Photocatalytic inactivation of E-Coli by ZnO-Ag nanoparticles under solar radiation

Porous and fluffy ZnO photocatalysts were successfully prepared via simple solution based combustion synthesis method. The photocatalytic inactivation of Escherichia coli bacteria was studied separately for both Ag substituted and impregnated ZnO under irradiation of natural solar light. A better understanding of substitution and impregnation of Ag was obtained by Raman spectrum and X-ray photoelectron analysis. The reaction parameters such as catalyst dose, initial bacterial concentration and effect of hydroxyl radicals via H2O2 addition were also studied for ZnO catalyst. Effective inactivation was observed with 0.25 g L-1 catalyst loading having 10(9) CFU mL(-1) bacterial concentration. With an increase in molarity of H2O2, photocatalytic inactivation was enhanced. The effects of different catalysts were studied, and highest bacterial killing was observed by Ag impregnated ZnO with 1 atom% Ag compared to Ag substituted ZnO. This enhanced activity can be attributed to effective charge separation that is supported by photoluminescence studies. The kinetics of reaction in the presence of different scavengers showed that reaction is significantly influenced by the presence of hole and hydroxyl radical scavenger with high efficiency.

Biofunctionalized surface-modified silver nanoparticles for gene delivery

Silver nanoparticles (AgNPs) find use in different biomedical applications including wound healing and cancer. We propose that the efficacy of the nanoparticles can be further augmented by using these particles for gene delivery applications. The objective of this work was to engineer biofunctionalized stable AgNPs with good DNA binding ability for efficient transfection and minimal toxicity. Herein, we report on the one-pot facile green synthesis of polyethylene glycol (PEG) stabilized chitosan-g-polyacrylamide modified AgNPs. The size of the PEG stabilized AgNPs was 38 +/- 4 nm with a tighter size distribution compared to the unstabilized nanoparticles which showed bimodal distribution of particle sizes of 68 +/- 5 nm and 7 +/- 4 nm. To enhance the efficiency of gene transfection, the Arg-Gly-Asp-Ser (RGDS) peptide was immobilized on the silver nanoparticles. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 +/- 4% and 30 +/- 3% in HeLa and A549 cells, respectively, and significantly higher than 34 +/- 3% and 23 +/- 2%, respectively, with the use of polyethyleneimine (25 kDa). These nanoparticles were found to induce minimal cellular toxicity. Differences in cellular uptake mechanisms with RGDS immobilization resulting in improved efficiency are elucidated. This study presents biofunctionalized AgNPs for potential use as efficient nonviral carriers for gene delivery with minimal cytotoxicity toward augmenting the therapeutic efficacy of AgNPs used in different biomedical products.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (>= 1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB. (C) 2015 Published by Elsevier B.V.

A self-assembling polycationic nanocarrier that exhibits exceptional gene transfection efficiency

The lack of an efficient and safe carrier is a major impediment in the field of gene therapy. Although gelatin (GT), a naturally derived polymer, is widely used in drug delivery applications, it is unable to bind DNA efficiently. In this study, a novel polycationic gene carrier was prepared by conjugation of low molecular weight polyethyleneimine (LPEI) with GT through 4-bromonaphthaleic anhydride as a coupling agent to avoid self crosslinking. Self-assembly of LPEI conjugated GT (GT-LPEI) with plasmid DNA (pDNA) yielded nanoparticles with high gene complexation ability to form similar to 250 nm cylindrical nanoparticles with a zeta potential of similar to 27 mV. GT-LPEI showed exceptionally high transfection efficiency (> 90%) in various mammalian cells including primary stem cells with minimal cytotoxicity. The transfection efficiency of GT-LPEI significantly surpassed that of many commercial reagents. The high gene transfection expression was confirmed in vivo. Thus, GT-LPEI is shown to be a promising nonviral carrier for potential use in gene therapy.

A novel gene THSD7A is associated with obesity

Body mass index (BMI) is a non-invasive measurement of obesity. It is commonly used for assessing adiposity and obesity-related risk prediction. Genetic differences between ethnic groups are important factors, which contribute to the variation in phenotypic effects. India inhabited by the first out-of-Africa human population and the contemporary Indian populations are admixture of two ancestral populations; ancestral north Indians (ANI) and ancestral south Indians (ASI). Although ANI are related to Europeans, ASI are not related to any group outside Indian-subcontinent. Hence, we expect novel genetic loci associated with BMI. In association analysis, we found eight genic SNPs in extreme of distribution (P <= 3.75 x 10(-5)), of which WWOX has already been reported to be associated with obesity-related traits hence excluded from further study. Interestingly, we observed rs1526538, an intronic SNP of THSD7A; a novel gene significantly associated with obesity (P = 2.88 x 10(-5), 8.922 x 10(-6) and 2.504 x 10(-9) in discovery, replication and combined stages, respectively). THSD7A is neural N-glycoprotein, which promotes angiogenesis and it is well known that angiogenesis modulates obesity, adipose metabolism and insulin sensitivity, hence our result find a correlation. This information can be used for drug target, early diagnosis of obesity and treatment.

Roles for Transcription Factors Sp1, NF-kappa B, IRF3, and IRF7 in Expression of the Human IFNL4 Gene

The expression of a biologically active human IFN4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFN4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFN4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

Background: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. Results: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited similar to 2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by similar to 3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by similar to 10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. Conclusions: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.