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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 (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 (CCA1) we included the subscales of ADI and
ADOS as clinical measures and in the second (CCA2)
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 CCA2 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 CCA1 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 CCA1 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
CCA1 (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 CCA1 analyses (ADI&ADOS), and the bottom
row for the CCA2 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 CCA1 analyses (ADI&ADOS), and the bottom
row for the CCA2 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 CCA1 analyses (ADI&ADOS), and the bottom
row for the CCA2 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 CCA2 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 CCA2 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 CCA1, 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 CCA2, 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 CCA1, and the cerebellum,
frontal lobe, and again precentral gyrus, and parietal
and occipital lobes in CCA2. 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/s13229-020-00389-4. 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 NOWLEDGEMENTS 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/10.1080/0300443001650104 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/10.1080/0300443001650104 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/psycharchives.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://timssandpirls.bc.edu/timss2019/methods/
chapter-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://timssandpirls.bc.edu/timss2019/methods/
chapter-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/bul0000330 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/10.1080/10409289.2017.1331662 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/10.1080/09243453.2013.806334 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/dev0001281 Henrich, J., Heine, S. J., & Norenzayan, A. (2010). The weirdest people in the world? Behavioral and Brain Science
https://doi.org/10.1017/S0140525X0999152X e, S. J., & Norenzayan, A. (2010). The weirdest people in the world? Behavioral and Brain Sciences, 33(2–3), 61–83. org/10.1017/S0140525X0999152X 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://timss2019.org/international-database/. 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
89X14553660 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
89X14553660 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
89X14553660 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/10.1080/0256854010
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/ipp0000102 g
g
y
p
nal Perspectives in Psychology: Research, Practice, Consultation, 8(1), 1–13. https://doi.org/10.1037/ipp0000102 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://timssandpirls.bc.edu/timss2019/methods/chapter-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/10.1080/09669761003693926 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/s10566-019-09505-9 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/10.1080/10409289.2015.968243 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://timssandpirls.bc.edu/timss2019/methods/chapter-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://timssandpirls.bc.edu/timss2019/frameworks/ 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/10.1080/0256854010
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://timssandpirls.bc.edu/timss2019/methods/chapter-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/jamanetworkopen.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/ciac161 (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/eid2607.200885 (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.scitotenv.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/ciaa905 (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.scitotenv.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.scitotenv.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.scitotenv.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.scitotenv.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/10.1063/5.0076495 (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/10.1063/5.0076495 (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/10.1183/13993003.02339-2018 (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.2104547118 (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.scitotenv.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/atmos12030309 (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.jviromet.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.scitotenv.2021.146967 (2021). p
g
j
0. “Real-time RT-PCR Primers and Probes for COVID-19 | CDC.” https://www.cdc.gov/coronavirus/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.2020.25.3.2000045 (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.scitotenv.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.buildenv.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.scitotenv.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.scitotenv.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.scitotenv.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/thoraxjnl-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/jamanetworkopen.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/10.1101/2020.12.30.20248890 (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.nanolett.0c04465 (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/nejmoa2001017 (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.2112703118 (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.jaerosci.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.buildenv.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.buildenv.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.buildenv.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/10.1080/15459620590918466 (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.0c05229 (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.0c05229 (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/acsnano.0c03252 (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/
10.1101/2022.02.10.22270797 (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/
10.1101/2022.02.10.22270797 (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.jaerosci.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://creativecommons.org/licenses/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://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
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 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]: AR m2
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 α1 is approximately equal to α2; 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 ρs (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 ρs 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/m2) (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/m2)
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/m2)
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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 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 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 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 0.844,
0.805; PCC 0.919, 0.897; SRC 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 0.919, p-value
<0.0001) and the validation dataset (Pearson’s r 0.897,
p-value <0.0001). Te equation of the model trained on the
entire training dataset was y 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 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 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 0.844)
and
the
“any-item”
subset
(EVS 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 0.844, 0.847; PCC 0.919, 0.920;
SRC 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 0.898,
p-value <0.0001) and SRC (Spearman’s r 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 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 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/general-surgery-curriculum-aug-
2021-approved-oct-20v3.pdf 1. ISCP (2021) General surgery curriculum [Available from: https://
www.iscp.ac.uk/media/1103/general-surgery-curriculum-aug-
2021-approved-oct-20v3.pdf 2. NCEPOD (2007) Trauma: who cares? [Available from: https://
www.ncepod.org.uk/2007report2/Downloads/SIP_summary.pdf 2. NCEPOD (2007) Trauma: who cares? [Available from: https://
www.ncepod.org.uk/2007report2/Downloads/SIP_summary.pdf 3. TraumaSteeringGroup (2018) A trauma system for Ireland [Avail-
able from: https://www.hse.ie/eng/about/who/acute-hospitals-
division/trauma-services/further-information-and-documentation/
report-of-the-trauma-steering-group.pdf 3. TraumaSteeringGroup (2018) A trauma system for Ireland [Avail-
able from: https://www.hse.ie/eng/about/who/acute-hospitals-
division/trauma-services/further-information-and-documentation/
report-of-the-trauma-steering-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://creativecommons.org/licenses/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
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.