Route of Arsenic Exposure Differentially Impacts the Expression of Genes Involved in Gut-Mucosa-Associated Immune Responses and Gastrointestinal Permeability.

First-pass metabolism alters arsenic biotransformation and its immunomodulatory activities. This study aims to determine the mRNA expression of intestinal-immunity- and permeability-associated genes, levels of cytokine/chemokines and levels of immunoglobulin isotypes when CD-1 mice were exposed to a single dose of intravenous (IV) sodium arsenite (50 µg/kg body weight (BW)) and to compare these responses to exposure via oral gavage (OG) (50 µg/kg BW). Samples were collected at 1, 4, 24 and 48 h post IV exposure and 24 and 48 h post OG. Sodium arsenite IV exposure led to a transient modulation of mRNA expression and protein levels of immunity-related genes involved in inflammation/apoptotic pathways and production of cytokines/chemokines, whereas it also led to downregulated expression of genes encoding tight junction, focal adhesion, and gap junction proteins, which are responsible for maintaining cell permeability. Oral exposure perturbed fewer cell-permeability-related genes at 24 and 48 h post exposure. At 24 h post exposure, OG decreased IgA and IgG2b levels; however, IV exposure significantly increased IgG2b, IgG3 and IgA in ileal tissue. Earlier, we showed significant downregulation of mRNA expression of genes involved in the immune-related pathways during OG in the intestinal mucosa of the same animals. Cumulatively, these results provide evidence that the exposure route of a xenobiotic can differentially impact the intestinal responses due to the impact of first-pass metabolism.

Lung microbiome, gut-lung axis and chronic obstructive pulmonary disease.

PURPOSE OF REVIEW: To review recent data on the microbiome of the lungs and how it changes with the evolution of COPD. To explore initial data with respect to COPD and the gut-lung axis. An expanded understanding of the pathogenesis of COPD may lead to new therapeutic targets. RECENT FINDINGS: Intermittent pulmonary seeding is essential to health. The lung inflammation of chronic obstructive pulmonary disease (COPD) appears to change the lung milieu such that there is a shift in the microbiome of the lung as COPD progresses. Current data contain internal contradictions, but viewed in to suggest that the lung microbiome participates in the ongoing process of inflammation and destruction (in contrast to the role of the 'healthy lung' microbiome). Gut and lung 'communicate' and share some functions. COPD is associated with increased intestinal permeability (a dysfunction associated with inflammation). COPD has an impact upon the gastrointestinal microbiome. The gastrointestinal tract may, thus play a role in the progression of COPD. SUMMARY: Lung injury/inflammation alters the milieu of the lung and favors an evolving microbiome, which reflects and probably participates in the processes of inflammation and injury. There is some evidence that the gastrointestinal tract participates in that inflammatory process.

Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure.

The gastrointestinal tract (GIT) is exposed to xenobiotics, including drugs, through both: local (oral) and systemic routes. Despite the advances in drug discovery and in vitro pre-clinical models, there is a lack of appropriate translational models to distinguish the impact of these routes of exposure. Changes in intestinal permeability has been observed in different gastrointestinal and systemic diseases. This study utilized one such xenobiotic, arsenic, to which more than 200 million people around the globe are exposed via their food, drinking water, work environment, soil, and air. The purpose of this study was to establish an in vitro model to mimic gastrointestinal tract exposure to xenobiotics via oral or intravenous routes. To achieve this, we compared the route (mimicking oral and intravenous exposure to GIT and the dose response (using threshold approach) of trivalent and pentavalent inorganic arsenic species on the permeability of in vitro cultured polarized T84 cells, an example of intestinal epithelial cells. Arsenic treatment to polarized T84 cells via the apical and basolateral compartment of the trans-well system reflected oral or intravenous routes of exposure in vivo, respectively. Sodium arsenite, sodium arsenate, dimethyl arsenic acid sodium salt (DMAV), and disodium methyl arsonate hydrate (MMAV) were assessed for their effects on intestinal permeability by measuring the change in trans-epithelial electrical resistance (TEER) of T-84 cells. Polarized T-84 cells exposed to 12.8 µM of sodium arsenite from the basolateral side showed a marked reduction in TEER. Cytotoxicity of sodium arsenite, as measured by release of lactate dehydrogenase (LDH), was increased when cells were exposed via the basolateral side. The mRNA expression of genes related to cell junctions in T-84 cells was analyzed after exposure with sodium arsenite for 72 h. Changes in TEER correlated with mRNA expression of focal-adhesion-, tight-junction- and gap-junction-related genes (upregulation of Jam2, Itgb3 and Notch4 genes and downregulation of Cldn2, Cldn3, Gjb1, and Gjb2). Overall, exposure to sodium arsenite from the basolateral side was found to have a differential effect on monolayer permeability and on cell-junction-related genes as compared to apical exposure. Most importantly, this study established a preclinical human-relevant in vitro translational model to assess the changes in permeability and cytotoxicity during exposure, mimicking oral or intravenous routes.

Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding, Adhesion and Proliferation of Epithelial Cells.

Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations (1, 10, and 100 µg/mL). mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion (GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3-IL23-IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.

Human Intestinal Tissue Explant Exposure to Silver Nanoparticles Reveals Sex Dependent Alterations in Inflammatory Responses and Epithelial Cell Permeability.

Consumer products manufactured with antimicrobial silver nanoparticles (AgNPs) may affect the gastrointestinal (GI) system. The human GI-tract is complex and there are physiological and anatomical differences between human and animal models that limit comparisons between species. Thus, assessment of AgNP toxicity on the human GI-tract may require tools that allow for the examination of subtle changes in inflammatory markers and indicators of epithelial perturbation. Fresh tissues were excised from the GI-tract of human male and female subjects to evaluate the effects of AgNPs on the GI-system. The purpose of this study was to perform an assessment on the ability of the ex vivo model to evaluate changes in levels of pro-/anti-inflammatory cytokines/chemokines and mRNA expression of intestinal permeability related genes induced by AgNPs in ileal tissues. The ex vivo model preserved the structural and biological functions of the in-situ organ. Analysis of cytokine expression data indicated that intestinal tissue of male and female subjects responded differently to AgNP treatment, with male samples showing significantly elevated Granulocyte-macrophage colony-stimulating factor (GM-CSF) after treatment with 10 nm and 20 nm AgNPs for 2 h and significantly elevated RANTES after treatment with 20 nm AgNPs for 24 h. In contrast, tissues of female showed no significant effects of AgNP treatment at 2 h and significantly decreased RANTES (20 nm), TNF-α (10 nm), and IFN-γ (10 nm) at 24 h. Smaller size AgNPs (10 nm) perturbed more permeability-related genes in samples of male subjects, than in samples from female subjects. In contrast, exposure to 20 nm AgNPs resulted in upregulation of a greater number of genes in female-derived samples (36 genes) than in male-derived samples (8 genes). The ex vivo tissue model can distinguish sex dependent effects of AgNP and could serve as a translational non-animal model to assess the impacts of xenobiotics on human intestinal mucosa.

Alteration in the mRNA expression of genes associated with gastrointestinal permeability and ileal TNF-α secretion due to the exposure of silver nanoparticles in Sprague-Dawley rats.

BACKGROUND: Silver ions from silver nanoparticles (AgNP) or AgNPs themselves itself that are ingested from consumer health care products or indirectly from absorbed food contact material can interact with the gastrointestinal tract (GIT). The permeability of the GIT is strictly regulated to maintain barrier function and proper nutrient absorption. The single layer intestinal epithelium adheres and communicates actively to neighboring cells and the extracellular matrix through different cell junctions. In the current study, we hypothesized that oral exposure to AgNPs may alter the intestinal permeability and expression of genes controlling cell junctions. Changes in cell junction gene expression in the ileum of male and female rats administered different sizes of AgNP for 13-weeks were assessed using qPCR. RESULTS: The results of this study indicate that AgNPs have an altering effect on cell junctions that are known to dictate intestinal permeability. mRNA expression of genes representing tight junction (Cldn1, Cldn5, Cldn6, Cldn10 and Pecam1), focal adhesion (Cav1, Cav2, and Itgb2), adherens junction (Pvrl1, Notch1, and Notch2), and hemidesmosome (Dst) groups were upregulated significantly in females treated with 10 nm AgNP, while no change or downregulation of same genes was detected in male animals. In addition, a higher concentration of pro-inflammatory cytokine, TNF-α, was noticed in AgNP-treated female animals as compared to controls. CONCLUSIONS: This study proposes that interaction of silver with GIT could potentially initiate an inflammatory process that could lead to changes in the gastrointestinal permeability and/or nutrient deficiencies in sex-specific manner. Fully understanding the mechanistic consequences of oral AgNP exposure may lead to stricter regulation for the commercial usage of AgNPs and/or improved clinical therapy in the future.

