [20,25,29-31]. Like 3-APPiA, lesoga- beran is inactivated centrally by GAT-dependent trans- port [25]. The purpose of this study was to evaluate the Figure 1 A representative trace of coughing evoked by a citric acid challenge to an awake guinea pig is depicted. Abstract Agonists of the metabotropic GABAB receptor such as baclofen have been evaluated for their utility in targeting a number of peripheral disorders thought to involve aberrant reflexes and sensations including pain, overactive * Correspondence: bjc@jhmi.edu 1Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224, USA Full list of author information is available at the end of the article Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Page 2 of 7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 effects of lesogaberan and other GABAB receptor ago- nists on citric acid induced coughing in guinea pigs. bladder, hiccups, tetanus/spasticity, and headache [3-10]. GABAB receptor agonists have also been evaluated for their effects on airways hyperresponsiveness, GERD and cough [11-20]. Although clinical benefit has been reported in these latter studies, a sedative effect of baclofen has also been noted [21,22]. Ideally, an effective treatment for cough would prevent cough through direct effects on sen- sory nerves innervating the airways and independent of any significant CNS-dependent side effects. A therapy that targets both cough and GERD would be especially desi- rable, given the association between these conditions. Methods Inspiratory (I) efforts produce a negative pressure in the chamber, with expiratory (E) efforts producing positive pressures. These traces were used to measure respiratory rate at the outset of each experiment (breaths/ min), the time to first cough following initiation of the citric acid challenges, the Peak to Peak (P-P) pressures associated with cough (measured by comparing the P-P pressures associated with coughing, expressed as a percentage of the P-P pressures measured at eupnea), the total number of coughs evoked by each dose of citric acid and the total number of coughs evoked cumulatively by all doses of citric acid studied. Figure 1 A representative trace of coughing evoked by a citric acid challenge to an awake guinea pig is depicted. Inspiratory (I) efforts produce a negative pressure in the chamber, with expiratory (E) efforts producing positive pressures. These traces were used to measure respiratory rate at the outset of each experiment (breaths/ min), the time to first cough following initiation of the citric acid challenges, the Peak to Peak (P-P) pressures associated with cough (measured by comparing the P-P pressures associated with coughing, expressed as a percentage of the P-P pressures measured at eupnea), the total number of coughs evoked by each dose of citric acid and the total number of coughs evoked cumulatively by all doses of citric acid studied. Figure 1 A representative trace of coughing evoked by a citric acid challenge to an awake guinea pig is depicted. Inspiratory (I) efforts produce a negative pressure in the chamber, with expiratory (E) efforts producing positive pressures. These traces were used to measure respiratory rate at the outset of each experiment (breaths/ min), the time to first cough following initiation of the citric acid challenges, the Peak to Peak (P-P) pressures associated with cough (measured by comparing the P-P pressures associated with coughing, expressed as a percentage of the P-P pressures measured at eupnea), the total number of coughs evoked by each dose of citric acid and the total number of coughs evoked cumulatively by all doses of citric acid studied. Page 3 of 7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Canning et al. Methods Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Cough was defined visually and by the characteristic pressure changes in the chamber, reflecting an enhanced inspiratory effort followed by a forceful expiratory effort, with expiratory pressure changes ≥500% of the pres- sures associated with expiration during eupnea, all oc- curring in less than 1 second. Coughing is readily differentiated from enhanced breaths (sighs), often asso- ciated with stimuli inducing bronchospasm, which have a roughly symmetric pressure signature (with equally enhanced inspiratory and expiratory pressures) and slower cycle (Figure 1). In these healthy, young animals and with the cough evoked by citric acid aerosols, the tus- sive responses evoked were presumed and indeed likely to be the result of a direct effect of the citric acid on airway sensory nerves. By extension, any antitussive effects of the compounds studied were likely to be the result of a direct effect on cough, and not the consequence of any effects on underlying pathology (e.g. GERD). Figure 2 GABAB receptor agonists have differential effects on respiratory rate in awake guinea pigs. Each bar represents the mean ± sem of 5–15 experiments. An asterisk (*) indicates that the treatment reduced respiratory rate relative to that measured in animals pretreated with vehicle (p < 0.05). The results are presented as a mean ± sem of n experi- ments, where n is the number of guinea pigs studied. Guinea pigs were challenged only once (to each dose of citric acid) in this unpaired experimental design. Differ- ences amongst group means were evaluated by 1 way analysis of variance. A p-value of less than 0.05 was con- sidered significant. Figure 2 GABAB receptor agonists have differential effects on respiratory rate in awake guinea pigs. Each bar represents the mean ± sem of 5–15 experiments. An asterisk (*) indicates that the treatment reduced respiratory rate relative to that measured in animals pretreated with vehicle (p < 0.05). Figure 2 GABAB receptor agonists have differential effects on respiratory rate in awake guinea pigs. Each bar represents the mean ± sem of 5–15 experiments. An asterisk (*) indicates that the treatment reduced respiratory rate relative to that measured in animals pretreated with vehicle (p < 0.05). acid had a characteristic pattern of single, powerful coughs, occasionally 2 on consecutive breaths, but never the paroxysmal coughing we reported previously in stu- dies of bradykinin evoked coughing [32,33]. No signs of tachyphylaxis were apparent in these studies. Methods Thus, if a robust cough response was evoked by a lower concentra- tion of citric acid, subsequent challenge with a higher dose of citric acid still evoked coughing. Results Baseline respiratory rate in control animals averaged 96 ± 1 breaths/ min (n = 15). At a dose of 3 mg/ kg, both baclo- fen and SKF97541 reduced baseline respiratory rate (78 ± 7 and 69 ± 3 breaths/ min, respectively; n = 5-6; p < 0.05) and produced obvious signs of sedation and le- thargy. By contrast, 3 mg/ kg 3-APPiA and lesogaberan, even up to a dose of 10 mg/ kg, were without effect on respiratory rate (98 ± 2 and 97 ± 1 breaths/min, re- spectively; n = 7-8; p > 0.1) and produced no other overt signs of sedation or lethargy (Figure 2). Citric acid inhalation evoked concentration-dependent coughing in the awake guinea pigs. No animals in any of the treatment groups coughed in response to the citric acid vehicle (water) inhalation, while 56/61 animals coughed at least once to 0.3 M citric acid challenge. The highest concentration of citric acid was not always well- tolerated. Five-minute challenges with 1 M citric acid were interrupted in 2/15 control experiments due to labored breathing. All but 3 of 41 animals pretreated with baclofen, 3-APPiA or lesogaberan completed the 1 M citric acid challenges. The coughing evoked by citric Reagents Baclofen and citric acid were purchased from Sigma (St. Louis, MO). AstraZeneca (Mölndal, Sweden) provided lesogaberan, 3-APPiA and SKF97541. All reagents were dissolved in 0.9% NaCl solution. Baclofen, 3-APPiA and lesogaberan all inhibited citric acid induced coughing (Figure 3). The effects of all of these drugs were found to be dose-dependent. Lesoga- beran was equipotent to baclofen in these studies (but, as mentioned above, without coincident sedative effects and respiratory depression). The sedating effects of SKF97541 were so profound that its antitussive actions were not extensively studied. Baclofen, 3-APPiA and leso- gaberan all slightly reduced the percentage of animals coughing to any given dose of citric acid, and reduced the number of coughs evoked by given doses of citric acid. None of the drugs studied had any pronounced effects on the time to onset of coughing, nor on the peak pressures produced during the cough (Table 1). Discussion Lesogaberan is a GABAB receptor agonist with a limited CNS side effect profile. This compound has been evalu- ated as a treatment for GERD, a primary cause of chronic cough [20,25,29-31]. In the present study, leso- gaberan was found to be an effective antitussive agent, preventing citric acid evoked coughing in conscious Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Page 4 of 7 Figure 3 Effects of GABAB receptor agonists on citric acid evoked coughing. Each bar represents the mean ± sem of 3–15 experiments. An asterisk (*) indicates a significant reduction in the number of cumulative coughs relative to vehicle control (p < 0.05). Figure 3 Effects of GABAB receptor agonists on citric acid evoked coughing. Each bar represents the mean ± sem of 3–15 experiments. An asterisk (*) indicates a significant reduction in the number of cumulative coughs relative to vehicle control (p < 0.05). guinea pigs in a dose-dependent manner but without co- incident sedative effects or respiratory depression. We found this compound was as potent and effective as baclofen, a GABAB receptor agonist used clinically for the treatment of several disorders. Unlike lesogaberan, however, the antitussive effects of baclofen were accom- panied by undesirable side effects, including sedation, res- piratory depression and a trend towards a decrease in peak cough pressures. Based on these results, we conclude guinea pigs in a dose-dependent manner but without co- incident sedative effects or respiratory depression. We found this compound was as potent and effective as baclofen, a GABAB receptor agonist used clinically for the treatment of several disorders. Unlike lesogaberan, however, the antitussive effects of baclofen were accom- panied by undesirable side effects, including sedation, res- piratory depression and a trend towards a decrease in peak cough pressures. Based on these results, we conclude that lesogaberan may hold promise for the treatment of acute and chronic cough and may have a better side effect profile than baclofen. The four GABAB receptor agonists used in this study are essentially identical in receptor pharmacological pro- file. All are selective GABAB receptor agonists, with SKF97541, 3-APPiA and lesogaberan sharing structural similarities. The data are presented as the %animals coughing or as the mean ± sem of n experiments where n is a single animal studied in a non paired experimental design. The time to first cough was measured during challenges with 0.3 M citric acid. The peak to peak (P-P) pressures associated with cough were measured for the first 5 coughs evoked during challenge, regardless of citric acid concentration. An asterisk (*) indicates a statistically significant difference from that observed in vehicle treated animals (p < 0 05) Discussion Baclofen and SKF97541 are not transported, whereas both 3-APPiA and lesogaberan are substrates for GAT. Thus, the site of action for the latter 2 compounds is thought to be limited to peripheral loca- tions and perhaps central locations with limited GAT ex- pression [19,25]. The marked respiratory depression and lethargy induced by SKF97541 and baclofen but not by lesogaberan and 3-APPiA we observed are consistent with the known susceptibility to uptake of these compounds. yet, there is no evidence to suggest their activation would prevent action potential discharge. Interestingly, however, there are several studies showing direct inhibi- tory effects of GABAB receptor agonists on the excitabi- lity of vagal afferent nerves, including those innervating the stomach and esophagus [47-50]. Non-neuronal sites of action for GABAB receptor ago- nists are also possible. Acid evoked cough may be mast cell dependent, and mast cell activation may trigger or at least modulate subsequently evoked cough [51,52]. GABAB receptor agonists can attenuate allergen-induced inflammation, perhaps through effects on mast cell acti- vation [53,54]. GABAB receptors on non-neural airway cells has also been documented, including airway smooth muscle and epithelium [55,56], and we have described a transduction pathway dependent upon acti- vation of chemosensory epithelial cells [57]. Endogenous GABA in the lung may also regulate cough [58]. Finally, GABAB receptor agonists have also been shown to at- tenuate parasympathetic-cholinergic responses in the airways, which might also indirectly attenuate cough responses [59-61]. In general, however, these non-neural influences on cough would seem more likely to be mo- dulatory than essential to evoked cough. Bronchospasm is not an effective stimulus for cough, and bronchodila- tors have modest, variable effects on evoked cough [44]. Our results confirm and extend the studies by Bolser et al. [27,28,34], who reported that both baclofen and 3-APPiA prevented coughing in guinea pigs and cats. Because 3-APPiA was more effective administered pe- ripherally than centrally, the authors concluded that GABAB receptor agonists prevent cough at least in part through peripheral sites of action. But a central site of action of these drugs cannot be entirely dismissed. We found that intraperitoneally administered baclofen inhi- bits cough [33], but we also reported that baclofen microinjected into the nTS inhibits cough in anesthe- tized guinea pigs [35]. Bolser et al. concluded that baclofen works primarily via central effects [28,34]. Discussion Similarly, Callaway and King [36] found that baclofen inhalation prevented citric acid induced alterations in respiratory pattern (possibly by inhibiting broncho- spasm) but was without effect on citric acid induced cough. Perhaps cell groupings in the brainstem, access- ible to peripherally administered agonists and on the fringe of the blood brain barrier, are targeted by these drugs. For a central site of action to be viable, agonists such as 3-APPiA and lesogaberan must penetrate brain- stem regions relevant to cough and attain sufficient con- centrations to prevent or blunt synaptic transmission. There is conflicting evidence relating to blood-borne ac- cess and GABA uptake mechanisms in nTS [37-43]. g GABAB receptor agonists may hold promise in the treatment of GERD due to their ability to prevent TLESR, the primary physiologic process responsible for reflux [15,17,18,62,63]. Compounds such as lesogaberan may offer a distinct advantage over conventional thera- peutic approaches to GERD inasmuch as proton pump inhibitors and histamine H2 receptor antagonists do not prevent reflux, but rather, reduce the acid content of gastric fluid. While acid is thought to be the major trig- ger of symptoms and pathology in GERD, other compo- nents of refluxate such as pepsin likely contribute to the pathophysiology of this disease [64,65]. GERD is also a major cause of chronic cough [1,2]. Indeed, cough may be the only presenting symptom of GERD in some patients. The ability of compounds such as lesogaberan to prevent coughing evoked directly from the airways is another potential therapeutic advantage of GABAB re- ceptor agonists in the treatment of GERD. These attri- butes along with the limited side effect profile of lesogaberan relative to that of baclofen provides impetus for continued study of this compound as a treatment for both GERD and chronic cough. GABAB receptor activation might prevent coughing through peripheral inhibitory effects on bronchopul- monary vagal afferent nerves. The vagal afferent nerves regulating cough in guinea pigs are C-fibers arising from the jugular ganglia, and cough receptors, terminating in the larynx, trachea and mainstem bronchi and with cell bodies in the nodose ganglia [32,44]. Both of these vagal afferent nerve subtypes are responsive to acid but largely insensitive to changes in airway luminal pressure, stretch or airway smooth muscle contraction. The cough recep- tors are, however, insensitive to capsaicin. Given that Bolser et al. Discussion Key to their differential side effect profile, however, is their susceptibility to uptake by GABA Table 1 Effects of GABAB agonists on the percentage of animals coughing, time to onset of coughing and on peak expiratory pressures during cough %Animals Coughing to Citric Acid Treatment n Time to 1st Cough (min) -P Pressures (%control) 0.01M 0.1M Vehicle control 15 2.0±03 1144±102% 27% (4/ 15) 93% (14/ 15) 0.3 mg/ kg baclofen 3 2.4±0.6 837±187% 100% (3/ 3) 100% (3/ 3) 3 mg/ kg baclofen 6 1.4±0.3 705±110% 0% (0/ 6)* 50% (3/ 6) 3 mg/ kg SKF97541 5 2.7±1.0 852±92% 0% (0/ 5)* 60% (3/ 5) 0.3 mg/ kg 3-APPiA 4 2.8±0.7 762±121% 0% (0/ 5)* 80% (4/ 5) 3 mg/ kg 3-APPiA 7 3.3±0.7 986±120% 0% (0/ 7)* 57% (4/ 7) 1 mg/ kg lesogaberan 5 1.7±0.2 1426±142% 0% (0/ 5)* 80% (4/ 5) 3 mg/ kg lesogaberan 8 2.1±0.6 1100±144% 13% (1/ 8) 63% (5/ 8) 10 mg/ kg lesogaberan 8 3.0±0.6 855±99% 0% (0/ 8)* 50% (4/ 8) The data are presented as the %animals coughing or as the mean ± sem of n experiments where n is a single animal studied in a non paired experimental design. The time to first cough was measured during challenges with 0.3 M citric acid. The peak to peak (P-P) pressures associated with cough were measured for the first 5 coughs evoked during challenge, regardless of citric acid concentration. An asterisk (*) indicates a statistically significant difference from that observed in vehicle treated animals (p < 0.05). Table 1 Effects of GABAB agonists on the percentage of animals coughing, time to onset of cough expiratory pressures during cough GABAB agonists on the percentage of animals coughing, time to onset of coughing and on peak es during cough The data are presented as the %animals coughing or as the mean ± sem of n experiments where n is a single animal studied in a non paired experimental design. The time to first cough was measured during challenges with 0.3 M citric acid. The peak to peak (P-P) pressures associated with cough were measured for the first 5 coughs evoked during challenge, regardless of citric acid concentration. An asterisk (*) indicates a statistically significant difference from that observed in vehicle treated animals (p < 0.05). Page 5 of 7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 transporters [23-25]. Discussion studied the antitussive effects of baclofen and 3-APPiA with capsaicin as the tussive stimulus, the available data would suggest that if GABAB receptor agonists are working peripherally, they act at least in part through effects on airway vagal C-fiber terminals [27,28]. In fact, there is functional evidence to suggest that the vagal C-fibers regulating cough express GABAB receptors on their peripheral terminals [45,46], but as Competing interests AL is an employee of AstraZeneca. References 1. Morice AH, Fontana GA, Sovijarvi AR, Pistolesi M, Chung KF, Widdicombe J, O'Connell F, Geppetti P, Gronke L, De Jongste J, Belvisi M, Dicpinigaitis P, Fischer A, McGarvey L, Fokkens WJ, Kastelik J: ERS Task Force. The diagnosis and management of chronic cough. Eur Respir J 2004, 24(3):481–492. 24. Lehmann A, Antonsson M, Aurell-Holmberg A, Blackshaw L, Brändén L, Elebring T, Jensen J, Kärrberg L, Mattsson J, Nilsson K, Oja S, Saransaari P, von Unge S: Different in vitro and in vivo profiles of substituted 3-aminopropylphosphinate and 3-aminopropyl(methyl)phosphinate GABA(B) receptor agonists as inhibitors of transient lower oesophageal sphincter relaxation. Br J Pharmacol 2012, 165(6):1757–1772. 2. Irwin RS, Baumann MH, Bolser DC, Boulet LP, Braman SS, Brightling CE, Brown KK, Canning BJ, Chang AB, Dicpinigaitis PV, Eccles R, Glomb WB, Goldstein LB, Graham LM, Hargreave FE, Kvale PA, Lewis SZ, McCool FD, McCrory DC, Prakash UB, Pratter MR, Rosen MJ, Schulman E, Shannon JJ, Smith Hammond C, Tarlo SM: American College of Chest Physicians (ACCP). Diagnosis and management of cough executive summary: ACCP evidence- based clinical practice guidelines. Chest 2006, 129(1 Suppl):1S–23S. 25. Lehmann A, Antonsson M, Aurell Holmberg A, Blackshaw LA, Branden L, Brauner-Osborne H, Christiansen B, Dent J, Elebring T, Jacobson BM, Jensen J, Mattsson JP, Nilsson K, Oja SS, Page AJ, Saransaari P, von Unge S: AZD3355, a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action. J Pharmacol Exp Ther 2009, 331(2):504–512. 3. Müller H, Börner U, Zierski J, Hempelmann G: Intrathecal baclofen in tetanus. Lancet 1986, 1(8476):317–318. 26. Ong J, Kerr DI: The gamma-aminobutyric acid uptake inhibitor NO-711 potentiates 3-aminopropylphosphinic acid-induced actions in rat neocortical slices. Eur J Pharmacol 1998, 347(2–3):197–200. , ( ) 4. Launois S, Bizec JL, Whitelaw WA, Cabane J, Derenne JP: Hiccup in adults: an overview. Eur Respir J 1993, 6(4):563–575. 4. Launois S, Bizec JL, Whitelaw WA, Cabane J, Derenne JP: Hiccup in adults: an overview. Eur Respir J 1993, 6(4):563–575. 27. Bolser DC, Aziz SM, DeGennaro FC, Kreutner W, Egan RW, Siegel MI, Chapman RW: Antitussive effects of GABAB agonists in the cat and guinea-pig. Br J Pharmacol 1993, 110(1):491–495. 5. Hering-Hanit R, Gadoth N: Baclofen in cluster headache. Headache 2000, 40(1):48–51. 6. Meythaler JM, Guin-Renfroe S, Brunner RC, Hadley MN: Intrathecal baclofen for spastic hypertonia from stroke. Stroke 2001, 32(9):2099–2109. 28. References Bolser DC, DeGennaro FC, O'Reilly S, Chapman RW, Kreutner W, Egan RW, Hey JA: Peripheral and central sites of action of GABA-B agonists to inhibit the cough reflex in the cat and guinea pig. Br J Pharmacol 1994, 113(4):1344–1348. 7. Pehrson R, Lehmann A, Andersson KE: Effects of gamma-aminobutyrate B receptor modulation on normal micturition and oxyhemoglobin induced detrusor overactivity in female rats. J Urol 2002, 168(6):2700–2705. 29. Alstermark C, Amin K, Dinn SR, Elebring T, Fjellström O, Fitzpatrick K, Geiss WB, Gottfries J, Guzzo PR, Harding JP, Holmén A, Kothare M, Lehmann A, Mattsson JP, Nilsson K, Sundén G, Swanson M, von Unge S, Woo AM, Wyle MJ, Zheng X: Synthesis and pharmacological evaluation of novel gamma-aminobutyric acid type B (GABAB) receptor agonists as gastroesophageal reflux inhibitors. J Med Chem 2008, 51(14):4315–4320. 8. Enna SJ, McCarson KE: The role of GABA in the mediation and perception of pain. Adv Pharmacol 2006, 54:1–27. 9. Brennan PM, Whittle IR: Intrathecal baclofen therapy for neurological disorders: a sound knowledge base but many challenges remain. Br J Neurosurg 2008, 22(4):508–519. 10. Miyazato M, Sasatomi K, Hiragata S, Sugaya K, Chancellor MB, de Groat WC, Yoshimura N: GABA receptor activation in the lumbosacral spinal cord decreases detrusor overactivity in spinal cord injured rats. J Urol 2008, 179(3):1178–1183. 30. Brändén L, Fredriksson A, Harring E, Jensen J, Lehmann A: The novel, peripherally restricted GABAB receptor agonist lesogaberan (AZD3355) inhibits acid reflux and reduces esophageal acid exposure as measured with 24-h pHmetry in dogs. Eur J Pharmacol 2010, 634(1–3):138–141. 11. Dicpinigaitis PV, Spungen AM, Bauman WA, Absgarten A, Almenoff PL: Inhibition of bronchial hyperresponsiveness by the GABA-agonist baclofen. Chest 1994, 106(3):758–761. 31. Boeckxstaens GE, Denison H, Jensen JM, Lehmann A, Ruth M: Translational gastrointestinal pharmacology in the 21st century: 'the lesogaberan story'. Curr Opin Pharmacol 2011, 11(6):630–633. 12. Dicpinigaitis PV: Use of baclofen to suppress cough induced by angiotensin-converting enzyme inhibitors. Ann Pharmacother 1996, 30(11):1242–1245. 32. Canning BJ, Mazzone SB, Meeker SN, Mori N, Reynolds SM, Undem BJ: Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs. J Physiol 2004, 557(Pt 2):543–558. 13. Dicpinigaitis PV, Dobkin JB: Antitussive effect of the GABA-agonist baclofen. Chest 1997, 111(4):996–999. 14. Dicpinigaitis PV, Dobkin JB, Rauf K, Aldrich TK: Inhibition of capsaicin- induced cough by the gamma-aminobutyric acid agonist baclofen. J Clin Pharmacol 1998, 38(4):364–367. 33. Funding h d This study was funded by AstraZeneca and by a grant from the National Institutes of Health (HL083192). 20. Boeckxstaens GE, Rydholm H, Lei A, Adler J, Ruth M: Effect of lesogaberan, a novel GABA-receptor agonist, on transient lower esophageal sphincter relaxations in male subjects. Aliment Pharmacol Ther 2010, 31(11):1208–1217. Received: 23 March 2012 Accepted: 7 September 2012 Published: 1 October 2012 Received: 23 March 2012 Accepted: 7 September 2012 Published: 1 October 2012 Received: 23 March 2012 Accepted: 7 September 2012 Published: 1 October 2012 23. Clausen RP, Madsen K, Larsson OM, Frølund B, Krogsgaard-Larsen P, Schousboe A: Structure-activity relationship and pharmacology of gamma-aminobutyric acid (GABA) transport inhibitors. Adv Pharmacol 2006, 54:265–284. Authors’ contributions BC d d d d BC designed and conducted many of the experiments, interpreted the results and wrote multiple sections of the manuscript. NM conducted many of the experiments, analyzed the data and created the graphic summaries of Page 6 of 7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 the results. AL designed the experiments, interpreted the results and wrote multiple sections of the manuscript. All authors read and approved the final manuscript. 18. Zhang Q, Lehmann A, Rigda R, Dent J, Holloway RH: Control of transient lower oesophageal sphincter relaxations and reflux by the GABA(B) agonist baclofen in patients with gastro-oesophageal reflux disease. Gut 2002, 50(1):19–24. 19. Lehmann A: GABAB receptors as drug targets to treat gastroesophageal reflux disease. Pharmacol Ther 2009, 122(3):239–245. Author details 1J h H ki 1Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224, USA. 2AstraZeneca R&D Mölndal, SE-431 83, Mölndal, Sweden. 21. Choo YM, Kim GB, Choi JY, Park JH, Park JH, Yang CW, Kim YS, Bang BK: Severe respiratory depression by low-dose baclofen in the treatment of chronic hiccups in a patient undergoing CAPD. Nephron 2000, 86(4):546–547. 22. Marshall FH: Is the GABA B heterodimer a good drug target? J Mol Neurosci 2005, 26(2–3):169–176. References Smith JA, Young EC, Saulsberry L, Canning BJ: Anti-tussive effects of memantine in guinea pigs. Chest, 141(4):996–1002. 34. Bolser DC, Hey JA, Chapman RW: Influence of central antitussive drugs on the cough motor pattern. J Appl Physiol 1999, 86(3):1017–1024. 15. Lehmann A, Antonsson M, Bremner-Danielsen M, Flärdh M, Hansson- Brändén L, Kärrberg L: Activation of the GABA(B) receptor inhibits transient lower esophageal sphincter relaxations in dogs. Gastroenterology 1999, 117(5):1147–1154. 35. Canning BJ, Mori N: Encoding of the cough reflex in anesthetized guinea pigs. Am J Physiol Regul Integr Comp Physiol 2011, 300(2):R369–R377. gy 16. Dicpinigaitis PV, Grimm DR, Lesser M: Baclofen-induced cough suppression in cervical spinal cord injury. Arch Phys Med Rehabil 2000, 81(7):921–923. gy 16. Dicpinigaitis PV, Grimm DR, Lesser M: Baclofen-induced cough suppression in cervical spinal cord injury. Arch Phys Med Rehabil 2000, 81(7):921–923. 36. Callaway JK, King RG: Effects of inhaled alpha 2-adrenoceptor and GABAB receptor agonists on citric acid-induced cough and tidal volume changes in guinea pigs. Eur J Pharmacol 1992, 220(2–3):187–195. 37. Simon JR, DiMicco SK, Aprison MH: Neurochemical studies of the nucleus of the solitary tract, dorsal motor nucleus of the vagus and the hypoglossal nucleus in rat: topographical distribution of 17. Lidums I, Lehmann A, Checklin H, Dent J, Holloway RH: Control of transient lower esophageal sphincter relaxations and reflux by the GABA(B) agonist baclofen in normal subjects. Gastroenterology 2000, 118(1):7–13. Page 7 of 7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 Canning et al. Cough 2012, 8:7 http://www.coughjournal.com/content/8/1/7 60. Chapman RW, Danko G, Rizzo C, Egan RW, Mauser PJ, Kreutner W: Prejunctional GABA-B inhibition of cholinergic, neurally-mediated airway contractions in guinea-pigs. Pulm Pharmacol 1991, 4(4):218–224. glutamate uptake, GABA uptake and glutamic acid decarboxylase activity. Brain Res Bull 1985, 14(1):49–53. 38. Gross PM, Wall KM, Pang JJ, Shaver SW, Wainman DS: Microvascular specializations promoting rapid interstitial solute dispersion in nucleus tractus solitarius. Am J Physiol 1990, 259(6 Pt 2):R1131–R1138. 61. Chapman RW, Hey JA, Rizzo CA, Bolser DC: GABAB receptors in the lung. Trends Pharmacol Sci 1993, 14(1):26–29. 62. Blackshaw LA, Staunton E, Lehmann A, Dent J: Inhibition of transient LES relaxations and reflux in ferrets by GABA receptor agonists. Am J Physiol 1999, 277(4 Pt 1):G867–G874. 39. Ikegaki N, Saito N, Hashima M, Tanaka C: Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry. Brain Res Mol Brain Res 1994, 26(1–2):47–54. 63. doi:10.1186/1745-9974-8-7 44. Canning BJ, Mori N, Mazzone SB: Vagal afferent nerves regulating the cough reflex. Respir Physiol Neurobiol 2006, 152(3):223–242. 45. Belvisi MG, Ichinose M, Barnes PJ: Modulation of non-adrenergic, non-cholinergic neural bronchoconstriction in guinea-pig airways via GABAB-receptors. Br J Pharmacol 1989, 97(4):1225–1231. 46. Ray NJ, Jones AJ, Keen P: GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro Br J Pharmacol 1991, 102(4):801–804. 47. Page AJ, Blackshaw LA: GABA(B) receptors inhibit mechanosensitivity of primary afferent endings. J Neurosci 1999, 19(19):8597–8602. 48. Blackshaw LA, Smid SD, O'Donnell TA, Dent J: GABA(B) receptor- mediated effects on vagal pathways to the lower oesophageal sphincter and heart. Br J Pharmacol 2000, 130(2):279–288. 49. Partosoedarso ER, Young RL, Blackshaw LA: GABA(B) receptors on vagal afferent pathways: peripheral and central inhibition. Am J Physiol Gastrointest Liver Physiol 2001, 280(4):G658–G668. 50. Smid SD, Young RL, Cooper NJ, Blackshaw LA: GABA(B)R expressed on vagal afferent neurones inhibit gastric mechanosensitivity in ferret proximal stomach. Am J Physiol Gastrointest Liver Physiol 2001, 281(6):G1494–G1501. 51. Lai YL, Lin TY: Mast cells in citric acid-induced cough of guinea pigs. Toxicol Appl Pharmacol 2005, 202(1):18–24. 52. Mazzone SB, Undem BJ: Cough sensors. V. Pharmacological modulation of cough sensors. Handb Exp Pharmacol 2009, 187:99–127. 53. Gentilini G, Franchi-Micheli S, Mugnai S, Bindi D, Zilletti L: GABA-mediated inhibition of the anaphylactic response in the guinea-pig trachea. Br J Pharmacol 1995, 115(3):389–394. 54. Tohda Y, Ohkawa K, Kubo H, Muraki M, Fukuoka M, Nakajima S: Role of GABA receptors in the bronchial response: studies in sensitized guinea-pigs. Clin Exp Allergy 1998, 28(6):772–777. 55. Osawa Y, Xu D, Sternberg D, Sonett JR, D'Armiento J, Panettieri RA, Emala CW: Functional expression of the GABAB receptor in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2006, 291(5):L923–L931. 56. Mizuta K, Osawa Y, Mizuta F, Xu D, Emala CW: Functional expression of GABAB receptors in airway epithelium. Am J Respir Cell Mol Biol 2008, 39(3):296–304. References Staunton E, Smid SD, Dent J, Blackshaw LA: Triggering of transient LES relaxations in ferrets: role of sympathetic pathways and effects of baclofen. Am J Physiol Gastrointest Liver Physiol 2000, 279(1):G157–G162. 40. Evans JE, Frostholm A, Rotter A: Embryonic and postnatal expression of four gamma-aminobutyric acid transporter mRNAs in the mouse brain and leptomeninges. J Comp Neurol 1996, 376(3):431–446. 64. Fock KM, Talley NJ, Fass R, Goh KL, Katelaris P, Hunt R, Hongo M, Ang TL, Holtmann G, Nandurkar S, Lin SR, Wong BC, Chan FK, Rani AA, Bak YT, Sollano J, Ho KY, Manatsathit S: Asia-Pacific consensus on the management of gastroesophageal reflux disease: update. J Gastroenterol Hepatol 2008, 23(1):8–22. 41. Wang QP, Guan JL, Pan W, Kastin AJ, Shioda S: A diffusion barrier between the area postrema and nucleus tractus solitarius. Neurochem Res 2008, 33(10):2035–2043. 42. Maolood N, Meister B: Protein components of the blood–brain barrier (BBB) in the brainstem area postrema-nucleus tractus solitarius region. J Chem Neuroanat 2009, 37(3):182–195. 65. Kahrilas PJ: Clinical practice. Gastroesophageal reflux disease. N Engl J Med 2008, 359(16):1700–1707. 65. Kahrilas PJ: Clinical practice. Gastroesophageal reflux disease. N Engl J Med 2008, 359(16):1700–1707. 43. Batten TF, Pow DV, Saha S: Co-localisation of markers for glycinergic and GABAergic neurones in rat nucleus of the solitary tract: implications for co-transmission. J Chem Neuroanat 2010, 40(2):160–176. doi:10.1186/1745-9974-8-7 Cite this article as: Canning et al.: Antitussive effects of the peripherally restricted GABAB receptor agonist lesogaberan in guinea pigs: comparison to baclofen and other GABAB receptor-selective agonists. Cough 2012 8:7. doi:10.1186/1745-9974-8-7 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: • 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: • 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: 57. Krasteva G, Canning BJ, Hartmann P, Veres TZ, Papadakis T, Mühlfeld C, Schliecker K, Tallini YN, Braun A, Hackstein H, Baal N, Weihe E, Schütz B, Kotlikoff M, Ibanez-Tallon I, Kummer W: Cholinergic chemosensory cells in the trachea regulate breathing. Proc Natl Acad Sci USA 2011, 108(23):9478–9483. • Convenient online submission 58. Xiang YY, Wang S, Liu M, Hirota JA, Li J, Ju W, Fan Y, Kelly MM, Ye B, Orser B, O'Byrne PM, Inman MD, Yang X, Lu WY: A GABAergic system in airway epithelium is essential for mucus overproduction in asthma. Nat Med 2007, 13(7):862–867. 59. Tamaoki J, Graf PD, Nadel JA: Effect of gamma-aminobutyric acid on neurally mediated contraction of guinea pig trachealis smooth muscle. J Pharmacol Exp Ther 1987, 243(1):86–90.
https://openalex.org/W2169093495
https://europepmc.org/articles/pmc3543808?pdf=render
English
null
Outcome Measures of Chinese Herbal Medicine for Hypertension: An Overview of Systematic Reviews
Evidence-based complementary and alternative medicine
2,012
cc-by
5,478
Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 697237, 7 pages doi:10.1155/2012/697237 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 697237, 7 pages doi:10.1155/2012/697237 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 697237, 7 pages doi:10.1155/2012/697237 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 697237, 7 pages doi:10.1155/2012/697237 Jie Wang and Xingjiang Xiong Department of Cardiology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China Correspondence should be addressed to Xingjiang Xiong 5administration@163 com Department of Cardiology, Guang’anmen Hospital, China Academy of Chinese Medica epartment of Cardiology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, Chi Correspondence should be addressed to Xingjiang Xiong, 5administration@163.com Received 11 November 2012; Accepted 10 December 2012 Academic Editor: Tabinda Ashfaq Copyright © 2012 J. Wang and X. Xiong. 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. The aim of this overview was to summarize the outcome measures of Chinese herbal medicine (CHM) for the treatment of hypertension based on available systematic reviews (SRs), so as to evaluate the potential benefits and advantages of CHM on hypertension. Methods. Literature searches were conducted in the Cochrane Database of Systematic Reviews, MEDLINE, and 4 databases in Chinese. SRs of CHM for hypertension were included. Two independent reviewers (J. Wang and X. J. Xiong) extracted the data. Results. 10 SRs were included. 2 SRs had primary endpoints, while others focused on secondary endpoints to evaluate CHM for hypertension such as blood pressure (BP) and Traditional Chinese Medicine (TCM) syndrome. 6 SRs have reported the adverse effects, whereas the other 4 SRs have not mentioned it at all. Many CHM appeared to have significant effect on improving BP, TCM syndrome, and so on. However, most SRs failed to make a definite conclusion for the effectiveness of CHM for hypertension due to poor evidence. Conclusion. Primary endpoints have not been widely used currently. The benefits of CHM for hypertension need to be confirmed in the future with randomized controlled trials (RCTs) of more persuasive primary endpoints and high-quality SRs. 2. Methods Literature searches were conducted in the Cochrane Database of Systematic Review (October, 2012), MED- LINE (2002–2012), Chinese National Knowledge Infrastruc- ture (CNKI, 2002–2012), Chinese Biomedical Literature Database (CBM, 2002–2012), Chinese Scientific Journal Database (VIP, 2002–2012), and Wanfang Databases (2002– 2012). All of those searches ended on October 10, 2012. CNKI, CBM, VIP, and Wanfang were four main databases in China. All of the databases in Chinese were searched to retrieve the maximum possible number of systematic reviews or meta-analyses of CHM for hypertension because CHMs are mainly used and researched in China. We searched papers from 2002 to 2012 for high-quality RCTs and SRs mainly focusing on the recent ten years. The strategy below was used to search The Cochrane Library and adapted appropriately for use in different electronic databases: #1 herb∗; #2 medic∗; #3 (#1 and #2); #4 Chinese; #5 (#3 or #4); #6 blood pressure; #7 hypertension; #8 high blood pressure; #9 (#6 or #7 or #8); #10 (#5 and #9). Two reviewers (J. Wang and X. J. Xiong) independently scanned the relevance of all references based on title and abstract of each record. If the information included a systematic review or a meta-analysis of CHM for hypertension, the full paper was obtained for further assessment. Papers were excluded when problems occurred with: repeat publication, methodological studies, quality assessment report, research on acupuncture, qigong, mas- sage, or other treatments (Figure 1). y 8 SRs were concerned with essential hypertension, and the other 2 were related to elderly hypertension. We also retrieved the related clinical trials for further analysis. These clinical trials in SRs were mainly conducted in China. The methodological quality of clinical trials was assessed independently with criteria from the Cochrane Handbook for Systematic Review of Interventions, Version 5.1.0 (J. Wang and X. J. Xiong) [37]. The items included random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (per- formance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other bias. It was found out that although the original trials included all claimed “RCTs” or “quasi-RCTs”, only few of them were typical RCTs. Almost all the trials mentioned that “patients were randomized into two groups” without detailed information about ran- domization. So, it is hard to judge whether randomization was conducted properly and really. Most of them have not mentioned allocation concealment and double-blind. 1. Introduction with unique theory of diagnosis and treatment [10–15]. In recent years, with the popularity and prevalence of Chinese medicine (CM), there has been a growing interest in Chinese herbal medicine (CHM) for patients with hypertension both in China and the West [16–20]. Until now a number of clinical studies of CHM reported the clinical effectiveness in hypertensive patients ranging from case reports and case series to controlled observational studies and randomized clinical trials. However, the evidence needs to be reviewed systematically [21]. Hypertension is one of the most common and important health problems affecting millions of people throughout the world and about 20% of the adult population in many countries [1]. It could lead to severe complications, such as hypertensive cardiovascular disease, hypertensive renal disease, and atherosclerotic complications including stroke, coronary heart disease, renal insufficiency, and heart failure [2]. However, hypertension in most individuals remains untreated or uncontrolled [3]. Effective treatment of hyper- tension is limited by availability, cost, and adverse effects of antihypertensive medications. Some hypertension-related symptoms could not be completely relieved by conventional medicine. Hypertension is the major cause of morbidity and mortality and is the third highest risk factor for lifetime burden worldwide [4]. Therefore, some patients have turned to complementary and alternative therapies (or traditional medicine), especially Chinese medicine (CM) [5–9], hoping that such treatments might improve their symptoms. Chi- nese medicine (CM) has a history for more than 2500 years As the evidence gathering tools, systematic reviews (SRs) of randomized controlled trials (RCTs) are considered to provide the best evidence about the effectiveness of interven- tions [22, 23]. Physicians and policy makers need evidence from SRs for decision making and policy making. Patients and researchers also need such information to support shared decisions and to set priorities for research. Recently, an increasing number of SRs about CHM for hypertension have been reported. However, few of them have shown that CHM was definitely effective for hypertension due to the weak evidence. There is a need for combining multiple Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 2 Evidence-Based Complementary and Alternative Medicine 2 reviews into overviews to provide users with easily available information. In addition, when people making decisions about health care look for guidance from research, the outcomes reported are key, which also plays an important role in drawing a more persuasive conclusion [24]. 1. Introduction However, there is a general lack of consensus regarding the choice of outcomes in particular clinical settings, which affect trial design, conduct, analysis, and reporting [25]. The aim of this overview was to summarize the outcome measures of CHM for treatment of hypertension based on available SRs both in English and Chinese, so as to display the current situation and evaluate the potential benefits and advantages of CHM on hypertension. of systematic reviews and meta-analyses. It requires each reviewer to follow the research process and include a flow diagram providing information about the number of studies identified, included, and excluded through database searching and other sources, and reasons for excluding them such as duplicates. Information of each included SRs was imported into PRISMA statement for analysis. One author (J. Wang or X. J. Xiong) independently extracted data from each included review using predefined criteria and discussed the data with the other author to reach a consensus when there is a disagreement. 3. Results After primary search of 6 databases, 182 articles were screened out from electronic and manual searches (as shown in Figure 1), and the majority were excluded due to obvious ineligibility which including irrelevant titles and abstracts (some papers were found from more than one database). After reading the titles and abstracts, a majority of them were excluded. 170 articles were excluded because of duplicates, nonclinical studies, case reports, and research on acupunc- ture, moxibustion, cupping, qigong, Tai Chi, and other treat- ments. Then 12 full articles were retrieved for more detailed evaluation. Due to methodological study and quality assess- ment report, 2 out of them were excluded respectively based on the assessment tool. In the end, 10 SRs were reviewed [27– 36]. All the SRs were conducted in China with 1 in English and 9 in Chinese. 9 SRs from the Chinese electronic databases were published between 2006 and 2012. Since 2011, the num- ber of SRs increased markedly. Only 1 SR from the Cochrane Database of Systematic Review was published in 2012 [31]. 2. Methods Most of adverse effects of CHM were mentioned as “none obvious,” “low adverse effect” or even “no adverse effect.” The reported adverse reactions in control groups were more severe than in treatment groups. Adverse events reported in 4 SRs [27, 30, 32, 34], including headache, dizziness, cough, dry stool, and diarrhea. Thus, adverse reactions of CHM should be highlighted in systematic reviews, and the safety of CHMs needs to be monitored rigorously and reported appropriately in the future clinical trials. Articles excluded: total = 2 Methodological studies: 1 Quality assessment report: 1 Figure 1: Flow-chart of SRs selection. The Cochrane Collaboration is an international organi- zation which aims to prepare and maintain rigorous sys- tematic reviews in order to help people make well-informed decisions about health care [46]. As we know that Cochrane reviews are regarded as the highest standard of evidence with a greater methodological quality [47]. Outcome measures of the included SRs in Cochrane Database of Systematic Reviews are more credible than non-Cochrane reviews [48, 49]. They adopt primary endpoints, secondary endpoints, and safety as outcome measures. Unfortunately, in our paper, only one SR about Tianma Gouteng Yin for essential hypertension was retrieved from Cochrane Library [31]. The authors of the SR identified no study which met the inclusion criteria for review. As no trials could be identified for the review, no conclusions can be made about the role of Tianma Gouteng Yin in the treatment of essential hypertension. When referring to non-Cochrane reviews, primary endpoints and adverse effects are seldom taken as outcome measures in most SRs. mixture, and four were about calming the liver therapy such as Pingjiangyin capsule, Pinggan jiangya capsule, Niuhuang jiangya tablet, and Tiaopingkang tablet. Among these 10 SRs, 3 kinds of CHM were reviewed, including capsules, pellets, and herbal decoction as follows: Niuhuang Jiangya preparation (n = 1) [27]; Tianma Gouteng Yin (n = 2) [30, 31]; herbal decoction (n = 7) [28, 29, 32–36]. The characteristics of 10 SRs were summarized in Table 1. As shown Table 1, 2 SRs analyzed primary endpoints and the remaining nine SRs all focused on secondary endpoints to evaluate CHM for hypertension. This is mainly due to whether there was detailed information in the original research or not. 4 primary endpoints were analyzed in 2 SRs including essential hypertension and elderly hypertension. 2. Methods That is to say, the claimed RCTs may not be true RCTs actually. Therefore, most of the trials in the SRs were of low quality. However, only 10 RCTs were of high quality: three were concerned with replenishing spleen and kidney therapy, one was related to promoting blood circulation and removing blood stasis therapy, two were associated with clearing heat therapy such as Bidens bipinnata L. and Qinre jiangya g g Outcome measures included primary endpoints and secondary endpoints. Primary endpoints include mortality, stroke, coronary heart disease, and hypertensive renal dam- age. Secondary endpoints mainly indicate blood pressure, the level of blood lipids, pulse pressure (PP), quality of life (QOL), and Traditional Chinese Medicine (TCM) syndrome. In addition, PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) was used as an assessment tool to evaluate the quality of the included SRs [26]. As shown in the article written by Moher et al. [26], the checklist consists of 27 items in 7 key areas and a four-phase flow diagram in order to help authors improve the reporting of systematic reviews and meta-analyses. It describes the preferred way to present the title, abstract, introduction, methods, results, discussion, and funding sections in detail Evidence-Based Complementary and Alternative Medicine Potentially relevant articles identified through search strategy for more detailed evaluation: total = 182 Full articles retrieved for more detailed evaluation: total = 12 Articles excluded: duplicates; nonclinical studies; case reports; research on acupuncture, moxibustion, cupping, qigong, Tai Chi and other treatments: total = 170 Articles excluded: total = 2 Methodological studies: 1 Quality assessment report: 1 Articles identified for inclusion in the review: total = 10 Figure 1: Flow-chart of SRs selection. 3 root causes, and no toxic and side effects [40–42]. Even it is widely accepted that it is safe to use herbal medicines for various diseases in China. However, along with the development of pharmacology study, there are more and more reports of liver toxicity and other adverse events associated with CHM [43–45], so this paper makes the analysis on the adverse effects of CHM for hypertension. In this paper, adverse effects are ignored. 6 SRs [27, 30, 32– 34, 36] have reported the adverse effects, whereas the other 4 SRs [28, 29, 31, 35] have not mentioned it at all. Only 2 trials in the 1 SR [34] had long-term data on adverse effects. 2. Methods 1 SR about Niuhuang Jiangya preparation showed no effect on the mortality, stroke, coronary heart disease, and hypertensive renal damage [27]. The other 1 SR about herbal products appeared to be effective on improving hypertensive renal damage [36]. Blood pressure was the most common secondary endpoint in the SRs. All the included SRs reported blood pressure changes. Among them, 8 SRs showed improvement in blood pressure, but the other 2 SRs showed insufficient evidence [27, 31]. 5 SRs analyzed TCM syndrome changes [32–36]. There are 3 SRs that reported Triglycerides (TG) [28], pulse pressure (PP) [35], and quality of life (QOL) [36], respectively. Many CHMs appear to be effective on improving signs and symptoms, level of blood lipids, and so forth. Some SRs also reflected that CHM may be effective to prevent progression to severe complications of hypertension. However, due to poor methodological quality in the majority of included trials, most SRs could not draw confirmative con- clusions on the beneficial effect of CHM for hypertension. In addition, it was found out that most of the included SRs were generally of low quality according to PRISMA statement. Review methods were not fully reported in most SRs. The characteristics of included clinical trials were not described with detailed information in 5 SRs [29, 30, 32–34]. No flow-chart of information through the different phases of a systematic review was provided. Sensitivity analysis, subgroup analysis, and potential publication bias were not analyzed sufficiently in the reviews. Convincing outcome measures were lacked in most SRs. 4. Discussion (2008) [27] 3/3 B NA hypertensive renal damage (1) Elderly hypertension (1) Herbal products Han (2012) [36] 4/45 A NA Secondary endpoints Blood pressure (10) Essential hypertension (8) Niuhuang Jiangya preparation Wang (2008) [27] 3/3 B NA Herbal products Hu (2009) [28] 24/24 A L Herbal products Ren (2006) [29] 11/11 A H Tianma Gouteng Yin Dong (2011) [30] 6/6 A L Tianma Gouteng Yin Zhang (2012) [31] 0/0 B NA Pinggan qianyang Xu (2012) [32] 8/8 A H Buyi shenqi Shi (2012) [33] 5/5 A H Buyi pishen Liu (2011) [34] 13/15 A L Elderly isolated systolic hypertension (1) Herbal products Li (2012) [35] 17/17 A L Elderly hypertension (1) Herbal products Han (2012) [36] 45/45 A NA Triglycerides (1) Essential hypertension (1) Herbal products Hu (2009) [28] 4/24 A L Pulse pressure (1) Elderly isolated systolic hypertension (1) Herbal products Li (2012) [35] 4/17 A L Quality of life (1) Elderly hypertension (1) Herbal products Han (2012) [36] 4/45 A NA TCM syndrome (5) Essential hypertension (3) Pinggan qianyang Xu (2012) [32] 3/8 A H Buyi shenqi Shi (2012) [33] 4/5 A H Buyi pishen Liu (2011) [34] 9/15 A L Elderly isolated systolic hypertension (1) Herbal products Li (2012) [35] 6/17 A L Elderly hypertension (1) Herbal products Han (2012) [36] 4/45 A NA Notes: Pinggan qianyang: calming the liver and suppressing liver-yang to patients with hyperactivity of liver yang syndrome; Buyi shenqi: replenishing kidney qi to patients with kidney qi deficiency syndrome; Buyi pishen: replenishing spleen and kidney to patients with spleen and kidney deficiency syndrome; A: CHM may be or appear to be effective; B: the evidence is insufficient and inclusive; H: high; L: low; NA: not mentioned. Notes: Pinggan qianyang: calming the liver and suppressing liver-yang to patients with hyperactivity of liver yang syndrome; Buyi shenqi: replenishing kidney qi to patients with kidney qi deficiency syndrome; Buyi pishen: replenishing spleen and kidney to patients with spleen and kidney deficiency syndrome; A: CHM may be or appear to be effective; B: the evidence is insufficient and inclusive; H: high; L: low; NA: not mentioned. at endpoint. Most of the included SRs have not reported the mortality rate or the incidence of complications. The primary endpoints are seldom used due to the difficulty of clinical implementation, limitations of the research funding and other reasons. On the contrary, secondary endpoints are most commonly adopted in clinical trials. 4. Discussion In our overview, the primary endpoints and secondary endpoints are all used to evaluate the efficacy of CHM for hypertension. It is widely known that the primary goal of essential hypertension treatment is to reduce mortality, or prevent progression to severe complications such as stroke, coronary heart disease, heart failure, and hypertensive renal damage. However, there is a lack of data on the final indicator Adverse effects, providing a guideline to both doctors and patients for reasonable medication, should also be regarded as an essential outcome measure in clinical trials [38, 39]. However, there is a widespread misunderstanding of CHM. Most people, especially in East Asia, think that the application of TCM has a long history, natural origination, good health care effects, efficacy of treating symptoms and Evidence-Based Complementary and Alternative Medicine 4 Table 1: Outcome measures of CHM for hypertension in systematic reviews. Table 1: Outcome measures of CHM for hypertension in systematic reviews. Outcome measures (number of SR) Condition (number of SR) CHM First author Number of RCTs/total Conclusion Risk of publication bias Primary endpoints Mortality, stroke, coronary heart disease, and hypertensive renal damage (1) Essential hypertension (1) Niuhuang Jiangya preparation Wang et al. References [1] K. Sliwa, S. Stewart, and B. J. Gersh, “Hypertension: a global perspective,” Circulation, vol. 123, no. 24, pp. 2892–2896, 2011. [2] S. MacMahon, M. H. Alderman, L. H. Lindholm, L. Liu, R. A. Sanchez, and Y. K. Seedat, “Blood-pressure-related disease is a global health priority,” The Lancet, vol. 371, no. 9623, pp. 1480–1482, 2008. [3] P. M. Kearney, M. Whelton, K. Reynolds, P. Muntner, P. K. Whelton, and J. He, “Global burden of hypertension: analysis of worldwide data,” Lancet, vol. 365, no. 9455, pp. 217–223, 2005. [4] A. V. Chobanian, G. L. Bakris, H. R. Black et al., “Seventh report of the joint national committee on prevention, detec- tion, evaluation, and treatment of high blood pressure,” Hypertension, vol. 42, no. 6, pp. 1206–1252, 2003. [5] H. Xu and K. J. Chen, “Complementary and alternative medi- cine: is it possible to be mainstream?” Chinese Journal of Inte- grative Medicine, vol. 18, no. 6, pp. 403–404, 2012. [6] A. Weil, “The state of the integrative medicine in the U.S. and Western World,” Chinese Journal of Integrative Medicine, vol. 17, no. 1, pp. 6–10, 2011. [7] C. Keji and X. Hao, “The integration of traditional Chinese medicine and Western medicine,” European Review, vol. 11, no. 2, pp. 225–235, 2003. [8] F. Cheung, “TCM: made in China,” Nature, vol. 480, no. 7378, supplement, pp. S82–S83, 2011. More specifically, the following deficiencies in this overview should be taken into consideration before rec- ommending the conclusion. Firstly, both the majority of included SRs and the original clinical trials are of low quality due to poorly designed and low-quality methodology. Secondly, as CHM is mainly used in China, SRs published in Chinese and English are retrieved. However, electronic databases in other languages have been omitted. Thirdly, unpublished studies and many negative randomized, double- blind, and controlled trials have not been taken into account for further analysis. [9] H. Xu and K. Chen, “Integrative medicine: the experience from China,” Journal of Alternative and Complementary Medi- cine, vol. 14, no. 1, pp. 3–7, 2008. [10] N. Robinson, “Integrative medicine—traditional Chinese medicine, a model?” Chinese Journal of Integrative Medicine, vol. 17, no. 1, pp. 21–25, 2011. [11] K. J. Chen, “Where are we going?” Chinese Journal of Integra- tive Medicine, vol. 16, no. 2, pp. 100–101, 2010. [12] G. Dobos and I. Evidence-Based Complementary and Alternative Medicine and the National Natural Science Foundation Project of China (90209011). Ever since 1999 when the first Cochrane review of CHM was published [50], there is an increasing number of similar systematic reviews/meta-analysis. Thus, it is necessary to systematically identify and assess the quality of these reviews. The methodology and reporting quality of systematic reviews/meta-analyses of CHM have attracted great attention [51–54]. According to PRISMA statement, the quality of the current included SRs is judged as generally poor, especially those published in Chinese journals. Reviews had methodological and reporting flaws that could have influenced the reviews validity. The deficiencies mainly lies in searching literature, reporting of characteristics of included and excluded studies, extracting relevant data, evaluating primary trials’ quality, and merging data. Also, the report of less persuasive outcome measures in most of the SRs has reduced the validity of the conclusions. So, in future, reviewers should attach more importance to the method of performing SR and receive relevant training of skills in reporting to reduce the amount of bias in their reviews. Researchers of clinical trials in TCM should also pay more attention to experimental design and methodological quality and improve the reporting quality according to the Consolidated Standards of Reporting Trials (CONSORT) statement [55], so as to improve the quality of TCM clinical research and ensure truth and reliability of conclusions. Although CHM appeared to be effective for hypertension in clinical use, most SRs were inconclusive that CHM had a definite effect for hypertension due to the poor evidence. References Tao, “The model of western integrative medicine: the role of Chinese medicine,” Chinese Journal of Integrative Medicine, vol. 17, no. 1, pp. 11–20, 2011. In summary, although both primary and secondary endpoints were all used to evaluate the effectiveness of CHM for hypertension, primary endpoints have not widely been used currently. Although this overview may show potential effectiveness of CHM for hypertension in terms of some outcome measures, most SRs failed to draw a confirmative conclusion for recommendation on the beneficial effect of CHM in hypertensive patients due to poor evidence. The benefits of CHM for hypertension still need to be confirmed in the future with more rigorous RCTs of more persuasive primary endpoints and high-quality SRs. [13] D. Eisenberg, “Reflections on the past and future of integrative medicine from a lifelong student of the integration of Chinese and Western medicine,” Chinese Journal of Integrative Medi- cine, vol. 17, no. 1, pp. 3–5, 2011. [14] X. G. Sun, W. K. Wu, and Z. P. Lu, “Chinese integrative medicine: translation toward person centered and balanced medicine,” Chinese Journal of Integrative Medicine, vol. 18, no. 1, pp. 3–6, 2012. [15] J. Wang, P. Q. Wang, and X. J. Xiong, “Current situation and re-understanding of syndrome and formula syndrome in Chinese medicine,” Internal Medicine, vol. 2, no. 3, 2012. Conflict of Interests [16] H. Xu and K. J. Chen, “Integrating traditional medicine with biomedicine towards a patient-centered healthcare system,” Chinese Journal of Integrative Medicine, vol. 17, no. 2, pp. 83– 84, 2011. All authors declare that they have no conflict of interests. 4. Discussion The outcome measures from all the included SRs are mainly blood pressure and TCM symptom. It is probably related to the feasibility and operability either in inpatients or outpatients in small sample size and short-term clinical trials. Although it is helpful to reduce future cardiovascular risk to some extent by decreasing blood pressure and improving TCM symptoms, primary endpoints are widely recognized as more persuasive outcome measures when evaluating the efficacy of CHM for hypertension. Moreover, adverse effect, which is also very important in evaluating the safety of CHM, should be taken as outcome measures too. All of these problems affect the generation of high-level evidence of CHM for hypertension. 5 Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine Acknowledgments [17] J. Wang and X. J. Xiong, “Current situation and perspectives of clinical study in integrative medicine in China,” Evidence- Based Complementary and Alternative Medicine, vol. 2012, Article ID 268542, 11 pages, 2012. This work was supported in part by the National Basic Research Program of China (973 Program, 2003CB517103) Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 6 [18] R. A. Bell, C. K. Suerken, J. G. Grzywacz, W. Lang, S. A. Quandt, and T. A. Arcury, “CAM use among older adults age 65 or older with hypertension in the United States: general use and disease treatment,” Journal of Alternative and Complementary Medicine, vol. 12, no. 9, pp. 903–909, 2006. [34] L. Liu and Y. L. Li, “Systematic review on treatment of essential hypertension from spleen and kidney deficiency,” Zhonghua Zhong Yi Yao Za Zhi, vol. 26, no. 8, pp. 1700–1703, 2011 (Chinese). [35] D. N. Li and C. H. Yang, “Effects of Chinese medicine on elderly isolated systolic hypertension: a meta-analysis,” Liaon- ing Zhong Yi Za Zhi, vol. 39, no. 5, pp. 812–815, 2012 (Chinese). [19] E. Ernst, “Complementary/alternative medicine for hyperten- sion: a mini-review,” Wiener Medizinische Wochenschrift, vol. 123, pp. 386–391, 2005. [36] S. H. Han, Evaluation of integrated Chinese and western medi- cine in treatment of hypertension in the elderly and their life quality, [M.S. thesis], China Academy of Chinese Medical Sci- ences, Beijing, China, 2011. [20] J. J. Park, S. Beckman-Harned, G. Cho, D. Kim, and Hangon Kim, “The current acceptance, accessibility and recognition of Chinese and Ayurvedic medicine in the United States in the public, governmental, and industrial sectors,” Chinese Journal of Integrative Medicine, vol. 18, no. 6, pp. 405–408, 2012. [37] J. P. T. Higgins and S. Green, “Cochrane handbook for system- atic reviews of interventions,” version 5. 1. 0, The Cochrane Collaboration, 2009, http://www.cochrane-handbook.org/. [21] J. Wang and X. J. Xiong, “Control strategy on hypertension in Chinese medicine,” Evidence-Based Complementary and Alter- native Medicine, vol. 2012, Article ID 284847, 6 pages, 2012. [38] M. Clarke, “Standardising outcomes for clinical trials and systematic reviews,” Trials, vol. 8, article no. 39, 2007. [22] H. Xu and K. J. Chen, “Making evidence-based decisions in the clinical practice of integrative medicine,” Chinese Journal of Integrative Medicine, vol. 16, no. 6, pp. 483–485, 2010. [39] E. Veitch, “The science of outcomes: how far have we come?” http://blogs.plos.org/speakingofmedicine/2011/07/15/the-sci- ence-of-outcomes-how-far-have-we-come/. [23] M. Y. Liu and K. J. Acknowledgments Chen, “Convergence: the tradition and the modern,” Chinese Journal of Integrative Medicine, vol. 18, no. 3, pp. 164–165, 2012. [40] K. J. Chen, “Clinical service of Chinese medicine,” Chinese Journal of Integrative Medicine, vol. 14, no. 3, pp. 163–164, 2008. [24] P. Williamson and M. Clarke, “The COMET (Core Outcome Measures in Effectiveness Trials) Initiative: its role in improv- ing Cochrane Reviews,” Cochrane Database of Systematic Reviews , vol. 5, Article ID ED000041, 2012. [41] K. Chan, “Some aspects of toxic contaminants in herbal med- icines,” Chemosphere, vol. 52, no. 9, pp. 1361–1371, 2003. [42] L. Zhang, J. B. Yan, X. M. Liu et al., “Pharmacovigilance prac- tice and risk control of traditional Chinese medicine drugs in China: current status and future perspective,” Journal of Eth- nopharmacology, vol. 140, no. 3, pp. 519–525, 2012. [25] R. M. Smyth, J. J. Kirkham, A. Jacoby, D. G. Altman, C. Gamble, and P. R. Williamson, “Frequency and reasons for outcome reporting bias in clinical trials: interviews with trial- ists,” BMJ, vol. 342, Article ID c7153, 2011. [43] H. Xu and K. J. Chen, “Herb-drug interaction: an emerging issue of integrative medicine,” Chinese Journal of Integrative Medicine, vol. 16, no. 3, pp. 195–196, 2010. [26] D. Moher, A. Liberati, J. Tetzlaff, and D. G. Altman, “Preferred reporting items for systematic reviews and Meta-analyses: the PRISMA statement,” PLOS Medicine, vol. 6, no. 7, Article ID e1000097, 2009. [44] J. Wang, R. van der Heijden, S. Spruit et al., “Quality and safe- ty of Chinese herbal medicines guided by a systems biology perspective,” Journal of Ethnopharmacology, vol. 126, no. 1, pp. 31–41, 2009. [27] H. Wang, H. C. Shang, J. H. Zhang et al., “Niuhuang Jiangya preparation for treatment of essential hypertension: a system- atic review,” Liaoning Zhong Yi Za Zhi, vol. 35, no. 5, pp. 649– 652, 2008 (Chinese). [45] D. Melchart, K. Linde, S. Hager, D. Shaw, and R. Bauer, “Liver enzyme elevations in patients treated with traditional Chinese medicine,” Journal of the American Medical Association, vol. 282, no. 1, pp. 28–29, 1999. [28] Y. X. Hu, Quantitative analysis of clinical controlled trials of traditional Chinese medicine and systematic evaluation of ran- domized controlled trials involving traditional Chinese medicine for essential hypertension, [M.S. thesis], Guangzhou University of Chinese Medicine, Guangzhou, China, 2009. [46] S. Green, J. P. T. Higgins, P. Alderson et al., Cochrane Hand- book, version 5.0.1, The Cochrane Library, 2008. Acknowledgments [47] The Cochrane Collaboration, “Cochrane reviews,” 2011, http://www.cochrane.org/cochrane-reviews/. [29] Y. Ren, A. H. Ou, X. Z. Lin, and Y. R. Lao, “Meta-analysis of traditional Chinese medicine for essential hypertension,” Shanxi Zhong Yi, vol. 27, no. 7, pp. 794–796, 2006 (Chinese). [48] J. Luo and H. Xu, “Outcome measures of Chinese herbal medicine for coronary heart disease: an overview of system- atic reviews,” Evidence-Based Complementary and Alternative Medicine, vol. 2012, Article ID 927392, 9 pages, 2012. [30] D. X. Dong, S. L. Yao, N. Yu, and B. Yang, “Systematic review and meta-analysis of Tianma Gouteng Yin combined with enalapril for essential hypertension,” Zhongguo Zhong Yi Ji Zheng, vol. 20, no. 5, pp. 762–764, 2011 (Chinese). [49] Y. Qiu, H. Xu, and D. Z. Shi, “Traditional Chinese herbal products for coronary heart disease: an overview of Cochrane Reviews,” Evidence-Based Complementary and Alternative Medicine, vol. 2012, Article ID 417387, 2012. [31] H. W. Zhang, J. Tong, G. Zhou, H. Jia, and J. Y. Jiang, “Tianma Gouteng Yin formula for treating primary hypertension,” Cochrane Database of Systematic Reviews, no. 6, Article ID CD008166, 2012. [50] J. Hu, J. H. Zhang, W. Zhao, Y. L. Zhang, L. Zhang, and H. C. Shang, “Cochrane systematic reviews of Chinese herbal medi- cines: an overview,” PLoS One, vol. 6, no. 12, Article ID e28696, 2011. [32] W. J. Xu and Y. L. Li, “Systematic review of clinical evidence about calm the liver and subdue yang therapy on the hyper- tension disease with syndrome of upper hyperactivity of liver yang,” Zhonghua Zhong Yi Yao Za Zhi, vol. 27, no. 3, pp. 736– 739, 2012 (Chinese). [51] B. Ma, J. Guo, G. Qi et al., “quality and reporting character- istics of systematic reviews of traditional Chinese medicine interventions published in Chinese journals,” PLoS One, vol. 6, no. 5, Article ID e20185, 2011. [33] M. Shi and Y. H. Zhang, “Systematic review of replenishing kidney qi method for essential hypertension with kidney qi deficiency syndrome,” Shandong Zhong Yi Za Zhi, vol. 31, no. 4, pp. 236–238, 2012 (Chinese). [52] J. He, L. Du, G. Liu et al., “Quality assessment of reporting of randomization, allocation concealment, and blinding in tradi- tional chinese medicine RCTs: a review of 3159 RCTs identified 7 Evidence-Based Complementary and Alternative Medicine 7 Evidence-Based Complementary and Alternative Medicine from 260 systematic reviews,” Trials, vol. 12, article no. 122, 2011. [53] Z. Junhua, S. Hongcai, G. Xiumei et al., “Methodology and reporting quality of systematic review/meta-analysis of tradi- tional Chinese medicine,” Journal of Alternative and Comple- mentary Medicine, vol. 13, no. 8, pp. 797–805, 2007. [54] J. Wang, K. W. Yao, X. C. Yang et al., “Chinese patent medicine liu wei di huang wan combined with antihypertensive drugs, a new integrative medicine therapy, for the treatment of essential hypertension: a systematic review of randomized controlled trials,” Evidence-Based Complementary and Alterna- tive Medicine, vol. 2012, Article ID 714805, 7 pages, 2012. [55] K. F. Schulz, D. G. Altman, and D. Moher, “CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials,” PLoS Medicine, vol. 7, no. 3, Article ID e1000251, 2010.
