To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signal-sensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TLC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C218, forms an inhibitory intramolecular disulfide bond with C207 that must be isomerized to form the active C207-C207 intermolecular bond. We then found bile salt-dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cholerae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation.T he human pathogen Vibrio cholerae is the causative agent of the diarrheal disease cholera. The Vibrio life cycle begins with a freeswimming phase in aquatic environments. Human infection normally starts with the ingestion of food or water contaminated with V. cholerae. As it colonizes small intestines of a host and only when it colonizes, V. cholerae produces an array of virulence factors, including cholera toxin (CT), which causes the diarrhea characteristic of cholera and toxin-coregulated pili (TCP), type IV pili required for intestinal colonization both in animal models and in human volunteers (1, 2).Bacterial pathogens have evolved highly sophisticated signal transduction systems to coordinately control the expression of virulence determinants to better infect their hosts. Extensive in vitro studies have revealed details of V. cholerae virulence gene regulation (3): AphA and AphB proteins activate transcription of the transmembrane transcription factor TcpP, which in turn activates toxT transcription together with ToxR, which then completes the cascade by activating toxin and TCP production (Fig. 1A). However, all of the experiments to date used artificial in vitro conditions to induce virulence factor production, leaving unanswered which microenvironmental signals in the intestines activate the V. cholerae virulence cascade. It has been reported that certain environmental conditions such as temperature, oxygen concentra...

Ammonium transporters play a key functional role in nitrogen uptake and assimilation in microorganisms and plants; however, little is known about their structural counterpart in mammals. Here, we report the molecular cloning and biochemical characterization of Rh type B glycoproteins, human RhBG and mouse Rhbg, two new members of the Rh family with distinct tissue specificities. The RhBG orthologues possess a conserved 12-transmembrane topology and most resemble bacterial and archaeal ammonium transporters. Human RHBG resides at chromosome 1q21.3, which harbors candidate genes for medullary cystic kidney disease, whereas mouse Rhbg is syntenic on chromosome 3. Northern blot and in situ hybridization revealed that RHBG and Rhbg are predominantly expressed in liver, kidney, and skin, the specialized organs involving ammonia genesis, excretion, or secretion. Confocal microscopy showed that RhBG is located in the plasma membrane and in some intracellular granules. Western blots of membrane proteins from stable HEK293 cells and from mouse kidney and liver confirmed this distribution. N-Glycanase digestion showed that RhBG/Rhbg has a carbohydrate moiety probably attached at the NHS motif on exoloop 1. Phylogenetic clustering, tissuespecific expression, and plasma membrane location suggest that RhBG homologous proteins are the long sought major ammonium transporters in mammalians.

Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.

The separation of acetylene and carbon dioxide is an essential but challenging process owing to the similar molecular sizes and physical properties of the two gas molecules. Notably, these molecules usually exhibit different orientations in the pore channel. We report an adsorption site selective occupation strategy by taking advantage of differences in orientation to sieve the C2H2 from CO2 in a judiciously designed amine‐functionalized metal–organic framework, termed CPL‐1‐NH2. In this material, the incorporation of amino groups not only occupies the adsorption sites of CO2 molecules and shields the interaction of uncoordinated oxygen atom and CO2 molecules resulting in a negligible adsorption amount and a decrease in enthalpy of adsorption but also strengthened the binding affinity toward C2H2 molecules. This material thus shows an extremely high amount of C2H2 at low pressure and a remarkably high C2H2/CO2 IAST selectivity (119) at 1 bar and 298 K.

Hepatitis A virus (HAV), a classic nonenveloped virus, has recently been found to be released mainly in the form of quasi-enveloped HAV (eHAV) by hijacking host endosomal sorting complexes required for transport (ESCRT) complexes. Unlike the nonenveloped virion, eHAV contains the viral protein pX on the surface of the HAV capsid as an extension of VP1. How HAV capsids acquire the host envelope and whether the pX protein is involved in this process were previously unknown. Here, we analyse the role of pX in foreign protein secretion in exosome-like extracellular vesicles (EVs) and the formation of eHAV. Fusion of pX to eGFP guided eGFP into exosome-like EVs through directing eGFP into multivesicular bodies (MVBs), and apoptosis-linked gene 2-interacting protein X (ALIX) release was significantly enhanced. Coimmunoprecipitation (co-IP) demonstrated the interaction between pX and the ALIX V domain. Removal of the C-terminal half of pX abolished eHAV release and reduced the interaction between the HAV virion and ALIX. Finally, the C-terminal half of pX alone was sufficient for loading eGFP into EVs by interacting with ALIX. In conclusion, the C-terminal part of pX is important for eHAV production and may have potential for large protein complex loading into exosome-like EVs for therapeutic purposes.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.

The electrochemical CO 2 reduction reaction (CO 2 RR) over Cu-based catalysts shows great potential for converting CO 2 into multicarbon (C 2 + ) fuels and chemicals. Herein, we introduce an A 2 M 2 O 7 structure into a Cu-based catalyst through a solid-state reaction synthesis method. The Cu 2 P 2 O 7 catalyst is electrochemically reduced to metallic Cu with a significant structure evolution from grain aggregates to highly porous structure under CO 2 RR conditions. The reconstructed Cu 2 P 2 O 7 catalyst achieves a Faradaic efficiency of 73.6 % for C 2 + products at an applied current density of 350 mA cm À 2 , remarkably higher than the CuO counterparts. The reconstructed Cu 2 P 2 O 7 catalyst has a high electrochemically active surface area, abundant defects, and low-coordinated sites. In situ Raman spectroscopy and density functional theory calculations reveal that CO adsorption with bridge and atop configurations is largely improved on Cu with defects and low-coordinated sites, which decreased the energy barrier of the CÀ C coupling reaction for C 2 + products.

Prolonged exposure of 3T3-L1 adipocytes to insulin increases GLUT1 protein content while diminishing GLUT4. These changes arise in part from changes in mRNA transcription. Here we examined whether there are also specific effects of insulin on GLUT1 and GLUT4 mRNA translation. Insulin enhanced association of GLUT1 mRNA with polyribosomes and decreased association with monosomes, suggesting increased translation. Conversely, insulin arrested the majority of GLUT4 transcripts in monosomes. Insulin inactivates the translational suppressor eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) through the mammalian target of rapamycin (mTOR). Hence, we examined the effect of rapamycin on GLUT1 mRNA translation and protein expression. Rapamycin abrogated the insulin-mediated increase in GLUT1 protein synthesis through partial inhibition of GLUT1 mRNA translation and partial inhibition of the rise in GLUT1 mRNA. 4E-BP1 inhibited GLUT1 mRNA translation in vitro. Because phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), in concert with mTOR, inactivate 4E-BP1, we explored their role in GLUT1 protein expression. Cotransfection of cytomegalovirus promoter-driven, hemagglutinin epitope-tagged GLUT1 with dominant inhibitory mutants of PI3K or PKB inhibited the insulin-elicited increase in hemagglutinin-tagged GLUT1 protein. These results unravel the opposite effects of insulin on GLUT1 and GLUT4 mRNA translation. Increased GLUT1 mRNA translation appears to occur via the PI3K/PKB/mTOR/ 4E-BP1 cascade.