The dawn of directed acyclic graphs in primary care research and education.

Dramatical increase in articles mentioning "directed acyclic graph."

A Small Compound, HYGIC, Promotes Hypocotyl Growth Through Ectopic Ethylene Response.

Plant seedlings adjust the growth of the hypocotyl in response to surrounding environmental changes. Genetic studies have revealed key players and pathways in hypocotyl growth, such as phytohormones and light signaling. However, because of genetic redundancy in the genome, it is expected that not-yet-revealed mechanisms can be elucidated through approaches different from genetic ones. Here, we identified a small compound, HYGIC (HG), that simultaneously induces hypocotyl elongation and thickening, accompanied by increased nuclear size and enlargement of cortex cells. HG-induced hypocotyl growth required the ethylene signaling pathway activated by endogenous ethylene, involving CONSTITUTIVE PHOTOMORPHOGENIC 1, ETHYLENE INSENSITIVE 2 (EIN2) and redundant transcription factors for ethylene responses, ETHYLENE INSENSITIVE 3 (EIN3) and EIN3 LIKE 1. By using EBS:GUS, a transcriptional reporter of ethylene responses based on an EIN3-binding-cis-element, we found that HG treatment ectopically activates ethylene responses at the epidermis and cortex of the hypocotyl. RNA-seq and subsequent gene ontology analysis revealed that a significant number of HG-induced genes are related to responses to hypoxia. Indeed, submergence, a representative environment where the hypoxia response is induced in nature, promoted ethylene-signaling-dependent hypocotyl elongation and thickening accompanied by ethylene responses at the epidermis and cortex, which resembled the HG treatment. Collectively, the identification and analysis of HG revealed that ectopic responsiveness to ethylene promotes hypocotyl growth, and this mechanism is activated under submergence.

Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

Male intimate partner violence (IPV) victims in Japan: Associations of types of harm, sociodemographic characteristics, and depression trait.

Aim: The purpose of this study was to examine factors affecting depression trait among male intimate partner violence (IPV) victims in Japan utilizing a multiple linear regression analysis. Methods: A web-based questionnaire survey was conducted. Male IPV victims living in Japan were recruited to answer the questionnaire on the website on February 25-26, 2021. A total of 16,414 subjects were enrolled, of whom 1466 respondents were included in the study. Other than IPV exposure, information about sociodemographic characteristics, past traumatic experiences and psychiatric history was collected. The Domestic Violence Screening Inventory (DVSI), a 20-item questionnaire regarding IPV exposure, and the Patient Health Questionnaire-9 (PHQ-9) were used to determine the intensity and the type of IPV harm and to screen for depression, respectively. Results: The victims were more frequently subject to psychological abuse than to physical violence. Based on PHQ-9 scores, 10.7% of respondents exhibited moderate to severe depression. In the DVSI score, 79.2% of respondents required "observation and support." The lowest level of academic attainment (junior high school), positive psychiatric history, foregoing divorce to avoid adverse childhood experiences of their offspring, childhood exposure to domestic violence, younger age, having no children, and experience of school bullying were shown to be significantly associated with depression trait. Conclusion: Male IPV harm has a multilayered complexity. The sociodemographic characteristics and experiences of victims' own have a greater impact on depression trait than direct violent harm, suggesting that the violence-focused support might be inadequate for male victims. Comprehensive supports are urgently needed.

Importance of the Q/N-rich segment for protein stability of endogenous mouse TDP-43.

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.

Universal Virucidal Activity of Calcium Bicarbonate Mesoscopic Crystals That Provides an Effective and Biosafe Disinfectant.