Irreversible effects of trichloroethylene on the gut microbial community and gut-associated immune responses in autoimmune-prone mice.

The developing immune system is particularly sensitive to immunotoxicants. This study assessed trichloroethylene (TCE)-induced effects on the gut microbiome and cytokine production during the development in mice. Mice were exposed to TCE (0.05 or 500 µg/mL) at the levels that approximate to environmental or occupational exposure, respectively. Mice were subjected to a continuous developmental exposure to these doses encompassing gestation, lactation and continuing directly in the drinking water postnatally for 154 days (PND154) or PND259. To observe persistence of the effect TCE was removed from the drinking water in a subset of mice on PND154 and were provided regular drinking water until the study terminus (PND259). Abundance of total tissue-associated bacteria reduced only in mice exposed to TCE until PND259. The ratio of Firmicutes/Bacteroidetes did not alter during this continuos exposure; however, cessation of high-dose TCE at PND154 resulted in the increased abundance Bacteroidetes at PND259. Furthermore, high-dose TCE exposure until PND259 resulted in a lower abundance of the genera Bacteroides and Lactobaccilus and increased abundance of genus Bifidobactrium and bacterial family Enterobacteriaceae. TCE exposure until PND154 showed significant changes in the production of interleukin-33; that might play a dual role in maintaining the balance and homeostasis between commensal microbiota and mucosal health. At PND259, interleukin-3, granulocyte-macrophage colony-stimulating factor and Eotaxin were altered in both, the continuous exposure and cessation groups, whereas only a cessation group had a higher level of KC that may facilitate infiltration of neutrophils. The irreversible effects of TCE after a period of exposure cessation suggested a unique programming and potential toxicity of TCE even at the environmental level exposure.

Drug resistance in Mycobacterium tuberculosis and targeting the l,d-transpeptidase enzyme.

Tuberculosis (TB) is a disease that has afflicted mankind for thousands of years, but in the last seven decades, much progress has been made in anti-TB therapy. Early drugs, such as para-aminosalicylic acid, streptomycin, isoniazid, and rifamycins were very effective in combatting the disease, giving rise to the hope that TB would be eradicated from the face of the earth by 2010. Despite that optimism, TB continues to kill more than a million people annually worldwide. A major reason for our inability to contain TB is the emergence drug resistance in Mycobacterium tuberculosis. This commentary is based on our recent publication on the structure of l,d-transpeptidase enzyme, relevant to drug resistance. As a background, we briefly outline the history and development of anti-TB therapy. Based on the crystal structure, we suggest a potential direction for designing more potent drugs against TB.

In vitro test systems to determine tetracycline residue binding to human feces.

The use of antimicrobials, such as tetracycline, in food-producing animals may result in antimicrobial drug residues (ADR) in edible tissues from treated animals and contribute to the emergence of antibiotic resistant bacteria. The Veterinary International Conference on Harmonization (VICH) document (VICH GL36(R)/FDA-CVM Guidance for Industry#159) provides guidance on evaluating the safety of veterinary ADR in the human foods as related to effects on the human intestinal microbiome. One recognized research gap is a need for additional data and testing requirements to determine the fraction of an oral dose of ADR available to intestinal microorganisms. In the present study, we address this need by examining the binding of tetracycline to human feces using chemical and microbiological assays. High-performance liquid chromatography and liquid chromatography mass spectrometry assays showed that 25% (w/v) diluted steam sterilized feces dosed with 0.15 and 1.5 µg/ml tetracycline had binding of 58.2 ±â€¯10.8% and 56.9 ±â€¯9.1%, respectively. Tetracycline binding to fecal slurries gave similar results. Microbiological assays with two reference bacterial strains validated the results of the chemical assays. Based on data from chemical and microbiological assays methods, the fraction of dose available to microorganisms was 0.418 and 0.431 of the 0.15 and 1.5 µg/ml tetracycline treatments, respectively. This study also proposes factors to be considered when designing and conducting experiments to determine the percent of an antimicrobial agents that is available to microorganisms in the gastrointestinal tract.