https://openalex.org/W4362671131
https://link.springer.com/content/pdf/10.1007/s11229-023-04132-3.pdf
English
null
Correction to: Closure, deduction and hinge commitments
Synthese
2,023
cc-by
297
Synthese (2023) 201:140 https://doi.org/10.1007/s11229-023-04132-3 Synthese (2023) 201:140 https://doi.org/10.1007/s11229-023-04132-3 CORRECTION Published online: 6 April 2023 © The Author(s) 2023 Published online: 6 April 2023 © The Author(s) 2023 Published online: 6 April 2023 © The Author(s) 2023 Synthese (2018) 198:3533-3551 https://doi.org/10.1007/s11229-018-1679-x Xiaoxing Zhang zhang@phare.normalesup.org 1 Paris-Sorbonne University, Paris, France Correction to: Closure, deduction and hinge commitments Xiaoxing Zhang1 Synthese (2018) 198:3533-3551 https://doi.org/10.1007/s11229-018-1679-x The article Closure, deduction and hinge commitments, written by Xiaoxing Zhang, was originally published Online First without Open Access. After publication in vol­ ume 198, issue S15, pages 3533–3551, the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to ©The Author(s) 2023 and the article is forthwith distributed under the terms of the Creative Commons Attribution. 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/. Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The online version of the original article can be found at https://doi.org/10.1007/s11229-018-1679-x Xiaoxing Zhang zhang@phare.normalesup.org 3
https://openalex.org/W4291463784
https://link.springer.com/content/pdf/10.1007/s11845-022-03117-4.pdf
English
null
Competency in trauma surgery: a national survey of trainees and consultants
Irish journal of medical science
2,022
cc-by
4,764
Abstract Background  The current sparsity of surgical trainees’ exposure to training in operative trauma surgery is multifactorial. This concern has been addressed in the revised Intercollegiate Surgical Curriculum Programme (ISCP) for general and vascular surgery (2021). In the lead up to its implementation, we aimed to assess both trainee and consultant confidence levels as a surrogate reflection in the core competency operative skills in general emergency trauma surgery, identify individual experi- ence in commonly performed trauma procedures and gauge interest in a career in trauma surgery. Method  An online survey was circulated to general surgery and vascular surgery trainees and consultants. Self-reported competencies were assessed using a 1–10 confidence rating scale. Most questions were based on competencies in emergency trauma surgery as set out by the ISCP. Results  Out of 251 surgical trainees and consultants, 119 responded to our survey (47.4% response rate). Less than half (44.1%; n = 52) of respondents had experienced a trauma thoracotomy. Respondents scored ‘somewhat’ or ‘not at all’ com- petent in the majority of competencies assessed. Conclusion  Self-reported competencies in operative trauma skills across all subgroups were sub-standard with incremental levels of perceived competence proportional to years of surgical training. Our data supports the necessity of the new curricu- lum, in addition to modern training pathways with direct exposure to operative trauma surgery involving dedicated trauma centres and networks, and responsibility of training pathways in the provision of training trauma surgery. Keywords  Intercollegiate Surgical Curriculum Programme · Thoracotomy · Trauma skills · Trauma surgery rds  Intercollegiate Surgical Curriculum Programme · Thoracotomy · Trauma skills · Trauma surgery Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 https://doi.org/10.1007/s11845-022-03117-4 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 https://doi.org/10.1007/s11845-022-03117-4 ORIGINAL ARTICLE ORIGINAL ARTICLE Competency in trauma surgery: a national survey of trainees and consultants Michael Flanagan1   · Jessica Ryan1 · Gary Bass2 · Kevin Barry3 · Oscar Traynor4 · Morgan McMo Received: 6 April 2022 / Accepted: 23 July 2022 / Published online: 15 August 2022 © Crown 2022 * Michael Flanagan michaelflanagan@rcsi.ie 1 Department of Surgery, University Hospital Waterford, Waterford, Ireland 2 Division of Traumatology, Surgical Critical Care and Emergency Surgery, Penn Medicine, Philadelphia, PA, USA Participants Data was collected between 31 January and 5 February 2021 using an anonymised online survey (SurveyMonkey®, Sur- veyMonkey Inc., San Mateo, CA). Specialty training admin- istrators working at the Royal College of Surgeons in Ireland (RCSI) assisted with the survey distribution to relevant con- sultant surgeons and trainees to ensure General Data Protec- tion Regulation (GDPR) compliance and using their respec- tive Internet Protocol addresses only. Two-hundred fifty-one surgeons were contacted (100 general surgery consultants, 76 general surgery trainees, 34 vascular consultants and 50 vascular trainees), via email with an invitation to partici- pate including an explanation of the survey with the relevant link. Completion of the survey was interpreted as informed consent, and no compensation was offered in reward for participation. This study has been reported in line with the Strengthening the reporting of cohort, cross-sectional and case–control studies in surgery (STROCSS) criteria [7]. Introduction Finally, participants were asked whether they felt that Trauma and Emergency Surgery should be considered a separate subspecialty within surgical training, and whether or not they themselves were interested in being a Trauma and Emergency surgeon. participants were asked if they had ever been involved in a trauma thoracotomy, and its location (emergency depart- ment/trauma bay or operating theatre). The full list of ques- tions can be found in Appendix 1. Finally, participants were asked whether they felt that Trauma and Emergency Surgery should be considered a separate subspecialty within surgical training, and whether or not they themselves were interested in being a Trauma and Emergency surgeon. Data analysis Results were analysed using Predictive Analytics Software (PASW 18.0.2, SPSS Inc., Chicago, IL, USA). Descriptive data are presented as absolute frequencies and percent- ages. Continuous data are presented as means and standard deviations. For analysis, pre-CCST (ST3-8) and post-CCST (post-CCST non-consultant, consultant) respondents were grouped together and compared. Comparative analyses of quantitative data were performed using Student’s t-test for continuous variables. All tests of significance were 2-tailed, with p < 0.05 indicating statistical significance. Survey questions A total of 119 surgical trainees and consultants responded, giving a response rate of 47.4%. One third were female (n = 76), and the majority (n = 71/114; 61.7%) had graduated more than 10 years prior (Table 2). A total of 63 out of 119 respondents (52.9%) were specialist trainees (ST3–ST8), and the remainder had completed their training. Of the con- sultant-level respondents, the majority (n = 41/51; 80.4%) had been in practice for greater than 5 years. Colorectal and vascular surgery respondents accounted for 43/115 (37.4%) and 30/115 (26.1%) of subspecialties, respectively (Table 3). Almost all (95.6%) respondents were practicing in Ireland at the time of the survey. Appendix 1 outlines the survey questions posed. Demo- graphics data collected included age, gender, subspecialty and country of current practice. Information on level of training was also collected; including years since gradua- tion, training stage and years since appointment at consultant grade. For training stage, the UK and Irish grading system was used, which comprises specialist trainees (levels ST3 through to ST8), non-consultant post-specialist training and consultant level. Self-reported competencies were assessed using a 1–10 confidence rating scale (Table 1). A larger 10-point Lik- ert scale was used to increase the variance as compared to smaller 7-point or 5-point Likert scales, to obtain a higher degree of measurement precision and better detect changes in sequential responses among the respondents [8]. Ques- tions were developed using a selection of 26 technical skill-based competencies taken from the most recent Gen- eral Surgical Curriculum from the ISCP [1]. Additionally, Introduction care, reflecting the growing need to train and upskill sur- geons in the generality of emergency surgery [1]. This need is partly in response to the success of the UK Trauma Net- work [2] and is particularly relevant to Ireland as the new trauma service awaits implementation [3]. There are justifi- able concerns amongst both trainees and trainers with regard to the lack of exposure to emergency surgery, particularly trauma with a resultant potential for substandard competen- cies in operative trauma procedures that may be encountered as a newly appointed consultant surgeon. This is in part due to the increasingly non-operative nature of injury manage- ment [4], a reduction in surgical exposure associated with the European Working Time Directive [5] and a failure to establish high-volume centralised major trauma care in cer- tain jurisdictions that would to facilitate focused training pathways in operative trauma skills [6]. The intercollegiate surgical curriculum programme for general surgery has been recently revised and updated with added emphasis on emergency general surgery and trauma 1 Department of Surgery, University Hospital Waterford, Waterford, Ireland 2 Division of Traumatology, Surgical Critical Care and Emergency Surgery, Penn Medicine, Philadelphia, PA, USA 3 Department of Surgical Affairs, National Surgical Training Programmes, Royal College of Surgeons in Ireland, Dublin, Ireland To better understand current competencies in trauma surgery among trainees and consultants, both general and vascular surgery trainees and consultants were surveyed on a 4 Department of Surgical Affairs, Royal College of Surgeons in Ireland, Dublin, Ireland 5 Imperial College London, London, UK (0121 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1304 Table 1   The 1–10 competency rating scale Score Competency 1 Not at all competent 2–4 Somewhat competent 5–7 Quite competent 8–10 Highly competent broad range of competencies in trauma surgery as prescribed by the new curriculum. The aim of this study was to assess the individuals’ confidence level as a surrogate reflection of competency level in general emergency trauma surgery prior to commencement of the new Intercollegiate Surgical Curriculum Programme [1]. In addition, we wanted to gauge interest in the speciality of trauma surgery among trainees and consultants, which may inform stakeholders in potential workforce arrangements. participants were asked if they had ever been involved in a trauma thoracotomy, and its location (emergency depart- ment/trauma bay or operating theatre). The full list of ques- tions can be found in Appendix 1. 1 3 Discussion This study demonstrates incremental levels of perceived competence in operative trauma skills proportional to the years of surgical training and into consultant-level practice. Higher levels of competency were reported for those pro- cedures that are mirrored with similar routine elective or non-trauma emergency work, such as small-bowel resection, with lower levels of competency for the more severe injuries or those not routinely performed outside of the specialty of trauma surgery (e.g. thoracotomy). Less than half of respondents reported having performed or been involved in a trauma thoracotomy, which is an expected competency skill for exsanguinating injury, especially penetrating chest trauma. A total of 15 out of 62 respondents involved in a trauma thoracotomy (24.2%) were of training grade only. A trauma thoracotomy is a life-saving skill in which all surgeons taking acute call should be competent, in addition to the complex decision making around it, as reflected in the new ISCP curriculum. Recent analysis of registry data on resuscitative thoracotomy identified that in 76.8% (109/142) of cases the procedure was performed in the emergency department [9], further highlighting the need for surgical expertise and appropriate training. Guidelines and consensus statement documents outline the accepted and selective indications for which emergency department thora- cotomy is appropriate and indications for which survival is poor, e.g., blunt trauma [10, 11]. CCST Certificate of Completion of Specialist Training, ST year of surgical training CCST Certificate of Completion of Specialist Training, ST year of surgical training department/trauma bay thoracotomy. Only 15 of 62 (24.2%) trainees had any experience of a trauma thoracotomy. A total of 75 of 116 respondents (64.6%) felt that trauma and emergency surgery should be considered a separate subspe- cialty within general surgery training. A total of 36 of 116 respondents (31.3%) expressed an interest in Trauma and Emergency surgery as their subspecialty interest of choice. Of the 26 competencies surveyed, the majority of respondents scored as ‘somewhat’ or ‘not at all’ competent, with only 10 reporting a mean score of 5 or greater. No competency had a mean score consistent with a high degree of competence (i.e. 8–10) with that particular injury or skill (Table 1). The lowest scoring (score < 3) competencies were head of pancreas resection for trauma, management of a ret- rohepatic IVC injury, repair of an injured kidney and man- agement of a combined pancreatico-duodenal injury. Trauma experience Less than half (n = 52/118; 44.1%) of respondents had expe- rienced a trauma thoracotomy, and of those that did only 25 of 52 respondents (48.1%) had experienced an emergency 3 3 3 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1305 Irish Journal of Medical Science (1971 ) (2023) 192:1303 1309 Table 2   Participant demographics CCST Certificate of Completion of Specialist Training, ST year of surgical training N (%) Gender Male 76 (66) Female 36 (31.3) Prefer not to say 3 (2.6) Years since graduation   < 5 8 (6.9) 5–9 35 (31.3) 10–14 21 (18.3) 15–20 15 (13)  > 20 35 (30.4) Stage of training ST3–5 or equivalent 40 (33.6) ST6–8 or equivalent 23 (19.3) Post-CCST non-consultant 5 (4.2) Consultant 51 (42.9)   < 5 years appointed 10 (19.6)   > 5 years appointed 41 (80.4) Irish Journal of Medical Science (1971 ) (2023) 192:1303 1309 Table 2   Participant demographics CCST Certificate of Completion of Specialist Training, ST year of surgical training N (%) Gender Male 76 (66) Female 36 (31.3) Prefer not to say 3 (2.6) Years since graduation   < 5 8 (6.9) 5–9 35 (31.3) 10–14 21 (18.3) 15–20 15 (13)  > 20 35 (30.4) Stage of training ST3–5 or equivalent 40 (33.6) ST6–8 or equivalent 23 (19.3) Post-CCST non-consultant 5 (4.2) Consultant 51 (42.9)   < 5 years appointed 10 (19.6)   > 5 years appointed 41 (80.4) highest scoring (score > 7) competencies were management of a bowel injury (both large and small bowel) and trauma laparotomy (Table 4). When pre- and post-CCST respond- ents were compared, post-CCST respondents scored signifi- cantly higher in every self-reported competency (Table 5). Scores of the post-CCST respondents were sub-standard in a number of self-reported trauma competencies. highest scoring (score > 7) competencies were management of a bowel injury (both large and small bowel) and trauma laparotomy (Table 4). When pre- and post-CCST respond- ents were compared, post-CCST respondents scored signifi- cantly higher in every self-reported competency (Table 5). Scores of the post-CCST respondents were sub-standard in a number of self-reported trauma competencies. Discussion The As predicted, trainees are lacking in competence for those procedures that they are less frequently exposed to, yet many of these could be described as ‘expected skills’ for Table 3   Subspecialty by level of training CCST Certificate of Completion of Specialist Training N (%) Subspecialty Pre-CCST Post-CCST Total Upper gastrointestinal/bariatric 7 (11.9) 3 (5.4) 10 (8.7) Hepatobiliary 3 (5.1) 1 (1.8) 4 (3.5) Colorectal 23 (39.0) 20 (35.7) 43 (37.4) Breast/endocrine 8 (13.6) 10 (17.9) 18 (15.6) Vascular 13 (22) 17 (30.4) 30 (26.1) General/trauma/emergency 5 (8.5) 5 (8.9) 10 (8.7) 1 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1306 Table 4   Self-reported competency in the surgical management of an unstable trauma patient * Highest percentage allocation; **Mean score rated as quite competent or above N (%) Not at all competent Somewhat competent Quite competent Highly competent Mean competency score (SD) 1 Performing a thoracotomy/clamshell thoracotomy 28 (28.3) 34 (34.3)* 27 (27.2) 10 (1) 3.7 (2.5) 2 Suturing the heart/cardiac repair for trauma 35 (35.3)* 35 (35.3)* 23 (23.2) 6 (6.1) 3.1 (2.3) 3 Performing a trauma laparotomy 2 (2.1) 14 (14.4) 30 (30.9) 51 (52.5)* 7.1 (2.4)** 4 Performing a blind trauma laparotomy 6 (6.2) 16 (16.5) 36 (37.1) 39 (40.2)* 6.4 (2.5)** 5 Packing the abdomen for major traumatic haemorrhage 4 (4.1) 16 (16.3) 32 (32.6) 46 (46.9)* 6.7 (2.4)** 6 Managing a small-bowel injury for trauma 4 (4.1) 9 (9.2) 23 (23.7) 61 (62.9)* 7.6 (2.5)** 7 Managing a large-bowel injury for trauma 5 (5.1) 10 (10.2) 25 (25.5) 58 (59.2)* 7.4 (2.7)** 8 Managing a rectal injury for trauma 9 (9.1) 25 (25.2) 27 (27.2) 38 (38.3)* 6 (3.1)** 9 Managing a gastric injury for trauma 8 (8.2) 21 (21.4) 39 (39.8)* 30 (30.6) 5.8 (2.5)** 10 Performing a trauma splenectomy 6 (6.1) 17 (17.3) 27 (27.5) 48 (49)* 6.6 (2.7)** 11 Packing an injured liver 5 (5) 22 (22.2) 41 (41.4)* 31 (31.3) 6 (2.5)** 12 Extraperitoneal pelvic packing via laparotomy 14 (14.4) 30 (30.9) 34 (35)* 19 (19.6) 5 (2.7)** 13 Extraperitoneal pelvic packing via pelvis only 20 (20.4) 34 (34.7)* 31 (31.6) 13 (13.3) 4.3 (2.7) 14 Performing a trauma nephrectomy 28 (28.6) 40 (40.8)* 20 (20.4) 10 (10.2) 3.6 (2.6) 15 Performing repair of an injured kidney (e.g. Discussion mesh repair) 47 (47.9)* 36 (36.7) 11 (11.2) 4 (4.1) 2.5 (2) 16 Operative management of a pancreatic injury 36 (36.4) 40 (40.4)* 16 (16.2) 7 (7.1) 3 (2.3) 17 Tail of pancreas resection for trauma 30 (30.6) 33 (33.7)* 25 (25.5) 10 (10.2) 3.7 (2.7) 18 Head of pancreas resection for trauma 62 (63.9)* 23 (23.7) 9 (9.2) 3 (3.1) 2.1 (2) 19 Managing a duodenal injury for trauma 19 (19.2) 41 (41.4)* 28 (28.3) 11 (11.1) 4.1 (2.5) 20 Managing a combined pancreatico- duodenal injury for trauma 43 (43.4)* 42 (42.4) 10 (10.1) 4 (4) 2.6 (2.1) 21 Retroperitoneal exposure for trauma 14 (14.1) 46 (46.5)* 27 (27.2) 12 (12.1) 4.2 (2.6) 22 Operative management of an infrarenal IVC injury 28 (28.6) 37 (37.7)* 22 (22.4) 11 (11.2) 3.6 (2.7) 23 Operative management of a retrohepatic IVC injury 49 (50)* 36 (36.7) 11 (11.2) 2 (2) 2.4 (1.8) 24 Operative management of a suprarenal aortic injury 39 (40.6)* 32 (33.3) 18 (18.7) 7 (7.3) 3 (2.3) 25 Operative management of a infrarenal aortic injury 23 (23.7) 34 (35)* 23 (23.7) 17 (17.5) 4.2 (2.8) 26 Performing a vascular shunt for arterial trauma 35 (35.7)* 31 (31.6) 13 (13.3) 19 (19.4) 3.6 (3) * Highest percentage allocation; **Mean score rated as quite competent or above Ireland, where centralisation of trauma services has yet to be implemented. Therefore surgeons without sub-specialty knowledge and skill in trauma surgery are much more likely to be deficient in the management of complex polytrauma. a newly appointed surgeon managing complex polytrauma as part of acute call. It is clear from our data that a signifi- cant deficiency in trauma operative skills exists for many of the injuries that a newly appointed consultant surgeon will encounter within a wider trauma network or major trauma centre. This is especially concerning for those practicing in i Competence in certain key trauma procedures requires development during training and must be maintained 1 3 3 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1307 Table 5   Self-reported competency in the surgical management of an unstable trauma patient: a comparison between pre-CCST and post-CCST Pre-CCST Post-CCST p Value Table 5   Self-reported competency in the surgical management of an unstable trauma patient: a comparison between pre-CCST and post-CCST CCST Certificate of Completion of Specialist Training. Discussion The level of significance is p < 0.05, and all p values reached significance Pre-CCST Post-CCST p Value Mean (SD) 1 Performing a thoracotomy/clamshell thoracotomy 2.73 (1.89) 4.57 (2.72)  < 0.001 2 Suturing the heart/cardiac repair for trauma 2.21 (1.63) 4.02 (2.53)  < 0.001 3 Performing a trauma laparotomy 6.09 (2.01) 8.04 (2.34)  < 0.001 4 Performing a blind trauma laparotomy (i.e. in the absence of imaging) 5.13 (2.21) 7.61 (2.23)  < 0.001 5 Packing the abdomen for major traumatic haemorrhage 5.32 (2.25) 8 (1.86)  < 0.001 6 Managing a small-bowel injury for trauma 6.52 (2.53) 8.67 (1.99)  < 0.001 7 Managing a large-bowel injury for trauma 6.19 (2.62) 8.52 (2.15)  < 0.001 8 Managing a rectal injury for trauma 4.73 (2.71) 7.18 (2.94)  < 0.001 9 Managing a gastric injury for trauma 5.06 (2.33) 6.6 (2.52)  < 0.001 10 Performing a trauma splenectomy 5.1 (2.37) 8 (2.18) 0.003 11 Packing an injured liver 4.9 (2.22) 6.98 (2.38)  < 0.001 12 Extraperitoneal pelvic packing via laparotomy 3.4 (1.98) 6.55 (2.37)  < 0.001 13 Extraperitoneal pelvic packing via pelvis only 3 (1.91) 5.58 (2.67)  < 0.001 14 Performing a trauma nephrectomy 2.63 (1.86) 4.52 (2.83)  < 0.001 15 Performing repair of an injured kidney (e.g. mesh repair) 1.79 (1.1) 3.1 (2.41)  < 0.001 16 Operative management of a pancreatic injury 2.13 (1.56) 3.84 (2.6)  < 0.001 17 Tail of pancreas resection for trauma 2.67 (1.83) 4.66 (2.99)  < 0.001 18 Head of pancreas resection for trauma 1.54 (1.21) 2.63 (2.43) 0.007 19 Managing a duodenal injury for trauma 3.15 (2) 4.98 (2.69)  < 0.001 20 Managing a combined pancreatico-duodenal injury for trauma 1.88 (1.39) 3.22 (2.36) 0.001 21 Retroperitoneal exposure for trauma 3.02 (1.84) 5.29 (2.68)  < 0.001 22 Operative management of an infrarenal IVC injury 2.23 (1.62) 5 (2.76)  < 0.001 23 Operative management of a retrohepatic IVC injury 1.68 (1.15) 2.98 (2.12)  < 0.001 24 Operative management of a suprarenal aortic injury 1.91 (1.37) 3.98 (2.61)  < 0.001 25 Operative management of a infrarenal aortic injury 3.06 (2.24) 5.3 (2.92)  < 0.001 26 Performing a vascular shunt for arterial trauma 2.79 (2.49) 4.47 (3.32) 0.006 e surgical management of an unstable trauma patient: a comparison between pre-CCST and post-CCST the revised ISCP curriculum. A recent survey of UK trainees has proposed the introduction of a live large animal course to facilitate trauma training [12]. References 1. ISCP (2021) General surgery curriculum [Available from: https://​ www.​iscp.​ac.​uk/​media/​1103/​gener​al-​surge​ry-​curri​culum-​aug-​ 2021-​appro​ved-​oct-​20v3.​pdf 1. ISCP (2021) General surgery curriculum [Available from: https://​ www.​iscp.​ac.​uk/​media/​1103/​gener​al-​surge​ry-​curri​culum-​aug-​ 2021-​appro​ved-​oct-​20v3.​pdf 2. NCEPOD (2007) Trauma: who cares? [Available from: https://​ www.​ncepod.​org.​uk/​2007r​eport2/​Downl​oads/​SIP_​summa​ry.​pdf 2. NCEPOD (2007) Trauma: who cares? [Available from: https://​ www.​ncepod.​org.​uk/​2007r​eport2/​Downl​oads/​SIP_​summa​ry.​pdf 3. TraumaSteeringGroup (2018) A trauma system for Ireland [Avail- able from: https://​www.​hse.​ie/​eng/​about/​who/​acute-​hospi​tals-​ divis​ion/​trauma-​servi​ces/​furth​er-​infor​mation-​and-​docum​entat​ion/​ report-​of-​the-​trauma-​steer​ing-​group.​pdf 3. TraumaSteeringGroup (2018) A trauma system for Ireland [Avail- able from: https://​www.​hse.​ie/​eng/​about/​who/​acute-​hospi​tals-​ divis​ion/​trauma-​servi​ces/​furth​er-​infor​mation-​and-​docum​entat​ion/​ report-​of-​the-​trauma-​steer​ing-​group.​pdf 4. Bulinski P, Bachulis B, Naylor DF Jr et al (2003) The changing face of trauma management and its impact on surgical resident training. J Trauma 54(1):161–163 5. Fitzgerald JE, Caesar BC (2012) The European Working Time Directive: a practical review for surgical trainees. Int J Surg 10(8):399–403 6. Mohan HM, Mullan D, McDermott F et al (2015) Saving lives, limbs and livelihoods: considerations in restructuring a national trauma service. Ir J Med Sci 184(3):659–666 7. Agha RA, Borrelli MR, Vella-Baldacchino M et al (2017) The STROCSS statement: Strengthening the Reporting of Cohort Studies in Surgery. Int J Surg 46:198–202 Our study is a snapshot survey of trainees and qualified surgeons regarding their competency/confidence manag- ing a major trauma operative case load. The results of this nationwide survey of trauma competencies in consultants and trainees have wider implications for the provision of trauma surgery services. There are clearly concerns with regards to a deficiency of experience in our system to date, which is important to address ahead of the roll out of the major trauma network in Ireland. A number of means are suggested to address low levels of competency in trauma procedures, such as trauma simulation and trauma immer- sion in high-volume centres. It is likely that a solution will come from a combination of both. Over 30% of respondents reported that they would be interested in being a trauma surgeon. It is the responsibility of training bodies, key 8. Bayer W (1994) The measurement imperative. Market Res 9. DuBose JJ, Morrison J, Moore LJ et al (2020) Does clamshell thoracotomy better facilitate thoracic life-saving procedures without increased complication compared with an anterolateral approach to resuscitative thoracotomy? Results from the Ameri- can Association for the Surgery of Trauma Aortic Occlusion for Resuscitation in Trauma and Acute Care Surgery Registry. J Am Coll Surg 231(6):713–719 e1 10. Tabiner N (2019) Resuscitative thoracotomy for traumatic cardiac arrest: clinical evidence and clinical governance. Resuscitation 139:200 11. Discussion Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s11845-​022-​03117-4. Funding  Open Access funding provided by the IReL Consortium. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s11845-​022-​03117-4. It is important to note the level of interest amongst the respondents in a career dedicated to the subspecialty of trauma and emergency surgery. Unlike the USA, there is currently no dedicated pathway for a unique career in trauma surgery in Ireland, notwithstanding attempts to address this in the UK, with the Training Interface Group (TIG) trauma fellowship. The TIG fellowships were established in 2019 to meet the increased demand for trauma-competent surgeons within the UK trauma network and as a pathway to acquire operative skills [4]. This training pathway includes both ‘resuscitative’ and ‘operative’ pathways to match the skills acquisition across a broad spectrum of training for dedicated personnel within the major trauma network. Funding  Open Access funding provided by the IReL Consortium. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. There are a number of limitations to our current study. Vascular and general trainees and consultants were surveyed on a broad spectrum of trauma procedures, some of which are particular to subspecialty training and experience, poten- tially skewing results in favour of the particular consultant/ trainee’s chosen field. Our chosen Likert type scale was not validated in a pilot study with a smaller cohort prior to circu- lation to the total consultant and trainee body [14, 15]. The author’s definition of competence was not shared with the survey respondent prior to survey completion and thus the respondents’ view of competence may differ from that of the author or other respondents. Discussion The use of the word competence and its relationship with confidence in performing a trauma procedure is difficult to either define or measure. A system- atic review and qualitative discourse analysis to critically analyse language used to measure general surgery trainees’ confidence or readiness to practice concluded that robust methods of measuring self-efficacy be a primary focus rather than confidence or competence which are difficult to define and measure [16].i Discussion Among 54 trainee respond- ents to the survey, 90% agreed or strongly agreed that a live large animal course with exposure to major intraoperative haemorrhage is beneficial. However, the ethics and costs that come with such a course may be prohibitive. Cadevaric dissection provides a high-fidelity training experience incor- porating equipment, environment and behavioural factors that are difficult to replicate in a synthetic model although evidence currently does not demonstrate superiority over lower fidelity models [13]. throughout consultant practice for all surgeons taking acute care admissions. The revised ISCP curriculum acknowl- edges this and that certain skills and capabilities are best learned and maintained within the formal setting of a spe- cific taught course (e.g. general management of the multi- ply injured patient). There is only one mandated course in the revised curriculum [1] but multiple other highly rec- ommended courses are available. Acquiring certain clini- cal trauma skills to the level of competency sufficient to manage severe injury is challenging due to its infrequency. Successful course completion that has a high level of gov- ernance associated with it may be invaluable. Examples of courses that meet the required learning outcomes as out- lined by ISCP include the Definitive Surgical Trauma Skills (DSTS) course, Advanced Trauma Life Support (ATLS), and the European Trauma Course. However, only one of these (DSTS) teaches advanced operative trauma skills and the associated complex decision making required. i A number of developments are underway to address com- petencies in key trauma operative skills. The implementation and rollout of the new ISCP Curriculum aims to re-establish “Emergency/Elective competencies across the generality of surgery” as a primary focus during higher specialist training. The successful reconfiguration of trauma care within the UK [2] and that planned in Ireland [3] has implications on how trauma surgery training is provided and how a career The role of simulation in addressing the paucity of trauma operative training has also been addressed within 1 3 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1308 government stakeholders, and trainees to define the role and career pathway of the trauma surgeon in the provision of the trauma care. in trauma surgery will mature going forward [3]. Having a defined and dedicated trauma network provides improved exposure to and training in the optimal management of the severely injured patient, including key operative damage control skills. 13. James HK, Chapman AW, Pattison GTR et al (2019) Systematic review of the current status of cadaveric simulation for surgical training. Br J Surg 106(13):1726–1734 g g ( ) 14. Eysenbach G (2004) Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHER- RIES). J Med Internet Res 6(3):e34 ) ( ) 15. Jones TL, Baxter MA, Khanduja V (2013) A quick guide to survey research. Ann R Coll Surg Engl 95(1):5–7 13. James HK, Chapman AW, Pattison GTR et al (2019) Systematic review of the current status of cadaveric simulation for surgical training. Br J Surg 106(13):1726–1734 14. Eysenbach G (2004) Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHER- RIES). J Med Internet Res 6(3):e34 15. Jones TL, Baxter MA, Khanduja V (2013) A quick guide to survey research. Ann R Coll Surg Engl 95(1):5–7 References Aseni P, Rizzetto F, Grande AM et al (2021) Emergency Depart- ment Resuscitative Thoracotomy: indications, surgical procedure and outcome. A narrative review Am J Surg 221(5):1082–1092 12. Brown RJ, Hosgood SA, Praseedom R, Nicholson ML (2021) Exposure to and training in the control of major intraopera- tive haemorrhage: a survey of surgical trainees. The Bullitin 28–31 3 3 Irish Journal of Medical Science (1971 -) (2023) 192:1303–1309 1309 16. Elfenbein DM (2016) Confidence crisis among general surgery residents: a systematic review and qualitative discourse analysis. JAMA Surg 151(12):1166–1175 Publisher's Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3
W2990985755.txt
https://revistaenfermagematual.com.br/index.php/revista/article/download/571/537
pt
Acolhimento aos pacientes e familiares atendidos no ambulatório de oncologia: um relato de experiência
Revista Enfermagem Atual In Derme
2,019
cc-by
3,723
110 R E L ATO D E E X P E R I Ê N C I A Acolhimento aos pacientes e familiares atendidos no ambulatório de oncologia: um relato de experiência Reception to patients and families served in clinic for oncology: an experience report Lucimere Maria dos Santos1 • Wbiratan de Lima Souza2 • Geisiane de Souza Santos3 • Eliane Ramos Pereira4 • Rose Mary Costa Rosa Andrade Silva5 • Cristina Lavoyer Escudeiro6 RESUMO O objetivo deste estudo é relatar as experiências vivenciadas por uma enfermeira e sua equipe, referente ao atendimento prestado aos pacientes e seus familiares em um ambulatório de oncologia clínica de um hospital especializado na cidade do Rio de Janeiro. Trata-se de relato de experiência vivenciado no decorrer das práticas profissionais. O primeiro contato do paciente e seus familiares no ambulatório acontece na recepção, no momento da entrega do cartão de consulta ou dos encaminhamentos, para os pacientes de primeira vez. Quando o paciente chega à recepção com qualquer queixa, imediatamente a equipe de enfermagem recebe a informação da necessidade do mesmo e realiza o atendimento, onde são aferidos os sinais vitais e o enfermeiro realiza a avaliação a partir exame físico associado ao diálogo, a fim de identificar as necessidades de saúde e a prioridade para o atendimento médico a partir das queixas descritas pelo paciente e/ou familiar. Concluí-se que experiências vivenciadas no ambulatório de oncologia clínica contribuíram de formas satisfatórias para compreensão de gerenciar os serviços e as ações da equipe de enfermagem, no que se refere acolhimento aos pacientes e familiares, bem como para tomada de decisão. Palavras-chave: Humanização da Assistência; Neoplasias; Cuidados de Enfermagem. ABSTRACT The goal of this study is to describe experiences lived by a nurse and her team, in reference to the assistance provided to the patients and their families at an oncology ambulatory, on a specialized hospital in Rio de Janeiro city. It’s a report about the experiences lived in the course of professional practices. The first contact from patients and their families with the ambulatory begins at the reception, the moment of delivery the consult card or the medical referrals for the first-way patients. When the patient arrives with any complaint, the nursing team immediately receive the information of the patient needs and do the assistance, where the vital signs are checked, the nurse do a physical assessment associated to dialogue in order to identify the health needs and priorities to the medical care from the complaints reported by the patient and/or a family member. Conclusion: The experiences lived at the oncology ambulatory helped satisfactorily the understanding to manage the services and actions of the nursing team, when referencing to the patients and family reception, as like the decision make. Keywords: Attendance Humanization; Neoplasms; Nursing Care. NOTA 1 Enfermeira. Mestra do Programa de Mestrado Profissional em Enfermagem Assistencial (MPEA) da Escola de Enfermagem Aurora de Afonso Costa (EEAAC) da Universidade Federal Fluminense (UFF). Enfermeira do Instituto Nacional do Câncer (INCA – Ambulatório de Oncologia Clínica e Hematologia). Duque de Caxias - RJ, Brasil. E-mail: lucimere_santos@hotmail.com. 2 Enfermeiro. Mestrando do Programa de Mestrado Profissional em Enfermagem Assistencial (MPEA) da Escola de Enfermagem Aurora de Afonso Costa (EEAAC) da Universidade Federal Fluminense (UFF). Professor do Centro Universitário Tiradentes (UNIT-Maceió). Professor da Universidade Estadual de Ciências da Saúde de Alagoas (UNCISAL). Professor substituto da Universidade Federal de Alagoas (UFAL-Maceió). Enfermeiro Assistencial do Hospital de Emergência Dr. Daniel Houly (HEDH). Membro do Núcleo de Estudo e Pesquisa em Cidadania e Gerência e Enfermagem (NECIGEN). Maceió - AL, Brasil. E-mail: wbiratansouza@yahoo.com.br. 3 Enfermeira. Mestranda do Programa de Mestrado Profissional em Enfermagem Assistencial (MPEA) da Escola de Enfermagem Aurora de Afonso Costa (EEAAC) da Universidade Federal Fluminense (UFF). Enfermeira do Hospital Estadual Roberto Chabo. Membro do Núcleo de Estudo e Pesquisa em Cidadania e Gerência e Enfermagem (NECIGEN). Saquarema - RJ, Brasil. E-mail: grisianesantos10@hotmail.com. 4 Enfermeira. Pós-Doutorado em Enfermagem. Coordenadora e Docente do Programa de Mestrado Profissional em Enfermagem Assistencial (MPEA), Professora do Curso de Graduação (EEAAC/UFF). São Gonçalo – RJ. Brasil. E-mail: elianeramos.uff@gmail.com. 5 Professora Drª, Universidade Federal Fluminense, Escola de Enfermagem Aurora de Afonso Costa, Departamento de Enfermagem Médico- Cirúrgica, Programa de PósGraduação Mestrado Profissional em Enfermagem Assistencial. Niterói – RJ, Brasil. E-mail: roserosauff@gmail.com 6 Enfermeira. Doutora em Enfermagem. Coordenadora do curso de Graduação em Enfermagem da Escola de Enfermagem Aurora de Afonso Costa (EEAAC) da Universidade Federal Fluminense (UFF). Docente Permanente do MPEA (EEAAC/UFF). Membro do NECIGEN. Niterói – RJ. Brasil. E-mail: cristinalescudeiro@gmail.com. REVISTA ENFERMAGEM ATUAL | 2017; 81 Acolhimento nA o R a mTb Iu G l a tO ó r iO o dRe I oGn cI oN l oA g iLa Introdução O acolhimento como ato ou efeito de acolher expressa, em suas várias definições, uma ação de aproximação, um “estar com” e um “estar perto de”. Essa atitude implica estar em relação com algo ou alguém. É exatamente nesse sentido, de ação de “estar com” ou “estar perto de”, que o acolhimento se afirma como uma das diretrizes de maior relevância ética/estética/política da Política Nacional de Humanização do SUS. Sendo este, um modo de operar os processos de trabalho em saúde, de forma a atender a todos que procuram os serviços de saúde, ouvindo seus pedidos e assumindo no serviço uma postura capaz de acolher, escutar e dar respostas mais adequadas aos usuários.1 Com a elevada expectativa de vida da população brasileira e o aumento do número de idosos em nosso país, observa-se também a elevação dos índices de doenças crônicas degenerativas e, dentre estas, o câncer, que vêm ganhando importância no perfil epidemiológico tornandose um problema de saúde pública, haja vista o número de novos casos, que vem sendo diagnosticados no país e no mundo.2 O número estimado para 2014/2015 de acordo com o Instituto Nacional de Câncer (INCA) é de aproximadamente 576 mil novos casos de câncer no Brasil, incluindo os casos de pele não melanoma, que é o tipo mais incidente para ambos os sexos (182 mil casos novos), seguido de próstata (69 mil), mama feminina (75 mil), cólon e reto (33 mil), pulmão (27 mil), estômago (20 mil) e colo do útero (15 mil). Duas tendências importantes foram apontadas: a redução na incidência dos casos novos de câncer do colo do útero e de pulmão em homens. Esses resultados estão relacionados as iniciativas para prevenção e detecção precoce do câncer do colo do útero e das ações de prevenção ao tabagismo no Brasil.2 O INCA é um centro de alta complexidade de referência no atendimento a paciente com diagnóstico de neoplasia maligna situado no estado do Rio de Janeiro. O hospital possui diversos setores para atendimento hospitalar, desde o atendimento emergencial, internações, serviços de cuidados paliativos até as consultas ambulatoriais. Os ambulatórios são distribuídos pelas áreas internas e externas da instituição que se dividem de acordo com os tipos de câncer como exemplo: câncer de cabeça e pescoço, câncer de bexiga, câncer de próstata e etc, obedecendo as especificidades e a modalidade de tratamento. A entrada do paciente na instituição para a primeira avaliação acontece através do Sistema de Regulação de Vagas (SISREG) que é definido como um Sistema online desenvolvido pelo Departamento de Informática do Sistema Único de Saúde do Ministério da Saúde. O sistema é um software, disponibilizado pelo Ministério da Saúde para o gerenciamento de todo Complexo Regulatório, que vai da rede básica de saúde à internação hospitalar, visando a humanização dos serviços e maior controle do fluxo.3 Após a avaliação realizada pelo médico na primeira consulta, o paciente pode abrir matrícula no respectivo ambulatório ou ser encaminhado para o setor de triagem que após avaliação médica o mesmo pode ser conduzido para o ambulatório de oncologia clínica e assim abrir matrícula. O fluxo de atendimento no referido ambulatório acontece de duas formas: através de um encaminhamento médico aos pacientes já matriculados na instituição, submetidos ou não ao tratamento cirúrgico e os que necessitam dar continuidade ao tratamento fazendo uso de quimioterapias (método que utiliza compostos químicos, no tratamento de doenças causadas por agentes biológicos) ou radioterapias (é o emprego de feixes de radiações ionizantes para destruir células tumorais) ou ambas, sendo empregada concomitantemente. O câncer é uma das doenças crônicas mais temidas pelas pessoas e se caracteriza como um conjunto de sentimentos carregados de significados. O diagnóstico do câncer promove nos pacientes e seus familiares, uma sensação de catástrofe, desordem, desespero e muito sofrimento, pois o que percebemos é a relação do diagnóstico com o medo da morte, além de todas as comorbidades advindas da doença, bem como do tratamento, que promove várias reações adversas.4-6 O paciente com câncer não deve ser avaliado, somente, como mais um caso. Nesse aspecto, precisa ser empreendida uma visão holística, procurando compreendê-lo nas suas várias relações, proporcionando uma abordagem profissional humanizada e profundamente solidária, geradora não só de saúde, mas, principalmente, de vida.7 O entendimento da saúde como condição para a cidadania sugere a dinamização de ações que potencializam o bem-estar e a qualidade de vida das pessoas. Nesta perspectiva, estruturar serviços e ações em saúde que vão ao encontro das necessidades das pessoas, torna-se cada vez mais um desafio, para enfermagem, pois a medida que os determinantes sociais, biológicos, políticos, culturais, entre outros, interagem com complexidade crescente, trazendo repercussões, não apenas de imediato, mas em longo prazo.8 A enfermagem se torna imprescindível, no que se refere à promoção da assistência, a fim de identificar as necessidades dos pacientes e seus familiares, oferecendolhes um atendimento humanizado e acolhedor. A sala de espera de uma unidade ambulatorial deve ser vista, pelos profissionais de saúde, como um espaço propício para promover, o diálogo, acolhimento e vínculo, entre os pacientes, seus acompanhantes e os serviços de saúde. O processo de educação e de confiabilidade pode estimular aos pacientes a responsabilidade do autocuidado, gerando a interpretação de que muitas situações são preveníveis. É nesse momento, que os profissionais de enfermagem têm a oportunidade de detectar problemas de saúde, que podem ser expressados através das falas ou de expressões REVISTA ENFERMAGEM ATUAL | 2017; 81 111 112 Santos LM, Souza WL, Santos GS, Pereira ER, Silva RMCRA, Escudeiro CL corporais e faciais dos pacientes como, por exemplo, os sinais e sintomas da dor. A partir desse contato, é possível também aos profissionais tirar dúvidas e promover a educação em saúde, sendo assim, possível compreender essa clientela de forma integral e humanizada atendendo aos princípios do Sistema Único de Saúde.9 de espera, os pacientes e seus familiares passam por medos e incertezas em relação aos resultados de exames, atendimentos e tantas outras situações que geram expectativas em relação ao tratamento. Diante disso, este estudo objetivou relatar as experiências vivenciadas por uma enfermeira e sua equipe de enfermagem, referente ao atendimento acolhedor prestado aos pacientes e seus familiares em um ambulatório de oncologia clínica de um hospital especializado na cidade do Rio de Janeiro. Diante disso, evidenciou-se a importância do acolhimento aos pacientes e familiares, no ambulatório de oncologia clínica e como uma grande contribuição para o serviço foi implantado o “Projeto Sala de Espera” no ambulatório de oncologia clínica e hematologia: Otimizando o tempo dos pacientes acompanhantes e fortalecendo, assim, o manejo e atendimento no ambulatório de oncologia clínica. Metodologia Resultados e Discussão Trata-se de um relato de experiência, descrito por uma enfermeira que atua em um ambulatório de oncologia clinica e hematologia de um hospital especializado, referente ao atendimento prestado pela mesma e sua equipe de enfermagem. O ambulatório de oncologia clínica tem uma característica diferenciada aos outros, pois os pacientes atendidos, em sua maioria encontram-se em situações de complicações, que giram em torno das reações adversas ao tratamento ou ao avanço do processo patológico. Estes apresentam condições clínicas ruins e importantes comorbidades como: debilidades físicas, comprometimentos hemodinâmicos, além do medo, da ansiedade e muitas dúvidas. Todas essas condições podem levar o paciente a apresentar alguma intercorrência a qualquer momento do período em que o mesmo aguarda o atendimento. O ambulatório de oncologia clínica e hematologia realizam atendimentos de segunda a sexta feira, de 7 às 19 horas. Atende em média trezentos e cinquenta a quatrocentos pacientes por mês e conta com dez (10) ambulatórios de Oncologia Clínica. Um (1) consultório atende os pacientes que chegam ao setor apresentando intercorrências clínicas, os pacientes que necessitam ser avaliados pelo médico para checagem do exame de sangue para liberar a quimioterapia e os outros nove (9) consultórios, atendem os pacientes com consultas previamente agendadas. O ambulatório de hematologia conta com três (3) consultórios, um (1) atende pacientes de consultas extras e dois (2) atendem pacientes com consultas marcadas. Temos também uma sala para realização de atendimentos e/ou procedimentos de enfermagem tais como: repouso, soroterapia, preparo e administração de medicamentos, realizações de curativos, entre outros. Os pacientes que necessitam de internação e estão apresentando alguma comorbidades ficam nesta sala aguardando a liberação do leito. Contamos também com uma sala de procedimentos médicos, onde são realizados: punção lombar, biópsia de medula óssea e mielograma. Todos esses fatores acabam desencadeando um grau de ansiedade e estresse elevado, não somente aos pacientes como aos seus acompanhantes. O volume de atendimentos e procedimentos realizados no ambulatório, o estado de saúde mais comprometido dos pacientes e o grau de complexidade, requer dos profissionais médicos e de enfermagem um atendimento bastante criterioso. Todos esses fatores geram um tempo de espera maior, e, consequentemente, leva a ansiedade e ociosidade, não só aos pacientes, bem como aos acompanhantes. O período de espera nos ambulatórios é momento de angústia e ansiedade, um tempo que poderia ser melhor aproveitado, pois nesse período de permanência na sala REVISTA ENFERMAGEM ATUAL | 2017; 81 Vale ressaltar que as repercussões supracitadas podem acontecer na recepção, sala de observação e/ou sala de procedimentos, por isso, a equipe multiprofissional, deve estar sempre atenta e preparada para atuar em diversas situações. O primeiro contato dos pacientes e seus familiares no ambulatório acontece na recepção, no momento da entrega do cartão de consulta ou dos encaminhamentos, para os pacientes de primeira vez. As recepcionistas são profissionais bastante comprometidos com o serviço e realizam um atendimento de qualidade, estes possuem uma sintonia importante com os profissionais de enfermagem. Assim quando o paciente chega à recepção com qualquer queixa, imediatamente a equipe de enfermagem recebe a informação da necessidade do mesmo e realiza o atendimento. O paciente e seu familiar são direcionados à sala de enfermagem, sendo recebido pelo enfermeiro e o técnico de enfermagem, onde são aferidos os sinais vitais. O enfermeiro realiza a avaliação a partir do exame físico associado ao diálogo, a fim de identificar as necessidades de saúde e de possível necessidade de prioridade para o atendimento médico a partir das queixas descritas pelo paciente e/ou familiar. A assistência de enfermagem não está voltada apenas para solucionar os problemas clínicos e imediatos, mas, sim em realizar um atendimento holístico visando atender as necessidades integrais de saúde do cliente e seus familiares. Acolhimento nA o R a mTb Iu G l a tO ó r iO o dRe I oGn cI oN l oA g iLa A equipe de enfermagem, fornece as orientações educativas no que se refere a realização de vários procedimentos específicos ao tratamento no qual o paciente esteja sendo submetido no período tais como: administração de medicamento por via subcutânea, por via oral, retal, realizações dos curativos das lesões cutâneas, o cuidado com a integridade da pele, enfim, vários são os procedimentos e cuidados que o paciente oncológico realiza no cenário domiciliar. A equipe de enfermagem em cumplicidade com a equipe interdisciplinar em saúde elaborou estratégias para contribuir com a identificação e o manejo das principais repercussões hemodinâmicas, favorecendo um equilíbrio de estabilidade. O acolhimento é realizado com orientações direcionadas aos pacientes e familiares, referente aos processos fisiopatológicos, das possibilidades de instabilidades ou repercussões hemodinâmicas previsíveis, fortalecendo assim as habilidades do autocuidado e de uma assistência de qualidade, estimulando o empoderamento do conhecimento e um cuidar holístico, além do funcionamento e fluxo do atendimento no ambulatório. A ação profissional da enfermeira oncológica engloba uma grande diversidade de atividades, com variados graus de complexidade, cabendo-lhe igual empenho na realização de qualquer uma delas, tais como o atendimento aos clientes na recepção ou porta de entrada, resolutividade nas intercorrências, bem como habilidades e competências na tomada de decisão.10 Esta estratégia de um atendimento mais acolhedor, realizada pela equipe interdisciplinar, garantiu a qualidade da assistência prestada aos pacientes e seus familiares, com a prevenção das reações inesperadas no período de espera para o atendimento médico, bem como maior adesão ao tratamento e a continuidade do cuidado em domicílio. Diante disso, face às situações de urgência e emergência na rotina, percebe-se que as principais complicações giram em torno das reações adversas ao tratamento ou ao avanço do processo patológico, podendo apontar como as principais: dor, febre, náuseas, vômitos, diarreias, constipações, crises convulsivas, lipotímias, hipotensões e raramente parada cardiorrespiratória. Algumas das intercorrências supracitadas levam os profissionais de enfermagem a um pensamento crítico reflexivo sobre o atendimento ambulatorial, no que se refere a situações inusitadas, inesperadas, conflitantes que podem surgir no período em que os pacientes aguardam ser chamados para o atendimento médico. Sabe-se que por muitas vezes os acompanhantes e profissionais não estão preparados habitualmente para lidar com essas intercorrências em um ambiente ambulatorial. Essas situações fortalecem ainda mais a necessidade do acolhimento holístico e humanizado, entendendo que a ordem de chegada nem sempre será a única prioridade a ser avaliada, mas a possibilidade de um olhar clínico direcionado para investigação de possíveis instabilidades hemodinâmicas, podendo então dar ao profissional de saúde o pensamento crítico para tomada de decisão. No ambulatorial, os pacientes instáveis ou com possibilidade de instabilidade são atendidos prioritariamente, ou são levados para a sala de observação, onde a equipe de enfermagem de forma holística atua efetivamente para restaurar as funções corporais do paciente. Avaliar os riscos e a vulnerabilidade implica estar atento tanto ao grau de sofrimento físico quanto psíquico, pois muitas vezes o usuário que chega andando, sem sinais visíveis de problemas físicos, mas muito angustiado, pode estar mais necessitado de atendimento e com maior grau de risco e vulnerabilidade do que outros pacientes aparentemente mais necessitados.1 Nesse momento aproveita-se para estreitar a relação, pois acredita-se que essa ação seja capaz de promover a confiança entre pacientes, familiares e os profissionais, bem como fortalecer seus valores e crenças na possibilidade de eficácia e maior adesão ao tratamento. O cuidado humanizado significa tornar humano, dar condições humanas a pessoa que esta necessitando de cuidados, não de forma técnica, mecanizada, mas como processo vivencial, sendo assim a relação do profissional de enfermagem com o paciente oncológico e seus familiares, no ambulatório de oncologia clínica e hematologia, busca promover segurança e bem-estar tanto individual quanto coletivo.11 Conclusão As experiências vivenciadas no ambulatório de oncologia clínica contribuíram de formas satisfatórias na compreensão de gerenciar os serviços e as ações da equipe de enfermagem, no que se refere acolhimento aos pacientes e familiares, pois permitiu apontar estratégias que facilitaram a logística de atendimento, contribuindo para uma assistência de qualidade, humanizada, integral e eficaz. Assim, a equipe interdisciplinar, necessita de uma comunicação terapêutica eficiente entre profissionalpaciente/familiares e monitorização constante das repercussões hemodinâmicas enquanto os pacientes aguardam atendimento, visto que as instabilidades acontecem de forma imprevisível. Portanto, as contribuições do estudo vão além do contexto humanístico, mas transcendem a uma avaliação clínica para tomada de decisão, onde neste contexto oncológico o tempo pode ser crucial para uma assistência integral que possa garantir a vida e/ou a qualidade de vida, favorecendo dessa forma um acolhimento baseado nos princípios norteadores das políticas públicas de saúde. Frente às lacunas nos modelos de atenção e gestão dos serviços de saúde pública, no que se refere ao acesso e ao modo como o usuário é acolhido, sugere-se estudos que destaquem a abordagem do cuidado humanizado REVISTA ENFERMAGEM ATUAL | 2017; 81 113 114 Santos LM, Souza WL, Santos GS, Pereira ER, Silva RMCRA, Escudeiro CL e acolhedor aos pacientes oncológicos e seus familiares em atendimento ambulatorial, possibilitando o acesso ao conhecimento necessário à sua situação de saúde, em busca de promoção da autonomia para o autocuidado, bem como da qualidade de vida. Referências 1. Ministério da Saúde (BR). Secretaria de Atenção à Saúde. Núcleo Técnico da Política Nacional de Humanização. Acolhimento nas práticas de produção de saúde. Brasília (DF): MS; 2010. 2. Ministério da Saúde (BR) - Instituto Nacional de Câncer José Alencar Gomes da Silva. Coordenação de Prevenção e Vigilância. Estimativa 2014: Incidência de Câncer no Brasil/ Instituto Nacional de Câncer José Alencar Gomes da Silva, Coordenação de Prevenção e Vigilância. Rio de Janeiro: INCA, 2014. 3. Sistema de regulação de vagas – SISREG - Data SUS – Manoal operador solicitante. SISREG III solicitação de agendamento de consultas e procedimentos – Brasil setembro – 2008. 4. Zillmer JGV, Schwartz E, Muniz RM.O olhar da enfermagem sobre as práticas de cuidado de famílias rurais à pessoa com câncer. Rev. esc. enferm. USP [Internet]. 2012, 4(6):1371-1378. ISSN 0080-6234. Disponível em: http://dx.doi.org/10.1590/ S0080-62342012000600013. 5. Guerrero GP; Zago MMF, Sawada NO, Pinto MH. Relação entre espiritualidade e câncer: perspectiva do paciente. Revista Brasileira de Enfermagem [Internet]. 2011, 64(1):5359. Disponível em: http://www.scielo.br/pdf/reben/v64n1/ v64n1a08.pdf. 6. Salimena AMO, Teixeira SR, Amorim TV. O vivido dos enfermeiros no cuidado ao paciente oncológico. Cogitare Enferm [Internet]. 2013 Jan/Mar; 18(1):142-7. Disponível em: http://revistas.ufpr.br/cogitare/article/view/31320. 7. Silva RCV, Cruz EA. Planejamento da assistência de enfermagem ao paciente com câncer: reflexão teórica sobre as dimensões sociais. Esc. Anna Nery [Internet]. 2011, 15(1):180-185. ISSN 1414-8145. Disponível em: http:// dx.doi.org/10.1590/S1414-81452011000100025. 8. Reis FV, Brito JR, Santos JN, Oliveira MG. Educação em saúde na sala de espera – relato de experiência Educação em saúde na sala de espera – relato de experiência. Rev Med Minas Gerais [Internet]. 2014; 24(1): s32-s36. Disponível em: rmmg.org/exportar-pdf/549/v24s1a05.pdf. 9. Carneiro ACLL, Souza V, Godinho LK, Faria ICM, Silva KL, Gazzinelli MF. Educação para a promoção da saúde no contexto da atenção primária. Rev Panam Salud Publica [Internet]. 2012; 31(2):115–20. Disponível: http://www. scielosp.org/pdf/rpsp/v31n2/a04v31n2.pdf. 10. Machado GC; Ouro ETR; Santana FPG. Acolhimento de enfermagem em oncologia: enfrentamento da doença pelos pacientes em Feira de Santana-BA. Revista Brasileira de Saúde Funcional, [Internet]. 2015; 2(1):33-45. Disponível em: http://www.seer-adventista.com.br/ojs/index.php/RBSF/ article/view/696. 11. Oliveira NES, Oliveira LMAC, Lucchese R, Alvarenga GC, Brasil VV. Humanização na teoria e na prática: a construção do agir REVISTA ENFERMAGEM ATUAL | 2017; 81 de uma equipe de enfermeiros. Rev. Eletr. Enf. [Internet]. 2013 abr/jun; 15(2):334-43. Disponível em: http://dx.doi. org/10.5216/ree.v15i2.17916.
https://openalex.org/W2950614078
https://www.frontiersin.org/articles/10.3389/fimmu.2018.02096/pdf
English
null
Activation of RIG-I-Mediated Antiviral Signaling Triggers Autophagy Through the MAVS-TRAF6-Beclin-1 Signaling Axis
Frontiers in immunology
2,018
cc-by
10,661
Activation of RIG-I-Mediated Antiviral Signaling Triggers Autophagy Through the MAVS-TRAF6-Beclin-1 Signaling Axis Na-Rae Lee 1, Junsu Ban 1, Noh-Jin Lee 1, Chae-Min Yi 1, Ji-Yoon Choi 1, Hyunbin Kim 2,3, Jong Kil Lee 1, Jihye Seong 2,3, Nam-Hyuk Cho 4,5, Jae U. Jung 6 and Kyung-Soo Inn 1,2* 1 Department of Fundamental Pharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul, South Korea, 2 KHU-KIST Department of Converging Science and Technology, Graduate School, Kyung Hee University, Seoul, South Korea, 3 Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul, South Korea, 4 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, South Korea, 5 Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, South Korea, 6 Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States ORIGINAL RESEARCH published: 12 September 2018 doi: 10.3389/fimmu.2018.02096 ORIGINAL RESEARCH published: 12 September 2018 doi: 10.3389/fimmu.2018.02096 Edited by: Autophagy has been implicated in innate immune responses against various intracellular pathogens. Recent studies have reported that autophagy can be triggered by pathogen recognizing sensors, including Toll-like receptors and cyclic guanosine monophosphate-adenosine monophosphate synthase, to participate in innate immunity. In the present study, we examined whether the RIG-I signaling pathway, which detects viral infections by recognizing viral RNA, triggers the autophagic process. The introduction of polyI:C into the cytoplasm, or Sendai virus infection, significantly induced autophagy in normal cells but not in RIG-I-deficient cells. PolyI:C transfection or Sendai virus infection induced autophagy in the cells lacking type-I interferon signaling. This demonstrated that the effect was not due to interferon signaling. RIG-I-mediated autophagy diminished by the deficiency of mitochondrial antiviral signaling protein (MAVS) or tumor necrosis factor receptor-associated factor (TRAF)6, showing that the RIG-I- MAVS-TRAF6 signaling axis was critical for RIG-I-mediated autophagy. We also found that Beclin-1 was translocated to the mitochondria, and it interacted with TRAF6 upon RIG-I activation. Furthermore, Beclin-1 underwent K63-polyubiquitination upon RIG- I activation, and the ubiquitination decreased in TRAF6-deficient cells. This suggests that the RIG-I-MAVS-TRAF6 axis induced K63-linked polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism. Autophagy has been implicated in innate immune responses against various intracellular pathogens. Recent studies have reported that autophagy can be triggered by pathogen recognizing sensors, including Toll-like receptors and cyclic guanosine monophosphate-adenosine monophosphate synthase, to participate in innate immunity. In the present study, we examined whether the RIG-I signaling pathway, which detects viral infections by recognizing viral RNA, triggers the autophagic process. The introduction of polyI:C into the cytoplasm, or Sendai virus infection, significantly induced autophagy in normal cells but not in RIG-I-deficient cells. PolyI:C transfection or Sendai virus infection induced autophagy in the cells lacking type-I interferon signaling. This demonstrated that the effect was not due to interferon signaling. RIG-I-mediated autophagy diminished by the deficiency of mitochondrial antiviral signaling protein (MAVS) or tumor necrosis factor receptor-associated factor (TRAF)6, showing that the RIG-I- MAVS-TRAF6 signaling axis was critical for RIG-I-mediated autophagy. We also found that Beclin-1 was translocated to the mitochondria, and it interacted with TRAF6 upon RIG-I activation. Furthermore, Beclin-1 underwent K63-polyubiquitination upon RIG- I activation, and the ubiquitination decreased in TRAF6-deficient cells. Edited by: This suggests that the RIG-I-MAVS-TRAF6 axis induced K63-linked polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism. Reviewed by: Ingvild Bjellmo Johnsen, Norwegian University of Science and Technology, Norway Christine McDonald, Cleveland Clinic, Lerner Research Institute, United States *Correspondence: Kyung-Soo Inn innks@khu.ac.kr Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 07 June 2018 Accepted: 24 August 2018 Published: 12 September 2018 Keywords: autophagy, RIG-I, MAVS, TRAF6, polyubiquitination, Beclin-1, innate immunity Edited by: Timothy B. Niewold, New York University, United States Immunoblotting and co-IP Besides the SLRs, the innate immune system utilizes a limited number of sensors including Toll-like receptors (TLRs), Nod-like receptors, cyclic guanosine monophosphate- adenosine monophosphate synthase (cGAS), and RIG-I-like receptors (RLRs) to detect various pathogen-associated molecular patterns (10–14). Among these sensors, RIG-I and MDA5 recognize viral RNAs to mount an antiviral immune response. Upon recognition, RIG-I and MDA5 translocate to mitochondria to interact with the mitochondrial antiviral signaling protein (MAVS)/IPS-1/Cardif, a downstream mitochondrial signaling protein. The subsequent recruitment of signaling molecules, including tumor necrosis factor receptor-associated factor (TRAF)3 and TRAF6, results in the activation of transcription factors, such as IRF3/7, NF- κB, and AP-1, leading to the production of type-I interferon (IFN). Immunoblotting and co IP The cells were transfected with the indicated plasmids and treated as described. The cells were collected and lysed with Triton X-100 lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100) containing protease inhibitor cocktail (Pierce, #78430). After centrifugation, the cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. Primary antibodies used were as follows: anti- LC3 (Cosmo Bio, #CTB-LC3-1-50), anti-SQSTM1/P62 (Abcam, #ab56416, Cambridge, UK), anti-phospho-IRF3 (Cell Signaling Technology, #4947), anti-phospho-NF-κB (Cell Signaling Technology, #3031), anti-β-actin (Santa Cruz Biotechnology, #sc-47778, Dallas, TX, USA), anti-ubiquitin P4D1 (Cell Signaling Technology, #3936), anti-Flag (Sigma-Aldrich, #F7435, Saint Louis, MO, USA), anti-Beclin-1 (Cell Signaling Technology, #3738), anti-HA (Santa Cruz Biotechnology, #sc-7392), anti- V5 (Cell Signaling Technology, #13202), anti-K63-linked ubiquitin (Cell Signaling Technology, #5621), anti-VPS34 (Cell Signaling Technology, #4263), anti-ATG14 (Cell Signaling Technology, #96752), anti-Ambra-1 (Cell Signaling Technology, #12250), anti-UVRAG (Cell Signaling Technology, #5320), anti-GST (Abcam, #ab19256, Cambridge, UK), anti-TRAF6 (Cell Signaling Technology, #8028), anti-Cox4 (Santa Cruz Biotechnology, #133478), anti-β-tubulin (Cell Signaling Technology, #2146). For IP, the clarified cell lysates were incubated with the indicated antibodies for 12 h at 4◦C, followed by further incubation with protein A/G resin for 2 h. For IP Flag-tagged proteins, the cell lysates were incubated with anti-Flag M2 affinity resin (Sigma-Aldrich, #A2220) or anti-V5 affinity resin (Sigma-Aldrich, #A7345) for 12 h at 4◦C. After extensive washing with lysis buffer, the bound proteins were suspended in 1 × sample buffer and analyzed by SDS-PAGE and immunoblotting. As autophagy can be activated by infection with pathogens, it is conceivable that the innate immune sensors regulate the autophagic process upon recognition of pathogens. Citation: Lee N-R, Ban J, Lee N-J, Yi C-M, Choi J-Y, Kim H, Lee JK, Seong J, Cho N-H, Jung JU and Inn K-S (2018) Activation of RIG-I-Mediated Antiviral Signaling Triggers Autophagy Through the MAVS-TRAF6-Beclin-1 Signaling Axis. Front. Immunol. 9:2096. doi: 10.3389/fimmu.2018.02096 Keywords: autophagy, RIG-I, MAVS, TRAF6, polyubiquitination, Beclin-1, innate immunity September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 1 Induction of Autophagy by RIG-I Lee et al. MATERIALS AND METHODS Cells, Viruses, and Plasmids Cells, Viruses, and Plasmids The RIG-I+/+ (WT), RIG-I−/−(KO), Atg3 WT and Atg3 KO mouse embryonic fibroblasts (MEFs) were described previously (22, 23). The MAVS KO and IFNAR KO MEFs were generated from the MAVS KO mice (kindly provided by Dr. Shizuo Akira at the Osaka University) and IFNAR KO mice (kindly provided by Dr. Heung Kyu Lee at the Korea Advanced Institute of Science and Technology). The TRAF6 KO MEFs were kindly provided by Dr. Yoon-Jae Song at the Gachon University. Beclin-1 stable knock-down A549 cells were generated as described previously (24). N2a and BV- 2 cells were described preciously (25). HEK293T, HEK293A, Raw264.7, Huh7, Huh7.5, Vero, and MEF cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin/streptomycin (100 U/mL). Sendai virus (SeV; Cantell strain) was purchased from Charles River Laboratories. Influenza A virus was prepared and infected as described previously (26). Autophagy is a process that sequesters cytosolic regions and delivers their contents to the lysosomes for subsequent degradation. Both extracellular stimuli, such as starvation and hypoxia, and intracellular stresses, including the accumulation of damaged organelles, induce autophagy to degrade long-lived proteins and damaged organelles in order to recycle and maintain cell homeostasis (1). As autophagy is triggered by infection with intracellular pathogens, such as bacteria and viruses, it is recognized as part of the innate immune system to control and eliminate infections by engulfing and degrading intracellular pathogens (i.e., xenophagy) (2–4). Recent extensive studies have revealed that several autophagic adaptors, such as sequestosome 1 (SQSTM1/p62), optineurin, and nuclear dot protein 52 kDa, specifically recognize the intracellular presence of bacteria, including Salmonella, Shigella, Listeria, and Mycobacteria, and induce autophagy (5–8). Adaptor proteins, referred to as sequestosome 1/p62-like receptors (SLRs), directly recognize ubiquitinated microbes as their targets to induce autophagy. They are now regarded as a class of pattern recognition receptors of the innate immune system (9). Frontiers in Immunology | www.frontiersin.org Autophagosome Staining by Cyto-ID and LC3 Antibodies Scientific, #31556; Thermo Scientific, #62-6511) according to the instruction of the manufacturer. The colocalization images were examined under an Olympus FV-1000 confocal microscope. HEK293T cells were transfected with 2 µg of polyI:C or infected with SeV at 200 HA units/mL, and collected after 12 h. Subsequently, Cyto-ID autophagy reagent (Enzo, #ENZ-51031, Farmingdale, NY, USA) staining was performed according to the instruction of the manufacturer. Briefly, the cells were washed twice with 1 × assay buffer and treated with diluted Cyto- ID green stain solution. The cells were incubated for 30 min at 37◦C, and then washed and incubated for 20 min with 4% paraformaldehyde. For LC3 staining, the cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% tritonX-100 buffer. Then cells were stained with LC3 antibody (Cell Signaling Technology, #3868) for 2 h at 37◦C and stained with anti-rabbit IgG FITC reagent. The cells were then washed three times and analyzed by fluorescence microscopy to observe punctated forms of autophagosomes. Immunoblotting and co-IP Indeed, the activation of innate immune signaling pathways triggered by innate immune sensors, including TLR4 and cGAS, activates autophagy, indicating that the innate immune system modulates autophagy directly (15–17). Besides participating in the innate immune system by directly eliminating pathogens, autophagy also plays crucial roles in regulating this system to prevent excessive responses (18–20). A recent study showed that the absence of autophagy amplified RIG-I signaling due to increased mitochondrial MAVS and reactive oxygen species from damaged mitochondria, implicating autophagy in RLR signaling (21). However, it has not been reported whether RIG-I-mediated antiviral signaling directly regulates autophagy. Herein, we show that the activation of RIG-I by its ligands provokes autophagy in a downstream MAVS-TRAF6 signaling axis-dependent manner. September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 2 Induction of Autophagy by RIG-I Lee et al. Activation of the RIG-I Signaling Pathway Activates Autophagy To investigate whether the recognition of viral RNA by RIG- I can trigger autophagy, HEK293T cells were transfected with the RIG-I agonist, polyI:C, a synthetic double-stranded RNA analog, or infected with Sendai virus (SeV). Both polyI:C transfection and SeV infection increased the level of LC3-II, a lipidated form of LC3 (Figure 1A). In both the experimental settings, the change was evident 2 h after stimulation (Figure 1A). Increased formation of autophagic vesicles was also observed in the polyI:C-transfected and SeV-infected cells compared with that in mock-infected cells (Figure 1B), as determined by Cyto-ID that can stain both autophagosomes and autolysosomes specifically (27). The increased autophagosome formation by polyI:C transfection or Statistical Analysis Data are represented as mean ± standard error of the mean (SEM) unless otherwise indicated, and were analyzed by Student’s unpaired two-tailed t test using GraphPad Prism 5 software. A value of ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005 was considered significant. Transmission Electron Microscopy Transmission Electron Microscopy HEK293T cells were transfected with 10 µg polyI:C using Lipofectamine 2000, or infected with SeV at 200 HA units/mL, and incubated for 12 h. The WT and MAVS KO MEF cells were transfected with polyI:C (10 µg), pEBG vector (2.5 µg), or pEBG- RIG-IN (5 µg) using Lipofectamine 2000, and further incubated for 12 h. The cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4◦C for 24 h. The cells were then embedded in epoxy resin and polymerized at 38◦C for 12 h, followed by further incubation at 60◦C for 48 h. Thin sections, cut using an ultramicrotome (MT-XL, RMC Products), were collected on a copper grid and stained with 4% lead citrate and saturated 4% uranyl acetate. The samples were examined at 80 kV with a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan) at the Seoul National University Hospital Medical Research Institute (Seoul, Korea). Double membrane vesicles measuring 0.3 to 2.0 µm in diameter were defined as autophagosomes. Mitochondria Isolation HEK293T cells (1 × 107) were transfected with the pEBG vector or pEBG-RIG-IN and incubated for 24 h. The cells were washed with phosphate-buffered saline and mitochondria were isolated using a kit (Thermo Scientific, #89874) according to the instruction of the manufacturer instructions. The cytosolic and mitochondrial fractions were analyzed by SDS-PAGE and immunoblotting. Cox4 and β-tubulin served as markers for the cytosolic and mitochondrial fractions, respectively. RNA Isolation and Reverse-Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) ( q ) Expression of genes encoding IFN-β and ISG15 was analyzed by RT-qPCR using a CFX Connect real-time PCR system (Bio- Rad). Total RNAs were isolated from harvested cells using an RNA isolation kit (GeneAll) according to the manufacturer’s instructions. cDNAs were generated from RNAs (1 µg) using an RT kit (Enzynomics) and subjected to quantitative PCR. Human IFN-β mRNA was amplified using the primer pair 5- AAGAGTTACACTGCCTTTGCCATC-3 (forward) and 5-CAC TGTCTGCTGGTGGAGTTCATC-3 (reverse). Human ISG15 mRNA was amplified using the primer pair 5-CCTCTGAG CATCCTGGT-3 (forward) and 5-AGGCCGTACTCCCCCAG- 3 (reverse). IFN-β and ISG15 mRNA levels were normalized to those of human β-actin, which was amplified with the primer pair 5-TGGAATCCTGTGGCATCCATGAAAC-3 (forward) and 5-TAAAACGCAGCTCAGTAACAGTCCG-3 (reverse). Mouse IFN-β mRNA was amplified using the primer pair 5-CCTCT GAGCATCCTGGT-3 (forward) and 5- AGGCCGTACTCCC CCAG-3 (reverse). Mouse ISG15 mRNA was amplified using the primer pair 5-AGCTCCAAGAAAGGACGAACAT-3 (forward) and 5-GCCCTGTAGGTGAGGTTGATCT-3 (reverse). IFN-β and ISG15 mRNA levels were normalized to those of mouse β- actin, which was amplified with the primer pair 5-TGGAATCCT GTGGCATCCAT-3 (forward) and 5-TAAAACGCAGCTCAGT AACA-3 (reverse). MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling g g The role of MAVS, a downstream mitochondrial signaling molecule, in inducing autophagy was investigated. The LC3- II level was increased by the ectopic expression of MAVS in HEK293T cells (Figure 5A). However, the introduction of polyI:C into the MAVS-deficient MEFs failed to increase LC3 lipidation (Figure 5B). Consistently, no significant increase in autophagosome formation was observed by electron microscopy in the MAVS-deficient MEFs upon transfection with polyI:C. In contrast, a significant increase in the number of autophagosomes was observed in WT MEFs (Figure 5C). Furthermore, there was no significant decrease in p62 upon SeV infection in the cycloheximide (CHX)-treated MAVS-deficient MEFs, whereas, a significant decrease in the p62 level was observed in the CHX-treated WT MEFs (Figure 5D). These results indicate that RIG-I or MDA5 induce autophagy flux via their downstream MAVS. Given that MAVS recruits TRAF6 to activate downstream signaling and TRAF6 activates TLR4-mediated autophagy, it is worth testing the hypothesis that TRAF6 plays a crucial role in RIG-I-mediated autophagy. Indeed, LC3-II formation following SeV infection or polyI:C transfection was significantly lower in TRAF6-deficient than in WT MEFs (Figures 6A,B). Consistently, LC3 puncta formation by transfection with polyI:C or RIG-IN was significantly lower in the TRAF6-defective MEFs than in the WT MEFs (Figure 6C). In addition, there was a significant decrease in the level of p62 in the SeV-infected The negative regulatory role of autophagy in RIG-I signaling was confirmed using Beclin-1 knock-down cells and Atg3 knock-out cells. Beclin1 knock-down cells and atg3 deficient cells showed higher mRNA levels of IFN-β and ISG15 upon transfection of polyI:C or ectopic expression of constitutively active form of RIG-I (N-terminal 2Cards domain; RIG-IN) compared to WT cells (Figures 2E,F). To further confirm that the triggering of autophagy by an RIG-I agonist was due to the activation of RIG-I signaling, the effect of expressing a constitutively active form of RIG-I on autophagy was examined. The ectopic expression of RIG-IN or MDA5-2Card domains (MDA5-N) increased LC3 lipidation (Figure 3A). The activation of RIG-I signaling by ectopic expression also increased autophagosomes, as determined by cyto-ID staining (Figure 3B). The ectopic expression of RIG-IN or MDA5-N also increased LC3-functa formation, indicating that both RIG-I and MDA5 can modulate autophagy (Figure 3C). The level of p62 decreased in the RIG-IN-expressing cells in a time-dependent manner (Figure 3D). Immunofluorescence Staining and Confocal Microscopy HEK293A cells were transfected with polyI:C or treated with SeV as described. The cells were moved to fibronectin-coated confocal dish and incubated for 12 h. The cells were stained with mitotracker (Thermo Scientific, #M7512, Rockford, IL, USA) according to the instruction of the manufacturer and fixed with 4% paraformaldehyde for 15 min, permeabilized using 0.1% tritonX-100 for 10 min and blocked with 5% BSA. The cells were then stained with the primary and secondary antibodies (Thermo September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 3 Lee et al. Induction of Autophagy by RIG-I SeV infection was further confirmed by transmission electron microscopy. As shown in Figure 1C, the number of dense black double-membrane structure vesicles increased in the polyI:C transfected or SeV infected cells. The formation of LC3 puncta by polyI:C or SeV infection also contributed to the modulation of autophagy by RIG-I signaling (Figure 1D). Not only the increase of autophagy flux but also the blockade of autophagosomal maturation can be resulted in accumulation of LC3-II. Thus, LC3-II formation upon RIG-I activation was examined with or without bafilomycinA1, an inhibitor of the late phase of autophagy. BafilomycinA1 treatment resulted in further increase of LC3-II formation in polyI:C transfected or SeV infected cells compared to mock-treated samples. LC3-II formation was increased by PolyI:C transfection or SeV infection in a time-dependent manner regardless of bafilomycinA1 treatment (Figure 1E). Furthermore, the level of p62 decreased in a time-dependent manner in the polyI:C- transfected or SeV-infected cells, whereas the level was not noticeably changed in untreated samples (Figure 1F). These results indicated that the increased level of LC3-II was due to increased autophagy and not due to decreased phagolysosome formation. The induction of autophagy by polyI:C or SeV was examined in cells defective in type-I interferon signaling to test whether RIG-I-mediated autophagy was due to type-I interferon signaling induced by RIG-I signaling. The transfection of polyI:C triggered LC3 lipidation in type-I interferon receptor-deficient MEFs, suggesting that RIG-I signaling can induce autophagy flux in a type-I interferon-independent manner (Figure 3F). Furthermore, polyI:C transfection or SeV infection also increased LC3-II in Vero cells, which are defective in interferon signaling (Figure 3G). Autophagy Induction by SeV Infection or PolyI:C Transfection Requires Functional RIG-I The influence of RIG-I-mediated signaling on the induction of autophagy was further examined using cells deficient in RIG- I activity. We used Huh7.5 human hepatoma cells that were derived from Huh7 cells. These cells lose their RIG-I activity due to a mutation in RIG-I (T55I). As expected, LC3-II formation increased by the transfection of Huh7 but not by Huh7.5 cells with polyI:C (Figure 4A). However, the infection of Huh7.5 cells with influenza A virus activated autophagy to a level comparable to Huh7 cells. This suggested that influenza A virus triggered autophagy via other signaling pathways, such as TLR4, in the absence of RIG-I signaling (Figure 4B). Consistent with the Huh7.5 data, LC3 lipidation induced by polyI:C transfection or SeV infection in RIG-I-deficient MEFs was noticeably reduced compared to what was observed in WT MEFs (Figures 4C,D). The induction of autophagy by RIG-I activation was also observed in different types of cells. The transfection of polyI:C was resulted in increased LC3 lipidation and LC3 puncta formation in Raw264.7 murine monocytic cells (Figures 2A,B). Phosphorylation of IRF3 served as a control to show the activation of RIG-I signaling. Consistently, increment of LC3 lipidation by polyI:C transfection or SeV infection was easily detected in N2a mouse neuroblastoma cells and BV-2 mouse microglial cells (Figures 2C,D), suggesting that the induction of autophagy by RIG-I activation is not a cell- or species-specific phenomenon. Frontiers in Immunology | www.frontiersin.org MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling The bottom panels show the relative p62 expression levels. Data are represented as mean ± SE from untreated samples (gray bars) and polyI:C-transfected or SeV-infected samples (black bars). A value of *p < 0.05, **p < 0.005. vs. not treated samples. FIGURE 1 | RIG-I activation invokes autophagy. (A) HEK293T cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) (200 HA U/mL) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3 (LC3-I and LC3-II), phospho-IRF3 (p-IRF3), and β-actin. (B) HEK293T cells were transfected with polyI:C or infected with SeV as in (A) and incubated for 12 h. Autophagosomes were labeled with cytoID-green reagent and observed by fluorescence microscopy. The bottom panels show staining with Hoechst dye to visualize nuclei of the cells. (C) HEK293T cells were transfected with polyI:C (10 µg) or infected with SeV (200 HA U/mL) for 12 h. The cells were harvested, fixed, and subjected to transmission electron microscopy to observe autophagic vesicles (red arrows in the lower panels). The bottom panels show enlarged view of the boxed regions in the top panels. The bottom graph show that means of autophagic vacuoles in a cell determined from 5 different images. The data are presented as mean ± SE. (D) HEK293T cells were mock-treated, transfected with polyI:C or infected with SeV as in (A). After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. The number of puncta was counted and analyzed using the image J software. The bottom panel shows the mean number of LC3 puncta in a cell. The data are presented as mean ± SE. (E) HEK293T cells were transfected with polyI:C or infected with SeV and treated with or without bafilomycin (50 nM) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3 and β-actin. The ratios of LC3II/I was determined by densitometry and presented below. (F) HEK293T cells were transfected with polyI:C (2 µg) or infected with SeV (200 HAU/mL) and treated with cycloheximide (CHX, 100 ng/mL) for indicated hours. The levels of p62 were analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. The bottom panels show the relative p62 expression levels. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling Moreover, the inhibition of lysosomal degradation by the treatment with chloroquine (CQ) significantly increased the effect of RIG-IN on LC3 lipidation, confirming that the accumulation of LC3-II by RIG-I signaling was not due to the suppression of autophagy flux (Figure 3E). September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 4 Lee et al. Induction of Autophagy by RIG-I FIGURE 1 | RIG-I activation invokes autophagy. (A) HEK293T cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) (200 HA U/mL) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3 (LC3-I and LC3-II), phospho-IRF3 (p-IRF3), and β-actin. (B) HEK293T cells were transfected with polyI:C or infected with SeV as in (A) and incubated for 12 h. Autophagosomes were labeled with cytoID-green reagent and observed by fluorescence microscopy. The bottom panels show staining with Hoechst dye to visualize nuclei of the cells. (C) HEK293T cells were transfected with polyI:C (10 µg) or infected with SeV (200 HA U/mL) for 12 h. The cells were harvested, fixed, and subjected to transmission electron microscopy to observe autophagic vesicles (red arrows in the lower panels). The bottom panels show enlarged view of the boxed regions in the top panels. The bottom graph show that means of autophagic vacuoles in a cell determined from 5 different images. The data are presented as mean ± SE. (D) HEK293T cells were mock-treated, transfected with polyI:C or infected with SeV as in (A). After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. The number of puncta was counted and analyzed using the image J software. The bottom panel shows the mean number of LC3 puncta in a cell. The data are presented as mean ± SE. (E) HEK293T cells were transfected with polyI:C or infected with SeV and treated with or without bafilomycin (50 nM) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3 and β-actin. The ratios of LC3II/I was determined by densitometry and presented below. (F) HEK293T cells were transfected with polyI:C (2 µg) or infected with SeV (200 HAU/mL) and treated with cycloheximide (CHX, 100 ng/mL) for indicated hours. The levels of p62 were analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling The cell lysates were analyzed by immunoblotting using the indicated antibodies as primary antibodies. (D) BV-2 murine microglial cells were transfected with polyI:C and incubated for indicated hours. The cell lysates were analyzed by immunoblotting using indicated antibodies. Each experiment was repeated three or more times and representative data are shown. (E) Control (pGIPZ) or Beclin-1 knockdown (BECN KD) A549 cells were transfected with polyI:C (upper) or RIG-IN (bottom) and incubated for indicated hours. mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. (F) Atg3 wildtype (Atg3 WT) or Atg3 knockout (Atg3 KO) MEFs were transfected with polyI:C (upper) or RIG-IN (bottom). mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. Data are represented as means ± SE. **p < 0.005. ***p < 0.0005. FIGURE 2 | Induction of autophagy by RIG-I in different types of cells. (A) Raw264.7 murine macrophage cells were transfected with polyI:C (2 µg) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3, phospho-IRF3, and β-actin. (B) Raw264.7 cells were mock-treated, transfected with polyI:C (2 µg), infected with SeV (200 HAU/ml) or RIG-IN (100ng) for 8 h. After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. (C) N2a murine hypothalamus cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) for 0, 4, or 8 h. The cell lysates were analyzed by immunoblotting using the indicated antibodies as primary antibodies. (D) BV-2 murine microglial cells were transfected with polyI:C and incubated for indicated hours. The cell lysates were analyzed by immunoblotting using indicated antibodies. Each experiment was repeated three or more times and representative data are shown. (E) Control (pGIPZ) or Beclin-1 knockdown (BECN KD) A549 cells were transfected with polyI:C (upper) or RIG-IN (bottom) and incubated for indicated hours. mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. (F) Atg3 wildtype (Atg3 WT) or Atg3 knockout (Atg3 KO) MEFs were transfected with polyI:C (upper) or RIG-IN (bottom). mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. Data are represented as means ± SE. **p < 0.005. ***p < 0.0005. G | ( ) FIGURE 2 | Induction of autophagy by RIG-I in different types of cells. (A) Raw264.7 murine macrophage cells were transfected with polyI:C (2 µg) for the indicated hours. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling Data are represented as mean ± SE from untreated samples (gray bars) and polyI:C-transfected or SeV-infected samples (black bars). A value of *p < 0.05, **p < 0.005. vs. not treated samples. September 2018 | Volume 9 | Article 2096 5 Frontiers in Immunology | www.frontiersin.org Lee et al. Lee et al. Induction of Autophagy by RIG-I FIGURE 2 | Induction of autophagy by RIG-I in different types of cells. (A) Raw264.7 murine macrophage cells were transfected with polyI:C (2 µg) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3, phospho-IRF3, and β-actin. (B) Raw264.7 cells were mock-treated, transfected with polyI:C (2 µg), infected with SeV (200 HAU/ml) or RIG-IN (100ng) for 8 h. After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. (C) N2a murine hypothalamus cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) for 0, 4, or 8 h. The cell lysates were analyzed by immunoblotting using the indicated antibodies as primary antibodies. (D) BV-2 murine microglial cells were transfected with polyI:C and incubated for indicated hours. The cell lysates were analyzed by immunoblotting using indicated antibodies. Each experiment was repeated three or more times and representative data are shown. (E) Control (pGIPZ) or Beclin-1 knockdown (BECN KD) A549 cells were transfected with polyI:C (upper) or RIG-IN (bottom) and incubated for indicated hours. mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. (F) Atg3 wildtype (Atg3 WT) or Atg3 knockout (Atg3 KO) MEFs were transfected with polyI:C (upper) or RIG-IN (bottom). mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. Data are represented as means ± SE. **p < 0.005. ***p < 0.0005. FIGURE 2 | Induction of autophagy by RIG-I in different types of cells. (A) Raw264.7 murine macrophage cells were transfected with polyI:C (2 µg) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3, phospho-IRF3, and β-actin. (B) Raw264.7 cells were mock-treated, transfected with polyI:C (2 µg), infected with SeV (200 HAU/ml) or RIG-IN (100ng) for 8 h. After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. (C) N2a murine hypothalamus cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) for 0, 4, or 8 h. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling The cell lysates were analyzed by immunoblotting using antibodies for LC3, phospho-IRF3, and β-actin. (B) Raw264.7 cells were mock-treated, transfected with polyI:C (2 µg), infected with SeV (200 HAU/ml) or RIG-IN (100ng) for 8 h. After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. (C) N2a murine hypothalamus cells were transfected with polyI:C (2 µg) or infected with Sendai virus (SeV) for 0, 4, or 8 h. The cell lysates were analyzed by immunoblotting using the indicated antibodies as primary antibodies. (D) BV-2 murine microglial cells were transfected with polyI:C and incubated for indicated hours. The cell lysates were analyzed by immunoblotting using indicated antibodies. Each experiment was repeated three or more times and representative data are shown. (E) Control (pGIPZ) or Beclin-1 knockdown (BECN KD) A549 cells were transfected with polyI:C (upper) or RIG-IN (bottom) and incubated for indicated hours. mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. (F) Atg3 wildtype (Atg3 WT) or Atg3 knockout (Atg3 KO) MEFs were transfected with polyI:C (upper) or RIG-IN (bottom). mRNA levels of IFN-β and ISG15 were analyzed by RT-qPCR. Data are represented as means ± SE. **p < 0.005. ***p < 0.0005. September 2018 | Volume 9 | Article 2096 6 Frontiers in Immunology | www.frontiersin.org Lee et al. Induction of Autophagy by RIG-I Lee et al. Induction of Autophagy by RIG-I FIGURE 3 | A constitutively active form of RIG-I triggers autophagy. (A) HEK293T cells were transfected with constitutively active N-terminal Card domains of RIG-I (RIG-IN) or MDA5 (MDA5-N). LC3 lipidation was analyzed by immunoblotting using anti-LC3 and anti-β-actin antibodies. (B) HEK293T cells were transfected with vector or RIG-IN and incubated for 24 h. Autophagosomes were stained with Cyto-ID reagent and observed under a fluorescence microscope. The bottom panels show staining with Hoechst dye to visualize nuclei of the cells. (C) HEK293A cells were transfected with RIG-IN or MDA5-N. Eighteen hours after transfection, the cells were fixed and stained with anti-LC3 antibody and FITC-labeled secondary antibody and subjected to fluorescence microscopy. The numbers of puncta was counted and analyzed using the image J software. The right panel shows the mean number of LC3 puncta in a cell. The data are presented as mean ± standard error of the mean. (D) HEK293T cells were transfected with RIG-IN for 0, 6, 9, 12, and 24 h. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling The levels of p62 were analyzed by immunoblotting. Densitometric analysis was performed using 3 results from independent experiments. **p < 0.005. vs. 0h control. (E) HEK293T cells were transfected with vector or RIG-IN for 0 or 24 h with or without chloroquine (CQ) treatment (20 µM) for 12 h before harvest. The cells were subjected to immunoblotting using an anti-LC3 antibody. (F) Type-I interferon receptor (INFR)-deficient mouse embryonic fibroblast cells were transfected with 2 µg polyI:C and incubated for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting. (G) Vero cells were transfected with 2 µg polyI:C or infected with 200 HA U/mL SeV for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. FIGURE 3 | A constitutively active form of RIG-I triggers autophagy. (A) HEK293T cells were transfected with constitutively active N-terminal Card domains of RIG-I (RIG-IN) or MDA5 (MDA5-N). LC3 lipidation was analyzed by immunoblotting using anti-LC3 and anti-β-actin antibodies. (B) HEK293T cells were transfected with vector or RIG-IN and incubated for 24 h. Autophagosomes were stained with Cyto-ID reagent and observed under a fluorescence microscope. The bottom panels show staining with Hoechst dye to visualize nuclei of the cells. (C) HEK293A cells were transfected with RIG-IN or MDA5-N. Eighteen hours after transfection, the cells were fixed and stained with anti-LC3 antibody and FITC-labeled secondary antibody and subjected to fluorescence microscopy. The numbers of puncta was counted and analyzed using the image J software. The right panel shows the mean number of LC3 puncta in a cell. The data are presented as mean ± standard error of the mean. (D) HEK293T cells were transfected with RIG-IN for 0, 6, 9, 12, and 24 h. The levels of p62 were analyzed by immunoblotting. Densitometric analysis was performed using 3 results from independent experiments. **p < 0.005. vs. 0h control. (E) HEK293T cells were transfected with vector or RIG-IN for 0 or 24 h with or without chloroquine (CQ) treatment (20 µM) for 12 h before harvest. The cells were subjected to immunoblotting using an anti-LC3 antibody. (F) Type-I interferon receptor (INFR)-deficient mouse embryonic fibroblast cells were transfected with 2 µg polyI:C and incubated for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting. MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling (G) Vero cells were transfected with 2 µg polyI:C or infected with 200 HA U/mL SeV for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. September 2018 | Volume 9 | Article 2096 7 Frontiers in Immunology | www.frontiersin.org Lee et al. Lee et al. Induction of Autophagy by RIG-I FIGURE 4 | Functional RIG-I is required for polyI:C and Sendai virus (SeV)-mediated autophagy activation. (A) Huh7 human hepatoma cells and Huh7-derived Huh7.5 cells with defective RIG-I activity were transfected with 2 µg polyI:C. LC3 lipidation was analyzed by immunoblotting using anti-LC3 and anti-β-actin antibodies. (B) Huh7 and Huh7.5 cells were infected with influenza A PR8 (Flu-PR8) (multiplicity of infection = 1) as indicated. LC3 lipidation was analyzed as in (A). LC3 lipidation in wild-type (WT) and RIG-I knock-out (KO) mouse embryonic fibroblasts transfected with 2 µg polyI:C (C) or infected with 200 HA U/mL SeV (D). LC3 lipidation was analyzed as in (A). Each experiment was repeated three or more times and representative data are shown. FIGURE 4 | Functional RIG-I is required for polyI:C and Sendai virus (SeV)-mediated autophagy activation. (A) Huh7 human hepatoma cells and Huh7-derived Huh7.5 cells with defective RIG-I activity were transfected with 2 µg polyI:C. LC3 lipidation was analyzed by immunoblotting using anti-LC3 and anti-β-actin antibodies. (B) Huh7 and Huh7.5 cells were infected with influenza A PR8 (Flu-PR8) (multiplicity of infection = 1) as indicated. LC3 lipidation was analyzed as in (A). LC3 lipidation in wild-type (WT) and RIG-I knock-out (KO) mouse embryonic fibroblasts transfected with 2 µg polyI:C (C) or infected with 200 HA U/mL SeV (D). LC3 lipidation was analyzed as in (A). Each experiment was repeated three or more times and representative data are shown. Beclin-1 Undergoes K63-Linked Polyubiquitination Upon Rig-I Activation WT cells but not in the TRAF6 knock-out (KO) MEFs (Figure 6D). As TRAF6 is an E3-ubiquitin ligase, and K63-linked polyubiquitination of Beclin-1 modulates its function in autophagy, we hypothesized that the interaction between Beclin- 1 and TRAF6 might lead to K63-linked polyubiquitination of Beclin-1. Indeed, the ubiquitination of Beclin-1 was increased by the ectopic expression of RIG-IN and further increased by the treatment with CQ, as determined by IP of the overexpressed Beclin-1 and immunoblotting using an anti-ubiquitin antibody (Figures 8A,B). MAVS-TRAF6 Signaling Axis Is Required for RIG-I-Mediated Signaling Immunoblotting using the K63-linked ubiquitin-specific antibody showed that the K63-ubiquitination of Beclin-1 and its interaction with VPS34 were also increased by RIG-IN expression and CQ treatment (Figure 8C). PolyI:C transfection also increased the ubiquitination of Beclin-1 within 30 min (Figure 8D). Frontiers in Immunology | www.frontiersin.org TRAF6 Associates With Beclin-1 Upon RIG-I Activation Induction of Autophagy by RIG I FIGURE 5 | Mitochondrial antiviral signaling protein (MAVS) is required for the induction of autophagy. (A) HEK293T cells were transfected with MAVS and harvested after 0, 6, and 12 h. LC3 lipidation was analyzed by immunoblotting using an anti-LC3 antibody. (B) The wild-type (WT) and MAVS knockout (KO) mouse embryonic fibroblasts (MEFs) were transfected with 2 µg polyI:C for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting as in (A). (C) The WT and MAVS KO MEFs were transfected with 10 µg polyI:C for 12 h. The cells were observed by transmission electron microscopy. The bottom panels show enlarged view of the boxed regions in the top panels. The bottom graph show that means of autophagic vacuoles in a cell. The data are presented as mean ± SE. (D) The WT and MAVS KO MEFs were infected with 200 HA U/mL SeV and treated with cycloheximide (CHX, 100 ng/mL) for 0, 6, or 12 h. The level of p62 was analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. FIGURE 5 | Mitochondrial antiviral signaling protein (MAVS) is required for the induction of autophagy. (A) HEK293T cells were transfected with MAVS and harvested after 0, 6, and 12 h. LC3 lipidation was analyzed by immunoblotting using an anti-LC3 antibody. (B) The wild-type (WT) and MAVS knockout (KO) mouse embryonic fibroblasts (MEFs) were transfected with 2 µg polyI:C for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting as in (A). (C) The WT and MAVS KO MEFs were transfected with 10 µg polyI:C for 12 h. The cells were observed by transmission electron microscopy. The bottom panels show enlarged view of the boxed regions in the top panels. The bottom graph show that means of autophagic vacuoles in a cell. The data are presented as mean ± SE. (D) The WT and MAVS KO MEFs were infected with 200 HA U/mL SeV and treated with cycloheximide (CHX, 100 ng/mL) for 0, 6, or 12 h. The level of p62 was analyzed by immunoblotting. Each experiment was repeated three or more times and representative data are shown. Beclin-1 undergoes K63-linked polyubiquitination upon RIG-I activation in a TRAF6-dependent manner to facilitate autophagy flux. antigen presentation and (2) secretion of immune mediators (27). TRAF6 Associates With Beclin-1 Upon RIG-I Activation TRAF6 interacts with Beclin-1 to activate TLR4-mediated autophagy. Thus, we explored the possible interaction between TRAF6 and Beclin-1 upon RIG-I activation. The interaction between overexpressed Beclin-1 and endogenous TRAF6 was detected by the co-immunoprecipitation (co-IP) assay. The interaction increased significantly by the ectopic expression of RIG-IN (Figure 7A). The transfection of polyI:C into V5- Beclin-1-expressing cells, followed by co-IP experiment showed an increase in the interaction of Beclin-1 with the autophagy initiation complex components, including VPS34, ATG14, and Ambra-1, indicating that Beclin-1 autophagy initiation complex formation is triggered by RIG-I activation (Figure 7B). Furthermore, the co-IP assay showed an association between endogenous Beclin-1 and TRAF6 upon transfection of polyI:C. This assay also demonstrated that VPS34 interacted with Beclin- 1 upon polyI:C transfection (Figure 7C). These results suggest that Beclin-1 associates with VPS34 and TRAF6 to facilitate autophagy upon RIG-I activation. Thus, we examined whether Beclin-1 migrated to mitochondria upon RIG-I activation to interact with TRAF6, which can recruit mitochondrial MAVS. As shown in Figure 7D, the translocation of Beclin-1 to mitochondria was detected in the cells overexpressing RIG-IN, indicating that Beclin-1 can be recruited to mitochondria upon activation of RIG-I signaling. Increased localization of Beclin-1 in the mitochondria upon RIG-IN expression or SeV infection was also confirmed by co-localization of Beclin-1 with mitotracker using confocal microscopy (Figures 7E,F). To further confirm the K63-linked polyubiquitination of Beclin-1 upon RIG-I activation, we used a K63-only ubiquitin mutant where all lysines, except K63, were mutated to arginine and thereby restricting the polyubiquitination to K63. The PolyI:C transfection of HEK293T cells ectopically expressing the HA-tagged K63-only ubiquitin mutant with Beclin-1 showed increased K63-linked polyubiquitination of Beclin-1 (Figure 8E). In addition, the ubiquitination of endogenous Beclin-1 upon polyI:C transfection was observed by IP and immunoblotting (Figure 8F). To determine whether TRAF6 was required for RIG-I-mediated Beclin-1 ubiquitination, the WT and TRAF6 KO MEFs were analyzed. As shown in Figure 8G, there was no significant change in the Beclin-1 ubiquitination level in the TRAF6 KO MEFs upon SeV infection, whereas, increased ubiquitination was detected in the WT MEFs. Collectively, these results show that September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 8 Lee et al. Lee et al. Induction of Autophagy by RIG-I ee et al. TRAF6 Associates With Beclin-1 Upon RIG-I Activation Autophagy is triggered by infection with a diverse range of viruses suggesting there is crosstalk between the innate immune recognition of viral infection and the autophagy pathway (27). Recent studies have revealed that the innate immune sensors, including TLRs and cGAS, can trigger the autophagic process (16, 17). Regarding RLRs, a deficiency of autophagy augments RIG-I-mediated type-I interferon and some autophagic proteins, such as the ATG5-ATG12 complex, that inhibit the RIG-I signaling pathway. This suggests a DISCUSSION In addition to its basal roles in maintaining cellular homeostasis, autophagy has been implicated in immunity. Autophagy affects the (1) innate and adaptive immune systems by directly eliminating pathogens, controlling inflammation, and facilitating September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 9 Lee et al. Induction of Autophagy by RIG-I FIGURE 6 | Tumor necrosis factor receptor-associated factor (TRAF)6 is required for the induction of autophagy. The WT and TRAF6 KO MEFs were transfected with 2 µg polyI:C (A) or infected with 200 HA U/mL SeV (B) for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting using an anti-LC3 antibody. (C) The TRAF6 WT and KO MEFs were transfected with polyI:C (2 µg/well) or RIG-IN (100 ng/well). Eighteen hours after transfection, LC3-puncta was visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody. The number of puncta was counted and analyzed using the image J software. The right panel shows the mean number of LC3 puncta in a cell. Data presented as mean ± standard error of the mean. (D) The WT and TRAF6 KO MEFs were infected with 200 HA U/mL SeV and treated with CHX (100 ng/mL) for 0, 6, or 12 h, then subjected to immunoblotting using an anti-p62 antibody. Each experiment was repeated three or more times and representative data are shown. FIGURE 6 | Tumor necrosis factor receptor-associated factor (TRAF)6 is required for the induction of autophagy. The WT and TRAF6 KO MEFs were transfected with 2 µg polyI:C (A) or infected with 200 HA U/mL SeV (B) for 0, 4, or 8 h. LC3 lipidation was analyzed by immunoblotting using an anti-LC3 antibody. (C) The TRAF6 WT and KO MEFs were transfected with polyI:C (2 µg/well) or RIG-IN (100 ng/well). Eighteen hours after transfection, LC3-puncta was visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody. The number of puncta was counted and analyzed using the image J software. The right panel shows the mean number of LC3 puncta in a cell. Data presented as mean ± standard error of the mean. (D) The WT and TRAF6 KO MEFs were infected with 200 HA U/mL SeV and treated with CHX (100 ng/mL) for 0, 6, or 12 h, then subjected to immunoblotting using an anti-p62 antibody. Each experiment was repeated three or more times and representative data are shown. Frontiers in Immunology | www.frontiersin.org DISCUSSION Eighteen hours after transfection, the cells were stained with Mitotracker and anti-V5 antibody as described in the Materials and Methods. Fixed dishes were observed by confocal microscopy. The right panel shows the intensity of Mitotracker (Red) and Beclin-1 (Green) along the white line of the left panel images. (F) HEK293A cells were transfected with V5-Beclin-1. Sixteen hours after transfection, the cells were mock-infected or infected with SeV for 4h. The localization of Beclin-1 was analyzed by confocal microscopy as in (E). Each experiment was repeated three or more times and representative data are shown. FIGURE 7 | RIG-I activation increases the interaction between tumor necrosis factor receptor-associated factor (TRAF)6 and Beclin-1. (A) HEK293T cells were transfected with Flag-Beclin-1 (Flag-BECN1) and pEBG (GST, -) or pEBG-RIG-IN (RIG-IN, +). Thirty-six hours after transfection, the cell lysates were subjected to co-immunoprecipitation (co-IP) using a M2 anti-Flag antibody-coated resin. The sepharose resin (Sep) served as a negative control. Whole cell lysates (WCL) and samples from co-IP were analyzed by immunoblotting using the indicated antibodies. (B) HEK293T cells were transfected with V5-Beclin-1. Twelve hours after transfection, polyI:C was transfected into the cells at different time points and incubated for the indicated hours. The cell lysates were subjected to co-IP using anti-V5 antibody, followed by immunoblotting using the indicated antibodies. (C) HEK293T cells were transfected with polyI:C and harvested at 0, 0.5, 1, 2, 4, and 6 h. The cell lysates were subjected to co-IP with an anti-Beclin-1 (BECN1) antibody and analyzed by immunoblotting using the indicated antibodies. (D) HEK293T cells were transfected with Flag-BECN1, pEBG (GST), or pEBG-RIG-IN and incubated for 24 h. The mitochondrial and cytoplasmic fractions were separated as described in the Materials and Methods and subjected to immunoblotting using the indicated antibodies. (E) HEK293A cells were transfected with GST control vector or GST-RIG-IN together with V5-Beclin-1. Eighteen hours after transfection, the cells were stained with Mitotracker and anti-V5 antibody as described in the Materials and Methods. Fixed dishes were observed by confocal microscopy. The right panel shows the intensity of Mitotracker (Red) and Beclin-1 (Green) along the white line of the left panel images. (F) HEK293A cells were transfected with V5-Beclin-1. Sixteen hours after transfection, the cells were mock-infected or infected with SeV for 4h. The localization of Beclin-1 was analyzed by confocal microscopy as in (E). Each experiment was repeated three or more times and representative data are shown. DISCUSSION of a synthetic RNA (polyI:C), SeV infection, or forced expression of a constitutively active form of RIG-I, clearly indicates the importance of RIG-I signaling. Moreover, the RIG-I ligands failed to induce autophagy in cells defective in RIG-I signaling. The main effect of RIG-I-mediated antiviral signaling is the production of type-I interferon, which can facilitate autophagy (31, 32). Surprisingly, activating RIG-I increased autophagy in type-I interferon signaling-defective cells, indicating that RIG- I signaling-mediated autophagy is independent from type-I interferon-mediated autophagy. negative regulatory role of autophagy in the RIG-I-mediated signaling pathway (21, 27–29). Recent studies have showed that polyI:C transfection activates autophagy and MAVS maintains mitochondrial homeostasis via autophagy (30). However, it is not clear whether RIG-I activation by the recognition of a pathogen- associated molecular pattern regulates the autophagic process directly. y The results of the present study provide evidence that RIG- I triggers autophagic flux upon recognition of its ligands. The induction of autophagy following the intracellular introduction September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 10 Lee et al. Lee et al. Induction of Autophagy by RIG-I FIGURE 7 | RIG-I activation increases the interaction between tumor necrosis factor receptor-associated factor (TRAF)6 and Beclin-1. (A) HEK293T cells were transfected with Flag-Beclin-1 (Flag-BECN1) and pEBG (GST, -) or pEBG-RIG-IN (RIG-IN, +). Thirty-six hours after transfection, the cell lysates were subjected to co-immunoprecipitation (co-IP) using a M2 anti-Flag antibody-coated resin. The sepharose resin (Sep) served as a negative control. Whole cell lysates (WCL) and samples from co-IP were analyzed by immunoblotting using the indicated antibodies. (B) HEK293T cells were transfected with V5-Beclin-1. Twelve hours after transfection, polyI:C was transfected into the cells at different time points and incubated for the indicated hours. The cell lysates were subjected to co-IP using anti-V5 antibody, followed by immunoblotting using the indicated antibodies. (C) HEK293T cells were transfected with polyI:C and harvested at 0, 0.5, 1, 2, 4, and 6 h. The cell lysates were subjected to co-IP with an anti-Beclin-1 (BECN1) antibody and analyzed by immunoblotting using the indicated antibodies. (D) HEK293T cells were transfected with Flag-BECN1, pEBG (GST), or pEBG-RIG-IN and incubated for 24 h. The mitochondrial and cytoplasmic fractions were separated as described in the Materials and Methods and subjected to immunoblotting using the indicated antibodies. (E) HEK293A cells were transfected with GST control vector or GST-RIG-IN together with V5-Beclin-1. DISCUSSION A crucial role of TRAF6 during autophagy induced by the innate immune system has been shown by several studies. In macrophages, activated CD40 recruits TRAF6 and induces Beclin-1-dependent autophagy to eliminate infected Toxoplasma gondii (33, 34). In addition, TLR4 signaling requires TRAF6- mediated Beclin-1 ubiquitination to induce autophagy. This suggests that the recruitment and activation of TRAF6 by innate immune signaling may lead to the ubiquitination of Beclin-1 and formation of downstream signaling complexes, including VPS34 (17). Our study using MAVS- and TRAF6-deficient cells has proven the crucial role of the MAVS-TRAF6 signaling axis in the RIG-I-dependent pathway. We demonstrated that TRAF6 is required for RIG-I-mediated autophagy, and Beclin-1 is translocated to the mitochondria, and it interacts with TRAF6 upon RIG-I activation. These September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 11 Lee et al. Induction of Autophagy by RIG-I FIGURE 8 | RIG-I activation increases K63-linked polyubiquitination of Beclin-1. (A) HEK293T cells were transfected with Flag-Beclin-1 (Flag-BECN1) and pEBG (GST) or pEBG-RIG-IN, and incubated for 24 h. The cell lysates were subjected to immunoprecipitation (IP) using M2 anti-Flag resin and analyzed by immunoblotting using the indicated antibodies. (B) HEK293T cells were transfected with Flag-BECN1 and pEBG-RIG-IN, and treated with or without 20 µM chloroquine (CQ). The cell lysates were analyzed by IP and immunoblotting as in (A). (C) HEK293T cells were transfected with the indicated plasmids and incubated in the presence or absence of 20 µM CQ for 12 h. Lys63 (K63)-linked polyubiquitination of Beclin-1 was analyzed by IP and immunoblotting with the indicated antibodies. (D) HEK293T cells were transfected with Flag-Beclin-1. After 24 h, the cells were transfected with polyI:C and incubated for 0, 0.5, 1, 2, 4, and 6 h. The cell lysates were analyzed by IP and immunoblotting. (E) HEK293T cells were transfected with V5-Beclin-1 and HA-K63-only ubiquitin mutant plasmids. After 24 h, the cells were transfected with polyI:C and incubated for 0, 3, or 6 h. The cell lysates were analyzed by IP and immunoblotting. (F) HEK293T cells were transfected with polyI:C and incubated for 0, 2, 4, and 8 h. The cell lysates were subjected to IP using the anti-Beclin-1 (BECN1) antibody and analyzed by immunoblotting. (G) Wild-type (WT) and TRAF6 knock-out (TRAF6 KO) MEFs were transfected with Flag-BECN1. After 24 h, the cells were infected with SeV and incubated for 0, 4, or 8 h. Frontiers in Immunology | www.frontiersin.org REFERENCES 14. Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev. (2009) 227:54–65. doi: 10.1111/j.1600-065X.2008.00727.x 1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell (2004) 6:463–77. doi: 10.1016/S1534-5807(04)00099-1 1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell (2004) 6:463–77. doi: 10.1016/S1534-5807(04)00099-1 15. Xu Y, Jagannath C, Liu XD, Sharafkhaneh A, Kolodziejska KE, Eissa NT. Toll- like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity (2007) 27:135–44. doi: 10.1016/j.immuni.2007.05.022 2. Kirkegaard K, Taylor MP, Jackson WT. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol. (2004) 2:301–14. doi: 10.1038/nrmicro865 2. Kirkegaard K, Taylor MP, Jackson WT. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol. (2004) 2:301–14. doi: 10.1038/nrmicro865 16. Liang Q, Seo GJ, Choi YJ, Kwak MJ, Ge J, Rodgers MA, et al. Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses. Cell Host Microbe (2014) 15:228–38. doi: 10.1016/j.chom.2014.01.009 3. Levine B. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell (2005) 120:159–62. doi: 10.1016/j.cell.2005.01.005 3. Levine B. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell (2005) 120:159–62. doi: 10.1016/j.cell.2005.01.005 17. Shi CS, Kehrl JH. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci Signal. (2010) 3:ra42. doi: 10.1126/scisignal.2000751 4. Jo EK, Yuk JM, Shin DM, Sasakawa C. Roles of autophagy in elimination of intracellular bacterial pathogens. Front Immunol. (2013) 4:97. doi: 10.3389/fimmu.2013.00097 p gy of intracellular bacterial pathogens. Front Immunol. (2013) 4:97. doi: 10.3389/fimmu.2013.00097 g 18. Saitoh T, Akira S. Regulation of inflammasomes by autophagy. J Aller Clin Immunol. (2016) 138:28–36. doi: 10.1016/j.jaci.2016.05.009 5. Thurston TL, Ryzhakov G, Bloor S, von Muhlinen N, Randow F. The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat Immunol. (2009) 10:1215–21. doi: 10.1038/ni.1800 19. Konno H, Konno K, Barber GN. Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell (2013) 155:688–98. doi: 10.1016/j.cell.2013.09.049 6. Mostowy S, Sancho-Shimizu V, Hamon MA, Simeone R, Brosch R, Johansen T, et al. p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways. J Biol Chem. (2011) 286:26987–95. doi: 10.1074/jbc.M111.223610 20. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature (2011) 469:323–35. doi: 10.1038/nature09782 21. FUNDING This work was supported by the Basic Science Research Program grants through the National Research Foundation of Korea (NRF), which was funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1A2B4005596) and by the Medical Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017R1A5A201476). Collectively, RIG-I activation leads to Beclin-1 K63- polyubiquitination and mitochondrial translocation to induce autophagy in a MAVS-TRAF6-dependent manner. Given that autophagy can suppress RIG-I-mediated interferon production, it seems likely that RIG-I-induced autophagy serves as a negative-feedback mechanism to prevent an excessive response. It would be interesting to examine whether viral components, such as nucleic acids, proteins, or viral particles in the cytoplasm, can be targeted for the autophagic elimination process upon RIG-I activation. AUTHOR CONTRIBUTIONS to induce autophagy. In a previous study, Beclin-1 was shown to interact with MAVS to suppress RIG-I-mediated interferon production in an ATG-5-independent manner (37). Our data showing the mitochondrial translocation of Beclin-1 upon RIG-I activation suggest that this may have the dual effect of suppressing the RIG-I interaction with MAVS and inducing autophagy to negatively regulate RIG-I signaling. to induce autophagy. In a previous study, Beclin-1 was shown to interact with MAVS to suppress RIG-I-mediated interferon production in an ATG-5-independent manner (37). Our data showing the mitochondrial translocation of Beclin-1 upon RIG-I activation suggest that this may have the dual effect of suppressing the RIG-I interaction with MAVS and inducing autophagy to negatively regulate RIG-I signaling. N-RL, JB, N-JL, C-MY, J-YC, and HK performed the experiments. N-RL, K-SI, JKL, JS analyzed the data. N-RL, K-SI, N-HC, and JJ designed the study and wrote the manuscript. ACKNOWLEDGMENTS We would like to thank professor Yoon-Jae Song (Gachon University, Seoul) for providing TRAF6 KO MEFs. DISCUSSION Induction of Autophagy by RIG-I DISCUSSION The cell lysates were subjected to IP and immunoblotting. Each experiment was repeated three or more times and representative data are shown. FIGURE 8 | RIG-I activation increases K63-linked polyubiquitination of Beclin-1. (A) HEK293T cells were transfected with Flag-Beclin-1 (Flag-BECN1) and pEBG (GST) or pEBG-RIG-IN, and incubated for 24 h. The cell lysates were subjected to immunoprecipitation (IP) using M2 anti-Flag resin and analyzed by immunoblotting using the indicated antibodies. (B) HEK293T cells were transfected with Flag-BECN1 and pEBG-RIG-IN, and treated with or without 20 µM chloroquine (CQ). The cell lysates were analyzed by IP and immunoblotting as in (A). (C) HEK293T cells were transfected with the indicated plasmids and incubated in the presence or absence of 20 µM CQ for 12 h. Lys63 (K63)-linked polyubiquitination of Beclin-1 was analyzed by IP and immunoblotting with the indicated antibodies. (D) HEK293T cells were transfected with Flag-Beclin-1. After 24 h, the cells were transfected with polyI:C and incubated for 0, 0.5, 1, 2, 4, and 6 h. The cell lysates were analyzed by IP and immunoblotting. (E) HEK293T cells were transfected with V5-Beclin-1 and HA-K63-only ubiquitin mutant plasmids. After 24 h, the cells were transfected with polyI:C and incubated for 0, 3, or 6 h. The cell lysates were analyzed by IP and immunoblotting. (F) HEK293T cells were transfected with polyI:C and incubated for 0, 2, 4, and 8 h. The cell lysates were subjected to IP using the anti-Beclin-1 (BECN1) antibody and analyzed by immunoblotting. (G) Wild-type (WT) and TRAF6 knock-out (TRAF6 KO) MEFs were transfected with Flag-BECN1. After 24 h, the cells were infected with SeV and incubated for 0, 4, or 8 h. The cell lysates were subjected to IP and immunoblotting. Each experiment was repeated three or more times and representative data are shown. data suggest that RIG-I may share TRAF6-dependent downstream signaling with TLR4 signaling to promote autophagy. Notably, Beclin-1 interacts simultaneously with VPS34 and TRAF6 upon polyI:C transfection, suggesting the possible role of TRAF6 in the formation of an active Beclin-1 complex. Several recent studies have showed that the K63-linked polyubiquitination of Beclin-1 is crucial for autophagy activation and can be targeted by cellular proteins to modulate autophagy (17, 35, 36). Our data suggest that the TRAF6-mediated K63-polyubiquitination of Beclin-1 upon RIG-I activation may activate the Beclin1-VPS34 complex September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 12 Lee et al. REFERENCES Tal MC, Sasai M, Lee HK, Yordy B, Shadel GS, Iwasaki A. Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling. Proc Natl Acad Sci USA. (2009) 106:2770–5. doi: 10.1073/pnas.0807694106 7. von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein A, Bloor S, Rutherford TJ, et al. LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Molecul Cell (2012) 48:329–42. doi: 10.1016/j.molcel.2012.08.024 22. Gack MU, Shin YC, Joo CH, Urano T, Liang C, Sun L, et al. TRIM25 RING- finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature (2007) 446:916–20. doi: 10.1038/nature05732 8. Wild P, Farhan H, McEwan DG, Wagner S, Rogov VV, Brady NR, et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science (2011) 333:228–33. doi: 10.1126/science.1205405 23. Ko Y, Choi JH, Ha NY, Kim IS, Cho NH, Choi MS. Active escape of Orientia tsutsugamushi from cellular autophagy. Infect Immun. (2013) 81:552–9. doi: 10.1128/IAI.00861-12 9. Deretic V. Autophagy as an innate immunity paradigm: expanding the scope and repertoire of pattern recognition receptors. Curr Opin Immunol. (2012) 24:21–31. doi: 10.1016/j.coi.2011.10.006 24. Yang Y, He S, Wang Q, Li F, Kwak MJ, Chen S, et al. Autophagic UVRAG promotes UV-induced photolesion repair by activation of the CRL4(DDB2) E3 ligase. Molecul Cell (2016) 62:507–19. doi: 10.1016/j.molcel.2016.04.014 10. Thompson MR, Kaminski JJ, Kurt-Jones EA, Fitzgerald KA. Pattern recognition receptors and the innate immune response to viral infection. Viruses (2011) 3:920–40. doi: 10.3390/v3060920 25. Kim N, Yoo HS, Ju YJ, Oh MS, Lee KT, Inn KS, et al. Synthetic 3’,4’-Dihydroxyflavone exerts anti-neuroinflammatory effects in BV2 microglia and a mouse model. Biomol Ther. (2018) 26:210–7. doi: 10.4062/biomolther.2018.008 11. Chen Q, Sun L, Chen ZJ. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat Immunol. (2016) 17:1142–9. doi: 10.1038/ni.3558 26. Choi MS, Heo J, Yi CM, Ban J, Lee NJ, Lee NR, et al. A novel p38 mitogen activated protein kinase (MAPK) specific inhibitor suppresses respiratory syncytial virus and influenza a virus replication by inhibiting virus-induced p38 MAPK activation. Biochem Biophys Res Commun. (2016) 477:311–6. doi: 10.1016/j.bbrc.2016.06.111 12. Dempsey A, Bowie AG. Innate immune recognition of DNA: a recent history. Virology (2015) 479-80:146–52. doi: 10.1016/j.virol.2015.03.013 13. Elinav E, Strowig T, Henao-Mejia J, Flavell RA. Regulation of the antimicrobial response by NLR proteins. Immunity (2011) 34:665–79. REFERENCES doi: 10.1016/j.immuni.2011.05.007 September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 13 Induction of Autophagy by RIG-I Lee et al. 27. Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol. (2013) 13:722–37. doi: 10.1038/nri3532 34. Subauste CS, Andrade RM, Wessendarp M. CD40-TRAF6 and autophagy- dependent anti-microbial activity in macrophages. Autophagy (2007) 3:245–8. doi: 10.4161/auto.3717 28. O’Sullivan TE, Geary CD, Weizman OE, Geiger TL, Rapp M, Dorn GW II, et al. Atg5 is essential for the development and survival of innate lymphocytes. Cell Rep. (2016) 15:1910–9. doi: 10.1016/j.celrep.2016.0 4.082 35. Xia P, Wang S, Du Y, Zhao Z, Shi L, Sun L, et al. WASH inhibits autophagy through suppression of Beclin 1 ubiquitination. EMBO J. (2013) 32:2685–96. doi: 10.1038/emboj.2013.189 36. Xu D, Shan B, Sun H, Xiao J, Zhu K, Xie X, et al. USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1. Genes Dev. (2016) 30:1718–30. doi: 10.1101/gad.285122.116 29. Jounai N, Takeshita F, Kobiyama K, Sawano A, Miyawaki A, Xin KQ, et al. The Atg5 Atg12 conjugate associates with innate antiviral immune responses. Proc Natl Acad Sci USA. (2007) 104:14050–5. doi: 10.1073/pnas.07040 14104 37. Jin S, Tian S, Chen Y, Zhang C, Xie W, Xia X, et al. USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1. EMBO J. (2016) 35:866–80. doi: 10.15252/embj.201593596 30. Sun X, Sun L, Zhao Y, Li Y, Lin W, Chen D, et al. MAVS maintains mitochondrial homeostasis via autophagy. Cell Discov. (2016) 2:16024. doi: 10.1038/celldisc.2016.24 31. Schmeisser H, Fey SB, Horowitz J, Fischer ER, Balinsky CA, Miyake K, et al. Type I interferons induce autophagy in certain human cancer cell lines. Autophagy (2013) 9:683–96. doi: 10.4161/auto.23921 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 32. Dong G, You M, Fan H, Ding L, Sun L, Hou Y. STS-1 promotes IFN-alpha induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells. Eur J Immunol. (2015) 45:2377–88. doi: 10.1002/eji.2014 45349 Copyright © 2018 Lee, Ban, Lee, Yi, Choi, Kim, Lee, Seong, Cho, Jung and Inn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Immunology | www.frontiersin.org September 2018 | Volume 9 | Article 2096 REFERENCES 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. 33. Andrade RM, Wessendarp M, Gubbels MJ, Striepen B, Subauste CS. CD40 induces macrophage anti-Toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J Clin Invest. (2006) 116:2366–77. doi: 10.1172/JCI28796 September 2018 | Volume 9 | Article 2096 Frontiers in Immunology | www.frontiersin.org 14
https://openalex.org/W4312809792
https://periodicals.karazin.ua/humanenviron/article/download/18231/16616
Ukrainian
null
IT Technology for Establishing Borders of Reserved Territories in the Conditions of Land Reform in Ukraine
Lûdina ì dovkìllâ. Problemi neoekologìï
2,021
cc-by
5,568
ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 DOI: https://doi.org/10.26565/1992-4224-2021-36-09 УДК (UDC): 502.4 ___________________________________________________________________________ © Максименко Н. В., Пересадько В. А., Сінна О. І., Клєщ А. А., Баскакова Л. В., 2021 This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0. Як цитувати: Максименко Н. В., Пересадько В. А., Сінна О. І., Клєщ А. А., Баскакова Л. В. IT-технологія встановлення меж заповідних територій в умовах земельної реформи в Україні. Людина та довкілля. Про- блеми неоекології. 2021. Вип. 36. С. 111-122. https://doi.org/10.26565/1992-4224-2021-36-09 In cites: Maksymenko, N. V., Peresadko, V. A., Sinna, O. I., Klieshch, A. A. & Baskakova, L.V. (2021). IT tech- nology of establishing borders of reserved territories in the conditions of land reform in Ukraine. Man and Environ- ment. Issues of Neoecology, (36), 111-122. https://doi.org/10.26565/1992-4224-2021-36-09 DOI: https://doi.org/10.26565/1992-4224-2021-36-09 УДК (UDC): 502.4 Н. В. МАКСИМЕНКО1, д-р географ. наук, проф., завідувачка кафедри екологічного моніторингу та заповідної справи e-mail: maksymenko@karazin.ua ORCID ID: https://orcid.org/0000-0002-7921-9990 В. А. ПЕРЕСАДЬКО1, д-р географ. наук, проф., декан факультету геології, географії, рекреації і туризму e-mail: vilinaperesadko@gmail.com ORCID ID: https://orcid.org/0000-0002-2439-2788 О. І. СІННА1, канд. географ. наук, доцент кафедри фізичної географії та картографії e-mail: o.sinna@physgeo.com ORCID ID: https://orcid.org/0000-0002-7693-7348 А. А. КЛЄЩ1, канд. географ. наук, доцент кафедри екологічного моніторингу та заповідної справи e-mail: klieshch@karazin.ua ORCID ID: https://orcid.org/0000-0003-1379-1043 Л. В. БАСКАКОВА1, доцент кафедри екології та менеджменту довкілля e-mail: baskakova@karazin.ua 1Харківський національний університет імені В. Н. Каразіна майдан Свободи, 4, м. Харків, Україна 61022 IT-ТЕХНОЛОГІЯ ВСТАНОВЛЕННЯ МЕЖ ЗАПОВІДНИХ ТЕРИТОРІЙ В УМОВАХ ЗЕМЕЛЬНОЇ РЕФОРМИ В УКРАЇНІ Вступ Відповідно до системи землеустрою України, що регламентована Земельним кодексом України та законом України «Про землеустрій», плани землеустрою розроб- ляються на окремі адміністративні одиниці чи їх частини. З точки зору Проектування природоохоронних територій доцільним є використання планів землеустрою мініма- льної адміністративної одиниці — сільської ради. Особливо важливо це на етапі відве- дення земель, що відбувається при пого- дженні з сільськими головами та землевла- сниками. Тому території природних парків доцільно розглядати в межах кожної сільсь- кої ради окремо. ГІС-технологій, що дозволяють порівнюва- ти та співставляти космічні знімки та старі плани землеустрою і проводити уточнення, визначати площі кожної ділянки, створюва- ти нові плани землеустрою території. Окремо тема невпорядкованості пи- тань землеустрою торкнулася природно- заповідного фонду (ПЗФ) України. Наразі, більшість територій та об’єктів ПЗФ не мають необхідної земплевпорядної докуме- нтації й не винесені у Державний земель- ний кадастр. Відповідно до частини 4 статті 7 Закону України «Про природно- заповідний фонд України», межі територій та об’єктів природно-заповідного фонду встановлюються в натурі відповідно до законодавства. До встановлення меж тери- торій та об’єктів природно-заповідного фонду в натурі їх межі визначаються відпо- відно до їх Проектів створення територій. При виникненні спірних ситуацій щодо місцеположення певних земельних ділянок відносно територій ПЗФ, відсутність меж ПЗФ у державному кадастрі потребує залу- чення науковців у досудових та судових розслідуваннях – з метою адекватної для картографічної візуалізації меж земельних ділянок і меж ПЗФ відного один одного. Хоча для конкретних заповідних територій таке дослідження має виконуватися окремо, однак може бути запропоновано та апробо- вано єдиний алгоритм дій для досягнення результату. Землеустрій заповідних територій має забезпечити диференційований режим охо- рони, відтворення та використання природ- них комплексів шляхом виділення чотирьох функціональних зон: заповідної зони, зони регульованої рекреації, зони стаціонарної рекреації та господарської зони. Актуалізація землевпорядних схем – одна з найсуттєвіших проблем, що стоїть перед структурними підрозділами Держав- ного комітету України із земельних ресур- сів на всіх адміністративних рівнях. За ін- формацією «Харківського інституту земле- устрою» переважна більшість схем землев- порядкування адміністративних одиниць (крім населених пунктів) Харківської обла- сті була складена ще в 80х роках ХХ століт- тя, тому потребують повного поновлення. Поновлення схем землеустрою можна виконувати в польових умовах з проведен- ням топографічного знімання території і уточнення типу землекористування кожно- го наділу. Також уточнення схем землеуст- рою можна проводити в камеральних умо- вах з використанням космічних знімків. Сучасні знімки середньої та високої роз- дільної здатності дають досить повну та оперативну інформацію про сучасні межі земельних ділянок, типи землекористуван- ня в їх межах. ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 Існування лише паперової схеми контуру у Проекті створення національного природного парку не дозволяє однозначно трактувати межі об’єкту. Існування лише паперової схеми контуру у Проекті створення національного природного парку не дозволяє однозначно трактувати межі об’єкту. Мета. Розробка технології встановлення меж заповідних територій. Методи. Картографічні з використанням геоінформаційної системи ArcGIS Результати. Базовим картографічним документом для здійснення дослідження стала сканована карта масштабу 1:50 000 із відображенням меж НПП «Гомільшанські ліси» із Проекту створення національного природного парку (НПП). Основною проблемою була відсутність на карті елементів її математичної основи. ІТ-технологія геоприв’язки сканованої карти меж парку в середовищі ГІС включає наступні дії: прив’язка аркушів топографічної карти масштабу 1:50000 у середовищі геоінформаційної системи ArcGIS, збереження відповідних геоприв’язаних, трансформованих копій зображення; візуалізація цифрових геоданих із геометрі- єю та координатами точок меж лісових ділянок (кварталів); завантаження сканованої карти меж НПП із Про- екту створення без географічної прив’язки та виконання процедури послідовного додавання контрольних точок прив’язки; трансформація зображення та збереження результату у форматі geotiff. Після цього створено полігональний векторний шар формату *shp і оцифровано територію парку згідно Проекту НПП. Результатом дослідження стала карта меж НПП «Гомільшанські ліси» у форматі geotiff, географічно прив’язана в середо- вищі ArcGIS, а також векторний шар території НПП, укладений за нею. Висновки. Використання розробленої технології дозволяє визначити розташування будь-яких зе- мельних ділянок по відношенню до національного природного парку, знаючи їх координати або підван- тажуючи у ГІС-Проект Публічну кадастрову карту України. КЛЮЧОВІ СЛОВА: топографічна карта, геопривязка, візуалізація, векторний шар, націо- нальний природний парк, Гомільшанські ліси Як цитувати: Максименко Н. В., Пересадько В. А., Сінна О. І., Клєщ А. А., Баскакова Л. В. IT-технологія встановлення меж заповідних територій в умовах земельної реформи в Україні. Людина та довкілля. Про- блеми неоекології. 2021. Вип. 36. С. 111-122. https://doi.org/10.26565/1992-4224-2021-36-09 In cites: Maksymenko, N. V., Peresadko, V. A., Sinna, O. I., Klieshch, A. A. & Baskakova, L.V. (2021). IT tech- nology of establishing borders of reserved territories in the conditions of land reform in Ukraine. Man and Environ- ment. Issues of Neoecology, (36), 111-122. https://doi.org/10.26565/1992-4224-2021-36-09 ~ 111 ~ Результати дослідження з’являється на Публічній кадастровій карті [10] значно пізніше, ніж території приватної забудови. В усіх законодавчих актах України в галузі земельних відносин зазначаються правила, норми і процедура відведення зе- мель [1-4, 6, 7], у тому числі і земель приро- дно-заповідного фонду (ПЗФ) України, до якого наряду з заповідниками, заказниками, пам’ятками природи входять національні природні та регіональні ландшафтні парки [5]. Але в силу ряду обставин законодавчого, технічного і ментального характеру – межі об’єктів ПЗФ часто порушуються і виника- ють колізії, які потребують втручання судо- вої системи. уд Найскладніша ситуація склалась із об’єктами природно-заповідного фонду. Формально порядок відведення земельних ділянок під об’єкти природно-заповідного фонду визначається Земельним Кодексом України (2001 рік). При цьому повноважен- ня сільських, селищних, міських, районних і обласних рад у даному питанні регулюється Законом України «Про місцеве самовряду- вання в Україні» [11]. Під час підготовки планувальних документів на Проект ство- рення, обов'язково враховуються вимоги законів України «Про основи містобудуван- ня» [12], «Про планування і забудову тери- торій» [13], а також вимоги містобудівних норм і правил. Крім того, при створенні при- родно-заповідної території враховуються й інші законодавчі акти: кодекси України – Лісовий (2006 рік), Водний (1995 рік), зако- ни України – «Про туризм» (1995 рік), «Про охорону культурної спадщини» (2000 рік), а також ряд відповідних міжнародних конвен- цій – про біорізноманіття, Рамсарська, Берн- ська, про всесвітню спадщину тощо. Так, до причин законодавчого і техні- чного характеру відносять неврегульованість законодавчої бази та її невідповідність євро- пейським правовим нормам – нечіткість процедури встановлення меж ділянок різно- го призначення [9], недосконалість кадрової підготовки інженерів-землевпорядників чи/та геодезистів [8], неврегульованість під- ходів до процедури встановлення меж діля- нок між геодезистами і землевпорядниками, неоднозначність у виборі систем координат (СК), оскільки традиційно в землевпорядку- ванні застосовувались СК 1963 р., тоді як геодезисти при проведенні топографо- геодезичних робіт зі встановлення меж ви- користовують більш сучасну, більш точну і адаптовану до загальносвітової системи – УСК 2000 р. І третя причина, яка частково випливає з неврегульованості законодавчої бази, а частково з особливостей менталітету, а саме: а) коли посадовець чи наближена до владних структур особа не зважає на закон- но встановлені межі тих чи інших територій; б) коли процедура встановлення меж одних територій настільки затягується, що Таким чином, реалізація земельної ре- форми в Україні потребує здійснення захо- дів, спрямованих насамперед на підвищення правової обізнаності та організованості зем- лекористувачів, а також на виправлення не- доліків при встановленні меж ділянок. Вступ Актуалізацію планів землеу- строю доцільно проводити з використанням Враховуючи те, що на сьогодні в Ук- раїні не існує офіційно затвердженої Мето- дики державної експертизи для визначення меж територій, що знаходяться під об’єктами природно-заповідного фонду України, а також визначення відповідних конфліктів земле- та природокористування, метою роботи э розробка алгоритму гео- прив’язки сканованих карт меж об’єктів ПЗФ в середовищі ГІС і його апробація на прикладі НПП «Гомільшанські ліси», а та- кож подальшого їх використання у спірних ситуаціях. ~ 112 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 Методика дослідження і використані матеріали Дослідження виконано на програм- ному забезпеченні, а саме: ліцензовані та вільні геоінформаційні системи ArcGIS (ESRI) із модулями ArcCatalog, ArcMap, ArcGlobe, ArcScene. • лист-відповідь від Державного підпри- ємства «Харківська державна лісовпорядна експедиція» №104 від 20.02.2020 р. із коор- динатами точок меж лісових ділянок (квар- талів) за визначеним переліком; Для апробації розробленого алгори- тму на прикладі НПП «Гомільшанські ліси» використано: • топографічні карти масштабу 1:50000, надані Прокуратурі військовою картографі- чною частиною; • матеріали Проекту створення націона- льного природного парку «Гомільшанські ліси», у тому числі – картографічні додатки у сканованому вигляді; • технічна документація, зокрема Проек- ти землеустрою щодо відведення земельних ділянок та обмінні файли Державного земе- льного кадастру. Результати дослідження Частково держава почала процес упо- рядкування меж ділянок, помилки геометрії яких викликані невідповідністю систем ко- ординат, що використовувалися у різні роки різними службами. На разі, виконується ~ 113 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 виправлення на публічній кадастровій карті викликане неспівпаданням меж ділянок, встановлених за СК 63 і УСК 2000. І хоча це порівняно невеликі території (неспівпадання до 6 м), але може викликати значні пробле- ми в населення України. Безумовно, така ситуація вимагає негайного реагування для врегулювання спорів з приводу перетину, «накладання» меж приватних ділянок між сусідніми територіями. Інша справа з влас- никами ділянок, які свідомо порушили межі ПЗФ. Проти таких осіб порушуються кримі- нальні впровадження і виникає нагальна потреба в доказовій базі. Науковці факульте- ту геології, географії, рекреації і туризму та навчально-наукового інституту екології Ха- рківського національного університету імені В.Н. Каразіна у порядку волонтерської до- помоги виконують замовлення для обласних прокуратур України щодо встановлення меж ділянок ПЗФ і приватних ділянок, межі яких збігаються з межами заповідних територій. землеустрою території: підготовчий, базовий та завершальний [14]. На підготовчому етапі виконується комплекс робіт спрямованих на збирання інформації про природні умови та ресурси, історико - культурні об’єкти, соціально- економічний розвиток території парку та регіону його розташування. На даному етапі наноситься межа НПП з урахуванням при- родних особливостей та господарського ви- користання території, формуються основні підходи до зонування території та прово- диться попереднє зонування. На даному етапі створюються карти розташування те- риторії та ділянок НПП в масштабі 1:100 000, сучасного використання території в масштабі 1:50 000 – 1:25 000 та карта функ- ціонального зонування території в масштабі 1:50 000 – 1:25 000. Останні дві карти ство- рюються на основі планів землеустрою. Са- ме на цих планах відображається сучасне використання територій в господарській діяльності. Карта функціонального зонуван- ня території також створюється на основі плану землеустрою, адже принципове зна- чення при проведенні зонування території має рівень антропогенного навантаження та інтенсивність господарської діяльності в минулому та на момент створення природ- ного парку. Вибір території дослідження зумовле- но запитом Прокуратури Харківської області до Харківського національного університету імені В.Н. Каразіна, за яким шляхом комі- сійного дослідження пропонувалося з’ясу- вати, чи входять певні земельні ділянки, передані у приватну власність, до складу НПП «Гомільшанські ліси». Слід зазначити, що відповідно до пункту Г частини 4 статті 84 ЗК України, до земель державної власно- сті, які не можуть передаватись у приватну власність, належать землі під об'єктами при- родно-заповідного фонду. Результати дослідження При цьому землі, які перебували у державній власності на момент створення об’єкту ПЗФ та знаходяться в його межах згідно Проекту створення, тобто під об’єктом ПЗФ, заборо- нено передавати у приватну власність. Не зважаючи на це, є не поодинокі випадки, коли на території об’єкту ПЗФ здійснювала- ся сільськогосподарська діяльність, «вирос- тали» приватні будівлі, при чому на ділян- ках, для яких оформлено кадастрову доку- ментацію. Основною причиною цього вва- жаємо незавершеність законодавчої проце- дури створення об’єкту ПЗФ із передачею землі та внесенням відповідних даних у Державний земельний кадастр. Для віднов- лення громадської справедливості досить часто виникає потреба встановлення меж об’єкту ПЗФ, ґрунтуючись іноді лише на паперових картосхемах, що містяться у про- екті створення, та їх співставлення з актуа- льною кадастровою ситуацією та незаконно виділеними земельними ділянками. Оскільки на теперішній час немає єдиної методики такої роботи, авторами розроблено і апробо- вано на прикладі національного природного парку «Гомільшанські ліси» (рис.1) алго- ритм встановлення меж парку, визначення координат поворотних точок і співвіднесен- ня незаконно приватизованих ділянок з ме- жами парку. Існуючі межі парку, що відо- бражаються на матеріалах аерофотозйомки показані на рис.2. Базовим картографічним документом для здійснення дослідження стала сканована карта масштабу 1:50 000 із відображенням меж НПП «Гомільшанські ліси» із Проекту створення. Основним проблемним моментом є те, що дана карта не містить елементів математичної основи (кілометрової сітки, підписів координат), тому географічна прив’язка даного зображення у ГІС могла бути здійснена лише шляхом візуального визначення контрольних точок прив’язки за елементами картографічного зображення об’єктів території, що можуть бути чітко ідентифіковані на карті Проекту створення парку та інших офіційних картографічних матеріалів, що містять відомі координати та джерела походження. Зокрема, серед елеме- нтів географічної основи карти меж НПП у Проекті створення можна зазначити елемен- ти гідрографії, автошляхи, межі лісових ква- рталів тощо. При цьому, територія охоплен- ня карти включає як безпосередньо терито- рію у межах НПП, так і прилеглі ділянки, що збільшує можливості більш точної прив’язки зображення. р Алгоритм геоприв’язки сканованої ка- рти меж парку в середовищі ГІС включав наступні дії: Національний природний парк «Гомі- льшанські ліси» створено Указом Президен- та України від 06.09.2004 №1047/2004 на території Зміївського та Первомайського районів Харківської області. Площу НПП «Гомільшанські ліси» установлено в розмірі 14314,8 га, у тому числі 3377,3 га земель, які мають бути надані йому у постійне користу- вання та 10937,5 га земель, що включаються до його складу без вилучення у землекорис- тувачів. Результати дослідження На базовому етапі виконуються осно- вні польові та камеральні роботи. Прово- диться функціональне зонування території, поділ її на квартали, виділення, філіали, об- ходи, таксація та оцінка лісів, земель, визна- чається рекреаційний потенціал парку. Уто- чнюється попередня карта функціонального зонування території в масштабі 1:50 000 – 1:25 000. У результаті виконання низки дослі- джень даної тематики нами розроблено ал- горитм визначення меж територій природно- заповідного фонду за їх Проектами ство- рення та співставлення з межами земельних ділянок, що передані в приватну власність, але при цьому можуть знаходитись у межах ПЗФ. Одним із останніх був запит на прове- дення такого дослідження відносно НПП «Гомільшанські ліси», що знаходиться в Харківській області України. На завершальному етапі здійснюється підготовка Проекту організації території НПП з відповідними планово-картогра- фічними матеріалами. Доопрацьований Про- ект організації території НПП схвалюється науково-технічною нарадою НПП, погоджу- ється землекористувачами та землевласни- ками. На етапі погодження з землекористу- вачами та землевласниками плани землеуст- рою є вкрай необхідними. Нанесені на план землеустрою межі парку та межі функціона- льних зон надаються землевласникам на підпис. Саме за цими планами потім відбу- вається відведення земель під НПП та вилу- чення земель у землевласників і передачу їх в постійне користування парку згідно з по- Відповідно до Положення «Про Про- ект організації території НПП, охорони, від- творення та рекреаційного використання його природних комплексів і об'єктів», за- твердженого Міністерством охорони навко- лишнього природного середовища від 06.07.2005р. проектування територій НПП здійснюється в три етапи на основі планів ~ 114 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 дослідною установою «Український науко- во-дослідний інститут екологічних проблем» відповідно до статті 52 Закону України «Про природно-заповідний фонд України» розро- блено Проект створення цього національно- го природного парку, яким визначено тери- торію парку, а також його функціональне зонування. З урахуванням того, що до тепе- рішнього часу землевпорядна документація щодо встановлення меж вказаного об’єкту ПЗФ не розроблена та не затверджена, межі в натурі не встановлені, то межі НПП у дос- лідженні визначались відповідно до Проекту створення. ложеннями Закону України «Про природно- заовідний фонд». Таким чином, кожен етап базується на використанні планів землеустрою території Проектування НПП. В дійсності ж, при створенні об’єкту ПЗФ, після підписання Указу Президента України, відведення земель в натурі часто не здійснювалось і землевпорядна документа- ція щодо встановлення меж об’єкту ПЗФ не розроблялась та не затверджувалась. Результати дослідження Перед виданням Указу Науково- 1) прив’язка аркушів М-37-85-А (Таранівка) та М-37-85-Б (Лиман) топогра- фічної карти масштабу 1:50 000, надані вій- ськовою картографічною частиною у сере- довищі геоінформаційної системи ArcGIS, збереження відповідних геоприв’язаних, трансформованих копій зображення; 2) візуалізація цифрових геоданих, наданих ДП «Харківська державна лісовпо- рядна експедиція» із геометрією та коорди- натами точок меж лісових ділянок (кварта ~ 115 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 Рис. 1 – Розтащування дослідної території Fig. 1 – Location of the research area Рис. 1 – Розтащування дослідної території Fig. 1 – Location of the research area лів), які знаходяться в межах території, відо- браженої на карті Проекту створення НПП «Гомільшанські ліси» (з межами території парку та прилеглими ділянками місцевості); ти та векторних даних щодо розміщення та конфігурації лісових кварталів 4) трансформація зображення та збереження результату після внесення всіх опорних точок прив’язки. 4) трансформація зображення та збереження результату після внесення всіх опорних точок прив’язки. 3) завантаження сканованої карти меж НПП із Проекту створення без геогра- фічної прив’язки та виконання процедури послідовного додавання контрольних точок прив’язки, позначаючи точку відомого об’єкту на негеоприв’язаній карті та скеро- вуючи, де ця точка має знаходитися згідно географічно прив’язаних топографічної кар- За результатами виконання відповід- ного алгоритму маємо карту меж НПП «Го- мільшанські ліси» у форматі geotiff, геогра- фічно прив’язану в середовищі ArcGIS у прямокутній системі координат Pulkovo 1942, зона 7N. Слід зазначити, що масштаб вихідної карти 1:50 000, а також застарілість ~ 116 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 Рис. 2 – Межі НПП «Гомільшанські ліси» на матеріалах аерофотозйомки Fig. 2 – Borders of the Gomilshansky Forests National Park on aerial photography materials Рис. 2 – Межі НПП «Гомільшанські ліси» на матеріалах аерофотозйомки Fig. 2 – Borders of the Gomilshansky Forests National Park on aerial photography materia 1) у середовищі ГІС створено полігона- льний шейп-файл території парку та на ос- нові геоприв’язаної растрової карти з Проек- ту створення парку викреслено межі НПП; 1) у середовищі ГІС створено полігона- льний шейп-файл території парку та на ос- нові геоприв’язаної растрової карти з Проек- ту створення парку викреслено межі НПП; географічної основи, на якій вона укладена, яка являє собою плани землеустрою, орієн- товно укладені в 80-х роках ХХ століття, дозволяє говорити про певну неточність географічної прив’язки. Так, згідно таблиці опорних точок прив’язки загальна середньо- квадратична похибка складає близько 60 метрів. Результати дослідження Тому при векторізації меж НПП у створеному файлі *shp було враховано та відкореговано окремі місцеположення по диференціації реальних географічних об’єктів на місцевості, а також по більш точ- ним та актуальним, ніж вихідна основа, да- ним про межі лісових кварталів. Отже, нара- зі можна стверджувати, що точність відо- браження меж НПП «Гомільшанські ліси», отримана авторами у процесі дослідження, задовольняє його первинним вимогам. у р р у р 2) застосовано інструмент створення ок- ремого точкового шару вершин полігону; 3) застосовано інструмент визначення координат точок у межах шару у прямокут- ній системі координат Pulkovo 1942, зона 7N (координати поворотних точок представлено окремими файлами, що додаються в елект- ронному вигляді) (рис.3). Слід зазначити, що використання Pulkovo 1942 певним чином не відповідає сучасним вимогам, однак використання саме цієї системи координат у процесі роботи визначається необхідним на початкових етапах роботи. Це зумовлено тим, що пер- винні матеріали, на яких створювалися карти у Проектах створення об’єктів ПЗФ, а також топографічні основи, що є офіційним карто Для визначення координат поворотних точок меж території НПП «Гомільшанські ліси» виконано наступні дії: ~ 117 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 Рис. 3 – Фрагмент базової карти НПП Гомільшанські ліси з поворотними точками прив’язки меж Fig. 3 – Fragment of the basic map of the Gomilshansky Forests National Park with turning points Рис. 3 – Фрагмент базової карти НПП Гомільшанські ліси з поворотними точками прив’язки меж Fig. 3 – Fragment of the basic map of the Gomilshansky Forests National Park with turning points Рис. 3 – Фрагмент базової карти НПП Гомільшанські ліси з поворотними точками прив’язки меж Рис. 3 – Фрагмент базової карти НПП Гомільшанські ліси з поворотними точками прив’язки меж Fig. 3 – Fragment of the basic map of the Gomilshansky Forests National Park with turning point прив’язки меж Fig. 3 – Fragment of the basic map of the Gomilshansky Forests National Park with turning points р Fig. 3 – Fragment of the basic map of the Gomilshansky Forests National Park with turning рні дані меж можуть бути трансформовані у середовищі ГІС в інші системи координат, у тому числі – в місцеві, за умови наявності їх базових параметрів. графічним джерелом, у більшості випадків укладені саме у цій системі координат, тож технічно більш коректним є використання саме вихідних параметрів при прив’язці гео- зображення. У подальшому отримані векто- Ви Висновки Не зважаючи на відсутність нормати- вно врегульованих дій стосовно створених об’єктів ПЗФ, а саме: не здійснення відве- дення земель в натурі, не розроблення і не затвердження землевпорядної документації щодо встановлення меж об’єкту ПЗФ, ціл- ком можливо із застосуванням запропонова- ного алгоритму встановлювати межі запо- відних територій згідно Проектів створення. Це може бути попереднім, менш точним, але більш швидким етапом винесення меж на довідкові й оглядові сервіси, на Публічну кадастрову карту України, що дозволить у тому числі реалізувати концептуальні ідеї земельної реформи в Україні, а також більш критично та оперативно слідкувати за ста- ном землекористування у межах заповідних територій. Звичайно, наступним етапом обов’язково має бути реалізована повноцін- на, передбачена законодавством процедура створення Проектів землеустрою та передачі землі тим заповідним територіям, що мають статус юридичної особи. Отримані результати стосовно конкре- тного об’єкту природно-заповідного фонду – національного природного парку «Гомоль- шанські ліси» безпосередньо демонструють земельні ділянки у межах НПП, що були ~ 118 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 незаконно надані й для яких можуть бути здійснені процедури вилучення у нових вла- сників. Для загального ознайомлення з ситу- ацією можна підвантажити довідково шар Публічної кадастрової карти в якості WMF- сервісу до Проекту ArcMap, а в подальшому використати офіційно отримані з Державно- го земельного кадастру обмінні файли із поворотними координатами точок спірних земельних ділянок. координати їх поворотних точок слід додати в Проект ArсMap, на їх основі створити по- лігональні шари земельних ділянок. Співста- влення в єдиному програмному середовищі ArcGIS геоприв’язаного зображення вихід- ної карти меж НПП «Гомільшанські ліси», векторізованого шару меж НПП та відповід- них полігонів земельної ділянки дозволяє чітко відповісти на запитання, де по відно- шенню до парку розташована конкретна земельна ділянка, і гарантовано надавати для приватизації лише ті ділянки, що знаходять- ся поза межами національного парку. Для встановлення розташування вже наданих або таких, що планується надати, земельних ділянок по відношенню до НПП, Конфлікт інтересів Автори заявляють, що конфлікту інтересів щодо публікації цього рукопису немає. Крім того, автори повністю дотримувались етичних норм, включаючи плагіат, фальсифіка- цію даних та подвійну публікацію. Список використаної літератури Рекомендована до друку 20.12.2021 IT TECHNOLOGY FOR ESTABLISHING BORDERS OF RESERVED TERRITORIE IN THE CONDITIONS OF LAND REFORM IN UKRAINE The existence of only a paper outline scheme in the National Nature Park Project does not allow to unambiguously interpret the boundaries of the object. Список використаної літератури 1. Закон України Про Державний земельний кадастр. URL: https://zakon.rada.gov.ua/law р р р р p g 2. Закон України Про землеустрій. URL: https://zakon.rada.gov.ua/laws/show/858-15 р р у р p g 3. Закон України Про наукову і науково-технічну експертизу. URL: https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Закон України Про національну інфраструктуру геопросторових даних. URL: https://zakon.rada.gov.ua/laws/show/554-20 3. Закон України Про наукову і науково-технічну експертизу. URL: https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 3. Закон України Про наукову і науково-технічну експертизу. URL: https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Закон України Про національну інфраструктуру геопросторових даних. URL: https://zakon.rada.gov.ua/laws/show/554-20 5. Закон України Про природно-заповідний фонд України. 2017. URL: https://zakon.rada.gov.ua/laws/show/2456-12/ed20170903 6 Закон України Про топографо геодезичну і картографічну діяльність 2021 URL: https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Закон України Про національну інфраструктуру геопросторових даних. URL: https://zakon.rada.gov.ua/laws/show/554-20 5 Закон України Про природно-заповідний фонд України 2017 URL: 4. Закон України Про національну інфраструктуру геопросторових даних. URL: https://zakon.rada.gov.ua/laws/show/554-20 p g 6. Закон України Про топографо-геодезичну і картографічну діяльність. 2021 URL: https://zakon.rada.gov.ua/laws/show/353-14 p g 7. Земельний Кодекс України. URL: https://zakon.rada.gov.ua/laws/show/2768-14 8. Мартин А.Г., Аврамчук Б.О. Регулювання землеустрою у Європейському Союзі: напрями адаптації для України. Землеустрій, кадастр і моніторинг земель. 2018. № 1. С. 4-13. http://dx.doi.org/10.31548/zemleustriy2018.01.001 9. Новаковський Л. Сучасні проблеми регулювання земельних відносин в Україні. Землевпорядний віс- ник. 2013. № 6. С. 2-6. 11. Закон України. Про місцеве самоврядування в Україні. (2021). URL: https://zakon.rada.gov.ua/laws/show/280/97-%D0%B2%D1%80#Text 13. Закон України Про регулювання містобудівної діяльності територій. 2011. URL: https://zakon.rada.gov.ua/laws/show/1699-14 p g 14. Положення про Проект організації території національного природного парку, охорони, відтворення та рекреаційного використання його природних комплексів і об'єктів. 2014. URL: https://zakon.rada.gov.ua/laws/show/z0831-05#Text p g 14. Положення про Проект організації території національного природного парку, охорони, відтворення та рекреаційного використання його природних комплексів і об'єктів. 2014. URL: https://zakon.rada.gov.ua/laws/show/z0831-05#Text Стаття надійшла до редакції 29.11.2021 Рекомендована до друку 20.12.2021 Стаття надійшла до редакції 29.11.2021 Рекомендована до друку 20.12.2021 ~ 119 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 N. V. MAKSYMENKO1, DSc (Geography), Prof., Head of the Department of Environmental Monitoring and Protected Area e-mail: maksymenko@karazin.ua ORCID ID: https://orcid.org/0000-0002-7921-9990 V. A. PERESADKO1, DSc (Geography), Prof., Dean of the Faculty of Geology, Geography, Recreation and Tourism e-mail: vilinaperesadko@gmail.com ORCID ID: https://orcid.org/0000-0002-2439-2788 O. I. SINNA1, Ph.D. (Geography), Associate Professor of the Department of Physical Geography and Cartography e-mail: o.sinna@physgeo.com ORCID ID: https://orcid.org/0000-0002-7693-7348 A. A. KLIESHCH1, Ph.D. (Geography), Associate Professor of the Department of Environmental Monitoring and Protected Area e-mail: klieshch@karazin.ua ORCID ID: https://orcid.org/0000-0003-1379-1043 L. V. BASKAKOVA1, Associate Professor of the Department of Ecology and Environmental Management e-mail: baskakova@karazin.ua 1V. N. Karazin Kharkiv National University, Svobody sq. 4, 61022, Kharkiv, Ukraine IT TECHNOLOGY FOR ESTABLISHING BORDERS OF RESERVED TERRITORIES IN THE CONDITIONS OF LAND REFORM IN UKRAINE N. V. MAKSYMENKO1, DSc (Geography), Prof., Head of the Department of Environmental Monitoring and Protected Area e-mail: maksymenko@karazin.ua ORCID ID: https://orcid.org/0000-0002-7921-9990 V. A. PERESADKO1, DSc (Geography), Prof., Dean of the Faculty of Geology, Geography, Recreation and Tourism e-mail: vilinaperesadko@gmail.com ORCID ID: https://orcid.org/0000-0002-2439-2788 O. I. SINNA1, Ph.D. (Geography), Associate Professor of the Department of Physical Geography and Cartography e-mail: o.sinna@physgeo.com ORCID ID: https://orcid.org/0000-0002-7693-7348 A. A. KLIESHCH1, Ph.D. (Geography), Associate Professor of the Department of Environmental Monitoring and Protected Area e-mail: klieshch@karazin.ua ORCID ID: https://orcid.org/0000-0003-1379-1043 L. V. BASKAKOVA1, Associate Professor of the Department of Ecology and Environmental Management e-mail: baskakova@karazin.ua 1V. N. Karazin Kharkiv National University, Svobody sq. 4, 61022, Kharkiv, Ukraine IT TECHNOLOGY FOR ESTABLISHING BORDERS OF RESERVED TERRITORIES IN THE CONDITIONS OF LAND REFORM IN UKRAINE The existence of only a paper outline scheme in the National Nature Park Project does not allow to unambiguously interpret the boundaries of the object. Purpose. Development of technique for establishing the boundaries of protected areas. Results. The basic cartographic document for the study was a scanned map at a scale of 1:50 000 showing the boundaries of the Gomilshansky Forests National Park from the National Nature Park (NNP) Project. The main problem was the lack of elements of its mathematical basis on the map. The IT technology of geo-linking the scanned map of the park boundaries in the GIS environment includes the following actions: linking topographic map sheets at a scale of 1: 50,000 in the ArcGIS geographic information system, saving the relevant geo-linked, transformed copies of the image; visualization of digital geodata with geometry and coordinates of points of boundaries of forest areas (quarters); download the scanned map of the boundaries of the NNP from the Project of creation without geographical reference and perform the procedure of sequential addition of control points of reference; image transformation and saving the result in geotiff format. After that, a polygonal vector layer of * shp format was created and the park territory was digitized according to the NNP Project. The result of the research was a map of the boundaries of the Gomilshansky Forests National Park in geotiff format, geographically linked in the ArcGIS environment, as well as a vector layer of the National Park's territory based on it. y Conclusions. The use of the developed technology allows to determine the location of any land plots in relation to the national nature park, knowing their coordinates or uploading the Public Cadastral Map of Ukraine to the GIS project. p p j KEY WORDS: topographic map, geolocation, visualization, vector layer, national nature park, Gomilshansky forests KEY WORDS: topographic map, geolocation, visualization, vector layer, national nature park, Gomilshansky forests References 1. Law of Ukraine About the State Land Cadastre. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/3613-17 2. Law of Ukraine About Land Management. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/858-15 3. Law of Ukraine About Scientific and Scientific-Technical Examination. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Law of Ukraine on the national infrastructure of geospatial data. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/554-20 References 1. Law of Ukraine About the State Land Cadastre. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/3613-17 2. Law of Ukraine About Land Management. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/858-15 3. Law of Ukraine About Scientific and Scientific-Technical Examination. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Law of Ukraine on the national infrastructure of geospatial data. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/554-20 4. References 1. Law of Ukraine About the State Land Cadastre. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/3613-17 2. Law of Ukraine About Land Management. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/858-15 3. Law of Ukraine About Scientific and Scientific-Technical Examination. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/51/95-%D0%B2%D1%80 4. Law of Ukraine on the national infrastructure of geospatial data. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/554-20 The existence of only a paper outline scheme in the National Nature Park Project does not allow to unambiguously interpret the boundaries of the object. Law of Ukraine on the national infrastructure of geospatial data. (2020). Retrieved from https://zakon.rada.gov.ua/laws/show/554-20 ~ 120 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 5. Law of Ukraine about the nature reserve fund of Ukraine. (2017). Retrieved from https://zakon.rada.gov.ua/laws/show/2456-12/ed20170903 p g 6. Law of Ukraine about topographic, geodetic and cartographic activities. (22021).. Retrieved from https://zakon.rada.gov.ua/laws/show/353-14 p g 6. Law of Ukraine about topographic, geodetic and cartographic activities. https://zakon.rada.gov.ua/laws/show/353-14 p g 7. Land Code of Ukraine. Retrieved from :https://map.land.gov.ua/ p p g 8. Martin, A.G., & Avramchuk , B.O. (2018). Land management in the European Union: areas of adaptation for Ukraine. Land management, cadastre and land monitoring, (1), 4–13. http://dx.doi.org/10.31548/zemleustriy2018.01.001 p g y 9. Novakovsky, L. (2013). Modern problems of land relations in Ukraine. Land M 9. Novakovsky, L. (2013). Modern problems of land relations in Ukraine. Land Management Bulletin, (6), 2-6. 10. Law of Ukraine Public cadastral map.. Retrieved from https://map.land.gov.ua/ 10. Law of Ukraine Public cadastral map.. Retrieved from https://map.land.gov.ua/ 11. Law of Ukraine About local self-government in Ukraine. Retrieved from 11. Law of Ukraine About local self-government in Ukraine. Retrieved from https://zakon.rada.gov.ua/laws/show/280/97#Text Law of Ukraine On the basics of urban planning. (20 12. Law of Ukraine On the basics of urban planning. (2020). URL: https://zakon.rada.gov.ua/laws/show/2780-12 p g p g 13. Law of Ukraine About planning and building of territories. (2021). Retrieved from https://zakon.rada.gov.ua/laws/show/1699-14 p g 14. Regulations on the Project of organization of the territory of the national natural park, protection, reproduction and recreational use of its natural complexes and objects. (2014). Retrieved from https://zakon.rada.gov.ua/laws/show/z0831-05#Text 14. Regulations on the Project of organization of the territory of the national natural park, protection, reproduction and recreational use of its natural complexes and objects. (2014). Retrieved from https://zakon.rada.gov.ua/laws/show/z0831-05#Text The article was received by the editors 29.11.2021 The article was received by the editors 29.11.2021 The article is recommended for printing 20.12.2021 The article is recommended for printing 20.12.2021 Существование только бумажной схемы контура в Проекте создания национального природного парка не позволяет однозначно трактовать границы объекта. Существование только бумажной схемы контура в Проекте создания национального природного парка не позволяет однозначно трактовать границы объекта. Цель. Разработка технологии установления границ заповедных территорий. М К ф ф й Методы. Картографические с использованием геоинформационной системы ArcGIS Результаты. Базовым картографическим документом для осуществления исследования стала ска- нированная карта масштаба 1:50 000 с отображением границ НПП «Гомольшанские леса» из Проекта создания национального природного парка (НПП). Основной проблемой было отсутствие на карте эле- ментов ее математической основы. ИТ-технология геопривязки сканированной карты границ парка в среде ГИС включает следующие действия: привязка листов топографической карты масштаба 1:50000 в среде геоинформационной системы ArcGIS, сохранение соответствующих геопривязанных, трансформи- рованных копий изображения; визуализация цифровых геоданных с геометрией и координатами точек ~ 121 ~ ISSN 1992-4224 Людина та довкілля. Проблеми неоекології, 2021, Випуск 36 границ лесных участков (кварталов); загрузка сканированной карты границ НПП из Проекта создания без географической привязки и выполнения процедуры последовательного добавления контрольных точек привязки; трансформация изображения и сохранение результата в формате geotiff. После этого создан полигональный векторный слой формата *shp и оцифрована территория парка согласно Проекту НПП. Результатом исследования стала карта границ НПП «Гомольшанские леса» в формате geotiff, гео- графически привязанная в среде ArcGIS, а также векторный слой территории НПП, заключенный за ней. Выводы. Использование разработанной технологии позволяет определить расположение любых земельных участков по отношению к национальному природному парку, зная их координаты или под- гружая в ГИС-проект Публичную кадастровую карту Украины. ру р у у д р у р у р КЛЮЧЕВЫЕ СЛОВА: топографическая карта, геопривязка, визуализация, векторный слой, национальный природный парк, Гомольшанские леса ру р у у д р у р у р КЛЮЧЕВЫЕ СЛОВА: топографическая карта, геопривязка, визуализация, векторный слой, национальный природный парк, Гомольшанские леса Статья поступила в редакцию 29.11.2021 у р Статья рекомендована к печати 20.12.2021 ~ 122 ~