We investigated the virucidal effects in solution of a new type of disinfectant, calcium bicarbonate mesoscopic crystals, designated CAC-717, against various types of virus. CAC-717 in solution is alkaline (pH 12.4) and has a self-electromotive force that generates pulsed electrical fields. Upon application to human skin, the pH of the solution becomes 8.4. CAC-717 contains no harmful chemicals and is thus non-irritating and harmless to humans and animals. Its virucidal effects were tested against six types of animal virus: enveloped double-strand (ds)-DNA viruses, non-enveloped ds-DNA viruses, non-enveloped single strand (ss)-DNA viruses, enveloped ss-RNA viruses, non-enveloped ss-RNA viruses, and non-enveloped ds-RNA viruses. The treatment resulted in a reduction in viral titer of at least 3.00 log10 to 6.38 log10. Fetal bovine serum was added as a representative organic substance. When its concentration was ≥20%, the virucidal effect of CAC-717 was reduced. Real-time PCR revealed that CAC-717 did not reduce the quantity of genomic DNA of most of the DNA viruses, but it greatly reduced that of the genomic RNA of most of the RNA viruses. CAC-717 may therefore be a useful biosafe disinfectant for use against a broad range of viruses.

The impact of the number of electroconvulsive therapy sessions on relapse in major depressive disorder.

OBJECTIVE: Electroconvulsive therapy (ECT) is an effective treatment of major depressive disorder (MDD). However, high relapse rates after ECT represent clinical problems. To date, influence of number of ECT sessions on relapse rate remains to be elucidated. We evaluated associations between number of ECT sessions and relapse rate. METHODS: This retrospective review collected clinical data of 53 patients with MDD who received ECT. They underwent a 1-year follow-up after their last ECT session. We performed survival analysis to evaluate associations between number of ECT sessions and time until rehospitalisation or suicide. RESULTS: The patients were divided into a higher number of ECT group (≧8 sessions) and lower number of ECT group (<8 sessions). No significant difference was found regarding the patients' clinical and demographic data. Survival analysis using log-rank test revealed that the cumulative survival rate in the higher number of ECT group (79%) was higher compared with the lower number of ECT group (49%) (p = 0.042). CONCLUSION: Patients who underwent a higher number of ECT had improved survival rate compared with those who received a lower number. Therefore, additional sessions might be necessary, even in patients who achieved remission within seven ECT sessions, to prevent relapse.Key pointsHigh rate of relapse after ECT is a key problem.Impact of the Number of ECT sessions on relapse remains to be elucidated.In the present study, the patients with MDD who underwent eight or more sessions of ECT showed significant lower relapse rate compared with those who received less than eight sessions.

PACT/PRKRA and p53 regulate transcriptional activity of DMRT1.

The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

An Inpatient Observational Study: Characteristics And Outcomes Of Avoidant/Restrictive Food Intake Disorder (ARFID) In Children And Adolescents In Japan.

PURPOSE: To determine the clinical characteristics and course of severe avoidant/restrictive food intake disorder (ARFID) in hospitalized children and adolescents and compare them with those of patients with restricting-type anorexia nervosa (R-AN). PATIENTS AND METHODS: We conducted a retrospective chart review of inpatients diagnosed with ARFID or R-AN based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, at Jichi Children's Medical Center Tochigi between April 1, 2007 and March 31, 2017. We compared the characteristics of the ARFID and R-AN patients at admission, during hospitalization, and after discharge. RESULTS: Both the ARFID (n=13) and R-AN (n=79) patients required hospitalization for their medically unstable state. The features of ARFID group included concern about the aversive consequences of eating and avoidance of eating due to sensory concerns. Significant differences were found at admission between ARFID and R-AN groups in age (10.7 vs 12.7 years), family history of mental disorders (46.2% vs 17.7%), comorbid developmental disorders (6 vs 3 cases), and the time from onset to admission (3.9 vs 6.3 months). The body weight status, % ideal body weight (%IBW), % expected body weight (%EBW), <75% IBW rate, and <75% EBW rate did not differ significantly between the two groups at admission or discharge. The duration of post-discharge outpatient follow-up treatment did not differ significantly between ARFID and R-AN groups (15.3 vs 18.4 months); however, ARFID group recovery rate was significantly higher than that of R-AN group (77% vs 43%). The reasons that the patients with ARFID had significantly better outcomes than the R-AN patients remain unclear. Compared to those in previous studies, the present patients were younger and demonstrated better outcomes. Our results indicate that the body weight status is similar between ARFID and R-AN patients, but the ARFID patients achieved better outcomes. CONCLUSION: These findings suggest that early onset in childhood, early disease recognition, and early intervention are important factors for achieving better outcomes for patients with ARFID.

