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BackgroundPrimary healthcare providers play a critical role in diagnosing and managing digestive disorders. Standardized clinical care guidelines have been developed, but with limited and inconsistent implementation. An evidence-based gastroenterology clinical care pathway (GUTLINK) has been proposed in one region of Canada; however, little is known in the medical literature about potential barriers to pathway implementation within primary care. We aimed to identify behavioral and environmental barriers and facilitators to implementation of evidence-based care pathways for undifferentiated lower gastrointestinal tract symptoms in primary care.MethodsOne-on-one semi-structured interviews were conducted with primary healthcare providers between September 2021 and May 2022. Interview script development was guided by the COM-B framework. Interviews were transcribed and data were analyzed using an inductive thematic analysis approach.ResultsA total of 15 primary healthcare provider interviews were conducted. Several key barriers to GUTLINK implementation were identified in all three domains of the COM-B framework. Key barriers included Capability (e.g., Physician Knowledge and Access to Allied Health), Opportunity (e.g., Access to diagnostic tools), and Motivation (e.g., Comfort with managing cases and optimism). Some of these barriers have not previously been identified in medical literature.ConclusionsEvidence-based clinical care pathways have the potential to support access to quality gastroenterology care, yet primary healthcare providers in this study identified several barriers to implementation. Potential solutions exist at the individual and clinic levels (e.g., greater education, improved provider-specialist communication), but must be supported with systems-level changes (e.g., increased funding for gastrointestinal care and e-Health platforms) to support pathway implementation and improve quality of care.

IntroductionIntegrated care is effective in reducing all-cause mortality in patients with atrial fibrillation (AF) in primary care, though time and resource intensive. The aim of the current study was to assess whether integrated care should be directed at all AF patients equally.MethodsThe ALL-IN trial (n = 1,240 patients, median age 77 years) was a cluster-randomized trial in which primary care practices were randomized to provide integrated care or usual care to AF patients aged 65 years and older. Integrated care comprised of (i) anticoagulation monitoring, (ii) quarterly checkups and (iii) easy-access consultation with cardiologists. For the current analysis, cox proportional hazard analysis with all clinical variables from the CHA2DS2-VASc score was used to predict all-cause mortality in the ALL-IN trial. Subsequently, the hazard ratio and absolute risk reduction were plotted as a function of this predicted mortality risk to explore treatment heterogeneity.ResultsUnder usual care, after a median of 2 years follow-up the absolute risk of all-cause mortality in the highest-risk quarter was 31.0%, compared to 4.6% in the lowest-risk quarter. On the relative scale, there was no evidence of treatment heterogeneity (p for interaction = 0.90). However, there was substantial treatment heterogeneity on the absolute scale: risk reduction in the lowest risk- quarter of risk 3.3% (95% CI -0.4% - 7.0) compared to 12.0% (95% CI 2.7% - 22.0) in the highest risk quarter.ConclusionWhile the relative degree of benefit from integrated AF care is similar in all patients, patients with a high all-cause mortality risk have a greater benefit on an absolute scale and should therefore be prioritized when implementing integrated care.

Objectives Cardiovascular conditions were shown to be predictive of clinical deterioration in hospitalised patients with coronavirus disease 2019 (COVID-19). Whether this also holds for outpatients managed in primary care is yet unknown. The aim of this study was to determine the incremental value of cardiovascular vulnerability in predicting the risk of hospital referral in primary care COVID-19 outpatients. Design Analysis of anonymised routine care data extracted from electronic medical records from three large Dutch primary care registries. Setting Primary care. Participants Consecutive adult patients seen in primary care for COVID-19 symptoms in the ‘first wave’ of COVID-19 infections (March 1 2020 to June 1 2020) and in the ‘second wave’ (June 1 2020 to April 15 2021) in the Netherlands. Outcome measures A multivariable logistic regression model was fitted to predict hospital referral within 90 days after first COVID-19 consultation in primary care. Data from the ‘first wave’ was used for derivation (n = 5,475 patients). Age, sex, the interaction between age and sex, and the number of cardiovascular conditions and/or diabetes (0, 1, or ≥2) were pre-specified as candidate predictors. This full model was (i) compared to a simple model including only age and sex and its interaction, and (ii) externally validated in COVID-19 patients during the ‘second wave’ (n = 16,693). Results The full model performed better than the simple model (likelihood ratio test pConclusions For patients with COVID-19 symptoms managed in primary care, the risk of hospital referral was on average 5.1%. Older, male and cardiovascular vulnerable COVID-19 patients are more at risk for hospital referral.

