Development of multivariable models to predict perinatal depression before and after delivery using patient reported survey responses at weeks 4-10 of pregnancy.

BACKGROUND: Perinatal depression is estimated to affect ~ 12% of pregnancies and is linked to numerous negative outcomes. There is currently no model to predict perinatal depression at multiple time-points during and after pregnancy using variables ascertained early into pregnancy. METHODS: A prospective cohort design where 858 participants filled in a baseline self-reported survey at week 4-10 of pregnancy (that included social economics, health history, various psychiatric measures), with follow-up until 3 months after delivery. Our primary outcome was an Edinburgh Postnatal Depression Score (EPDS) score of 12 or more (a proxy for perinatal depression) assessed during each trimester and again at two time periods after delivery. Five gradient boosting machines were trained to predict the risk of having EPDS score > = 12 at each of the five follow-up periods. The predictors consisted of 21 variables from 3 validated psychometric scales. As a sensitivity analysis, we also investigated different predictor sets that contained: i) 17 of the 21 variables predictors by only including two of the psychometric scales and ii) including 143 additional social economics and health history predictors, resulting in 164 predictors. RESULTS: We developed five prognostic models: PND-T1 (trimester 1), PND-T2 (trimester 2), PND-T3 (trimester 3), PND-A1 (after delivery 1) and PND-A2 (delayed onset after delivery) that calculate personalised risks while only requiring that women be asked 21 questions from 3 validated psychometric scales at weeks 4-10 of pregnancy. C-statistics (also known as AUC) ranged between 0.69 (95% CI 0.65-0.73) and 0.77 (95% CI 0.74-0.80). At 50% sensitivity the positive predictive value ranged between 30%-50% across the models, generally identifying groups of patients with double the average risk. Models trained using the 17 predictors and 164 predictors did not improve model performance compared to the models trained using 21 predictors. CONCLUSIONS: The five models can predict risk of perinatal depression within each trimester and in two post-natal periods using survey responses as early as week 4 of pregnancy with modest performance. The models need to be externally validated and prospectively tested to ensure generalizability to any pregnant patient.

Perinatal depressive symptoms often start in the prenatal rather than postpartum period: results from a longitudinal study.

Depressive symptoms during and after pregnancy confer risks for adverse outcomes in both the mother and child. Postpartum depression is traditionally diagnosed after birth of the child. Perinatal depression is a serious, prevalent heterogeneous syndrome that can occur during the period from conception through several months after childbirth. Onset and course are not well understood. There is a paucity of longitudinal studies of the disorder that include the antenatal period in population-based samples. We used an Internet panel of pregnant women recruited in 2 cohorts; 858 ascertained in the first and 322 ascertained in the third trimesters of pregnancy. We recruited the second cohort in order to assure sufficient sample to examine depressive symptoms later in pregnancy and in the postpartum period. Assessments included standard psychometric measures, health history, and pregnancy experience. The Edinburgh Postnatal Depression Scale was used for the assessment of depressive symptoms. Nearly 10% of women entered the pregnancy with depressive symptoms. Prevalence was about the same at 4 weeks and 3 months postpartum. During pregnancy, prevalence increased to 16% in the third trimester. Among incident cases, 80% occurred during pregnancy, with 1/3 occurring in the first trimester. We describe predictors of incident depressive symptoms and covariates associated with time-to-onset which include health history (psychiatric and medical) and social support covariates. The majority of incident depressive symptoms occur during pregnancy rather than afterward. This finding underscores the mandate for mental health screening early in pregnancy and throughout gestation. It will be important to find safe and effective interventions that prevent, mitigate, or delay the onset of depressive symptoms that can be implemented during pregnancy.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice.

Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D'D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf(-/-) mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D'D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf(-/-) mice. Although the VWF D'D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D'D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D'D3 and VWF D'D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D'D3/D'D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.

Genetic variants in PLG, LPA, and SIGLEC 14 as well as smoking contribute to plasma plasminogen levels.