https://openalex.org/W2972563063
https://europepmc.org/articles/pmc6734418?pdf=render
English
null
Childhood MMR vaccination and the incidence rate of measles infection: a ten year longitudinal cohort study of American children born in the 1990s
BMC pediatrics
2,019
cc-by
8,282
Geier et al. BMC Pediatrics (2019) 19:325 https://doi.org/10.1186/s12887-019-1710-5 Geier et al. BMC Pediatrics (2019) 19:325 https://doi.org/10.1186/s12887-019-1710-5 Open Access © 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. Childhood MMR vaccination and the incidence rate of measles infection: a ten year longitudinal cohort study of American children born in the 1990s David A. Geier1,2, Janet K. Kern1,2* and Mark R. Geier1,2 David A. Geier1,2, Janet K. Kern1,2* and Mark R. Geier1,2 Abstract Background: Measles (rubeola) is a highly contagious disease with significant morbidity/mortality. Measles-Mumps- Rubella (MMR) is a live-attenuated vaccine used in the United States (US) since the early 1970s to prevent measles infection. This retrospective longitudinal cohort study examined childhood MMR vaccination effectiveness (VE) on preventing diagnosed measles cases. Methods: The Independent Healthcare Research Database (IHRD) is composed of non-identifiable linked eligibility and claim healthcare records prospectively generated from the Florida Medicaid system. The SAS system was utilized to examine a cohort of 101,736 persons eligible for Florida Medicaid from 1990 to 2009 and continuously eligible with ≥10 outpatient office visits during the 120-month period following birth. There were 32,870 persons (224,492 person-years) in the cohort receiving a single dose of childhood MMR vaccine (vaccinated) and 43,538 persons (434,637 person-years) in an unvaccinated cohort (no exposures to measles-containing vaccine). The frequency of diagnosed measles (ICD-9 code: 055xxx) was examined. Cox proportional hazards models evaluated MMR vaccination and diagnosed measles over time. Results: MMR vaccinated cohort members were at significantly reduced risk of measles in the unadjusted (VE = 83.6, 95% CI = 67.2–91.8%) and adjusted (VE = 80.7, 95% CI = 61.5–83.9%) models as compared to the unvaccinated cohort. VE = 80% among younger MMR recipients (12–15 months), whereas VE = 90% among older MMR recipients (16–20 months) as compared to the unvaccinated cohort. Conclusion: Routine childhood MMR vaccination significantly reduced the incidence rate of childhood measles infections, and the VE was greater in the older recipients (16–20 months) than in the younger recipients (12–15 months). Keywords: Cohort, Measles, MMR vaccine, Pediatric, Rubeola Background occur in epidemic cycles and virtually all people acquired measles before adulthood in the US prior to the imple- mentation of the national measles vaccine program in 1963. It was described prior to the national measles vaccine program in the US, annually about 500,000 cases of measles were reported, of whom 500 persons died, 48, 000 were hospitalized, and about 1000 cases of encephal- itis with permanent brain damage were observed [2]. As described by the United States (US) Centers for Dis- ease Control and Prevention (CDC), measles (rubeola) is highly contagious (90% of exposed susceptible persons develop measles) rash illness that is transmitted by direct contact with respiratory droplets or airborne spread between person to person [1]. Measles was observed to In the US, measles vaccination was initially recom- mended for administration at 9 months in 1963, 12 months in 1965, and 15 months in 1967 [1]. During the * Correspondence: jkern@dfwair.net 1Institute of Chronic Illnesses, Inc, 14 Redgate Ct, Silver Spring, MD 20905, USA 2CoMeD, Inc, 14 Redgate Ct, Silver Spring, MD 20905, USA * Correspondence: jkern@dfwair.net 1Institute of Chronic Illnesses, Inc, 14 Redgate Ct, Silver Spring, MD 20905, USA 2CoMeD, Inc, 14 Redgate Ct, Silver Spring, MD 20905, USA Study participants Fi 1 y p p Figure 1 presents a schematic flowchart of the IHRD data examined in the present study. A cohort of 8,440, 941 persons of all ages with no changes or missing gen- ders or dates of birth and eligible at specific times for Florida Medicaid from July 1990 through June 2009 was initially evaluated in this study. Among this cohort, a total of 1,871,728 persons were eligible for Florida Medicaid from their date of birth and among those persons a total of 193,453 persons were continuously eligible for Florida Medicaid for 120 months following birth. Finally, among the cohort of 193,453 persons continuously eligible for Florida Medicaid for 120 months following birth, a sub-cohort of 101,736 persons with ≥10 outpatient office visits during the 120-month period following birth was identified. Since, the late 1980s/early 1990s, the Advisory Commit- tee on Immunization Practices (ACIP), the American Academy of Pediatrics (AAP), and American Academy of Family Practitioners (AAFP) have recommended that the first dose of MMR vaccine should be given to children aged 12 through 15 months (with administration between 6 and 12 months under special circumstances) [1]. The measles vaccine program in the US was so success- ful against measles infections that it is the largest country in the world to have ended endemic measles transmission [6]. Therefore, it would be ethically unacceptable to con- duct placebo-controlled trials to assess measles vaccine effectiveness in the US, and as a result, current epidemio- logical research on the effectiveness of MMR vaccine needs to focus on retrospective studies of populations to optimize protection by modifying immunization schedules [7]. Independent healthcare research database (IHRD) Independent healthcare research database (IHRD) The Independent Healthcare Research Database (IHRD) is composed of non-identifiable healthcare records generated from the Florida Medicaid system. The data contained within the IHRD were obtained from the Agency for Health Care Administration (AHCA) of the state of Florida and included eligibility and claim files. It is possible to link a person’s eligibility and claim records by a unique recipient identifier code. The eligibility records included detailed information for each person regarding their month and year of enrollment, gender, date of birth, and county level residency. The claims records included detailed information for each person regarding their diagnosis status using the International Vaccination status h The exposure variable examined in this study was identi- fied from the healthcare procedure codes filed on claims for each cohort member examined. The procedure codes examined, included: measles vaccination (codes: 9945, W1941, 90705), MMR vaccine (codes: 90707, W1943, 9948), measles and rubella vaccine (code: 90708), mea- sles, mumps, rubella, and varicella (MMRV) vaccine (code: 90710). Only persons receiving a single dose of MMR vaccine were included in the vaccinated cohort (codes: 90707, W1943, or 9948) and persons were con- sidered unvaccinated, if they did not receive any mea- sles-containing vaccine (codes: 9945, W1941, 90705, 90707, W1943, 9948, 90708, or 90710) during the study period examined. All persons receiving more than 1 dose of MMR vaccination or other measles-containing vac- cines were excluded from the present study. Among those vaccinated with MMR, the date of service for the first claim in chronological order with a procedure code specifying MMR vaccine was assumed to be the date of vaccine administration. Overall, it was observed that 32, 916 persons were in the MMR vaccinated cohort and 43, 538 persons were in the unvaccinated cohort. The purpose of the present retrospective longitudinal cohort study was to examine the vaccine effectiveness of childhood MMR vaccination to reduce the incidence rate of childhood measles infections in the US during the 1990s/2000s. In addition, this study examined the relationship between the age of childhood MMR vaccin- ation and its impact on the effectiveness of the vaccine. Page 2 of 11 Page 2 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Geier et al. BMC Pediatrics (2019) 19:325 Code for Disease, 9th revision (ICD-9) codes, healthcare procedure codes (medical, dental, etc.), and administered drugs using National Drug Codes (NDC). The data in the IHRD were assembled and accessed under approval by the Liberty Institutional Review Board (IRB) (Deland, FL). The SAS system for Windows, version 9.4 (Cary, NC, USA) was used to examine the IHRD. 1970s combined measles-mumps-rubella (MMR) vaccine was introduced in the US [3, 4]. The MMR vaccine used in the US since the 1980s is the M-M-R® II vaccine (Merck & Co, Inc., Whitehouse Station, NJ), and is a sterile lyophilized preparation of (1) ATTENUVAX® (Measles Virus Vaccine Live), a more attenuated line of measles virus, derived from Enders’ attenuated Edmon- ston strain and propagated in chick embryo cell culture; (2) MUMPSVAX® (Mumps Virus Vaccine Live), the Jeryl Lynn™(B level) strain of mumps virus propagated in chick embryo cell culture; and (3) MERUVAX® II (Ru- bella Virus Vaccine Live), the Wistar RA 27/3 strain of live attenuated rubella virus propagated in WI-38 human diploid lung fibroblasts [5]. Outcomes The outcome variable examined in this study was identi- fied from the ICD-9 codes filed on claims for each cohort member examined. All measles-related diagnoses (code: 055xxx) were examined, including: measles (055), Page 3 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Geier et al. BMC Pediatrics Fig. 1 A schematic flowchart of the data examined in the present study. Persons in the MMR vaccinated cohort received only 1 dose of MMR vaccine and includes only persons diagnosed with measles post-MMR vaccine administration (43 persons were excluded with measles diagnoses pre-MMR vaccine administration and 3 persons were diagnosed measles within 30 days of vaccine administration, which are most likely measles vaccine-associated adverse events) Fig. 1 A schematic flowchart of the data examined in the present study. Persons in the MMR vaccinated cohort received only 1 dose of MMR vaccine and includes only persons diagnosed with measles post-MMR vaccine administration (43 persons were excluded with measles diagnoses pre-MMR vaccine administration and 3 persons were diagnosed measles within 30 days of vaccine administration, which are most likely measles vaccine-associated adverse events) vaccination were included in the final MMR vaccinated cohort. As a result, the overall size of the MMR vaccinated cohort was reduced to 32,870 persons. post-measles encephalitis (code: 055.0), post-measles pneumonia (code: 055.1), post-measles otitis media (code: 055.2), measles with other specified complications (code: 055.7), measles keratoconjunctivitis (code: 055.71), mea- sles with other specified complications (code: 055.79), measles with unspecified complication (code: 055.8), and measles without mention of complication (code: 055.9). No information was available regarding whether measles cases were laboratory confirmed or not. Persons were considered to have measles if they had any of the measles- related diagnoses and persons were not considered to have measles if they did not have any of the measles-related diagnoses during the study period examined. Among those with a measles diagnosis, the date of service for the first claim in chronological order with a measles diagnosis was assumed to be the date of onset of measles infection. Only those persons either not diagnosed with measles or diagnosed with measles more than 30 days post-MMR Statistical analyses In all statistical analyses, the statistical package in SAS was utilized, and a two-sided p-value < 0.05 was considered statistically significant. The null hypothesis was that MMR vaccination would have no impact on the incidence rate of measles diagnoses. It was also assumed in this study that chances of exposure to wild type measles virus were equal in the vaccinated and unvaccinated cohorts. In order to evaluate the relationship between MMR vac- cination and diagnosed measles, person-years of follow-up were calculated in the vaccinated and unvaccinated co- horts examined in this study. In the unvaccinated cohort, person-years of follow-up began on the date of birth and continued until the end of eligibility (a maximum of 120 Geier et al. BMC Pediatrics (2019) 19:325 Page 4 of 11 Page 4 of 11 months after birth) or until the date of the first measles diagnosis. In the vaccinated cohort, person-years of fol- low-up began on the date of MMR vaccine administration and continued until the end of eligibility (a maximum of 120 months after birth) or until the date of the first mea- sles diagnosis. cohort contributing a total of 224,492 person-years and 43,538 persons in the unvaccinated cohort contributing a total of 434,637 person-years. The gender distribution was similar in the MMR vaccinated cohort (male/female ratio: 1.13) and unvaccinated cohort (male/female ratio = 1.14). In addition, overall mean dates of birth were similar in both the vaccinated and unvaccinated cohorts. g A regression analysis of diagnosed measles cases based on the Cox proportional hazards model was used to evalu- ate overtime in years the potential relationship between MMR vaccination and the outcome of a measles diagno- sis. Ties in the failure times were handled using the exact method. In addition, an evaluation of the potential impact of young (< 1 year-old) persons in the unvaccinated cohort being diagnosed with measles at an age prior to the earli- est age when MMR vaccine is first recommended at 12 months-old was undertaken. MMR vaccine effectiveness was examined in modeling by only counting cases of measles diagnosed at ≥12 months-old in the unvaccinated cohort and only examining persons receiving MMR vac- cine at ≥12 months-old in the vaccinated cohort. Finally, modeling was conducted to evaluate the potential impact of age of MMR vaccine administration (≥12 months, ≥ 16 months, and ≥20 months) on the effectiveness of the vaccine to prevent cases of measles. Statistical analyses All models were con- structed without adjustment for covariates (Model I) and with adjustment for the covariates of gender (categorical variable), date of birth (continuous variable), and county of residence (as a continuous variable) (Model II). Overall, vaccine effectiveness was determined as ((1 – hazard ratio) × 100). Table 2 shows the demographic characteristics of the 85 persons diagnosed with measles examined this study. Slightly more males than females were diagnosed with measles (male/female ratio = 1.15), but the ratio was consistent with those observed in the vaccinated and unvaccinated cohorts examined. The mean date of birth among persons diagnosed with measles was slightly earlier in chronological time in the unvaccinated cohort (1993) as compared to vaccinated cohort (1994). Most diagnosed cases of measles (> 90%) were without complications. Table 3 reveals the Cox proportional hazards model results examining the impact of childhood MMR vaccin- ation on the incidence rate of diagnosed measles. It was observed regardless of the age of childhood MMR vac- cination that the vaccine effectiveness was 83.6% (95% confidence interval = 67.2 to 91.8%) in the unadjusted and 80.7% (95% confidence interval = 61.5 to 90.4%) in the adjusted models. MMR vaccine effectiveness remained significant when only counting cases of mea- sles diagnosed at ≥1 year-old in the unvaccinated cohort and only examining persons receiving MMR vaccine at ≥1 year-old in the vaccinated cohort in the unadjusted (vaccine effectiveness = 70.9, 95% confidence interval = 37.8 to 86.4%) and adjusted (vaccine effectiveness = 65.4, 95% confidence interval = 25.6 to 83.9%) models. ICD-9 International Code of Disease, 9th revision, MMR Measles, mumps, rubella, std. Standard deviation a All persons examined in this study were enrolled from their date of birth for 120 consecutive months. All persons had non-changing dates of birth and gender status. All persons had ≥10 outpatient office visits b Persons received only 1 dose of MMR vaccine and includes only persons diagnosed with measles post-MMR vaccine administration (43 persons were excluded with measles diagnoses pre-MMR vaccine administration and 3 persons were diagnosed measles within 30 days of vaccine administration, which are most likely measles vaccine-associated adverse events) c d d f l Results Table 1 displays the demographic characteristics of the population of persons examined in this study. Overall, there were a total 32,870 persons in the MMR vaccinated Figure 2 is a Cox proportional hazards survival plot evaluating the incidence of measles cases diagnosed over Table 1 Demographic characteristics of the persons examined in this studya Parameter Examined MMR Vaccinated Cohortb (n = 32,870) Unvaccinated Cohortc (n = 43,538) Person-Years 224,492 434,637 Gender (%) Male 17,468 (53.14%) 23,202 (53.29%) Female 15,402 (46.86%) 20,336 (46.71%) Date of Birth mean ± std. (range) 1995 ± 2.5 (1990–1999) 1994 ± 2.5 (1990–1999) Number Diagnosed with Measles (ICD-9 Code: 055xxx) 9 76 Number Diagnosed with Measles (ICD-9 Code: 055xxx) ICD-9 International Code of Disease, 9th revision, MMR Measles, mumps, rubella, std. Standard deviation a All persons examined in this study were enrolled from their date of birth for 120 consecutive months. All persons had non-changing dates of birth and gender status. All persons had ≥10 outpatient office visits b Persons received only 1 dose of MMR vaccine and includes only persons diagnosed with measles post-MMR vaccine administration (43 persons were excluded with measles diagnoses pre-MMR vaccine administration and 3 persons were diagnosed measles within 30 days of vaccine administration, which are most likely measles vaccine-associated adverse events) c f c Persons received no doses of any measles-containing vaccine Page 5 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Table 2 Demographic summary of the persons diagnosed with measles examined in this studya Parameter Examined All Persons Diagnosed with Measles (n = 85) Vaccinated Persons Diagnosed with Measles (n = 9)c Unvaccinated Persons with Diagnosed Measles (n = 76)d Gender (%) Male 45 (53.41%) 5 (55.56%) 40 (52.63%) Female 40 (47.06%) 4 (44.44%) 36 (47.37%) Date of Birth mean ± std. (range) 1993 ± 2.1 (1990–1999) 1994 ± 1.26 (1992–1996) 1993 ± 2.2 (1990–1999) Age at Measles Diagnosis mean ± std. (range) 1.65 ± 1.69 (0.15–7.08) 1.27 ± 1.72 (0.15–5.39) 1.69 ± 1.7 (0.18–7.08) Year of Measles Diagnosis mean ± std. (range) 1995 ± 2.78 (1991–2003) 1996 ± 1.9 (1993–1999) 1994 ± 2.8 (1991–2003) Measles Diagnosis-Associated Complications No Complications 78 (91.77%) 8 (88.89%) 70 (92.11%) Complications Specifiedb 5 (5.88%) 1 (11.11%) 4 (5.26%) Unknown Complication Status 2 (2.35%) 0 (0%) 2 (2.63%) aAll persons examined in this study were enrolled from their date of birth for 120 consecutive months. Results The unvac- cinated cohort revealed the most significant increases in the number of measles cases diagnosed in the first 2 years of follow-up in the present study. This was then followed by a period of still increasing numbers of mea- sles case diagnoses from the 2nd to the 6th year of fol- low-up, but at a slower rate than the initial 2-year period of follow-up. Finally, in the last period from the 6th to 10th year of follow-up, there were relatively few measles cases diagnosed as compared to the previous follow-up periods. Results All persons had non-changing dates of birth and gender status. All persons had ≥10 outpatient office visits bThis includes persons with post-measles otitis media (n = 2), measles keratoconjunctivitis (n = 1), measles with other specified complications (n = 1), measles with unspecified complication (n = 1) cPersons received only 1 dose of MMR vaccine and includes only persons diagnosed with measles post-MMR vaccine administration (43 persons were excluded with measles diagnoses pre-MMR vaccine administration and 3 persons were diagnosed measles within 30 days of vaccine administration, which are most likely measles vaccine-associated adverse events) dPersons received no doses of measles-containing vaccine when examining the MMR vaccinated cohort at the older ages examined with receipt at ≥16 months-old or ≥ 20 months-old vaccine effectiveness was about 90% or more. the period of years persons were followed in the MMR vaccinated cohort and the unvaccinated cohort. The plot reveals that in the initial period post-MMR vaccination (< 6 months) there were a greater number of measles cases diagnosed than in the unvaccinated cohort, but in the later periods post-MMR vaccination (> 6 months) there relatively few measles cases diagnosed as compared to the initial 6-month follow-up period in the MMR vac- cinated cohort or the unvaccinated cohort. The unvac- cinated cohort revealed the most significant increases in the number of measles cases diagnosed in the first 2 years of follow-up in the present study. This was then followed by a period of still increasing numbers of mea- sles case diagnoses from the 2nd to the 6th year of fol- low-up, but at a slower rate than the initial 2-year period of follow-up. Finally, in the last period from the 6th to 10th year of follow-up, there were relatively few measles cases diagnosed as compared to the previous follow-up periods. the period of years persons were followed in the MMR vaccinated cohort and the unvaccinated cohort. The plot reveals that in the initial period post-MMR vaccination (< 6 months) there were a greater number of measles cases diagnosed than in the unvaccinated cohort, but in the later periods post-MMR vaccination (> 6 months) there relatively few measles cases diagnosed as compared to the initial 6-month follow-up period in the MMR vac- cinated cohort or the unvaccinated cohort. Discussion The results of this retrospective longitudinal cohort study of prospectively collected healthcare data provide important and compelling new epidemiological quantita- tive data regarding the vaccine effectiveness of the child- hood MMR vaccine routinely administered to American children. Further, the vaccine effectiveness of childhood MMR vaccination remained when considering covariates such as gender, date of birth, and county of residence. In considering the results observed in the present study with previous studies, the Cochrane Collaboration recently published an extensive review examining MMR vaccine effectiveness [7]. Of three MMR vaccine effect- iveness studies examined by the Cochrane Collaboration, all three were cohort studies that observed childhood MMR vaccine administration was significantly effective in preventing clinical cases of measles [8–10]. In addition, the Cochrane Collaboration review also described that vaccine effectiveness = 97% for MMR vaccine administered in US vaccine programs [11]. Table 4 reveals the impact of the age of MMR vaccine administration on vaccine effectiveness in comparison to the unvaccinated cohort. Among those persons receiving MMR vaccine at the younger ages examined with receipt at ≥12 months-old was the least effective with a vaccine effectiveness ranging from about 80 to 85%. By contrast, Page 6 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Table 3 Cox proportional hazards model results examining the relationship between MMR vaccination and diagnosed measles Model Variable Hazard Ratio (95% CI) VE (95% CI) p-value χ2 I Vaccinateda vs Unvaccinatedb (all ages) 0.164 (0.082 to 0.328) 83.6% (67.2 to 91.8%) < 0.0001 26.2 Vaccinated vs Unvaccinated (≥12 months-old)c 0.291 (0.136 to 0.622) 70.9% (37.8 to 86.4%) 0.0015 10.1 II Vaccinated vs Unvaccinated (all ages) 0.193 (0.096 to 0.385) 80.7% (61.5% to 90.4) < 0.0001 21.7 Gender (Female vs Male) 1.01 (0.659 to 1.544) 0.97 0.002 County of Residence 0.993 (0.982 to 1.004) 0.24 1.40 Date of Birth 0.999 (0.999 to 0.999) < 0.0001 30.2 Vaccinated vs Unvaccinated (≥12 months-old)c 0.346 (0.161 to 0.744) 65.4% (25.6 to 83.9%) 0.00065 7.39 Gender (Female vs Male) 0.959 (0.544 to 1.692) 0.89 0.02 County of Residence 0.997 (0.982 to 1.011) 0.65 0.21 Date of Birth 0.999 (0.999 to 0.999) < 0.0001 18.8 Italicized results are statistically significant. Discussion Model I = unadjusted, Model II = adjusted for gender, county of residence, and date of birth CI Confidence interval, VE Vaccine effectiveness aPersons received only 1 dose of MMR vaccine bPersons received no doses of measles-containing vaccine cOnly persons diagnosed with measles at ≥12 months-old were included in the unvaccinated cohort and only examining persons receiving MMR vaccine at ≥12 months-old in the vaccinated cohort, so as to ensure direct overlap in ages with the vaccinated and unvaccinated cohorts Table 3 Cox proportional hazards model results examining the relationship between MMR vaccination and diagnosed measles y bPersons received no doses of measles-containing vaccine cOnly persons diagnosed with measles at ≥12 months-old were included in the unvaccinated cohort and only examining persons receiving MMR vaccine at ≥12 months-old in the vaccinated cohort, so as to ensure direct overlap in ages with the vaccinated and unvaccinated cohorts Fig. 2 A Cox proportional hazards survival plot evaluating cases of measles diagnosed over the period of follow-up in the MMR vaccinated cohort1 (1) in comparison to the unvaccinated cohort2 (0). 1 Persons received only 1 dose of MMR vaccine. 2 Persons received no doses of measles-containing vaccine Fig. 2 A Cox proportional hazards survival plot evaluating cases of measles diagnosed over the period of follow-up in the MMR vaccinated cohort1 (1) i i h i d h 2 (0) 1 P i d l 1 d f MMR i 2 P i d d f Fig. 2 A Cox proportional hazards survival plot evaluating cases of measles diagnosed over the period of follow-up in the MMR vaccinated cohort1 (1) in comparison to the unvaccinated cohort2 (0). 1 Persons received only 1 dose of MMR vaccine. 2 Persons received no doses of measles-containing vaccine Page 7 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Geier et al. Discussion BMC Pediatrics Table 4 An evaluation of the impact of the age of MMR vaccine administration on the effectiveness of the vaccine to prevent cases of measles Model Variable Hazard Ratio (95% CI) VE (95% CI) p-value χ2 I Vaccinateda vs Unvaccinatedb (≥12 months-old) 0.147 (0.071 to 0.305) 85.3% (69.5 to 92.9%) < 0.0001 26.5 Vaccinated vs Unvaccinated (≥16 months-old) 0.100 (0.036 to 0.273) 90% (72.7 to 86.4%) < 0.0001 20.2 Vaccinated vs Unvaccinated (≥20 months-old) 0.061 (0.015 to 0.247) 93.9% (75.3 to 98.5%) < 0.0001 15.3 II Vaccinated vs Unvaccinated (≥12 months-old) 0.173 (0.083 to 0.360) 82.7% (64 to 91.7%) < 0.0001 22.1 Gender (Female vs Male) 1.032 (0.673 to 1.584) 0.89 0.02 County of Residence 0.994 (0.983 to 1.005) 0.27 1.20 Date of Birth 0.999 (0.999 to 0.999) < 0.0001 30 Vaccinated vs Unvaccinated (≥16 months-old) 0.111 (0.040 to 0.303) 88.9% (69.7 to 96%) < 0.0001 18.4 Gender (Female vs Male) 1.109 (0.718 to 1.715) 0.64 0.22 County of Residence 0.995 (0.984 to 1.007) 0.42 0.66 Date of Birth 0.999 (0.999 to 0.999) < 0.0001 28.1 Vaccinated vs Unvaccinated (≥20 months-old) 0.070 (0.017 to 0.287) 93% (71.3 to 98.3%) < 0.0001 13.7 Gender (Female vs Male) 1.027 (0.658 to 1.602) 0.91 0.01 County of Residence 0.996 (0.985 to 1.008) 0.49 0.47 Date of Birth 0.999 (0.999 to 1.000) < 0.0001 27.4 Italicized results are statistically significant. Model I = unadjusted, Model II = adjusted for gender, county of residence, and date of birth CI Confidence interval, VE Vaccine effectiveness a Persons received only 1 dose of MMR vaccine b Persons received no doses of measles containing vaccine Table 4 An evaluation of the impact of the age of MMR vaccine administration on the effectiveness of the vaccine to prevent cases of measles Italicized results are statistically significant. Model I = unadjusted, Model II = adjusted for gender, county of residence, and date of birth CI Confidence interval, VE Vaccine effectiveness a Persons received only 1 dose of MMR vaccine b Persons received no doses of measles-containing vaccine The results of the present study also revealed that vac- cine effectiveness was greatest for those children admin- istered a single dose of MMR vaccine between 16 and 20 months of age as compared to those receiving a single dose of MMR vaccine between 12 and 15 months. Discussion It is also worth considering in countries where the risk of measles infection is high and low MMR vaccine uptake, especially for the second dose of MMR vaccine, that administration of MMR vaccine at ≥15 months maybe a means to improve long-term protection against measles infection for many children. those vaccinated with MMR vaccine at younger ages had undetectable or low levels of measles antibody 5–6 years post-immunization [20]. As a consequence, there is a potential delicate balance to weigh between ensuring the best possible long-term immunological response to MMR vaccine administration in vaccine recipients ver- sus the potential of measles exposure and measles-asso- ciated disease among children. It would seem, at least in the US, in more recent years with the end of endemic measles transmission that for most children administration of MMR vaccine at ≥15 months would be more appropriate than < 15 months, although the impact maybe limited because most children will subsequently receive a second childhood dose of MMR vaccine. It is also worth considering in countries where the risk of measles infection is high and low MMR vaccine uptake, especially for the second dose of MMR vaccine, that administration of MMR vaccine at ≥15 months maybe a means to improve long-term protection against measles infection for many children. examined were generated completely separate from the current study design. The healthcare providers submit- ting claims for MMR vaccine administration and measles diagnoses were most likely not thinking about the possible relationship between MMR vaccination and measles diagnoses. The study design utilized to examine the IHRD was another important strength of the present study. All persons examined in this study were eligible for Florida Medicaid from birth for 120 months (no gaps in eligibil- ity were allowed). In addition, in order to ensure that the cohort of persons examined was actively utilizing healthcare services from the Florida Medicaid system, all persons examined in this study had to have ≥10 out- patient office visit claims submitted (that averages to at least one outpatient office visit per person per year). These requirements helped to significantly reduce possible enrollment factors or differences in healthcare- seeking behaviors among the persons examined in this study. Discussion y Another interesting aspect of the present study was that there were three measles disease diagnoses made within 30 days post-MMR vaccine (these were excluded from analyses undertaken in the present study to deter- mine MMR vaccine effectiveness). It was previously re- ported the usual incubation period of measles is 8 to 12 days [21]. All three measles diagnoses appeared to occur within the biologically appropriate time for the incuba- tion and manifestation of measles infection (one on day 11 and two on day 13), and as a result are most likely MMR vaccine-associated adverse events. It is unclear whether any previous epidemiological studies were large enough to observe this phenomenon following child- hood MMR vaccine administration, but a previous day- to-day reactogenicity study of MMR vaccine versus a placebo administered to 14 to 18 months-old in a twin study revealed apparently mild measles-associated symp- toms commenced 5 to 7 days post-MMR vaccine admin- istration and peaked on day 10 [21]. The results of the present study support that MMR vaccine-associated measles adverse events are rare with a rate of 0.91 per 10,000 recipients (95% confidence interval = 0.19 to 2.67 per 10,000 recipients) within 30 days of MMR vaccin- ation. It would be interesting in future studies to use microbiological tests to determine whether such poten- tial measles adverse events are truly vaccine-associated or the result of wild-type measles infections. Further, vaccination status was determined with preci- sion for each person because detailed information regard- ing procedure codes and dates of service for claims submitted on behalf of each person were examined. In order for a person to become a member of the vaccinated cohort, the claims records for a person revealed that only a single dose of MMR vaccine was administered. Those persons with procedure codes specifying other measles- containing vaccine(s) or multiple doses of MMR vaccine were excluded from the present analyses. Similarly, members of the unvaccinated cohort were confirmed to have no claims submitted on their behalf specifying receipt of any type of measles-containing vaccine. The outcome status was also determined with precision for each person because detailed information regarding outcomes using ICD-9 diagnosis coding and dates of ser- vice for claims submitted on behalf of each person were examined. In order for a person to be recognized as having a measles diagnosis, the initial date of service speci- fying a measles diagnosis (055xxx) was identified. Discussion Finally, the use of Cox proportional hazards survival plot modeling to evaluate cases of measles diagnosed over a period of many years in the vaccinated and unvaccinated cohorts allowed for us to draw inferences regarding the relationship between vaccination and out- comes as a function of follow-up time. It is possible that a potential limitation of this study was that the findings observed were the result of statistical chance or cofounders/unknown biases in the data. Statis- tical chance seems unlikely given that a limited number of statistical tests were performed, and most results were highly statistically significant. In addition, it was observed that the significant effects observed in unadjusted models remained significant even when adjusting for potential Discussion This type of age dependent MMR vaccine effectiveness was described in previous epidemiological studies [18]. The ACIP reported that measles vaccine is at least 95% effective for children vaccinated at ≥15 months of age, whereas a lower efficacy was observed for children vacci- nated between 12 and 14 months of age. They commen- ted that measles vaccine efficacy maybe lower because of trans-placental maternal antibody persists beyond the first birthday in some children, which, interferes with re- sponse to vaccination [19]. The biological plausibility of age dependent MMR vaccine effectiveness is supported by a previous study that revealed higher proportion of The results observed in this study regarding the vaccine effectiveness of childhood MMR to significantly reduce the incidence rate of measles cases for many years post-vaccin- ation are biologically plausible. For example, it was reported as early as 1971 that among 715 children (with no initial antibody to measles) that MMR vaccination induced a posi- tive measles antibody response in 96% of the children [12]. It was subsequently reported that among children administered a single dose of MMR vaccine at 15 months- old and evaluated for antibodies against measles at 6–7 years-old that > 90% were still positive for antibodies against measles [13]. Other studies in different populations revealed that measles antibodies may persistent more than a decade post-MMR vaccination [14, 15]. In addition, it was even revealed in long-term longitudinal cohort studies of measles vaccine recipients that persistent antibodies were observed in > 90% of the persons examined [16, 17]. Page 8 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Page 8 of 11 those vaccinated with MMR vaccine at younger ages had undetectable or low levels of measles antibody 5–6 years post-immunization [20]. As a consequence, there is a potential delicate balance to weigh between ensuring the best possible long-term immunological response to MMR vaccine administration in vaccine recipients ver- sus the potential of measles exposure and measles-asso- ciated disease among children. It would seem, at least in the US, in more recent years with the end of endemic measles transmission that for most children administration of MMR vaccine at ≥15 months would be more appropriate than < 15 months, although the impact maybe limited because most children will subsequently receive a second childhood dose of MMR vaccine. Strengths/limitations It is possible that there may be differences in the chances of wild type measles virus exposure in different geographical areas over different years. This potential phenomenon should be further examined in future studies. aPersons received only 1 dose of MMR vaccine bPersons received no doses of measles-containing vaccine An additional potential limitation of this study was that measles cases were not uniformly diagnosed during the study period from 1990 to 2009. As revealed in Table 5, it was observed that most cases of measles were diagnosed in the early 1990s period, and by the 2000s virtually no cases of measles were diagnosed, regardless of vaccination status. This phenomena most probably re- flects increasing “herd immunity” from increasing MMR vaccine coverage in the overall population. As described previously about “herd immunity” [22], the consequence is that the chance of exposure to measles throughout the study period examined significantly decreased regardless of vaccination status, and as a result, this may have reduced the vaccine effectiveness observed in the present study in comparison with previous studies examining measles vaccine effectiveness. Namely, unvaccinated persons were deriving a benefit of protection against measles infection from vaccinated persons. It would be interesting in future studies to evaluate the impact of increasing “herd immunity” on population measles disease patterns. The ACIP recommends that a second dose of MMR vac- cination should be administered during childhood [1]. It is possible that additional doses of MMR vaccination may further improve vaccine effectiveness to prevent cases of measles. It is recommended that future studies further explore the impact of additional doses of MMR vaccination on vaccine effectiveness to prevent measles cases. The ACIP recommends that a second dose of MMR vac- cination should be administered during childhood [1]. It is possible that additional doses of MMR vaccination may further improve vaccine effectiveness to prevent cases of measles. It is recommended that future studies further explore the impact of additional doses of MMR vaccination on vaccine effectiveness to prevent measles cases. Another potential limitation of the present study was that the better vaccine effectiveness associated with older age of receiving MMR vaccination might associated with missing data on the first vaccination (i.e., perhaps the first documented MMR vaccination was really a second MMR vaccination, because the first was not docu- mented). Strengths/limitations An important strength of this study was that retrospect- ive observations made in the IHRD were derived from eligibility and claims records prospectively generated as part of the routine healthcare provided for persons in the Florida Medicaid system. Therefore, the data Geier et al. BMC Pediatrics (2019) 19:325 Page 9 of 11 Table 5 A summary of diagnosed measles cases by year of diagnosis Year of Diagnosis All Measles Cases MMR Vaccinated Cohorta Diagnosed with Measles Unvaccinated Cohortb Diagnosed with Measles 1991 18 0 18 1992 14 0 14 1993 11 1 10 1994 11 2 6 1995 8 1 7 1996 8 2 6 1997 6 1 5 1998 5 1 4 1999 4 1 3 2000 2 0 2 2001 0 0 0 2002 0 0 0 2003 1 0 1 2004 0 0 0 2005 0 0 0 2006 0 0 0 2007 0 0 0 2008 0 0 0 2009 0 0 0 Total 85 9 76 aPersons received only 1 dose of MMR vaccine bPersons received no doses of measles-containing vaccine covariates such as gender, date of birth, and county of residence. The results observed this study were consistent with previous epidemiological observations on different populations and were biologically plausible. Table 5 A summary of diagnosed measles cases by year of diagnosis It is also possible that some of the persons examined in the IHRD may have had symptoms of measles that were so slight that they were not noted by their healthcare pro- viders, or healthcare providers may have misdiagnosed or misclassified vaccination status for some persons. How- ever, these potential limitations, while possible, should not have affected the results appreciably because it is uncer- tain how differential application would have occurred in the vaccinated and unvaccinated cohorts examined. Im- portantly, any misclassification with respect to diagnostic or vaccination status, would in all likelihood bias the find- ings towards the null hypothesis because persons exam- ined would have been put into the wrong vaccination and/ or diagnostic category and result in diminished statistical power to establish the accurate relationship between vaccination and outcomes. Another potential limitation of this study was that limited information was available regarding the area of residence of persons over the multiple years of this study. It was assumed that chances of exposure to wild type measles virus were equal in the vaccinated and un- vaccinated cohorts examined. AAFP: American Academy of Family Practitioners; AAP: American Academy of Pediatrics; ACIP: Advisory Committee on Immunization Practices; AHCA: Agency for Health Care Administration; CA: California; FL: Florida; ICD-9: International Code for Disease, 9th revision; IHRD: Independent Healthcare Research Database; IRB: Institutional Review Board; MMR: Measles-mumps-rubella; NDC: National Drug Codes; NJ: New Jersey; US: United States; USA: United States of America; VE: Vaccination effectiveness; WA: Washington References 1. McLean HQ, Fiebelkorn AP, Temte JL, Wallace GS. Centers for Disease Control and Prevention. Prevention of measles, rubella, congenital rubella syndrome and mumps, 2013: summary recommendations of the advisory committee on immunization practices (ACIP). MMWR Recomm Rep. 2013; 62(RR-04:1–34. 1. McLean HQ, Fiebelkorn AP, Temte JL, Wallace GS. Centers for Disease Control and Prevention. Prevention of measles, rubella, congenital rubella syndrome and mumps, 2013: summary recommendations of the advisory committee on immunization practices (ACIP). MMWR Recomm Rep. 2013; 62(RR-04:1–34. 2. Bloch AB, Orenstein WA, Stetler HC, et al. Health impact of measles vaccination in the United States. Pediatrics. 1985;76:524–32. 2. Bloch AB, Orenstein WA, Stetler HC, et al. Health impact of measles vaccination in the United States. Pediatrics. 1985;76:524–32. 3. Redd SC, Markowitz LE, Katz SL. Measles vaccine. In: Plotkin SA, Orenstein WA, editors. Vaccines. Philadelphia: WB Saunders; 1999. p. 222–66. vaccination in the United States. Pediatrics. 1985;76:524–32. 3. Redd SC, Markowitz LE, Katz SL. Measles vaccine. In: Plotkin SA, Orenstein WA, editors. Vaccines. Philadelphia: WB Saunders; 1999. p. 222–66. 3. Redd SC, Markowitz LE, Katz SL. Measles vaccine. In: Plotkin SA, Orenstein WA, editors. Vaccines. Philadelphia: WB Saunders; 1999. p. 222–66. 4. Schwarz AJ, Jackson JE, Ehrenkranz NJ, Ventura A, Schiff GM, Walters VW. Clinical evaluation of a new measles-mumps-rubella trivalent vaccine. Am J Dis Child. 1975;129(12):1408–12. 5. MERCK & CO., INC., M-M-R-II, Measles Mumps, and Rubella Virus Vaccine Live. http://www.merck.com/product/usa/pi_circulars/m/mmr_ii/mmr_ii_pi. pdf. Revised 5/2017; Accessed 18 July 2018. Overall, the results of the present study support the ongoing successful use of routine MMR vaccination as an important public health tool to reduce the incidence rate of diagnosed measles. Future studies should con- tinue to monitor how long and how robust the protect- ive effects of MMR vaccine(s) can be expected to persist. 6. Strebel PM, Henao-Restrepo AM, Hoekstra E, Olive JM, Papania MJ, Cochi SL. Global measles elimination efforts: the significance of measles elimination in the United States. J Infect Dis. 2004;189(Suppl 1):S251–7. 6. Strebel PM, Henao-Restrepo AM, Hoekstra E, Olive JM, Papania MJ, Cochi SL. Global measles elimination efforts: the significance of measles elimination in the United States. J Infect Dis. 2004;189(Suppl 1):S251–7. 7. Demicheli V, Rivetti A, Debalini MG, Di Pietrantonj C. Vaccines for measles, mumps, and rubella in children. Cochrane Database Syst Rev. 2012;15(2): CD004407. 7. Demicheli V, Rivetti A, Debalini MG, Di Pietrantonj C. Vaccines for measles, mumps, and rubella in children. Abbreviations AAFP A i AAFP: American Academy of Family Practitioners; AAP: American Academy of Pediatrics; ACIP: Advisory Committee on Immunization Practices; AHCA: Agency for Health Care Administration; CA: California; FL: Florida; ICD-9: International Code for Disease, 9th revision; IHRD: Independent Healthcare Research Database; IRB: Institutional Review Board; MMR: Measles-mumps-rubella; NDC: National Drug Codes; NJ: New Jersey; US: United States; USA: United States of America; VE: Vaccination effectiveness; WA: Washington 9. Marolla F, Baviera G, Cacciapuoti CV, Cannavavo R, Clemente A, et al. A field study on vaccine efficacy against mumps of three MMR vaccines [Efficacia verso la parotite di tre diversi vaccini a tripla componente: studio sul campo]. Riv Ital Pediatr. 1998;24(3):466–72. 9. Marolla F, Baviera G, Cacciapuoti CV, Cannavavo R, Clemente A, et al. A field study on vaccine efficacy against mumps of three MMR vaccines [Efficacia verso la parotite di tre diversi vaccini a tripla componente: studio sul campo]. Riv Ital Pediatr. 1998;24(3):466–72. 10. Ong G, Rasidah N, Wan S, Cutter J. Outbreak of measles in primary school students with high first dose MMR vaccination coverage. Singap Med J. 2007;48(7):656–61. 10. Ong G, Rasidah N, Wan S, Cutter J. Outbreak of measles in primary school students with high first dose MMR vaccination coverage. Singap Med J. 2007;48(7):656–61. 11. Orenstein WA, Bernier RH, Dondero TJ, Hinman AR, Marks JS, Bart KJ, et al. Field evaluation of vaccine efficacy. Bull World Health Org. 1985;63(6):1055–68. 11. Orenstein WA, Bernier RH, Dondero TJ, Hinman AR, Marks JS, Bart KJ, et al. Field evaluation of vaccine efficacy. Bull World Health Org. 1985;63(6):1055–68. Consent for publication Not applicable. Consent for publication Not applicable. Funding Thi This research was funded by the non-profit organizations, CoMeD, Inc. and Institute of Chronic Illnesses, Inc. Dr. Mark Geier and Mr. David Geier are directors of the non-profit Institute of Chronic Illnesses, Inc. and the non-profit CoMeD, Inc. The funding organizations did not influence the design, data collection, analyses, data interpretation, or the writing of this manuscript. 16. Kremer JR, Schneider F, Muller CP. Waning antibodies in measles and rubella vaccines-a longitudinal study. Vaccine. 2006;24(14):2594–601. 17. Kato H, Mori M, Oba M, Kawahara H, Kaneko T. Persistence and half-lives of anti-measles and anti-rubella antibodies in Japanese hospital workers: a longitudinal study. Intern Med. 2016;55(18):2587–94. 18. Orenstein WA, Markowitz L, Preblud SR, Hinman AR, Tomasi A, Bart KJ. Appropriate age for measles vaccination in the United States. Dev Biol Stand. 1986;65:13–21. Acknowledgements N 12. Stokes J Jr, Weibel RE, Villarejos VM, Arguedas JA, Buynak EB, Hilleman MR. Trivalent combined measles-mumps-rubella vaccine. Findings in clinical- laboratory studies. JAMA. 1971;218(1):57–61. 12. Stokes J Jr, Weibel RE, Villarejos VM, Arguedas JA, Buynak EB, Hilleman MR. Trivalent combined measles-mumps-rubella vaccine. Findings in clinical- laboratory studies. JAMA. 1971;218(1):57–61. Authors’ contributions DAG l d d 13. Gil Miguel A, Astasio Arbiza P, Ortega Molina P, Dominguez Rojas V, Gonzalez Lope A. Seroprevalence of antibodies against measles, rubella, mumps and varicella among school children in Madrid. An Esp Pediatr. 1999;50(5):459–62. DAG conceptualized and designed the study, carried out the initial analyses, drafted the initial manuscript, and approved the final manuscript as submitted. JKK critically reviewed and revised the manuscript, and approved the final manuscript as submitted. MRG conceptualized and designed the study, drafted the initial manuscript, and approved the final manuscript as submitted. All authors read and approved the final manuscript. 14. Seagle EE, Bednarczyk RA, Hill T, Fiebelkorn AP, Hickman CJ, Icenogle JP, Belongia EA, McLean HQ. Measles, mumps, and rubella antibody patters of persistence and rate of decline following the second dose of the MMR vaccine. Vaccine. 2018;36(6):818–26. 15. Davidkin I, Jokinen S, Broman M, Leinikki P, Peltola H. Persistence of measles, mumps, and rubella in an MMR-vaccinated cohort: a 20-year follow-up. J Infect Dis. 2008;197(7):950–6. Received: 7 September 2018 Accepted: 3 September 2019 Received: 7 September 2018 Accepted: 3 September 2019 Ethics approval and consent to participate Ethics approval and consent to participate The data in the IHRD were assembled and accessed under approval by the Liberty Institutional Review Board (IRB) (Deland, FL). The data in the IHRD were assembled and accessed under approval by the Liberty Institutional Review Board (IRB) (Deland, FL). References Cochrane Database Syst Rev. 2012;15(2): CD004407. 8. Marin M, Nguyen HQ, Langidrik JR, Edwards R, Briand K, Papania MJ, et al. Measles transmission and vaccine effectiveness during a large outbreak on a densely populated island: implications for vaccination policy. Clin Infect Dis. 2006;42(3):315–9. 8. Marin M, Nguyen HQ, Langidrik JR, Edwards R, Briand K, Papania MJ, et al. Measles transmission and vaccine effectiveness during a large outbreak on a densely populated island: implications for vaccination policy. Clin Infect Dis. 2006;42(3):315–9. Competing interests Th th d l th This retrospective cohort study of prospectively collected healthcare data from the IHRD provides new evidence con- sistent with and extending results from previous epidemio- logical studies revealing that routine childhood MMR vaccination in the US significantly reduced the incidence rate of diagnosed measles cases. Furthermore, MMR vac- cine administration at 16–20 months-old was associated with greater vaccine effectiveness relative to MMR vaccine administration at 12–15 months-old. It was also revealed that on rare occasions in the 30-day post-MMR vaccine administration period about 1 in 10,000 doses of MMR vac- cine developed MMR vaccine-associated measles adverse events. Finally, the results observed in this study help to establish that the IHRD is an important epidemiological resource to help quantitatively evaluate important public health issues. The authors declare that they have no competing interests. Received: 7 September 2018 Accepted: 3 September 2019 Strengths/limitations This would appear to be unlikely, since the ages examined for receipt of MMR vaccination were all before the second birthday, and the ACIP does not recommend administration of a second dose of MMR vaccine at such a young age [1]. A final potential limitation of the present study was that it is unknown how many cases were reported to the national notifiable disease system from Florida in this time period compared to what was observed in the A still further potential limitation of the present study was that only persons receiving a single dose of MMR vaccination were examined in the vaccinated cohort. Page 10 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Geier et al. BMC Pediatrics (2019) 19:325 Florida Medicaid system. It is possible that there may be some sort of selection bias, although it would probably not have a large impact on the observations made in this study. Ethics approval and consent to participate Availability of data and materials This database is publicly available through the US Medicaid system. This database is publicly available through the US Medicaid system. Page 11 of 11 Geier et al. BMC Pediatrics (2019) 19:325 Geier et al. BMC Pediatrics (2019) 19:325 19. Centers for Disease Control (CDC). Measles prevention. MMWR Suppl. 1989; 38(9):1–18. 20. Boulianne N, Serres GD, Ratnam S, Ward BJ, Joly JR, Duval B. Measles, mumps, and rubella antibodies in children 5-6 years after immunization: effect of vaccine type and age at vaccination. Vaccine. 1995;13(16):1611–6. 20. Boulianne N, Serres GD, Ratnam S, Ward BJ, Joly JR, Duval B. Measles, mumps, and rubella antibodies in children 5-6 years after immunization: effect of vaccine type and age at vaccination. Vaccine. 1995;13(16):1611–6. 21. Virtanen M, Petola H, Paunio M, Heinonen OP. Day-to-day reactogenicity and the healthy vaccine effect of measles-mumps-rubella vaccination. Pediatrics. 2000;106(5):E62. 21. Virtanen M, Petola H, Paunio M, Heinonen OP. Day-to-day reactogenicity and the healthy vaccine effect of measles-mumps-rubella vaccination. Pediatrics. 2000;106(5):E62. 22. Fing P, Earnes K, Heymann DL. “Herd immunity”: a rough guide. Clin Infect Dis. 2011;52(7):911–6. 22. Fing P, Earnes K, Heymann DL. “Herd immunity”: a rough guide. Clin Infect Dis. 2011;52(7):911–6. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
W2970931287.txt
https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-019-7518-2
en
Why did I stop? And why did I restart? Perspectives of women lost to follow-up in option B+ HIV care in Dar es Salaam, Tanzania
BMC public health
2,019
cc-by
8,528
Sariah et al. BMC Public Health (2019) 19:1172 https://doi.org/10.1186/s12889-019-7518-2 RESEARCH ARTICLE Open Access Why did I stop? And why did I restart? Perspectives of women lost to follow-up in option B+ HIV care in Dar es Salaam, Tanzania Adellah Sariah1* , Joan Rugemalila2, Joyce Protas3, Eric Aris4, Helen Siril5, Edith Tarimo6 and David Urassa7 Abstract Background: Despite an increased uptake of option B+ treatment among HIV- positive pregnant and breastfeeding women, retaining these women in care is still a major challenge. Previous studies have identified factors associated with loss to follow-up (LTFU) in HIV care, however, the perspectives from HIV-positive pregnant and breastfeeding women regarding their LTFU in option B+ needs further exploration. We explored reasons for LTFU and motivation to resume treatment among HIV-positive women initiated in option B+ in an Urban setting. Methods: A descriptive qualitative study was conducted at three public care and treatment clinics (CTC) (Buguruni health center, Sinza hospital, and Mbagala Rangitatu health center) in Dar es Salaam, Tanzania between February and May 2017. In-depth interviews were conducted with 30 HIV-positive pregnant and breastfeeding women who were lost to follow up in the option B+ regimen. Analysis of data followed content analysis that was performed using NVivo 10 computer-assisted qualitative data analysis software. Results: Eleven women were lost to follow-up and did not resume Option B+, while 19 had resumed treatment. The study indicated a struggle with long term disease amongst HIV-positive pregnant and breastfeeding women initiated in option B+ treatment. The reported reasons contributing to LTFU among these women appeared in three categories. The contribution of LTFU in the first category namely health-related factors included medication side effects and lack of disease symptoms. The second category highlighted the contribution of psychological factors such as loss of hope, fear of medication side effects and HIV-related stigma. The third category underscored the influence of socio-economic statuses such as financial constraints, lack of partner support, family conflicts, nondisclosure of HIV-positive status, and religious beliefs. Motivators to resume treatment after LTFU included support from health care providers and family members, a desire to protect the unborn child from HIV-infection and a need to maintain a healthy status. Conclusion: The study has highlighted the reasons for LTFU and motivation to resume treatment among women initiated in Option B+. Our results provide further evidence on the need for future interventions to focus on these factors in order to improve retention in life-long treatment. Keywords: Loss to follow up, Reasons, Option B + , Pregnant, Breastfeeding, HIV-positive women, Tanzania * Correspondence: adellahsariah@gmail.com 1 Department of Mental Health and Psychiatric Nursing, Hubert Kairuki Memorial University (HKMU), 70 Chwaku Street-Mikocheni, P.O. Box 65300, Dar es Salaam, Tanzania 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. Sariah et al. BMC Public Health (2019) 19:1172 Background The WHO sustainable development goal (SDG) number three emphasizes healthy lives and the promotion of well-being for all ages [1]. Treatment for people living with HIV including HIV-positive pregnant and breastfeeding women fall under this SDG. The use of Option A and B treatment regimens for prevention of mother to child transmission (PMTCT) was recommended by the World Health Organization (WHO) prior to the introduction of Option B+. However, with the maintenance of viral suppression, Option B+ has proved to have important advantages over both options A and B including protection against mother to child transmission (MTCT of HIV) in subsequent pregnancies, prevention against sexual transmission to discordant couples and avoiding on and off antiretroviral treatment (ART) regimen [2]. Option B+ was introduced in Tanzania in 2013 by the Ministry of Health, Community Development, Gender, Elderly and Children (MoHCDGEC) to facilitate achievement of elimination of mother to child HIV transmission (eMTCT) goals. The treatment regimen requires that all HIV-infected pregnant and breastfeeding women are initiated on ART for life as soon as they are diagnosed, regardless of CD4 count and WHO clinical staging and 6 weeks of daily nevirapine for the infant [2]. The MoHCDGEC recommends the use of a fixed drug combination of Tenofovir/ Lamivudine/ Efavirenz (TLE) taken as one tablet a day for life. All HIV-infected pregnant women are advised to take option B+ at the beginning of the antenatal clinic and ought to continue for life. So far, Option B+ has been rolled out in Dar es Salaam and all the other regions in the country. The life-long treatment has displayed remarkable results in resourceconstrained settings; dramatically increasing the number of pregnant and breastfeeding women enrolled in ART [3]. Various reasons have been shown to elucidate why women starting ART do not attend their follow-up appointments. For instance, qualitative studies from other countries have identified factors contributing to loss to follow-up (LTFU) under the Option B+ program including lack of motivation to adhere to the life-long medication after a healthy delivery, fear of drug side effects, uncertainty on their abilities to overcome challenges of life-long medication [4, 5] and denial or lack of disclosure of HIV-positive status [6]. The former studies that implemented Option B+, [7] reported increases in ART coverage among HIV-infected pregnant women [8, 9]. However, retention in care is required for optimal prevention of negative maternal treatment outcomes and LTFU [10, 11]. Protocol changes from Option A to B and to the current B+ may have modified some of the factors associated with ART adherence or made them more pronounced. For instance, studies have reported increased problems with stigma, fear of disclosure and lack of support (emanating Page 2 of 11 from the need to store and take antiretroviral (ARV) treatment more often) as well as health care system-related factors (the necessity for frequent visits to the clinic to get medication, and the limited resources and infrastructure) that pose challenges in ART implementation [6]. These problems (stigma, and fear of disclosure) continue to impede the uptake of PMTCT across sub-Saharan Africa [12]. Success in option B+ depends on the proportion of women who adhere to treatment and are retained in care, however, this is challenged by a high LTFU rate [8, 13]. Information regarding reasons for LTFU since the initiation of Option B+ in Tanzania is still scarce. Among a few available information is a report from MoHCDGEC which showed that, immediately after the introduction of Option B+, retention in care and treatment ranged from 100 to 64% on the 1st and 3rd month respectively [14]. This indicates a persistently increased rate of LTFU. We, therefore, sought to explore the reasons for LTFU among HIV-positive pregnant and breastfeeding women initiated on Option B+ in Dar es Salaam, Tanzania. Methods Design A descriptive qualitative study was carried out between February and May 2017 to explore reasons for LTFU in Option B+ among HIV-positive pregnant and breastfeeding women. Setting The study was conducted at three (1 from each of the three districts) Management and Development for Health (MDH) supported public HIV care and treatment clinics (CTC) in Dar es Salaam, Tanzania. The facilities included Buguruni health center in Ilala district; Sinza hospital in Kinondoni district; and Mbagala Rangitatu health center in Temeke district. The MDH care and treatment program was established in 2004. The program provides infrastructure, laboratory, and technical support to the five districts. Previously, the Dar es Salaam region had three districts (Temeke, Ilala, and Kinondoni). In 2016, two more districts (Ubungo and Kigamboni) were established, bringing the number of districts to five in the region. Sampling and recruitment of participants Participants and the health facilities were purposively selected. The selection of health facilities was based on high patient volume. Inclusion criteria during recruitment of study participants included HIV-positive women with 18 and above years of age who were either LTFU and did not resume, or LTFU and had resumed Option B+ treatment. Participants were defined as LTFU if they had not attended the clinic for ≥180 days since the last visit [15]. Seriously ill participants and those who could not express themselves well were excluded from our Sariah et al. BMC Public Health (2019) 19:1172 study. The CTC sites determine whether patients are transferred to another clinic, either through clinic referral or self-referral to another clinic. Self-referral here means that a patient transfers oneself to another clinic rather than being referred by the health care provider. Additionally, the CTC sites usually track patients according to the guidelines that have been set by the clinic. This tracking enables health care providers to identify patients who missed appointments and attempt to re-engage them into care or identify if they had transferred to another clinic [15]. Before the recruitment of participants and data collection, two research assistants and two nurses from each of the selected clinics received a brief training that was facilitated by the principal investigator (PI). During the training, the research assistants and nurses received a brief introduction about the study objectives, purpose and ethical issues, including the informed consent process. In addition, the research assistants were trained on how to conduct indepth interviews (IDIs), where they reviewed the data collection tool and practiced how to use it. Materials for the training included a review guide for informed consent [16], and a guide for conducting IDIs [17]. Nurses were also trained on how to identify potential participants basing on the predetermined inclusion and exclusion criteria. The nurses played an important role in identifying potential participants from the clinic files. Potential participants who met the inclusion criteria were contacted by phone. Upon identification, nurses contacted the participants, explained the purpose and benefits of the study and asked them for consent to participate in the study as well as permission to use their personal information by researchers. An appointment date for an in-depth interview (IDI) was set depending on each participant’s convenience. Data collection procedures The IDIs took place at the selected clinics. One room was specifically allocated for the interviews. Participation in the study was voluntary. Before the interviews, the trained social scientists provided a full explanation to potential participants about the purpose of the study, duration and the procedures involved. This included information about voluntary participation in IDIs, audio recording procedures, the confidentiality of participants’ information and the freedom to withdraw from the study at any time without any loss of entitled services. Participants were ensured that the audio recorded interviews would not contain any of their identifying information, such as individual names. Instead, they would be identified by code numbers throughout the interviews. Participants were guaranteed that the audio recorded responses would be kept confidential and only the research team would have access to that information. Additionally, participants were ensured that any information included in the report would not identify them Page 3 of 11 as respondents. Participants who agreed to participate in the study were requested to complete the written informed consent forms. IDIs were used to obtain individual perspectives on reasons for LTFU in option B+ and motivation to resume treatment after LTFU. A semi-structured topic guide in Swahili language was developed by the researcher. Before data collection, the interview guide was reviewed by PMTCT content experts to ensure that it contained all the relevant questions. It was then piloted among 5 HIV-positive women (who were not recruited for the main study) in one of the CTCs. The pilot study revealed areas that needed to be reformed. For instance, some of the questions had to be rephrased to ensure clarity during the IDIs. We revised the guide to obtain the final version for the study. Two trained social scientists (MA and KA) conducted the interviews. IDIs were audio recorded using a digital recorder and the duration for each interview ranged from 30 to 60 min. Privacy was maintained at all times during the interviews. Interviews were conducted until saturation was achieved. Saturation was reached when no new information was emerging from the interviews [18]. A maximum of two IDIs were conducted each day, data was collected for 3 weeks. A reimbursement equivalent to $2.5 was provided to each study participant as compensation for their time and travel to the clinic. Data analysis Audio-recorded Swahili interviews were transcribed to text in computer files and then translated into English. The PI conducted the initial transcript examination, to obtain an overall sense of the content of responses from participants regarding various issues. Analysis of data followed qualitative content analysis [19]. Transcribed data was first served in a word processing program, i.e., Microsoft Word. Then the researcher imported the transcribed data into NVivo 10 computer-assisted qualitative data analysis software. We identified meaning units from each transcript and condensed them. We then developed a list of codes which were shared among the PI and co-authors. The team discussed, refined and agreed on a set of codes relevant for the aims of the study. The discussion provided an avenue for the addition of new codes, which were incorporated in the list of developed codes. We finally developed a common codebook to aid the analysis of data. The team used negotiation to resolve disagreements about any issue concerning the identified codes and the coding process. Each transcript was coded to gather material into nodes (categories), sub-categories and finally, the major theme emerged. An example of the content analysis process is shown in Tables 1 and 2. Sariah et al. BMC Public Health (2019) 19:1172 Page 4 of 11 Table 1 Example of content analysis process (Meaning unit, condensed meaning unit, and codes) Meaning Unit Condensed meaning unit Codes I stopped using medication because I had no money for bus fare and my husband left home. That is why I didn’t swallow pills for 3 months. He left home because he is not HIV-infected. The medications make me sick; I feel better without them. Actually, I do not have any symptoms, so why should I take medications?! NZo7 I stopped taking medication because my husband left me, I had no money for transport, the medications make me feel bad, and I am asymptomatic. -Poor financial status -Lack of financial support -Lack of social support -Medication side effects -Absence of disease symptoms I stopped taking the medication because I was not sick. How can I take drugs while I am not sick? Is that possible?... The separation from my husband also played a big part in my stopping medication. He left me because he tested HIVnegative. Therefore, I used the medication for some time, but then my legs began to feel numb …. I worry that using drugs for life may cause other problems. NZ04 I stopped the medication because I was not sick. My husband left me because he is HIV-negative. The medication caused numbness in my legs, and I feared about the problems I could get from using long-term medication -Absence of disease symptoms -Family problems -Medication side effects -Fear of long-term effects of medications Even though there were times did not have money for transport, I walked to the clinic to get my medication. I never thought of quitting the treatment because I wanted to protect my child. BN1 I did not have money for transport but I walked to the clinic for my medication. I wanted to protect my child Poor financial status A need to protect the child Results The study involved 30 HIV-positive women (12 pregnant and 18 breastfeeding). Their ages ranged between 24 and 41 years. The majority [18] had one to two children, 19 were employed, 24 had primary school level of education, and 13 were married. Eleven reported having stopped using ART (LTFU and did not resume) while 19 had resumed treatment after LTFU. Duration on ART for these women ranged from 1 to 7 years and the majority [15] had been on ART for about two to 3 years (Table 3). Themes One main theme and four major categories emerged from this study (Table 4). Participants’ quotes are presented in the text using code numbers. Several factors were found to play a role in LTFU among HIV-positive pregnant and breastfeeding women. Participants mentioned reasons contributing to their LTFU in Option B+ as well as factors that motivated them to resume treatment. The reasons for LTFU were categorized into health-related, psychological and socio-economic factors; while motivation to resume treatment was categorized into a need to stay healthy, a need to protect the child from HIV infection and social support from family members and health care providers. There were no differences in the reasons for LTFU between breastfeeding and pregnant women. However, a major difference was observed on the motivation to resume treatment. For instance, pregnant women mainly resumed treatment because they wanted to protect their unborn children from HIV infection. On the other hand, breastfeeding women resumed treatment because they needed to maintain a healthy status, free from opportunistic infections. Health-related factors Participants expressed health challenges that made them stop attending Option B+ services. Common heath concerns reported included medication side effects, larger pill sizes, and absence of disease symptoms. Medication side effects Table 2 Example of content analysis process (codes, categories, and theme) Codes Categories Theme Medication side effects Health-related factors Struggling with a life-long disease Absence of disease symptoms Family problems Socio-economic factors Poor financial status Lack of financial support Lack of social support Fear of long-term effects of medication Most participants could not tolerate the side effects of ART medication, particularly the first-timers. Some complained about the large pill size and how it was difficult to take them regularly as prescribed. Additionally, others worried about the outcome of their financial status because they could not continue engaging in income-generating activities due to medication side effects. A 29-year-old single woman had 2 children, was LTFU, and did not resume treatment. She had been on ART treatment under Option B+ regimen for 3 years. She reported: Psychological factors A need to protect the child Motivation to resume treatment “I stopped because the medication made me feel very bad after taking them. Firstly, the tablet was too big to swallow; it would stick on my throat for hours. Sariah et al. BMC Public Health (2019) 19:1172 Page 5 of 11 Table 3 Characteristics of study participants Variable Number Percentage 20–29 7 23.3 30–39 22 73.3 ≥ 40 1 3.3 Single 11 36.7 Married 13 43.3 Separated 3 10.0 Age (years) Marital status Widowed 1 3.3 Divorced 2 6.7 Employed 25 83.3 Unemployed 5 16.7 Occupation Level of education No formal education 1 3.3 Primary school 24 80.0 Form II dropout 2 6.7 Secondary school 3 10.0 No child 2 6.7 1–2 18 60.0 3–4 10 33.3 0–1 1 3.3 2–3 16 53.3 4–5 11 36.7 ≥6 2 6.7 0–1 1 3.3 2–3 15 50.0 4–5 10 33.3 ≥6 4 13.3 Completely LTFU 11 36.7 Resumed treatment 19 63.3 Parity Secondly, it made me feel dizzy the whole day. So, I slept most of the time and could not go to work, this could have led to poverty.” BN06 Participants expressed a variety of physical and mental disturbances when they used Option B+ medications. Common side effects including dizziness, drowsiness, confusion, and blurring of vision were reported. A 30year-old single woman with one child was LTFU and did not resume treatment. She had been on ART under Option B+ regimen for 3 years. She described her experience as follows: “Whenever I took the medication, my eyes could not see properly anymore, my vision was getting weaker every day, and my head felt so heavy. I felt sick to the extent that I regretted taking the medication in the first place.” BN05 Another participant was a 39-year-old married breastfeeding woman who had three children and had been on ART under Option B+ regimen for 4 years. She was also LTFU and did not resume treatment. She added: “I used to take my medication at 10 pm every night. But I would feel drunk like I had taken alcohol and I felt confused when I woke up in the middle of the night. I had to stop taking the dose.” BN02 ART use duration (years) Duration since HIV diagnosis (years) LTFU status LTFU Loss to follow-up, ART Antiretroviral treatment, HIV Human Immunodeficiency Virus Table 4 Emerged categories and the main theme from the study Categories Theme Health-related factors Struggling with a life-long disease Socio-economic factors Psychological factors Motivation to resume treatment Asymptomatic status Participants claimed that they did not see the point of taking medications when they felt well, with no disease symptoms. A 30-year-old single woman with one child was LTFU and did not resume treatment. She expressed: “Yes, like I said I was fine until I began taking the medication. I had no signs of sickness, and I did not understand why I was supposed to take the dose while I have been fine all along.” BN05 Other participants had been on option B+ medication ever since they were diagnosed with HIV during pregnancy. They continued taking the medication after delivery and throughout the breastfeeding period, despite the absence of disease symptoms. However, they stopped taking the medication as soon as their babies tested negative for HIV infection. A 35-year-old widow who had resumed treatment and had three children stated: “I tested my baby, and she was HIV-negative, then I thought I was fit and able to carry on with my daily activities, so I decided to stop taking the medication.” NZ05 Sariah et al. BMC Public Health (2019) 19:1172 Psychological factors Loss of hope Participants reported a loss of hope in the course of option B+ treatment, which compelled them to stop taking the medication. Some of the participants lost hope because they were abandoned by people who were expected to provide care