Background The quality of evidence about the effectiveness of non-pharmaceutical health interventions is often low, but little is known about the effects of communicating indications of evidence quality to the public. Methods In two blinded, randomised, controlled, online experiments, US participants (total n = 2140) were shown one of several versions of an infographic illustrating the effectiveness of eye protection in reducing COVID-19 transmission. Their trust in the information, understanding, feelings of effectiveness of eye protection, and the likelihood of them adopting it were measured. Findings Compared to those given no quality cues, participants who were told the quality of the evidence on eye protection was ‘low’, rated the evidence less trustworthy (p = .001, d = 0.25), and rated it as subjectively less effective (p = .018, d = 0.19). The same effects emerged compared to those who were told the quality of the evidence was ‘high’, and in one of the two studies, those shown ‘low’ quality of evidence said they were less likely to use eye protection (p = .005, d = 0.18). Participants who were told the quality of the evidence was ‘high’ showed no statistically significant differences on these measures compared to those given no information about evidence quality. Conclusions Without quality of evidence cues, participants responded to the evidence about the public health intervention as if it was high quality and this affected their subjective perceptions of its efficacy and trust in the provided information. This raises the ethical dilemma of weighing the importance of transparently stating when the evidence base is actually low quality against evidence that providing such information can decrease trust, perception of intervention efficacy, and likelihood of adopting it.

INTRODUCTION:Exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations. OBJECTIVES:To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. STUDY DESIGN:88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006-2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations. RESULTS:By mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p

OBJECTIVE:After one previous caesarean section (CS), pregnant women can deliver by elective repeat CS or have a trial of labor which can end in a vaginal birth after caesarean (VBAC) or an unplanned CS. Despite guidelines describing women's rights to make an informed choice, trial of labor and VBAC rates vary greatly worldwide. Many women are inadequately informed due to caregivers' fear of an increase in CS rates in a high VBAC rate setting. We compared counseling with a decision aid (DA) including a prediction model on VBAC to care as usual. We hypothesize that counselling with the DA does not decrease VBAC rates. In addition, we aimed to study the effects on unplanned CS rate, patient involvement in decision-making and elective repeat CS rates. METHODS:We performed a prospective cohort study. From 2012 to 2014, 483 women in six hospitals, where the DA was used (intervention group), were compared with 441 women in six matched hospitals (control group). Women with one previous CS, pregnant of a singleton in cephalic presentation, delivering after 37 weeks 0 days were eligible for inclusion. RESULTS:There was no significant difference in VBAC rates between the intervention (45%) and control group (46%) (adjusted odds ratio 0,92 (95% Confidence interval 0.69-1.23)). In the intervention group more women (42%) chose an elective repeat CS compared to the control group (31%) (adjusted odds ratio 1.6 (95% Confidence interval 1.18-2.17)). Of women choosing trial of labor, in the intervention group 77% delivered vaginally compared to 67% in the control group, resulting in an unplanned CS adjusted odds ratio of 0,57 (0.40-0.82) in the intervention group. In the intervention group, more women reported to be involved in decision-making (98% vs. 68%, P< 0.001). CONCLUSIONS:Implementing a decision aid with a prediction model for risk selection suggests unchanged VBAC rates, but 40% reduction in unplanned CS rates, increase in elective repeat CS and improved patient involvement in decision-making.

Holocene climate variability in the Mediterranean Basin is often cited as a potential driver of societal change, but the mechanisms of this putative influence are generally little explored. In this paper we integrate two tools-agro-ecosystem modeling of potential agricultural yields and spatial analysis of archaeological settlement pattern data-in order to examine the human consequences of past climatic changes. Focusing on a case study in Provence (France), we adapt an agro-ecosystem model to the modeling of potential agricultural productivity during the Holocene. Calibrating this model for past crops and agricultural practices and using a downscaling approach to produce high spatiotemporal resolution paleoclimate data from a Mediterranean Holocene climate reconstruction, we estimate realistic potential agricultural yields under past climatic conditions. These serve as the basis for spatial analysis of archaeological settlement patterns, in which we examine the changing relationship over time between agricultural productivity and settlement location. Using potential agricultural productivity (PAgP) as a measure of the human consequences of climate changes, we focus on the relative magnitudes of 1) climate-driven shifts in PAgP and 2) the potential increases in productivity realizable through agricultural intensification. Together these offer a means of assessing the scale and mechanisms of the vulnerability and resilience of Holocene inhabitants of Provence to climate change. Our results suggest that settlement patterns were closely tied to PAgP throughout most of the Holocene, with the notable exception of the period from the Middle Bronze Age through the Early Iron Age. This pattern does not appear to be linked to any climatically-driven changes in PAgP, and conversely the most salient changes in PAgP during the Holocene cannot be clearly linked to any changes in settlement pattern. We argue that this constitutes evidence that vulnerability and resilience to climate change are strongly dependent on societal variables.