Plasminogen is the precursor of the serine protease plasmin, a central enzyme of the fibrinolytic system. Plasma levels of plasminogen vary by almost 2-fold among healthy individuals, yet little is known about its heritability or genetic determinants in the general population. In order to identify genetic factors affecting the natural variation of plasminogen levels, we performed a genome-wide association study and linkage analysis in a sample of 3456 young healthy individuals who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS). Heritability of plasminogen levels was 48.1% to 60.0%. Tobacco smoking and female sex were associated with higher levels of plasminogen. In the meta-analysis, 11 single-nucleotide polymorphisms (SNPs) in 2 regions reached genome-wide significance (P < 5.0E-8). Of these, 9 SNPs were near the PLG or LPA genes on Chr6q26, whereas 2 were on Chr19q13 and 5' upstream of SIGLEC14. These 11 SNPs represented 4 independent signals and collectively explained 6.8% of plasminogen level variation in the study populations. The strongest association was observed for a nonsynonymous SNP in the PLG gene (R523W). Individuals bearing an additional copy of this allele had an average decrease of 13.4% in plasma plasminogen level.

Leveraging Digital Technology in Conducting Longitudinal Research on Mental Health in Pregnancy: Longitudinal Panel Survey Study.

BACKGROUND: Collecting longitudinal data during and shortly after pregnancy is difficult, as pregnant women often avoid studies with repeated surveys. In contrast, pregnant women interact with certain websites at multiple stages throughout pregnancy and the postpartum period. This digital connection presents the opportunity to use a website as a way to recruit and enroll pregnant women into a panel study and collect valuable longitudinal data for research. These data can then be used to learn new scientific insights and improve health care. OBJECTIVE: The objective of this paper is to describe the approaches applied and lessons learned from designing and conducting an online panel for health care research, specifically perinatal mood disorders. Our panel design and approach aimed to recruit a large sample (N=1200) of pregnant women representative of the US population and to minimize attrition over time. METHODS: We designed an online panel to enroll participants from the pregnancy and parenting website BabyCenter. We enrolled women into the panel from weeks 4 to 10 of pregnancy (Panel 1) or from weeks 28 to 33 of pregnancy (Panel 2) and administered repeated psychometric assessments from enrollment through 3 months postpartum. We employed a combination of adaptive digital strategies to recruit, communicate with, and build trust with participants to minimize attrition over time. We were transparent at baseline about expectations, used monetary and information-based incentives, and sent personalized reminders to reduce attrition. The approach was participant-centric and leveraged many aspects of flexibility that digital methods afford. RESULTS: We recruited 1179 pregnant women-our target was 1200-during a 26-day period between August 25 and September 19, 2016. Our strategy to recruit participants using adaptive sampling tactics resulted in a large panel that was similar to the US population of pregnant women. Attrition was on par with existing longitudinal observational studies in pregnant populations, and 79.2% (934/1179) of our panel completed another survey after enrollment. There were 736 out of 1179 (62.4%) women who completed at least one assessment in both the prenatal and postnatal periods, and 709 out of 1179 (60.1%) women who completed the final assessment. To validate the data, we compared participation rates and factors of perinatal mood disorders ascertained from this study with prior research, suggesting reliability of our approach. CONCLUSIONS: A suitably designed online panel created in partnership with a digital media source that reaches the target audience is a means to leverage a conveniently sized and viable sample for scientific research. Our key lessons learned are as follows: sampling tactics may need to be adjusted to enroll a representative sample, attrition can be reduced by adapting to participants' needs, and study engagement can be boosted by personalizing interactions with the flexibility afforded by digital technologies.

SEC23A rescues SEC23B-deficient congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDAII) results from loss-of-function mutations in SEC23B. In contrast to humans, SEC23B-deficient mice deletion do not exhibit CDAII but die perinatally with pancreatic degeneration. Here, we demonstrate that expression of the full SEC23A protein (the SEC23B paralog) from the endogenous regulatory elements of Sec23b completely rescues the SEC23B-deficient mouse phenotype. Consistent with these data, while mice with erythroid-specific deletion of either Sec23a or Sec23b do not exhibit CDAII, we now show that mice with erythroid-specific deletion of all four Sec23 alleles die in mid-embryogenesis with features of CDAII and that mice with deletion of three Sec23 alleles exhibit a milder erythroid defect. To test whether the functional overlap between the SEC23 paralogs is conserved in human erythroid cells, we generated SEC23B-deficient HUDEP-2 cells. Upon differentiation, these cells exhibited features of CDAII, which were rescued by increased expression of SEC23A, suggesting a novel therapeutic strategy for CDAII.

