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Type I interferons (IFNα, IFNβ) are key regulators of innate and adaptive immunity, modulating the severity of both viral and bacterial infections. While type I IFN signaling leads to improved outcomes in viral infections, its role in bacterial infections is more contextual and depends on the specific pathogen and route of infection. Given the limited evidence on whether type I IFN signaling affects enteric bacterial pathogens, we investigated the role of this signaling pathway in Salmonella enterica serovar Typhimurium (S. typhimurium)-induced colitis. Comparing mice deficient in IFNAR1- the common receptor for IFNα and IFNβ- with wild-type mice, we found that type I IFN signaling leads to more rapid death, more severe colonic inflammation, higher serum levels of pro-inflammatory cytokines, and greater bacterial dissemination. Specific ablation of plasmacytoid dendritic cells (pDCs), which are prominent producers of type I IFNs in antiviral responses, did not alter survival after infection. This result established that pDCs do not play a major role in the pathogenesis of S. typhimurium colitis. Flow cytometric analysis of macrophages and conventional dendritic cells (cDCs) during active colitis demonstrated an increase in CD11c- macrophages and CD103+ cDCs in the colon of Ifnar1-/- animals. Interestingly, cells expressing the anti-inflammatory cytokine receptor IL-10R are more abundant within these subsets in Ifnar1-/- than in wild-type mice. Moreover, blockade of IL-10R in Ifnar1-/- mice increased their susceptibility to S. typhimurium colitis, suggesting that altered numbers of these immunoregulatory cells may underlie the difference in disease severity. This cross-talk between type I IFN and IL-10R signaling pathways may represent a key host cellular mechanism to investigate further in order to unravel the balance between pathogenic inflammation and homeostasis of the colon. Taken together, our data clearly demonstrate that type I IFN signaling is pathogenic in S. typhimurium colitis.

Interleukin (IL)-1 signaling plays a critical role in intestinal immunology. Here, we report that the major population of intestinal lamina propria lymphocytes expressing IL-1 receptor 1 (IL-1R1) is the lymphoid tissue inducer (LTi)-like cell, a type of innate lymphoid cell. These cells are significant producers of IL-22, and this IL-22 production depends on IL-1R1 signaling. LTi-like cells are required for defense against Salmonella enterica serovar Typhimurium. Moreover, colonic LTi-like cell numbers depend on the presence of the intestinal microbiota. LTi-like cells require IL-1R1 for production of protective cytokines and confer protection in infectious colitis, and their cell numbers in the colon depend upon having a microbiome.

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m(2) (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m(2) (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m(2); p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m(2) for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.

Recent studies have identified links between phospholipid composition and altered cellular functions in animal models of obesity, but the involvement of phospholipid biosynthesis genes in human obesity are not well understood. We analyzed the transcript of four phospholipid biosynthesis genes in adipose and muscle from 170 subjects. We examined publicly available genome-wide association data from the GIANT and MAGIC cohorts to investigate the association of SNPs in these genes with obesity and glucose homeostasis traits, respectively. Trait-associated SNPs were genotyped to evaluate their roles in regulating expression in adipose. In adipose tissue, expression of PEMT, PCYT1A, and PTDSS2 were positively correlated and PCYT2 was negatively correlated with percent fat mass and body mass index (BMI). Among the polymorphisms in these genes, SNP rs4646404 in PEMT showed the strongest association (p = 3.07E-06) with waist-to-hip ratio (WHR) adjusted for BMI. The WHR-associated intronic SNP rs4646343 in the PEMT gene showed the strongest association with its expression in adipose. Allele "C" of this SNP was associated with higher WHR (p = 2.47E-05) and with higher expression (p = 4.10E-04). Our study shows that the expression of PEMT gene is high in obese insulin-resistant subjects. Intronic cis-regulatory polymorphisms may increase the genetic risk of obesity by modulating PEMT expression.