This paper presents a reassessment of the archaeological record at Leang Burung 2, a key early human occupation site in the Late Pleistocene of Southeast Asia. Excavated originally by Ian Glover in 1975, this limestone rock-shelter in the Maros karsts of Sulawesi, Indonesia, has long held significance in our understanding of early human dispersals into 'Wallacea', the vast zone of oceanic islands between continental Asia and Australia. We present new stratigraphic information and dating evidence from Leang Burung 2 collected during the course of our excavations at this site in 2007 and 2011-13. Our findings suggest that the classic Late Pleistocene modern human occupation sequence identified previously at Leang Burung 2, and proposed to span around 31,000 to 19,000 conventional 14C years BP (~35-24 ka cal BP), may actually represent an amalgam of reworked archaeological materials. Sources for cultural materials of mixed ages comprise breccias from the rear wall of the rock-shelter-remnants of older, eroded deposits dated to 35-23 ka cal BP-and cultural remains of early Holocene antiquity. Below the upper levels affected by the mass loss of Late Pleistocene deposits, our deep-trench excavations uncovered evidence for an earlier hominin presence at the site. These findings include fossils of now-extinct proboscideans and other 'megafauna' in stratified context, as well as a cobble-based stone artifact technology comparable to that produced by late Middle Pleistocene hominins elsewhere on Sulawesi.

[This corrects the article DOI: 10.1371/journal.pone.0193025.].

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit.

Routine physical examinations might be of value in HIV-infected patients, but the yield is unknown. We determined the diagnoses that would have been missed without performing annual routine physical examinations in HIV-infected patients with stable disease.Data were collected from the medical records of 299 HIV-1-infected patients with CD4 count >350 cells/mm3 if not using combination antiretroviral therapy (cART), or CD4 count >100 cells/mm3 and undetectable viral load if using cART. We defined the diagnoses that would have been missed without performing routine physical examinations on annual check-ups in 2010. Exclusion criteria were hepatitis B/C co-infection, start/ switch of cART < 24 weeks, pregnancy, and transgenderism.215 patients (72%) had positive findings: lipodystrophy (30%), lymphadenopathy (16%) and hypertension (8.4%) were the most common. Two-thirds of all findings were not new or were based on complaints indicating a physical examination even if not routinely scheduled. For 24 patients (8.0%) the routine physical examination led to the finding of a new diagnosis: six-all men who have sex with men (MSM)-had a concurrent sexually transmitted infection, eight had hypertension, and ten others had a large variety of diagnoses. A total atrioventricular block with bradycardia was the most clinically relevant finding.Annual physical examinations of HIV-infected patients with stable disease brought few new diagnoses that would have been missed without performing a routine examination. Our results suggest that standard assessments could be restricted to six-monthly measuring blood pressure in all patients and annually performing anogenital and digital rectal examination on MSM.

BACKGROUND:There is an ongoing discussion on the rising CS rate worldwide. Suboptimal guideline adherence may be an important contributor to this rise. Before improvement of care can be established, optimal CS care in different settings has to be defined. This study aimed to develop and measure quality indicators to determine guideline adherence and identify target groups for improvement of care with direct effect on caesarean section (CS) rates. METHOD:Eighteen obstetricians and midwives participated in an expert panel for systematic CS quality indicator development according to the RAND-modified Delphi method. A multi-center study was performed and medical charts of 1024 women with a CS and a stratified and weighted randomly selected group of 1036 women with a vaginal delivery were analysed. Quality indicator frequency and adherence were scored in 2060 women with a CS or vaginal delivery. RESULTS:The expert panel developed 16 indicators on planned CS and 11 indicators on unplanned CS. Indicator adherence was calculated, defined as the number of women in a specific obstetrical situation in which care was performed as recommended in both planned and unplanned CS settings. The most frequently occurring obstetrical situations with low indicator adherence were: 1) suspected fetal distress (frequency 17%, adherence 46%), 2) non-progressive labour (frequency 12%, CS performed too early in over 75%), 3) continuous support during labour (frequency 88%, adherence 37%) and 4) previous CS (frequency 12%), with adequate counselling in 15%. CONCLUSIONS:We identified four concrete target groups for improvement of obstetrical care, which can be used as a starting point to reduce CS rates worldwide.

