Alpha-tocopherol exerts protective function against the mucotoxicity of particulate matter in amphibian and human goblet cells

Exposure to particulate matter (PM) in ambient air is known to increase the risk of cardiovascular disorders and mortality. The cytotoxicity of PM is mainly due to the abnormal increase of reactive oxygen species (ROS), which damage cellular components such as DNA, RNA, and proteins. The correlation between PM exposure and human disorders, including mortality, is based on long-term exposure. In this study we have investigated acute responses of mucus-secreting goblet cells upon exposure to PM derived from a heavy diesel engine. To this end, we employed the mucociliary epithelium of amphibian embryos and human Calu-3 cells to examine PM mucotoxicity. Our data suggest that acute exposure to PM significantly impairs mucus secretion and results in the accumulation of mucus vesicles in the cytoplasm of goblet cells. RNA-seq analysis revealed that acute responses to PM exposure significantly altered gene expression patterns; however, known regulators of mucus production and the secretory pathway were not significantly altered. Interestingly, pretreatment with alpha-tocopherol nearly recovered the hyposecretion of mucus from both amphibian and human goblet cells. We believe this study demonstrates the mucotoxicity of PM and the protective function of alpha-tocopherol on mucotoxicity caused by acute PM exposure from heavy diesel engines

Intrinsic antimicrobial resistance determinants in the superbug Pseudomonas aeruginosa

Antimicrobial-resistant bacteria pose a serious threat in the clinic. This is particularly true for opportunistic pathogens that possess high intrinsic resistance. Though many studies have focused on understanding the acquisition of bacterial resistance upon exposure to antimicrobials, the mechanisms controlling intrinsic resistance are not well understood. In this study, we subjected the model opportunistic superbug Pseudomonas aeruginosa to 14 antimicrobials under highly controlled conditions and assessed its response using expression-and fitness-based genomic approaches. Our results reveal that gene expression changes and mutant fitness in response to sub-MIC antimicrobials do not correlate on a genomewide scale, indicating that gene expression is not a good predictor of fitness determinants. In general, fewer fitness determinants were identified for antiseptics and disinfectants than for antibiotics. Analysis of gene expression and fitness data together allowed the prediction of antagonistic interactions between antimicrobials and insight into the molecular mechanisms controlling these interactions. IMPORTANCE Infections involving multidrug-resistant pathogens are difficult to treat because the therapeutic options are limited. These infections impose a significant financial burden on infected patients and on health care systems. Despite years of antimicrobial resistance research, we lack a comprehensive understanding of the intrinsic mechanisms controlling antimicrobial resistance. This work uses two fine-scale genomic approaches to identify genetic loci important for antimicrobial resistance of the opportunistic pathogen Pseudomonas aeruginosa. Our results reveal that antibiotics have more resistance determinants than antiseptics/disinfectants and that gene expression upon exposure to antimicrobials is not a good predictor of these resistance determinants. In addition, we show that when used together, genomewide gene expression and fitness profiling can provide mechanistic insights into multidrug resistance mechanisms.open

Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung

The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.open6

Xenopus: An alternative model system for identifying muco-active agents

The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagent

Analysis of Porcine Model of Fecal-Induced Peritonitis Reveals the Tropism of Blood Microbiome

Recent studies have suggested the existence of a blood microbiome in the healthy host. However, changes in the blood microbiome upon bloodstream infection are not known. Here, we analyzed the dynamics of the blood microbiome in a porcine model of polymicrobial bacteremia induced by fecal peritonitis. Surprisingly, we detected bacterial populations in the bloodstream even before the infection, and these populations were maintained over time. The native blood microbiome was notably taxonomically different from the fecal microbiome that was used to induce peritonitis, reflecting microbial tropism for the blood. Although the population composition after the infection was similar to that of the native blood microbiome, new bacterial strains entered the bloodstream upon peritonitis induction as clinical symptoms relevant to sepsis developed. This indicates that the bacteria detected in the blood before peritonitis induction were derived from the blood rather than a contamination. Comparison of the functional pathways enriched in the blood and fecal microbiomes revealed that communication and stress management pathways are essential for the survival of the blood microbiome

Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB-pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB-rapamycin-FKBP25 was determined at 1.67-angstrom resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling.ope

Extracellular matrixes and neuroinflammation

The extracellular matrix is a critical component of every human tissue. ECM not only functions as a structural component but also regulates a variety of cellular processes such as cell migration, differentiation, proliferation, and cell death. In addition, current studies suggest that ECM is critical for the pathophysiology of various human diseases. ECM is composed of diverse components including several proteins and polysaccharide chains such as chondroitin sulfate, heparan sulfate, and hyaluronic acid. Each component of ECM exerts its own functions in cellular and pathophysiological processes. One of the interesting recent findings is that ECM is involved in inflammatory responses in various human tissues. In this review, we summarized the known functions of ECM in neuroinflammation after acute injury and chronic inflammatory diseases of the central nerve systems

Prenatal selective serotonin reuptake inhibitor (SSRI) exposure induces working memory and social recognition deficits by disrupting inhibitory synaptic networks in male mice

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed antidepressant drugs in pregnant women. Infants born following prenatal exposure to SSRIs have a higher risk for behavioral abnormalities, however, the underlying mechanisms remains unknown. Therefore, we examined the effects of prenatal fluoxetine, the most commonly prescribed SSRI, in mice. Intriguingly, chronic in utero fluoxetine treatment impaired working memory and social novelty recognition in adult males. In the medial prefrontal cortex (mPFC), a key region regulating these behaviors, we found augmented spontaneous inhibitory synaptic transmission onto the layer 5 pyramidal neurons. Fast-spiking interneurons in mPFC exhibited enhanced intrinsic excitability and serotonin-induced excitability due to upregulated serotonin (5-HT) 2A receptor (5-HT2AR) signaling. More importantly, the behavioral deficits in prenatal fluoxetine treated mice were reversed by the application of a 5-HT2AR antagonist. Taken together, our findings suggest that alterations in inhibitory neuronal modulation are responsible for the behavioral alterations following prenatal exposure to SSRIs

GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base

The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein ??1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliog

Sperm is epigenetically programmed to regulate gene transcription in embryos.

For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.We thank: T. Jenuwein and N. Shukeir for anti-H3K27me3 antibody; A. Bannister, J. Ahringer and E. Miska for comments on the manuscript; Gurdon group members for reading the manuscript; The International Xenopus laevis Genome Project Consortium (the Harland, Rokhsar, Taira labs and others) for providing unpublished genome and gene annotation information. M.T. is supported by WT089613 and by MR/K011022/1. V.G. and P.Z. are funded by AICR 10-0908. A.S. is supported by MR/K011022/1. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship. E.M.M. is supported by National Institutes of Health, National Science Foundation, Cancer Prevention Research Institute of Texas, and the Welch Foundation (F1515). J.J. and J.B.G. are supported by WT101050/Z/13/Z. S.E. acknowledges Boehringer Ingelheim Fond fellowship. A.H.F.M.P. is supported by the Swiss National Science Foundation (31003A_125386) and the Novartis Research Foundation. All members of the Gurdon Institute acknowledge the core support provided by CRUK C6946/A14492 and WT092096.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via https://doi.org/10.1101/gr.201541.11