An in vitro study to assess the impact of tetracycline on the human intestinal microbiome.

The human intestinal microbiome, a generally stable ecosystem, could be potentially altered by the ingestion of antimicrobial drug residues in foods derived from animals. Data and the scientific published literature on the effects of antimicrobial residues on the human intestinal microbiome are reviewed by national regulatory authorities as part of the human food safety evaluation of veterinary antimicrobial agents used in food-producing animals. In this study, we determined if tetracycline, at low residue concentrations, could impact the human intestinal microbiome structure and the resistance-gene profile, following acute and subchronic exposure. The effects of 0.15, 1.5, 15, and 150 µg/ml of tetracycline, after 24 h and 40 days of exposure, in 3% human fecal suspensions, collected from three individuals (A, B, and C) were investigated using in vitro batch cultures. Results were variable, with either no change or minor changes in total bacterial 16S rRNA gene copies after exposure of fecal samples to tetracycline, because of the inter-individual variation of human gastrointestinal tract microbiota. Bacterial community analysis using rRNA-based pyrosequencing revealed that Firmicutes and Bacteroidetes were the predominant phyla in the three fecal samples; the ratio of phylotypes varied among individuals. The evaluation of bacterial community changes at the genus level, from control to tetracycline-treated fecal samples, suggested that tetracycline under the conditions of this study could lead to slight differences in the composition of intestinal microbiota. The genus Bacteroides (of the Bacteroidetes) was consistently altered from 1.68 to 5.70% and 4.82-8.22% at tetracycline concentrations of 0.15 µg/ml or above at both time points for individual A, respectively, and increased 5.13-13.50% and 10.92-22.18% for individual B, respectively. Clostridium family XI increased 3.50-25.34% in the presence of tetracycline at 40 days for individual C. Principal Component Analysis (PCA) confirmed the pyrosequencing findings of inter-individual variability of the ratio of phylotypes and the effect of tetracycline. Among the 23 tetracycline resistance genes (TRGs) screened, four tet genes (tetO, Q, W, and X) were major TRGs in control and tetracycline-dosed fecal samples. A variable to slight increase of copy number of TRGs appeared to be related to tetracycline treatment, interindividual variability and duration of exposure. Despite, the inherent variability of the intestinal microbiota observed among or within individuals, this pilot study contributes to the knowledge base of the impact of low residue concentrations of tetracycline on the human intestinal microbiome on the potential for antimicrobial resistance.

Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins.

Nanomaterial synthesis and handling in a non-sterile environment can result in the final product becoming contaminated with bacterial endotoxin or lipopolysaccharides (LPB). During toxicological testing, the effects caused by endotoxin-contaminated nanomaterials can be misinterpreted in the end-point analysis (such as cytotoxicity and immune responses) and could result in erroneous conclusions. The objective of this study was twofold: (i) to test different carbon-based nanomaterials (CBNs) [pristine graphene and multi-wall carbon nanotubes (MWCNTs)] for the presence of endotoxin and develop strategies for depyrogenation, and (ii) to compare the immune response exhibited by macrophages after exposure to native CBNs versus depyrogenated CBNs. The gel-clot limulus amebocyte lysate (LAL) and chromogenic-based LAL assays were used to detect endotoxins. Results revealed that the CBNs contained greater amounts of endotoxin than are approved by major regulatory agencies (0.5 EU ml-1 ). Three repeated cycles of autoclaving reduced the endotoxin in the test materials. Macrophages were incubated with pyrogenated and depyrogenated pristine graphene and MWCNTs to test differences in phagocytosis, cytotoxicity, and expression of genes involved in macrophage activation. The uptake of depyrogenated CBNs was significantly reduced as compared with pyrogenated CBNs. Exposure of macrophages to depyrogenated CBNs resulted in a distinct pattern of gene expression for TLR signaling, NOD-like receptor signaling, and downstream signal transduction molecules. Furthermore, macrophages exposed to both types of CBNs showed the downregulation of TLR5 and NLRC4 inflammasomes. The results of this study reaffirm that assessment of endotoxin and other bacterial contamination is critical when evaluating the cellular toxicity of nanomaterials. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Size and dose dependent effects of silver nanoparticle exposure on intestinal permeability in an in vitro model of the human gut epithelium.