Acetyl Coenzyme A carboxylase β (ACACB) is the rate-limiting enzyme in fatty acid oxidation, and continuous fatty acid oxidation in Acacb knock-out mice increases insulin sensitivity. Systematic human studies have not been performed to evaluate whether ACACB variants regulate gene expression and insulin sensitivity in skeletal muscle and adipose tissues. We sought to determine whether ACACB transcribed variants were associated with ACACB gene expression and insulin sensitivity in non-diabetic African American (AA) and European American (EA) adults.ACACB transcribed single nucleotide polymorphisms (SNPs) were genotyped in 105 EAs and 46 AAs whose body mass index (BMI), lipid profiles and ACACB gene expression in subcutaneous adipose and skeletal muscle had been measured. Allelic expression imbalance (AEI) was assessed in lymphoblast cell lines from heterozygous subjects in an additional EA sample (n = 95). Selected SNPs were further examined for association with insulin sensitivity in a cohort of 417 EAs and 153 AAs.ACACB transcribed SNP rs2075260 (A/G) was associated with adipose ACACB messenger RNA expression in EAs and AAs (p = 3.8×10(-5), dominant model in meta-analysis, Stouffer method), with the (A) allele representing lower gene expression in adipose and higher insulin sensitivity in EAs (p = 0.04). In EAs, adipose ACACB expression was negatively associated with age and sex-adjusted BMI (r = -0.35, p = 0.0002).Common variants within the ACACB locus appear to regulate adipose gene expression in humans. Body fat (represented by BMI) may further regulate adipose ACACB gene expression in the EA population.

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress-induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

Many different kind of fluids in a wide variety of industries exist, such as two-phase and three-phase. Various combinations of them can be expected and gas-oil-water is one of the most common flows. Measuring the volume fraction of phases without separation is vital in many aspects, one of which is financial issues. Many methods are utilized to ascertain the volumetric proportion of each phase. Sensors based on measuring capacity are so popular because this kind of sensor operates seamlessly and autonomously without necessitating any form of segregation or disruption for measuring in the process. Besides, at the present moment, Artificial intelligence (AI) can be nominated as the most useful tool in several fields, and metering is no exception. Also, three main type of regimes can be found which are annular, stratified, and homogeneous. In this paper, volume fractions in a gas-oil-water three-phase homogeneous regime are measured. To accomplish this objective, an Artificial Neural Network (ANN) and a capacitance-based sensor are utilized. To train the presented network, an optimized sensor was implemented in the COMSOL Multiphysics software and after doing a lot of simulations, 231 different data are produced. Among all obtained results, 70 percent of them (161 data) are awarded to the train data, and the rest of them (70 data) are considered for the test data. This investigation proposes a new intelligent metering system based on the Multilayer Perceptron network (MLP) that can estimate a three-phase water-oil-gas fluid's water volume fraction precisely with a very low error. The obtained Mean Absolute Error (MAE) is equal to 1.66. This dedicates the presented predicting method's considerable accuracy. Moreover, this study was confined to homogeneous regime and cannot measure void fractions of other fluid types and this can be considered for future works. Besides, temperature and pressure changes which highly temper relative permittivity and density of the liquid inside the pipe can be considered for another future idea.

Type I interferons (IFNα, IFNβ) are key regulators of innate and adaptive immunity, modulating the severity of both viral and bacterial infections. While type I IFN signaling leads to improved outcomes in viral infections, its role in bacterial infections is more contextual and depends on the specific pathogen and route of infection. Given the limited evidence on whether type I IFN signaling affects enteric bacterial pathogens, we investigated the role of this signaling pathway in Salmonella enterica serovar Typhimurium (S. typhimurium)–induced colitis. Comparing mice deficient in IFNAR1- the common receptor for IFNα and IFNβ- with wild-type mice, we found that type I IFN signaling leads to more rapid death, more severe colonic inflammation, higher serum levels of pro-inflammatory cytokines, and greater bacterial dissemination. Specific ablation of plasmacytoid dendritic cells (pDCs), which are prominent producers of type I IFNs in antiviral responses, did not alter survival after infection. This result established that pDCs do not play a major role in the pathogenesis of S. typhimurium colitis. Flow cytometric analysis of macrophages and conventional dendritic cells (cDCs) during active colitis demonstrated an increase in CD11c- macrophages and CD103+ cDCs in the colon of Ifnar1-/- animals. Interestingly, cells expressing the anti-inflammatory cytokine receptor IL-10R are more abundant within these subsets in Ifnar1-/- than in wild-type mice. Moreover, blockade of IL-10R in Ifnar1-/- mice increased their susceptibility to S. typhimurium colitis, suggesting that altered numbers of these immunoregulatory cells may underlie the difference in disease severity. This cross-talk between type I IFN and IL-10R signaling pathways may represent a key host cellular mechanism to investigate further in order to unravel the balance between pathogenic inflammation and homeostasis of the colon. Taken together, our data clearly demonstrate that type I IFN signaling is pathogenic in S. typhimurium colitis. [ABSTRACT FROM AUTHOR]