Selection on cis-regulatory variation at B4galnt2 and its influence on von Willebrand factor in house mice.

The RIIIS/J inbred mouse strain is a model for type 1 von Willebrand disease (VWD), a common human bleeding disorder. Low von Willebrand factor (VWF) levels in RIIIS/J are due to a regulatory mutation, Mvwf1, which directs a tissue-specific switch in expression of a glycosyltransferase, B4GALNT2, from intestine to blood vessel. We recently found that Mvwf1 lies on a founder allele common among laboratory mouse strains. To investigate the evolutionary forces operating at B4galnt2, we conducted a survey of DNA sequence polymorphism and microsatellite variation spanning the B4galnt2 gene region in natural Mus musculus domesticus populations. Two divergent haplotypes segregate in these natural populations, one of which corresponds to the RIIIS/J sequence. Different local populations display dramatic differences in the frequency of these haplotypes, and reduced microsatellite variability near B4galnt2 within the RIIIS/J haplotype is consistent with the recent action of natural selection. The level and pattern of DNA sequence polymorphism in the 5' flanking region of the gene significantly deviates from the neutral expectation and suggests that variation in B4galnt2 expression may be under balancing selection and/or arose from a recently introgressed allele that subsequently increased in frequency due to natural selection. However, coalescent simulations indicate that the heterogeneity in divergence between haplotypes is greater than expected under an introgression model. Analysis of a population where the RIIIS/J haplotype is in high frequency reveals an association between this haplotype, the B4galnt2 tissue-specific switch, and a significant decrease in plasma VWF levels. Given these observations, we propose that low VWF levels may represent a fitness cost that is offset by a yet unknown benefit of the B4galnt2 tissue-specific switch. Similar mechanisms may account for the variability in VWF levels and high prevalence of VWD in other mammals, including humans.

The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.

Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

Using the Sediment Quality Triad to characterize baseline conditions in the Anacostia River, Washington, DC, USA.

The Sediment Quality Triad (SQT) consists of complementary measures of sediment chemistry, benthic community structure, and sediment toxicity. We applied the SQT at 20 stations in the tidal portion of the Anacostia River from Bladensburg, MD to Washington, DC to establish a baseline of conditions to evaluate the effects of management actions. Sediment toxicity was assessed using 10-day survival and growth tests with the freshwater amphipod, Hyalella azteca and the midge, Chironomus dilutus. Triplicate grabs were taken at each station for benthic community analysis and the Benthic Index of Biotic Integrity (B-IBI) was used to interpret the data. Only one station, #92, exhibited toxicity related to sediment contamination. Sediments from this station significantly inhibited growth of both test species, had the highest concentrations of contaminants, and had a degraded benthic community, indicated by a B-IBI of less than 3. Additional sediment from this station was tested and sediment toxicity identification evaluation (TIE) procedures tentatively characterized organic compounds as the cause of toxicity. Overall, forty percent of the stations were classified as degraded by the B-IBI. However, qualitative and quantitative comparisons with sediment quality benchmarks indicated no clear relationship between benthic community health and contaminant concentrations. This study provides a baseline for assessing the effectiveness of management actions in the Anacostia River.

Erratum to: Effects-based spatial assessment of contaminated estuarine sediments from Bear Creek, Baltimore Harbor, MD, USA.

The original publication of this paper contains an error. The correct image of figure 5 is shown in this paper.

Correction to: Effects-based spatial assessment of contaminated estuarine sediments from Bear Creek, Baltimore Harbor, MD, USA.

The authors of the article have informed the Journal that an author, Dr. Sacoby Wilson of the University of Maryland School of Public Health, was inadvertently omitted from the published version of their manuscript due to a miscommunication regarding authorship criteria.

Effects-based spatial assessment of contaminated estuarine sediments from Bear Creek, Baltimore Harbor, MD, USA.