BACKGROUND: The antimicrobial activity of silver nanoparticles (AgNP) has led to interest in their use in consumer products such as food contact materials, utensils, and storage containers. Incorporation of these materials into items intended for food processing and storage suggests that consumer use of these products could result in gastrointestinal exposure to AgNP, should the nanoparticles migrate from the product. The health impact of AgNP exposure is unknown, especially effects related to intestinal epithelial permeability and barrier function. This study examined the effects of AgNP exposure of different sizes (10, 20, 75 and 110 nm) and doses (20 and 100 µg/mL) on the permeability of T84 human colonic epithelial cells, which serve as an in vitro model of the human gut epithelium. RESULTS: Results showed that effects of AgNP on the T84 epithelial cells were size- and dose-dependent, with the 10 nm AgNP causing the most significant changes. Changes in permeability of the epithelial cell monolayer, as measured by transepithelial electrical resistance, after exposure to 10 nm AgNP were most dramatic at the highest dose (100 µg/mL), but also observed at the lower dose (20 µg/mL). AgNP could be visualized inside cells using transmission electron microscopy and silver was detected in basal wells using inductively coupled plasma-mass spectrometry. Exposure to AgNP significantly affected the expression of genes involved in anchoring tight junctions, cellular proliferation and signaling, endocytosis, and cell-cell adhesion, with the 10 nm AgNP having the greatest effect. CONCLUSIONS: The results of this study show that small-size AgNP have significant effects on intestinal permeability in an in vitro model of the human gastrointestinal epithelium. Such effects have the potential to compromise the integrity of the intestinal epithelium and this disruption of barrier function could have health consequences for the gastrointestinal tract.

Dose and Size-Dependent Antiviral Effects of Silver Nanoparticles on Feline Calicivirus, a Human Norovirus Surrogate.

OBJECTIVES: Silver nanoparticles (AgNPs) as antibacterial agents are incorporated in many consumer products, while the use as antiviral agents is an ongoing area of research. We evaluated the antiviral properties of AgNPs of variable sizes (10, 75, and 110 nm) and doses (25, 50, and 100 µg/mL) at different contact time points against feline calicivirus (FCV), a surrogate for norovirus. MATERIALS AND METHODS: Antiviral effects of the AgNPs were determined by comparing the infectivity of FCV, the appearance of cytopathic effects (CPEs), and the integrity of the viral capsid protein in viral suspension treated with AgNPs with the untreated controls. RESULTS: The 10 nm AgNPs at 50 and 100 µg/mL concentrations inactivated the FCV beyond the limit of detection, resulting in a decrease of up to 6.5 log10 viral titer, prevented development of CPEs, and reduction in the western blot band signal of the viral capsid protein. No significant antiviral effect was observed for the 75 and 110 nm AgNPs. Conclusions and Applications: These results demonstrate that the antiviral effects of AgNPs are both size and dose dependent, thus potential applications of AgNPs as antiviral agents to prevent contamination of foodborne viruses need to consider size and dose effects.

Crystal structure of Mycobacterium tuberculosis polyketide synthase 11 (PKS11) reveals intermediates in the synthesis of methyl-branched alkylpyrones.

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.

Early Developmental Exposure to Triclosan Impacts Fecal Microbial Populations, IgA and Functional Activities of the Rat Microbiome.