We propose a method to predict when a woman will develop breast cancer (BCa) from her lifestyle and health history features. To address this objective, we use data from the Alberta's Tomorrow Project of 18,288 women to train Individual Survival Distribution (ISD) models to predict an individual's Breast-Cancer-Onset (BCaO) probability curve. We show that our three-step approach-(1) filling missing data with multiple imputations by chained equations, followed by (2) feature selection with the multivariate Cox method, and finally, (3) using MTLR to learn an ISD model-produced the model with the smallest L1-Hinge loss among all calibrated models with comparable C-index. We also identified 7 actionable lifestyle features that a woman can modify and illustrate how this model can predict the quantitative effects of those changes-suggesting how much each will potentially extend her BCa-free time. We anticipate this approach could be used to identify appropriate interventions for individuals with a higher likelihood of developing BCa in their lifetime.

Do health and economic shocks exacerbate prejudice towards racial/ethnic minority groups? We investigate this question in the wake of the COVID-19 pandemic by collecting nationally representative survey data with an embedded experiment. Results show that priming COVID-19 salience has an immediate impact: compared to the control group, respondents in the treatment group reported increased prejudice towards East Asian and Hispanic colleagues. East Asians in the treatment group faced higher prejudicial responses from Americans living in counties with higher COVID-19 infections and those who lost jobs due to COVID-19, and fewer prejudicial responses in counties with a higher concentration of Asians. These results point to the salience of COVID-19 fueled health and economic insecurities in shaping prejudicial attitudes, specifically towards East Asians. County-level socioeconomic factors did not moderate the increased prejudicial attitudes toward Hispanics in the workplace. These findings highlight a dimension of prejudice, intensified during the pandemic, which has been largely underreported and therefore missing from the current discourse on this important topic.

Introduction Cambodia aims to eliminate malaria by 2025, however tackling Plasmodium vivax (P.v) presents multiple challenges. The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency has prevented the deployment of 8-aminoquinolones for “radical cure”, due to the risk of severe haemolysis. Patients with P. vivax have therefore continued to experience recurrent relapses leading to cumulative health and socioeconomic burden. The recent advent of point of care testing for G6PD deficiency has made radical cure a possibility, however at the time of the study lack of operational experience and guidance meant that they had not been introduced. This study therefore aimed to design, implement and evaluate a new care pathway for the radical cure of P.vivax. Methods This implementation study took place in Pursat province, Western Cambodia. The interventions were co-developed with key stakeholders at the national, district, and local level, through a continuous process of consultations as well as formal meetings. Mixed methods were used to evaluate the feasibility of the intervention including its uptake (G6PD testing rate and the initiation of primaquine treatment according to G6PD status); adherence (self-reported); and acceptability, using quantitative analysis of primary and secondary data as well as focus group discussions and key informant interviews. Results The co-development process resulted in the design of a new care pathway with supporting interventions, and a phased approach to their implementation. Patients diagnosed with P.v infection by Village Malaria Workers (VMWs) were referred to local health centres for point-of-care G6PD testing and initiation of radical cure treatment with 14-day or 8-week primaquine regimens depending on G6PD status. VMWs carried out follow-up in the community on days 3, 7 and 14. Supporting interventions included training, community sensitisation, and the development of a smartphone and tablet application to aid referral, follow-up and surveillance. The testing rate was low initially but increased rapidly over time, reflecting the deliberately cautious phased approach to implementation. In total 626 adults received G6PD testing, for a total of 675 episodes. Of these 555 occurred in patients with normal G6PD activity and nearly all (549/555, 98.8%) were initiated on PQ14. Of the 120 with deficient/intermediate G6PD activity 61 (50.8%) were initiated on PQ8W. Self-reported adherence was high (100% and 95.1% respectively). No severe adverse events were reported. The pathway was found to be highly acceptable by both staff and patients. The supporting interventions and gradual introduction were critical to success. Challenges included travel to remote areas and mobility of P.v patients. Conclusion The new care pathway with supporting interventions was highly feasible with high levels of uptake, adherence and acceptability in this setting where high prevalence of G6PD deficiency is high and there is a well-established network of VMWs. Scaling up of the P.v radical cure programme is currently underway in Cambodia and a decline in reduction in the burden of malaria is being seen, bringing Cambodia a step closer to elimination.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 μM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer's therapy, was shown to have antiviral activity between 10 to 66 μM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