and offer support in the important areas of their lives. For example, others were neglected by their male partners after a positive HIV diagnosis, leaving them alone with no psychological support. These women felt that they could not continue with the life-long treatment unless they received support from their significant others. Other women were burdened by the poor financial status and medication side effects which made them feel hopeless using the life-long treatment. A 28-year-old single breastfeeding woman who had resumed treatment and had two children stated: “I quit taking medications because of the stress I went through. I was not feeling well …. And I was confused because the person I depended on, and who can help as well as give me courage, was not with me. I just saw myself as a dying person … ” NZ02 A 29-year-old single woman with two children was LTFU and did not resume treatment. She said: “To be honest, I lost hope in the treatment because it made me feel even worse than before.” BN06 A 33-year-old married woman who had resumed treatment and had one child added: “Yes, my income was so bad that I thought it is better to die even though it’s not my time yet. I lost hope back then.” NZ08 Fear of medication side effects While some participants expressed their fear of developing drug resistance as a negative outcome of long-term use of medication, others feared the immediate unpleasant and disturbing side effects of Option B+ medication. A 39-year-old married woman had three children, was LTFU and did not resume treatment expressed: “I fear what might happen when the treatment gets used to the body. It will fail to work and the time will come when no other medication will be useful to the body anymore.” BN02 A single woman aging 30 years had one child, was LTFU and did not resume treatment. She reported her situation as follows: Page 6 of 11 “… What got me worried was the way I felt sick after taking the dose. I said to myself, I was very fine before I began the treatment, but now this new treatment is going to kill me before my time. I should stop it.” BN05 HIV-related stigma Majority of participants reported that they stopped taking option B+ medication because they were scared of being identified and labeled as HIV-positive by their family members and neighbors, which could have led to stigma and discrimination. Some had to travel to the farthest CTCs away from their residencies just to avoid meeting people who would recognize them. Others decided to stop attending the CTC all together because they could not bear the thought of being identified by familiar people at the clinic. A 30-year-old married breastfeeding woman who had resumed treatment and had two children stated: “I developed fear and shame around people as I didn’t want anybody to know my status. Whoever enters the clinic gets the idea about your HIV status, so I decided not to go there anymore. However, after a long time, I started going to Vikindu CTC which is quite far from where I live. Nobody knows me there.” GL05 Fear was also reported by a 34-year-old married woman who had four children. She was LTFU and did not resume treatment. She said: “I could not take the medication in the presence of my mother-in-law; I was afraid she would discriminate against me and tell everyone about my HIV status.” GL10 Socio-economic factors Financial constraints Participants had different views regarding social issues that contributed to their LTFU. Some of the participants were unemployed, and they could not afford the bus fare to attend the CTC. Others complained about the inability to buy food because of the poor economic status. With a shortage of food, they could not take their medication as required. A 32-year-old single breastfeeding woman who had one child and had resumed treatment explained her situation: “Yes, you may wake up early in the morning and eat nothing until 11:00 a.m. just because of economic hardship. Hence you cannot take the medication in an empty stomach.” NZ03 Sariah et al. BMC Public Health (2019) 19:1172 Participants reported that they did not get financial support from their families or male partners which resulted in poor or non-attendance to the CTC. They also expressed their experience with the long-term treatment as they struggled to take care of themselves and their significant others amidst poor financial status. A 35year-old married breastfeeding woman with three children was LTFU and did not resume treatment. She expressed why she dropped out of care as follows: “I don’t understand my husband, he knows about my HIV-positive status but refuses to give me money for transport to the clinic, he insists that I stay home.” GL09 A 37-year-old single woman who had three children and had resumed treatment commented: “… … life is hard sister, there are other people I have to care for. For example, my children, even though they come from different fathers, I am grateful they not infected. But, raising them alone while still taking care of myself is a very difficult task. I am a single mother.” GL03 Lack of partner support In addition to financial problems, others felt they needed their male partner’s emotional support as they engaged in Option B+ care and treatment. However, nobody was there to comfort them during the most difficult period. Some of the participants declared that they were prohibited from attending the CTC. Therefore, lack of emotional support plus economic constraints made treatment follow-up worse. A 30-year-old woman who was separated from her husband had one child and had resumed treatment, stated: “I stopped using medications because my husband left me and I had no bus fare to get me to the clinic. He left me because he was HIV-negative. I, therefore, didn't take medication for three consecutive months.” NZ07 Another 35-year-old married breastfeeding woman who had three children was LTFU and did not resume treatment, said: “My husband is the greatest challenge, I can’t say much, but in short, he forbids me to attend the clinic” GL09 Family conflicts Participants reported how family conflicts have contributed to their LTFU in the Option B+. For instance, Page 7 of 11 conflicts between a patient and her in-laws led to anger that finally resulted in stopping the treatment. Furthermore, an HIV-negative diagnosis for some of the participants’ male partners brought conflicts in the family which led to couple separation and spouse neglect. Once the couple was separated, the women could not afford to cater for their daily needs including food and transport fee to the clinic, hence they dropped out of the Option B+ treatment. A 28-year-old married pregnant woman with no child was LTFU and did not resume treatment. She expressed: “I stopped because first, I traveled to my in-laws and I didn’t want my mother-in-law to know about my health status. Second, I lost my clinic card, and third, my mother-in-law wanted to break my marriage. This made me very angry; I decided to stop taking the dose completely.” BN03 A woman of 30 years of age with one child, had resumed treatment after LTFU. She expressed how her HIV-positive status led to a separation from a husband who was HIV-negative. She stated: “I stopped using medications because my husband left me and I had no bus fare to get me to the clinic. He left me because he was HIV-negative. I, therefore, didn’t take medication for three consecutive months.” NZ07 Lack of HIV-disclosure Out of fear of rejection and HIV-related stigma, some of the participants explained that they stopped taking their option B+ medication because they had not told their male partners or other family members about their HIVpositive status. The lack of disclosure made it difficult to continue taking the medication or attending the clinic without being noticed or asked questions by their male partners or other family members. A 28-year-old married woman who had resumed treatment and had one child said: “I was afraid to disclose my status to my husband, but then, every month I had to go to the clinic for followup. He always asked where I was going, so it was hard for me to attend the clinic most of the time. When I finished the pills, I was given that month, I stopped and I never went to the clinic again.” NZ01 Another woman who was 39 years of age with three children, was LTFU and did not resume treatment reported: Sariah et al. BMC Public Health (2019) 19:1172 “I used to stay at my mother-in-law’s place because she assisted me with the baby when I gave birth. It was very difficult for me to take the dose regularly as prescribed because I had not told her about my HIV-positive status. It reached a time when I had to quit the dose.” BN02 Religious beliefs Religious faith was mentioned as one of the reasons that made other participants stop taking their medications and attending the CTC. These women believed that only prayers could cure them of HIV infection. This was expressed by a 32-year-old single woman who had resumed treatment and had one child. She said: “When I was diagnosed with HIV, I started using ARVs. After birth, the baby was tested and was found HIV-negative. My husband never accepted that I was HIV-positive; he insisted that we are people of God. So, he convinced me to stop taking the ARVs, and I did. I started believing in prayers.” NZ10 Motivation to resume treatment HIV-positive women who were once LTFU felt motivated to resume treatment because they received adequate psychological support from their families and health care providers. In addition, the need to maintain a healthy status, as well as protect their children from HIV infection were among the factors that made these women decide to resume treatment. The need to protect the child from HIV infection Some of the HIV-positive women who were once lost to follow-up expressed that their decision to resume treatment was triggered by their pregnancy status. This state made them feel that they needed to protect their unborn children from HIV infection. A 36-year-old divorced pregnant woman with three children expressed: “Even though there were times I did not have money for transport; I walked to the clinic to get my medication. I never thought of quitting the treatment because I wanted to protect my child from HIV.”BN1 The need to maintain a healthy status Majority of women who were once LTFU resumed treatment because they wanted to keep themselves healthy, free from opportunistic infections. Some had already experienced how it feels to get opportunistic infections and they did not want to go through that again. Others Page 8 of 11 had not gotten sick yet, but they wanted to remain healthy and free from infections. A 35-year-old widowed woman with three children expressed: “My baby tested HIV-negative and felt that I was fit to carry out my daily activities. I decided to stop taking my medication … … As days passed, I started feeling tired most of the time and I then developed diarrhea. That’s when I decided to come to the clinic where I started taking my medication.” NZ05 A 37-year-old single woman with three children reported: “I will always listen to what health care providers tell me. I will make sure that I take my medications because they keep me healthy. I will surely keep participating in the treatment, as it is for my own goodness.” GL03 Social support from the family and healthcare providers Encouragement from family members played an important role in women’ decision to start engaging in the lifelong treatment after stopping. A single, 28-year-old pregnant woman with two children said: “I quit the medications because of the stress and what I went through. I also did not feel well, but once I told my mother, she encouraged me to go back to the clinic, where the nurse counselor gave me hope to live again.” NZ02 Additionally, reminders and emphasis about the importance of treatment adherence and keeping ART clinic appointments by health care providers influenced women to restart treatment after LTFU. A married, 34-year-old pregnant woman with two children expressed: “ … . the nurses are very friendly and they keep patients’ records well. As for me, I once stopped the treatment, but I was reminded to continue and I did. I am currently expecting another child.” GL07 Discussion Our study has revealed the experiences of HIV-positive pregnant and breastfeeding women as they struggle to survive while embarking in the life-long ART treatment. Common reasons for LTFU in Option B+ and factors that motivate these women to resume treatment were identified. The reasons for LTFU included health-related, psychological and social-economic factors. Motivation to Sariah et al. BMC Public Health (2019) 19:1172 resume treatment was divided into the need to maintain a healthy status, protect the child from HIV infection and support from the family and healthcare providers. Health-related factors The findings demonstrated how HIV-positive women felt about ART medication. For example, their highest complaint was the issue of side effects. Similarly, previous studies have reported that women under option B+ experienced adverse medication side effects which compelled them to stop [5, 8, 14]. Apart from medication side effects, participants dropped out of the life-long treatment because they felt healthy and did not experience any disease symptoms. This is consistent with what was described in other studies [20, 21]. Additionally, similar to previous reports, some of these women dropped out of care after their children tested HIV-negative [5]. Page 9 of 11 The role of religious beliefs in promoting behaviors that reduce new HIV infections [31] and facilitating retention in ART [32, 33] has been cited. Inconsistent with our results, religious beliefs facilitated LTFU in Option B+. Participants in our study stopped taking medications or attending the clinics because they believed they would get cured through their religious beliefs and not ART medication. Similar findings have been reported elsewhere [10, 34]. Variations in efforts taken by religious bodies to promote health-seeking behaviors among their members may have contributed to the observed inconsistencies. Perhaps, future research could explore further on how religious beliefs may influence one’s participation in long-term ART treatment. Other reasons for LTFU included traveling and relocation, which have been cited in other HIV studies [10, 30]. Motivation to resume treatment Psychological factors Due to the fear of anticipated HIV-related stigma, participants had to travel to the farthest clinics to receive option B+ care, which required more funds for transportation. However, a study by Iroezi et al. [22] found that women preferred clinics that were closer to their homes. The different results may have resulted from the implementation of effective interventions to reduce HIV-related stigma in those communities. Such efforts may have made HIV-positive women feel free to visit the nearest clinics for follow-up care. Consistent with other studies, loss of hope was cited as a reason for LTFU [23–25]. Interventions that focus on educating these women about the benefits of remaining in care and adhering to the treatment as well as instilling hope through counseling may help reduce the rate of LTFU in Option B+. Socio-economic factors Similar to other studies, our results found that non-disclosure contributed to LTFU in option B+ treatment [12, 20, 22, 26–29]. Participants preferred to keep their HIV-positive status a secret because they feared stigma from family members, and neighbors. In the current Tanzanian society, the act of taking medication every day put the patient’s HIV-positive status at risk of being exposed, making matters worse [4]. Consistent with our findings, lack of disclosure related to HIV stigma has been reported in previous option B+ studies [22, 27, 29]. Similar to other studies, the lack of food resulting from financial constraints [22] was one of the reasons for LTFU. Participants also expressed a lack of support from their male partners [20, 26, 30]. For example, similar to previous reports [20], participants expressed how their male partners forbade their attendance to the clinics or even neglected them completely. Studies have documented facilitators to re-engagement in ART care. For instance, consistent with our findings, most pregnant women started ART after initially stopping because they had a desire to protect their unborn babies from HIV infection [35, 36]. Another common reason for re-starting ART was feeling sick. Similar studies have cited a declined health status as a facilitator for starting ART in women under the Option B+ program [20]. In addition to feeling sick, these women re-engaged in ART care because they wanted to remain healthy [35, 37]. A healthy status enabled them to support and care for their children [36, 37]. Starting ART after LTFU was believed to improve their survival by keeping them free from opportunistic infections [36, 37]. Similar to our findings, health care providers played an important role in encouraging women who were lost to follow-up to re-engage in ART care and treatment. Such efforts acted as a motivation to resume treatment [20, 35]. Consistent with our results, these women were also motivated to resume treatment when they received support from their male partners and other members of the family [35]. Limitations Our data represent views of study participants from three CTCs, each located in a different district of the urban environment in Dar es Salaam. Being a qualitative study, there is a limitation in the generalizability of our findings. However, the rich information obtained from IDIs with HIV-positive pregnant and breastfeeding has provided a deeper understanding of their experiences in the life-long treatment, therefore, generating important issues that require careful consideration in PMTCT programs. Conclusions Our findings provide insights on the common reasons for LTFU in women initiated on option B+ treatment. Sariah et al. BMC Public Health (2019) 19:1172 Medication side effects, feeling healthy, fear of HIV disclosure, fear of HIV-related stigma, and lack of financial and emotional support from the male partner were most commonly mentioned reasons for LTFU in option B+. Motivators for starting treatment after LTFU included support from health care providers and family members, a need to stay healthy, and a desire to protect the unborn child from HIV-infection. The study underscores the importance of strengthening HIV services by improving male partner support, promoting public awareness about negative effects of HIV-related stigma, early identification and proper management of women in ART care and treatment. Provision of psychological support for women LTFU, as well as those experiencing medication side effects, loss of hope and HIV-related fears, might help improve retention in the life-long treatment. Abbreviations CTC: Care and Treatment Clinic; HKMU: Hubert Kairuki Memorial University; IDIs: In-depth interviews; LTFU: Loss to follow-up; MDH: Management and Development for Health; MoHCDGEC: Ministry of Health, Community Development, Gender, Elderly, and Children; MUHAS: Muhimbili University of Health and Allied Sciences; UW: University of Washington Acknowledgments We are grateful to all of those with whom we have had the pleasure to work during the study. We specifically thank the study participants for their willingness to take part in this study and also appreciate the cooperation we received from nurses working at the CTCs. Our sincere gratitude to MDH, MUHAS, and HKMU for their cooperation and support throughout the study period. This research was supported by the Afya Bora Consortium (Health Resources & Service Administration U91HA06801B) and University of Washington Center for AIDS Research (CFAR), an NIH funded program (P30 AI027757) which is supported by the following NIH Institutes and Centers (NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA). Authors’ contributions AS conceived of the study, participated in its design, collected, analyzed and interpreted the data. AS, JP and JR wrote the first draft of the manuscript. HS, ET, EA, and DU helped to draft the manuscript. All authors revised and approved the final manuscript for publication. Funding This study was funded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number P30AI027757, through the University of Washington Center for AIDS Research Supplement for Afya Bora Fellowship. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Ethical approval to conduct the study was obtained from the National Institute for Medical Research (NIMR) #2280 and the University of Washington (UW) #50938. Participants were informed of study procedures; those who agreed to participate in the study were asked to complete a written informed consent form. One copy of the signed consent form remained with the researcher, and a second copy was given to the study participant. Consent for publication Not applicable Page 10 of 11 Competing interests The authors declare that they have no competing interests. Author details 1 Department of Mental Health and Psychiatric Nursing, Hubert Kairuki Memorial University (HKMU), 70 Chwaku Street-Mikocheni, P.O. Box 65300, Dar es Salaam, Tanzania. 2Department of Internal Medicine, Muhimbili National Hospital, P.O. Box 65000, Dar es Salaam, Tanzania. 3Department of Community Health Nursing, Hubert Kairuki Memorial University (HKMU), 70 Chwaku Street-Mikocheni, P.O. Box 65300, Dar es Salaam, Tanzania. 4Clinical Department, Management and Development for Health (MDH), P.O. Box 79810, Dar es Salaam, Tanzania. 5Department of Public Health Evaluation, Management and Development for Health (MDH), P.O. Box 79810, Dar es Salaam, Tanzania. 6Department of Nursing Management, Muhimbili University of Health and Allied Sciences (MUHAS), P.O. Box 65004, Dar es Salaam, Tanzania. 7Department of Community Health, School of Public Health and Social Sciences, Muhimbili University of Health and Allied Sciences (MUHAS), P.O. Box 65015, Dar es Salaam, Tanzania. Received: 20 March 2019 Accepted: 19 August 2019 References 1. Rosa W, editor. Transforming our world: The 2030 agenda for sustainable development. In: A new era in global health. New York: Springer Publishing Company; 2017. Available from: http://connect.springerpub.com/lookup/ doi/10.1891/9780826190123.ap02. Cited 4 Mar 2019 2. World Health Organisation. Programmatic update: use of antiretroviral drugs for treating pregnant women and preventing HIV infection in infants. Geneva: World Health Organisation; 2012. 3. UNICEF. Options B and B+: Key considerations for countries to implement and equity-focused approach. Eliminating new HIV infections among children and keeping mothers living with HIV alive and well. 2012. 4. Ngarina M, Tarimo EAM, Naburi H, Kilewo C, Mwanyika-Sando M, Chalamilla G, et al. Women’s preferences regarding infant or maternal antiretroviral prophylaxis for prevention of mother-to-child transmission of HIV during breastfeeding and their views on option B+ in Dar Es Salaam, Tanzania. PLoS One. 2014;9(1):e85310 Thorne C, editor. 5. Clouse K, Schwartz S, Rie AV, Bassett J, Yende N, Pettifor A. “What they wanted was to give birth; nothing Else”: barriers to retention in option B+ HIV care among postpartum women in South Africa. J Acquir Immune Defic Syndr. 2014;67(1):7. 6. Coutsoudis A, Goga A, Desmond C, Barron P, Black V, Coovadia H. Is option B+ the best choice? Lancet. 2013;381(9863):269–71. 7. Ahmed S, Kim MH, Abrams EJ. Risks and benefits of lifelong antiretroviral treatment for pregnant and breastfeeding women: a review of the evidence for the option B+ approach. Curr Opin HIV AIDS. 2013;8(5):474–89. 8. CDC. Impact of an innovative approach to prevent mother-to-child transmission of HIV — Malawi, July 2011–September 2012. Morb Mortal Wkly Rep. 2013;62:141–56. 9. Kim MH, Ahmed S, Hosseinipour MC, Giordano TP, Chiao EY, Kazembe PN, et al. The impact of option B+ on the antenatal PMTCT Cascade in Lilongwe, Malawi. J Acquir Immune Defic Syndr. 2015;68(5):7. 10. Tweya H, Gugsa S, Hosseinipour M, Speight C, Ng’ambi W, Bokosi M, et al. Understanding factors, outcomes and reasons for loss to follow-up among women in option B+ PMTCT programme in Lilongwe, Malawi. Tropical Med Int Health. 2014;19(11):1360–6. 11. Kim MH, Ahmed S, Preidis GA, Abrams EJ, Hosseinipour MC, Giordano TP, et al. Low rates of mother-to-child HIV transmission in a routine programmatic setting in Lilongwe, Malawi. PLoS One. 2013;8(5):e64979 Jhaveri R, editor. 12. Gourlay A, Birdthistle I, Mburu G, Iorpenda K, Wringe A. Barriers and facilitating factors to the uptake of antiretroviral drugs for prevention of mother-to-child transmission of HIV in sub-Saharan Africa: a systematic review. J Int AIDS Soc. 2013;16(1):18588. 13. Keehn E, Karfakis J. Current practices to improve uptake, retention, and adherence for option B+ in Malawi; 2014. 14. Ministry of Health and Social Welfare (MoHSW). National HIV and AIDS Response Report 2013 Tanzania Mainland. Dar es Salaam: Prime Minister’s Office; 2014. 15. Chi BH, Yiannoutsos CT, Westfall AO, Newman JE, Zhou J, Cesar C, et al. Universal definition of loss to follow-up in HIV treatment programs: a Sariah et al. BMC Public Health 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. (2019) 19:1172 statistical analysis of 111 facilities in Africa, Asia, and Latin America. PLoS Med. 2011;8(10):e1001111 Bartlett J, editor. Musmade P, Nijhawan L, Udupa N, Bairy K, Bhat K, Janodia M, et al. Informed consent: issues and challenges. J Adv Pharm Technol Res. 2013;4(3):134. Boyce C, Neale P. Conducting In-depth interviews. 2006;16. Moser A, Korstjens I. Series: practical guidance to qualitative research. Part 3: sampling, data collection, and analysis. Eur J Gen Pract. 2018;24(1):9–18. Graneheim UH, Lundman B. Qualitative content analysis in nursing research: concepts, procedures, and measures to achieve trustworthiness. Nurse Educ Today. 2004;24(2):105–12. Kim MH, Zhou A, Mazenga A, Ahmed S, Markham C, Zomba G, et al. Why did I stop? Barriers and facilitators to uptake and adherence to ART in option B+ HIV Care in Lilongwe, Malawi. PLoS One. 2016;11(2):e0149527 Ferrand RA editor. Kuznetsova AV, Meylakhs AY, Amirkhanian YA, Kelly JA, Yakovlev AA, Musatov VB, et al. Barriers and facilitators of HIV care engagement: results of a qualitative study in St. Petersburg, Russia. AIDS Behav. 2016;20(10):2433–43. Iroezi N, Mindry D, Kawale P, Chikowi G, Jansen P. A qualitative analysis of the barriers and facilitators to receiving care in a prevention of mother-tochild program in Nkhoma, Malawi. 2015;14. Tsega B, Srikanth A, Shewamene Z. Determinants of non-adherence to antiretroviral therapy in adult hospitalized patients, Northwest Ethiopia. Patient Prefer Adherence. 2015;373:373-80. Siril H, Fawzi MCS, Todd J, Wyatt M, Kilewo J, Ware N, et al. Hopefulness fosters affective and cognitive constructs for actions to cope and enhance quality of life among people living with HIV in Dar Es Salaam, Tanzania. J Int Assoc Provid AIDS Care. 2017;16(2):140–8. Ngarina M, Popenoe R, Kilewo C, Biberfeld G, Ekstrom AM. Reasons for poor adherence to antiretroviral therapy postnatally in HIV-1 infected women treated for their own health: experiences from the Mitra Plus study in Tanzania. BMC Public Health. 2013;13(1). Available from: http:// bmcpublichealth.biomedcentral.com/articles/10.1186/1471-2458-13-450. Cited 26 Oct 2018 Clouse K, Schwartz S, Van Rie A, Bassett J, Yende N, Pettifor A. “What they wanted was to give birth; nothing else”: barriers to retention in option B+ HIV care among postpartum women in South Africa. J Acquir Immune Defic Syndr. 2014;67(1):e12–8. Hodgson I, Plummer ML, Konopka SN, Colvin CJ, Jonas E, Albertini J, et al. A systematic review of individual and contextual factors affecting ART initiation, adherence, and retention for HIV-infected pregnant and postpartum women. Newell M-L. PLoS One. 2014;9(11):e111421. Webb R, Cullel M. Understanding the perspectives and/or experiences of women living with HIV regarding option B+ in Uganda and Malawi: GNP+, ICRW, ICW-EA, COWLHA; 2013. Flax VL, Hamela G, Mofolo I, Hosseinipour MC, Hoffman IF, Maman S. Factors influencing postnatal option B+ participation and breastfeeding duration among HIV-positive women in Lilongwe District, Malawi: a qualitative study. PLoS One. 2017;12(4):e0175590 Blackard J, editor. Gugsa S, Potter K, Tweya H, Phiri S, Sande O, Sikwese P, et al. Exploring factors associated with ART adherence and retention in care under option B+ strategy in Malawi: a qualitative study. PLoS One. 2017;12(6):e0179838 Okulicz JF, editor. Kagimu M, Guwatudde D, Rwabukwali C, Kaye S, Walakira Y, Ainomugisha D. Religiosity for promotion of behaviors likely to reduce new HIV infections in Uganda: a study among Muslim youth in Wakiso District. J Relig Health. 2013;52(4):1211–27. Pecoraro A, Mimiaga MJ, O’Cleirigh C, Safren SA, Blokhina E, Verbitskaya E, et al. Lost-to-care and engaged-in-care HIV patients in Leningrad oblast, Russian Federation: barriers and facilitators to medical visit retention. AIDS Care. 2014;26(10):1249–57. Pecoraro A, Pacciolla A, O’Cleirigh C, Mimiaga M, Kwiatek P, Blokhina E, et al. Proactive coping and spirituality among patients who left or remained in antiretroviral treatment in St Petersburg, Russian Federation. AIDS Care. 2016;28(3):334–8. Medved Kendrick H. Are religion and spirituality barriers or facilitators to treatment for HIV: a systematic review of the literature. AIDS Care. 2017;29(1):1–13. Buregyeya E, Naigino R, Mukose A, Makumbi F, Esiru G, Arinaitwe J, et al. Facilitators and barriers to uptake and adherence to lifelong antiretroviral Page 11 of 11 therapy among HIV infected pregnant women in Uganda: a qualitative study. BMC Pregnancy Childbirth. 2017;17(1):94. 36. McLean E, Renju J, Wamoyi J, Bukenya D, Ddaaki W, Church K, et al. ‘I wanted to safeguard the baby’: a qualitative study to understand the experiences of option B+ for pregnant women and the potential implications for ‘test-and-treat’ in four sub-Saharan African settings. Sex Transm Infect. 2017;93(Suppl 3):e052972. 37. Elwell K. Facilitators and barriers to treatment adherence within PMTCT programs in Malawi. AIDS Care. 2016;28(8):971–5. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2144249175
https://zenodo.org/records/1540997/files/article.pdf
German
null
Zur Bestimmung des Dicyandiamids im Kalkstickstoff
Angewandte Chemie
1,917
public-domain
3,077
(Wttellung der landwirtschaftlichen Versuchsstation Kempeu-Rheln.) Von G. H ~ Q E E und J. KEXN. (Eingeg. 9Jl. 1917.1 Es kameii nur groBere Mengen bei der K j e 1 d a h 1 schen Bestimmung zur Verbrennung: Tn allen Fiillen, wo die Bestimmung des Cyanamidstickstoffs in dern genannten Dungemittel nicht durchaus notwendig ist, wird die Trennung und Fiillung dieser Verbindung mit den nicht bjlligen Silbersalzen am besten soweit moglich, ganz verrnieden, zumal auch nach unseren Untersuchungen die Gefahr des Mitreinens von Di- cyandiamid beim Ausfallen des Cyanamidsilbers besteht. D i c y a n d i a m i d : Vorhanden in yo . . . 1,186 2,344 4,581 7,408 Gefunden in yo 1. . . 1.188 2,304 4,607 7,500 Gpfunden in yo 2. . . 1,103 2,263 4,568 7,430 Dem S t u t z e r schen Vorschlnge entsprechend, wird deshalb bei der alleinigen Dicyandiamidbestirnmung der alkoholische Auszug am einfachsten und zweckmiiligsten fur die Untersuchung Verwen- dung finden. Die Berechnungen sind bei den Versuchen auf Dicyandiamid und nicht auf Dicyandiamidstickstoff bezogen. g Da bekanntlich Calciumcyanamid in Alkohol so gut wie unloslich ist, wird der Auszug nur noch die geringen Mengen frcien Cyanamids enthalten. Zum Schlu&se sei noch erwiihnt, daB auch T r u n i n g e r nach einer kurzen Mitteilung in der Chemiker-Zeitung die C a r o sche Methode in gleicher Weise abgeiindert hat. B r i o u x fiillt in einem Teil der Losung das Cyanamid nach C a r o mit Silbersalz und Ammoniak und berechnet aus dem Silber- gehalt dea Niederschlages den Cyanamidstickstoff. In einem anderen Teil des Auszuges wird das Cyanamid und das Dicyandiamid mit Silbernitratlosung und Kalilauge gefiillt. Der N wird nach K j e 1 - d a h 1 bestimmt. Die Differenz ergibt den Gehltlt an Dicyandiamid- stickstoff. g g Nun hat A. S t u t z e re) vor kurzem in dieser Zeitschrift ein ab- geiindertes Verfahren der C a r o schen Methode mitgeteilt. Da wir die hier vorgeschriebene Arbeitsweise ebenfalh gepriift haben, seien einige Beobachtungen mitgeteilt, aus denen sich ergibt, da5 die Methode noch ergiinzender Untersuchungen bedarf, anderenfalls manche Differenzen und unrichtige Ergebnisse je nach der Arbeits- weise und vor allem je nach dem kiirzeren oder langeren Stehen- lassen der L&ungen unausbleiblich sind. (Wttellung der landwirtschaftlichen Versuchsstation Kempeu-Rheln.) Von G. H ~ Q E E und J. KEXN. (Eingeg. 9Jl. 1917.1 In Nr. 63 vom 8.,/8. 1916 dieser Zeitschrift haben wir einige Unter- suchungsergebnissc uber die Mange1 der C a r o schen Methode der Dicyandiamidbestimmung mitgeteilt. Es sei hierzu noch ergiinzend erwiihnt, d a l auch B r i o u XI) bereits im Jahre 1910 auf die Stick- stoffverluste aufmerksam gemacht hat, die beim Kochen der Dicyan- diamidsilberlosung entstehen. Er hat auch diejenige Modifikation vorgeschlagen, die an der hiesigen Versuchsstation in einer Abiinderung auf Grund unserer Untersuchungen zurzeit angewendet wird. Die Arbeiten des genannten Verfassers waren uns nicht bekaunt, weil die betreffende Zeitschrift hier nicht zur Verfdgung stcht. ( y ) , , , g Von der alkoholischen Losung wurden dann 20 ccm zur Bestim- mung verwendet. g D i c y a n d i a m i d . Vorhanden in yo . . . 1,186 2,344 1,581 7,408 Gefunden in Yo 1. . . 1,147 2,268 4,533 7,420 Gefunden in Yo 2. . . 1,276 2,240 4,600 7,280 Gefunden in yo 3. . . 1,110 2,272 4,572 7,340 Die noch in dem Kalkstickstoff vorhandenen Spuren Dicyan. diamid und Cyanamid sind selbstverstiindlich durch sorgfaltige blinde Bestimmungcn festgestellt und beriicksichtigt worden. g g Da also demnach die Festatellung, daB bei der Dicyandiamidbestim- mung nach C a r o Stickstoffverluste unvermeidlich sind, voil ver- schiedenen Seiten unabhiingig voneinander gemacht ist, kann wohl an der Richtigkeit dieser kein Zweifel bestehen. g g g Trotzdem nur 20 ccm der Losung zur Untersuchung verwendet sind, ist das Resultat ein durchaus befriedigendes. g Weil fiir die Beurteilung des Kalkstickstoffs als Diingemittel unter Umstiinden auch die Festatellung des Dicyandiamidgehaltes durchaus von Bedeutung ist, erscheint die Ausarbeitung einer exakten Unter- suchungsmethode, die schnell auszufdhren ist und keine zu gro5en Kosten verursacht, wohl zweckmalig. g Auch ein weiterer Versuch ergab dasselbe Resultat. Je 10 g Kalkstickstoff werdcn mit den doppelten Mengen Dicyandiamid, also 0,12, 0,24, 0,48 und 0,8 g versetzt, mit 200 ccm Alkohol in derselben Weise ausgezogen und 50 ccm vom Filtrat verwendet. Der prozen- tische Gehalt an Dicyandiamid ist derselbe. AnfaStctell. Hager und Kern: Zur Bestirnmung des Dicyandiamids im Kalkstickstoff. 53 80. Jahrgsng 1917.3 - Zeitschrift f U angewandte Chemie I 53 - Hager und Kern: Zur Bestirnmung des Dicyandiamids im Kalkstickstoff. AnfaStctell. 80. Jahrgsng 1917.3 I ) Annales de la Science aqron. franqaise et Btranghre, April 1910; nuch Lriefl. Mitteilung v. S. H a 1 s Christiania. 2, Angew. Chem. 29, 11, 417 [1916]. Angew. Chem. 1917. AufMt6tefl 0. Bd.) IU Nr. 17. Zur Bestimmung des Dicyandiamids im Kalk- stickstoff. euc t ac j e d a ve b a t. Die Differenz ergibt den Gehalt an Dicyandiamidstickstoff. g y Dan diese Arbeitsweise befriedigende Resultate ergibt, zeigen die folgenden Beleganalysen. Unzersetzter Kalkstickstoff wurde mit hochprozentigem Alkohol gereinigt, mit Aceton nachgewaschen und bei geringer Warme getrocknet. Von diesem Produkt wurden dann sofort je 5 g mit wechselnden Mengen Dicyandiamid von bekanntem Gehalt (GG,18y0 N) versetzt, und zwar mit O,OG, 0,12, 0,24 und 0,4 g. (Wttellung der landwirtschaftlichen Versuchsstation Kempeu-Rheln.) Von G. H ~ Q E E und J. KEXN. (Eingeg. 9Jl. 1917.1 I. Band, S. 63-56 I. Band, S. 63-56 riihren wird sofort filtriert, der Niederschlag ausgewaschen und feucht nach K j e 1 d a h 1 verbrannt. riihren wird sofort filtriert, der Niederschlag ausgewaschen und feucht nach K j e 1 d a h 1 verbrannt. M i t t c l z u r B e h a n d l u n g v o n W u n d e n u n d H a u t - 1; r a ii k h c i t c 11. M i t t c l z u r B e h a n d l u n g v o n W u n d e n u n d H a u t - 1; r a ii k h c i t c 11. M i t t c l z u r B e h a n d l u n g v o n W u n d e n u n d H a u t - 1; r a ii k h c i t c 11. 111. I v. v. Erstc LBsung . . . . . . 19,55y0 15,9204 :<,CJey;, Zweite Losung. . . . . . 14,L8,, 9,72 ,, 1,25,, Fur dicse Arzncimittclgriippe interrssivren in erster Linie die Ersatzmittel des Glycerins, das Tcgoclykol und Mollphorus. ,,T c g o g 1 y k o 1" ist das dem Glycerin chemisch und physi- kalisch nnhe stehende Athylenglykol, C,H,(OH),, das von Th. Gold- Schmidt A. G. in Esscn nach einem patentierten Verfahren zu billigem Preise hergestellt wird. Es eignct sich nach B a c h e mli) schr gut als Ersatz des Glycerins zu pharmazeutischen wid kosrnetischcn Zwecken. Ks ist cine neutrale, diclte, rtwas siiI3lich schmeckende Fliissigkeit, die sirh init Wasser uiid Alkohol in jedcin Vvrhiiltnis mischeii Isfit, in Ather hingegcn iiur nenig loslich ist. Srin spezi- fisches Gewicht betfigt. 1,12, srin Sirdepunkt. 198" und win Gefrirr- punkt - 13". Es ist sehr hygroskopisch und lost vielc organischc Stoffe. Wir Glycerin ist es praktisch ungiftig und kann innerlich, subcutan und rectal in entsprechcnden Dosen ohne Schaden gegeben werden. Auch zu Salben und Suppositorien kann es verarbeitet werden. Seine abfuhrende Wirkung bci rectaler Applikation wurdr bereita festgestellt. Die X1)meichungen sind also his auf Reihe V so grolJ, daB die er- haltenen Resultate durchaur falsch ausfallen miissen. Diese Versuche lehrcn, daR hei schnelleiii Arbeiten nnch der S t u t z e r schcn Methode geringe Mengen Dicyandiamid iiberhaupt nicht wider gefunden werdcn, und daR der Fehler mit steigenden nicyandianiidinengcn geringer wird, iucist aber so grol3 bleibt, daR die Resultate unrichtig ausfallcn. Dcr Grund licgt in der Anwesenheit des Ammoniaks. I)a.wclbe hiilt rben rrhcblichc Mengrri Dicyandia- niidsilber in Losung. Die Filtrate der Ltkungen wurden nun nach C: a r o bis zum Ver- schwindcn dcs Animoniakgeruches eingedampft, dic Niederschlnge abfiltricrt und nach I< j e 1 d a h 1 verbrannt. diesem Grunde haben wir einigc erganzende Versuche ausgefiihrt, aus dcnen sich das Folgende ergab: diesem Grunde haben wir einigc erganzende Versuche ausgefiihrt, aus dcnen sich das Folgende ergab: Verluste sind nicht mehr fcststellbar. In] Grgenteil, die Zahlen 'allen um ein geringes zu hoch Bus. Der Grund hiwfiir ist niit groBtrr Wahrscheinlichkeit die kolloide aniorphc Eigenschnft des Kiedcr- jchlages und dic durch die grol3e aktive Oberflachr brgcinstigtr Ad- jorption von Animoniak oder Ammonialrsalzcii. Durch .4uswaschrn mit reinern Wasscr lassen sich bekaiintlich adsorbirrtc Substanzen kaum entferncn. Es sri hier nur an die bekamiten Eigeiischaften des Mangansupcroxyds, des Eiscrihydrouyds und der amorphcn Kicselsaure erinnert. Genau nach Vorschrift von S t u t z e r wurden alkoholische Lo- sungen von bekanntem Gehalt an Dicyandiamid in Becherglaser gegeben, mit reincm Alkohol das Volumen zu 50 ccm erganxt, 180 ccni Waaser, 10 ccni Silberlosuiig und 10 ccm de8 vorgeschriebenen Ammo- niaks hinzugcfiigt. Jeder Versuch ist doppelt angesctzt worden, um Irrtiimer auszuschliel3en. Nach dem Umriihren wurden 200 ccm in Bccherglaser abpipcttiert und mit 50 ccm 10yoiger NaOH vcrsctzt. Da die Stutzersche Vorschrift nicht angibt., ob die Niederschlage tofort zu filtrieren sind oder nicht, also den gegebcnen Fehlerquellen keinr Riicksicht tragt, sind Unstimmigkviten nirht zu vermeiden. Je geringer der Dicyandiamidgchalt cler nntersuchten Kalkstickstoff- proben ist, um so grol3er fallen die Fehlrr aus, wenn nicht durcli langeres Stehenlassen fur ein Entweichen des die Fallung des Dicyandiamidsilbers hemmenden Arnmoniaks gesorgt wird. Aber iiuch dann wird die Genauigkeit und Sicherheit der S t u t z e r schen Modifikation noch sehr zu wiinsclien iibrig lassen. Sie kann a m rliesen Griinden nach unserrn ICrfahrungen fur die technische Untrrsuchung nicht in Fragc koinmen und strllt, wenigstcns in vorliegcnder Form, kaum einv 1'r.rbessvrnng drr urspriinglichen C a r o schen Mrthode clar. [a. 2.1 Die in den fiinf verschiedenen Versuchsreihen vorhandencn Di- cyandiamidinengen waren: I. 11. 111. IV. V. 10 mg 20 mg 40 mg 60 mg 120 mg In den 4 Losungen der Reihen I. und 11. entstanden keine Nieder- echlage. Bci Vcrsuchsreihc III., IV. uiid V. fiel weiBes Dicyandiamid- silber aus. Da in der Vorschrift der Mcthode kcinc Angabcn gemacht sind, ob dns Piltrieren sofort erfolgen, oder ob die Losung mit drni Niedewchlag vor dem Filtrieren langere Zeit stehcn soll, wurdc bei Reilic 1V. bis V. M i t t c l z u r B e h a n d l u n g v o n W u n d e n u n d H a u t - 1; r a ii k h c i t c 11. D i c y a n d i a m i d i m F i l t r a t : 111. 1v. V. Erste LBsung. . . . . . 4,83 mg 8,59 mg 4,30 mg Xweite LBsung . . . . . $41 ,, 6,76 ,, 1,G'J ,, g ,,M o 11 p h o r 11 s" (H. Schmitz, CSlri-hliingersdorf) stellt nach R1 e i r o w s k y Is) eine hocliprozrntige, besonders praparierte, fliissigc Raffinack dar, dit: Rohrducker und Invertzucker in bestimm- tem Verhiiltnis enthalt. l h s l'raparat ist dickfliissig und zeigt keinc Neigung zum Krystallisivrcm, auch tiann nicht, wenn es in dunner Schicht ausgestrichcn wirtl. Ks knnn fur Trockenpinseluiigen und zur Herstvllung von Zinklcim nntl Vrrbandstoffen an Stelle von Glycerin vrrwrndet wcrdvn. 11 e i r o \v s k y gibt liivrfiir eine Reihc von S~~ezialvorschrifteii an. In den Losungen der Reihe 11. bildete sich nun nach einstiindigem Stehen erst an der Oberflache, dann auch im Inneren der Losung ein weil3brauncr Niederschlag, dcr bei noch langerem Stehen g r o h wurdc. Bei Reihe I cntstand ein Niederschlag erst nach 4-5 Stunden. Es wurden die Losungen bis zurn nachsten Tage sich selbst iiberlassen, mid dann die entstandencn Niederschlage abfiltriert und wie oben weiter behandelt. Unter der Bezrichnung ,,S a 11 o" komint jetzt rim: Hypochlorit- losung in den HandellY) (Alfrcd U'asmuth, Coln), die schon vor Jahren von G e r 1 a c hZo) in zerstilubtrr Form zur Behandlung von Lungen- tuberkulose vcrsucht wordcn ist. Sic enthalt im Liter angeblich 74,36 g wasserfreies Salz, i,94 g Gesamtchlor, 1,59 g disponibles Chlor, 32,24 g Natriumoxyd und 28,32 g BorsaureanhydridZi). K ii s t e r und G ii 11 z 1 e rZ1) benutzten Sano in einer Verdiinnung, die O,Z% disponibleni Chlor cntsprach, in Form voii Nebeln im Inhalatorium bei Bronchitiden und bei Rachen- und Kehlkopfkatarrhen sowie bei Meningokokken- und Diphtheriebacillentragern mit Erfolg. Die G e f u 11 d e n: Erste Liisung. . . . . . 8,59 mg 19,31 mg Dicyandiamid Zweite L6sung . . . . . 8,58 ,, 17,16 ,, ,, V o r h a n d e n . . . . . 10,00 ,, 20,OO ,, , I Im Filtrat waren noch an Dicyandiamid vorhanden: Erste Losung. . . . . . . 1,61 mg 1,40 rug Dicyandiamid Zweite Losung . . . 17) Miinch. hlcd. Woclienschr. 53, 1475 [I'JlG]; Angcw. Chcm. Is) Bed. klin. Wochenschr. 53, 1306 [1916l. lU) Vgl. Angcw. Chem. 29, I, 260 [191G]. 20) Therap. Monatsh. 16, 391 L19021. 21) Die in letzter Zeit vie1 geriihnite U a k i II sche LBsung ist demnach ihrer quulitativen Zusamnienset,zmig nach nichts Keues. "2) Zentralbl. f. Bakteriol. Orig. 78, 451 [1916]. 30, 21 [1917]. nach halbstiindigem Stehenlassen untcr oftrrrni L'm- riihren filtrirrt und mit 250-300 ccm Wasser sorgfaltig der Xicdrr- sclilag gewascheri, wahrend die Glascr dcr Reihcn I. und 11. zur Rrob- achtung beiseite gcstellt wurden. g [a. 2.1 G ef u n d e n : Erste LBsung . . . 32,18 mg 50,45 mg 116,3 tng Zweite Liisung. . . 3 4 3 ,, 54,17 ,, 118,5 ,, V o r h a n d e n : 111. IV. V. 40 mg 60 mg 120 mg 111. I v. v. Die Fehler betragcn also auf 100 Teile Dicyandiamid llezogen: Erstc LBsung . . . . . . 19,55y0 15,9204 :<,CJey;, Zweite Losung. . . . . . 14,L8,, 9,72 ,, 1,25,, G ef u n d e n : Erste LBsung . . . 32,18 mg 50,45 mg 116,3 tng Zweite Liisung. . . 3 4 3 ,, 54,17 ,, 118,5 ,, 111. IV. V. p 21) Die in letzter Zeit vie1 geriihnite U a k i II sche LBsung ist demnach ihrer quulitativen Zusamnienset,zmig nach nichts Keues. g g , , [ 20) Therap. Monatsh. 16, 391 L19021. (Wttellung der landwirtschaftlichen Versuchsstation Kempeu-Rheln.) Von G. H ~ Q E E und J. KEXN. (Eingeg. 9Jl. 1917.1 Nach w r e n Erfahrungen hat sich die folgende Arbeitsweise ah leicht und mit befriedigender Genauigkeit aus fiihrbar erwiesen: 5 g Kalkstickstoff werden in einem trockenen Kolben mit 100 ccm Alkohol ( 95-96y0) iibergossen und mehrere Stunden unter ofterem Umschhtteln stehen gelaasen. Vom Filtrat werden je 40 ccm in zwei groBere Bechergliiser abpipttiert und mit ungefiihr 150 ccm Wasser verdiinnt. In der einen Lijsung erfolgt die Bestimmung des Cyan- amidstickstoffs durch Fallen mit Silbernitrat und Ammoniak. Die geringen Mengen Cyanamidsilber setzen sich nur sehr langsam ab. Um die Ausflockung zu beschleunigen, werden 2-3 Tropfen Natriumphosphatlijsung zugegeben. Da sich in der Wsung steta Kalksalze vorfinden, bildet sich amorphes Calciumphosphat, welches das Cyanamidsilber niederreilt, so d a l das Filtrieren bald erfolgen kann. Der abfiltrieh und gut ausgewaschene Niederschlag wird nach K j e 1 d a h 1 verbrannt. Durch Zugabe von wechselnden Mengen Ammoniak, Natronlauge und 10 ccm Silbernitratlijsung zu einer U u n g von 0,1666 g Di- cyandiamidstickstoff bei einem Geaamtvolumen der Fliissigkeit von 2-220 ccm suchte S t u t z e r dasjenige Mengenverhaltnis von Ammoniak zu Natronlauge empirisch festzustellen, bei dem unter den angegebenen Arbeitabedingungen der Stickstoffverlust auf ein zuliissigae Minimum baechrjinkt bleibt.. Bei Verwendung von 10 ccm 100/8gen Ammoniaks und 50 ccm 10%iger Lauge waren diese Verluste am geringsten und betrugen 0,9, 2,1, und 5,2%. Nun erscheinen UTIB die Differenzen unter den drei Paralleluntersuchungen bedenklich g m l zu sein. Die Schwan- kungen uberschreiten weit die Fehlergrenzen der K j e 1 d a h 1 sohen Methode und konnen ihre Ursache nur in unbekannten Umstiinden haben. Bevor aber diese Ursachen der abweichenden Resultate nicht erkannt und beseitigt sind, scheinen diese Versuche ohne weiteres keine sichere Grundlage fiir eine analytische Methode zu bieten. Als Beleg fiir die Genauigkeit der Methode werden nur zwei Parallel- analysen mit je 0,1333 g Dicyandiamidstickstoff mgefiihrt. Ver- suche mit wechselnden verschieden groaen Mengen fehlen. Aus In der zweiten Lijsung wird nach Zugabe von Phenolphthalein dascyanamid und dasDicgandiamid mit Silbernitratlosung und durah tropfenweisee Zuflielenlassen von 5 oder lO%iger Natronlauge bis zur deutlichen alkalischen Reaktion gefiillt. Nach lebhaftem Um- in Angew. Chem. 1917. AufMt6tefl 0. Bd.) IU Nr. 17. Vierteljahresberieht iiber neue Arzneimittel. Im Auftrage der Fachgruppe fur medizinisch - pharmazeutische Chemie des Vereins deutscher Clieniiker erstattet von J NEWNER. (S hl E S 60 ) V o r h a n d e n : 40 mg 60 mg 120 mg Die Fehler betragcn also auf 100 Teile Dicyandiamid llezogen: J NEWNE (SchluE von S. 60.) (SchluE von S. 60.) lU) Vgl. Angcw. Chem. 29, I, 260 [191G]. M i t t c l z u r B e h a n d l u n g v o n W u n d e n u n d H a u t - 1; r a ii k h c i t c 11. . . . 1,68 ,, 3.36 ,, Bei dem liingeren Stehen war der Gehalt der Liisungen an Ammo- niak geringer geworden. Infolgedessen fiel das Dicyandiamidsilber nach und nach aus. Eiri weiterer Versuch bestatigte diese Annahme. Neue Liisungeii der Reihen 111.-V. wurden vor dem Zusatze von 50 ccm NaOH 24 Stunden offen stehen gelassen, d a m wurde die h u g e zugegeben und nach zwei Stunden filtriert. Gefunden . . . 42,l mg 62,5 mg 123 mg Dicyandiamid Vorhanden . . 40,O ,, 60,O ,, 120 ,, ,, Gefunden . . . 42,l mg 62,5 mg 123 mg Dicyandiamid Vorhanden . . 40,O ,, 60,O ,, 120 ,, ,,
https://openalex.org/W2620416884
https://www.scielo.br/j/csp/a/qNpFjbvXDN6Sgj7qhKrKpDG/?lang=en&format=pdf
Spanish; Castilian
null
Gender differences and psychotropic polypharmacy in psychiatric patients in Brazil: a cross-sectional analysis of the PESSOAS Project
Cadernos de Saúde Pública
2,017
cc-by
7,658
Gender differences and psychotropic polypharmacy in psychiatric patients in Brazil: a cross-sectional analysis of the PESSOAS Project Diferenças de gênero e polifarmácia psicotrópica em pacientes psiquiátricos no Brasil: uma análise transversal do Projeto PESSOAS Juliana de Oliveira Costa 1 Maria das Graças Braga Ceccato 1 Ana Paula Souto Melo 2 Francisco de Assis Acurcio 1 Mark Drew Crosland Guimarães 3 Diferencias de género y polifarmacia psicotrópica en pacientes psiquiátricos en Brasil: un análisis transversal del Proyecto PESSOAS doi: 10.1590/0102-311X00168915 doi: 10.1590/0102-311X00168915 Correspondence J. O. Costa Faculdade de Farmácia, Universidade Federal de Minas Gerais. Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brasil. juliana.olic@gmail.com 1 Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. 2 Faculdade de Medicina, Universidade Federal de São João del-Rei, Divinópolis, Brasil. 3 Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. This article is published in Open Access under the Creative Commons Attribution license, which allows use, distribution, and reproduction in any medium, without restrictions, as long as the original work is correctly cited. Cad. Saúde Pública 2017; 33(4):e00168915 Abstract Correspondence J. O. Costa Faculdade de Farmácia, Universidade Federal de Minas Gerais. Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brasil. juliana.olic@gmail.com Juliana de Oliveira Costa 1 Maria das Graças Braga Ceccato 1 Ana Paula Souto Melo 2 Francisco de Assis Acurcio 1 Mark Drew Crosland Guimarães 3 3 Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. Introduction Mental disorders are among the five most important contributors to the global burden of disease and disability. Together with substance use disorders, they account for 7.4% of disability-adjusted life-years lost and were the leading cause of years lived with disability worldwide in 2010 1. In Brazil, the prevalence of at least one lifetime mental disorder was 44.8% in a large metropolitan area, with an estimated lifetime risk at age 75 of 57.7% 2. Gender plays a vital role in neurobiological aspects, psychosocial factors, and behavioral patterns in several psychiatric disorders. Gender differences have been reported in the prevalence, type of diagnosis, treatment and behavior among individuals with mental disorders 3, with higher prevalence of disorders among boys during childhood and among women during adulthood 1,4. Women have a higher prevalence of affective and anxiety disorders while men have higher rates of substance use and antisocial personality disorders 1,2,5,6,7,8. The use of psychotropic medicines has been the main strategy for treatment of these individuals. In Brazil, their utilization appears to be three times higher among women, especially benzodiazepines and antidepressants 9,10. Women also seek and use health services more often than men 10. Drug inter­ actions and adverse events commonly occur with these medications, and are responsible for the most common cause of hospitalizations due to adverse drug events 11. Polypharmacy has no uniform definition, and can be classified as the number of medicines taken simultaneously, corresponding to the use of two or more medicines or be evaluated as minor poly­ pharmacy (concurrent use of two to four medicines) and major polypharmacy (concurrent use of five or more medicines) 12. Concerns with polypharmacy include the possibility of cumulative toxicity and worsening adherence by increasing the complexity of treatment 13,14. In addition, they increase the costs of therapy with strategies that are not well established in the literature 14. The prescription of multiple drugs for patients with mental illnesses has been an increasingly common practice without clear clinical effectiveness and with a potential likelihood of adverse drug reactions, despite controversies 13,15. Accordingly, the aim of this study was to assess the prevalence and correlates of psychotropic polypharmacy, defined as the use of two or more psychotropic medi­ cines from the same or a different pharmacological class, with emphasis on gender differences, in a national representative sample of psychiatric patients under care in Brazil. Correspondence Faculdade de Farmácia, Universidade Federal de Minas Gerais. Av. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brasil. We aimed to estimate the prevalence and correlates of psychotropic poly­ pharmacy in Brazilian psychiatric patients by gender. Sociodemographic, behavioral and clinical data were obtained through face-to-face interviews and medical charts of 2,475 patients. Psychotropic polypharmacy was defined as the use of two or more psychotropic drugs and occurred in 85.7% of men (95%CI: 83.6%-87.6%) and 84.9% of women (95%CI: 82.8%-86.8%; p > 0.05). The mean number of psychotropic drugs/patient was 2.98 ± 1.23 and most common combinations included antipsychotics. Multivariate analysis showed that for both genders, previous hospitalization, severe mental illness, multi­ ple psychiatric diagnoses and an insufficient number of professionals in the health care unit was associated with psychotropic polypharmacy. However, other correlates such as inpatient care, use of non-psychotropic drugs, living in unstable conditions and current smoking vary among them. Psychotropic polypharmacy was a common practice in this national sample. The results highlighted the need for national guidelines to manage patients with mental illness, considering the difference among genders and disease severity, to re­ duce the burden of polyphamacy in this population. 1 Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. 2 Faculdade de Medicina, Universidade Federal de São João del-Rei, Divinópolis, Brasil. 3 Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil. Polypharmacy; Psychotropic Drugs; Mental Disorders; Gender and Health 2 2 Costa JO et al. Cad. Saúde Pública 2017; 33(4):e00168915 Events and explanatory variables Psychotropic polypharmacy, the event of interest in this study, was defined as the concurrent use of two or more psychotropic medicines registered in the medical charts at the time of data collection. Patients under psychotropic polypharmacy were compared with patients on monotherapy and those who did not use any medication. For the purposes of this analysis, the potential explanatory variables were grouped into individual and institutional characteristics. The individual characteristics investigated were: (a) demographic: age (≥ 40 or < 40 years old), marital status (single/divorced/widowed or married/stable union); (b) socioeconomic: education level (≥ 5 or < 5 years of study), place of residence (unstable or stable), health insurance (yes or no), family income in the last month (≤ USD 200 or > USD 200); (c) behavioral: lifetime cigarette smoking (current smokers or ex-smokers or non-smokers), lifetime alcohol or illicit drug use (yes or no); (d) medical information: self-reported previous hospitalization (yes or no), non-psychotropic medicine prescription (yes or no), main psychiatric diagnosis, number of psychiatric diagnoses, non-psychiatric comorbidities and number of non-psychiatric diagnoses. Explanatory characteristics related to the mental health service were type of service setting (psychiatric hospital or CAPS), number of profes­ sionals, and availability of medicines in the centers. Current place of residence was defined as unstable (living in shelters, hostels, hospital or on the streets) and stable (living in a house or apartment). Current smokers were defined as those who reported smoking at least one cigarette per day regardless of the length of smoking period at the time of the interview, and ex-smokers those who used to smoke, but were not smokers at the time of the interview. Lifetime alcohol or illicit drug use was defined as the use of alcohol or any illicit drug regardless of the frequency or dose. Previous hospitalization was defined as self-reported hospitalization before the date of the inter­ view. The main psychiatric diagnosis was extracted from the patient’s chart and described according to the 10th revision of the International Classification of Diseases (ICD-10) 21. In the case of more than one diagnosis, these were hierarchically classified according to severity, as follows (1) psychotic disorders, depression with psychotic symptoms and bipolar disorder, (2) depression and anxiety, (3) substance use disorder, and (4) other (e.g. dementia, mood disorders and personality and behavior, epilepsy). Events and explanatory variables On account of the small numbers, categories (2), (3) and (4) were grouped together as non-severe mental illness and (1) corresponded to severe mental illness 22. Psychotropic medicines were classified according to the Anatomical Therapeutic Chemical (ATC) classification of the World Health Organization 23. Characteristics related to the services were obtained from the director of each site. The number of professionals was classified as sufficient and insufficient, considering the Brazilian Ministry of Health recommendation to health services operation. Medicines were classified as sufficient or insufficient based on a qualitative assessment and perception of the availability and variability of medicine lists for prescription by health professionals at the service, as described previously 17. Methods This study is part of a national multicenter study among psychiatric patients in Brazil, as described in detail in previous publications 16,17,18. Briefly, we conducted a multicenter cross-sectional study in 2006 in 11 public psychiatric hospitals and 15 outpatient Psychosocial Care Centers (CAPS). CAPS are public mental health outpatient clinics created to progressively reduce psychiatric hospital care. They provide consultations, therapeutic and creative workshops, physical and recreational activities, art therapy, and medication on a daily basis during week days 19, while hospitals attend patients under closed regimens for an average of 20 days. Medications provided at CAPS include at least those of the Brazilian National List of Essential Medi­ cines (RENAME in portuguese), while local health services can provide other medications as needed. In 2006, the list included three options of antipsychotic agents (haloperidol, chlorpromazine, lithium carbonate), two anxiolytics, hypnotics and sedatives (diazepam, clonazepam), two antiparkinsonian (biperiden, levodopa+carbidopa), four antidepressants (fluoxetine, amitriptyline, nortriptyline, clo­ mipramine) and four antiepileptics (carbamazenine, valproatesodium, phenytoin and phenobarbital) 20. In addition to the list, the provision of medicines in the hospitals depends on each institution’s stan­ dard practices. We obtained a two-stage probability sample, proportional to the type of care (inpatient or outpa­ tient) and the national distribution of the reported AIDS cases by region. Eligibility criteria included adult patients (≥ 18 years old) under psychiatric care in hospitals or adult CAPS, who were able to answer the questionnaire and to sign the informed consent, as assessed by a mental health profes­ sional interviewer. We obtained demographic, socioeconomic, behavioral, and selected clinical data Cad. Saúde Pública 2017; 33(4):e00168915 PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 3 through a semi-structured interview. Medical data, including psychiatric diagnoses and prescriptions, was obtained from medical charts. Protocol, questionnaires and procedures were tested in a pilot study, described previously 18. The project was approved by the participating centers and by the Ethics Research Committee of the Minas Gerais Federal University (COEP/UFMG, Etic 125/05) and the National Ethics Research Committee of the Brazilian Ministry of Health (CONEP 592/2006). Statistical analysis A descriptive analysis was carried out followed by bivariate analysis, stratified by gender. Pearson`s Chi-square was used to assess statistical differences in proportions. The magnitude of the associations between explanatory variables and psychotropic polypharmacy was estimated by the odds ratio (OR) with a 95% confidence interval (95%CI) and the level of significance considered was 0.05. Multivariate analysis was conducted by logistic regression. Variables with p-values equal to or less than 0.20 in the bivariate analysis were selected to start multivariate modeling. The backward Cad. Saúde Pública 2017; 33(4):e00168915 4 Costa JO et al. stepwise procedure was conducted, i.e. modeling, began with all variables (full model) and sequential deleting of each variable. Only variables with p-values equal to or less than 0.05 remained in the final model. Missing data were excluded from analysis. The likelihood ratio test was used to compare models, and fit was evaluated using the Hosmer-Lemeshow test. Statistical analyses were performed using the SAS 9.4 (SAS Inst., Cary, USA) and Stata 13 (StataCorp LP, College Station, USA) softwares. Results A total of 2,475 (90%) participants were interviewed. The remaining 10% of the sample (288) did not participate mainly because of refusal (52%). There was no statistically significant difference between participants and non-participants with regard to age, sex, schooling and psychiatric diagnoses. Among the 2,475 participants, 1,277 (51.6%) were women. Psychotropic medicines were pre­ scribed for 96.0% of the patients with a mean number of 2.98 psychotropic medicines per patient (SD = 1.23: coefficient of variation = 41%, and median = 3.0). Psychotropic polypharmacy occurred in 85.3% of patients (N = 2,111; 95%CI: 83.8%-86.6%), 85.7% among men (95%CI: 83.6%-87.6%) and 84.9% among women (95%CI: 82.8%-86.8%; p > 0.05). Non-psychotropic medicines were also pre­ scribed for 31.8% (95%CI: 30.0%-33.6%) of the patients (Table 1). Table 1 Description of psychiatric patients studied. PESSOAS Project, Brazil, 2006 (N = 2,475). Description of psychiatric patients studied. PESSOAS Project, Brazil, 2006 (N = 2,475). Characteristics Total (N = 2,475) Men (n = 1,198) Women (n = 1,277) n * % n * % n * % Mental health service Service setting (psychiatric hospital ) 898 36.3 537 44.8 361 28.3 ** Socio-demographics Age (≥ 40 years) 1,351 54.6 604 50.5 747 58.5 ** Schooling (< 5 years) 1,206 49.8 598 51.2 608 48.5 Marital status (single/divorced/widowed) 1,662 67.2 913 76.3 749 58.7 ** Place of residence (unstable) 306 12.4 199 16.6 107 8.4 ** Health insurance (yes) 256 10.6 112 9.7 144 11.4 Monthly family income (> USD 200) 1,017 64.5 497 70.0 520 60.0 ** Behavior Current smokers (yes) 1,298 52.7 761 63.8 537 42.3 ** Ex-smokers (yes) 465 18.9 209 17.5 256 20.2 Ever used alcohol (yes) 1,591 64.7 924 77.5 667 52.7 ** Ever used any illicit drug (yes) 621 25.2 438 36.7 183 14.4 ** Clinical Previous hospitalization (yes) 1,434 58.3 790 66.3 644 50.8 ** Psychotropic polypharmacy (≥ 2 medicines) 2,111 85.3 1027 85.7 1,084 84.9 Non-psychotropic medicines prescribed (yes) 774 31.8 435 36.6 339 27.2 ** Psychiatric diagnosis (ICD-10) [severe mental illness] 1,403 56.7 729 60.9 674 52.8 ** Number of psychiatric diagnoses (> 1) 994 40.2 496 41.4 498 39.0 Number of non-psychiatric diagnoses (> 1) 189 7.8 88 7.6 101 8.1 ICD-10: 10th revision of the International Classification on Diseases 21. * Excluded missing values; ** Statistical significance p < 0.05. Note: severe mental illness: psychotic disorders, depression with psychotic symptoms and bipolar disorder. Non-severe mental illness: depression and anxiety, substance use disorder, dementia, other. Cad. Saúde Pública 2017; 33(4):e00168915 PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 5 Considering the mental health service, 36.3% of patients were treated in a psychiatric hospitals, with a greater proportion of men (44.8%; p < 0.05). Regarding socio-demographic characteristics, 54.6% were 40 years old or over – with women older than men on average (p < 0.05) – and 49.8% of patients had less than five years of schooling. Men were more often single, divorced or widowed (76.3% vs. 58.7%), had an unstable place of residence (16.6% vs. 8.4%), and had family incomes higher than USD 200 in the last month (70% vs. 60%). Only 10.6% of all patients had health insurance. Table 1 There was a high prevalence of current smoking and alcohol use among patients (52.7% and 64.7%, respectively) and about a quarter of them declared having used any illicit drug during their lifetime, with men presenting higher rates than women in all of those characteristics. With respect to clinical characteristics, the majority of patients had been previously hospitalized (58.3%) and had severe mental illness diagnosis (56.7%), both with higher prevalence among men (p < 0.05). Moreover, 40.2% of the patients had more than one psychiatric diagnosis, while only 7.8% had more than one non-psychiatric diagnosis (Table 1). Among the 26 services included, 65.4% were considered to have an insufficient number of profes­ sionals (CAPS = 80%; hospitals = 45.5%) and 19.2% to have an insufficient number of mental health medicine available (CAPS = 36.7%; hospitals = 9%) (data not shown in tables). The ten most commonly prescribed medicines were haloperidol (15.7%), biperiden (10.5%), diaz­ epam (8.9%), chlorpromazine (8.7%), carbamazepine (7.3%), promethazine (6.9%), clonazepam (6.4%), levomepromazine (4.4%), amitriptyline (3.9%), and fluoxetine (3.4%), accounting for about 75% of all prescription medicine records. Considering the second and third levels of the ATC classification system, the most prescribed medicines were psycholeptics (antipsychotic agents = 37%; anxiolytics = 10%; hypnotics and sedatives = 1%), followed by antiepileptics (17%), psychoanaleptics (antidepres­ sants = 12%) and antiparkinsonian (anticholinergic agents = 11%) (data not shown in tables). The same pattern was observed for patients in psychotropic polypharmacy. Antipsychotics were most often combined with other medicines, either in the same class (37.5%) or with other classes, including antiepileptics (33.7%), anticholinergics (33.3%), anxiolytics (22.2%), antidepressants (17.9%), and to a lesser extent with hypnotics and sedatives (2.4%) (Table 2). i * Included only patients on psychotropic polypharmacy (≥ 2 medicines prescribed). Stratified gender analysis The bivariate analysis indicated that for both genders, severe mental illness diagnoses were associated with a higher chance of psychotropic polypharmacy, as well as the number of psychiatric diagnoses and inpatient care (i.e., psychiatric hospital), and self-reported previous hospitalization (p < 0.05). An insufficient number of professionals in the services was negatively associated with a higher chance of polypharmacy for both genders (p < 0.05). Furthermore, for both genders being single, divorced or widowed, and lifetime illicit drug use were associated with higher psychotropic polypharmacy (p < 0.05). Having more than five years of schooling and having health insurance were associated with psy­ chotropic polypharmacy among men only. Among women, more than one non-psychiatric diagnosis and reporting current smoking or alcohol use were associated with psychotropic polypharmacy (p < 0.05). The prescription of non-psychotropic medicines was positively associated with psychotropic medicines among women, and negatively associated among men (p < 0.05) (Table 3). Multivariate analysis revealed that correlates of polypharmacy differed between men and women. For both genders, patients previously hospitalized, with severe mental illness diagnoses, and with more than one psychiatric diagnosis had a higher chance of psychotropic polypharmacy (p < 0.05). Instead, an insufficient number of professionals in the health care unit was negatively associated with psychotropic polypharmacy (p < 0.05). Among men, inpatient care was associated with higher psycho­ tropic polypharmacy, while unstable living and the prescription of any non-psychotropic medicines were negatively associated with psychotropic polypharmacy (p < 0.05). Finally, among women, the prescription of non-psychotropic medicines and lifetime cigarette smoking were associated with a higher chance of psychotropic polypharmacy (p < 0.05) (Table 4). Cad. Saúde Pública 2017; 33(4):e00168915 Costa JO et al. 6 Table 2 Saúde Pública 2017; 33(4):e00168915 PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 7 Men (n = 1,198) Women (n = 1,277) Total OR 95%CI p-value Total OR 95%CI p-value 604 0.79 0.57-1.09 0.150 747 1.28 0.94-1.74 0.117 593 1.00 530 1.00 598 0.67 0.48-0.94 0.019 * 608 0.82 0.61-1.12 0.219 569 1.00 645 1.00 913 1.44 1.01-2.07 0.043* 749 1.65 1.21-2.24 0.001* 284 1.00 528 1.00 199 0.71 0.47-1.06 0.094 107 1.45 0.78-2.70 0.237 997 1.00 1168 1.00 112 2.02 1.00-4.07 0.046 * 144 0.65 0.42-1.00 0.050 1,042 1.00 1,117 1.00 497 1.11 0.70-1.77 0.664 520 0.72 0.50-1.05 0.088 213 1.00 346 1.00 761 1.24 0.89-1.74 0.195 537 2.00 1.43-2.80 < 0.001 * 209 0.95 0.62-1.44 0.795 256 1.10 0.75-1.63 0.621 222 1.00 476 1.00 924 1.24 0.85-1.80 0.262 667 1.44 1.05-1.96 0.021* 269 1.00 598 1.00 438 1.75 1.22-2.51 0.002 * 183 3.47 1.80-6.69 < 0.001 * 755 1.00 1,090 1.00 790 1.91 1.38-2.65 < 0.001 * 644 4.71 3.26-6.78 < 0.001 * 402 1.00 623 1.00 435 0.70 0.51-0.98 0.037 * 339 2.78 1.76-4.39 < 0.001 * 753 1.00 909 1.00 729 2.74 1.96-3.81 < 0.001 * 674 5.20 3.61-7.50 0.000 * 469 1.00 603 1.00 496 1.74 1.23-2.47 0.002 * 498 4.95 3.22-7.62 < 0.001 * 702 1.00 779 1.00 88 0.73 0.41-1.29 0.273 101 3.47 1.39-8.67 0.005 * 1,077 1.00 1,148 1.00 e Centers; ICD-10: 10th revision of the International Classification of Diseases 21; OR: odds ratio. Table 2 Table 2 Description of the combinations of psychotropic medicines prescribed according to the ATC classification. PESSOAS Project, Brazil, 2006 (n = 2,111). Description of the combinations of psychotropic medicines prescribed according to the ATC classification. PESSOAS Project Brazil 2006 (n = 2 111) Description of the combinations of psychotropic medicines prescribed according to the ATC classification. PESSOAS Project, Brazil, 2006 (n = 2,111). ATC classification (3rd level) combination n * % Same class combination Antipsychotics 792 37.5 Antiepileptics 207 9.8 Antidepressants 74 3.5 Anxiolytics 2 0.1 Anticholinergics 1 0.0 Different classes combination Antipsychotics + Antiepileptics 712 33.7 Antipsychotics + Anticholinergics 703 33.3 Antipsychotics + Anxiolytics 469 22.2 Antipsychotics+ Antidepressants 377 17.9 Antiepileptics + Antidepressants 313 14.8 Antiepileptics + Anticholinergics 288 13.6 Antidepressants + Anxiolytics 253 12.0 Anxiolytics + Anticholinergics 204 9.7 Antiepileptics + Anxiolytics 197 9.3 Anticholinergics + Antidepressants 142 6.7 Antipsychotics + Hypnotics and sedatives 50 2.4 Antidepressants + Hypnotics and sedatives 27 1.3 Antiepileptics + Hypnotics and sedatives 25 1.2 Anxiolytics + Hypnotics and sedatives 22 1.0 Anticholinergics + Hypnotics and sedatives 17 0.8 ATC: Anatomical Therapeutic Chemical classification system 23. * Included only patients on psychotropic polypharmacy (≥ 2 medicines prescribed). Table 3 Bivariate analysis of psychotropic polypharmacy, stratified by gender, among patients with mental disorders. PESSOAS Project, Brazil, 2006 (N = 2,475). Characteristics Men (n = 1,198) Women (n = 1,277) Total OR 95%CI p-value Total OR 95%CI p-value Mental health service Service setting Psychiatric hospital 537 1.65 1.18-2.32 0.003 * 361 3.97 2.46-6.42 < 0.001 * CAPS 661 1.00 916 1.00 Number of professionals Insufficient 743 0.57 0.39-0.80 0.001* 355 0.62 0.42-0.90 0.011 * Sufficient 455 1.00 922 1.00 Number of medicines Insufficient 189 1.05 0.67-1.65 0.825 329 0.82 0.58-1.16 0.262 Sufficient 1,009 1.00 948 1.00 (continues) Cad. Table 2 Table 3 (continued) Characteristics Men (n = 1,198) Women (n = 1,277) Total OR 95%CI p-value Total OR 95%CI p-value Socio-demographics Age (years) ≥ 40 604 0.79 0.57-1.09 0.150 747 1.28 0.94-1.74 0.117 < 40 593 1.00 530 1.00 Schooling (years) < 5 598 0.67 0.48-0.94 0.019 * 608 0.82 0.61-1.12 0.219 ≥ 5 569 1.00 645 1.00 Marital status Single/Divorced/Widowed 913 1.44 1.01-2.07 0.043* 749 1.65 1.21-2.24 0.001* Married/Stable union 284 1.00 528 1.00 Place of residence Unstable 199 0.71 0.47-1.06 0.094 107 1.45 0.78-2.70 0.237 Stable 997 1.00 1168 1.00 Health insurance Yes 112 2.02 1.00-4.07 0.046 * 144 0.65 0.42-1.00 0.050 No 1,042 1.00 1,117 1.00 Monthly family income (USD) > 200 497 1.11 0.70-1.77 0.664 520 0.72 0.50-1.05 0.088 ≤ 200 213 1.00 346 1.00 Behavior Smoking Current smoker 761 1.24 0.89-1.74 0.195 537 2.00 1.43-2.80 < 0.001 * Ex-smoker 209 0.95 0.62-1.44 0.795 256 1.10 0.75-1.63 0.621 Non-smoker 222 1.00 476 1.00 Alcohol use (ever) Yes 924 1.24 0.85-1.80 0.262 667 1.44 1.05-1.96 0.021* No 269 1.00 598 1.00 Any illicit drug use (ever) Yes 438 1.75 1.22-2.51 0.002 * 183 3.47 1.80-6.69 < 0.001 * No 755 1.00 1,090 1.00 Clinical Self-reported previous hospitalization Yes 790 1.91 1.38-2.65 < 0.001 * 644 4.71 3.26-6.78 < 0.001 * No 402 1.00 623 1.00 Non psychotropic medicines prescribed Yes 435 0.70 0.51-0.98 0.037 * 339 2.78 1.76-4.39 < 0.001 * No 753 1.00 909 1.00 Psychiatric diagnosis (ICD-10) Severe mental illness 729 2.74 1.96-3.81 < 0.001 * 674 5.20 3.61-7.50 0.000 * Non-severe mental illness 469 1.00 603 1.00 Number of psychiatric diagnoses > 1 496 1.74 1.23-2.47 0.002 * 498 4.95 3.22-7.62 < 0.001 * ≤ 1 702 1.00 779 1.00 Number of non-psychiatric diagnoses > 1 88 0.73 0.41-1.29 0.273 101 3.47 1.39-8.67 0.005 * ≤ 1 1,077 1.00 1,148 1.00 95%CI: 95% confidence interval; CAPS: Psychosocial Care Centers; ICD-10: 10th revision of the International Classification of Diseases 21; OR: odds ratio. * Statistical significance p < 0.05. Note: excluded missing values. Severe mental illness: psychotic disorders, depression with psychotic symptoms and bipolar disorder. Non-severe mental illness: depression and anxiety, substance use disorder, dementia, other. Table 3 (continued) Characteristics Cad. Saúde Pública 2017; 33(4):e00168915 Costa JO et al. 8 Table 4 Multivariate analysis of psychotropic polypharmacy, stratified by sex, among patients with mental disorders. PESSOAS Project, Brazil, 2006 (N = 2,245). Table 2 Characteristics Men * (n = 1,078) Women ** (n = 1,167) OR (95%CI) p-value OR (95%CI) p-value Mental health service Service setting (psychiatric hospital) 2.00 (1.23-3.24) 0.005 *** - - Number of professionals (insufficient) 0.61 (0.39-0.96) 0.033 *** 0.61 (0.38-0.97) 0.035 *** Socio-demographics Place of residence (unstable) 0.50 (0.30-0.83) 0.007 *** - - Behavior Current smoker (yes) - - 1.91 (1.27-2.87) 0.002 *** Ex-smoker (yes) - - 1.54 (0.96-2.45) 0.072 Clinical Self-reported previous hospitalization (yes) 1.59 (1.10-2.29) 0.014 *** 2.56 (1.68-3.89) < 0.001 *** Psychiatric diagnosis (severe mental illness) 2.61 (1.81-3.77) < 0.001 *** 2.91 (1.93-4.39) < 0.001 *** Number of psychiatric diagnoses (> 1) 1.56 (1.07-2.27) 0.022 *** 3.90 (2.48-6.12) < 0.001 *** Non psychotropic medicines prescribed (yes) 0.58 (0.38-0.86) 0.009 *** 1.96 (1.19-3.18) 0.008 *** * Hosmer-Lemeshow test: χ2 = 10.71; df = 8; p = 0.219; ** Hosmer-Lemeshow test: χ2 = 6.45; df = 8; p = 0.597; *** Statistical significance p < 0.05. Note: excluded missing values. Severe mental illness: psychotic disorders, depression with psychotic symptoms and bipolar disorder. Non-severe mental illness: depression and anxiety, substance use disorder, dementia, other. Cad. Saúde Pública 2017; 33(4):e00168915 Discussion The literature describes primary psychiatric diagnosis as one of the most important factors associated with psychotropic polypharmacy: patients with severe mental illnesses such as schizophrenia, depression with psychotic symptoms, and bipolar disorders are at greater risk of polypharmacy and increased use of psychotropic medicines 13,26,30,32,33. Patients with multiple psychiatric diagnoses also had increased chances of polypharmacy. These findings are corroborated by other studies, which pointed to an association between polypharmacy and markers of more severe illness, such as the number of psychiatric diagnoses 15, non-psychiatric comorbidities 12, and previous hospitalization 34. This could partially be explained by actual need of different drugs or monotherapy-refractory conditions that require changes in prescription regimens. Furthermore, similar to other countries, the reduction in the number of psychiatric hospital beds in Brazil may have resulted in discharge of patients with clinically severe conditions, meaning there is a greater need of medicalization in order to facilitate social interaction 13,31, despite the lack of evidence to support these inferences 27. We observed an independent association between psychotropic polypharmacy and current ciga­ rette smoking among women only. Studies in the general population have shown the association between smoking and the simultaneous use of four or more medicines 35 and also with psychotropic medicines 24. In addition, previous studies among psychiatric patients indicate an association between current smoking and substance use disorder diagnoses, but not with ex-smokers 36. Current smokers with substance use disorder diagnoses may be more refractory to smoking cessation and potentially in need of more adjuvant therapy. Whether this is only the case among women needs further investi­ gation and studies are needed to clarify this association. Among men, being currently hospitalized was associated with higher psychotropic polypharmacy, while having an unstable place of residence (living in shelters, hostels, streets, hospital), as well as hav­ ing a prescription for non-psychotropic medicines were associated with less psychotropic polyphar­ macy. We did not find any study that considered these two treatment settings (hospital and CAPS). However, a Brazilian study conducted in 2005 found that elderly patients with length of hospitalization of between 12 and 35 months had less polypharmacy than newly institutionalized patients, probably because at the time of admission these patients had greater severity and instability of the disease 37. Minor antipsychotic polypharmacy in hospitalized and homeless patients occurred in in-patient facilities in Italy in 2002-2003 26. Discussion The use of psychotropic medicines and the occurrence of psychotropic polypharmacy were common therapeutic approaches in this nationwide sample of patients with mental illness in Brazil. The pro­ portion of polypharmacy in this population was high and similar among genders, despite the fact that factors independently associated with psychotropic polypharmacy varied among them. Cross-sectional studies have shown that the use of psychotropic medicines is quite common in the general population. In Brazil, the prevalence of psychotropic use ranged from 6.5% to 13.3% 9,10,24, with an emphasis on the use of antidepressants, which were more common among women 9. In addi­ tion, only 16% of patients with a mental disorder were treated with psychotropic agents 9, contrasting with the high rate of prescriptions of these medicines in our study (96%). These differences may be due to the population, since our sample contains more severe cases than in the general population, and also due to the source of information obtained about the use of medicines (self-reported vs. medical chart register) and the diagnoses evaluated (self-reported vs. medical chart as registered by physi­ cians). Moreover, the difficulty of general practitioners to diagnose and treat patients with mental disorders in primary care settings and barriers of access to specialized health services 25 may also help to explain these differences. Studies have shown that psychotropic polypharmacy among patients with psychiatric disorders varies from 10% to 93% 13,26,27,28,29,30, in line with the psychotropic polypharmacy prevalence of 85.3% in our national sample. A trend of enhancing polypharmacy among psychiatric patients has been observed, with a decline in monotherapy strategy over the years from 47.8% in the 1970s, to 19.6% in the 1990s 31. This can also be observed in the mean number of psychotropic medications prescribed per individual. A combined analysis of 28 studies involving 23,428 patients showed a mean of 2.47 (1.5 to 4.0) psychotropic medicines/institutionalized patient 31. In recent studies this number is even higher (2.8) 28, as was observed in our study (2.98). Cad. Saúde Pública 2017; 33(4):e00168915 PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 9 Regarding risk factors relating to psychotic polypharmacy, we found that severe mental illness diagnoses were associated with psychotic polypharmacy for both men and women and almost 60% of the patients had severe mental illness diagnosis. Cad. Saúde Pública 2017; 33(4):e00168915 Discussion These studies are consistent with our findings, in which patients in current treatment at the hospitals, with greater severity of illness, showed greater polypharmacy, while those living in unstable conditions had lower polypharmacy. In our study, the most prescribed medicines were haloperidol, biperiden, diazepam, and chlor­ promazine, as reported in East Asia for schizophrenic patients 27. Antipsychotics were the most prescribed medicines (37%) and the most commonly used combinations involved these agents, as observed in other studies 13,28,30. However, antipsychotic polypharmacy is quite controversial in the literature. It has been associated with higher prescriptions of adjunctive medications such as anticholinergic and anti-parkinsonian medicines 26,27; higher daily dosages and higher rates of adverse events 14. The negative association between insufficiency of psychiatric professionals and lower poly­ pharmacy deserves attention. It should be noted that proper diagnostic assessment and the need for psychotropic prescription is better ascertained by trained psychiatric professionals. It is likely that this association is only a marker for a lack of psychiatrists, thus patients are not being evaluated for the use of psychotropic medicines. We should also note that the variable ‘insufficient availability of medicines’ was also found to be in the same direction in the univariate analysis, though it was not statistically significant. Regarding the availability of psychotropic medicines, in 80.8% of the Brazilian psychiatric ser­ vices this availability was considered sufficient. However, for most services (92.3%) the standardized medicine list included mainly first generation antipsychotic agents (e.g. haloperidol, chlorpromazine, lithium carbonate) 17. According to a recent Chinese study, the reduced availability of therapeu­ tic classes can potentially be associated with polypharmacy utilization 38. The updated RENAME did not include antipsychotic agents, but did include two anti-parkinson drugs (cabergoline and Cad. Saúde Pública 2017; 33(4):e00168915 Costa JO et al. 10 10 Costa JO et al. 10 Costa JO et al. levodopa+benserazide) and one hypnotics and sedative – midazolam. Alternatively, second-gener­ ation antipsychotics (clozapine, risperidone, olanzapine, quetiapine, and ziprasidone), other anti- parkinson drugs (amantadine, bromocriptine, entacapone, pramipexole, selegiline, tolcapone), other antiepileptics (ethosumide, gabapentin, lamotrigine, primidone, topiramate, trihexyphenidyl, viga­ bratrin) and the anxiolytic clobazam are provided by the Brazilian Unified National Health System (SUS) through the specialized component of pharmaceuticals in line with specific criteria 39. Discussion Another barrier to the quality of care in mental health that can contribute to the occurrence of psychotropic polypharmacy is the lack of a national guidelines for prescribing practices in psychiat­ ric care, considering the medicine-based evidence principles. These guidelines should address when polypharmacy is appropriated, for example, in short periods to control the exacerbation of symptoms, to prevent adverse events after increasing the dose of a single agent, or in patients that have been refractory to monotherapy 26,40,41. Case management, continuing education, treatment algorithms, reminders and feedbacks, and pharmaceutical care are other approaches that can be used to avoid inappropriate psychotropic prescribing 41. Some limitations of the study should be pointed out. We were not able to address polypharmacy with regard to its duration and reasons for using more than one medication, and this should be fur­ ther explored in future studies. The definition of psychotropic polypharmacy as the use of two or more psychotropic medicines led to a high rate of this event in this population, and therefore, the effect size of the associated factors may be potentially overestimated by the use of logistic regression. Psychiatric diagnoses were collected from medical charts and there was a limit of seven psychiatric medications that could be collected. Also, because the sample size was not a priori calculated to study polypharmacy stratified by gender, we may have had limited power to detect differences among selected variables with small numbers in both groups. Finally, the study design limits the establish­ ment of direct causality. The associations observed are only indicators of potential causal effect and they may contain residual confounding due to unmeasured variables. In addition, due to the scarcity of literature in the field regarding this population, comparisons are limited and other studies are needed to confirm these findings. Cad. Saúde Pública 2017; 33(4):e00168915 Acknowledgments This study was carried out by the UFMG with tech­ nical and financial support from the Ministry of Health/Secretariat of Health Surveillance/Depart­ ment of STDs, AIDS and Viral Hepatitis through the International Technical Cooperation Project 914/ BRA/1101 between the Brazilian Government and the United Nations Educational, Scientific and Cul­ tural Organization (UNESCO). 