Triclosan (TCS), a broad-spectrum antibacterial chemical, is detected in human urine, breast milk, amniotic fluid, and feces; however, little is known about its impact on the intestinal microbiome and host mucosal immunity during pregnancy and early development. Pregnant female rats were orally gavaged with TCS from gestation day (GD) 6 to postpartum (PP) day 28. Offspring were administered TCS from postnatal day (PND) 12 to 28. Studies were conducted to assess changes in the intestinal microbial population (16S-rRNA sequencing) and functional analysis of microbial genes in animals exposed to TCS during pregnancy (GD18), and at PP7, PP28 and PND28. Microbial abundance was compared with the amounts of TCS excreted in feces and IgA levels in feces. The results reveal that TCS decreases the abundance of Bacteroidetes and Firmicutes with a significant increase in Proteobacteria. At PND28, total Operational Taxonomic Units (OTUs) were higher in females and showed correlation with the levels of TCS and unbound IgA in feces. The significant increase in Proteobacteria in all TCS-treated rats along with the increased abundance in OTUs that belong to pathogenic bacterial communities could serve as a signature of TCS-induced dysbiosis. In conclusion, TCS can perturb the microbiome, the functional activities of the microbiome, and activate mucosal immunity during pregnancy and early development.

Evaluation of gender differences in the pharmacokinetics of oral zileuton nanocrystalline formulation using a rat model.

Zileuton is a leukotriene inhibitor used to treat asthma. As a BCS class II drug it exhibits challenges with solubility which likely impact its absorption. As patient gender significantly impacts the pharmacokinetics of many drugs, this study aimed to investigate potential gender-based pharmacokinetic differences after oral zileuton administration in rats. Male and female Sprague Dawley rats received single oral gavage doses of pure zileuton as an active pharmaceutical ingredient (30 mg/kg body weight (bw)), physical mixture (PM; at 30 mg/kg bw of the formulation contains zileuton, kollidon VA64 fine, dowfax2A1 and trehalose), and nanocrystalline formulation of zileuton (NfZ; at 30 mg/kg bw of the formulation). Plasma, tissue, and urine concentrations were quantified using high performance liquid chromatography (HPLC). Noncompartmental pharmacokinetic analysis showed higher zileuton levels in the plasma of female versus male rats across all evaluated forms of zileuton (API, PM, and NfZ). Female rats demonstrated higher peak plasma concentrations (Cmax) and increased area under the plasma concentration-time curve (AUC) relative to males, regardless of formulation. These findings reveal substantial gender disparities in the pharmacokinetics of zileuton in the rat model. This study emphasizes the critical need to evaluate gender differences during preclinical drug development to enable gender-based precision dosing strategies for equivalent efficacy/safety outcomes in male and female patients. Additional studies are warranted to investigate underlying mechanisms of such pharmacokinetic gender divergences.

Evaluation of virulence and antimicrobial resistance in Salmonella enterica serovar Enteritidis isolates from humans and chicken- and egg-associated sources.

Salmonella enterica serovar Enteritidis is a leading cause of salmonellosis throughout the world and is most commonly associated with the consumption of contaminated poultry and egg products. Salmonella Enteritidis has enhanced ability to colonize and persist in extraintestinal sites within chickens. In this study, 54 Salmonella Enteritidis isolates from human patients (n=28), retail chicken (n=9), broiler farms (n=9), and egg production facilities (n=8) were characterized by antimicrobial susceptibility testing, plasmid analysis, genetic relatedness using XbaI and AvrII pulsed-field gel electrophoresis (PFGE), and the presence of putative virulence genes. Nine isolates were evaluated for their abilities to invade and survive in intestinal epithelial and macrophage cell lines. Overall, 56% (n=30) of isolates were resistant to at least one antimicrobial agent tested, yet no isolates showed resistance to more than three antimicrobials. All isolates carried a common ∼55-kb plasmid, with some strains containing additional plasmids ranging from 3 to 50 kb. PFGE analysis revealed five XbaI and AvrII clusters. There were significant overlaps in the PFGE patterns of the isolates from human, chicken, and egg houses. All isolates tested PCR positive for iacP, purR, ttrB, spi4H, rmbA, sopE, invA, sopB, spvB, pagC, msgA, spaN, orgA, tolC, and sifA, and negative for iss, virB4, and sipB. Of the isolates selected for virulence testing, those containing the iron acquisition genes, iutA, sitA, and iucA, and ∼50-kb plasmids demonstrated among the highest levels of macrophage and epithelial cell invasion, which may indicate their importance in pathogenesis.