Estuarine sediments in regions with prolonged histories of industrial activity are often laden to significant depths with complex contaminant mixtures, including trace metals and persistent organic pollutants. Given the complexity of assessing risks from multi-contaminant exposures, the direct measurement of impacts to biological receptors is central to characterizing contaminated sediment sites. Though biological consequences are less commonly assessed at depth, laboratory-based toxicity testing of subsurface sediments can be used to delineate the scope of contamination at impacted sites. The extent and depth of sediment toxicity in Bear Creek, near Baltimore, Maryland, USA, was delineated using 10-day acute toxicity tests with the estuarine amphipod Leptocheirus plumulosus, and chemical analysis of trace metals and persistent organic pollutants. A gradient of toxicity was demonstrated in surface sediments with 21 of 22 tested sites differing significantly from controls. Effects were most pronounced (100% lethality) at sites proximate to a historic industrial complex. Sediments from eight of nine core samples to depths of 80 cm were particularly impacted (i.e., caused significant lethality to L. plumulosus) even in locations overlain with relatively non-toxic surface sediments, supporting a conclusion that toxicity observed at the surface (top 2 cm) does not adequately predict toxicity at depth. In seven of nine sites, toxicity of surface sediments differed from toxicity at levels beneath by 28 to 69%, in five instances underestimating toxicity (28 to 69%), and in two instances overestimating toxicity (44 to 56%). Multiple contaminants exceeded sediment quality guidelines and correlated positively with toxic responses within surface sediments (e.g., chromium, nickel, polycyclic aromatic hydrocarbon (PAH), total petroleum hydrocarbon). Use of an antibody-based PAH biosensor revealed that porewater PAH concentrations also increased with depth at most sites. This study informs future management decisions concerning the extent of impact to Bear Creek sediments, and demonstrates the benefits of a spatial approach, relying primarily on toxicity testing to assess sediment quality in a system with complex contaminant mixtures.

Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the ADAMTS13 locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of ADAMTS13 (ß, 6.7%; P = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (ß, -22.3%; P = 1.2E-30) and rs3124762 (ß, 3.5%; P = 8.9E-9) close to ADAMTS13 and rs4075970 (ß, 2.4%; P = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around ADAMTS13 (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous ADAMTS13 variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.

Loving Nature From the Inside Out: A Biophilia Matrix Identification Strategy for Designers.

OBJECTIVE: The development of the Biophilic Design Matrix (BDM) was to aid designers or other specialists in identifying and quantifying biophilic features through a visual inventory of interior spaces. BACKGROUND: With mounting evidence to support the healing attributes of biophilic environments, we propose a method to identify biophilic content within interior spaces. Such a strategy offers much promise to the advancement of restorative environments. METHODS: The BDM was based on Stephen Kellert's biophilic design attribute list and modified to be appropriate for interior environments, specifically children's healthcare spaces. A photo-ethnographic documentation method of 24 child life play spaces within a South Atlantic state was used to determine whether the BDM could reliably reveal biophilic features (listed as attributes by Kellert in 2008). RESULTS: This matrix appears useful in documenting biophilia within the pediatric healthcare context, attesting to the usability and functionality of the BDM for this special population. Specifically, the BDM revealed that biophilic attributes were constantly present in some spaces while others were completely absent. When a biophilic attribute was present, the BDM indicated that they varied considerably in type and occurrence. Thus, use of the BDM in the hospital areas designed for patient recreation and play successfully provided a visual inventory of biophilic features as well as the frequency of application. CONCLUSIONS: Further use of the BDM as a tool for strategizing biophilic feature inclusion can thus increase the connections available with nature in the interior, beneficial for optimizing health and wellness.

A field test and comparison of acute and chronic sediment toxicity tests with the estuarine amphipod Leptocheirus plumulosus in Chesapeake Bay, USA.

