Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin⁺ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.Embryonic hematopoietic development in the mouse proceeds through defined stages. The first hematopoietic cells of the erythroid lineage develop in the extraembryonic yolk sac at embryonic day 7.5 (E7.5; Moore and Metcalf, 1970). At E9, hematopoietic stem cell (HSC) activity from the yolk sac and paraaortic splanchnopleura (PSp) can be detected when transplanted into neonatal mice (Yoder et al., 1997; Arora et al., 2014). HSCs that engraft lethally irradiated adult recipients emerge in the aorta–gonad–mesonephros (AGM) region at E10-11 (Medvinsky and Dzierzak, 1996; North et al., 2002). These HSCs later colonize additional organs required for adult hematopoiesis and possess the ability to reconstitute multiple hematopoietic lineages.The development of the murine circulatory system at E8.5 coincides with the development of more definitive hematopoietic compartments, including HSCs and the lymphoid lineage. Accordingly, recent studies have linked biomechanical forces, such as blood flow–induced shear stress, to hematopoietic development (Adamo et al., 2009; North et al., 2009). In these studies, genetic mutants lacking intravascular circulation were used to demonstrate the reduction in hematopoietic emergence in the AGM and nitric oxide signaling was implicated in blood flow–dependent AGM hematopoiesis (Adamo et al., 2009; North et al., 2009; Wang et al., 2011). Although chimeric analysis demonstrated a cell autonomous requirement for nitric oxide signaling in zebrafish (North et al., 2009), whether this pathway directly promotes hematopoiesis remains an open question due to the vasodilatory effect of nitric oxide donors and their effects on smooth muscle. Apart from these observations, signaling pathways responsible for flow-dependent hematopoietic induction have not been characterized.Activation of protein kinase A (PKA) and its downstream target cAMP response element-binding protein (CREB) by exogenous shear stress has been observed in diverse cell types, including chondrocytes and osteocytes (Cherian et al., 2003; Ogawa et al., 2014). The classic mechanism of PKA activation involves the binding of a ligand to a G protein–coupled receptor and activation of adenylyl cyclase, which converts ATP into the second messenger cyclic AMP (cAMP). The binding of cAMP to PKA releases catalytic subunits that phosphorylate CREB in the nucleus. In differentiating mouse embryonic stem cells (mESCs), PKA–CREB has been linked to endothelial and hematopoietic differentiation via binding of CREB to the Etv2 promoter, which up-regulates pro-hematopoietic factors such as Gata2 and Scl/Tal1 (Yamamizu et al., 2012). Moreover, the PKA–CREB signaling pathway has been explored in the context of the prostaglandin E₂ signaling pathway in zebrafish, where it promotes AGM hematopoiesis via activation of the Wnt pathway (Goessling et al., 2009). However, whether this pathway is conserved in the mouse is unclear, especially given conflicting reports on Wnt signaling in AGM hematopoiesis (Ruiz-Herguido et al., 2012; Chanda et al., 2013). Prostaglandin E₂ also directly activates several pathways including PI3K–AKT and ERK–MAPK, which makes it difficult to conclude that PKA–CREB is the sole mediator of the pro-hematopoietic effects of this molecule (Alfranca et al., 2006). Given the shear-responsiveness of the PKA–CREB pathway and its implication in early embryonic hematopoiesis in other species, we investigated the possible role of shear stress–activated PKA–CREB signaling during AGM hematopoiesis in the mouse.We first verified that this pathway is activated by shear stress in VE-cadherin⁺ endothelial cells and present in the murine AGM, specifically in the cells lining the dorsal aorta. We then conducted a bioinformatics-based screen using microarray data on CREB overexpression and CREB chromatin immunoprecipitation-sequencing (ChIP-Seq) data using data available at Encyclopedia of DNA Elements (ENCODE) and elsewhere to identify regulators of CREB function in hematopoietic cells (Esparza et al., 2008; Jolma et al., 2010; Pencovich et al., 2011; Raney et al., 2011; Trompouki et al., 2011; Martens et al., 2012). Using insight gained from bioinformatics, we discover that the bone morphogenetic protein (BMP) signaling pathway acts downstream of PKA–CREB signaling in regulating AGM hematopoiesis. Finally, we show that this is a blood flow–dependent pathway by demonstrating the abrogation of PKA–CREB–BMP signaling axis in Ncx1-null embryos, which lack a heartbeat and intravascular flow. Our data thus document a blood-flow dependent pathway regulating hematopoietic development.     

