Design,synthesis, DNA-binding affinity,cytotoxicity, apoptosis,and cell cycle arrest of Ru(II) polypyridyl complexes

A novel polypyridyl ligand CNPFIP (CNPFIP = 2-(5(4-chloro-2-nitrophenyl)furan-2-yl)-1H-imidazo[4,5f][1,10]phenanthroline) and its mononuclear Ru(II) polypyridyl complexes of [Ru(phen)₂CNPFIP]²⁺(1) (phen = 1,10-phenanthroline), [Ru(bpy)₂CNPFIP]²⁺(2) (bpy = 2,2′-bipyridine), and [Ru(dmb)₂CNPFIP]²⁺(3) (dmb = 4,4′-dimethyl-2,2′-bipyridine) have been synthesized successfully and characterized thoroughly by elemental analysis, UV/Vis, IR, NMR, and ESI-MS. The interaction of the Ru(II) complexes with calf thymus DNA (CT-DNA) was investigated by absorption titration, fluorescence, viscosity measurements. The experimental results suggest that three complexes bind to CT-DNA through an intercalative mode and the DNA-binding affinity of complex 1 is greater than that of complexes 2 and 3. The photocleavage of plasmid pBR322 DNA by ruthenium complexes 1, 2, and 3 was investigated. We have also tested three complexes for their antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria. The in vitro cytotoxicity of these complexes was evaluated by MTT assay, and complex 1 shows higher cytotoxicity than 2 and 3 on HeLa cells. The induced apoptosis and cell cycle arrest of HeLa cells were investigated by flow cytometry for 24 h. The molecular docking of ruthenium complexes 1, 2, and 3 with the active site pocket residues of human DNA TOP1 was performed using LibDock.     

Mechanism of Multivalent Nanoparticle Encounter with HIV-1 for Potency Enhancement of Peptide Triazole Virus Inactivation

Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.     

Environmental Factors Associated with High Fly Densities and Diarrhea in Vellore,India

Diarrhea causes significant morbidity and mortality in Indian children under 5 years of age. Flies carry enteric pathogens and may mediate foodborne infections. In this study, we characterized fly densities as a determinant of infectious diarrhea in a longitudinal cohort of 160 urban and 80 rural households with 1,274 individuals (27% under 5 years of age) in Vellore, India. Household questionnaires on living conditions were completed at enrollment. Fly abundance was measured during the wet and dry seasons using fly ribbons placed in kitchens. PCRs for enteric bacteria, viruses, and protozoa were performed on 60 fly samples. Forty-three (72%) fly samples were positive for the following pathogens: norovirus (50%), Salmonella spp. (46.7%), rotavirus (6.7%), and Escherichia coli (6.7%). Ninety-one episodes of diarrhea occurred (89% in children under 5 years of age). Stool pathogens isolated in 24 of 77 (31%) samples included E. coli, Shigella spp., Vibrio spp., Giardia, Cryptosporidium, and rotavirus. Multivariate log-linear models were used to explore the relationships between diarrhea and fly densities, controlling for demographics, hygiene, and human-animal interactions. Fly abundance was 6 times higher in rural than urban sites (P < 0.0001). Disposal of garbage close to homes and rural living were significant risk factors for high fly densities. The presence of latrines was protective against high fly densities and diarrhea. The adjusted relative risks of diarrheal episodes and duration of diarrhea, associated with fly density at the 75th percentile, were 1.18 (95% confidence interval [CI], 1.03 to 1.34) and 1.15 (95% CI, 1.02 to 1.29), respectively. Flies harbored enteric pathogens, including norovirus, a poorly documented pathogen on flies.     

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.     

Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation

Lignocellulose-derived microbial inhibitors such as furfural and 5-hydroxymethyl furfural adversely affect fermentation of lignocellulosic biomass hydrolysates to fuels and chemicals due to their toxicity on fermenting microbes. To harness the potential of lignocellulose as a cheap source of fermentable sugars, in situ detoxification of furfural and other lignocellulose-derived microbial inhibitors is essential. To enhance in situ detoxification and tolerance of furfural by Clostridium beijerinckii NCIMB 8052 during acetone-butanol-ethanol (ABE) fermentation, the effect of glycerol on NADH/NADPH generation and ABE production by furfural (4, 5, and 6 g/L)-challenged cultures was investigated in this study. In all instances, beneficial outcomes were observed. For example, the fermentation medium supplemented with glycerol and subjected to 5 g/L furfural elicited up to 1.8- and 3-fold increases, respectively, in NADH and NADPH levels in C. beijerinckii 8052 relative to the control culture. These critical changes are the likely underpinnings for the glycerol-mediated 2.3-fold increase in the rate of detoxification of 5 g/L furfural, substrate consumption, and ABE production compared to the unsupplemented medium. Collectively, these results demonstrate that increased intracellular NADH/NADPH in C. beijerinckii 8052 due to glycerol utilization engenders favorable effects on many aspects of cellular metabolism, including enhanced furfural reduction and increased ABE production.     

Biodegradation of nicotine by a novel nicotine-degrading bacterium, Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates

Tobacco wastes that contain nicotine alkaloids are harmful to human health and the environment. In the investigation, a novel nicotine-biodegrading bacterium TND35 was isolated and identified as Pseudomonas plecoglossicida on the basis of phenotypic, biochemical characteristics and 16S rRNA sequence homology. We have studied the nicotine biodegradation potential of strain TND35 by detecting the intermediate metabolites using an array of approaches such as HPLC, GC–MS, NMR and FT-IR. Biotransformation metabolites, N-methylmyosmine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and other three new intermediate metabolites namely, 3,5-bis (1-methylpyrrolidin-2-yl) pyridine, 2,3-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol and 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one have been identified. Interestingly, these intermediate metabolites suggest that the strain TND35 employs a novel nicotine biodegradation pathway, which is different from the reported pathways of Aspergillus oryzae 112822, Arthrobacter nicotinovorans pAO1, Agrobacterium tumefaciens S33 and other species of Pseudomonas. The metabolite, HPB reported in this study can also be used as biochemical marker for tobacco related cancer studies.     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

How Did the TB Patients Reach DOTS Services in Delhi? A Study of Patient Treatment Seeking Behavior

Setting

Revised National Tuberculosis Control Programme (RNTCP), Delhi, India.

Objective

To ascertain the number and sequence of providers visited by TB patients before availing treatment services from DOTS; to describe the duration between onset of symptoms to treatment.

Study design

A cross sectional, qualitative study. Information was gathered through in-depth interviews of TB patients registered during the month of Oct, 2012 for availing TB treatment under the Revised National TB Control Programme from four tuberculosis diagnosis and treatment centers in Delhi.

Results

Out of the 114 patients who registered, 108 participated in the study. The study showed that informal providers and retail chemists were the first point of contact and source of clinical advice for two-third of the patients, while the rest sought medical care from qualified providers directly. Most patients sought medical care from more than two providers, before being diagnosed as TB. Female TB patients and patients with extra-pulmonary TB had long mean duration between onset of symptoms to initiation of treatment (6.3 months and 8.4 months respectively).

Conclusion

The pathways followed by TB patients, illustrated in this study, provide valuable lessons on the importance of different types of providers (both formal and informal) in the health system in a society like India and the delays in the diagnosis and treatment of tuberculosis.     

Thermodynamics of coupled folding in the interaction of archaeal RNase P proteins RPP21 and RPP29

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg2?-dependent cleavage of the 5' leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5-RPP30 and RPP21-RPP29. Nuclear magnetic resonance studies of the Pyrococcus furiosus RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔC(p)), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess ΔC(p). We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔC(p). These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies.