• Glucose metabolism enhances hematopoietic stem cell formation and function in the vertebrate embryo • Glucose metabolism modulates hif1a activity via mitochondrial generation of reactive oxygen species to impact HSC-relevant gene expressionMany pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrialderived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1a (hif1a). Epistasis assays demonstrated that hif1a regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability. (Blood. 2013;121(13):2483-2493

The E3 ubiquitin ligase β-TRCP, acting in concert with casein kinase I, drives ubiquitination and degradation of VEGFR2, and renders human papillary thyroid cancer cells resistant to the VEGFR2 inhibitor sorafenib.

Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens which threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), HIV-1, and Lactobacillus crispatus, prior to being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. Cytokines and chemokines analysis in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans.

SUMMARY The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here, we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver-versus-pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell fate decisions and outgrowth of the embryonic endodermal anlagen.

Summary The genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood, however less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report 17β-estradiol (E2) influences the production of runx1+ HSPCs in the AGM region by antagonizing VEGF-signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development (12–24hpf) significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity and specification of scl+ HE by decreasing the expression of VEGFAa and downstream arterial Notch-pathway components; heat-shock induction of VEGFAa/Notch rescued E2-mediated hemato-vascular defects. Conversely, repression of endogenous E2-activity increased somitic VEGF expression and vascular-target regulation, shifting the assignment of arterial/venous fate and HE localization; blocking E2-signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest yolk-derived estrogen sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral limits of VEGF regulation.

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb+ HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.

IntroductionThe use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals.Materials and MethodsA set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed.ResultsNone of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR-145 correlated with nadir CD4+ T cell count.DiscussionNo associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection.

The mechanisms that fungi use to co-regulate subsets of genes specifically associated with morphogenic states represent a basic unsolved problem in fungal biology. Candida albicans is an important model of fungal differentiation both for rapid interconversion between yeast and hyphal growth forms and for white/opaque switching mechanisms. The Sundstrom lab is interested in mechanisms regulating hypha-specific expression of adhesin genes that are critical for C. albicans hyphal growth phenotypes and pathogenicity. Early studies on hypha-specific genes such as HWP1 and ALS3 reported 5’ intergenic regions that are larger than those typically found in an average promoter and are associated with hypha-specific expression. In the case of HWP1, activation and repression involves a 368 bp region, denoted the HWP1 control region (HCR), located 1410 bp upstream of its transcription start site. In previous work we showed that HCR confers developmental regulation to a heterologous ENO1 promoter, indicating that HCR by itself contains sufficient information to couple gene expression to morphology. Here we show that the activation and repression mediated by HCR are localized to distinct HCR regions that are targeted by the transcription factors Nrg1p and Efg1p. The finding that Efg1p mediates both repression via HCR under yeast morphological conditions and activation conditions positions Efg1p as playing a central role in coupling HWP1 expression to morphogenesis through the HCR region. These localization studies revealed that the 120 terminal base pairs of HCR confer Efg1p-dependent repressive activity in addition to the Nrg1p repressive activity mediated by DNA upstream of this subregion. The 120 terminal base pair subregion of HCR also contained an initiation site for an HWP1 transcript that is specific to yeast growth conditions (HCR-Y) and may function in the repression of downstream DNA. The detection of an HWP1 mRNA isoform specific to hyphal growth conditions (HWP1-H) showed that morphology-specific mRNA isoforms occur under both yeast and hyphal growth conditions. Similar results were found at the ALS3 locus. Taken together, these results, suggest that the long 5’ intergenic regions upstream of hypha-specific genes function in generating mRNA isoforms that are important for morphology-specific gene expression. Additional complexity in the HWP1 promoter involving HCR-independent activation was discovered by creating a strain lacking HCR that exhibited variable HWP1 expression during hyphal growth conditions. These results show that while HCR is important for ensuring uniform HWP1 expression in cell populations, HCR independent expression also exists. Overall, these results elucidate HCR-dependent mechanisms for coupling HWP1-dependent gene expression to morphology uniformly in cell populations and prompt the hypothesis that mRNA isoforms may play a role in coupling gene expression to morphology in C. albicans.