Predominance of KPC-3 in a Survey for Carbapenemase-Producing Enterobacteriaceae in Portugal

Among the 2,105 Enterobacteriaceae tested in a survey done in Portugal, 165 were nonsusceptible to carbapenems, from which 35 (26 Klebsiella pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes, and 3 Enterobacter cloacae isolates and 1 Klebsiella oxytoca isolate) were confirmed to be carbapenemase producers by the presence of 30 Tn4401d-bla_KPC-3, 4 intI3-bla_GES-5, and one intI1-bla_VIM-2 gene, alone or in combination with other bla genes. The dissemination of bla_KPC-3 gene carried by an IncF plasmid suggests lateral gene transfer as a major mechanism of dissemination.     

Susceptibilities of Genotype 1a, 1b,and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier {"type":"clinical-trial","attrs":{"text":"NCT01532973","term_id":"NCT01532973"}}NCT01532973.)     

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.     

Lineage fate of ductular reactions in liver injury and carcinogenesis

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.     

Contribution of Clinically Derived Mutations in ERG11 to Azole Resistance in Candida albicans

In Candida albicans, the ERG11 gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations in ERG11 that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. Although ERG11 mutations have been observed in clinical isolates, the specific contributions of individual ERG11 mutations to azole resistance in C. albicans have not been widely explored. We sequenced ERG11 in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation in ERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinct ERG11 alleles were recovered, with 10 ERG11 alleles containing a single amino acid substitution. We then characterized 19 distinct ERG11 alleles by introducing them into the wild-type azole-susceptible C. albicans SC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations in ERG11 are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.     

Detection of Abnormal Glucose Tolerance in Africans Is Improved by Combining A1C With Fasting Glucose: The Africans in America Study

OBJECTIVE

Abnormal glucose tolerance is rising in sub-Saharan Africa. Hemoglobin A_1c by itself and in combination with fasting plasma glucose (FPG) is used to diagnose abnormal glucose tolerance. The diagnostic ability of A1C in Africans with heterozygous variant hemoglobin, such as sickle cell trait or hemoglobin C trait, has not been rigorously evaluated. In U.S.-based Africans, we determined by hemoglobin status the sensitivities of 1) FPG ≥5.6 mmol/L, 2) A1C ≥ 5.7% (39 mmol/mol), and 3) FPG combined with A1C (FPG ≥5.6 mmol/L and/or A1C ≥5.7% [39 mmol/mol]) for the detection of abnormal glucose tolerance.

RESEARCH DESIGN AND METHODS

An oral glucose tolerance test (OGTT) was performed in 216 African immigrants (68% male, age 37 ± 10 years [mean ± SD], range 20–64 years). Abnormal glucose tolerance was defined as 2-h glucose ≥7.8 mmol/L.

RESULTS

Variant hemoglobin was identified in 21% (46 of 216). Abnormal glucose tolerance occurred in 33% (72 of 216). When determining abnormal glucose tolerance from the OGTT (2-h glucose ≥7.8 mmol/L), sensitivities of FPG for the total, normal, and variant hemoglobin groups were 32%, 32%, and 33%, respectively. Sensitivities for A1C were 53%, 54%, and 47%. For FPG and A1C combined, sensitivities were 64%, 63%, and 67%. Sensitivities for FPG and A1C and the combination did not vary by hemoglobin status (all P > 0.6). For the entire cohort, sensitivity was higher for A1C than FPG and for both tests combined than for either test alone (all P values ≤ 0.01).

CONCLUSIONS

No significant difference in sensitivity of A1C by variant hemoglobin status was detected. For the diagnosis of abnormal glucose tolerance in Africans, the sensitivity of A1C combined with FPG is significantly superior to either test alone.     

Finding the elusive and causative autoantibody: An atypical case of autoimmune hemolytic anemia

An isolated IgA‐mediated autoimmune hemolytic anemia can present a diagnostic challenge. When a routine direct antiglobulin test (DAT) is negative but clinical suspicion remains high, further testing with monospecific antisera should be performed. As with IgG‐mediated WAIHA, steroids are first‐line treatment, though splenectomy is often required to achieve a durable treatment response.