A 28-d partial life-cycle test with the estuarine amphipod Leptocheirus plumulosus was developed in response to the need for an assay to mimic chronic exposure to sediment-associated contaminants. To ensure that toxicity tests have environmental relevance, it is essential to evaluate the relationship between laboratory responses and field measures of contamination. Consequently, one objective of the study was to compare the results of the chronic sediment toxicity test with L. plumulosus to gradients of sediment contamination and the in situ benthic community in its native Chesapeake Bay. Chronic tests were conducted by two laboratories, the Army Corps of Engineers Waterways Experiment Station ([WES]; Vicksburg, MS, USA) and the University of Maryland ([UM] College Park, MD, USA) using different feeding regimes, providing the opportunity to evaluate the effect of this variable on response sensitivity. A second objective was to compare the relative sensitivity of acute and chronic tests with L. plumulosus with field-collected sediments. Overall, there was good agreement between the toxicological response of acute and chronic tests with L. plumulosus and field measures of contamination. Survival in the acute test and chronic test conducted by WES was negatively correlated with concentrations of sediment-associated contaminants. Survival in acute exposures was significantly reduced in sediments from 8 of 11 stations. Indigenous L. plumulosus were found only at two of the three stations that did not exhibit acute toxicity. An unexpected finding was the difference in responsiveness of the two chronic tests. Survival in tests conducted by UM and WES was significantly reduced in sediments from 4 and 6 of 11 stations, respectively. No additional sublethal toxicity was detected in the UM chronic test, but the WES test detected reproductive effects at two additional stations. We believe the observed differences were related to the test diet used. Partly as a result of our findings, the recommended diet for the L. plumulosus chronic test was changed in the final methods document.

Expression of the blood-group-related glycosyltransferase B4galnt2 influences the intestinal microbiota in mice.

Glycans on mucosal surfaces have an important role in host-microbe interactions. The locus encoding the blood-group-related glycosyltransferase ß-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) is subject to strong selective forces in natural house-mouse populations that contain a common allelic variant that confers loss of B4galnt2 gene expression in the gastrointestinal (GI) tract. We reasoned that altered glycan-dependent intestinal host-microbe interactions may underlie these signatures of selection. To determine whether B4galnt2 influences the intestinal microbial ecology, we profiled the microbiota of wild-type and B4galnt2-deficient siblings throughout the GI tract using 16S rRNA gene pyrosequencing. This revealed both distinct communities at different anatomic sites and significant changes in composition with respect to genotype, indicating a previously unappreciated role of B4galnt2 in host-microbial homeostasis. Among the numerous B4galnt2-dependent differences identified in the abundance of specific bacterial taxa, we unexpectedly detected a difference in the pathogenic genus, Helicobacter, suggesting Helicobacter spp. also interact with B4galnt2 glycans. In contrast to other glycosyltransferases, we found that the host intestinal B4galnt2 expression is not dependent on presence of the microbiota. Given the long-term maintenance of alleles influencing B4galnt2 expression by natural selection and the GI phenotypes presented here, we suggest that variation in B4galnt2 GI expression may alter susceptibility to GI diseases such as infectious gastroenteritis.

Poultry litter-induced endocrine disruption in fathead minnow, sheepshead minnow, and mummichog laboratory exposures.

Animal feeding operations in the United States produce more than 500 million tons of manure annually. Disposal of poultry waste via application as fertilizer results in substantial runoff of poultry litter-associated contaminants (PLAC). Of particular concern are sex steroids, 17ß-estradiol, estrone and testosterone, responsible for sex differentiation and development of reproductive structures. In a series of laboratory assays, mature male and mixed-sex larval/juvenile fish were continuously exposed to environmentally relevant PLAC solutions. Effects on gonads were assessed histologically, and vitellogenin (VTG) induction was measured as a gauge of estrogenicity. Twenty-one-day exposures to laboratory-generated PLAC solutions routinely induced VTG in mature male Pimephales promelas. Vitellogenesis in Fundulus heteroclitus only occurred at the highest tested PLAC concentration, and Cyprinodon variegatus were unresponsive at any tested concentration. All species produced considerable VTG in response to a 17ß-estradiol-positive control. A pronounced feminization was seen in P. promelas when exposed to PLAC as larvae but not when exposed as juveniles. Runoff from a poultry litter-amended field cropped under standard agronomic practices induced significant VTG in male P. promelas. Results indicate that environmentally relevant PLAC concentrations exhibit endocrine activity sufficient to induce VTG production in male fish and possibly affect sex ratios in resident fish populations.

pak2a mutations cause cerebral hemorrhage in redhead zebrafish.

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd(mi149)), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.

Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.