CD4+CD25+LAG3+ T Cells With a Feature of Th17 Cells Associated With Systemic Lupus Erythematosus Disease Activity.

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple immune cell subsets. We analyzed immune cell subsets in human peripheral blood mononuclear cells (PBMC) in order to identify the cells that are significantly associated with SLE disease activity and treatment. The frequencies of various subsets of CD4+ T cells, B cells, monocytes and NK cells in PBMC were assessed in 30 healthy controls (HC), 30 rheumatoid arthritis (RA) patients and 26 SLE patients using flow cytometry. The correlations between subset frequencies in SLE and clinical traits including Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) were examined. Changes in subset frequencies after the treatment in SLE patients were investigated. We focused on CD25+LAG3+ T cells and investigated their characteristics, including cytokine secretion, mRNA expression and suppression capacity. We assessed correlations between CD25+LAG3+ T cells and SLEDAI by Spearman's rank correlation coefficient. CD25+LAG3+ T cells were significantly increased in SLE whereas there were few in RA and HC groups. CD25+LAG3+ T cell frequencies were significantly correlated with SLEDAI and were increased in patients with a high SLEDAI score (> 10). CD25+LAG3+ T cells produced both IL-17 and FOXP3, expressed mRNA of both FOXP3 and RORC and lacked suppressive capacity. CD25+LAG3+ T cells were associated with disease activity of SLE. CD25+LAG3+ T cells had features of both CD25+FOXP3+ regulatory T cells (CD25+ Treg) and Th17. CD25+LAG3+ T cells could be associated with the inflammatory pathophysiology of SLE.

Urinary tract bleeding from a urethral caruncle mimicking genital tract bleeding.

A 69-year-old Japanese woman with a post-hysterectomy status came to our primary care clinic. She presented with vaginal bleeding for the past 3 days which had developed after defecation. There was a palpable mass measuring approximately 2 cm on pelvic exam; however, heavy bleeding prevented in-depth observation. CT and MRI scans revealed that the mass was inside the urethral meatus and not in the vagina. She underwent surgical resection of the urethral tumour, and the pathological report showed no malignancy. A final diagnosis of urethral caruncle was made. Vaginal bleeding is commonly encountered in the primary care practice and is usually attributed to gynaecological diseases. However, patients and physicians may falsely regard urinary or gastrointestinal tract bleeding as one involving the genital tract. We present a case wherein vaginal bleeding was initially considered but was later identified to be due to a urethral caruncle.

A gene module associated with dysregulated TCR signaling pathways in CD4+ T cell subsets in rheumatoid arthritis.

We analyzed the transcriptome of detailed CD4+ T cell subsets including them after abatacept treatment, and examined the difference among CD4+ T cell subsets and identified gene sets that are closely associated disease activity and abatacept treatment. Seven CD4+ T cell subsets (naive, Th1, Th17, Th1/17, nonTh1/17, Tfh and Treg) were sorted from PBMCs taken from 10 RA patients and 10 healthy controls, and three RA patients donated samples before and 6 months after abatacept treatment. Paired-end RNA sequencing was performed using HiSeq 2500. A total of 149 samples except for 12 outliers were analyzed. Overview of expression pattern of RA revealed that administration of abatacept exerts a large shift toward the expression pattern of HC. Most of differentially expressed gene (DEG) upregulated in RA (n = 1776) were downregulated with abatacept treatment (n = 1349). Inversely, most of DEG downregulated in RA (n = 1860) were upregulated with abatacept treatment (n = 1294). This DEG-based analysis revealed shared pathway changes in RA CD4+ T cell subsets. Knowledge-based pathway analysis revealed the upregulation of activation-related pathways in RA that was substantially ameliorated by abatacept. Weighted gene co-expression network analysis (WGCNA) evaluated CD4+ T cells collectively and identified a gene module that consisted of 227 genes and was correlated with DAS28-CRP (Spearman's rho = 0.46, p = 4 × 10-9) and abatacept administration (Spearman's rho = -0.91, p = 5 × 10-57). The most highly connected 30 genes of this module included ZAP70 and JAK3, and pathway analysis of this module revealed dysregulation of the TCR signaling pathway network, which was ameliorated by abatacept.

Interleukin-10-producing LAG3+ regulatory T cells are associated with disease activity and abatacept treatment in rheumatoid arthritis.

BACKGROUND: Regulatory T cells (Tregs) play a role in the suppression of inflammation in autoimmune diseases, and lymphocyte activation gene 3 (LAG3) was reported as a marker of interleukin (IL)-10-producing Tregs. We aimed to clarify the function of human IL-10-producing CD4+CD25-LAG3+ T cells (LAG3+ Tregs) and their association with rheumatoid arthritis (RA). METHODS: LAG3+ Tregs of human peripheral blood mononuclear cells (PBMCs) were cultured with B cells and follicular helper T cells to examine antibody suppression effects. The frequency of LAG3+ Tregs was evaluated in peripheral blood samples from 101 healthy donors and 85 patients with RA. In patients treated with abatacept, PBMC samples were analyzed before and after treatment. Naive CD4+ T cells were sorted and cultured in the presence of abatacept, followed by flow cytometric analysis and function assays. RESULTS: LAG3+ Tregs produced high amounts of IL-10 and interferon-γ, and they suppressed B-cell antibody production more strongly than CD25+ Tregs. Cell-to-cell contact was required for the suppressive function of LAG3+ Tregs. The frequency of LAG3+ Tregs was lower in patients with RA, especially those with higher Clinical Disease Activity Index scores. LAG3+ Tregs significantly increased after 6 months of abatacept treatment, whereas CD25+ Tregs generally decreased. Abatacept treatment in vitro conferred LAG3 and EGR2 expression on naive CD4+ T cells, and abatacept-treated CD4+ T cells exhibited suppressive activity. CONCLUSIONS: IL-10-producing LAG3+ Tregs are associated with the immunopathology and therapeutic response in RA. LAG3+ Tregs may participate in a mechanism for the anti-inflammatory and immune-modulating effects of targeted therapy for costimulation.

Polygenic burdens on cell-specific pathways underlie the risk of rheumatoid arthritis.

Recent evidence suggests that a substantial portion of complex disease risk alleles modify gene expression in a cell-specific manner. To identify candidate causal genes and biological pathways of immune-related complex diseases, we conducted expression quantitative trait loci (eQTL) analysis on five subsets of immune cells (CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells and monocytes) and unfractionated peripheral blood from 105 healthy Japanese volunteers. We developed a three-step analytical pipeline comprising (i) prediction of individual gene expression using our eQTL database and public epigenomic data, (ii) gene-level association analysis and (iii) prediction of cell-specific pathway activity by integrating the direction of eQTL effects. By applying this pipeline to rheumatoid arthritis data sets, we identified candidate causal genes and a cytokine pathway (upregulation of tumor necrosis factor (TNF) in CD4+ T cells). Our approach is an efficient way to characterize the polygenic contributions and potential biological mechanisms of complex diseases.

Personal status of general health checkups and medical expenditure: A large-scale community-based retrospective cohort study.

BACKGROUND: We sought to clarify the association between the personal utilization of general health checkups (GHCs) and medical expenditures (MEs) in a middle-aged Japanese population. METHODS: A retrospective cohort study was conducted. Subjects were 33,417 residents (15,819 males and 17,598 females) aged 48 years or older in 2010 who were invited to undergo GHCs every year. Official records on GHCs from 2002 to 2007 and MEs from 2008 to 2010 were provided by Soka City, Saitama Prefecture, Japan. The utilization of GHCs was divided into zero times (non-utilizers), 1-3 times (low-frequency utilizers), and 4-6 times (high-frequency utilizers). Tweedie distributions in the generalized linear model were used to analyze the association between MEs and the subgroups of GHC utilization after adjustment for age and sex. RESULTS: Of the 33,417 subjects, 20,578 (61.6%) were non-utilizers, 5,777 (17.3%) were low-frequency utilizers, and 7,062 (21.1%) were high-frequency utilizers, based on the attendance to GHCs from 2002 to 2007. Compared with the non-utilizers, the high-frequency utilizers showed significantly higher outpatient MEs (JPY394,700 vs. JPY373,100). The low- and high-frequency utilizers showed significantly lower inpatient MEs (JPY224,000 and JPY181,500 vs. JPY309,300) and total MEs (JPY610,600 and JPY580,700 vs. JPY689,600) than the non-utilizers based on the pooled data from 2008 to 2010. CONCLUSIONS: This study suggests that the outpatient MEs rise when annual GHCs are increasingly attended (not including the GHC cost), but inpatient and total MEs are lower. To reduce MEs, increasing the rates of attendance at GHCs by the general public may be important.

Enhanced gut homing receptor expression of unswitched memory B cells in rheumatoid arthritis.

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Identification of tonsillar CD4+CD25-LAG3+ T cells as naturally occurring IL-10-producing regulatory T cells in human lymphoid tissue.

IL-10-producing regulatory T cells (IL-10-producing Tregs) are one of the regulatory T cell subsets characterized by the production of high amounts of IL-10, the lack of FOXP3 expression and the strong immunosuppressive capabilities. IL-10-producing Tregs have been primarily reported as induced populations thus far, in part because identifying naturally occurring IL-10-producing Tregs was difficult due to the lack of definitive surface markers. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue that we have identified as being expressed on IL-10 producing Tregs. In human PBMC, LAG3 combined with CD49b efficiently identifies IL-10-producing Tregs. However, naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue have not been described. In this report, we identified CD4+CD25-LAG3+ T cells in human tonsil. This T cell subset produced high amounts of IL-10 and expressed low levels of FOXP3. Surface markers and microarray analysis revealed that this is a distinct tonsillar CD4+ T cell subset. CD4+CD25-LAG3+ T cells expressed interleukin 10 (IL10), PR/SET domain 1 (PRDM1), and CD274 at high levels and chemokine receptor 5 (CXCR5) at low levels. CD4+CD25-LAG3+ T cells suppressed antibody production more efficiently than CD4+CD25+ T cells, and CD4+CD25-LAG3+ T cells induced B cell apoptosis. Moreover, analysis of humanized mice revealed that this cell subset suppressed a graft-versus-host disease (GVHD) reaction in vivo. Our study reveals the existence of naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue and their function in immune regulation.

Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4(+) T cells, and disease activity.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4(+)CD4(+) T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4(+)CD4(+) T cells. Moreover, the frequency of memory CXCR4(+)CD4(+) T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4(+)CD4(+) T cells. Clinically, a higher frequency of memory CXCR4(+)CD4(+) T cells predicted a better response to CTLA4-Ig. Memory CXCR4(+)CD4(+) T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

Sickness absence and return to work among Japanese stroke survivors: a 365-day cohort study.

OBJECTIVES: The objective of this study was to investigate the cumulative return to work (RTW) rate and to clarify the predictors of the time to full-time RTW (full RTW) and resignation among Japanese stroke survivors, within the 365-day period following their initial day of sickness absence due to stroke. SETTING: This study was based on tertiary prevention of occupational health in large-scaled Japanese companies of various industries. PARTICIPANTS: The participants in this study were 382 Japanese workers who experienced an episode of sickness leave due to clinically certified stroke diagnosed between 1 January 2000 and 31 December 2011. Data were obtained from an occupational health register. Participants were followed up for 365 days after the start day of the first sickness absence. The cumulative RTW rates by Kaplan-Meier estimates and predictors for time to full RTW and resignation by Cox regression were calculated. RESULTS: A total of 382 employees had their first sickness absence due to stroke during the 12-year follow-up period. The cumulative full RTW rates at 60, 120, 180 and 365 days were 15.1%, 33.6%, 43.5% and 62.4%, respectively. Employees who took sick leave due to cerebral haemorrhage had a longer time to full RTW (HR, 0.50; 95% CI 0.36 to 0.69) than those with cerebral infarction. Older employees (over 50 years of age) demonstrated a shorter time to resignation than younger employees (HR, 3.30; 95% CI 1.17 to 9.33). Manual workers had a longer time to resignation than non-manual workers (HR, 0.24; 95% CI 0.07 to 0.78). CONCLUSIONS: Cumulative RTW rates depended on the subtype of stroke, and older age was a predictor of resignation.