BackgroundA widely held concern of screening is that its psychological harms may outweigh the benefits of early detection and treatment. This study describes pregnant women's perceptions of possible harms and benefits of mental health screening and factors associated with identifying screening as harmful or beneficial.MethodsThis study analyzed a subgroup of women who had undergone formal or informal mental health screening from our larger multi-site, cross-sectional study. Pregnant women >16 years of age who spoke/read English were recruited (May-December 2013) from prenatal classes and maternity clinics in Alberta, Canada. Descriptive statistics were generated to summarize harms and benefits of screening and multivariable logistic regression identified factors associated with reporting at least one harm or affirming screening as a positive experience (January-December 2014).ResultsOverall study participation rate was 92% (N = 460/500). Among women screened for mental health concerns (n = 238), 63% viewed screening as positive, 69% were glad to be asked, and 87% took it as evidence their provider cared about them. Only one woman identified screening as a negative experience. Of the 6 harms, none was endorsed by >7% of women, with embarrassment being most cited. Women who were very comfortable (vs somewhat/not comfortable) with screening were more likely to report it as a positive experience.LimitationsWomen were largely Caucasian, well-educated, partnered women; thus, findings may not be generalizable to women with socioeconomic risk.ConclusionsMost women perceived prenatal mental health screening as having high benefit and low harm. These findings dispel popular concerns that mental health screening is psychologically harmful.

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.

West Nile virus (WNV) has caused outbreaks and sporadic infections in Central, Eastern and Mediterranean Europe for over 45 years. Most strains responsible for the European and Mediterranean basin outbreaks are classified as lineage 1. In recent years, WNV strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as Italy, Hungary and Greece, while mass mortality among birds was not reported. This study characterizes three European strains of WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in terms of in vitro replication kinetics on neuroblastoma cells, LD50 values in C57BL/6 mice, median day mortality, cumulative mortality, concentration of virus in the brain and spinal cord, and the response to infection in the brain. Overall, the results indicate that strains circulating in Europe belonging to both lineage 1 and 2 are highly virulent and that Ita09 and 578/10 are more neurovirulent compared to the FIN strain.

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15-19 weeks' gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5-e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus.

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation.Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6(-/-) mice may provide a feasible model to study the role of human HerC5 in antiviral responses.

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the 'masculinization programming window' (MPW; e15.5-e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5-e18.5 (MPW), e13.5-e20.5 or e19.5-e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R² = -0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R² = -0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis.

Family-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 3' end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detection of known and unknown paramyxoviruses, and screening of large sample collections from humans and animals.

Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal 'masculinization programming window'. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ~3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders.

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

BACKGROUND:Identifying the molecular mechanisms and neural circuits that control learning and memory are major challenges in neuroscience. Mammalian MAGI/S-SCAM is a multi-PDZ domain synaptic scaffolding protein that interacts with a number of postsynaptic signaling proteins and is thereby thought to regulate synaptic plasticity [1], [2], [3]. PRINCIPAL FINDINGS:While investigating the behavioral defects of C. elegans nematodes carrying a mutation in the single MAGI ortholog magi-1, we have identified specific neurons that require MAGI-1 function for different aspects of associative learning and memory. Various sensory stimuli and a food deprivation signal are associated in RIA interneurons during learning, while additional expression of MAGI-1 in glutamatergic AVA, AVD and possibly AVE interneurons is required for efficient memory consolidation, i.e. the ability to retain the conditioned changes in behavior over time. During associative learning, MAGI-1 in RIA neurons controls in a cell non-autonomous fashion the dynamic remodeling of AVA, AVD and AVE synapses containing the ionotropic glutamate receptor (iGluR) GLR-1 [4]. During memory consolidation, however, MAGI-1 controls GLR-1 clustering in AVA and AVD interneurons cell-autonomously and depends on the ability to interact with the beta-catenin HMP-2. SIGNIFICANCE:Together, these results indicate that different aspects of associative learning and memory in C. elegans are likely carried out by distinct subsets of interneurons. The synaptic scaffolding protein MAGI-1 plays a critical role in these processes in part by regulating the clustering of iGluRs at synapses.

In the vertebrates, the BMP/Smad1 and TGF-beta/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.

A key question in developmental biology is how growth factor signals are integrated to generate pattern. In this study we investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, we found that Mad phosphorylation by GSK3 and MAPK occurred in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information.