6. 6. Pedersen CB, Mors O, Bertelsen A, Waltoft BL, Agerbo E, McGrath JJ, et al. A comprehensive nationwide study of the incidence rate and life­ time risk for treated mental disorders. JAMA Psychiatry 2014; 71:573-81. 7. Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S, et al. Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey. Arch Gen Psy­ chiatry 1994; 51:8-19. References 1. Whiteford HA, Degenhardt L, Rehm J, Baxter AJ, Ferrari AJ, Erskine HE, et al. Global burden of disease attributable to mental and substance use disorders: findings from the Global Burden of Disease Study 2010. Lancet 2013; 382:1575-86. 1. Whiteford HA, Degenhardt L, Rehm J, Baxter AJ, Ferrari AJ, Erskine HE, et al. Global burden of disease attributable to mental and substance use disorders: findings from the Global Burden of Disease Study 2010. Lancet 2013; 382:1575-86. J. O. Costa carried out the data analysis and inter­ pretation, critical revision of relevant content and approval of final version for publication, as well as overseeing all stages of the research to guarantee the precision and integrity of all elements of the study. M. G. B. Ceccato carried out the data interpretation, write-up of the article and approval of final version for publication, as well as overseeing all stages of the research to guarantee the precision and integrity of all elements of the study. A. P. S. Melo contrib­ uted towards the data analysis and interpretation, article write-up and approval of the final version for publication. F. A. Acurcio contributed towards the project design, critical revision of relevant content and approval of the final version for publication. M. D. C. Guimarães contributed towards the proj­ ect design, data analysis and interpretation, criti­ cal revision of relevant content and approval of the final version for publication. 2. Viana MC, Andrade LH. Lifetime prevalence, age and gender distribution and age-of-onset of psychiatric disorders in the São Paulo Met­ ropolitan Area, Brazil: results from the São Paulo Megacity Mental Health Survey. Rev Bras Psiquiatr 2012; 34:249-60. 3. Afifi M. Gender differences in mental health. Singapore Med J 2007; 48:385-91. 4. Gispert R, Rajmil L, Schiaffino A, Herdman M. Sociodemographic and health-related corre­ lates of psychiatric distress in a general popula­ tion. Soc Psychiatry Psychiatr Epidemiol 2003; 38:677-83. 5. 5. Bijl RV, De Graaf R, Ravelli A, Smit F, Volle­ bergh WA; Netherlands Mental Health Survey and Incidence Study. Gender and age-specific first incidence of DSM-III-R psychiatric dis­ orders in the general population. Results from the Netherlands Mental Health Survey and In­ cidence Study (NEMESIS). Soc Psychiatry Psy­ chiatr Epidemiol 2002; 37:372-9. Conclusion In this nationwide sample of psychiatric patients, psychotropic medicine utilization and psychotropic polypharmacy were common practices, with differences in the set of associated factors among gen­ ders. There is a need for developing national guidelines to manage patients with mental illness, which should consider the role of gender and disease severity. Other strategies such as case management, continuing education and pharmaceutical care can also contribute to the reduction of the burden of polyphamacy in this population. PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 11 Conflict of interest The use of drug mono­ therapy in psychiatric inpatient treatment. Prog Neuropsychopharmacol Biol Psychiatry 2002; 26:547-51. 18. Guimarães MD, Oliveira HN, Campos LN, San­ tos CA, Gomes CE, Oliveira SB, et al. Reliability and validity of a questionnaire on vulnerability to sexually transmitted infections among adults with chronic mental illness: PESSOAS Project. Rev Bras Psiquiatr 2008; 30:55-9. 32. Bernardo M, Coma A, Ibáñez C, Zara C, Bari JM, Serrano-Blanco A. Antipsychotic polyphar­ macy in a regional health service: a population- based study. BMC Psychiatry 2012; 12:42. y y y 33. Morrato EH, Dodd S, Oderda G, Haxby DG, Allen R, Valuck RJ. Prevalence, utilization pat­ terns, and predictors of antipsychotic poly­ pharmacy: experience in a multistate Medic­ aid population, 1998-2003. Clin Ther 2007; 29:183-95. 19. Departamento de Ações Programáticas Estraté­ gicas, Secretarias de Atenção à Saúde, Ministé­ rio da Saúde. Saúde mental no SUS: os centros de atenção psicossocial. Brasília: Ministério da Saúde; 2004. (Série F. Comunicação e Educação em Saúde). 34. Suokas JT, Suvisaari JM, Haukka J, Korhonen P, Tiihonen J. Description of long-term polyphar­ macy among schizophrenia outpatients. Soc Psychiatry Psychiatr Epidemiol 2013; 48:631-8. 20. Departamento de Assistência Farmacêutica e Insumos Estratégicos, Secretaria de Ciência, Tecnologia e Insumos Estratégicos, Ministério da Saúde. Relação Nacional de Medicamentos Essenciais – RENAME. 5a Ed. Brasília: Minis­ tério da Saúde; 2007. 35. Pappa E, Kontodimopoulos N, Papadopoulos AA, Tountas Y, Niakas D. Prescribed-drug uti­ lization and polypharmacy in a general popu­ lation in Greece: association with sociodemo­ graphic, health needs, health-services utiliza­ tion, and lifestyle factors. Eur J Clin Pharmacol 2011; 67:185-92. 21. World Health Organization. ICD-10: interna­ tional statistical classification of diseases and related health problems, 10th revision. Geneva: World Health Organization; 2004. 36. Barros FC, Melo AP, Cournos F, Cherchiglia ML, Peixoto ER, Guimarães MD. Cigarette smoking among psychiatric patients in Brazil. Cad Saúde Pública 2014; 30:1195-206. 22. Schinnar AP, Rothbard AB, Kanter R, Jung YS. An empirical literature review of definitions of severe and persistent mental illness. Am J Psy­ chiatry 1990; 147:1602-8. y 23. WHO Collaborating Centre for Drug Statistics Methodology; Norwegian Institute of Pub­ lic Health. ATC/DDD index database. http:// www.whocc.no/atc_ddd_index/ (accessed on 18/Apr/2013). 37. Lucchetti G, Granero AL, Pires SL, Gorzoni ML. Fatores associados à polifarmácia em ido­ sos institucionalizados; Factors associated to polypharmacy in institutionalized elderly. Rev Bras Geriatr Gerontol 2010; 13:51-8. 38. Conflict of interest The authors declare no conflict of interests. 8. Jané-Llopis E, Matytsina I. Mental health and alcohol, drugs and tobacco: a review of the co­ morbidity between mental disorders and the use of alcohol, tobacco and illicit drugs. Drug Alcohol Rev 2006; 25:515-36. 9. Quintana MI, Andreoli SB, Moreira FG, Ri­ beiro WS, Feijo MM, Bressan RA, et al. Epide­ miology of psychotropic drug use in Rio de Ja­ neiro, Brazil: gaps in mental illness treatments. PLoS One 2013; 8:e62270. 10. Lima MC, Menezes PR, Carandina L, Cesar CL, Barros MB, Goldbaum M. Transtornos mentais comuns e uso de psicofármacos: impacto das condições socioeconômicas. Rev Saúde Pública 2008; 42:717-23. 11. Hahn M, Braus DF. Psychiatric polypharmacy: hazard through drug-drug-interaction and pos­ sibilities for prevention. Versicherungsmedizin 2012; 64:127-31. 12. Viola R, Csukonyi K, Doró P, Janka Z, Soós G. Reasons for polypharmacy among psychiatric patients. Pharm World Sci 2004; 26:143-7. 13. De las Cuevas C, Sanz EJ. Polypharmacy in psy­ chiatric practice in the Canary Islands. BMC Psychiatry 2004; 4:18. 14. Tranulis C, Skalli L, Lalonde P, Nicole L, Stip E. Benefits and risks of antipsychotic polyphar­ macy: an evidence-based review of the litera­ ture. Drug Saf 2008; 31:7-20. Cad. Saúde Pública 2017; 33(4):e00168915 Costa JO et al. 12 Costa JO et al. 12 15. Mojtabai R, Olfson M. National trends in psy­ chotropic medication polypharmacy in office- based psychiatry. Arch Gen Psychiatry 2010; 67:26-36. 28. Jordanova V, Maric NP, Alikaj V, Bajs M, Cavic T, Iosub D, et al. Prescribing practices in psy­ chiatric hospitals in Eastern Europe. Eur Psy­ chiatry 2011; 26:414-8. 16. Guimarães MD, Campos LN, Melo AP, Carmo RA, Machado CJ, Acurcio FA, et al. Prevalence of HIV, syphilis, hepatitis B and C among adults with mental illness: a multicenter study in Bra­ zil. Rev Bras Psiquiatr 2009; 31:43-7. 29. Sarkar P, Chakraborty K, Misra A, Shukla R, Swain SP. Pattern of psychotropic prescription in a tertiary care center: a critical analysis. In­ dian J Pharmacol 2013; 45:270-3. 30. Adeponle AB, Obembe AO, Adeyemi SO, Su­ leiman GT. Polypharmacy in psychiatric out- patient practice in northern Nigeria. Afr J Psy­ chiatry (Johannesbg) 2007; 10:215-8. 17. Melo APS, Acúrcio FA, Cherchiglia ML, Gui­ marães CC, Veloso MDCG. Avaliação de servi­ ços de saúde mental: assistência e prevenção às doenças sexualmente transmissíveis no contex­ to do Projeto PESSOAS. Rev Méd Minas Gerais 2007; 17(1/2, Suppl 4):S240-8. 31. Rittmannsberger H. Resumen Os autores tiveram como objetivo estimar a pre­ valência da polifarmácia psicotrópica e fatores associados entre pacientes psiquiátricos brasileiros, segundo o gênero. Foram obtidos dados sociodemo­ gráficos, comportamentais e clínicos por meio de entrevistas presenciais e prontuários médicos de 2.475 pacientes. A polifarmácia psicotrópica foi definida como o uso de dois ou mais medicamentos psicotrópicos, e foi constatada em 85,7% dos ho­ mens (IC95%: 83,6%-87,6%) e 84,9% das mulhe­ res (IC95%: 82,8%-86,8%; p > 0,05). A média de medicamentos psicotrópicos por paciente foi 2,98 ± 1,23, e as combinações mais comuns incluíam os antipsicóticos. A análise multivariada mostrou que, para ambos os gêneros, internação hospita­ lar prévia, doença psiquiátrica grave, múltiplos diagnósticos psiquiátricos e número insuficiente de profissionais na unidade de saúde foram as­ sociados à polifarmácia psicotrópica. Entretanto, fatores como cuidados hospitalares, uso de drogas não-psicotrópicas, condições de vida instáveis e ta­ bagismo atual variaram de acordo com o gênero. A polifarmácia psicotrópica foi uma prática comum nessa amostra nacional. Os resultados evidenciam a necessidade de diretrizes nacionais para o ma­ nejo de pacientes com transtornos mentais, con­ siderando a diferença entre gêneros e a gravidade da doença, para reduzir a carga da polifarmácia nessa população de pacientes. El objetivo de los autores fue estimar la prevalencia de la polifarmacia psicotrópica y factores asocia­ dos entre pacientes psiquiátricos brasileños, según con el género. Se obtuvieron datos sociodemográ­ ficos, comportamentales y clínicos, a través de en­ trevistas presenciales y registros médicos de 2.475 pacientes. La polifarmacia psicotrópica se definió como el uso de dos o más medicamentos psicotró­ picos, y fue constatada en un 85,7% de los hombres (IC95%: 83,6%-87,6%) y en un 84,9% de las mu­ jeres (IC95%: 82,8%-86,8%; p > 0,05). La media de medicamentos psicotrópicos por paciente fue de 2,98 ± 1,23, y las combinaciones más comunes in­ cluían los antipsicóticos. El análisis multivariado mostró que, para ambos géneros, el internamiento hospitalario previo, enfermedad psiquiátrica gra­ ve, múltiples diagnósticos psiquiátricos, y número insuficiente de profesionales en la unidad de salud, estuvieron asociados a la polifarmacia psicotrópi­ ca. Asimismo, otros correlatos, tales como cuidados hospitalarios, uso de medicamentos no-psicotrópi­ cos, condiciones de vida inestables y tabaquismo actual variaron de acuerdo con el género. La po­ lifarmacia psicotrópica fue una práctica común en esa muestra nacional. Conflict of interest Li Q, Xiang YT, Su YA, Shu L, Yu X, Chiu HF, et al. Antipsychotic polypharmacy in schizo­ phrenia patients in China and its association with treatment satisfaction and quality of life: findings of the third national survey on use of psychotropic medications in China. Aust N Z J Psychiatry 2015; 49:129-36. 24. Rodrigues MA, Facchini LA, Lima MS. Modifi­ cações nos padrões de consumo de psicofárma­ cos em localidade do Sul do Brasil. Rev Saúde Pública 2006; 40:107-14. 25. Ballester DA, Filippon AP, Braga C, Andreoli SB. The general practitioner and mental health problems: challenges and strategies for medical education. São Paulo Med J 2005; 123:72-6. 39. Departamento de Assistência Farmacêutica e Insumos Estratégicos, Secretaria de Ciência, Tecnologia e Insumos Estratégicos, Ministério da Saúde. Relação Nacional de Medicamentos Essenciais – RENAME. 9a Ed. Brasília: Minis­ tério da Saúde; 2015. 26. Santone G, Bellantuono C, Rucci P, Picardi A, Preti A, Girolamo G. Patient characteristics and process factors associated with antipsychotic polypharmacy in a nationwide sample of psy­ chiatric inpatients in Italy. Pharmacoepidemiol Drug Saf 2011; 20:441-9. 40. Sernyak MJ, Rosenheck R. Clinicians’ reasons for antipsychotic coprescribing. J Clin Psychia­ try 2004; 65:1597-600. 27. Sim K, Su A, Fujii S, Yang SY, Chong MY, Un­ gvari GS, et al. Antipsychotic polypharmacy in patients with schizophrenia: a multicentre comparative study in East Asia. Br J Clin Phar­ macol 2004; 58:178-83. 41. Kukreja S, Kalra G, Shah N, Shrivastava A. Polypharmacy in psychiatry: a review. Mens Sana Monogr 2013; 11:82-99. Cad. Saúde Pública 2017; 33(4):e00168915 PSYCHOTROPIC POLYPHARMACY BY GENDER IN BRAZIL: PESSOAS PROJECT 13 Polifarmacia; Psicotrópicos; Trastornos Mentales; Género y Salud Cad. Saúde Pública 2017; 33(4):e00168915 Submitted on 14/Oct/2015 Final version resubmitted on 28/Mar/2016 Approved on 19/Aug/2016 Resumen Los resultados destacan la necesidad de directrices nacionales para cuidar de pacientes con trastornos mentales, considerando la diferencia entre géneros y la gravedad de la enfer­ medad, para reducir la carga de la polifarmacia en esa población de pacientes. Polimedicação; Psicotrópicos; Transtornos Mentais; Gênero e Saúde Polifarmacia; Psicotrópicos; Trastornos Mentales; Género y Salud Cad. Saúde Pública 2017; 33(4):e00168915
https://openalex.org/W2004501086
https://bmchealthservres.biomedcentral.com/counter/pdf/10.1186/1472-6963-14-11
English
null
Determinants of financial performance of home-visit nursing agencies in Japan
BMC health services research
2,014
cc-by
7,311
Abstract Background: Japan has the highest aging population in the world and promotion of home health services is an urgent policy issue. As home-visit nursing plays a major role in home health services, the Japanese government began promotion of this activity in 1994. However, the scale of home-visit nursing agencies has remained small (the average numbers of nursing staff and other staff were 4.2 and 1.7, respectively, in 2011) and financial performance (profitability) is a concern in such small agencies. Additionally, the factors related to profitability in home-visit nursing agencies in Japan have not been examined multilaterally and in detail. Therefore, the purpose of the study was to examine the determinants of financial performance of home-visit nursing agencies. Methods: We performed a nationwide survey of 2,912 randomly selected home-visit nursing agencies in Japan. Multinomial logistic regression was used to clarify the determinants of profitability of the agency (profitable, stable or unprofitable) based on variables related to management of the agency (operating structure, management by a nurse manager, employment, patient utilization, quality control, regional cooperation, and financial condition). Results: Among the selected home-visit nursing agencies, responses suitable for analysis were obtained from 1,340 (effective response rate, 46.0%). Multinomial logistic regression analysis showed that both profitability and unprofitability were related to multiple variables in management of the agency when compared to agencies with stable financial performance. These variables included the number of nursing staff/rehabilitation staff/patients, being owned by a hospital, the number of cooperative hospitals, home-death rate among terminal patients, controlling staff objectives by nurse managers, and income going to compensation. Conclusions: The results suggest that many variables in management of a home-visit nursing agency, including the operating structure of the agency, regional cooperation, staff employment, patient utilization, and quality control of care, have an influence in both profitable and unprofitable agencies. These findings indicate the importance of consideration of management issues in achieving stable financial performance in home-visit nursing agencies in Japan. The findings may also be useful in other countries with growing aging populations. Keywords: Home-visit nursing agency, Financial performance, Profit, Management positioned as one major service. The government started to promote home-visit nursing as health policy (medical insurance) in 1994 and as social policy (long- term care insurance) in 2000. Since then, there have been many positive revisions of both policies with re- gard to home-visit nursing [2,3]. * Correspondence: sfukui@redcross.ac.jp 1Department of Community Health Nursing, Graduate School of Nursing, The Japanese Red Cross University, Tokyo, Japan 4Graduate School of Nursing, The Japanese Red Cross University, 4-1-3 Hiroo, Shibuya-ku, Tokyo 150-0012, Japan Full list of author information is available at the end of the article Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Open Access Abstract However, the number of home-visit nursing agencies has remained at about 6,000 for the last decade (6,047 in 2011) [4]. This is low compared to other community-based services covered by long-term care insurance (e.g. 28,016 home-help ser- vices and 28,527 day care services in 2011) [5]. In addition, most home-visit nursing agencies have few © 2014 Fukui 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. © 2014 Fukui 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. Determinants of financial performance of home- visit nursing agencies in Japan Sakiko Fukui1,4*, Kazuhiro Yoshiuchi2, Junko Fujita1 and Sumie Ikezaki3 Background Japan demographically became the oldest country in the world in 2004 (19.5% at age 65+) and this rapid aging trend is likely to continue in the future (39.6% at age 65+ by 2050) [1]. The Japanese government has responded to this population shift by promoting home health ser- vices for the past two decades, with home-visit nursing Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Page 2 of 8 Page 2 of 8 staff (the average numbers of nursing staff and other staff per agency were 4.2 and 1.7, respectively in 2011) [6]. This small scale may a major reason for the small number of home-visit nursing agencies with stable fi- nancial performance [7-10]. found between continuous values and financial perform- ance, Dunnett multiple comparison was performed to compare agencies that were “profitable,” “stable,” and “unprofitable”. Next, to clarify the final determinants related to fi- nancial performance, multinominal logistic regression analysis was performed, using all variables in which a significant tendency or difference (p < .10) was found by univariate analysis. In the analysis, we examined the relationship of “profitable” and “un- profitable” agencies, with the “stable” agencies as a reference. After confirming a significant difference in variables of continuous values in the multinominal logistic regression analysis, categorical variables ob- tained by dividing the continuous value by the aver- age value were used to obtain useful indices for actual financial management. Since the relationship coefficients were high between the total number of staff members and the number of nursing staff, and between the total number of patients and the num- ber of patients covered by medical/long-term care insurance, the number of nursing staff and the num- ber of patients covered by medical/long-term care insurance were examined in multinominal logistic re- gression analysis, taking multicollinearity into ac- count. All data analyses were performed using SAS statistics software (version 9.2). To improve this situation, we have conducted nation- wide surveys of home-visit nursing agencies every 4 years [7-10]. We found that an agency’s profitability is influenced by variables associated with management of the agency, such as operating structure, management by a nurse manager, employment, patient utilization, quality control, regional cooperation, and financial condition. However, it is unclear which of these variables has the strongest impact on the agency’s profitability. Investigation variables We asked the agencies to return the questionnaire after explaining the objectives and details in a letter requesting participation in the study. In this letter, we also indicated that return of the questionnaire would be considered to indicate agreement to participation. In the questionnaire, the agencies were requested to pro- vide information on financial performance (profitable, stable, unprofitable or unclear) and variables in manage- ment of the agency, using the following seven categories: A) Operating structure; B) Management by a nurse man- ager; C) Employment; D) Patient utilization; E) Quality control; F) Regional cooperation; G) Financial condition during one month (September 2012). These variables (Table 1) were selected based on those found to affect fi- nancial performance (profitability) of home-visit nursing agencies in our studies in 2009-2011 [8-10]. Background Thus, in the current study we aimed to examine the determinants of financial performance (agency profitability) among variables related to management (operating structure, management by a nurse manager, employment, patient utilization, quality control, regional cooperation, and fi- nancial condition) in home-visit nursing agencies across Japan. Financial performance (profitability) of home-visit nursing agencies We sent the questionnaire to 2,993 home-visit nursing agencies, but 81 agencies did not receive the question- naire due to an unclear address. The final analysis was based on responses collected from 1,340 of the other 2,912 agencies (response rate: 46.0%). Among the 1,340 responding agencies, most showed profitability (582 [43.4%]), followed by 380 that were stable (28.4%), 242 unprofitable (18.1%), 101 unclear (7.5%), and 35 with no answer (2.6%) in September 2012. Therefore, we ana- lyzed a total of 1,204 agencies, excluding those with an unclear answer or no answer regarding profitability. Ethical consideration Th d f The study was performed after obtaining approval from the ethical committee of the Japanese Red Cross University (approval No: 2012–88). Participants and procedures The study surveyed 2,912 home-visit nursing agencies, which accounted for 50% of the randomly selected 5,824 agencies listed in the 47 prefectural databases across Japan that publish information about home-visit nursing agencies (as of September 2012). Questionnaires were sent to and collected from these home-visit nursing agencies from November to December 2012. Statistical analysis Patient utilization Number of patients using long-term care insurance 67.2 ± 49.5* 48.2 ± Number of patients using medical insurance 23.2 ± 21.1* 15.7 ± Total number of patients 89.6 ± 61.5* 62.8 ± Number of patients per nursing staffe 18.1 ± 16.8* 14.5 ± Percentage of patients covered by medical insurancee 25.6 ± 14.8 25.1 ± Number of new patients of medical insurance 3.4 ± 3.1* 2.4 ± Number of new patients of long-term care insurance 2.0 ± 2.7* 1.2 ± Percentage of new patientse 6.5 ± 4.8 6.4 ± Percentage of bedridden patientsf 28.8 ± 17.2 30.2 ± Percentage of patients who ended the service due to improvement 4.7 ± 6.1 6.1 ± Percentage of patients who ended the service due to hospitalized 6.0 ± 8.0 5.8 ± Percentage of home deaths among terminal patientsg 70.3 ± 30.8 67.1 ± Table 1 Relationship between financial performance (profitable, stable and unprofitable) and management of home-visit nursing agency (in September 2012) (n = 1204, 100%) Variables Profitable Stable Unprofitable n = 582 (48.3%) n = 380 (31.6%) n = 242 (20.1%) p value A) A. Operating structure Average ± Standard deviation or number of subjects (%) Establishment year of a home-visit nursing agency 2000 ± 5.5 2001 ± 5.8 2001 ± 5.7 0.37 Establishment year of a home-visit nursing agency (2011 or later) 28 (4.8) 25 (6.6) 21 (8.7) 0.11 Number of other home-visit nursing agencies managed by the same corporation 7.0 ± 11.3 5.5 ± 8.9 6.6 ± 10.3 0.53 Owned by a hospital 236 (42.0) 133 (37.8) 118 (50.9) 0.007** B. Management by a nurse manager Years of experience of nurse manager 7.0 ± 5.0* 6.2 ± 4.7 5.7 ± 4.7 0.001** Number of home visits per month by the nurse manager 52.0 ± 26.1 49.6 ± 25.0 39.8 ± 21.4 * <.001*** Experience of nurse manager in management training 421.0 (73.5) 252.0 (67.2) 147.0 (61.8) 0.003** Nurse manager’s capability with financial management 342 (59.4) 195 (52.3) 104 (44.4) <.001*** C. Statistical analysis Employment Number of nursing staff (converted to full-time staff) 5.7 ± 3.5* 4.5 ± 2.3 4.1 ± 2.0 <.001*** Number of rehabilitation staff (converted to full-time staff)a 1.4 ± 2.9* 0.9 ± 1.9 0.6 ± 1.6 <.001*** Total number of staff (converted to full-time staff)b 8.3 ± 5.7* 6.4 ± 4.2 5.3 ± 3.2* <.001*** Ratio of nursing staff within total staffc 74.5 ± 20.6* 78.0 ± 20.4 83.5 ± 18.4* <.001*** Ratio of rehabilitation staff within total staffc 13.0 ± 17.3* 10.5 ± 16.3 7.0 ± 14.1* <.001*** Ratio of full-time staffd 69.8 ± 21.6 71.9 ± 22.7 75.2 ± 22.6 0.007** D. Patient utilization Number of patients using long-term care insurance 67.2 ± 49.5* 48.2 ± 35.3 33.8 ± 24.7* <.001*** Number of patients using medical insurance 23.2 ± 21.1* 15.7 ± 16.2 11.0 ± 10.0* <.001*** Total number of patients 89.6 ± 61.5* 62.8 ± 43.4 44.5 ± 28.8* <.001*** Number of patients per nursing staffe 18.1 ± 16.8* 14.5 ± 8.8 11.3 ± 7.0* <.001*** Percentage of patients covered by medical insurancee 25.6 ± 14.8 25.1 ± 16.7 26.1 ± 19.2 0.78 Number of new patients of medical insurance 3.4 ± 3.1* 2.4 ± 2.4 1.9 ± 2.1* <.001*** Number of new patients of long-term care insurance 2.0 ± 2.7* 1.2 ± 1.9 1.4 ± 3.2 <.001*** Percentage of new patientse 6.5 ± 4.8 6.4 ± 6.5 7.8 ± 11.4 0.04* Percentage of bedridden patientsf 28.8 ± 17.2 30.2 ± 19.3 32.1 ± 21.6 0.09† Percentage of patients who ended the service due to improvement 4.7 ± 6.1 6.1 ± 8.6 7.6 ± 10.9* <.001*** Percentage of patients who ended the service due to hospitalized 6.0 ± 8.0 5.8 ± 5.5 7.0 ± 11.9 0.22 Percentage of home deaths among terminal patientsg 70.3 ± 30.8 67.1 ± 34.8 62.1 ± 35.3 0.04* Fukui et al. BMC Health Services Research 2014, 14:11 Page 3 of 8 http://www.biomedcentral.com/1472-6963/14/11 le, stable and unprofitable) and management of home-visit nursing agency (in September 2012) Statistical analysis First, the relationships between financial performance (profitable group, stable group, and unprofitable group) and each variable in management of the agency from the seven categories (A to G) were analyzed using univariate analyses. The relationships between financial performance and cat- egorical variables were analyzed by χ2 test, while one-way analysis of variance was performed to analyze relationships with continuous values. When a significant difference was Table 1 Relationship between financial performance (profitable, stable and unprofitable) and management of home-vis (n = 1204, 100%) Variables Profitable Stab n = 582 (48.3%) n = 380 ( A. Operating structure Average ± Standard deviation or number of Establishment year of a home-visit nursing agency 2000 ± 5.5 2001 ± Establishment year of a home-visit nursing agency (2011 or later) 28 (4.8) 25 (6 Number of other home-visit nursing agencies managed by the same corporation 7.0 ± 11.3 5.5 ± Owned by a hospital 236 (42.0) 133 (3 B. Management by a nurse manager Years of experience of nurse manager 7.0 ± 5.0* 6.2 ± Number of home visits per month by the nurse manager 52.0 ± 26.1 49.6 ± Experience of nurse manager in management training 421.0 (73.5) 252.0 (6 Nurse manager’s capability with financial management 342 (59.4) 195 (5 C. Employment Number of nursing staff (converted to full-time staff) 5.7 ± 3.5* 4.5 ± Number of rehabilitation staff (converted to full-time staff)a 1.4 ± 2.9* 0.9 ± Total number of staff (converted to full-time staff)b 8.3 ± 5.7* 6.4 ± Ratio of nursing staff within total staffc 74.5 ± 20.6* 78.0 ± Ratio of rehabilitation staff within total staffc 13.0 ± 17.3* 10.5 ± Ratio of full-time staffd 69.8 ± 21.6 71.9 ± D. D. Patient utilization Table 1 Relationship between financial performance (profitable, stable and unprofitable) and management of home-visit nursing agency (in September 2012) n = 1204, 100%) (Continued) E. Quality control (during the last year) Planned learning opportunities per year for all staff members 524 (90.0) 327 (86.0) 212 (87.6) 0.16 Conducting regularly of patient satisfaction survey 236 (41.3) 127 (34.2) 91 (39.1) 0.11 Conducting regularly of care monitoring among staff 262 (45.9) 139 (37.5) 90 (38.6) 0.02* Conducting regularly of control of staff objectives by nurse manager 323 (56.6) 136 (36.7) 94 (40.3) <.001*** Conducting regularly of visits of staff with nurse manager 166 (28.5) 112 (29.5) 67 (27.7) 0.89 F. Regional cooperation (during the last year) Number of hospitals with cooperation in the community 27.7 ± 23.1* 20.2 ± 17.4 14.7 ± 12.9* <.001*** Activities for regional contribution: Open lecture for citizens 74 (13.1) 38 (10.2) 18 (7.7) 0.12 Activities for regional contribution: Required to serve as instructors at external lectures 238 (42.2) 100 (26.9) 63 (26.9) <.001*** Activities for regional contribution: Participation in meetings of local home-care related committees 211 (37.4) 98 (26.3) 61 (26.1) <.001*** Activities for regional contribution: Received consultations from other health professionals 367 (65.1) 222 (59.7) 129 (55.1) 0.02* G. Financial condition Proportion of total income covered by long-term care insurance 64.8 ± 18.0 65.5 ± 18.9 65.3 ± 20.1 0.83 Proportion of total income covered by medical insurance 35.2 ± 18.0 34.5 ± 18.9 34.7 ± 20.1 0.83 Proportion of expenditures going toward compensation 75.2 ± 11.9 73.7 ± 11.2 80.2 ± 9.7* <.001*** Compensation of full-time nursing staff 4996046.4 ± 2251701.0* 4517809.1 ± 2236688.5 4634608.1 ± 2386036.2 0.02* When the variable was continuous values, we performed one-way analysis of variance. Furthermore, when significant correlation was confirmed between the financial performance and the variables, we performed Dunnett multiple comparison to compare the “stable” group with “profitable” and “unprofitable” groups. When variables were categorical data, we performed chi-square test. ***P<.001, **P<.01, *P<.05, †P<.10. :Total number of physiotherapists, occupational therapists and speech therapists. :Total number of nursing staff, rehabilitation staff and office staff. :Number of nursing staff or rehabilitation staff converted to full-time staff/number of staff converted to full-time staff. :Number of full-time staff/number of staff converted to full-time staff. :Ratio (%) calculated using the total number of patients as the denominator during the 1-month study period (September 2012). D. Patient utilization Ratio of independence in daily life C calculated using the total of J (independence), A (independence in a room), B (independence on the floor) and C (bedridden) as the denominator. :Number of patients died at home/number of patients who died after receiving terminal care during 6 months from April to September 2012. table, stable and unprofitable) and management of home-visit nursing agency (in September 2012 AWhen the variable was continuous values, we performed one-way analysis of variance. Furthermore, when significant correlation was confirmed between the financial performance and the variables, we performed Dunnett multiple comparison to compare the “stable” group with “profitable” and “unprofitable” groups. When variables were categorical data, we performed chi-square test. ***P<.001, **P<.01, *P<.05, †P<.10. a:Total number of physiotherapists, occupational therapists and speech therapists. b:Total number of nursing staff, rehabilitation staff and office staff. c:Number of nursing staff or rehabilitation staff converted to full-time staff/number of staff converted to full-time staff. d:Number of full-time staff/number of staff converted to full-time staff. e:Ratio (%) calculated using the total number of patients as the denominator during the 1-month study period (September 2012). f: Ratio of independence in daily life C calculated using the total of J (independence), A (independence in a room), B (independence on the floor) and C (bedridden) as the denominator. g:Number of patients died at home/number of patients who died after receiving terminal care during 6 months from April to September 2012. Determinants of financial performance in home-visit nurs- ing agencies: results of multinominal logistic regression analysis Table 2 shows the results of multi nominal logistic re- gression analysis of determinants of financial perform- ance in home-visit nursing agencies. Thirteen variables from all seven categories, A to G, were found to be de- terminants related to profitability, compared with stable agencies (neither profitable nor unprofitable). Profitable agencies were correlated to five variables: 1) hiring of ≥5 nursing staff (odds ratio: 1.65; 95% confidence interval: 1.16-2.34), 2) having ≥70 patients (1.94; 1.33-2.83), 3) care monitoring among staff as an approach to quality control (1.37; 1.03-1.82), 4) control of staff objectives by nurse managers (1.81; 1.36-2.42), and 5) requirement to serve as instructors at external lectures (1.40; 1.02-1.93), compared to stable agencies. Regarding employment, there was a higher number of profitable agencies with a larger number of nursing staff (converted to full-time staff), rehabilitation staff, and total staff (all p < .001). In unprofitable agencies, the ra- tio of nursing staff to total number of staff members was higher (p < .001) and the ratio of rehabilitation staff was lower (p < .001). The ratio of full-time staff was also higher in the unprofitable agencies (p = .007). Regarding patient utilization, the number of patients covered by long-term care insurance, the number of patients covered by medical insurance, the total number of patients, and the number of patients per nursing staff were all highest in profitable agencies, followed by stable and then unprofitable agencies (all p < .001). The num- bers of new patients covered by medical and long-term care insurance were significantly higher in profitable agencies, compared to the other agencies (p < .001; p < .001, respectively). On the other hand, the proportion of new patients was significantly higher in unprofitable agencies, compared to the other agencies (p = .04). The ratios of bedridden patients and patients who left the agency due to improvement were high or tended to be high in unprofitable agencies (p = .09; p < .001, respectively). The ratio of home deaths among terminal patients during a 6-month period was highest in pro- fitable agencies, followed by stable and unprofitable agencies (p = .04). G. Financial condition Proportion of total income covered by long-term care insurance Proportion of total income covered by long-term care insurance Proportion of total income covered by medical insurance Proportion of total income covered by medical insurance Proportion of expenditures going toward compensation Proportion of expenditures going toward compensation Compensation of full-time nursing staff Page 5 of 8 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 the agency: results of univariate analysis Table 1 shows the results of univariate analysis of relation- ship between profitability and management of the agency. Regarding the operating structure, the number of unprofit- able agencies was significantly higher among home-visit nursing agencies owned by a hospital (p = .007). Regarding the financial condition, although the pro- portion towards compensation was high in unpro- fitable agencies, costs (expenditures) for full-time nursing staff were significantly higher in profitable agencies, compared to the other agencies (p < .001; p = .02, respectively). Regarding management by a nurse manager, profitable agencies had managers with significantly longer experi- ence than stable and unprofitable agencies (p = 0.001). The managers of unprofitable agencies made fewer visits per month compared to those of stable and profitable agencies (p < .001). The managers of the profitable agen- cies also participated more in management training and had more capability with financial management, com- pared to those of stable and unprofitable agencies (p = 0.003; p < .001). Determinants of financial performance in home-visit nurs- ing agencies: results of multinominal logistic regression analysis Determinants of financial performance in home-visit nurs- ing agencies: results of multinominal logistic regression analysis Unprofitable agencies were correlated with eight vari- ables: 1) being owned by a hospital (1.83; 1.25-2.68), 2) visits of <50 times per month to patients’ homes by nurse managers (0.45; 0.31-0.66), 3) hiring of <1 rehabili- tation staff (0.34; 0.14-0.84), 4) having <10% rehabilita- tion staff (0.25; 0.11-0.58), 5) having <70 patients (0.52; 0.29-0.92), 6) <65% home deaths among terminal pa- tients (0.61; 0.41-0.89), 7) having <20 cooperative hospi- tals (0.62; 0.39-0.97), and 8) having ≥75% income going to compensation (3.83; 2.47-5.94), compared to stable agencies. Relationship between profitability and management of the agency: results of univariate analysis to have received requests to serve as instructors at exter- nal lectures (p < .001), participated in meetings of local home-care related committees (p < .001), and received consultations from other health professionals (p = .02). Discussion In this study, we clarified the relationship between prof- itability of home-visit nursing agencies and variables re- lated to management of the agency. Regarding quality control, we found that the ratios of care monitoring among staff and control of staff objec- tives by nurse managers were higher in profitable agen- cies than in stable and unprofitable agencies (p = .02; p < .001, respectively). First, we found that half of the home-visit nursing agencies were profitable (48.3%), while 31.6% and 20.1% were stable and unprofitable, respectively. Compared to our previous survey on the financial management of home-visit nursing agencies in Japan over the past four years, the number of profitable agencies has remained relatively similar at about 50% (53.5% in 2009, 56.9% in 2010, 52.6% in 2011, 48.4% in 2012), while unprofitable agencies have decreased by about 10% (31.2% in 2009, 29.6% in 2010, 24.9% in 2011, 18.1% in 2012), and stable Regarding regional cooperation, profitable agencies had the most cooperative hospitals, followed by stable and unprofitable agencies (p < .001). Regarding activities for regional contribution, compared to stable and un- profitable agencies, profitable agencies were more likely Page 6 of 8 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 h d b f [ ] h f h Table 2 Determinants of financial performance in home-visit nursing agencies: results of multinominal logistic regression analysisA) Profitable Unprofitable Variables B) Odds ratio (95% Confidence Interval) A. Operating structure Owned by a hospital 1.06 (0.78–1.44) 1.83 (1.25–2.68)** B. Management by a nurse manager Years of experience of nurse manager (≥6 years) 1.18 (0.89–1.58) 0.75 (0.52–1.09) Number of home visits per month by the nurse manager (≥50 times per month) 1.20 (0.90–1.59) 0.45 (0.31–0.66)*** Experience of nurse manager in management training (Having) 0.96 (0.71–1.31) 0.86 (0.59–1.25) Nurse manager’s capability with financial management (Having) 1.14 (0.85–1.51) 0.79 (0.55–1.14) C. Employment Number of nursing staff (converted to full-time staff) (≥5) 1.65 (1.16–2.34)** 0.70 (0.43–1.14) Number of rehabilitation staff (converted to full-time staff) (≥1)a 1.16 (0.62–2.18) 0.34 (0.14–0.84)* Ratio of nursing staff within total staff (≥75%)b 0.85 (0.59–1.21) 1.09 (0.69–1.72) Ratio of rehabilitation staff within total staff (≥10%)b 1.12 (0.63–1.99) 0.25 (0.11–0.58)** Ratio of full-time staff (≥75%)c 0.84 (0.63–1.12) 1.08 (0.75–1.55) D. F. Regional cooperation (during the last year) Number of hospitals with cooperation in the community (≥20) Activities for regional contribution: Required to serve as instructors at external lectures Activities for regional contribution: Participation in meetings of local home-care related committe Activities for regional contribution: Received consultations from other health professionals E. Quality control (during the last year) Conducting regularly of care monitoring among staff Conducting regularly of control of staff objectives by nurse manager p g ( , , y p y ) ( ) ( R2=0.26, Adjusted R2=0.30. AWe examined the association of “profitable” and “unprofitable” agencies, with the “stable” agencies as a reference. BIndependent variables were used with p values less than 0.10 by the univariate analysis, and numarical variables were divided into two by the average. ***:P<.001, **:P<.01, *:P<.05. a:Total number of physiotherapists, occupational therapists and speech therapists. b:Number of nursing care staff or rehabilitation staff converted to full-time staff/number of staff converted to full-time staff. c:Number of full-time staff/number of staff converted to full-time staff. d:Ratio (%) calculated using the total number of patients as the denominator during the 1-month study period (September 2012). e:Ratio of independence in daily life C calculated using the total of J (independence), A (independence in a room), B (independence on the floor) and C (bedridden) as the denominator. f:Number of patients died at home/number of patients who died after receiving terminal care during 6 months from April to September 2012. Discussion Patient utilization Total number of patients (≥70) 1.94 (1.33–2.83)*** 0.52 (0.29–0.92)* Number of patients per nursing staff (≥70) 1.18 (0.85–1.66) 0.79 (0.51–1.24) Percentage of new patients (≥7%)d 1.31 (0.97–1.77) 1.07 (0.73–1.56) Percentage of bedridden patients (≥30%)e 1.00 (0.76–1.33) 0.84 (0.59–1.20) Percentage of home deaths among terminal patients (≥65%)f 1.11 (0.83–1.49) 0.61 (0.41–0.89)* E. Quality control (during the last year) Conducting regularly of care monitoring among staff 1.37 (1.03–1.82)* 1.10 (0.76–1.59) Conducting regularly of control of staff objectives by nurse manager 1.81 (1.36–2.42)*** 1.10 (0.76–1.61) F. Regional cooperation (during the last year) Number of hospitals with cooperation in the community (≥20) 1.11 (0.80–1.55) 0.62 (0.39–0.97)* Activities for regional contribution: Required to serve as instructors at external lectures 1.40 (1.02–1.93)* 1.37 (0.89–2.11) Activities for regional contribution: Participation in meetings of local home-care related committees 1.27 (0.93–1.74) 1.12 (0.74–1.71) Activities for regional contribution: Received consultations from other health professionals 0.87 (0.65–1.17) 0.83 (0.58–1.20) G. Financial condition Proportion of expenditures going toward compensation (≥75%) 1.11 (0.83–1.48) 3.83 (2.47–5.94)*** Compensation of full-time nursing staff (≥4,500,000 yen per year) 1.01 (0.75–1.37) 1.05 (0.71–1.56) R2=0.26, Adjusted R2=0.30. AWe examined the association of “profitable” and “unprofitable” agencies, with the “stable” agencies as a reference. BIndependent variables were used with p values less than 0.10 by the univariate analysis, and numarical variables were divided into two by the average. ***:P<.001, **:P<.01, *:P<.05. a:Total number of physiotherapists, occupational therapists and speech therapists. b:Number of nursing care staff or rehabilitation staff converted to full-time staff/number of staff converted to full-time staff. c:Number of full-time staff/number of staff converted to full-time staff. d:Ratio (%) calculated using the total number of patients as the denominator during the 1-month study period (September 2012). e:Ratio of independence in daily life C calculated using the total of J (independence), A (independence in a room), B (independence on the floor) and C (bedridden) as the denominator. f:Number of patients died at home/number of patients who died after receiving terminal care during 6 months from April to September 2012. Table 2 Determinants of financial performance in home-visit nursing agencies: results of multinominal logistic regression analysisA) inants of financial performance in home-visit nursing agencies: results of multinominal logistic i A) Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 The 2012 revisions included the expanded use of cooperative in- struction costs upon hospital discharge by home-visit nurs- ing agencies [14] and the revision of nursing care benefits included additional cooperative instruction costs upon hos- pital discharge [13]. Such revisions of benefits have been made to medical insurance and long-term care insurance policies. Thus, stable financial management of home-visit nursing agencies could be achieved by cooperating not only with limited organizations in the same corporation, but also with many other local organizations. Regarding the financial condition, the relationship be- tween earnings and profitability, which was suggested to be significant in univariate analysis, was eliminated in multivariate analysis. Agencies with 75% or higher of their earnings going to expenditures would not be profit- able. According to a survey on home-visit nursing in 1999 by the Ministry of Health, Labour and Welfare of Japan, compensation accounts for 84.9% of expenditures in home-visit nursing agencies [16]. Although these data are somewhat old, this is the last detailed survey and is relevant as the average number of nursing staff in a home-visit nursing agency has not changed much be- tween 1999 (4.4 [total staff: 4.6]) and 2011 (4.6 [total staff: 5.9]) [16]. In the public hospital reform guidelines published in 2007 by the Ministry of Internal Affairs and Communications, the target compensation rate of public hospital staff (for medical income) was determined to be 54.8% in private hospitals and 65.6% in general public hospitals with >50 beds [17]. While the target compen- sation rate in home-visit nursing agencies differs from that in hospitals because of the difference in scale, it might be more important for the agencies to manage by setting objectives, similarly to the efforts of hospitals. In this study, the objective compensation rate was deter- mined to be 75% to avoid loss in profits in home-visit nursing agencies. This may be used as a financial man- agement target, in addition to the targets of 5 or more nursing staff and 70 or more patients. Regarding the management system, we found that agencies with managers with <50 patient visits per month were not profitable. Our 2011 study revealed that nurse managers of large-scale agencies had longer work- ing hours and total working time [8]. Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 both large in profitable agencies, as suggested in previ- ous studies [11,12]. These results show that profitable agencies tend to have more nursing staff and patients. We also found that unprofitable agencies had an average of <1 rehabilitation staff (converted to full-time rehabili- tation staff) and that these staff made up <10% of the total staff. Thus, it is possible that profitable agencies have a surplus due to employment of rehabilitation staff. In the latest 2012 revisions to rehabilitation support by home-visit nursing agencies in medical and long-term care insurance, time division of home visits by rehabili- tation staff from a home-visit nursing agency was short- ened and the cost was reduced [13], suggesting the need for further discussion of the roles and functions of re- habilitation staff at home-visit nursing agencies. terminal phase until death. Therefore, the present compen- sation system may be considered appropriate regarding pa- tients in the terminal phase. Regarding quality control and regional cooperation, we found that agencies which regularly conduct care moni- toring among staff and allow control of staff objectives by nurse managers, as well as agencies involved in exter- nal lectures for the health-care professionals in the com- munity, would be profitable. The profitable agencies may be more likely to try to implement aggressive quality control because they understand the importance of qual- ity control for nursing care, which directly affects the evaluation of the agency, as shown by previous studies [11,12]. In addition to these internal efforts, profitable agencies may have become well known in their commu- nities and are entrusted to provide regional contribu- tions and to dispatch information to external organizations. It could be interpreted that these agencies can secure a large number of patients, including those with severe conditions such as end-stage diseases, and could attract more nursing staff. This may lead to im- proved quality of care by nursing staff with greater ex- perience, producing a virtuous cycle that may provide for profitability. Our results also showed that agencies owned by a hos- pital and with <20 cooperative hospitals tended to be un- profitable, compared to financially stable agencies. G. Financial condition Proportion of expenditures going toward compensation (≥75%) Compensation of full-time nursing staff (≥4,500,000 yen per year) R2=0.26, Adjusted R2=0.30. AWe examined the association of “profitable” and “unprofitable” agencies, with the “stable” agencies as a reference. BIndependent variables were used with p values less than 0.10 by the univariate analysis, and numarical variables were divided into two by the average. ***:P<.001, **:P<.01, *:P<.05. a:Total number of physiotherapists, occupational therapists and speech therapists. b:Number of nursing care staff or rehabilitation staff converted to full-time staff/number of staff converted to full-time staff. c:Number of full-time staff/number of staff converted to full-time staff. d:Ratio (%) calculated using the total number of patients as the denominator during the 1-month study period (September 2012). e:Ratio of independence in daily life C calculated using the total of J (independence), A (independence in a room), B (independence on the floor) and C (bedridden) as the denominator. f:Number of patients died at home/number of patients who died after receiving terminal care during 6 months from April to September 2012. support the management of those agencies yet to achieve profitability. agencies have increased by 20% from 10% to 30% [7-10]. Therefore, half of the agencies can still not achieve good financial performance, regardless of the recent positive revisions to the insurance cost of home-visit nursing care by the Japanese government. Concrete financial management policies should thus be discussed to Secondly, we clarified the characteristics of both prof- itable and unprofitable agencies by comparing them with those of stable agencies. Regarding the scale of each agency, the numbers of nursing staff and patients were Page 7 of 8 Page 7 of 8 References 1. OECD Factbook 2009: Economic, Environmental and Social Statistics. ISBN 92- 64-05604-1. OECD; 2009. http://www.oecd-ilibrary.org/sites/factbook-2009- en/01/02/01/index.html?contentType=&itemId=/content/chapter/factbook- 2009-3-en&containerItemId=/content/serial/18147364&accessItemIds= &mimeType=text/html (Tables: Population aged 65 and over). (accessed July 2013). 2. Kashiwagi M, Tamiya N, Sato M, Yano E: Factors associated with the use of home-visit nursing services covered by the long-term care insurance in rural Japan: a cross-sectional study. BMC Geriatr 2013, 13:1. doi:10.1186/ 1471-2318-13-1. 3. Naruse T, Taguchi A, Kuwahara Y, Nagata S, Watai I, Murashima S: Relationship between perceived time pressure during visits and burnout among home visiting nurses in Japan. Jpn J Nurs Sci 2012, 9:185–194. 4. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Home-visit nursing services (in Japanese). http://www.mhlw.go.jp/stf/shingi/ 2r9852000001uo3f-att/2r9852000001uo71.pdf (accessed July 2013). 5. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Summary of survey of providers of long-term care services; 2012. http://www.mhlw.go.jp/ toukei/saikin/hw/kaigo/service11/dl/gaikyo.pdf (accessed December 2013). 6. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Status of workers in the survey of providers of long-term care services; 2012. http:// www.mhlw.go.jp/toukei/saikin/hw/kaigo/service11/dl/kekka-gaiyou_04.pdf (accessed December 2013). 3. Naruse T, Taguchi A, Kuwahara Y, Nagata S, Watai I, Murashima S: Relationship between perceived time pressure during visits and burnout among home visiting nurses in Japan. Jpn J Nurs Sci 2012, 9:185–194. Relationship between perceived time pressure during visits and burnout among home visiting nurses in Japan. Jpn J Nurs Sci 2012, 9:185–194. 4. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Home-visit nursing services (in Japanese). http://www.mhlw.go.jp/stf/shingi/ 2r9852000001uo3f-att/2r9852000001uo71.pdf (accessed July 2013). 4. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Home-visit nursing services (in Japanese). http://www.mhlw.go.jp/stf/shingi/ 2r9852000001uo3f-att/2r9852000001uo71.pdf (accessed July 2013). p y 5. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Summary of survey of providers of long-term care services; 2012. http://www.mhlw.go.jp/ toukei/saikin/hw/kaigo/service11/dl/gaikyo.pdf (accessed December 2013). 6. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Status of workers in the survey of providers of long-term care services; 2012. http:// www.mhlw.go.jp/toukei/saikin/hw/kaigo/service11/dl/kekka-gaiyou_04.pdf (accessed December 2013). 6. Minister’s Secretariat, Ministry of Health, Labour and Welfare: Status of workers in the survey of providers of long-term care services; 2012. http:// www.mhlw.go.jp/toukei/saikin/hw/kaigo/service11/dl/kekka-gaiyou_04.pdf (accessed December 2013). Received: 6 August 2013 Accepted: 6 January 2014 Published: 9 January 2014 Acknowledgements This work was supported by the Japanese government Enterprise for Health Promotion for the Elderly, Ministry Health, Labor and Welfare. We thank the home health professionals from the home-visit agencies for their cooperation in this study. We are also grateful to Dr. Junichi Shimizu and Ms. Yumi Yokota for their research assistance. 17. Ministry of Internal Affairs and Communications: The public hospital reform guidelines in 2007. http://www.soumu.go.jp/main_sosiki/c-zaisei/hospital/ pdf/191225_guideline.pdf (accessed July 2013). 17. Ministry of Internal Affairs and Communications: The public hospital reform guidelines in 2007. http://www.soumu.go.jp/main_sosiki/c-zaisei/hospital/ pdf/191225_guideline.pdf (accessed July 2013). doi:10.1186/1472-6963-14-11 Cite this article as: Fukui et al.: Determinants of financial performance of home-visit nursing agencies in Japan. BMC Health Services Research 2014 14:11. Authors’ contributions All authors contributed to the conception and design of the study and the acquisition of data. SF analyzed data and all authors contributed to interpretation of data. All authors contributed to drafting the article or revising it critically for important intellectual content, and all gave final approval of the submitted version. 14. Revisions of the services covered by medical insurance in 2012; 2013. http:// www.mhlw.go.jp/bunya/iryouhoken/iryouhoken15/dl/h24_01-03.pdf. 14. Revisions of the services covered by medical insurance in 2012; 2013. http:// www.mhlw.go.jp/bunya/iryouhoken/iryouhoken15/dl/h24_01-03.pdf. 15. Fukui S: A nationwide survey on the end-of-life care provided by home- visit nursing in Japan. Bull Soc Insur (Shakai Hoken Junpo) 2012, 2488:16–23. In Japanese. 16. Japanese Visiting Nurse Foundation: The survey of home-visit nursing in 1999. (in Japanese). http://www1.mhlw.go.jp/toukei/rkango99_8/sec04.html (accessed July 2013). 16. Japanese Visiting Nurse Foundation: The survey of home-visit nursing in 1999. (in Japanese). http://www1.mhlw.go.jp/toukei/rkango99_8/sec04.html (accessed July 2013). Conclusions The relationship between financial performance and man- agement was examined in a nationwide survey of 2,912 ran- domly selected home-visit nursing agencies in Japan from November to December 2012. Data were analyzed from a total of 1,340 agencies. Multinomial logistic regression ana- lyses revealed multiples management variables related to profitable and unprofitable agencies, compared to agencies with stable financial performance. These variables included the number of nursing staff/rehabilitation staff/patients, be- ing owned by a hospital, the number of cooperative hospi- tals, home-death rate among terminal patients, control of staff objectives by nurse managers, and income going to compensation. Quality assurance of management in home- visit nursing agencies in Japan is important for achievement of a stable financial performance. 7. Fukui S: Annual report in 2012 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2013. http:// www.murc.jp/uploads/2013/05/koukai130621_c2.pdf. 7. Fukui S: Annual report in 2012 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2013. http:// www.murc.jp/uploads/2013/05/koukai130621_c2.pdf. 8. Fukui S: Annual report in 2011 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2012. http:// www.zenhokan.or.jp/pdf/surveillance/H23-1-2.pdf. 9. Fukui S: Annual report in 2010 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2011. in Japanese. 10. Fukui S: Annual report in 2009 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2010. http:// www.zenhokan.or.jp/pdf/surveillance/H21-1-2.pdf. 10. Fukui S: Annual report in 2009 of strengthening quantity and quality of home- visit nursing agencies in Japan granted by government enterprise for health promotion for the elderly, Ministry Health, Labor and Welfare; 2010. http:// www.zenhokan.or.jp/pdf/surveillance/H21-1-2.pdf. 11. Fujita J, Fukui S: A nationwide survey on the activity of quality control for home-visit nursing agencies in Japan. Community Care 2012, 14(12):66–70. In Japanese. 11. Fujita J, Fukui S: A nationwide survey on the activity of quality control for home-visit nursing agencies in Japan. Community Care 2012, 14(12):66–70. In Japanese. 12. Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Page 8 of 8 Page 8 of 8 to the cross-sectional nature of the study. Agencies with a nurse manager who was not aware of the importance of management and did not make an effort to maintain qual- ity of care in the agency might not have responded to the questionnaire. If the response rate was higher, the determi- nants of profitability might have been revealed more clearly. In addition, a longitudinal study design would allow estab- lishment of causality between the financial performance and management of home-visit nursing agencies. However, there have been no studies to examine factors related to profitability in home-visit nursing agencies in Japan multi- laterally and in detail. Thus, we believe this study will con- tribute to future development of home health services in Japan and in other countries with a similar rapid aging trend. Conclusions Tsuno Y, Fukui S, Ikezaki S: Can be compatible with maintaining a quality of care and profitability in home-visit nursing agencies in Japan. Home-visit Nurs and Care. 2011, 16(11):948–951. In Japanese. 12. Tsuno Y, Fukui S, Ikezaki S: Can be compatible with maintaining a quality of care and profitability in home-visit nursing agencies in Japan. Home-visit Nurs and Care. 2011, 16(11):948–951. In Japanese. Competing interests N f h h h None of the authors has any conflict of interest for the past three years. 13. Revisions of the services covered by long-term care insurance in 2012; 2013. http://www.mhlw.go.jp/topics/kaigo/housyu/dl/b01.pdf. Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Taken together, we speculated that nurse managers of large-scale agencies had a larger amount of work due to the increase in the number of visits and due to dealing with daily opera- tions. On the other hand, managers should decrease home visits as much as possible as such work can be done by other staff, and more time should be reserved for management duties, which only managers can complete. Further discussion is thus needed to establish a system that ensures profitability even when the num- ber of visits by managers is decreased. Regarding patient utilization, poor financial performance was observed in agencies with less than 65% of home deaths among patients who died in the past 6 months. In 2011, we proposed two indices to evaluate the potential of a home-visit nursing agency: the number of home deaths (≥5 per year) and the rate of home deaths (≥50% per year) among terminal patients in a home-visit nursing agency [15]. This study also confirmed the relationship between profitability and these indices, as agencies with a 65% or higher rate of home deaths among terminal patients were considered to have the potential to support patients in the The limitations of this study include a low response rate and the inability to demonstrate a causal relationship due Fukui et al. BMC Health Services Research 2014, 14:11 http://www.biomedcentral.com/1472-6963/14/11 Author details 1 1Department of Community Health Nursing, Graduate School of Nursing, The Japanese Red Cross University, Tokyo, Japan. 2Department of Stress Sciences and Psychosomatic Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 3Department of Community Health Nursing, Graduate School of Nursing, The Chiba University, Chiba, Japan. 4Graduate School of Nursing, The Japanese Red Cross University, 4-1-3 Hiroo, Shibuya-ku, Tokyo 150-0012, Japan. Received: 6 August 2013 Accepted: 6 January 2014 Published: 9 January 2014
https://openalex.org/W4376867470
https://www.researchsquare.com/article/rs-2939824/latest.pdf
English
null
Ultra-broadband illusion acoustics for space and time camouflages
Research Square (Research Square)
2,023
cc-by
5,918
Ultra-broadband illusion acoustics for space and time camouflages Chenkai Liu  NJU Chu Ma  University of Wisconsin-Madison Yun Lai  Nanjing University https://orcid.org/0000-0002-0040-9274 Nicholas Fang  University of Hong Kong https://orcid.org/0000-0001-5713-629X Article Keywords: Posted Date: May 17th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2939824/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is NO Competing Interest. Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is NO Competing Interest. Version of Record: A version of this preprint was published at Nature Communications on September 14th, 2024. See the published version at https://doi.org/10.1038/s41467-024-49856-z. Page 1/16 Abstract Invisibility cloaks that can suppress wave scattering by objects have attracted a tremendous amount of interest in the past two decades. In comparison to prior methods that were severely limited by narrow bandwidths, here we present a practical strategy to suppress sound scattering across an ultra-broad spectrum by leveraging illusion metamaterials. Consisting of a collection of subwavelength tunnels with precisely crafted internal structures, this illusion metamaterial has the ability to guide acoustic waves around the obstacles and accurately recreate the incoming wavefront on the exit surface. Remarkably, two ultra-broadband illusionary effects are produced: “disappearing space” and “time shift”. Sound scatterings are removed at all frequencies below a limit determined by the tunnel width, as confirmed by full-wave simulations and acoustic experiments. Our approach establishes a metamaterial platform that enables the long-desired ultra-broadband sound manipulation such as acoustic camouflage and reverberation control, opening up exciting new possibilities in practical applications. Introduction Invisibility cloaks1,2, the long-cherished desire of human being, have become possible with the advent of artificial metamaterials and transformation optics theory, among the fields of optics3–9, acoustics10–12, elastics13 and thermotics14. Invisibility is a special illusionary effect15–19 of “empty space”, while from the perspective of wave scattering20–22, such cloaks can significantly suppress the scattering by any objects. By manipulating acoustic waves with acoustic metamaterials23–27 and metasurfaces28, acoustic invisibility cloaks29–36 have been demonstrated, which have wide implications like acoustic stealth and reverberation engineering. Besides invisibility cloaks, many other approaches for scattering suppression have been proposed, such as zero-index media37–39, destructive interference40, topological effects41, and spatial dispersion parameters42, etc. Nevertheless, so far, all these approaches have a severe limitation, i.e. narrow operating bandwidth. This limitation originates in the causality principle and resonant nature of general metamaterials and structures, which seriously hindered its real-world applications. In this work, we reveal that this long-standing and seemly unsurmountable bandwidth limitation throughout various disciplines can be completely removed by leveraging a new class of illusion metamaterials that exhibit two ultra-broadband illusionary effects: “disappearing space” and “time shift”, instead of the “empty space”. Such illusion metamaterials consist of a collection of subwavelength acoustic tunnels arranged around the obstacles. Attributing to the equal acoustic path design of tunnels and the uniform geometric shape of the entry and exit surfaces, the wavefront of incident wave can be accurately reproduced on the exit surface over an ultra-broad spectrum from the quasistatic limit to an upper frequency limit determined by the tunnel width. Consequently, the space occupied by the metamaterial and obstacles seems to be completely “disappeared”, while at the same time, the time is “shifted” backward by a corresponding amount. With such extraordinary illusionary effects, the original sound scattering effects of obstacles are also eliminated in this unprecedented broad spectrum. Page 2/16 Page 2/16 Through full-wave simulations and acoustic experiments of a typical example, we demonstrate that the bandwidth of this ultra-broadband illusionary effects covers the entire regime from 1 kHz to 16 kHz. Our work replaces the traditional route of creating “empty space” by a new paradigm of creating “disappearing space” and “time shift”, enabling sound scattering suppression with the long-coveted extreme bandwidth and frequency-independent feature for the first time. Acoustic tunnels with internal protrusion structures To show the generality of our approach, the tunnel is designed to possess an arbitrary shape, as shown in Fig. 2a. Suppose the tunnel has a varying cross-section-width , and a horizontal length To achieve a tunable acoustic path, protrusion structures with a height of and a lattice constant of are added to the interior of the tunnel. The thickness of the tunnel shell and the protrusion is set as . When the width of the tunnel is much smaller than the wavelength, there is only one mode in the tunnel and the acoustic tunnel can be described by a transmission-line model. A collection of such tunnels leads to anisotropic metamaterials43–45 that guide sound along certain directions. Different from the simple tunnels utilized in the previous works, here the internal protrusion structures play a key role in modulating the total acoustic path of each tunnel. w a. h d t To illustrate this mechanism, we have performed full-wave simulations for the tunnel plotted in Figs. 2b- c. The geometric parameters are set as , and . The maximum and minimum width of the tunnel are set as and , respectively. A frequency of is chosen for demonstration. Figure 2b shows the acoustic pressure field distributions inside the tunnels with different geometric parameters of . It is clearly seen that the phase shift of the transmitted wave increases substantially when increases. We note that and simultaneously change along the propagation path, but the ratio of is fixed in one tunnel. Figure 2c presents the phase shift and the transmittance as a function of at . We can observe that the transmitted phase shift can be conveniently modulated to cover the range of by changing from 0 to 0.32, while the average transmittance is kept above , indicating that such type of tunnels can achieve a tunable acoustic path with a high efficiency. Investigation on some minor factors to the transmittance, including the incident angle of sound, the bending angle and the variation width of the tunnel, are discussed in the Supplemental Materials. The results demonstrate that the functionality of the tunnel is quite robust against these factors. We note that comparing with other designs based on phase manipulation, such as the zigzag structures37, the protrusion structures exhibits a better performance in impedance matching. Design of the ultra-broadband illusion metamaterial and experimental setup This illusion metamaterial is constructed by assembling an array of such tunnels to route around the obstacles and guide sound waves from the entry surface to the exit other. To create the ultra-broadband illusionary effect, there are two criteria. The first criterion is that the entry and exit surfaces of the illusion metamaterial should have exactly the same shape (dotted lines in Fig. 1b). The other criterion is that all the tunnels should have exactly the same acoustic path. If the two criteria are met by the illusion metamaterial, the wavefront of the incident acoustic field can be copied from the entry to the exit surface, therefore realizing the two illusionary effects of “disappearing space” and “time shift”. Figure 3a shows the schematic diagram of a typical example of the illusion metamaterial and the corresponding experimental setup. There are three rhombic hard obstacles arranged along the y direction in air. The entry and exit surfaces of the illusion metamaterial are both flat surfaces in this case. A total number of twenty tunnels are applied for each rhombic obstacle, as shown by the zoom-in figure in Fig. 3b. Because of the mirror symmetry, ten different sets of are designed, as shown in Fig. 3c. Such parameters can guarantee that the acoustic path is almost the same ( ) for all twenty tunnels, despite that they have obviously different lengths and bending angles. In experiment, the illusion metamaterial as well as the obstacles are both fabricated by using 3D printing techniques. A photo of the sample and the experimental setup is shown in Fig. 3d. The whole experiment is performed in a plate waveguide with a height of 3 cm. A cylindrical wave is emitted by a speaker located at a distance of away from the sample. A microphone is mounted on a horizontal translation stage to scan the transmitted signals located at the black dashed area shown in Fig. 3a in the plane. The measured area with the size of and is of away from the sample. Sound absorbing foams are placed around the platform to reduce the reflected waves and noise from the environment. h/w L = 22.5cm d1 = 20cm xy Δx = 10cm Δy = 20cm d2 = 5cm Acoustic tunnels with internal protrusion structures t = 1mm d = 5mm a = 20cm 8.95mm 3.95mm f = 6860Hz h/w h/w w h h/w Δφ |t|2 h/w f = 6860Hz 2π h/w 97% Interestingly, such a type of tunnels with a curved path can be approximated as straight tunnels with a slower and dispersion-free effective sound speed and this works in an ultra-broad spectrum. The transmission phase of the tunnel is described as , where is the wave number in the background (air) and is the acoustic path. Here, is the effective refractive φ = k0L k0 L = nra nr = φ/ (k0a) Page 3/16 Page 3/16 index. In Fig. 2d, we plot the calculated as a function of and the frequency for the tunnel in Fig. 2a. It is clearly seen that increases with the increase of , and is almost unchanged from 0.1 kHz to 15 kHz. When is small, the dispersion of is negligible in the frequency regime. We emphasize that this frequency-independent refractive index holds the key to the realization of ultra- broadband illusion. nr h/w f nr h/w h/w nr Ultra-broadband illusionary effect of “disappearing space” and sound scattering suppression Ultra-broadband illusionary effect of “disappearing space” and sound scattering suppression Ultra-broadband illusionary effect of “disappearing space” and sound scat In Fig. 4, we demonstrate the full-wave simulation and acoustic experimental results obtained by using the illusion metamaterial designed in Fig. 3. For the case of bare obstacles at (Fig. 4a), large scattering clearly occurs due to the huge impedance mismatch between obstacles and air, as confirmed by the shadows behind the obstacles. However, when the obstacles are embedded in the designed metamaterial (Fig. 4b), the impinging cylindrical waves are guided around the obstacles, and then reproduce the cylindrical wavefront of the incident waves in the transmission region. From the f = 4860Hz Page 4/16 wavefront in the transmission region, the point source seems to have moved forward from the real point of to a virtual point of . Such a position change of 20 cm corresponds to the side length of the illusion metamaterial. Therefore, an illusion of “disappearing space”, i.e. the whole space of the metamaterial with the embedded obstacles has completely disappeared, is proved. Moreover, the field on the incidence region is almost undisturbed, indicating that the reflection is negligibly small. The measured results, as shown in the right inset graphs of Figs. 4a and 4b, agree well with the full-wave simulations. (−30cm, 0cm) (−10cm, 0cm) To demonstrate the ultra-broadband property, the numerical and experimental results obtained for two more frequencies, i.e. and are shown in Figs. 4c-4f. We can observe that the illusionary effects of “disappearing space” maintain perfectly under these frequencies with the illusion metamaterial, proving the ultra-broadband property. f = 6860Hz f = 8860Hz To quantify the performance of illusion metamaterial in a broad spectrum, we calculate the normalized scattered pressure intensity, which is defined as , where are the scattered field and the empty field, respectively. The subscript and represent the transmission region γt,r = |ps|2/|p0|2 ps, p0 t r To quantify the performance of illusion metamaterial in a broad spectrum, we calculate the normalized scattered pressure intensity, which is defined as , where are the scattered field and the empty field, respectively. The subscript and represent the transmission region and incidence region , respectively. The scattered field is obtained by subtracting the empty field without the sample ( ) from the total field with the sample ( ). It should be noted that, due to the illusionary effects, the empty field in the transmission region is calculated by using the virtual point source located at . Ultra-broadband illusionary effect of “disappearing space” and sound scat On the other hand, the empty field for the incidence region is calculated by using the real point source located at . The results are plotted in Figs. 4g-4h. It is clearly seen that maintain almost zero with the illusion metamaterial in an ultra-broad spectrum . In contrast, are significantly larger in the case of bare obstacles. γt,r = |ps|2/|p0|2 ps, p0 t r (x ∈(10cm, 50cm)) (x ∈(−50cm, −10cm)) ps p0 p (−10cm, 0cm) (−30cm, 0cm) γt,r 1kHz~16kHz γt,r We emphasize that the wide bandwidth in this approach is far beyond those of any other approaches. Previous methods as “empty space”29–36 always have narrow bandwidths, except for ray optics cloaks46,47 that have ignored the phase difference and thus cannot be described as a scattering suppression device. Here, the illusion metamaterial works for all frequencies below an upper limit where more than one propagating mode occur inside the tunnels, which is determined by w. We note that this functionality is independent of the wavefront of the incident waves. More examples such as the Gaussian wave incidence and multiple point source incidence are also plotted in the Supplemental Materials, which clearly verify the universality of this principle. Illusionary effect of “time shift” We emphasize that besides the illusionary effect of “disappearing space”, there is, simultaneously, another illusionary effect of “time shift”. This illusionary effect can be demonstrated by considering a sound pulse propagating through a collection of obstacles with and without the illusion metamaterial, as shown in Fig. 5. A number of rhombic obstacles are arranged along the y direction. The pulse is a time- domain Gaussian signal with a broad bandwidth. Clearly, the scattering by the obstacles has produced Page 5/16 Page 5/16 intense reflected waves and significantly changed the wavefront in transmission in the case of bare obstacles, as shown in Fig. 5a. While with the illusion metamaterial (Fig. 5b), the reflected waves are much smaller, because of the good impedance matching between the metamaterial and background medium (free space). In the transmission region, the circular wavefront is perfectly maintained, as if emitted from a shifted virtual point source located at . Furthermore, by comparing the wavefronts in the incidence and transmission regions, it is seen that there is a “time shift” in the transmission region. This is because that although the equal acoustic path in the tunnels leads to the illusionary effect of “disappearing space”, but such a “disappeared space” still takes a finite time for the waves to pass. The value of the extra time shift is obtained as 0.66 ms, which is consistent with the results obtained from Fig. 5b. The zoom-in inset graphs on the right of Fig. 5b exhibi the details of how the wavefront of the sound waves are routed around the obstacles inside the metamaterial, thereby significantly reducing the scattering effect in Fig. 5a. More details are shown in Supplemental Materials. The length of time shift can be modulated by controlling the acoustic path. The corresponding videos are shown in Supplemental Videos. (−10cm, 0cm) tshift = L/c0 = Illusion metamaterial for a random collection of obstacles As a further demonstration of the robustness of this approach, we design a particular illusion metamaterial to suppress the sound scattering from three obstacles in a random arrangement. As shown in Fig. 6a, the illusion metamaterial is set in a square shape with a side length of , and is composed of 46 tunnels, whose geometric parameters are presented in the Supplemental Materials. Here, the uniform acoustic path in these tunnels is set as . The size of the rhombic obstacles is the same as above. From the field distributions plotted in Fig. 6b, it is clearly seen that when the obstacles are covered by the illusion metamaterial, the wavefronts in both the incidence and transmission regions become undistorted, as if the obstacles disappear. The illusionary effects of “disappearing space” of length and a “time shift” of are both observed in the transmission region. On the contrary, mussy scattering occurs in the case of bare obstacles. More field distributions are shown in the Supplemental Materials. Furthermore, the normalized scattered pressure intensity is calculated and shown in Figs. 6c-6d, which turns out to be enormously reduced in the regime of 1 kHz-16 kHz. 2.25a(a = 20cm) L = 47.5cm 45cm 1.38ms Discussion It is recognized that when the tunnel width is small, e.g. <1mm, dissipation effect cannot be ignored due to the viscous friction between the air and the hard boundaries. The influence of loss in principle can be reduced by applying relatively large tunnel widths as well as shorter lengths. On the other hand, the illusionary effects are impaired when the tunnels support more than one propagating mode, or when the wavelength is small enough to cause the diffraction effects20,21. Therefore, the operating bandwidth has an upper limit, which is around 16 kHz in our design. Interestingly, there is no lower limit for our design. Page 6/16 Causality is one of the essential reasons why previous illusions of “empty space” have a narrow bandwidth46. However, in this work, there is no similar constraint due to the existence of the “time shift”, which, remarkably, enables ultra-broad operating bandwidth. It’s worth mentioning that the illusion of “disappearing space” is also fundamentally different from that of “empty space” extensively studied in invisibility cloaks. Although we are not realizing an invisibility cloak here, nevertheless, the original wave scattering effects of obstacles are completely removed. The only tradeoff is the ultra-broadband illusionary effect of “disappearing space” and “time shift”. It is therefore an ideal way to extend the bandwidth of scattering suppression to the extreme limit. We should note that the potential applications of such illusion metamaterials are not limited to scattering suppression. Attributing the exceptional ultra-broad bandwidth and frequency-independent functionalities, this class of metamaterials may find wide and practical applications in general acoustics, such as reverberation control, sound field reproduction, interference manipulation, etc. Our work thus enables a powerful platform for the long-desired ultra-broadband sound manipulation. Data availability The data during the current study are available from the corresponding author upon reasonable request. The data during the current study are available from the corresponding au The data during the current study are available from the corresponding author upon reasonable request. Acknowledgments Methods Numerical simulations. The finite element software COMSOL Multiphysics is performed for the full-wave simulations with “Pressure Acoustics, Frequency Domain” and “Pressure Acoustics, Transient” modules. The mass density and sound velocity of air are set as and , respectively. The resin structures are treated as acoustically rigid materials. The plane wave radiation is used in Fig. 2, and the monopole point source is used in Figs. 4–6. Perfectly matched layers are adopted to reduce the reflections. 1.21kg/m3 343m/s Experimental measurements. All samples are fabricated with resin by using stereolithography 3D printing techniques (SLA, in precision). A sound speaker radiates eight periods of sound waves with single frequency we want. A microphone (PCB 130F20) scans the sound field located at the black dashed area shown in Fig. 3a. The scanning has resolution of and in total 231 points are scanned. The measurement platform is a 2D waveguide with a height of . The sound absorbing foams are set around the system to reduce the reflections. 0.1mm 1cm 30mm Acknowledgments C.L. thanks J. Luo for helpful discussions. Y.L. acknowledges the support of the National Key Research and Development Program of China (Grant No. 2020YFA0211400) and the National Natural Science Foundation of China (Grants No. 11974176) for this work. Page 7/16 Page 7/16 Author contributions and C.M. contributed equally to this work. Y.L. and N.X.F. supervised the project. C.L. and Y.L. C.L. and C.M. contributed equally to this work. Y.L. and N.X.F. supervised the project. C.L. and Y.L. conducted the analysis, simulations and sample fabrication. C.M. and N.X.F. conducted the experiment design and measurements. All the authors contributed to the data analysis and manuscript preparation. C.L. and C.M. contributed equally to this work. Y.L. and N.X.F. supervised the project. C.L. and Y.L. conducted the analysis, simulations and sample fabrication. C.M. and N.X.F. conducted the experiment design and measurements. All the authors contributed to the data analysis and manuscript preparation. Competing interests The authors declare that they have no competing interests. References Leonhardt, U. Optical Conformal Mapping. Science 312, 1777–1781 (2006) 2. Pendry, J. B., Schurig, D. & Smith, D. R. Controlling electromagnetic fields. Science 312, 1780–1782 (2006). 2. Pendry, J. B., Schurig, D. & Smith, D. R. Controlling electromagnetic fields. Science 312, 1780–1782 (2006). 3. Smith, D. R., Pendry, J. B. & Wiltshire, M. C. K. Metamaterials and negative refractive index. Science 305, 788–792 (2004). 3. Smith, D. R., Pendry, J. B. & Wiltshire, M. C. K. Metamaterials and negative refractive index. Science 305, 788–792 (2004). 4. Li, J. & Pendry, J. B. Hiding under the carpet: A new strategy for cloaking. Phys. Rev. Lett. 101, 203901 (2008). 5. Lai, Y., Chen, H., Zhang, Z. Q. & Chan, C. T. Complementary media invisibility cloak that cloaks objects at a distance outside the cloaking shell. Phys. Rev. Lett. 102, 093901 (2009). 6. Yu, N. et al. Light Propagation with Phase Reflection and Refraction. Science 334, 333–337 (2011). 6. Yu, N. et al. Light Propagation with Phase Reflection and Refraction. Science 334, 333–337 (2011). 7. Liu, Y. & Zhang, X. Metamaterials: A new frontier of science and technology. Chem. Soc. Rev. 40, 2494–2507 (2011). 7. Liu, Y. & Zhang, X. Metamaterials: A new frontier of science and technology. Chem. Soc. Rev. 40, 2494–2507 (2011). 8. Kadic, M., Bückmann, T., Schittny, R. & Wegener, M. Metamaterials beyond electromagnetism. Reports Prog. Phys. 76, 126501 (2013). 9. Alù, A. & Engheta, N. Achieving transparency with plasmonic and metamaterial coatings. Phys. Rev. E 72, 016623 (2005). S. A. & Schurig, D. One path to acoustic cloaking. New J. Phys. 9, 45 (2007) 10. Cummer, S. A. & Schurig, D. One path to acoustic cloaking. New J. Phys. 11. Chen, H. & Chan, C. T. Acoustic cloaking in three dimensions using acoustic metamaterials. Appl. Phys. Lett. 91, 183518 (2007). 12. Norris, A. N. Acoustic cloaking. Acoust. Today 11, 38–46 (2015). 13. Farhat, M., Guenneau, S. & Enoch, S. Ultrabroadband elastic cloaking in thin plates. Phys. Rev. Lett. 103, 024301 (2009). 14. Schittny, R., Kadic, M., Guenneau, S. & Wegener, M. Experiments on transformation thermodynamics: Molding the flow of heat. Phys. Rev. Lett. 110, 195901 (2013). 15. Lai, Y. et al. Illusion optics: The optical transformation of an object into another object. Phys. Rev. Lett. 102, 253902 (2009). 16. Pendry, J. All smoke and metamaterials. Nature 460, 579–580 (2009). 16. Pendry, J. All smoke and metamaterials. References Nature 460, 579–580 (2009). Page 8/16 Page 8/16 17. Chen, H., Chan, C. T. & Sheng, P. Transformation optics and metamaterials. Nat. Mater. 9, 387–396 (2010). 18. Kan, W. et al. Acoustic Illusion near boundaries of arbitrary curved geometry. Sci. Rep. 3, 1427 (2013) 18. Kan, W. et al. Acoustic Illusion near boundaries of arbitrary curved geometry. Sci. Rep. 3, 1427 (2013). 19. Zhang, Y., Luo, Y., Pendry, J. B. & Zhang, B. Transformation-Invariant Metamaterials. Phys. Rev. Lett. 123, 67701 (2019). 20. KELLER, J. B. Geometrical theory of diffraction. J. Opt. Soc. Am. 52, 116 (1962). 21. Ufimtsev, P. Y. Fundamentals of the Physical Theory of Diffraction. (John Wiley & Sons, Inc., Hoboken, New Jersey, 2007). 22. P. Sheng. Introduction to Wave Scattering, Localization and Mesoscopic Phenomena. (Springer, New York, 2006). 23. Liu, Z. et al. Locally resonant sonic materials. Science 289, 1734 (2000). 23. Liu, Z. et al. Locally resonant sonic materials. Science 289, 1734 (2000). 24. Fang, N. et al. Ultrasonic metamaterials with negative modulus. Nat. Mater. 5, 452–456 (2006). 25. Ma, G. & Sheng, P. Acoustic metamaterials: From local resonances to broad horizons. Sci. Adv. 2, e1501595 (2016). 26. Cummer, S. A., Christensen, J. & Alù, A. Controlling sound with acoustic metamaterials. Nat. Rev. Mater. 1, 16001 (2016). 27. Ge, H. et al. Breaking the barriers: Advances in acoustic functional materials. Natl. Sci. Rev. 5, 159– 182 (2018). 28. Assouar, B. et al. Acoustic metasurfaces. Nat. Rev. Mater. 3, 460–472 (2018). 28. Assouar, B. et al. Acoustic metasurfaces. Nat. Rev. Mater. 3, 460–472 (2 29. Zhang, S., Xia, C. & Fang, N. Broadband acoustic cloak for ultrasound waves. Phys. Rev. Lett. 106, 024301 (2011). 30. Zhu, X., Liang, B., Kan, W., Zou, X. & Cheng, J. Acoustic cloaking by a superlens with single-negative materials. Phys. Rev. Lett. 106, 014301 (2011). 31. Chen, Y. et al. Broadband solid cloak for underwater acoustics. Phys. Rev. B 95, 180104 (2017). 32. Zhao, W., Chu, H., Tao, Z. & Hang, Z. H. Acoustic transmissive cloaking using zero-index materials and metasurfaces. Appl. Phys. Express 12, 054004 (2019). 33. Li, H. et al. Ultrathin Acoustic Parity-Time Symmetric Metasurface Cloak. Research 2019, 8345683 (2019). 34. Popa, B. I., Zigoneanu, L. & Cummer, S. A. Experimental acoustic ground cloak in air. Phys. Rev. Lett. 106, 253901 (2011). 35. Zigoneanu, L., Popa, B. I. & Cummer, S. A. Three-dimensional broadband omnidirectional acoustic ground cloak. Nat. Mater. References 13, 352–355 (2014). 36. Yang, Y., Wang, H., Yu, F., Xu, Z. & Chen, H. A metasurface carpet cloak for electromagnetic, acoustic and water waves. Sci. Rep. 6, 20219 (2016). 37. Liang, Z. & Li, J. Extreme acoustic metamaterial by coiling up space. Phys. Rev. Lett. 108, 114301 (2012). Page 9/16 38. Dubois, M., Shi, C., Zhu, X., Wang, Y. & Zhang, X. Observation of acoustic Dirac-like cone and double zero refractive index. Nat. Commun. 8, 14871 (2017). 39. Xu, C. et al. Three-Dimensional Acoustic Double-Zero-Index Medium with a Fourfold Degenerate Dirac-like Point. Phys. Rev. Lett. 124, 074501 (2020). 40. Sanchis, L. et al. Three-dimensional axisymmetric cloak based on the cancellation of acoustic scattering from a sphere. Phys. Rev. Lett. 110, 124301 (2013). 41. Zhang, X., Xiao, M., Cheng, Y., Lu, M. H. & Christensen, J. Topological sound. Commun. Phys. 1, 97 (2018). 42. Liu, C. et al. Wide-Angle Broadband Nonreflecting Acoustic Metamaterial Fence. Phys. Rev. Appl. 13, 054012 (2020). 43. Li, J., Fok, L., Yin, X., Bartal, G. & Zhang, X. Experimental demonstration of an acoustic magnifying hyperlens. Nat. Mater. 8, 931–934 (2009). 44. Zhu, J. et al. A holey-structured metamaterial for acoustic deep-subwavelength imaging. Nat. Phys. 7, 52–55 (2011). 45. Xia, J. P., Sun, H. X. & Yuan, S. Q. Modulating Sound with Acoustic Metafiber Bundles. Sci. Rep. 7, 8151 (2017). 46. Chen, H. et al. Ray-optics cloaking devices for large objects in incoherent natural light. Nat. Commun. 4:, 2652 (2013). 47. Chen, X. et al. Macroscopic invisibility cloaking of visible light. Nat. Commun. 2:, 176 (2011). 47. Chen, X. et al. Macroscopic invisibility cloaking of visible light. Nat. Com Supplemental Videos The Supplemental Videos are not available with this version. The Supplemental Videos are not available with this version. Figures Page 10/16 Figure 1 Illustration of the illusion. Upon the incidence of a sound wave, a multiple scattering is generated by a random collection of obstacles, however, b when the obstacles are embedded in the illusion metamaterial, the backward reflection is removed and the wave can propagate around the obstacles with its wavefront undistorted. The arrows and the alternating solid and dashed lines represent sound wave and its wavefront, respectively. Figure 1 Illustration of the illusion. Upon the incidence of a sound wave, a multiple scattering is generated by a random collection of obstacles, however, b when the obstacles are embedded in the illusion metamaterial, the backward reflection is removed and the wave can propagate around the obstacles with its wavefront undistorted. The arrows and the alternating solid and dashed lines represent sound wave and its wavefront, respectively. Illustration of the illusion. Upon the incidence of a sound wave, a multiple scattering is generated by a random collection of obstacles, however, b when the obstacles are embedded in the illusion metamaterial, the backward reflection is removed and the wave can propagate around the obstacles with its wavefront undistorted. The arrows and the alternating solid and dashed lines represent sound wave and its wavefront, respectively. Page 11/16 Figure 2 Design of acoustic tunnels with tunable acoustic paths. a Schematic diagram of a curved tunnel with a varying cross-section. The grey portion represents the solid structure. A magnified view is displayed in the top-right corner. Arrows indicate the incidence and transmission of waves. b Simulated acoustic pressure field distributions that vary with the ratio of h/w, corresponding to phase shift ΔΦ=0, π, 2π. c Phase shift ΔΦ and transmittance |t|2 under normal incidence as a function of h/w Vertical grey dotted Figure 2 D i f ti t l ith t bl ti th S h ti di f d t l ith Figure 2 Figure 2 Design of acoustic tunnels with tunable acoustic paths. a Schematic diagram of a curved tunnel with a varying cross-section. The grey portion represents the solid structure. A magnified view is displayed in the top-right corner. Arrows indicate the incidence and transmission of waves. b Simulated acoustic pressure field distributions that vary with the ratio of h/w, corresponding to phase shift ΔΦ=0, π, 2π. c Phase shift ΔΦ and transmittance |t|2 under normal incidence as a function of h/w. Vertical grey dotted lines indicate the cases with ΔΦ=0, π, 2π. d Equivalent relative refractive index nr as a function of h/w and the frequency f. Page 12/16 Page 12/16 Figure 3 Design of the ultra-broadband illusion metamaterial and experimental setup. a Schematic diagram of the illusion metamaterial and experimental setup. A magnified view of the metamaterial is shown in b. The side length of the square-shaped unit is  a=20 cm. The diagonal lengths of the rhombic-shaped obstacle are represented by a and a/2, respectively. c Geometric parameter h/w for various tunnels in the metamaterial. d Picture of the experimental setup. Design of the ultra-broadband illusion metamaterial and experimental setup. a Schematic diagram of the illusion metamaterial and experimental setup. A magnified view of the metamaterial is shown in b. The side length of the square-shaped unit is  a=20 cm. The diagonal lengths of the rhombic-shaped obstacle are represented by a and a/2, respectively. c Geometric parameter h/w for various tunnels in the metamaterial. d Picture of the experimental setup. Design of the ultra-broadband illusion metamaterial and experimental setup. a Schematic diagram of the illusion metamaterial and experimental setup. A magnified view of the metamaterial is shown in b. The side length of the square-shaped unit is  a=20 cm. The diagonal lengths of the rhombic-shaped obstacle are represented by a and a/2, respectively. c Geometric parameter h/w for various tunnels in the metamaterial. d Picture of the experimental setup. Page 13/16 P 14/16 Figure 4 Ultra-broadband illusionary effect of “disappearing space” and sound scattering suppression. a,c,e Simulated acoustic pressure field distributions for the scenario of bare obstacles under a point source radiation at different operating frequencies of f = 4860Hz, 6860 Hz, 8860 Hz, and the corresponding experimental measurements (located in the black dashed area) are on the right. b,d,f Results for the scenario of obstacles covered by the illusion metamaterial. Figure 2 g-h Frequency dependence of the normalized scattered pressure intensity for the transmission region (γt) and reflection region (γr). Figure 4 Figure 4 ffect of “disappearing space” and sound scattering suppression. a,c,e Ultra-broadband illusionary effect of “disappearing space” and sound scat Page 14/16 Ultra-broadband illusionary effect of “disappearing space” and sound scattering suppression. a,c,e Simulated acoustic pressure field distributions for the scenario of bare obstacles under a point source radiation at different operating frequencies of f = 4860Hz, 6860 Hz, 8860 Hz, and the corresponding experimental measurements (located in the black dashed area) are on the right. b,d,f Results for the scenario of obstacles covered by the illusion metamaterial. g-h Frequency dependence of the normalized scattered pressure intensity for the transmission region (γt) and reflection region (γr). Page 14/16 Ultra-broadband illusionary effect of “disappearing space” and sound scattering suppression. a,c,e Simulated acoustic pressure field distributions for the scenario of bare obstacles under a point source radiation at different operating frequencies of f = 4860Hz, 6860 Hz, 8860 Hz, and the corresponding experimental measurements (located in the black dashed area) are on the right. b,d,f Results for the scenario of obstacles covered by the illusion metamaterial. g-h Frequency dependence of the normalized scattered pressure intensity for the transmission region (γt) and reflection region (γr). Page 14/16 Page 14/16 igure 5 ulse radiation and illusionary effect of “time shift”. a Snapshots of a sound pulse propagating through a ollection of obstacles with significant scattering. b Snapshots of a sound pulse propagating through the bstacles embedded in the illusion metamaterial. The inset graphs display magnified details of the wavefront inside the metamaterial. The numbers indicate the order of the snapshots, where Δt = 0.1 ms. Figure 5 Pulse radiation and illusionary effect of “time shift”. a Snapshots of a soun Pulse radiation and illusionary effect of “time shift”. a Snapshots of a sound pulse propagating through a collection of obstacles with significant scattering. b Snapshots of a sound pulse propagating through the obstacles embedded in the illusion metamaterial. The inset graphs display magnified details of the wavefront inside the metamaterial. The numbers indicate the order of the snapshots, where Δt = 0.1 ms. See Supplemental Videos. Pulse radiation and illusionary effect of “time shift”. a Snapshots of a sound pulse propagating through a collection of obstacles with significant scattering. b Snapshots of a sound pulse propagating through the obstacles embedded in the illusion metamaterial. The inset graphs display magnified details of the wavefront inside the metamaterial. Figure 4 The numbers indicate the order of the snapshots, where Δt = 0.1 ms. See Supplemental Videos. Page 15/16 Figure 6 Ultra-broadband illusion metamaterial for a random collection of obstacles. a Schematic of a specific illusion metamaterial composed of three rhombic obstacles arranged randomly. b Simulated acoustic pressure field distributions for the scenarios of obstacles with or without the metamaterial under a poi source radiation at an operating frequency of f = 6860Hz. The point source is located at (-20 cm, 0 cm) and the center of three obstacles are located at (10 cm, 10 cm), (35 cm, 0 cm) and (12.5 cm, -12.5 cm) respectively. c-d Frequency dependence of the normalized scattered pressure intensity for the transmission region (γt) and reflection region ( γr). Fi 6 Ultra-broadband illusion metamaterial for a random collection of obstacles. a Schematic of a specific Ultra-broadband illusion metamaterial for a random collection of obstacles Ultra-broadband illusion metamaterial for a random collection of obstacles. a Schematic of a specific illusion metamaterial composed of three rhombic obstacles arranged randomly. b Simulated acoustic amaterial for a random collection of obstacles. a Schematic of a specific Ultra broadband illusion metamaterial for a random collection of obstacles. a Schematic of a specific illusion metamaterial composed of three rhombic obstacles arranged randomly. b Simulated acoustic pressure field distributions for the scenarios of obstacles with or without the metamaterial under a point source radiation at an operating frequency of f = 6860Hz. The point source is located at (-20 cm, 0 cm), and the center of three obstacles are located at (10 cm, 10 cm), (35 cm, 0 cm) and (12.5 cm, -12.5 cm), respectively. c-d Frequency dependence of the normalized scattered pressure intensity for the transmission region (γt) and reflection region ( γr). SupplementalMaterials.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementalMaterials.docx Page 16/16 Page 16/16 Page 16/16
https://openalex.org/W4298030058
https://zenodo.org/records/4642138/files/14_2_1_Albesa_Martinez-Orti_Robles.pdf
es
Primeros datos sobre la superfamilia Clausilioidea (Gastropoda, Pulmonata) en la Comunidad Valenciana
Zenodo (CERN European Organization for Nuclear Research)
1,996
cc-by
2,247
IBERUS, 14 (2): 1-8, 1996 Primeros datos sobre la superfamilia Clausilioidea (Gastropoda, Pulmonata) en la Comunidad Valenciana First data on the superfamily Clausilioidea (Gastropoda, Pulmonata) in the Comunidad Valenciana (Spain) Joaquín ALBESA*, Alberto MARTÍNEZ-ORTÍ** y Fernando ROBLES* RESUMEN La superfamilia Clausilioidea está representada en la Comunidad Valenciana por dos especies: Clausilia (Clausilia) bidentata abietina Dupuy, 1849 y Cochlodina (Cochlodina) laminata (Montagu, 1803), que se han recogido en el norte de la provincia de Castellón. Se comentan e ilustran sus conchas y rádulas mediante fotografías con MEB, así como el aparato genital y las mandíbulas de los ejemplares recolectados de ambas especies. Las nuevas localidades representan un enclave aislado de estas especies, muy alejado de su área de distribución previamente conocida. ABSTRACT Two species of the superfamily Clausilioidea are present in the Comunidad Valenciana (Spain): Clausilia (Clausilia) bidentata abietina Dupuy, 1849 and Cochlodina (Cochlodina) laminata (Montagu, 1803), both collected in the North of the province of Castellón. SEM photographies of shell and radula are included and commented, as well as the genital system and jaws. The new locations represent isolated places of these species, too far away from their previously known distribution area. PALABRAS CLAVE: Gastropoda, Clausilioidea, distribución geográfica, Comunidad Valenciana, España. KEY WORDS: Gastropoda, Clausilioidea, geographic distribution, Comunidad Valenciana, Spain. INTRODUCCIÓN La superfamilia Clausilioidea, representada por varias especies en el Norte de la Península Ibérica, era prácticamente desconocida hasta ahora en el territorio de la Comunidad Valenciana. La única referencia que hemos localizado en la revisión bibliográfica es la cita de Clausilia rugosa en el catálogo de la colección PAETEL (1873), en la ambigua localidad «Valencia». El muestreo sistemático que están realizando los autores en la provincia de Castellón ha permitido recolectar dos especies de esta superfamilia en el norte de la provincia. Descri- * Departamento de Geología. **Departamento de Biología Animal. Facultad de Ciencias Biológicas. Universidad de Valencia. Dr. Moliner, 50. 46100 Burjassot (Valencia). 1 IBERUS, 14 (2), 1996 bimos a continuación las principales características de las mismas, que presentan especial interés desde el punto de vista biogeográfico. MATERIAL Y MÉTODOS El material examinado procede, en su mayor parte, de la provincia de Castellón. Se añaden también algunas locali- dades de Tarragona y Teruel, que forman parte del mismo enclave geográfico. Para el estudio de las rádulas, se sometió el bulbo a una hidrólisis a temperatura ambiente en una solución de potasa 2,5 M. Las conchas y protoconchas, se limpiaron con agua oxigenada al 10% hasta eliminar el periostraco. Para estudiar la ornamentación de la abertura, se levantó la porción final de la última vuelta, extrayéndose el clausilio. RESULTADOS Superfamilia CLAUSILIOIDEA Familia CLAUSILIDAE Subfamilia ALOPIINAE Cochlodina (Cochlodina) laminata (Montagu, 1803) Material examinado: Fuente de las Rocas, Vallibona (Castellón), 31TBE4697, 1120 m (1 ej. + 3 c.). Fredes (Castellón), 31TBF6210, 920 m (1 ej. + 8 c.), 11/11/91 y 13/11/92. Font Ferrera, La Sènia (Tarragona), 31TBF6312, 1000 m (3 c.), 11/11/91, 1/10/92 y 13/11/92. Beceite, Río Matarraña (Teruel) 31TBF6222, 650 m (5 c.), 3/3/82. El Parrizal de Beceite (Teruel), 31TBF6419, a más de 800 m (7 c.), 3/3/82. Descripción: La morfología de la concha (Figs. 1E, F, 2A-E), coincide con las descripciones y figuras aportadas por ADAM (1960), GERMAIN (1930), GITTENBERGER, BACKHUYS Y RIPKEN (1984) y GROSSU (1981). Las dimensiones de las conchas recolectadas varían entre 16 y 19,3 mm de longitud y entre 4 y 4,4 mm de diámetro. Presentan entre 111/4 y 12 vueltas de espira; la protoconcha ocupa entre 21/4 y 21/2 vueltas. Las características de la denticulación y la morfología del clausilio son las típicas de la especie. Las características del aparato genital (Fig. 1H) coinciden con los datos aporta- dos por GERMAIN (1930), GROSSU (1981) y STEENBERGER (1914). La mandíbula (Fig. 1G), no es muy sólida y transversalmente la recorren numerosos surcos finos, que no llegan a constituir costillas. La rádula (Figs. 2F, G), presenta una fórmula hemiradular de 25+C. El diente central, de menor tamaño que los primeros laterales, es tricúspide y el mesocono tiene un poco más del doble de la longitud de los ectoconos. Los dientes laterales poseen un ectocono pequeño que se va extendiendo a medida que se acercan a los márgenes laterales de la rádula. Los dientes marginales poseen un ectocono dividido hasta en cuatro cúspides, al igual que el mesocono. Subfamilia CLAUSILIINAE Clausilia (Clausilia) bidentata abietina Dupuy, 1849 Material examinado: Fuente de las Rocas, Vallibona (Castellón), 31TBE4697, 1120 m (25 ejemplares vivos + 40 conchas), 11/11/91, 1/10/92 y 13/11/92. Descripción: La variabilidad de la concha de Clausilia bidentata ha dado 2 origen a la descripción de numerosas especies y variedades que, tras las re- 1 mm 1 mm 1 mm 1 mm ALBESA ET AL.: La superfamilia Clausilioidea en la Comunidad Valenciana C A D B 60 µm G H E 60 µm F 2 mm 1 mm Figura 1. Clausilia (Clausilia) bidentata abietina. A: concha; B: clausilio; C: genitalia; D: mandíbula. Cochlodina (Cochlodina) laminata. E: concha; F: clausilio; G: mandíbula; H: genitalia. Figure 1. Clausilia (Clausilia) bidentata abietina. A: shell; B: clausilium; C: genital system; D: jaw. Cochlodina (Cochlodina) laminata. E: shell; F: clausilium; G: jaw; H: genital system. 3 IBERUS, 14 (2), 1996 B C A D E F G Figura 2. Cochlodina (Cochlodina) laminata. A: abertura; B: clausilio; C: láminas de la abertura; D: protoconcha y primeras vueltas de la teloconcha; E: ornamentación; F: dientes central y primer lateral; G: dientes marginales. Escala, A, D: 1,36 mm; B: 0,6 mm; C, E: 1,2 mm; F: 6,1 µm; E: 12 µm. Figure 2. Cochlodina (Cochlodina) laminata. A: aperture; B: clausilium; C: sheets in the aperture; D: protoconch and first teleoconch whorls; E: ornamentation; F: rachidian and first lateral teeth; G: marginal teeth. Scale bar, A, D: 1,36 mm; B: 0,6 mm; C, E: 1,2 mm; F: 6,1 µm; G: 12 µm. 4 ALBESA ET AL.: La superfamilia Clausilioidea en la Comunidad Valenciana A C B D E F G H Figura 3. Clausilia (Clausilia) bidentata abietina. A: abertura; B: clausilio; C: láminas de la abertura; D: protoconcha y primeras vueltas de la teloconcha; E: nuca; F: ornamentación; G: dientes central y primer lateral; H: dientes marginales. Escalas, A, D, F: 0,86 mm; B: 0,43 mm; C: 1 mm; E: 1,2 mm; G: 6 µm; H: 10 µm. Figure 3. Clausilia (Clausilia) bidentata abietina. A: aperture; B: clausilium; C: sheets in the aperture; D: protoconch and first teleoconch whorls; E: nape; F: ornamentation; G: rachidian and first lateral teeth; H: marginal teeth. Scale bars, A, D, F: 0,86 mm; B: 0,43 mm; C: 1 mm; E: 1,2 mm; G: 6 µm; H: 10 µm. 5 IBERUS, 14 (2), 1996 cientes revisiones de HOLYOAK Y SEDDON (1988) y NORDSIECK (1990), han sido consideradas como sinónimos posteriores de aquélla. Para la determinación hemos utilizado las descripciones y figuras de C. bidentata y de C. nigricans, uno de estos sinónimos, que proporcionan ADAM (1960), CASTILLEJO (1983), GERMAIN (1930), HOLYOAK Y SEDDON (1988) y NORDSIECK (1990). Las dimensiones de las conchas recolectadas varían entre 10,5 y 11,7 mm de longitud y entre 2,4 y 2,7 mm de diámetro. Aparece constituida por 11 a 12 vueltas de espira; la protoconcha tiene de 2 a 3 vueltas. La morfología general, ornamentación y denticulación de la abertura (Figs. 1A, B; 3A-F) coinciden con las de esta especie, de acuerdo con los datos suministrados por los autores citados. NORDSIECK (1990) acepta la existencia de dos subespecies en la Península Ibérica: C. bidentata bidentata y C. bidentata abietina, que se diferencia de la subespecie nominal por la presencia de una robusta quilla nucal característica. La comparación, utilizando el MEB, de la porción cervical de la última vuelta de nuestros ejemplares con los de C. b. bidentata recogidos en el País Vasco, permiten observar la existencia de una fuerte quilla nucal, que limita un surco más marcado en los primeros que en los segundos. Debido a ello, los incluimos en la subespecie C. b. abietina. El aparato genital (Fig. 1C) ha sido descrito por CASTILLEJO (1993), FACI (1991), HOLYOAK Y SEDDON (1988) y NORDSIECK (1990). NORDSIECK (1990) señala que en la mayor parte de los ejemplares por él estudiados, el músculo retractor del pene está bifurcado; sin embargo, en ninguno de los cinco ejemplares que nosotros hemos examinado aparece esta característica. FACI (1991) indica la existencia de un único músculo retractor, lo que coincide con nuestras observaciones; CASTILLEJO (1983) no menciona esta característica. En todos los ejemplares, excepto en uno, aparece el divertículo, que posee una longitud máxima de 3 mm y queda incrustado entre la próstata. La mandíbula (Fig. 1D), arqueada y bastante consistente, aparece recorrida por numerosos surcos, constituyendo aproximadamente unas 35 costillas. La rádula (Figs. 3G, H), cuya fórmula hemiradular es 15+C, posee un diente central de menor tamaño que los primeros laterales y cuyo mesocono es de tres veces la longitud de los ectoconos. Los dientes laterales poseen un ectocono extendido lateralmente, cuya longitud es la mitad del mesocono. Los dientes marginales poseen un ectocono con cuatro cúspides y un mesocono que llega a poseer hasta seis cúspides. DISCUSIÓN El interés principal del hallazgo de dos especies de clausílidos en el norte de la Comunidad Valenciana es de orden biogeográfico. Por una parte aumenta nuestro conocimiento de la fauna malacológica regional, a la que deben agregarse Cochlodina laminata y Clausilia bidentata abietina. A ello hay que añadir la primera cita de C. laminata en la provincia de Tarragona. Las localidades de Beceite (Teruel), donde C. laminata ha sido citada también por ALTIMIRA (1965), Font Ferrera (Tarragona), Vallibona y Fredes (Castellón) se encuentran muy próximas entre sí y configuran un amplio enclave aislado geográficamente. En la Figura 4A 6 puede comprobarse la singularidad de este enclave, muy alejado del área de distribución de C. laminata en el nordeste de la Península Ibérica. La dispersión de C. b. abietina es más difícil de establecer, dada la escasez de localidades concretas en las que esta subespecie ha sido separada de la subespecie nominal. De todas formas la bibliografía consultada (BECH, 1990; NORDSIECK, 1990; FACI, 1991) permite constatar que su área de distribución es similar, en la Península Ibérica, a la de C. laminata. Se conocen, no obstante, algunas poblaciones aisladas de C. bidentata, similares a las que estudiamos nosotros, como es el caso de la Sierra del Moncayo, en el ALBESA ET AL.: La superfamilia Clausilioidea en la Comunidad Valenciana A B Figura 4. A: mapa de distribución de Cochlodina (Cochlodina) laminata en el nordeste de la Península Ibérica. Superficie en negro: distribución en la vertiente sur de los Pirineos (según BECH, 1990 y FACI, 1991); asteriscos: localidades propias. B: mapa de la Provincia de Castellón con cuadrícula UTM de 10x10 km2, con indicación de las localidades muestreadas. Estrella: Clausilia bidentata abietina y Cochlodina laminata; asterisco: Cochlodina laminata. Figure 4. A: Cochlodina (Cochlodina) laminata distribution map in the northeastern Iberian peninsula. Black colour: distribution in the south side of the Pyrenees (after BECH, 1990 and FACI, 1991); asterisks: own records. B: Castellón province map with 10x10 km2 UTM coordinates, showing the sampled localities. Star: Clausilia bidentata abietina and Cochlodina laminata; asterisk: Cochlodina laminata. límite entre las provincias de Soria y Zaragoza (FACI, 1991). El origen de esta distribución discontinua puede atribuirse al hábitat característico de estas especies, por una parte, y a las condiciones orográficas y climáticas de la región, por otra. Las especies estudiadas caracterizan zonas de clima atlántico y, en el área mediterránea, sólo se encuentran en zonas montañosas de relativa altura, donde se dan las condiciones climáticas de humedad y frescor estival, adecuadas para su desarrollo. Estas condiciones son las que existen en las localidades donde hemos recogido estas especies y debieron darse en un área mucho más extensa en épocas glaciares. El calentamiento postglaciar debe de estar en el origen de la distribución actual de las mismas, al propiciar la aparición de áreas relictas con condiciones bioclimáticas adecuadas. AGRADECIMIENTOS Los autores agradecen a D. Agustín Tato, técnico del Servicio de Microscopía Electrónica de la Universitat de Valencia, su ayuda en la realización de las fotografías. 7 IBERUS, 14 (2), 1996 BIBLIOGRAFÍA ADAM, W., 1960. Faune de Belgique. Mollusques terrestres et dulcicoles. Institute royal des Sciences naturelles Belgique, Bruxelles, 403 pp. ALTIMIRA, C., 1965. Notas malacológicas. (3). Datos sobre tres moluscos de la familia Clausiliidae en Cataluña y Aragón. Miscel.lanea Zoologica, 2: 22. BECH, M., 1990. Fauna malacològica de Catalunya. Mol.luscs terrestres y d’aigua dolça. Treballs Institució Catalana d’Història Natural, 12: 1-229. CASTILLEJO, J., 1983. Caracoles terrestres de Galicia. IV: Especies de los Superórdenes Systellommatophora y Stylommatophora (Gastropoda, Pulmonata). Trabajos Compostelanos de Biología, 10: 53-85. FACI, G., 1991. Contribución al conocimiento de diversos moluscos terrestres y su distribución en la Comunidad Autónoma Aragonesa. Tesis Doctoral (inédita). Universidad de Zaragoza. 787 pp. GERMAIN, L., 1930. Mollusques terrestres et fluviatiles, Faune de France, 21: 1-447. Paris. GITTENBERGER, E., BACKHUYS, W. Y RIPKEN, TH. E. J., 1984. De landslakken van Nederland. Koninklijke Nederlandse Natuurhistorische Vereniging Uitgave, 37: 1-183. GROSSU, A. V., 1981. Gastropoda Romaniae. Ordo Stylommatophora. 3. Suprafamiliile Clausiliacea, Achatinacea. Bucuresti. 269 pp. HOLYOAK, D. T. Y SEDDON, M. B., 1988. Systematics and biogeography of some western european Clausilia (Gastropoda: Clausiliidae). Journal of Conchology, 33: 55-80. NORDSIECK, H., 1990. Revision der Gattung Clausilia Draparnaud, besonders der Arten in SW-Europa (Das Clausilia rugosaProblem) (Gastropoda: Stylommatophora: Clausilidae). Archiv für Molluskenkunde, 118: 133-179. PAETEL, F., 1873. Catalog der ConchylienSammlung, 172 pp. Berlin. STEENBERGER, C. M., 1914. Anatomie des Clausilies danoises. I. Les organes génitaux. Mindeskrift for Japetus Steenstrup, 29: 1-44. Recibido el 15-II-1993 Aceptado el 18-VI-1993 8
https://openalex.org/W2954315564
https://europepmc.org/articles/pmc6610097?pdf=render
English
null
The Route to ‘Chemobrain’ - Computational probing of neuronal LTP pathway
Scientific reports
2,019
cc-by
18,558
The Route to ‘Chemobrain’ - Computational probing of neuronal LTP pathway Received: 8 November 2018 Accepted: 19 June 2019 Published: xx xx xxxx Ammad Fahim   1, Zaira Rehman1, Muhammad Faraz Bhatti1, Nasar Virk1,4, Amjad Ali1, Amir Rashid3 & Rehan Zafar Paracha2 Chemotherapy causes deleterious side effects during the course of cancer management. The toxic effects may be extended to CNS chronically resulting in altered cognitive function like learning and memory. The present study follows a computational assessment of 64 chemotherapeutic drugs for their off-target interactions against the major proteins involved in neuronal long term potentiation pathway. The cancer chemo-drugs were subjected to induced fit docking followed by scoring alignment and drug-targets interaction analysis. The results were further probed by electrostatic potential computation and ligand binding affinity prediction of the top complexes. The study identified novel off-target interactions by Dactinomycin, Temsirolimus, and Everolimus against NMDA, AMPA, PKA and ERK2, while Irinotecan, Bromocriptine and Dasatinib were top interacting drugs for CaMKII. This study presents with basic foundational knowledge regarding potential chemotherapeutic interference in LTP pathway which may modulate neurotransmission and synaptic plasticity in patient receiving these chemotherapies. Cancer is a multifactorial disease harboring disorders on multiple levels, rendering a single drug to be therapeu- tically insufficient. Therefore, designing a single drug aiming multiple targets is a difficult proposition. Keeping this in view, multiple targeted combinatorial therapeutic regimens, to achieve enhanced therapeutic efficacy, have been the main stay of cancer treatment. However, employment of drug combinations, may present with increased drug toxicity and varied battery of side effects1. Hence, foreseeing the drug side effects and probable drug adverse reactions (ADRs) encountered by patients is difficult by the existing medical literature. This in turn significantly effects quality of life of patients during treatment and disguise the patient well-being even after the treatment is concluded. Such side effects may be because of drug promiscuity or off targets interactions inducing drug-drug or drug-target interactions2. As the wealth of information regarding disease state, particularly cancers and its ther- apies is growing, it is more evident that both on-target and off-target drug-protein interactions need to be taken in account as to predict drug specific side effect profiles. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 8 November 2018 Accepted: 19 June 2019 Published: xx xx xxxx www.nature.com/scientificreports/ p , p , p p y The signaling intricacies triggered by LTP induction is not merely linear in its path but rather involve complex interaction and crosstalk of other pathways emaciating into converging or diverging outcome which may end up in positive or negative neuro-signaling feedback loops17. Substantial evidence been put forwarded that CICI may be implicated to interfere LTP15,16.The investigative ground gets further complicated by considering the cancer patient specific clinico-pathological characteristic and the subsequent combination and dosage of chemotherapy administered10. As most of the chemotherapeutic agents have been implicated to affect neurons by more than one mechanism, the resultant outcome of CICI may be attributed to converging and synergistic neurotoxic insults. It is also rather interesting to note that chemotherapy which generally target rapidly dividing cells, can also target very slowly reproducing cells of CNS. y y p g Most of the drugs impart their therapeutic action by stimulating or inhibiting a disease target protein. However, they may be rendered to interact or bind with other proteins i.e. ‘off-target’ due to resemblances in protein binding topological state. These off-target interactions can be a probable eventuality by considering the fact that drugs rarely binds to its only actual target28. Such outcome can lead to high drug attrition rate29. If an off target is known to mediate a certain side effect, then this information can be potentially utilized to adjust dose, avoid drug side effect and improve management in patients for better clinical outcomes.hi gf g The spectrum of neurological deficits by CICI is encompassing almost all classes of chemotherapeutic agents which include alkylating agents, anthracyclines, DNA interfering agents, antitumor antibiotics, mitotic inhibi- tors, antimetabolites and anti-hormonal agents30–41. Paradoxically, the deleterious effects of these agents on brain cortical functions in general and neurodegeneration in particular are well reported14–16,31,42, however, the effect of these drugs by virtue of their off target interaction without actually killing neurons but altering their functional dynamics on a particular pathway remains elusive.h y p p y The emergence of newer biological, chemical and immunotherapeutic cancer treating agents, with various unconventional drug delivery mechanisms has created scientific plausibility to understand the role of these ther- apeutic agents and their cumulative outcomes to cell signaling. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Previous studies addressing ‘chemobrain’ can be broadly segregated in to clinical and preclinical experi- ments14,15. The clinical studies engross clinical assessment of cognitive function during and/or after chemother- apy via use of battery of neuropsychological test. Whilst, preclinical histological or behavioral studies investigate any potential connection between chemotherapy associated neurotoxicity and hippocampal neurodegeneration in cell lines and rodent models. Interestingly, neurodegeneration has not been the only backdrop of chemo- brain. There is aberrant neuronal signaling and altered long term potentiation (LTP), suggesting the chemother- apy may not be toxic enough to ensue neurodegeneration, but deleterious enough to impair default neuronal functionality16.h y The neurological processes of learning and memory storage are reliant on active inter synaptic connectivity strength and subsequently the involvement of larger active synapses will be leading to bigger synaptic efficiency in the form of ‘Long Term Potentiation’ (LTP). Mechanistically and temporally, LTP can be dichotomized into Early LTP (E-LTP), which initiates following inducing stimulus lasting from few minutes to approx. 4 hours, and Late LTP (L-LTP) spanning from few hours to days, dependent on new protein synthesis17. The LTP induc- tion and maintenance is stringently managed by ionotropic Glutamate receptors (iGluR)18.Aberrant activation of iGluRs may lead to fulminant neuronal death also known as glutamate excitotoxicity19. One of the key regulators of LTP are NMDA-R and AMPA-R. These are voltage gated cationic channels with Ca+2 and Na+ preferences respectively20. The case of iGluR being targeted by pharmacologic modulation in order to augment the excitatory neurotransmission, is more than two decades old21. The rationale for drug targeting lies in the functional impor- tance of LTP in facilitating learning and memory. The therapeutic benefit can be translated for psychiatric disor- ders22. Therefore, any off target interaction can interfere with NMDA-R transportation and re-sculpting neuronal synapse, thereby rejuvenating synaptic efficiency in general and E-LTP in particular. This resultantly, will affect L-LTP induction which is dependent on E-LTP. The NMDA-R are also known for their critical involvement in neural cell migration and neural tube formation during embryogenesis. So evidently, alteration in their function leads to neural tube defects23. Moreover, they have been targeted for therapeutic modulation for CNS disorders like Schizophrenia24, Depression25, Alzheimer’s disease26 and Epilepsy27. The Route to ‘Chemobrain’ - Computational probing of neuronal LTP pathway Interestingly, such off targets have been notifying their presence notably in the shape of cardiotoxicity attributable to hERG inhibition3 and hepatotoxicity attributably by CYP inhibition4.hf y Therapies for cancer can cause both central and peripheral toxicities leading to a wide differential of cognitive changes which may span from acute onset delirium like symptoms to more progressive degenerative changes and delayed neurological consequences termed as ‘Chemobrain’,‘Chemofog’, ‘Cancer related Cognitive Impairment’ or ‘Chemotherapy induced Cognitive Impairment’ (CICI)5–10. Normal human cognitive process constitutes var- ious important neurobiological processes of day to day life like attention, learning, memory, planning and deci- sion making11,12. Therefore, any interference may translate into neurodegenerative and psychiatric morbidity. The CICI can exhibit itself in varying forms ranging from but not limited to headache, seizures, acute or chronic encephalopathies, cerebrovascular disease, movement disorders and cranial neuropathies13. 1Atta ur Rahman School of Applied Biosciences, National University of Sciences and Technology (NUST), Islamabad, Pakistan. 2Research Centre for Modeling and Simulation, National University of Sciences and Technology (NUST), Islamabad, Pakistan. 3Department of Biochemistry, National University of Medical Sciences, Rawalpindi, Pakistan. 4Present address: EBS Universität für Wirtschaft und Recht, EBS Business School, Rheingaustrasse 1, Oestrich- Winkel, 65375, Germany. Correspondence and requests for materials should be addressed to A.F. (email: afahim. phdabs04asab@asab.nust.edu.pk) or M.F.B. (email: mfbhatti@asab.nust.edu.pk) or R.Z.P. (email: rehan@rcms. nust.edu.pk) entific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 1 www.nature.com/scientificreports/ The advent of kinase inhibitors as chemothera- peutic agents with better blood brain barrier (BBB) permeation43,44, intrathecal administration of chemotherapy for managing metastatic disease45, and increasing the porosity of BBB by methamphetamine administration46, markedly increase the exposure of neuronal microenvironment to chemotherapeutic drugs. It is interesting to note that the employment of in vivo experimental tools like yeast two hybrid system and mass spectrome- try coupled with tandem affinity purification to experimentally measure protein interactions comes with high false positive rate47. Keeping in view of such limitation, the in silico prediction tools such as Molecular Docking may provide useful illustration of given 3D drugs structure interactions with large protein datasets. Molecular Docking involves prediction of molecular mechanics among molecules by computation of polyatomic torsional angles, charges and geometry48. Docking results in generation of thousands of potential poses of association in which the pose with lowest energy score is predicted to be with best binding mode. The lowest energy scor- ing reflects binding compactness for a particular ligand conformation bearing a physical or empirical energy function49. Although docking is labor intensive, it not simply shows two interactable proteins but also how they interact50. Most of the studies on drug target interactions are deploying statistical machine learning algorithms to execute high throughput screening for large drug databases and for genome wide predictions51–55. Although such methodology is useful for the intended objective, specific study of actual interactions with the corresponding target protein and the overall implication on the related pathway may be missed which can serve as a useful infor- mation for preventing adverse drug reactions. Moreover, the same information can be used for polypharmacology which is already in clinical evaluation with reference to mTOR inhibitors for varying indications such as cancer chemotherapy and Autism spectrum disorders56–58. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 2 www.nature.com/scientificreports/ Figure 1. Overall workflow of identification of off-target interactions protocol. Three dimensional structures of NMDA, AMPA, PKA, ERK, CBP and CaMKII were downloaded from protein databank and the structures of chemotherapeutic drugs were obtained from PubChem. These structures were then energy minimized. The validation of docking protocol was done by removal of co-crystallize ligand and re-docking of ligand in molecular operating environment (MOE) followed by RMSD calculation. The docking of drugs with the proteins was then performed using induced fit docking protocol. www.nature.com/scientificreports/ Top scoring complexes from each protein were then subjected to interaction analysis by protein ligand interaction profiler (PLIP) and PyMOL as well as for electrostatic surface calculations by PyMOL. Binding affinity analysis was done using CSM-Lig server. The ligand interaction fingerprints were calculated using MOE. Figure 1. Overall workflow of identification of off-target interactions protocol. Three dimensional structures of NMDA, AMPA, PKA, ERK, CBP and CaMKII were downloaded from protein databank and the structures of chemotherapeutic drugs were obtained from PubChem. These structures were then energy minimized. The validation of docking protocol was done by removal of co-crystallize ligand and re-docking of ligand in molecular operating environment (MOE) followed by RMSD calculation. The docking of drugs with the proteins was then performed using induced fit docking protocol. Top scoring complexes from each protein were then subjected to interaction analysis by protein ligand interaction profiler (PLIP) and PyMOL as well as for electrostatic surface calculations by PyMOL. Binding affinity analysis was done using CSM-Lig server. The ligand interaction fingerprints were calculated using MOE. In spite of off-target interactions by Tamoxifen and its metabolites have been reported59, there is a general dearth of information objectively elaborating the underlying biology of side effects causing chemobrain attributed to LTP interference mediated by simultaneous exposure of chemotherapeutic agents. Therefore, this study aimed to understand the drug – protein interaction casted by various chemotherapeutic agents to the major cellular pro- teins involved in LTP pathway which may impact functionality of these proteins critical for learning and memory processes of brain. Experimental Sectionl p A brief workflow used for the identification of off-target interactions between LTP proteins and chemotherapeutic drugs is shown in Fig. 1. Selection of drugs. In order to study the off-target interactions, most commonly used FDA approved chem- otherapeutic agents were selected from National Cancer Institute (NCI) directory60. Among them are alkylating agents, anti-metabolites, alkaloids, anthracyclines, aromatase inhibitors, nucleoside analogues, anti-hormonal, and antibiotics agents (Fig. 2). The structure of 65 drugs were extracted from PubChem followed by energy mini- mization using MMF94x force field61 implemented in Molecular Operating Environment (MOE) version 2016.08. Database of drugs was then constructed for off-target interaction analysis. Selection of proteins to study off-target interactions. In order to study the off-target interactions of chemotherapeutic drugs on the cognition, long term potentiation pathway was selected (Fig. 3). The major regulators of LTP pathway are N-methyl-D-aspartate receptor (NMDA), α-amino-3-hydroxy-5-methyl -4-isoxazolepropionic acid receptor (AMPA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), CREB-binding protein, and extracellular signal–regulated kinase (ERK). ( ) g p g g ( ) Structurally, NMDARs are comprised of dimers of N subunits (N 1 & 2), which are linked together to form tetramers in a homo/hetero dimer fashion62,63. Each N subunit is harboring an amino-terminal domain (ATD), followed by a Ligand Binding Domain (LBD), Transmembrane Domain and finally the C-Terminal Domain (CTD) oriented intracellularly64. The ligand binding domain of NMDA composed of two subunits NR1 and either NR2A/B/C or D. Our study involved NR2A which is further composed of S1 (462–502 a.a) and S2 (664–720 a.a) similarly NR1 is contributing to LBD involved residues of S1(480–526 a.a) and S2 (682–740 a.a) subunits. y g ( ) ( ) In case of AMPA, the ligand binding domain falls into S1 (393–506 a.a) and S2 (632–733 a.a) domain. In case of AMPA the ZK1 antagonist bound to the ligand binding domain. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 3 www.nature.com/scientificreports/ Figure 2. Chemical structure of chemotherapeutic drugs used for identification of off-target interactions. Figure 2. Chemical structure of chemotherapeutic drugs used for identification of off-target interactions. g y PKA functions with the help of 2 regulatory and 2 catalytic subunits, assembling into a tetrameric holoen- zyme. PKA consists of CaMKII binding domain-A and B (CBD-A and CBD-B). the R1α comprises both CBDs with sequence from 91–379 a.a. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 Experimental Sectionl Within this region there are several sub domains with major allosteric or binding hot spots include, the N3A motif (residues 119–150 a.a), the β2–3 loop (residues 163–171 a.a), the base binding region (BBR) (residues 180–193 a.a), the phosphate binding cassette (PBC) (residues 199–211 a.a), and the hinge (residues 226–251 a.a). Another important domain in the PKA is the glycine rich loop (50–55 a.a) and the ribose binding pocket65,66. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 4 www.nature.com/scientificreports/ Figure 3. The post synaptic long term potentiation (LTP) pathway168–170*. *Adapted from Kegg. (Pathway hsa04270). “Human Long Term Potentiation (LTP)” Retrieved 01-07-2018, from https://www.genome.jp/dbget- bin/www_bget?hsa04720. Figure 3. The post synaptic long term potentiation (LTP) pathway168–170*. *Adapted from Kegg. (Pathway hsa04270). “Human Long Term Potentiation (LTP)” Retrieved 01-07-2018, from https://www.genome.jp/dbget- bin/www_bget?hsa04720. Human ERK2 is spanning on 360 amino acids. Structurally they are similar to other kinases which constitutes protein kinase domain (25–313 aa)67. This domain harbors α and β helices which further host glycine rich loop (32–37 a.a), hinge region (106–109) and ATP phosphate binding loop68. g g p p g p Computational studies on human CREB are scarcely available probably because of its unstructured and intrin- sically disordered behavior in solution69,70. This can render interaction prediction against CREB with computa- tional inaccuracies. Therefore, we restricted our docking analysis with CBP which itself is harboring intrinsically disordered regions. g Human CREB binding protein (CBP) comprises of 2442 amino acids, almost 50% of which is reported to be intrinsically disordered. CBP constitutes a histone acetyl transferase (HAT) domain, bromo domain (BRD), KID binding domain (KIX), plant homeodomain (PHD) and transcriptional activator zinc finger (TAZ) domain71–73. Protein structure preparation. The atomic coordinates of proteins, NMDA (pdb ID = 5KDT), AMPA (pdb ID = 5KBV), CaMKII (pdb ID = 3SOA), PKA (pdb ID = 4UJA), CBP (pdb ID = 4NR5), and ERK (pdb ID = 2OJJ), were retrieved from Protein Data Bank. The details of all the structures are shown in Table 1. Proteins retrieved from the Pdb contained water molecules and also the original ligand/substrate/inhibitor. Thus, for the preparation of the proteins structures for ligand docking, co-crystallized ligand and any water molecules present were removed followed by protonation and energy minimization using AMBER 99 force field in MOE. Docking protocol. The docking studies were performed using induced fit docking (IFD) protocol imple- mented in MOE version 2016.0874. Experimental Sectionl For each docking run, the active site was identified on the basis of interaction of co-crystallized ligand with each protein. The co-crystallize ligand 6RV had been bound to the ligand binding domain of NMDA. For docking the 5 Å area around the interacting residues of NMDA (S1 and S2 domain) with 6RV was selected as active site (Volgraf et al. 2016). In AMPA, the co-crystallize ligand had been ZK1 bound at the ligand binding domain. A 5 Å area around the important residues that involved in interaction with ZK1 was selected as active site for docking of library of compounds75. In case of CaMKII, the bound inhibitor had been Bosutinib, targeting its regulatory domain, hence the 5 Å area around the Bosutinib binding pocket was selected as active site for further docking studies76. In case of PKA, the inhibitor 4L7 had been bound to glycine rich loop and β2–3 loop (ribose pocket). The 5 Å area around the residues involved in interactions with 4L7 was used as active site for docking studies77. In ERK2, the co-crystallize ligand had been 82 A that bound to protein kinase and glycine rich loop. A 5 Å area around the protein kinase and glycine rich loop was selected as active site for docking of compound’s library78. In case of CBP, the bound ligand had been 2LL attached to its bromo domain. The 5 Å area around the important residues that involved in interactions was selected for docking studies. The docking calculation were performed using triangle match as placement method with London DG as scoring function and re-scoring was performed with GBVI/WSA dG79,80. This was followed by ranking of the lowest energy protein-ligand interaction poses. The complexes with most negative IFD scores were considered carrying favorable binding. The validation step of docking protocol was performed through re-docking of the same default ligand with RMSD calculations. After re-docking, the RMSD value of co-crystallized and re-docked ligand was calculated. After the validation of docking protocol through re-docking, the library of chemotherapeutic com- pounds were docked into the binding domains of NMDA, AMPA, PKA, CBP, CaMKII, and ERK using the same protocol. The protocol generated 30 conformational poses for each drug with all selected proteins. The poses were re-scored by using GBVI/WSA Dg scoring function. Binding affinity analysis. The scoring analysis of each protein with the studied drugs was performed using box-plot function in R-3.3.3 package. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ interaction profiler (PLIP) server81 and PyMOL (PyMOL Molecular Graphics System Version 2 0 Schrodinger Protein PDB ID Resolution (Å) Structure Title Ligand Specie Ref NMDA 5KDT 2.44 Structure of the human GluN1/ GluN2A LBD in complex with GNE0723 (1~{R},2~{R})-2-[7-[[5-chloranyl-3- (trifluoromethyl)pyrazol-1-yl]methyl]-5- oxidanylidene-2-(trifluoromethyl)-[1,3] thiazolo[3,2-a]pyrimidin-3-yl]cyclopropane-1- carbonitrile Homo sapiens 171 AMPA 5KBV 6.8 Cryo-EM structure of GluA2 bound to antagonist ZK200775 at 6.8 Angstrom resolution {[7-morpholin-4-yl-2,3-dioxo-6- (trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)- yl]methyl}phosphonic acid Rattus norvegicus 75 ERK 2OJJ 2.4 Crystal structure of ERK2 in complex with (S)-N-(1-(3-chloro-4- fluorophenyl)-2-hydroxyethyl)-4-(4- (3-chlorophenyl)-1H-pyrazol-3-yl)- 1H-pyrrole-2-carboxamide (s)-n-(1-(3-chloro-4-fluorophenyl)-2- hydroxyethyl)-4-(4-(3-chlorophenyl)-1h-pyrazol- 3-yl)-1h-pyrrole-2-carboxamide Homo sapiens 78 PKA 4UJA 1.93 Protein Kinase A in complex with an Inhibitor 7-{(3S,4R)-4-[(5-bromothiophen-2-yl)carbonyl] pyrrolidin-3-yl}quinazolin-4(3 H)-one Homo sapiens 77 CBP 4NR5 1.66 Crystal structure of the bromodomain of human CREBBP in complex with an isoxazolyl- benzimidazole ligand 5-(3,5-dimethyl-1,2-oxazol-4-yl)-1-[2- (morpholin-4-yl)ethyl]-2-(2-phenylethyl)-1H- benzimidazole Homo sapiens To be published CaMKII 3SOA 3.5 Full-length human CaMKII 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6- methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy] quinoline-3-carbonitrile Homo sapiens 76 Table 1. List of proteins used in the study for docking analysis. Figure 4. Box plot of docking scores generated by MOE. Y-axis represent the scores while X-axis represent the name of proteins. NMDA (N-methyl-D-aspartate receptor), AMPA (α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor), CaMKII (Ca2+/calmodulin-dependent protein kinase II), PKA (protein kinase A), ERK (extracellular signal–regulated kinase), and CBP (CREB-binding protein). Protein PDB ID Resolution (Å) Structure Title Ligand Specie Ref NMDA 5KDT 2.44 Structure of the human GluN1/ GluN2A LBD in complex with GNE0723 (1~{R},2~{R})-2-[7-[[5-chloranyl-3- (trifluoromethyl)pyrazol-1-yl]methyl]-5- oxidanylidene-2-(trifluoromethyl)-[1,3] thiazolo[3,2-a]pyrimidin-3-yl]cyclopropane-1- carbonitrile Homo sapiens 171 AMPA 5KBV 6.8 Cryo-EM structure of GluA2 bound to antagonist ZK200775 at 6.8 Angstrom resolution {[7-morpholin-4-yl-2,3-dioxo-6- (trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)- yl]methyl}phosphonic acid Rattus norvegicus 75 ERK 2OJJ 2.4 Crystal structure of ERK2 in complex with (S)-N-(1-(3-chloro-4- fluorophenyl)-2-hydroxyethyl)-4-(4- (3-chlorophenyl)-1H-pyrazol-3-yl)- 1H-pyrrole-2-carboxamide (s)-n-(1-(3-chloro-4-fluorophenyl)-2- hydroxyethyl)-4-(4-(3-chlorophenyl)-1h-pyrazol- 3-yl)-1h-pyrrole-2-carboxamide Homo sapiens 78 PKA 4UJA 1.93 Protein Kinase A in complex with an Inhibitor 7-{(3S,4R)-4-[(5-bromothiophen-2-yl)carbonyl] pyrrolidin-3-yl}quinazolin-4(3 H)-one Homo sapiens 77 CBP 4NR5 1.66 Crystal structure of the bromodomain of human CREBBP in complex with an isoxazolyl- benzimidazole ligand 5-(3,5-dimethyl-1,2-oxazol-4-yl)-1-[2- (morpholin-4-yl)ethyl]-2-(2-phenylethyl)-1H- benzimidazole Homo sapiens To be published CaMKII 3SOA 3.5 Full-length human CaMKII 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6- methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy] quinoline-3-carbonitrile Homo sapiens 76 Table 1. List of proteins used in the study for docking analysis. Table 1. List of proteins used in the study for docking analysis. Figure 4. Box plot of docking scores generated by MOE. Y-axis represent the scores while X-axis represent the name of proteins. Experimental Sectionl On the basis of docking scores, top five complexes for each protein with studied drugs were selected for interaction analysis. The interaction analysis was performed using protein ligand Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 5 www.nature.com/scientificreports/ NMDA (N-methyl-D-aspartate receptor), AMPA (α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor), CaMKII (Ca2+/calmodulin-dependent protein kinase II), PKA (protein kinase A), ERK (extracellular signal–regulated kinase), and CBP (CREB-binding protein). Figure 4. Box plot of docking scores generated by MOE. Y-axis represent the scores while X-axis represent the name of proteins. NMDA (N-methyl-D-aspartate receptor), AMPA (α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor), CaMKII (Ca2+/calmodulin-dependent protein kinase II), PKA (protein kinase A), ERK (extracellular signal–regulated kinase), and CBP (CREB-binding protein). interaction profiler (PLIP) server81 and PyMOL (PyMOL, Molecular Graphics System, Version 2.0 Schrodinger, LLC). To further verify the interactions between docked complexes, protein ligand interaction fingerprints (PLIF) were calculated using PLIF algorithm implemented in MOE79,80. PLIF summarizes the interactions like H-bonds, ionic and surface contacts on the basis of fingerprint scheme that is representative of ligand-protein complex79,80. In order to further probe off-target interactions by top scoring chemotherapeutic drugs, the binding affinity of the top scoring docked complexes were calculated using CSM-Lig82. CSM-Lig predict the binding affinity of a protein-small molecule complex based on structural signatures and machine learning algorithm82. Physicochemical attributes of binding region. The three dimensional illustration of charge distribution among molecules is depicted by Electrostatic potential maps, also known as electrostatic potential energy maps. These maps aid in determination of variably charged regions of a molecule which can help in identifying intermo- lecular interactions and molecular properties of small molecules83. To understand the binding surfaces of NMDA, AMPA, PKA, CBP, CaMKII, and ERK, electrostatic charge distribution were studied using APBS plugins in PyMol. hemical attributes of binding region.  The thre Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Box plot of docking scores generated by MOE. Y-axis represent the scores while X-axis represent chemotherapeutic drugs. Dactinomycin, temsirolimus and Everolimus are the drugs that are having minimum docking scores. Figure 5. Box plot of docking scores generated by MOE. Y-axis represent the scores while X-axis represent chemotherapeutic drugs. Dactinomycin, temsirolimus and Everolimus are the drugs that are having minimum docking scores. Figure 6. The re-docked pose of 6RV. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 6. The re-docked pose of 6RV. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Results i Results Interaction analysis with LTP proteins. The off-targets of drugs were identified on the basis of docking scores (lower the scores, strong is the interactions).h ( g ) The docking scores of all the studied protein are presented in the form of box plot (Fig. 4). According to box plot, the ERK protein (a protein kinase) is having the median score of −7.8 with 75% of data in the upper quartile and 25% of data in the lower quartile. The median score of NMDA (a receptor protein) is −7.4 with 75% of data in upper quartile and 25% of data in lower quartile. PKA is a protein kinase with median score of −7.1 with 70% and 30% of data in upper and lower quartile, respectively. Another receptor protein of LTP pathway is AMPA having the median score of −7.0. In AMPA, 70% of data is present in upper quartile and 30% of data is in lower quartile. CaMKII, a kinase protein is having different distribution of data with 25% of data is in upper quartile and 75% of data is in lower quartile with median score of −6.8. CBP is a nuclear protein with the highest median value of −6.2 and with equal distribution of data in both quartiles.h q q The scores of all the chemotherapeutic drugs on the basis of their interactions with all the studied proteins is shown in Fig. 5. According to median values, Dactinomycin is having the lowest median scores of −10.8 with 100% of data present in upper quartile. Temsirolimus is having a median score of −10.3 with almost equal data distribution in both the quartile. Everolimus is having the median score of −9.7 with 15% of data in lower quar- tile and 85% of data in upper quartile. Bromocriptine and Docetaxel are having the same median score of −9.0 Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 7 www.nature.com/scientificreports/ www.nature.com/scientificreports entificreports/ but with different distribution of data In Bromocriptine 60% of data in the lower quartile and 40% of data in Figure 7. Top scoring docking conformation of NMDA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Teniposide (purple). Glu N1 residues shown in white while Glu N2A residues shown in blue. Figure 7. Top scoring docking conformation of NMDA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Teniposide (purple). Results i Glu N1 residues shown in white while Glu N2A residues shown in blue. Figure 7. Top scoring docking conformation of NMDA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Teniposide (purple). Glu N1 residues shown in white while Glu N2A residues shown in blue. but with different distribution of data. In Bromocriptine, 60% of data in the lower quartile and 40% of data in upper quartile while in Docetaxel 75% of data in lower quartile and 25% of data in the upper quartile. Teniposide and Irinotecan having the median scores of −8.9 and −8.8, respectively. In, Teniposide 5% of data in upper quartile and 95% of data in lower quartile while in Irinotecan 85% of data in lower quartile and 15% of data in upper quartile. Paclitaxel, and Etoposide are having the median score of −8.6 but with different distribu- tion of data. In paclitaxel 10% of data is in upper quartile and 90% of data is in lower quartile, while etoposide 100% of data present at the median. The median score of Afatinib is −8.4 with equal distribution of data in both quartiles. The drugs with the median scores in the range of −5 to −6 are Asparaginase, Busulfan, Carmustine, Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drugs Binding Affinity (−log10(KD|Ki)) Protein Residue Distance (Å) Type of Interactions Dactinomycin 37.0 GLU 230B 3.61 Hydrophobic TYR 237A 3.74 Hydrophobic LEU 245B 3.61 Hydrophobic GLU 230B 2.20 H-bond ARG 287B 3.60 H-bond Temsirolimus 31.1 TYR 237A 3.94 Hydrophobic ILE 238A 3.80 Hydrophobic GLU 244B 3.73 Hydrophobic LEU 245B 3.50 Hydrophobic LEU 279A 3.82 Hydrophobic ASN 219A 3.44 H-bond ASP 282B 2.53 H-bond ARG 287B 3.50 H-bond Everolimus 31.3 ASN 219A 3.79 Hydrophobic SER 235A 3.66 Hydrophobic TYR 237A 3.32 Hydrophobic ILE 238A 3.94 Hydrophobic LEU 245B 3.09 Hydrophobic GLU 230B 1.63 H-bond LEU 245B 3.27 H-bond GLU 275A 3.11 H-bond Docetaxel 29.4 ALA 216A 3.59 Hydrophobic ASN 219A 3.86 Hydrophobic ILE 238A 2.65 H-bond ARG 287B 2.34 H-bond Teniposide −45.2 ILE 238A 3.97 Hydrophobic LEU 245B 3.76 Hydrophobic MET 278B 3.70 Hydrophobic ASN 219A 3.25 H-bond TYR 237A 3.25 H-bond ILE 238A 2.30 H-bond LEU 245B 2.89 H-bond Table 2 Interacting residues of NMDA with Dactinomycin Temsirolimus Everolimus Doc Table 2. Interacting residues of NMDA with Dactinomycin, Temsirolimus, Everolimus, Docetaxel, and Teniposide. Table 2. Interacting residues of NMDA with Dactinomycin, Temsirolimus, Everolimus, Docetaxel, and Teniposide. Figure 8. The re-docked conformation of ZK1. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 8. The re-docked conformation of ZK1. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 8. The re-docked conformation of ZK1. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 9 www.nature.com/scientificreports/ www.nature.com/scientificreports ntificreports/ Figure 9. Top ranking docking poses of AMPA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Paclitaxel (silver); (D) Vincristine (golden); and (E) Irinotecan (maroon). Figure 9. Top ranking docking poses of AMPA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Paclitaxel (silver); (D) Vincristine (golden); and (E) Irinotecan (maroon). igure 9. Top ranking docking poses of AMPA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) aclitaxel (silver); (D) Vincristine (golden); and (E) Irinotecan (maroon). Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Decitabine, Exemestane, Fludarabine, Gemcitabine, Ifosfamide, Letrozole, Lomustine, Melphalan, Mitomycin, Prednisone, Streptozocin, Temzolomide, Thalidomide, Thioguanine, and Thiotepa. The drugs with median scores above −4.5 are Fluorouracil, Hydroxyurea, Mercaptopurine. Interaction of Drugs with NMDA. The re-docking of co-crystallize ligand was performed into the binding pocket of NMDA with overall score of −7.3 Kcal/mol and RMSD value 3.8 Å. www.nature.com/scientificreports/ Figure 6 is showing the original and re-docked conformation of 6RV into the binding pocket of NMDA. After the successful re-docking, the dock- ing of library of compounds were performed using the same protocol. For each compound, 30 conformations were explored and the top scoring docking poses of each compound were further used for studies. The details of docking scores of each compound is shown in Supplementary Fig. S1. Top five docking complexes were further evaluated for ligand protein interactions (Fig. 7). The docking scores of Dactinomycin, Temsirolimus, Everolimus, Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 10 www.nature.com/scientificreports/ The co-crystallized ligand is shown in cyan wh the re-docked ligand is shown in purple. of 37.0. Temsirolimus and Everolimus are having the binding affinity values of 31.3 and 31.1, respectively. All five drugs have shown interactions with Tyr 237 A, Leu 245B, and ILE 238 A, involved in hydrophobic interactions while Arg 287B involved in H-bonding (Table 2). of 37.0. Temsirolimus and Everolimus are having the binding affinity values of 31.3 and 31.1, respectively. All five drugs have shown interactions with Tyr 237 A, Leu 245B, and ILE 238 A, involved in hydrophobic interactions while Arg 287B involved in H-bonding (Table 2). Interaction of Drugs with AMPA. The validity of docking protocol was done through re-docking of ZK1 (co-crystallize ligand) into the active site of AMPA. ZK1 was re-docked with energy value of −6.99 Kcal/mol and RMSD of 1.5 Å (Fig. 8). Using the same docking protocol, the docking of our library of compounds into the ligand binding domain (S1 (393–503 a.a) and S2 (632–773 a.a)) of AMPA, were performed. For each com- pound 30 different docking conformations were generated (Fig. S2) and the top scoring docking conformations were further explored for ligand binding interactions. The ligands bind to the ligand binding domain with a much higher affinity compared to that of the allosteric site. This is shown by the differences in docking scores. In the ligand binding domain, the co-crystallize inhibitor binds with a binding strength of −6.99 Kcal/mol. While Dactinomycin, Temsirolimus, Paclitaxel, Vincristine, and Irinotecan binds with scores of −11.8, −11.2, −9.9, −9.5, and −9.1 Kcal/mol, respectively. The ligand binding affinities are comparable to the docking scores with Temsirolimus is having highest affinity for AMPA and Irinotecan is the least. Individual ligand binding interac- tions are shown in Fig. 9 and Table 3. All five drugs showing the hydrophobic interactions with Tyr450 and Leu 498 while H-bonding with Ser 654 and Glu 705. nteraction of Drugs with PKA. The crystal structure of PKA was retrieved with 4L7 as co-crystallized ligand L7 was re-docked into the binding pocket of PKA with binding affinity of −6.1 Kcal/mol (Fig. 10).h fi The library of chemotherapeutic drugs were docked into the binding pocket of PKA and 30 conformations per compound were generated. The detail of docking scores of all the compounds is shown in Fig. S3. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drugs Binding Affinity (−log10(KD|Ki)) Protein Residue Distance (Å) Type of Interactions Dactinomycin 37.1 LEU 410 3.91 Hydrophobic TYR 450 3.29 Hydrophobic LEU 498 2.54 Hydrophobic LEU 650 3.87 Hydrophobic SER 654 2.75 H-bond THR 655 2.13 H-bond GLU 705 2.80 H-bond Temsirolimus 38.2 GLU 402 3.90 Hydrophobic TYR 450 3.65 Hydrophobic PRO 478 478 Hydrophobic THR 482 3.93 Hydrophobic PRO 494 2.99 Hydrophobic LEU 498 2.78 Hydrophobic GLU 705 3.77 Hydrophobic MET 708 2.32 Hydrophobic LYS 730 3.30 Hydrophobic TYR 732 3.85 Hydrophobic SER 654 2.82 H-bond GLU 705 2.98 H-bond GLY 731 2.89 H-bond Paclitaxel 36.1 TYR 405 3.03 Hydrophobic TYR 450 3.80 Hydrophobic THR 480 3.56 Hydrophobic LEU 498 3.66 Hydrophobic PHE 658 3.71 Hydrophobic GLU 705 3.29 Hydrophobic LYS 730 3.00 Hydrophobic SER 654 2.42 H-bond THR 686 2.77 H-bond Vincristine 34.4 GLU 402 3.56 Hydrophobic TYR 450 3.11 Hydrophobic LEU 650 3.38 Hydrophobic PHE 658 3.99 Hydrophobic LYS 730 3.01 Hydrophobic SER 654 1.74 H-bond THR 655 3.15 H-bond THR 686 1.87 H-bond GLU 705 2.66 H-bond TYR 732 2.95 H-bond Irinotecan 35.1 GLU 402 2.71 Hydrophobic TYR 450 3.03 Hydrophobic THR 480 3.47 Hydrophobic LEU 498 3.35 Hydrophobic GLU 705 3.02 Hydrophobic MET 708 2.22 Hydrophobic LYS 730 3.55 Hydrophobic THR 686 1.80 H-bond TYR 450 3.52 Pi-stacking Table 3. Interacting residues of AMPA with Dactinomycin, Temsirolimus, Paclitaxel, Vinc Table 3. Interacting residues of AMPA with Dactinomycin, Temsirolimus, Paclitaxel, Vincristine, and Irinotecan Table 3. Interacting residues of AMPA with Dactinomycin, Temsirolimus, Paclitaxel, Vincristine, and Irinotecan. Docetaxel, and Teniposide are considerably lower in comparison to that of the bound inhibitor (6RV), thus dis- playing the superior binding affinity of chemotherapeutic drugs to NMDA. Dactinomycin binds with a score of −11, Temsirolimus with −10.4, Everolimus with −10.2, docetaxel with −8.9, and Teniposide with −8.8. The docking scores were further validated by calculating ligand binding affinities of top five complexes. Ligand bind- ing affinities are corresponding to the docking scores. The drug with the highest docking score is predicted to be with greater affinity for NMDA. Dactinomycin is having the highest binding affinity for NMDA with energy value Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 11 www.nature.com/scientificreports/ www.nature.com/scientificreports/ / p p / Figure 10. The original and re-docked conformation of 4L7. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 10. The original and re-docked conformation of 4L7. www.nature.com/scientificreports/ Among all the docked conformations, top five docking complexes were further studied for ligand binding interactions (Fig. 11; Table 4). On the basis of docking scores, it has been observed that the studied drugs are having better affinity for PKA compared to co-crystallized ligand. Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Bromocriptine bind with the PKA with scores of −10.7, −10.6, −9.7, −9.5, and −9.3 Kcal/mol, respectively. Ligand binding affinities of top five complexes are shown in Table 4. Dactinomycin is having the highest binding affinity for PKA with score of 39.1 while bromocriptine is having the least binding affinity for PKA. All the five drugs having hydrophobic interactions with Phe 54, Val 57, and H-bonding with Thr 51 in the glycine rich loop of PKA. In β2–3 loop, Lys 168 involved in either H-bonding or formed salt bridge with ligand atoms. In phosphate binding cassette, Pro 202 also involved in hydrophobic interactions. Interaction of Drugs with CaMKII. The co-crystallize ligand into the binding pocket of CaMKII is Bosutinib present in the regulatory domain of CaMKII. The Bosutinib was re-docked into the binding domain of CaMKII with binding score of −8.0 Kcal/mol (Fig. 12). Library of compounds were docked into the active site of CaMKII with binding energies ranging from −10 to −4 Kcal/mol (Fig. S4). On the basis of binding affinities, our analysis suggested Irinotecan, Bromocriptine, Dasatinib, Afatinib, and Imatinib were having better affinity for CaMKII with scores of −10.2, −10.2, −9.6, −9.3, and −9.2 Kacl/mol, respectively, compared to Bosutinib. Irinotecan and Bromocriptine are having the same docking scores but bromocriptine having the highest binding affinity for CaMKII compared to Irinotecan. Dasatinib, Imatinib and Afatinib are also having the binding affinities comparable to docking scores (Table 5). Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 12 www.nature.com/scientificreports/ / p p / Figure 11. Top five docking conformations of PKA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Bromocriptine (cyan). Figure 11. Top five docking conformations of PKA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Bromocriptine (cyan). Figure 11. Top five docking conformations of PKA with (A) Dactinomycin (green); (B) Temsirolimus (yellow); (C) Everolimus (beige); (D) Docetaxel (golden); and (E) Bromocriptine (cyan). www.nature.com/scientificreports/ All the five compounds showing interactions in the CaM binding domain where Lys 300, and Leu 308 involved in hydrophobic interactions while Arg 297 involved in H-bonding. Leu 221 in the kinase domain also showing hydrophobic and H-bond interactions with the compounds (Fig. 13; Table 5). Interaction of Drugs with ERK. The re-docked conformation of 82A and the original co-crystallize conformation is shown in Fig. 14. The 82A bound to ERK with energy value of −8.4 Kcal/mol and RMSD of 3.3 Å. The library of compounds were docked into the binding pocket of ERK and the detail scores of all the com- pounds is shown in Fig. S5. The results showed that the studied drugs are showing better affinity to ERK com- Interaction of Drugs with ERK. The re-docked conformation of 82A and the original co-crystallize conformation is shown in Fig. 14. The 82A bound to ERK with energy value of −8.4 Kcal/mol and RMSD of 3.3 Å.h h The library of compounds were docked into the binding pocket of ERK and the detail scores of all the com- pounds is shown in Fig. S5. The results showed that the studied drugs are showing better affinity to ERK com- pared to original co-crystallize ligand. Dactinomycin, Bromocriptine, Temsirolimus, Everolimus, and docetaxel having −11.0, −10.3, −10.2, −10.1, −10.0 Kcal/mol binding affinity for ERK. On the basis of ligand binding affinities Everolimus having the highest affinity for ERK. Dactinomycin and Temsirolimus are having the same binding affinity value of 37 for ERK (Table 6). The interactions of ERK with the drugs showed that Tyr 36, Val 39 involved in hydrophobic while Lys 151 involved in H-bonding with all the five drugs (Fig. 15; Table 6­)­. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 13 Discussionh The neurons of central and peripheral nervous system as well as oligodendrocytes are particularly susceptible to off target side effects pitched in by chemotherapy30,84. These off target effects may contemplate into revamping functions of both dividing and non-dividing cells both at central and peripheral levels. The proposed mecha- nisms put forwarded to explain these pathologies include faulty DNA repair mechanisms, blood brain barrier dysfunction, disordered immune regulation and impaired neurotransmitter signaling85. There is an increasing evidence that CICI disrupts neurogenesis particularly in adult hippocampus14,16. The implication of such refor- mation results in alteration of hippocampal neural circuitry which critically affects not only memory formation and learning acquisition, but also interregional articulation of anatomically distant but functionally cognate brain regions86. This can be further inferred from the reported clinical evidence of frontal cortical deficits by chemo- brain87. Therefore, synaptic plasticity functions mediated by neurons of hippocampus are at stake.h h y p p y y pp p The focus of the study is to investigate the interactions of various chemotherapeutic drugs against major pro- teins involved in LTP pathway, by docking algorithm as their interacting residues may provide useful insight into functional alteration which can be related to cognitive processes. The results derived from this study revealed that Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Teniposide are top 5 drugs interacting with NMDA res- idues located in Ligand Binding Domain (LBD) of GluN2 near Glutamate binding pocket (Fig. 7). Contextually, NMDARs have been focus of pharmacological modulation by virtue of allosteric modulators, however, limited by excessive off-target effects in lieu of excessive NMDAR inhibition. Notably, the competitive antagonists of two main substrates of NMDARs i.e. Glycine and Glutamate, targets LBD of GluN1 AND GluN2 respectively, while the Transmembrane Domain (TMD) is targeted by the channel blockers88. Interestingly, Dactinomycin has been reported to rescue retinal ganglion cells from NMDA mediated excitotoxicity, suggesting potential evi- dence of Dactinomycin in partial inhibition of NMDA receptor89. The mTOR inhibitor Everolimus has been tested in Phase 1 clinical trials for targeting glutamatergic signaling for Autism Spectrum Disorders (ASDs) and Temsirolimus have similarly been reported to be used in rodents for altering mTORpathway90–92. y p g p y Under physiological conditions, the simultaneous closure of GluN1/N2 aided by the binding energies in the advent of agonist (Glycine & Glutamate) binding puts the TMD to undergo conformational changes to open the channel93,94. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drugs Binding Affinity (−log10(KD|Ki)) Protein Residue Distance (Å) Type of Interactions Dactinomycin 39.1 PHE 54 3.03 Hydrophobic VAL 57 3.92 Hydrophobic LYS 72 3.55 Hydrophobic LEU 82 3.95 Hydrophobic GLU 170 3.57 Hydrophobic PHE 187 3.74 Hydrophobic PRO 202 3.93 Hydrophobic THR 51 2.12 H-bond SER 53 2.34 H-bond LYS 168 5.13 Salt bridge Temsirolimus 35.5 PHE 54 3.35 Hydrophobic VAL 57 3.32 Hydrophobic PHE 129 3.26 Hydrophobic PRO 202 3.72 Hydrophobic THR 51 2.47 H-bond LYS 72 2.33 H-bond LYS 168 2.56 H-bond LYS 168 5.48 Salt bridge Everolimus 37.3 PHE 54 3.21 Hydrophobic PHE 129 3.43 Hydrophobic GLU 170 3.72 Hydrophobic THR 51 3.96 Hydrophobic SER 53 3.28 H-bond ARG 133 1.94 H-bond LYS 168 3.18 H-bond Docetaxel 38.2 PHE 54 3.25 Hydrophobic VAL 57 3.52 Hydrophobic LEU 74 3.52 Hydrophobic PHE 129 3.21 Hydrophobic PHE 187 3.34 Hydrophobic PRO 202 3.73 Hydrophobic TYR 330 3.84 Hydrophobic LYS 168 2.39 H-bond GLU 170 3.17 H-bond LYS 72 4.87 Pi-stacking LYS 168 4.86 Salt bridge Bromocriptine 33.8 PHE 54 3.78 Hydrophobic LYS 72 3.45 Hydrophobic LEU 74 3.52 Hydrophobic GLU 170 3.84 Hydrophobic THR 51 2.66 H-bond LYS 72 1.59 H-bond LYS 168 3.70 Salt bridge GLU 170 5.34 Salt bridge Table 4. Interacting residues of PKA with Dactinomycin, Temsirolimus, Everolimus, Doce Bromocriptine Table 4. Interacting residues of PKA with Dactinomycin, Temsirolimus, Everolimus, Docetaxel, and Bromocriptine. Interaction of Drugs with CBP. In case of CBP the original docked ligand is 2LL which was re-docked with binding score of −6.0 Kcal/mol and RMSD of 3.1 Å (Fig. 16). The docking of library of compounds were per- formed and the scores of each docking conformation is shown in Fig. S6. On the basis of scores, it is obvious that CBP does not showing high affinity for the studied drugs when compared with NMDA, AMPA, ERK, PKA, and CaMKII. Electrostatic potential of proteins. The electrostatic potential of NMDA, AMPA, ERK, PKA, and CaMKII is shown in Fig. 17. On the basis of electrostatic potential the active site of PKA, and NMDA is more electronegative, in case of ERK it is more electronegative, in case of AMPA it is having the electroposi- tive as well as electronegative residues while in case of CaMKII it is almost neutral. The electropositivity and Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 14 www.nature.com/scientificreports/ Figure 12. The co-crystallize and re-docked conformation of Bosutinib. www.nature.com/scientificreports/ The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 12. The co-crystallize and re-docked conformation of Bosutinib. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. electronegativity favors the strong interactions of ligand with the proteins which is also evident with the binding affinity of Dactinomycin, Temsirolimus, and Everolimus. These drugs are showing highest binding affinity for NMDA, ERK, PKA, and AMPA. While in the neutral binding pocket of CaMKII, Irrinoteacn and Bromocriptine having the high binding affinity. electronegativity favors the strong interactions of ligand with the proteins which is also evident with the binding affinity of Dactinomycin, Temsirolimus, and Everolimus. These drugs are showing highest binding affinity for NMDA, ERK, PKA, and AMPA. While in the neutral binding pocket of CaMKII, Irrinoteacn and Bromocriptine having the high binding affinity. Plif analysis. The protein ligand interaction fingerprints were calculated using MOE. Dactinomycin, Temsirolimus and Everolimus are the drugs that are showing maximum affinity for NMDA, AMPA, PKA and ERK, hence the PLIF analysis were performed for all the three drugs in order to find any common functional group in all the compounds. On the basis of PLIF analysis it is revealed that due to large molecular structure of Dactinomycin, it has many interacting points with the active site residues of NMDA, AMPA, PKA, and ERK. It can involve in H-bonding due to the presence of carboxyl group as well as in hydrophobic interactions due to the presence of benzene ring (Fig. S7). Temsirolimus can also involve in H-bonding due to the presence of terminal hydroxyl group and also in hydrophobic interactions with the help of long alkane chain with the active site resi- dues of NMDA, PKA, AMPA, and ERK (Fig. S8). Everolimus is involve in H-bonding with the help of its hydroxyl and carboxyl group while its pyrimidine ring involved in hydrophobic interactions with NMDA, PKA and ERK (Fig. S9). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drugs Binding Affinity (−log10(KD|Ki)) Protein Residue Distance (Å) Type of Interactions Irinotecan 39.6 THR 176 3.21 Hydrophobic PRO 177 3.63 Hydrophobic LEU 221 2.33 Hydrophobic ARG 296 3.91 Hydrophobic LYS 300 3.70 Hydrophobic ALA 302 2.66 Hydrophobic LEU 308 2.93 Hydrophobic THR 176 2.73 H-bond TRP 214 2.10 H-bond ARG 296 1.78 H-bond ARG 297 2.04 H-bond LYS 300 2.74 H-bond LYS 137 4.48 Salt Bridge Bromocriptine 42.8 THR 176 3.65 Hydrophobic GLU 216 3.19 Hydrophobic GLN 218 2.81 Hydrophobic LEU 221 2.79 Hydrophobic ARG 297 3.80 Hydrophobic LYS 300 3.76 Hydrophobic VAL 306 3.41 Hydrophobic LEU 308 2.37 Hydrophobic TYR 179 2.14 H-bond LEU 308 1.04 H-bond LYS 300 5.35 Salt bridge Dasatinib 39.5 LEU 221 2.91 Hydrophobic LYS 300 3.78 Hydrophobic THR 310 3.88 Hydrophobic ILE 321 2.27 Hydrophobic GLU 139 3.53 H-bond GLU 139 4.82 Salt bridge Afatinib 38.3 ARG 297 3.83 Hydrophobic VAL 306 3.73 Hydrophobic LEU 308 2.38 Hydrophobic LYS 300 1.36 H-bond Leu 308 2.53 H-bond GLU 325 5.03 Salt bridge Imatinib 40.3 THR 176 3.82 Hydrophobic ASP 217 3.94 Hydrophobic GLN 218 3.91 Hydrophobic ARG 297 3.74 Hydrophobic LYS 300 3.41 Hydrophobic LEU 308 3.23 Hydrophobic THR 310 3.71 Hydrophobic ILE 321 3.06 Hydrophobic LYS 300 2.70 H-bond THR 310 2.77 H-bond GLU 139 3.34 Salt bridge T bl 5 I i id f C MKII i h I i B i i D i ib Af i Table 5. Interacting residues of CaMKII with Irinotecan, Bromocriptine, Dasatinib, Afatinib, and Imatinib. and Ile755. Interestingly, these are among the same residues which have shown interaction with Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Teniposide in our study. The residue Tyr754 had been proven to be of detrimental importance in site directed mutagenesis replacement with Lysine96. This replacement boosted the glutamate potency thus re-sculpting the conformational states of GluN2A and altering the general behavior of glutamate binding site in GluN2A. Perplexingly, the same residue along with Lys738, also effect the Glycine bind- ing site on GluN1. The neighboring residue Ile755 have also reported to modulate the binding cavity for negative NMDA allosteric modulators (NAMs)96. The functional implication of these findings favors a potential compet- itive antagonistic action with possible partial NMDA inhibition. Interestingly, peripheral NMDA receptors are and Ile755. Interestingly, these are among the same residues which have shown interaction with Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Teniposide in our study. Discussionh However, binding of NMDA-R competitive antagonist arrests the NMDA gating mechanism to an open cleft conformation of GluN1/N2 thus blocking the channel. This suggest that these drugs while interacting with residues on GluN1/N2 can interfere with NMDA gating mechanism. Specifically increased intracellular Ca+2 influx as a result of NMDA activation leading to increased intracellular Na+ by virtue of AMPA over activation may causes swelling of neuronal cell body95. However, it is difficult to deduce whether interactions by compounds mimicking competitive antagonists can induce allosteric modulation on the positive or negative side. The res- idues Tyr 754 and Ile755 have reported to influence the binding affinities of NMDA competitive antagonist. Moreover, the NAM NVP-AAM077 and ST3 binds cavity in GluN2A harboring non conserved residues Tyr 754 Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 15 www.nature.com/scientificreports/ The residue Tyr754 had been proven to be of detrimental importance in site directed mutagenesis replacement with Lysine96. This replacement boosted the glutamate potency thus re-sculpting the conformational states of GluN2A and altering the general behavior of glutamate binding site in GluN2A. Perplexingly, the same residue along with Lys738, also effect the Glycine bind- ing site on GluN1. The neighboring residue Ile755 have also reported to modulate the binding cavity for negative NMDA allosteric modulators (NAMs)96. The functional implication of these findings favors a potential compet- itive antagonistic action with possible partial NMDA inhibition. Interestingly, peripheral NMDA receptors are and Ile755. Interestingly, these are among the same residues which have shown interaction with Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Teniposide in our study. The residue Tyr754 had been proven to be of detrimental importance in site directed mutagenesis replacement with Lysine96. This replacement boosted the glutamate potency thus re-sculpting the conformational states of GluN2A and altering the general behavior of glutamate binding site in GluN2A. Perplexingly, the same residue along with Lys738, also effect the Glycine bind- ing site on GluN1. The neighboring residue Ile755 have also reported to modulate the binding cavity for negative NMDA allosteric modulators (NAMs)96. The functional implication of these findings favors a potential compet- itive antagonistic action with possible partial NMDA inhibition. Interestingly, peripheral NMDA receptors are Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 16 www.nature.com/scientificreports ntificreports/ Figure 13. Top five docking conformations of CaMKII with (A) Irinotecan (maroon); (B) Bromocriptine (cyan); (C) Dasatinib (olive green); (D) Afatinib (blue); and (E) Imatinib (brown). www.nature.com/scientificreports/ Figure 13. Top five docking conformations of CaMKII with (A) Irinotecan (maroon); (B) Bromocriptine (cyan); (C) Dasatinib (olive green); (D) Afatinib (blue); and (E) Imatinib (brown). Figure 13. Top five docking conformations of CaMKII with (A) Irinotecan (maroon); (B) Bromocriptine (cyan); (C) Dasatinib (olive green); (D) Afatinib (blue); and (E) Imatinib (brown). also drug targets to evade multidrug resistance in cancer by virtue of their ability to downregulate ABC transport- ers97. Such is case of compound MK-801, which noncompetitively antagonizes NMDA receptor98.h also drug targets to evade multidrug resistance in cancer by virtue of their ability to downregulate ABC transport- ers97. Such is case of compound MK-801, which noncompetitively antagonizes NMDA receptor98.h The AMPARs are another family members of iGluRs with structurally similar homo/hetero tetrameric organ- ization from GluA1-A4, however differing in its preferentially Na+ permeability, resulting in rapid postsynap- tic depolarization99,100. www.nature.com/scientificreports/ Interestingly, the AMPA antagonist DNQX targets the same Glu402- Thr 686 interaction114. The Gly731 residue participate in cleft closure from open to closed state115. The close receptor conformational state has been associated with full agonistic behavior while a partial agonist receptor renders incomplete receptor closure. Interestingly, the L605T mutants of LBD of iGluR2 showed contrary results.i All five top docking complexes exhibit interaction with residue Tyr450. Interestingly substitution of Tyr with Ala at this position has resulted in diminution of glutamate potency116. Similarly, off target interactions have also been observed at position Glu705, which had been reported to eliminate agonist binding on experimental muta- tion. Furthermore, experimental mutation by Armstrong et al., for residue Leu650 to Thr decrease the potency of AMPA receptors to Glutamate by 8.5 folds117.hhl y The GluA2 subunit of AMPA also harbors interacting residues Ser654, Thr655 and Phe658, which are flexible meta-interaction site and mutation in this region may alter binding kinetics culminating into aberrant AMPA receptor activation118. Residues Tyr450, Pro478,Thr480, Ser654 AND Glu705, all directly interact with ligand to mediate strong binding115. The GluA2 subunit critically regulates biophysical function by keeping in check the receptor kinetics and Ca+2 permeability. It is noteworthy that slowing down the AMPAR deactivation and sensitization by the use of allosteric modulators as Diazoxide119 and Aniracetam120 is tried in clinical studies for cognitive impairment and depression. Puzzlingly, slowing or blocking desensitization in transgenic animals led to lethal outcomes121. The cumulative endpoint from past studies related with our off target interaction suggested that the deactivation kinetics of AMPARs is highly variable and any untoward off-target interaction particularly within the regions of ligand binding can potentially enhance or delay AMPA deactivation thus dysregulating excitatory postsynaptic potential (EPSP). y p y p p Evolutionarily, kinases are generally conserved and receive a high competition for substrate binding par- ticularly from ATP in mM concentrations, thus decreasing their probability to be involved in off-target interac- tions122, however, with the development of tyrosine kinase inhibitors and their use particularly in cancer patients, off-target interactions are not very unlikely123. During LTP induction, the activity of NMDA leads to surge of intracellular Ca+2 triggering a biochemical cascade emanating at AMPAR mediated EPSC. This biochemical cas- cade is driven by CaMKII, an unusual kinase, capable of auto-phosphorylating itself (at residue T286 and T287), mediated by Ca+2 and Calmodulin124. www.nature.com/scientificreports/ The AMPARs have shown interactions with Dactinomycin, Temsirolimus, Paclitaxel, Vincristine, and Irinotecan with lowest binding energies among the current study’s selected drug dataset (Fig. 9). Related to our findings, the role of mTORinhibitor Temsirolimus in targeting AMPAR have been reported before90. To add further, Taxol has also been previously reported to selectively repress cationic influx in glu- tamate excitotoxicity101. Paclitaxel and Vincristine have been previously documented for their involvement in mediating chemotherapy induced peripheral neuropathy (CIPN), having toxic effect on dorsal root ganglion neurons1,102. However the CIPN damaging effect is also partly mediated by the induction of strong inflamma- tory component103. Similarly, Irinotecan have been documented previously for their reduction in excitatory Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 17 www.nature.com/scientificreports/ Figure 14. The co-crystallize and re-docked conformation of 82A. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 14. The co-crystallize and re-docked conformation of 82A. The co-crystallized ligand is shown in cyan while the re-docked ligand is shown in purple. Figure 14. The co-crystallize and re-docked conformation of 82A. The co-crystallized ligand is shown in cy while the re-docked ligand is shown in purple. neurotransmission by interfering with pre and post synaptic gene expression104,105. Moreover, clinical deteri- oration in cognitive decline in patients receiving irinotecan in combination with 5’Flourouracil has also been reported106. neurotransmission by interfering with pre and post synaptic gene expression104,105. Moreover, clinical deteri- oration in cognitive decline in patients receiving irinotecan in combination with 5’Flourouracil has also been reported106. p AMPARs have been tried with therapeutic inhibition in case of epilepsy107. Past studies also suggest that triggering multiple topological conformation in the face of activity dependent interactions in AMPARs alter its mobility which may leads to its desensitization108.hif The current study findings showed off target interaction of Temsirolimus, Vincristine and Irinotecan with Glu 402 residue and Thr686 of GluA2 subunit of AMPA. Previous crystal structure studies of AMPAR reported interaction of Glu402 residue of subunit with Thr 686 residue which are positioned at corners of binding cleft and contributes to the stability of closed conformational state109. Mutations at either of these residues result in fall of agonist binding affinities and efficacy110–113. Moreover, Hogner et al. reported that the inter-domain steric hin- drances or remodeling of Glu402-Thr686 interaction can readjust the course of domain closure, independent of ligand affinity109. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 T­ab­le 6.  Interacting residues of ERK with Dactinomycin, Bromocriptine, Temsirolimus, Everolimus, and Docetaxel. www.nature.com/scientificreports/ Moreover, it is further capable of holding its activated, the ‘autonomous state’, even though the initial stimulation is hold off i.e. more specifically speaking, even when the intracellular Ca+2 vanishes thus behaving like a ‘molecular switch’125. With reference to top interacting drugs with CaMKII, the list include Irinotecan, Bromocriptine, Dasatinib, Afatinib, and Imatinib (Fig. 13). Irinotecan has been previ- ously reported to enhance neurite outgrowth by structural similarity of Polysialic Acid126. Recently, the tyrosine kinase inhibitors have been shown to increase oxidative stress with resultant activation of CaMKII in cardiac fibroblasts127. Interestingly, Imatinib, the first approved kinase inhibitor had been known for remarkable selectiv- ity128–130 yet in our study it is one of the top contenders for off target interaction with CaMKII. The kinase inhibi- tors are however speculated to inhibit 10–100 kinases suggesting low selectivity130,131. p gg g y Structurally, CaMKII have 12 subunits, each having a carboxy terminal, the Hub region, followed by a ulatory segment which harbors PTM segments for phosphorylation, NAc-Glycosylation, oxidation at posi Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 18 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drugs Binding Affinity (−log10(KD|Ki)) Protein Residue Distance (Å) Type of Interactions Dactinomycin 37.7 ILE 31 3.63 Hydrophobic ALA 35 3.61 Hydrophobic TYR 36 3.96 Hydrophobic VAL 39 3.53 Hydrophobic GLU 53 3.49 H-bond ARG 67 2.18 H-bond LYS 151 4.00 Salt bridge Bromocriptine 32.7 VAL 39 3.47 Hydrophobic ALA 52 3.71 Hydrophobic LYS 54 3.88 Hydrophobic ILE 84 3.06 Hydrophobic LEU 156 3.97 Hydrophobic TYR 36 2.08 H-bond GLY 37 3.45 H-bond LYS 54 2.17 H-bond ASP 167 2.74 H-bond ASP 111 3.15 Halogen bond LYS 114 3.92 Halogen bond ARG 67 4.55 Salt bridge Temsirolimus 37.8 TYR 36 3.51 Hydrophobic VAL 39 3.34 Hydrophobic TYR 113 3.97 Hydrophobic LEU 156 3.70 Hydrophobic GLU 33 3.07 H-bond LYS 54 2.42 H-bond ARG 67 3.11 H-bond GLU 71 2.55 H-bond SER 153 2.98 H-bond LYS 114 5.29 Salt bridge LYS 151 3.93 Salt bridge Everolimus 41.2 VAL 39 3.49 Hydrophobic LEU 170 3.72 Hydrophobic GLU 33 2.89 H-bond TYR 36 1.83 H-bond LYS 54 2.68 H-bond TYR 64 3.08 H-bond GLU 71 1.90 H-bond ASP 167 3.31 H-bond LYS 151 3.21 Salt bridge Docetaxel 38.3 ILE 31 3.54 Hydrophobic ALA 35 3.76 Hydrophobic TYR 36 3.83 Hydrophobic VAL 39 3.50 Hydrophobic ALA 52 3.59 Hydrophobic ILE 56 3.49 Hydrophobic TYR 64 3.67 Hydrophobic ILE 84 3.26 Hydrophobic LEU 156 3.49 Hydrophobic ALA 35 3.09 H-bond LYS 54 2.73 H-bond LYS 151 2.21 H-bond ASN 154 3.42 H-bond ASP 167 2.57 H-bond LYS 54 5.04 Salt bridge ARG 67 4.76 Salt bridge Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 19 www.nature.com/scientificreports/ Figure 15. Top five docking conformations of ERK with (A) Dactinomycin (green); (B) Bromocriptine (cyan); (C) Temsirolimus (yellow); (D) Everolimus (beige); and (E) Docetaxel (golden). igure 15. Top five docking conformations of ERK with (A) Dactinomycin (green); (B) Bromocriptine (cyan); C) Temsirolimus (yellow); (D) Everolimus (beige); and (E) Docetaxel (golden). Thr287, Ser280 and Met 281 & 282 respectively76,132,133. The present work analysis suggests that chemotherapeu- tic drugs are exhibiting off targeting interactions in the regulatory segment which spans between the residues 273–317 and the kinase domain. Residues in this region and particularly the residue Thr 286 has been proven to be essential as knocking it down will abolish LTP induction with significant memory deficits134,135. www.nature.com/scientificreports/ Contrarily, taxanes have been previ- ously reported to pathologically enhance kinase activity promoting cell survival143,144.hfh subunit spans on multiple domains and harbors binding site for cAMP, dimerization-docking domain and a linker segment, while the catalytic subunit harbors two phosphorylation sites, one at residue Ser 338 which is near the C-terminal and the other is on residue Thr 197, coming under the activation loop/segment that serve as the docking site for protein substrate139. Recent literature suggest appearance of memory deficits and long term depression (LTD) on deletion of regulatory subunits140. The current investigative analysis reveals major off-target interaction from Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Bromocriptine (Fig. 11). In this frame of reference, Dactinomycin has been previously reported to inhibit kinase activity by interfering at the SH2 domain which is crucial for ligand induced activation141. Reduction of intracellular signal transduction by modulation of protein kinases have been reported with Bromocriptine142. Contrarily, taxanes have been previ- ously reported to pathologically enhance kinase activity promoting cell survival143,144.hfh y p p g y y p g The off-target interactions have been shown to be involving the residues Gly 50, Thr 51, Gly 52, Ser 53, which are part of the Glycine rich loop (spanning from residue 50–55), an integral part of ATP binding site, and the resi- due Glu 170, part of ribose pocket, which carries the phosphate binding cassette145. Interestingly, the Glycine rich loop is one of the targeted sites for the development of PKA inhibitors77. Moreover, off-target interactions have also been observed in the activation loop hooking residues Phe 187, Thr 201, Pro 202 and Glu 203. Notably, the activation loop when dephosphorylated, retains in intrinsically disordered conformation139. The residues from the catalytic loop have also been involved in off-target interaction.i f Two decades back, English and Sweatt reported for the first time, direct involvement of extracellular signal regulated kinase (ERK) in synaptic plasticity and later it was also established that ERK activation is detrimental for L-LTP146,147. This observation was further supplemented by the works of Gooney et al. who suggested require- ment of ERK activation for BDNF induced LTP148. ERK Kinase 1 and 2 which are important for LTP pathway, are related to mitogen activated protein kinase (MAPK) superfamily149. www.nature.com/scientificreports/ The impor- tant residues participating in off target interactions include Arg 296, Arg 297, Met307 which falls in regulatory segment more specifically in the CaM recognition sequence (residues 290–314) while the residue Glu 216 comes under kinase domain. The residue Arg 297 lies at the interface of regulatory and kinase domains and is involved in hydrophobic interactions with kinase domains of other subunits. Any interference in the CaM recognition sequence may results in alteration of Ca + 2 trapping which is very crucial for the autonomous phosphorylation activity of CaMKII. Moreover, another off-target interacting residue Met 307 is in very close proximity to the Calmodulin footprint which is on residue Thr 305 and 306 and phosphorylation of these residues will abrogate CaM/Calmodulin binding136 and binding with α-actinin which aids in CaMKII anchoring with NMDA137. g g g Another cellular kinase the Protein Kinase A (PKA), contributes to LTP induction. PKA since their discov- ery by Kreb and coworkers, have been known to be important second messenger after cAMP138. The regulatory Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 20 www.nature.com/scientificreports/ Figure 16. The co-crystallize and re-docked conformation of 2LL. The co-crystallize ligand is shown in cyan while re-docked ligand is shown in purple. Figure 16. The co-crystallize and re-docked conformation of 2LL. The co-crystallize ligand is shown in cyan while re-docked ligand is shown in purple. subunit spans on multiple domains and harbors binding site for cAMP, dimerization-docking domain and a linker segment, while the catalytic subunit harbors two phosphorylation sites, one at residue Ser 338 which is near the C-terminal and the other is on residue Thr 197, coming under the activation loop/segment that serve as the docking site for protein substrate139. Recent literature suggest appearance of memory deficits and long term depression (LTD) on deletion of regulatory subunits140. The current investigative analysis reveals major off-target interaction from Dactinomycin, Temsirolimus, Everolimus, Docetaxel and Bromocriptine (Fig. 11). In this frame of reference, Dactinomycin has been previously reported to inhibit kinase activity by interfering at the SH2 domain which is crucial for ligand induced activation141. Reduction of intracellular signal transduction by modulation of protein kinases have been reported with Bromocriptine142. www.nature.com/scientificreports/ Both human ERK1 and ERK2 share 84% identity and activated on parallel levels when stimulated150,151.h The results suggest Dactinomycin, Bromocriptine, Temosirolimus, Everolimus and Docetaxel among top low- est scorers of mediating off target interactions with ERK2 protein (Fig. 15). All of these interactions have been appeared in protein kinase domain (23–313 residue). All these drugs exhibited interactions in the Glycine rich residues spanning between 32–37 residues. Dactinomycin is associating through hydrophobic interactions on Ala35 and Tyr36, Temisrolimus via Tyr36 hydrophobically and through Hydrogen bonding on residue Glu33. Hydrogen bonding is also on residue Glu33 and Tyr36 by Everolimus while Docetaxel is interacting on residue Ala35 both through H-bonding and hydrophobically while residue Tyr36 is receiving only hydrophobic interac- tions. The Lys54 residue which mediates coupling of ATP phosphates to α helix is target of off-target Hydrogen bonding by Bromocriptine, Temosirolimus, Everolimus and Docetaxel. Temsirolimus and Everolimus, both are engaged to Glu71 via Hydrogen bonding. Everolimus and Docetaxel are interfering in the corner of activation segment on the residue Asp167 by hydrogen bonding. g p y y g g CBP are transcriptional coactivators known for gearing transcriptional expression of genes involved in cell survival. Moreover, CBP has also been linked to chromatin remodeling and in mediating acetyltransferase reac- tions72. CBP mutation in Rubinstein-Taybi Syndrome has shown to be critically detrimental in LTP pathway152. CREB and CBP are tools of cellular machinery imparting a central role in LTP and memory consolidation153. Specifically, CREB and CBP are positioned downstream in LTP signaling cascade and phosphorylation of Serine 133 on CREB results in its activation. This triggers CREB to interact with CBP154,155 via the same Ser 133 site, inducing gene expression.h g g p The results suggest Temsirolimus, Docetaxel and Everolimus to be binding with lowest energies, in the Bromo Domain region (BRD) of CBP, however, the scores are around −8 k cal/mol. The scores are less than the top dock- ing scores selected for studying interaction. Interestingly, the BRD has attracted particular target of interest for development of CBP BRD inhibitors156. Contextually, the CBP BRD inhibition has also been linked to modulate RGS4 expression, involved in GTPase activation, by the use of Temsirolimus157. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 21 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports ntificreports/ Figure 17. Electrostatic charge distribution at binding interfaces. (A) Electrostatic potential map of ERK complexed with Dactinomycin, Bromoriptine, Temsirolimus, Everolimus and Docetaxel. www.nature.com/scientificreports/ protein translation164,165. Therefore, interaction of mTOR inhibitors such as Everolimus & Temsirolimus can be detrimental. Since the chemotherapy drugs are primarily developed with the intention of targeting cancer cell proteins, the proteins with similar features are present in body elsewhere. The interaction of chemotherapeutic drugs with neuronal proteins suggest an area which is difficult to study directly on human tissues. g p ggfi y y Moreover, some chemotherapeutic drugs, the kinase inhibitors are in clinical evaluation for treating other neurological diseases58. Our study focused on some major proteins involved in LTP Pathway, however detailed elucidation of LTP dysregulation by chemotherapy requires investigation of all other proteins involved in LTP having other isoforms. Such is the case with Protein Kinase C zeta PKCζ166 which is also a very important protein in LTP pathway. The absence of crystal structures of PKCζ and the intrinsically disordered nature of CREB pro- tein167 limited our interaction analysis. y The spatiotemporal kinetics of LTP proteins affected by chemotherapy can also provide useful insight. Furthermore, receptor proteins involved in LTP like NMDA and AMPA are appareled with multitude of reg- ulatory sites imparting sensitive properties to afferent stimulus, therefore, our study’s result will be case of over simplification if we generalize our findings to all NMDAR or AMPAR functionally diversified subunits. The func- tional implication of the off targets corresponds to the importance of LTP pathway which is crucial in neuronal learning and memory processes. On a general note, although our study identified interactions which may be linked to aberrant LTP functioning, the mechanism of chemobrain is also well reported to be linked to cytokine dysregulation and other mechanism discussed above. Therefore, chemotherapy may be on an off-targeted route, be interacting with other pathways directly linked to the proposed mechanisms of causing chemobrain, and all of these etiologies can synergistically define the resultant disease outcome of cognitive decline. Having said that, explaining the intricacies of actual proteins and pathways involved in chemobrain by off target interactions is a relatively deserted area and our study in this context may add potential links to further explore not only the che- mobrain mechanisms but also to understand the LTP affected by drug target interactions. Conclusionshi The findings of this study suggest chemotherapy drugs to be interacting with LTP pathway proteins, which may modulate the induction and maintenance of E-LTP and L-LTP phases. As LTP is directly linked to synaptic medi- ation of learning acquisition and memory consolidation, the already reported aftermath of cognitive decline in chemobrain by altered LTP signaling is further objectively substantiated by this study. Moreover, regarding chemotherapy compounds in current clinical use, this study has provided novel aspects related to drug repur- posing and predictive toxicology, which can help in development of more effective yet more tolerable chemo- therapeutic drugs. However, further studies will be required to illustrate the agnostic and antagonistic effects of chemotherapy on LTP pathway. References Longitudinal Trajectory and Characterization of Cancer-Related Cognitive Impairment in a Nationwide Cohort Study. J Clin Oncol, JCO2018786624, https://doi.org/10.1200/JCO.2018.78.6624 (2018). g j j y 10. Janelsins, M. C. et al. Longitudinal Trajectory and Characterization of Cancer-Related Cognitive Impairment in a Nationwide Cohort Study. J Clin Oncol, JCO2018786624, https://doi.org/10.1200/JCO.2018.78.6624 (2018). 11. Millan, M. J. et al. Cognitive dysfunction in psychiatric disorders: characteristics, causes and the quest for Rev Drug Discov 11, 141–168, https://doi.org/10.1038/nrd3628 (2012). 11. Millan, M. J. et al. Cognitive dysfunction in psychiatric disorders: characteristics, causes and the quest for improved therapy Rev Drug Discov 11, 141–168, https://doi.org/10.1038/nrd3628 (2012). g y y ug Discov 11, 141–168, https://doi.org/10.1038/nrd3628 (2012). 12. Garcia-Carbonero, R. & Paz-Ares, L. Antibiotics and growth factors in the management of fever and neutropenia in cancer patients. Curr Opin Hematol 9, 215–221 (2002). 12. Garcia-Carbonero, R. & Paz-Ares, L. Antibiotics and growth factors in the management of fever and neutropenia in cancer patients. Curr Opin Hematol 9, 215–221 (2002). p p 13. Stone, J. B. & DeAngelis, L. M. Cancer-treatment-induced neurotoxicity–focus on newer treatments. Nat Rev Clin Oncol 13, 92–105, https://doi.org/10.1038/nrclinonc.2015.152 (2016). 13. Stone, J. B. & DeAngelis, L. M. Cancer-treatment-induced neurotoxicity–focus on newer treatments. Nat Rev Clin Oncol 13, 92–105, https://doi.org/10.1038/nrclinonc.2015.152 (2016). g 14. Dietrich, J., Prust, M. & Kaiser, J. Chemotherapy, cognitive impairment and hippocampal toxicity. Neuroscience 309, 224–232 https://doi.org/10.1016/j.neuroscience.2015.06.016 (2015). p g j ( ) 15. Seigers, R. & Fardell, J. E. Neurobiological basis of chemotherapy-induced cognitive impairment: a review of rodent rese Neurosci Biobehav Rev 35, 729–741, https://doi.org/10.1016/j.neubiorev.2010.09.006 (2011).f 16. Monje, M. & Dietrich, J. Cognitive side effects of cancer therapy demonstrate a functional role for adult neurogenesis. Behav Res 227, 376–379, https://doi.org/10.1016/j.bbr.2011.05.012 (2012). 16. Monje, M. & Dietrich, J. Cognitive side effects of cancer therapy demonstrate a functional role for adult neurogenesis. Behav Brain Res 227, 376–379, https://doi.org/10.1016/j.bbr.2011.05.012 (2012). 17 H E P S i l i i i h h h i h LTP C Bi l 8 R350 352 d i S0960 9822(98)70219 2 (1998) p g j 17. Huang, E. P. Synaptic plasticity: going through phases with LTP. Curr Biol 8, R350–352, doi:S0960-9822(98)70219-2 (1998). p p y g g g p Aricescu, A. R. A GluD Coming-Of-Age Story. Trends Neurosci 40, 18. Yuzaki, M. & Aricescu, A. R. A GluD Coming-Of-Age Story. T g g y 19. Won, S. J., Kim, D. Y. & Gwag, B. J. www.nature.com/scientificreports/ (B) Electrostatic potential map of NMDA complexed with Dactinomycin, Temsirolimus, Everolimus Docetaxel and Teniposide; (C) Electrostatic potential map of PKA complexed with Dactinomycin, Temsirolimus, Everolimus Docetaxel and Bromocriptine; (D) Electrostatic potential map of AMPA complexed with Dactinomycin, Temsirolimus, Paclitaxel, Vincristine and Irinotecan; (E) Electrostatic potential map of CaMKII complexed with Irinotecan, Bromocriptine, Dasatinib, Afatinib and Imatinib. Figure 17. Electrostatic charge distribution at binding interfaces. (A) Electrostatic potential map of ERK complexed with Dactinomycin, Bromoriptine, Temsirolimus, Everolimus and Docetaxel. (B) Electrostatic potential map of NMDA complexed with Dactinomycin, Temsirolimus, Everolimus Docetaxel and Teniposide; (C) Electrostatic potential map of PKA complexed with Dactinomycin, Temsirolimus, Everolimus Docetaxel and Bromocriptine; (D) Electrostatic potential map of AMPA complexed with Dactinomycin, Temsirolimus, Paclitaxel, Vincristine and Irinotecan; (E) Electrostatic potential map of CaMKII complexed with Irinotecan, Bromocriptine, Dasatinib, Afatinib and Imatinib. The molecular electrostatic potential analysis of the LTP pathway proteins (Fig. 17) reflected primarily elec- tronegative charge dominance in case of NMDA and PKA while electropositivity was observed more in case of ERK and AMPA. CaMKII stands in a position where the binding interfaces is not depicting overall charge elec- tropositivity or electronegativity which suggest its favor more towards hydrophobic interaction.h The molecular electrostatic potential analysis of the LTP pathway proteins (Fig. 17) reflected primarily elec- tronegative charge dominance in case of NMDA and PKA while electropositivity was observed more in case of ERK and AMPA. CaMKII stands in a position where the binding interfaces is not depicting overall charge elec- tropositivity or electronegativity which suggest its favor more towards hydrophobic interaction.h The development of kinase inhibitors have been increased tremendously during the last decade with more than 250 kinase inhibitors in the testable phase of clinical trials158 However, the selectivity of kinase inhibitors have been in question by invitro studies previously159. The current study findings are in alignment to the findings reported before, however, the current study identified potential offtarget interactions, suggesting new targets where the kinase inhibitors can be studied. Such is the case in mTOR signalling which has been associated with various neuropathlogies such as Autism, Epilepsy, Feeding behavior and age related synaptic alterations160–163. Moreover, mTOR pathway has been linked to LTP & Long term depression (LTD) pathways by virtue of synaptic Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 22 www.nature.com/scientificreports/ References 1. Park, S. B. et al. Chemotherapy-induced peripheral neurotoxicity: a critical analysis. CA Cancer J Clin 63, 419–437, https://doi org/10.3322/caac.21204 (2013).i 1. Park, S. B. et al. Chemotherapy-induced peripheral neurotoxicity: a critical analysis. CA Cancer J Clin 63, 419–437, https://doi. org/10.3322/caac.21204 (2013).i g ( ) 2. Bowes, J. et al. Reducing safety-related drug attrition: the use of in vitro pharmacological profiling. Nat Rev Drug Discov 11, 909–922, https://doi.org/10.1038/nrd3845 (2012). 2. Bowes, J. et al. Reducing safety-related drug attrition: the use of in vitro pharmacological profiling. Nat Rev Drug Discov 11 909–922, https://doi.org/10.1038/nrd3845 (2012). p g 3. Curran, M. E. et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795–803 doi:0092-8674(95)90358-5 (1995). 4. Cheng, F. et al. admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties. J Chem Inf Mode 52, 3099–3105, https://doi.org/10.1021/ci300367a (2012). 4. Cheng, F. et al. admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties. J Chem Inf Model 52, 3099–3105, https://doi.org/10.1021/ci300367a (2012). p g 5. Wang, X. M. et al. Chemobrain: a critical review and causal hypothesis of link between cytokines and epigenetic reprogramming associated with chemotherapy Cytokine 72 86 96 https://doi org/10 1016/j cyto 2014 12 006 (2015) g 5. Wang, X. M. et al. Chemobrain: a critical review and causal hypothesis of link between cytokines and epigenetic re associated with chemotherapy. Cytokine 72, 86–96, https://doi.org/10.1016/j.cyto.2014.12.006 (2015). 5. Wang, X. M. et al. Chemobrain: a critical review and causal hypothesis of link between cytokines and epigenetic reprogramming associated with chemotherapy. Cytokine 72, 86–96, https://doi.org/10.1016/j.cyto.2014.12.006 (2015). 5. Wang, X. M. et al. Chemobrain: a critical review and causal hypothesis of link between cytokines and epigenetic reprogramming associated with chemotherapy. Cytokine 72, 86–96, https://doi.org/10.1016/j.cyto.2014.12.006 (2015). py y p g j y 6. Hurria, A., Somlo, G. & Ahles, T. Renaming “chemobrain”. Cancer Invest 25, 373–377, doi:782023378 (2007). g 7. Gutmann, D. H. Clearing the Fog surrounding Chemobrain. Cell 176, 2–4, doi:S0092-8674(18)31641-6 (2019). 8. Weiss, B. Chemobrain: a translational challenge for neurotoxicology. Neurotoxicology 29, 891–898, https://doi.org/10.10 neuro.2008.03.009 (2008). 9. Argyriou, A. A., Assimakopoulos, K., Iconomou, G., Giannakopoulou, F. & Kalofonos, H. P. Either called “chemobrain” o “chemofog,” the long-term chemotherapy-induced cognitive decline in cancer survivors is real. J Pain Symptom Manage 41 126–139, https://doi.org/10.1016/j.jpainsymman.2010.04.021 (2011). p g j jp y ( ) 10. Janelsins, M. C. et al. www.nature.com/scientificreports/ Neuroreport 20 371–377, https://doi.org/10.1097/WNR.0b013e328324edcd (2009). 371–377, https://doi.org/10.1097/WNR.0b013e328324edcd (2009 p g 42. Cheung-Ong, K., Giaever, G. & Nislow, C. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology Chem Biol 20, 648–659, https://doi.org/10.1016/j.chembiol.2013.04.007 S1074-5521(13)00131-2 (2013). 43. Remon, J. & Soria, J. C. Improving brain penetration of kinase inhibitors in lung can Oncol 28, 196–198, https://doi.org/10.1093/annonc/mdw553 (2017).ffi 43. Remon, J. & Soria, J. C. Improving brain penetration of kinase inhibitors in lung cancer patients with oncogene dependency. Ann Oncol 28, 196–198, https://doi.org/10.1093/annonc/mdw553 (2017).ffi p g 44. Tan, J. et al. Tyrosine kinase inhibitors show different anti-brain metastases efficacy in NSCLC: A direct comparative analysis of icotinib gefitinib, and erlotinib in a nude mouse model. Oncotarget 8, 98771–98781, https://doi.org/10.18632/oncotarget.21936 (2017). gi g p g g 45. Clement, J. M. & Holle, L. M. Safe Administration of Intracerebral Spinal Fluid Chemotherapy: Time for Guidelines. J Oncol Prac 13, 713–718, https://doi.org/10.1200/JOP.2017.022038 (2017). p g 46. Kast, R. E. & Focosi, D. Three paths to better tyrosine kinase inhibition behind the blood-brain barrier in treating chronic myelogenous leukemia and glioblastoma with imatinib. Transl Oncol 3, 13–15 (2010). y g g 47. Deane, C. M., Salwinski, L., Xenarios, I. & Eisenberg, D. Protein interactions: two methods for assessment of the reliability of high throughput observations. Mol Cell Proteomics 1, 349–356 (2002).i throughput observations. Mol Cell Proteomics 1, 349–356 (2002 g 48. Lopes, P. E., Guvench, O. & MacKerell, A. D. Jr. Current status of protein force fields for molecular dynamics simulations. Methods Mol Biol 1215, 47–71, https://doi.org/10.1007/978-1-4939-1465-4_3 (2015). g 49. Huang, S. Y. & Zou, X. Advances and challenges in protein-ligand docking. Int J Mol Sci 11, 3016–3034, https://doi.org/10.3390 ijms11083016 (2010). j ( ) 50. Hue, M., Riffle, M., Vert, J. P. & Noble, W. S. Large-scale prediction of protein-protein interactions from structures. BMC Bioinformatics 11, 144, https://doi.org/10.1186/1471-2105-11-144 (2010).i 51. Koutsoukas, A. et al. In silico target predictions: defining a benchmarking data set and comparison of performance of the multiclass Naive Bayes and Parzen-Rosenblatt window. J Chem Inf Model 53, 1957–1966, https://doi.org/10.1021/ci300435j (2013). y J f p g j ( ) 52. Keiser, M. J. et al. Relating protein pharmacology by ligand chemistry. Nat Biotechnol 25, 197–206, doi:nbt1284 (2007). d l l ff d d k ll d d h 53. Cruz-Monteagudo, M. et al. Activity cliffs in drug discovery: Dr Jekyll or Mr Hyde? Drug Discov Today 19, 1069–1080, https://doi. org/10.1016/j.drudis.2014.02.003 (2014). g j 54. www.nature.com/scientificreports/ & Noble, M. CNS progenitor cells and oligodendrocytes are targets o chemotherapeutic agents in vitro and in vivo. J Biol 5, 22, https://doi.org/10.1186/jbiol50 (2006). 31. Nokia, M. S., Anderson, M. L. & Shors, T. J. Chemotherapy disrupts learning, neurogenesis and theta activity in the adult brain J Neurosci 36, 3521–3530, https://doi.org/10.1111/ejn.12007 (2012). p g j 32. Reiriz, A. B. et al. Cancer chemotherapy and cognitive function in rodent models: memory impairment induced by cyclophosphamide in mice. Clin Cancer Res 12, 5000; author reply 5000–5001, 12/16/5000 (2006).ff 33. Walker, E. A., Foley, J. J., Clark-Vetri, R. & Raffa, R. B. Effects of repeated administration of chemotherapeutic agents tamoxifen, methotrexate, and 5-fluorouracil on the acquisition and retention of a learned response in mice. Psychopharmacology (Berl) 217, 539–548, https://doi.org/10.1007/s00213-011-2310-8 (2011).il p g 34. ElBeltagy, M. et al. Fluoxetine improves the memory deficits caused by the chemotherapy agent 5-fluorouracil. Behav Brain Res 208, 112–117, https://doi.org/10.1016/j.bbr.2009.11.017 (2010). p g j ( ) 35. Christie, L. A. et al. Impaired cognitive function and hippocampal neurogenesis following cancer chemotherapy. Clin Cancer Res 18, 1954–1965, https://doi.org/10.1158/1078-0432.CCR-11-2000 (2012).l 36. Fardell, J. E., Vardy, J., Shah, J. D. & Johnston, I. N. Cognitive impairments caused by oxaliplatin and 5-fluorouracil chemotherapy are ameliorated by physical activity. Psychopharmacology (Berl) 220, 183–193, https://doi.org/10.1007/s00213-011-2466-2 (2012).f 36. Fardell, J. E., Vardy, J., Shah, J. D. & Johnston, I. N. Cognitive impairments caused by oxaliplatin and 5 fluorouracil chemotherapy are ameliorated by physical activity. Psychopharmacology (Berl) 220, 183–193, https://doi.org/10.1007/s00213-011-2466-2 (2012).f y p y y y p gy p g 37. Mustafa, S., Walker, A., Bennett, G. & Wigmore, P. M. 5-Fluorouracil chemotherapy affects spatial working memory and new neurons in the adult rat hippocampus. Eur J Neurosci 28, 323–330, https://doi.org/10.1111/j.1460-9568.2008.06325 (2008). pp p p g j 38. Mignone, R. G. & Weber, E. T. Potent inhibition of cell proliferation in the hippocampal dentate gyrus of mice by th chemotherapeutic drug thioTEPA. Brain Res 1111, 26–29, doi:S0006-8993(06)02003-8 (2006). 39. Yang, M. et al. Cyclophosphamide impairs hippocampus-dependent learning and memory in adult mice: Possible involvement of hippocampal neurogenesis in chemotherapy-induced memory deficits. Neurobiol Learn Mem 93, 487–494, https://doi. org/10.1016/j.nlm.2010.01.006 (2010). g j ( ) 40. Seigers, R. et al. Long-lasting suppression of hippocampal cell proliferation and impaired cognitive performance by methotrexate in the rat. Behav Brain Res 186, 168–175 (2008). doi:S0166-4328(07)00403-2. 41. Lau, B. W. et al. Intracerebroventricular infusion of cytosine-arabinoside causes prepulse inhibition disruption. References Cellular and molecular pathways of ischemic neuronal death. J Biochem Mol Biol 35, 6 (2002). 20. Platenik, J., Kuramoto, N. & Yoneda, Y. Molecular mechanisms associated with long-term consolidation of the NMDA signals Sci 67, 335–364, doi:S0024320500006329 (2000). ( ) 21. Staubli, U., Rogers, G. & Lynch, G. Facilitation of glutamate receptors enhances memory. Proc Natl Acad Sci USA 91, 777–781 (1994). Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 23 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 22. Carlsson, M. & Carlsson, A. Interactions between glutamatergic and monoaminergic systems within the basal ganglia–implication for schizophrenia and Parkinson’s disease. Trends Neurosci 13, 272–276 (1990). doi:0166-2236(90)90108-M. 22. Carlsson, M. & Carlsson, A. Interactions between glutamatergic and monoaminergic systems within the basal ganglia–implications for schizophrenia and Parkinson’s disease. Trends Neurosci 13, 272–276 (1990). doi:0166-2236(90)90108-M. 23. Sequerra, E. B., Goyal, R., Castro, P. A., Levin, J. B. & Borodinsky, L. N. NMDA Receptor Signaling Is Important for Neural Tube p 23. Sequerra, E. B., Goyal, R., Castro, P. A., Levin, J. B. & Borodinsky, L. N. NMDA Receptor Signaling Is Important for Neural Tube Formation and for Preventing Antiepileptic Drug-Induced Neural Tube Defects. J Neurosci 38, 4762–4773, https://doi.org/10.1523 JNEUROSCI.2634-17.2018 (2018).h 24. Balu, D. T. The NMDA Receptor and Schizophrenia: From Pathophysiology to Treatment. Adv Pharmacol 76, 351–382, https://doi. org/10.1016/bs.apha.2016.01.006 (2016). g p ( ) 25. Moskal, J. R. et al. GLYX-13, an NMDA receptor glycine site functional partial agonist enhances cognition and produces antidepressant effects without the psychotomimetic side effects of NMDA receptor antagonists. Expert Opin Investig Drugs 23, 243–254, https://doi.org/10.1517/13543784.2014.852536 (2014). p g 26. McKeage, K. Memantine: a review of its use in moderate to severe Alzheimer’s disease. CNS Drugs 23, 881–897, https://doi org/10.2165/11201020-000000000-00000 (2009). g 27. Hu, C., Chen, W., Myers, S. J., Yuan, H. & Traynelis, S. F. Human GRIN2B variants in neurodevelopmental disorders. J Pharmaco Sci 132, 115–121, doi:S1347-8613(16)30133-5 (2016).i ( ) ( ) 28. Xie, L., Kinnings, S. L. & Bourne, P. E. Novel computational approaches to polypharmacology as a means to define responses to individual drugs. Annu Rev Pharmacol Toxicol 52, 361–379, https://doi.org/10.1146/annurev-pharmtox-010611-134630 (2012).fi g p g p 29. Lim, H. et al. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing. PLoS Comput Biol 12, e1005135, https://doi.org/10.1371/journal.pcbi.1005135 (2016). g p g p 29. Lim, H. et al. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering d I A li i D R i PL S C Bi l 12 1005135 h //d i /10 1371/j l bi 1005135 (2016) 29. Lim, H. et al. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing. PLoS Comput Biol 12, e1005135, https://doi.org/10.1371/journal.pcbi.1005135 (2016). pp g p p g p p g j p 30. Dietrich, J., Han, R., Yang, Y., Mayer-Proschel, M. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 61. Halgren, T. MMFF VII. Characterization of MMFF94, MMFF94s, and other widely available force fields for conformational energies and for intermolecular- interaction energies and geometries. Journal of Computational Chemistry 20, 730–748, https://doi. org/10.1002/(SICI)1096-987X(199905)20:7<730::AID-JCC8>3.0.CO;2-T (1999). g ( ) ( ) J ( ) 62. Mayer, M. L., Westbrook, G. L. & Guthrie, P. B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones Nature 309, 261–263 (1984). , ( ) 63. Monyer, H. et al. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256, 1217–1221 (1992). ( ) 63. Monyer, H. et al. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256, 1217–1221 (1992) 64. Mayer, M. L. The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures. Biophys J 113, 2143–2151 doi:S0006-3495(17)30854-8 (2017). 65. Johnson, D. A., Akamine, P., Radzio-Andzelm, E., Madhusudan, M. & Taylor, S. S. Dynamics of cAMP-dependent protein kinase. Chem Rev 101, 2243–2270, doi:cr000226k (2001). 66. Masterson, L. R., Mascioni, A., Traaseth, N. J., Taylor, S. S. & Veglia, G. Allosteric cooperativity in protein kinase A. Proc Natl Acad Sci USA 105, 506–511, https://doi.org/10.1073/pnas.0709214104 (2008).i g 67. Kornev, A. P. & Taylor, S. S. Defining the conserved internal architecture of a protein kinase. Biochim Biophys Acta 1804, 440–444 https://doi.org/10.1016/j.bbapap.2009.10.017 (2010). 68. Knighton, D. R. et al. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414 (1991). 69. Radhakrishnan, I., Perez-Alvarado, G. C., Dyson, H. J. & Wright, P. E. Conformational preferences in the Ser133-phosphorylated and non-phosphorylated forms of the kinase inducible transactivation domain of CREB. FEBS Lett 430, 317–322, doi:S0014-5793(98)00680-2 (1998). 70. Richards, J. P., Bachinger, H. P., Goodman, R. H. & Brennan, R. G. Analysis of the structural properties of cAMP-respon element-binding protein (CREB) and phosphorylated CREB. J Biol Chem 271, 13716–13723 (1996). 71. Blobel, G. A. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95, 745–755 (200 71. Blobel, G. A. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95, 745–755 (2000). 72. Shiama, N. The p300/CBP family: integrating signals with transcription factors and chromatin. Trends Cell Biol 7, 230–236, doi:S0962-8924(97)01048-9 (1997). ( ) ( ) 73. Dyson, H. J. & Wright, P. E. Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6, 197–208, doi:nrm1589 (2005).if ( ) 74. Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A. & Farid, R. www.nature.com/scientificreports/ Novel procedure for modeling ligand/receptor induced fit effects J Med Chem 49, 534–553, https://doi.org/10.1021/jm050540c (2006). g j 75. Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy. Science 353, 83–86, https://doi.org/10.1126/science.aaf8411 (2016). 76. Chao, L. H. et al. A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin- dependent kinase I holoenzyme. Cell 146, 732–745, https://doi.org/10.1016/j.cell.2011.07.038 (2011). y p g j 77. Lauber, B. S. et al. Addressing the Glycine-Rich Loop of Protein Kinases by a Multi-Facetted Interaction Network: Inhibition o PKA and a PKB Mimic. Chemistry 22, 211–221, https://doi.org/10.1002/chem.201503552 (2016). 78. Aronov, A. M. et al. Flipped out: structure-guided design of selective pyrazolylpyrrole ERK inhibitors. J Med Chem 50, 1280–1287, https://doi.org/10.1021/jm061381f (2007). 79. Corbeil, C. R., Williams, C. I. & Labute, P. Variability in docking success rates due to dataset preparation. J Comput Aided Mol De 26, 775–786, https://doi.org/10.1007/s10822-012-9570-1 (2012).h p g 80. Labute, P. The generalized Born/volume integral implicit solvent model: estimation of the free energy of hydration using London dispersion instead of atomic surface area. J Comput Chem 29, 1693–1698, https://doi.org/10.1002/jcc.20933 (2008).i p p p 81. Salentin, S., Schreiber, S., Haupt, V. J., Adasme, M. F. & Schroeder, M. PLIP: fully auto Nucleic Acids Res 43, W443–447, https://doi.org/10.1093/nar/gkv315 (2015). 81. Salentin, S., Schreiber, S., Haupt, V. J., Adasme, M. F. & Schroeder, M. PLIP: fully automated protein-ligand interaction profiler Nucleic Acids Res 43, W443–447, https://doi.org/10.1093/nar/gkv315 (2015).fi 82. Pires, D. E. & Ascher, D. B. CSM-lig: a web server for assessing and comparing protein-small molecule affinities. Nucleic Acids Re 44, W557–561, https://doi.org/10.1093/nar/gkw390 (2016). p g g 83. Weiner, P. K., Langridge, R., Blaney, J. M., Schaefer, R. & Kollman, P. A. Electrostatic potential molecular surfaces. Proc Natl Acad Sci USA 79, 3754–3758, https://doi.org/10.1073/pnas.79.12.3754 (1982). p g p 84. Argyriou, A. A., Bruna, J., Marmiroli, P. & Cavaletti, G. Chemotherapy-induced peripheral neurotoxicity (CIPN): an update. Cri Rev Oncol Hematol 82, 51–77, https://doi.org/10.1016/j.critrevonc.2011.04.012 (2012). Rev Oncol Hematol 82, 51–77, https://doi.org/10.1016/j.critrevonc.2011.04.012 (2012). 85. Ahles, T. A. & Saykin, A. J. Candidate mechanisms for chemotherapy-induced cognitive changes. Nat Rev Cancer 7, 192–201, d i 2073 (2007) 85. Ahles, T. A. & Saykin, A. J. Candidate mechanisms for chemotherapy-induced cognitive changes. Nat Rev Cancer 7, 192–201 doi:nrc2073 (2007). ( ) 86. Takehara, K., Kawahara, S. & Kirino, Y. www.nature.com/scientificreports/ Time-dependent reorganization of the brain components underlying memory retention in trace eyeblink conditioning. J Neurosci 23, 9897–9905, doi:23/30/9897 (2003).hf y g 87. Winocur, G. et al. The effects of chemotherapy on cognitive function in a mouse model: a prospective study. Clin Cancer Res 18 3112–3121, https://doi.org/10.1158/1078-0432.CCR-12-0060 (2012). p g 88. Ogden, K. K. & Traynelis, S. F. New advances in NMDA receptor pharmacology. Trends Pharmacol Sci 32, 726–733, https://doi org/10.1016/j.tips.2011.08.003 (2011). g j p 89. Dreyer, E. B., Zhang, D. & Lipton, S. A. Transcriptional or translational inhibition blocks low dose NMDA-mediated cell death Neuroreport 6, 942–944 (1995).f p ( ) 90. Gordillo-Salas, M., Pilar-Cuellar, F., Auberson, Y. P. & Adell, A. Signaling pathways responsible for the rapid antidepressant-like effects p 90. Gordillo-Salas, M., Pilar-Cuellar, F., Auberson, Y. P. & Adell, A. Signaling pathways responsible for the rapid antidepressant-like e of a GluN2A preferring NMDA receptor antagonist Transl Psychiatry 8 84 https://doi org/10 1038/s41398 018 0131 9 (2018) p 90. Gordillo-Salas, M., Pilar-Cuellar, F., Auberson, Y. P. & Adell, A. Signaling pathways responsible for the rapid antidepressant-like effects of a GluN2A-preferring NMDA receptor antagonist. Transl Psychiatry 8, 84, https://doi.org/10.1038/s41398-018-0131-9 (2018). 91 G d i ki R H t M & L d C DSM 5 d ti t di d (ASD ) t it f id tif i ASD of a GluN2A preferring NMDA receptor antagonist. Transl Psychiatry 8, 84, https://doi.org/10.1038/s41398 018 0131 9 (2018). 91. Grzadzinski, R., Huerta, M. & Lord, C. DSM-5 and autism spectrum disorders (ASDs): an opportunity for identifying ASD subtypes Mol Autism 4 12 https://doi org/10 1186/2040-2392-4-12 (2013) p g p g y y p 91. Grzadzinski, R., Huerta, M. & Lord, C. DSM-5 and autism spectrum disorders (ASD subtypes. Mol Autism 4, 12, https://doi.org/10.1186/2040-2392-4-12 (2013). 91. Grzadzinski, R., Huerta, M. & Lord, C. DSM-5 and autism spectrum disorders (ASDs): an opportunity for identifying ASD subtypes. Mol Autism 4, 12, https://doi.org/10.1186/2040-2392-4-12 (2013).h 92. Lages, B. & Weiss, H. J. Inhibition of human platelet function in vitro and ex vivo by acetaminophen. Thromb Res 53, 603–613 (1989). 93. Kalbaugh, T. L., VanDongen, H. M. & VanDongen, A. M. Ligand-binding residues integrate affinity and efficacy in the NMDA receptor. Mol Pharmacol 66, 209–219, https://doi.org/10.1124/mol.66.2.209 (2004).t 92. Lages, B. & Weiss, H. J. Inhibition of human platelet function in vitro and ex vivo by acetaminophen. Thromb Res 53, 603–613 (1 g p y ph 93. Kalbaugh, T. www.nature.com/scientificreports/ Gonen, M. Predicting drug-target interactions from chemical and genomic kernels using Bayesian matrix factorization Bioinformatics 28, 2304–2310, https://doi.org/10.1093/bioinformatics/bts360 (2012). f g 55. Liu, Y., Wu, M., Miao, C., Zhao, P. & Li, X. L. Neighborhood Regularized Logistic Matrix Factorization for Drug-Target Interaction Prediction. PLoS Comput Biol 12, e1004760, https://doi.org/10.1371/journal.pcbi.1004760 (2016). 56. Sato, A. mTOR, a Potential Target to Treat Autism Spectrum Disorder. CNS Neurol Disord Drug Targets 15, 533–543 doi:CNSNDDT-EPUB-74924 (2016). 57. Royce, M. E. & Osman, D. Everolimus in the Treatment of Metastatic Breast Cancer. Breast Cancer (Auckl) 9, 73–79, https://doi org/10.4137/BCBCR.S29268 (2015). g 58. Mizuguchi, M. et al. Everolimus for epilepsy and autism spectrum disorder in tuberous sclerosis complex: EXIST-3 substudy in Japan. Brain Dev 41, 1–10 (2019). doi:S0387-7604(18)30176-1.f 59. Flynn, M., Heale, K. A. & Alisaraie, L. Mechanism of Off-Target Interactions and Toxicity of Tamoxifen and Its Metabolites. Chem Res Toxicol 30, 1492–1507, https://doi.org/10.1021/acs.chemrestox.7b00112 (2017). 60 National Cancer Institute, https://www cancer gov/about-cancer/treatment/drugs p g 60. National Cancer Institute, https://www.cancer.gov/about-cancer/treatment/drugs. p g 60. National Cancer Institute, https://www.cancer.gov/about-cancer/treatment/drugs. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 24 Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 www.nature.com/scientificreports/ Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28, 165–181, doi:S0896-6273(00)00094-5 (2000).i g g 15. Su, J. G. et al. Identification of functionally key residues in AMPA receptor with a thermodynamic method. J Phys Chem B 117 8689–8696, https://doi.org/10.1021/jp402290t (2013). p g jp 16. Holm, M. M. et al. A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study. J Biol Chem 280, 35469–35476 (2005). doi:M507800200. 117. Armstrong, N., Mayer, M. & Gouaux, E. Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes. Proc Natl Acad Sci USA 100, 5736–5741, https://doi.org/10.1073/pnas.1037393100 (2003). ( ) 118. Weston, M. C., Gertler, C., Mayer, M. L. & Rosenmund, C. Interdomain interactions in AMPA and kainate receptors regulate affinity for glutamate. J Neurosci 26, 7650–7658, doi:26/29/7650 (2006). fi y g 19. Yamada, K. A. & Rothman, S. M. Diazoxide blocks glutamate desensitization and prolongs excitatory postsynaptic currents in ra hippocampal neurons. J Physiol 458, 409–423 (1992). pp p y 120. Isaacson, J. S. & Nicoll, R. A. Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus. Proc Natl Acad Sci USA 88, 10936–10940 (1991). 121. O’Neill, M. J., Bleakman, D., Zimmerman, D. M. & Nisenbaum, E. S. AMPA receptor potentiators for the treatment of disorders. Curr Drug Targets CNS Neurol Disord 3, 181–194 (2004). 22. Roskoski, R. Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66, 105–143, https://doi.org/10.1016/j phrs.2012.04.005 (2012).h p 123. Abdel-Aziz, A. K., Mantawy, E. M., Said, R. S. & Helwa, R. The tyrosine kinase inhibitor, sunitinib malate, induces cognitive impairment in vivo via dysregulating VEGFR signaling, apoptotic and autophagic machineries. Exp Neurol 283, 129–141, https:// doi.org/10.1016/j.expneurol.2016.06.004 (2016). g j p 24. Lisman, J. E. A mechanism for memory storage insensitive to molecular turnover: a bistable autophosphorylating kinase. Proc Nat Acad Sci USA 82, 3055–3057 (1985). ( ) 25. Miller, S. G. & Kennedy, M. B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell 44, 861–870 (1986). doi:0092-8674(86)90008-5.h gg 126. Loers, G. et al. The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth signal via protein kinase C. J Neurochem 142, 392–406, https://doi.org/10.1111/jnc.14076 (2017).i 27. McMullen, C. et al. www.nature.com/scientificreports/ 02. Park, S. B. et al. Mechanisms underlying chemotherapy-induced neurotoxicity and the potential for neuroprotective strategies Curr Med Chem 15, 3081–3094 (2008). 102. Park, S. B. et al. Mechanisms underlying chemotherapy-induced neurotoxicity and the potential for neuroprotective strategies. Curr Med Chem 15, 3081–3094 (2008). 103 Materazzi S et al TRPA1 and TRPV4 mediate paclitaxel induced peripheral neuropathy in mice via a glutathione sensitive 03. Materazzi, S. et al. TRPA1 and TRPV4 mediate paclitaxel-induced peripheral neuropathy in mice via a glutathione-sensitive mechanism. Pflugers Arch 463, 561–569, https://doi.org/10.1007/s00424-011-1071-x (2012). l 04. Mabb, A. M. et al. Topoisomerase 1 inhibition reversibly impairs synaptic function. Proc Natl Acad Sci USA 111, 17290–17295 https://doi.org/10.1073/pnas.1413204111 (2014). https://doi.org/10.1073/pnas.1413204111 (2014). p g p ( ) 05. King, I. F. et al. Topoisomerases facilitate transcription of long genes linked to autism. Nature 501, 58–62, https://doi.org/10.1038 nature12504 (2013). 106. Aparicio, T. et al. Geriatric factors predict chemotherapy feasibility: ancillary results of FFCD 2001–02 phase III study in first-line chemotherapy for metastatic colorectal cancer in elderly patients. J Clin Oncol 31, 1464–1470, https://doi.org/10.1200/ JCO.2012.42.9894(2013). ( ) 107. Yelshanskaya, M. V. et al. Structural Bases of Noncompetitive Inhibition of AMPA-Subtype Ionotropic Glutamate Receptors by Antiepileptic Drugs. Neuron 91, 1305–1315, doi:S0896-6273(16)30504-9 (2016). p p g 108. Zachariassen, L. G. et al. Structural rearrangement of the intracellular domains during AMPA receptor activation. Proc Natl Acad Sci USA 113, E3950–3959, https://doi.org/10.1073/pnas.1601747113 (2016).i p g p 09. Hogner, A. et al. Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexe with the GluR2 ligand-binding core. J Mol Biol 322, 93–109, doi:S0022283602006502 (2002).tfifi g g 110. Robert, A., Armstrong, N., Gouaux, J. E. & Howe, J. R. AMPA receptor binding cleft mutations that alter affinity, efficacy, and recovery from desensitization. J Neurosci 25, 3752–3762, doi:25/15/3752 (2005). y 111. Abele, R., Keinanen, K. & Madden, D. R. Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A ki and mutagenetic analysis. J Biol Chem 275, 21355–21363, https://doi.org/10.1074/jbc.M909883199 (2000). g y p g j 12. Uchino, S., Sakimura, K., Nagahari, K. & Mishina, M. Mutations in a putative agonist binding region of the AMPA-selective glutamate receptor channel. FEBS Lett 308, 253–257, doi:0014-5793(92)81286-U (1992).l g 13. Mano, I., Lamed, Y. & Teichberg, V. I. A venus flytrap mechanism for activation and desensitization of alpha-amino-3-hydroxy-5 methyl-4-isoxazole propionic acid receptors. J Biol Chem 271, 15299–15302 (1996). 114. Armstrong, N. & Gouaux, E. www.nature.com/scientificreports/ L., VanDongen, H. M. & VanDongen, A. M. Ligand-binding residues integrate affinity and efficacy in the NMDA receptor. Mol Pharmacol 66, 209–219, https://doi.org/10.1124/mol.66.2.209 (2004).t p p g 94. Paganelli, M. A., Kussius, C. L. & Popescu, G. K. Role of cross-cleft contacts in NMDA receptor gating. PLoS One 8, e80953, https:// doi.org/10.1371/journal.pone.0080953 (2013). g j p ( ) 95. Choi, D. W. Ionic dependence of glutamate neurotoxicity. J Neurosci 7, 369–379 (1987). g j p 5. Choi, D. W. Ionic dependence of glutamate neurotoxicity. J Neu 96. Lind, G. E. et al. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for Glu over GluN2B subunits. Proc Natl Acad Sci USA 114, E6942–E6951, https://doi.org/10.1073/pnas.1707752114 (2017). 97 Fl h J I H b M H d M J & N i M D ABC i h j d ffl N 97. Fletcher, J. I., Haber, M., Henderson, M. J. & Norris, M. D. ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer 10, 147–156, https://doi.org/10.1038/nrc2789 (2010).h g 98. Ribeiro, M. P., Nunes-Correia, I., Santos, A. E. & Custodio, J. B. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells. Exp Cell Res 321, 288–296, https://doi.org/10.1016/j.yexcr.2013.11.002 (2014). p g j y 99. Traynelis, S. F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62, 405–496, https://doi org/10.1124/pr.109.002451 (2010). 100. Mayer, M. L. Emerging models of glutamate receptor ion channel structure and function. Structure 19, 1370–1380, https://doi. org/10.1016/j.str.2011.08.009 (2011). g j 01. Furukawa, K. & Mattson, M. P. Taxol stabilizes [Ca2+]i and protects hippocampal neurons against excitotoxicity. Brain Res 689 141–146 (1995). doi:0006-8993(95)00537-Z. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 25 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Dyson, H. J. & Wright, P. E. Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivator CREB-binding Protein (CBP) and p300. J Biol Chem 291, 6714–6722, https://doi.org/10.1074/jbc.R115.692020 (2016). g p p g j 168. Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, disease drugs. Nucleic Acids Res 45, D353–D361, https://doi.org/10.1093/nar/gkw1092 (2017). 169. Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28, 27–30 (2000). doi:gkd027. 169. Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28, 27–30 (2000). doi:gkd027. 170 Kanehisa M Sato Y Furumichi M Morishima K & Tanabe M New approach for understanding genome variations in KEGG 169. Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28, 27–30 (2000). doi:gkd027. 170. Kanehisa, M., Sato, Y., Furumichi, M., Morishima, K. & Tanabe, M. New approach for understanding genome variations in K Nucleic Acids Res 47, D590–D595, https://doi.org/10.1093/nar/gky962 (2019). 171. Volgraf, M. et al. Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design. J Med Chem 59, 2760–2779, https://doi.org/10.1021/acs.jmedchem.5b02010 (2016). www.nature.com/scientificreports/ Annu Rev Neurosci 21, 127–148, https://doi.org/10.1146 annurev.neuro.21.1.127 (1998). 55. Chrivia, J. C. et al. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365, 855–859, https://doi org/10.1038/365855a0 (1993). g ( ) 56. Hay, D. A. et al. Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains. J Am Chem Soc 136 9308–9319, https://doi.org/10.1021/ja412434f (2014). p g j 57. Weiler, M. et al. Suppression of proinvasive RGS4 by mTOR inhibition optimizes glioma treatment. Oncogene 32, 1099–1109 https://doi.org/10.1038/onc.2012.137 (2013). p g 58. Wu, P., Nielsen, T. E. & Clausen, M. H. FDA-approved small-molecule kinase inhibitors. Trends Pharmacol Sci 36, 422–439, https:/ doi.org/10.1016/j.tips.2015.04.005 (2015).h g j p 159. Klaeger, S. et al. The target landscape of clinical kinase drugs. Science 358, https://doi.org/10.1126/science.aan4368 (2017). 160 B T A ti t k t ti C O i N bi l 19 231 234 htt //d i /10 1016/j b 2009 06 003 (2009) 159. Klaeger, S. et al. The target landscape of clinical kinase drugs. Science 358, https://doi.org/10.1126/science.aan4368 (2017). 160. Bourgeron, T. A synaptic trek to autism. Curr Opin Neurobiol 19, 231–234, https://doi.org/10.1016/j.conb.2009.06.003 (2009). g y p p p g j 61. Ehninger, D. et al. Reversal of learning deficits in a Tsc2+/− mouse model of tuberous sclerosis. Nat Med 14, 843–848, https://do org/10.1038/nm1788 (2008). g 162. Flier, J. S. Neuroscience. Regulating energy balance: the substrate strikes back. Science 312, 861–864, doi:312/5775/861 (2006 . & Kennedy, B. K. TOR on the brain. Exp Gerontol 46, 155–163, htt 163. Garelick, M. G. & Kennedy, B. K. TOR on the brain. Exp Geron y p p g j g ( 164. Cammalleri, M. et al. Time-restricted role for dendritic activation of the mTOR-p70S6K pathway in the induction of late-p 164. Cammalleri, M. et al. Time-restricted role for dendritic activation of the mTOR-p70S6K pathway in the induction of late-phase long-term potentiation in the CA1. Proc Natl Acad Sci USA 100, 14368–14373, https://doi.org/10.1073/pnas.2336098100 (2003). , R p K p y p long-term potentiation in the CA1. Proc Natl Acad Sci USA 100, 14368–14373, https://doi.org/10.1073/pnas.2336098100 (2003). 65. Mameli, M., Balland, B., Lujan, R. & Luscher, C. Rapid synthesis and synaptic insertion of GluR2 for mGluR-LTD in the ventra tegmental area. Science 317, 530–533, doi:317/5837/530 (2007). g 66. Sacktor, T. C. et al. Persistent activation of the zeta isoform of protein kinase C in the maintenance of long-term potentiation. Proc Natl Acad Sci USA 90, 8342–8346 (1993). 67. Acknowledgements g We would like to acknowledge Dr. Umer Rashid, Department of Chemistry, COMSATS, Abbottabad, for providing working resources and NATIONAL UNVERSITY OF SCIENCES & TECHNOLOGY, ISLAMABAD, PAKISTAN for providing lab support. This research was funded by HIGER EDUCATION COMMISION (HEC), PAKISTAN under the 5000 Indigenous scholarship scheme. www.nature.com/scientificreports/ 141. Kim, H. K. et al. Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells. FEBS Lett 453, 174–178, doi:S0014-5793(99)00710-3 (1999). p 42. Matsuno, A. et al. Modulation of protein kinases and microtubule-associated proteins and changes in ultrastructure in female ra pituitary cells: effects of estrogen and bromocriptine. J Histochem Cytochem 45, 805–813, https://doi org/10.1177/002215549704500605 (1997).fi g 143. McDaid, H. M. et al. Enhancement of the therapeutic efficacy of taxol by the mitogen-activated protein kinase kinase inhibitor CI-1040 in nude mice bearing human heterotransplants. Cancer Res 65, 2854–2860, doi:65/7/2854 (2005). g p 144. Subbaramaiah, K., Hart, J. C., Norton, L. & Dannenberg, A. J. Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 AND p38 mitogen-activated protein kinase pathways. J Biol Chem 275, 14838–14845, doi:275/20/14838 (2000). 45. Zhang, P., Kornev, A. P., Wu, J. & Taylor, S. S. Discovery of Allostery in PKA Signaling. Biophys Rev 7, 227–238, https://doi org/10.1007/s12551-015-0170-x (2015). g 46. English, J. D. & Sweatt, J. D. Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271, 24329–24332 (1996). 147. English, J. D. & Sweatt, J. D. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem 272, 19103–19106 (1997). p 148. Gooney, M., Messaoudi, E., Maher, F. O., Bramham, C. R. & Lynch, M. A. BDNF-induced LTP in dentate gyrus is impaired age: analysis of changes in cell signaling events. Neurobiol Aging 25, 1323–1331, doi:S0197458004000417 (2004). 149. Sweatt, J. D. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 14, 311–317, https:/ org/10.1016/j.conb.2004.04.001 (2004).h 50. Weston, C. R. & Davis, R. J. The JNK signal transduction pathway. Curr Opin Genet Dev 12, 14–21, doi:S0959437X01002581 (2002). 151. Lefloch, R., Pouyssegur, J. & Lenormand, P. Total ERK1/2 activity regulates cell proliferation. Cell Cycle 8, 705–711, doi:7734 (2009).i 52. Alarcon, J. M. et al. Chromatin acetylation, memory, and LTP are impaired in CBP+/− mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration. Neuron 42, 947–959, https://doi.org/10.1016/j.neuron.2004.05.021 (2004). y y p g j 53. Kida, S. A Functional Role for CREB as a Positive Regulator of Memory Formation and LTP. Exp Neurobiol 21, 136–140, https:/ doi.org/10.5607/en.2012.21.4.136 (2012). 54. Silva, A. J., Kogan, J. H., Frankland, P. W. & Kida, S. CREB and memory. Author Contributionsh Statement: The following authors contributed under the headings as under; “conceptualization, A.F. and Z.R.; methodology, A.F., Z.R. and R.Z.P.; software, Z.R. and R.Z.P.; validation, A.F., Z.R., M.F.B. and R.Z.P.; formal analysis, A.F. and Z.R.; investigation, A.F., Z.R., and R.Z.P.; resources, A.F., Z.R., M.F.B., A.A., N.V., A.R. and R.Z.P.; data curation, A.F., Z.R., and R.Z.P.; writing—original draft preparation, A.F. and Z.R. writing—review and editing, A.F., Z.R., M.F.B., A.A., N.V., A.R. and R.Z.P.; visualization, M.F.B., A.A., N.V. and A.R.; supervision, M.F.B. and R.Z.P.; project administration, M.F.B., A.A., N.V., A.R. and; funding acquisition, A.F. and M.F.B.”. www.nature.com/scientificreports/ 12 Anti-cancer tyrosine kinase inhibitors increase oxidative stress in primary cardiac fibroblasts. Heart 104 A9–A9, https://doi.org/10.1136/heartjnl-2018-SCF.22 (2018). p g j 28. Bantscheff, M. et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Na Biotechnol 25, 1035–1044, doi:nbt1328 (2007).i 129. Rix, U. et al. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. Blood 110, 4055–4063, doi:blood-2007-07-102061 (2007).f g 30. Hantschel, O. Unexpected off-targets and paradoxical pathway activation by kinase inhibitors. ACS Chem Biol 10, 234–245, https:/ doi.org/10.1021/cb500886n (2015). g 31. Davis, M. I. et al. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 29, 1046–1051, https://doi.org/10.1038 nbt.1990 (2011). 132. Colbran, R. J. & Brown, A. M. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity. Curr Opin Neurobiol 14, 318–327, https://doi.org/10.1016/j.conb.2004.05.008 (2004). g j 33. Lisman, J., Yasuda, R. & Raghavachari, S. Mechanisms of CaMKII action in long-term potentiation. Nat Rev Neurosci 13, 169–182 https://doi.org/10.1038/nrn3192 (2012). p g 34. Lledo, P. M. et al. Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism. Proc Natl Acad Sci USA 92, 11175–11179 (1995). 35. Pi, H. J. et al. CaMKII control of spine size and synaptic strength: role of phosphorylation states and nonenzymatic action. Proc Nat Acad Sci USA 107, 14437–14442, https://doi.org/10.1073/pnas.1009268107 (2010). 136. Hell, J. W. CaMKII: claiming center stage in postsynaptic function and organization. Neuron 81, 249–265, https://doi.org/10.1016/j. neuron.2013.12.024 (2014). 37. Jalan-Sakrikar, N., Bartlett, R. K., Baucum, A. J. 2nd & Colbran, R. J. Substrate-selective and calcium-independent activation o CaMKII by alpha-actinin. J Biol Chem 287, 15275–15283, https://doi.org/10.1074/jbc.M112.351817 (2012). 138. Walsh, D. A., Perkins, J. P. & Krebs, E. G. An adenosine 3′,5′-monophosphate-dependant protein kinase from rabbit skeletal muscle. J Biol Chem 243, 3763–3765 (1968).t 139. Taylor, S. S., Zhang, P., Steichen, J. M., Keshwani, M. M. & Kornev, A. P. PKA: lessons learned after twenty years. Biochim Biophys Acta 1834, 1271–1278, https://doi.org/10.1016/j.bbapap.2013.03.007 (2013). p g j p p ( ) 40. Malmstrom, R. D., Kornev, A. P., Taylor, S. S. & Amaro, R. E. Allostery through the computational microscope: cAMP activation of a canonical signalling domain. Nat Commun 6, 7588, https://doi.org/10.1038/ncomms8588 (2015). Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 26 www.nature.com/scientificreports/ 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 Cre- ative 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 per- mitted 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/. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-45883-9.h Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 27 www.nature.com/scientificreports/ 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. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. fi 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 Cre- ative 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 per- mitted 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/. 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 Cre- ative 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 per- mitted 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/. Additional Information 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 Cre- ative 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 per- mitted 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/. © The Author(s) 2019 Scientific Reports | (2019) 9:9630 | https://doi.org/10.1038/s41598-019-45883-9 28