Interleukin (IL)-1 signaling plays a critical role in intestinal immunology. Here, we report that the major population of intestinal lamina propria lymphocytes expressing IL-1 receptor 1 (IL-1R1) is the lymphoid tissue inducer (LTi)-like cell, a type of innate lymphoid cell. These cells are significant producers of IL-22, and this IL-22 production depends on IL-1R1 signaling. LTi-like cells are required for defense against Salmonella enterica serovar Typhimurium. Moreover, colonic LTi-like cell numbers depend on the presence of the intestinal microbiota. LTi-like cells require IL-1R1 for production of protective cytokines and confer protection in infectious colitis, and their cell numbers in the colon depend upon having a microbiome. [ABSTRACT FROM AUTHOR]

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m2 (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m2 (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m2; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m2 for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN. [ABSTRACT FROM AUTHOR]

The present study provides first evidence on the role of plasma gelsolin in protecting pulmonary thromboembolism and thrombosis in a mouse model. Gelsolin is the most abundant actin depolymerizing protein in plasma and its significantly depleted values have been reported in metabolic disorders including cardiovascular diseases and myocardial infarction. Though gelsolin replacement therapy (GRT) has been shown to be effective in some animal models, no such study has been reported for thrombotic diseases that are acutely in need of bio-therapeutics for immediate and lasting relief. Here, using mice model and recombinant human gelsolin (rhuGSN), we demonstrate the antithrombotic effect of gelsolin in ferric chloride induced thrombosis in carotid artery and thrombin induced acute pulmonary thromboembolism. In thrombosis model, arterial occlusion time was significantly enhanced upon subcutaneous (SC) treatment with 8 mg of gelsolin per mice viz. 15.83 minutes vs. 8 minutes in the placebo group. Pertinently, histopathological examination showed channel formation within the thrombi in the carotid artery following injection of gelsolin. Fluorescence molecular tomography imaging further confirmed that administration of gelsolin reduced thrombus formation following carotid artery injury. In thrombin-induced acute pulmonary thromboembolism, mice pretreated with aspirin or gelsolin showed 100 and 83.33% recovery, respectively. In contrast, complete mortality of mice was observed in vehicle treated group within 5 minutes of thrombin injection. Overall, our studies provide conclusive evidence on the thrombo-protective role of plasma gelsolin in mice model of pulmonary thromboembolism and thrombosis.

Plasma gelsolin levels significantly decline in several disease conditions, since gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histopathological examinations of tissue revealed administration of gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of gelsolin can effectively reduce the pain and inflammation in mice model.

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-β signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-β in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-β in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-β followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-β treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-β together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-β induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut +TGF-β treated hGF cells. In concordance, areca nut along with TGF-β enhanced fibroblast activation as demonstrated by potentiation of αSMA, γSMA and collagen gel contraction by hGF cells. Furthermore, TGF-β secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

BACKGROUND: Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. PRINCIPAL FINDINGS: Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. CONCLUSIONS AND SIGNIFICANCE: The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.

BACKGROUND: Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells. METHODS: Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-(3)H] incorporation. RESULTS: CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo. CONCLUSION: This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug.

