Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.

Key pointsr The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown.r In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells. r The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction.Abstract The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective cation current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd 3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.

Notch signaling inhibits differentiation of endocrine cells in the pancreas and intestine. In a number of cases, the observed inhibition occurred with Notch activation in multipotential cells, prior to the initiation of endocrine differentiation. It has not been established how direct activation of Notch in endocrine precursor cells affects their subsequent cell fate. Using conditional activation of Notch in cells expressing Neurogenin3 or NeuroD1, we examined the effects of Notch in both organs, on cell fate of early endocrine precursors and maturing endocrine-restricted cells respectively. Notch did not preclude the differentiation of a limited number of endocrine cells in either organ when activated in Ngn3+ precursor cells. In addition, in the pancreas most Ngn3+ cells adopted a duct but not acinar cell fate; whereas in intestinal Ngn3+ cells, Notch favored enterocyte and goblet cell fates, while selecting against endocrine and Paneth cell differentiation. A small fraction of NeuroD1+ cells in the pancreas retain plasticity to respond to Notch, giving rise to intraislet ductules as well as cells with no detectable pancreatic lineage markers that appear to have limited ultrastructural features of both endocrine and duct cells. These results suggest that Notch directly regulates cell fate decisions in multipotential early endocrine precursor cells. Some maturing endocrine-restricted NeuroD1+ cells in the pancreas switch to the duct lineage in response to Notch, indicating previously unappreciated plasticity at such a late stage of endocrine differentiation.

Background & Aims The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from neurogenin3-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin. Methods We used Neurod1 and Neurog3 lineage analysis, and examined differentiation of serotonin-producing and ECL cells in stomach tissues of Neurod1-cre;ROSAtdTom, Tph1-CFP, c-Kitwsh/wsh, and Neurog3Cre;ROSAtdTom mice, by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We performed RNA-seq analysis of ECL cells. Results Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing Neurod1. Serotonin-secreting cells expressed a number of mast cell genes, but not genes associated with endocrine differentiation; they did not develop in c-Kitwsh/wsh mice and were labeled with transplanted bone marrow cells. RNA-seq analysis of ECL cells revealed high expression levels of many genes common to endocrine cells including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells. Conclusions Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) constitutes a key event in atherosclerosis, neointimal hyperplasia, and the response to vascular injury. Estrogen receptor ␣ (ER␣) mediates the protective effects of estrogen in injured blood vessels and regulates ligand-dependent gene expression in vascular cells. However, the molecular mechanisms mediating ER␣-dependent VSMC gene expression and VSMC proliferation after vascular injury are not well defined. Here, we report that the ER coactivator steroid receptor coactivator 3 (SRC3) is also a coactivator for the major VSMC transcription factor myocardin, which is required for VSMC differentiation to the nonproliferative, contractile state. The N terminus of SRC3, which contains a basic helix-loop-helix/Per-ARNT-Sim protein-protein interaction domain, binds the C-terminal activation domain of myocardin and enhances myocardinmediated transcriptional activation of VSMC-specific, CArGcontaining promoters, including the VSMC-specific genes SM22 and myosin heavy chain. Suppression of endogenous SRC3 expression by specific small interfering RNA attenuates myocardin transcriptional activation in cultured cells. The SRC3-myocardin interaction identifies a site of convergence for nuclear hormone receptor-mediated and VSMC-specific gene regulation and suggests a possible mechanism for the vascular protective effects of estrogen on vascular injury. steroid hormone receptors ͉ transcriptional coregulators ͉ vascular biology I n addition to an important role in growth and development of reproductive tissues, estrogen and its receptors regulate a variety of cardiovascular functions, including vascular tone and the response of blood vessels to injury (1, 2). The pleiotropic effects of estrogen are mediated through binding to its two cognate receptors, estrogen receptors ␣ (ER␣) and ␤ (ER␤), members of the nuclear hormone receptor (NHR) superfamily. NHRs are ligand-dependent transcription factors, regulating target gene expression by recruiting coactivator or corepressor complexes to their target promoters (3). Steroid receptor coactivator (SRC) 3 (SRC3) (RAC3/AIB1/TRAM1/pCIP) is a member of the p160 family of NHR coactivators including SRC1 and TIF2 (thyroid hormone receptor interacting factor 2 or SRC2) (4, 5). These molecules were initially identified as liganddependent nuclear receptor coactivators that interact with ligand-bound nuclear receptors to modulate target gene activation (3, 6-9). The SRC proteins recruit protein acetyltransferases such as cAMP response element-binding protein binding protein (CBP)/p300 and p/CAF (10-12) and protein methytransferases such as coactivator-associated arginine methyltransferase 1 (CARM-1) and protein arginine methyltransferase-1 (PRMT-1) to nuclear receptor target gene promoters to modify chromatin structure and/or assemble transcription initiation (13,14). Biochemical studies support that SRC complexes are essential for nuclear receptor activities. The SRC family of coactivators share a signature motif of LXXLL sequence...