Evaluation of mucosal immune profile associated with Zileuton nanocrystal-formulated BCS-II drug upon oral administration in Sprague Dawley rats.

Nanocrystal drug formulation involves several critical manufacturing procedures that result in complex structures to improve drug solubility, dissolution, bioavailability, and consequently the efficacy of poorly soluble Biopharmaceutics Classification System (BCS) II and IV drugs. Nanocrystal formulation of an already approved oral drug may need additional immunotoxic assessment due to changes in the physical properties of the active pharmaceutical ingredient (API). In this study, we selected Zileuton, an FDA-approved drug that belongs to BCS-II for nanocrystal formulation. To evaluate the efficacy and mucosal immune profile of the nanocrystal drug, 10-week-old rats were dosed using capsules containing either API alone or nanocrystal formulated Zileuton (NDZ), or with a physical mixture (PM) using flexible oral gavage syringes. Control groups consisted of untreated, or placebo treated animals. Test formulations were administrated to rats at a dose of 30 mg/kg body weight (bw) once a day for 15 days. The rats treated with NDZ or PM had approximately 4.0 times lower (7.5 mg/kg bw) API when compared to the micron sized API treated rats. At the end of treatment, mucosal (intestinal tissue) and circulating cytokines were measured. The immunological response revealed that NDZ decreased several proinflammatory cytokines in the ileal mucosa (Interleukin-18, Tumor necrosis Factor-α and RANTES [regulated upon activation, normal T cell expressed and secreted]). A similar pattern in the cytokine profile was also observed for the micron sized API and PM treated rats. The cytokine production revealed that there was a significant increase in the production of IL-1ß and IL-10 in the females in all experimental groups. Additionally, NDZ showed an immunosuppressive effect on proinflammatory cytokines both locally and systemically, which was similar to the response in micron sized API treated rats. These findings indicate that NDZ significantly decreased several proinflammatory cytokines and it displays less immunotoxicity, probably due to the nanocrystal formulation. Thus, the nanocrystal formulation is more suitable for oral drug delivery, as it exhibited better efficacy, safety, and reduced toxicity.

Structural analysis of VirD4 a type IV ATPase encoded by transmissible plasmids of Salmonella enterica isolated from poultry products.

Bacterial species have evolved with a wide variety of cellular devices, and they employ these devices for communication and transfer of genetic materials and toxins. They are classified into secretory system types I to VI based on their structure, composition, and functional activity. Specifically, the bacterial type IV secretory system (T4SS) is a more versatile system than the other secretory systems because it is involved in the transfer of genetic materials, proteins, and toxins to the host cells or other bacterial species. The T4SS machinery is made up of several proteins with distinct functions and forms a complex which spans the inner and outer membranes. This secretory machinery contains three ATPases that are the driving force for the functionality of this apparatus. At the initial stage of the secretion process, the selection of substrate molecules and processing occurs at the cytoplasmic region (also known as relaxosome), and then transfer mechanisms occur through the secretion complex. In this process, the VirD4 ATPase is the first molecule that initiates substrate selection, which is subsequently delivered to the secretory machinery. In the protein data bank (PDB), no structural information is available for the VirD4 ATPase to understand the functional property. In this manuscript, we have modeled VirD4 structure in the Gram-negative bacterium Salmonella enterica and described the predicted functional importance. The sequence alignment shows that VirD4 of S. enterica contains several insertion regions as compared with the template structure (pdb:1E9R) used for homology modeling. In this study, we hypothesized that the insertion regions could play a role in the flexible movement of the hexameric unit during the relaxosome processing or transfer of the substrate.

Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations.

The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS-ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix and in the conformation of the α3-α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4-6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS-ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS-ADP adopt different conformations depending upon the pH conditions of the crystallization solution.