The decline of freshwater fish biodiversity corroborates the trends of unsustainable pesticide usage and increase of disease incidence in the last few decades. Little is known about the role of nonlethal exposure to pesticide, which is not uncommon, and concurrent infection of opportunistic pathogens in species decline. Moreover, preventative measures based on current knowledge of stress biology and an emerging role for epigenetic (especially methylation) dysregulation in toxicity in fish are lacking. We herein report the protective role of lipotropes/methyl donors (like choline, betaine and lecithin) in eliciting primary (endocrine), secondary (cellular and hemato-immunological and histoarchitectural changes) and tertiary (whole animal) stress responses including mortality (50%) in pesticide-exposed (nonlethal dose) and pathogen-challenged fish. The relative survival with betaine and lecithin was 10 and 20 percent higher. This proof of cause-and-effect relation and physiological basis under simulated controlled conditions indicate that sustained stress even due to nonlethal exposure to single pollutant enhances pathogenic infectivity in already nutritionally-stressed fish, which may be a driver for freshwater aquatic species decline in nature. Dietary lipotropes can be used as one of the tools in resurrecting the aquatic species decline.

Earlier we identified wheat (Triticum aestivum L.) chromosome 3A as a major determinant of grain yield and its component traits. In the present study, a high-density genetic linkage map of 81 chromosome 3A-specific markers was developed to increase the precision of previously identified yield component QTLs, and to map QTLs for biomass-related traits. Many of the previously identified QTLs for yield and its component traits were confirmed and were localized to narrower intervals. Four novel QTLs one each for shoot biomass (Xcfa2262-Xbcd366), total biomass (wPt2740-Xcfa2076), kernels/spike (KPS) (Xwmc664-Xbarc67), and Pseudocercosporella induced lodging (PsIL) were also detected. The major QTLs identified for grain yield (GY), KPS, grain volume weight (GVWT) and spikes per square meter (SPSM) respectively explained 23.2%, 24.2%, 20.5% and 20.2% of the phenotypic variation. Comparison of the genetic map with the integrated physical map allowed estimation of recombination frequency in the regions of interest and suggested that QTLs for grain yield detected in the marker intervals Xcdo549-Xbarc310 and Xpsp3047-Xbarc356 reside in the high-recombination regions, thus should be amenable to map-based cloning. On the other hand, QTLs for KPS and SPSM flanked by markers Xwmc664 and Xwmc489 mapped in the low-recombination region thus are not suitable for map-based cloning. Comparisons with the rice (Oryza sativa L.) genomic DNA sequence identified 11 candidate genes (CGs) for yield and yield related QTLs of which chromosomal location of two (CKX2 and GID2-like) was confirmed using wheat aneuploids. This study provides necessary information to perform high-resolution mapping for map-based cloning and for CG-based cloning of yield QTLs.

Human mesenchymal stem cells (hMSCs) present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP) activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1) and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20) were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1) were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium "primed" MSCs for osteoblastic differentiation.

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.

Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy.

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encode the peroxisomal membrane protein. We conducted a genomic and protein expression study of susceptibility gene with its clinical and biochemical analysis. To the best of our knowledge this is the first preliminary comprehensive study in Indian population that identified novel mutations and SNPs in a relatively large group. We screened 17 Indian indigenous X-linked adrenoleukodystrophy cases and 70 controls for mutations and SNPs in the exonic regions (including flanking regions) of ABCD1 gene by direct sequencing with ABI automated sequencer along with Western blot analysis of its endogenous protein, ALDP, levels in peripheral blood mononuclear cells. Single germ line mutation was identified in each index case in ABCD1 gene. We detected 4 novel mutations (2 missense and 2 deletion/insertion) and 3 novel single nucleotide polymorphisms. We observed a variable protein expression in different patients. These findings were further extended to biochemical and clinical observations as it occurs with great clinical expression variability. This is the first major study in this population that presents a different molecular genetic spectrum as compared to Caucasian population due to geographical distributions of ethnicity of patients. It enhances our knowledge of the causative mutations of X-ALD that grants holistic base to develop effective medicine against X-ALD.