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Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

ObjectivesTo determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir.MethodsResistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list.ResultsFour hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84-3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience.ConclusionsViral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented.

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A characterization of the genome of Bacillus sp. strain C-125 is presented. Characterization was done using restriction endonucleases (AscI and Sse8387I) in combination with electrophoretic and hybridization analysis. Of 14 possible clones, 7 were genuine linker clones binding to one Enzyme A fragment. Two clones hybridized to several fragments and were assumed to have repetitive DNA sequences from the bacterial chromosome. The chromosome map compared well with a corresponding map of B. subtilis chromosomes. The map starting point was tentatively assigned to the 9A of 12A fragment.

The flagellin gene (hag) of the alkalophile Bacillus sp. C-125 was isolated and characterized to determine the possible structural adaptations of flagellin under alkaline conditions. Sequence analysis showed that the C-125 flagellin protein is highly similar to other known flagellins inthe N- and C-terminal regions. However, a high level of variation was observed in the central region of the protein. The changes in this region may be involved in alkaline adaptation of Bacillus sp. C-125.

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An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/ MEK/ ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo. Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions. [ABSTRACT FROM AUTHOR]

Background: Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods: Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results: We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions: Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally. [ABSTRACT FROM AUTHOR]