68Ga‐labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators

The dual interaction with integrins and neuropilin‐1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with ⁶⁸GaCl₃. Negatively charged ⁶⁸Ga‐DOTAGA‐iRGD and neutral ⁶⁸Ga‐NODAGA‐iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral ⁶⁸Ga‐NODAGA‐iRGD, but ⁶⁸Ga‐DOTAGA‐iRGD exhibited better tumor‐to‐blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.     

Isolation and characterization of biofilm-forming bacteria and associated extracellular polymeric substances from oral cavity

The current work deals with the studies on characterization of two biofilm-forming bacteria isolated from the oral cavity. The major constituent of biofilm other than bacterial cells is the extracellular polymeric substance (EPS) matrix, which is secreted by the bacterial cells themselves. Physical properties of biofilms such as attachment, mechanical strength, antibiotic resistance can be attributed to EPS matrix. Molecular phylogeny confirmed these two isolates as Pseudomonas aeruginosa and Bacillus subtilis. It was observed that cell attachment in both the strains was maximal when xylose was used as the sole carbon source. The EPS characterization result indicated the presence of a macromolecular complex constituting of carbohydrate, protein, lipids and nucleic acids. Test for biofilm formation in the presence of metal salts of iron and zinc showed moderate to high inhibition of biofilm formation. However, calcium, iron and copper have been found to enhance biofilm growth significantly. There was more than 50 % increase in biofilm growth by P. aeruginosa with an increase in calcium concentration up to 80 ppm (Two tailed t-test P?<?0.05), whereas ≥ 15 % increase in biofilm growth by B. subtilis was observed in the presence of 80 ppm of calcium. However, variations were significant (Two way ANOVA, P?<?0.01) between different metals in different concentrations. In this study, attempts have been made to examine the effect of different carbon sources and physiological conditions on biofilm growth.     

Molecular Epidemiology and Complete Genome Characterization of H1N1pdm Virus from India

Background

Influenza A virus is one of world’s major uncontrolled pathogen, causing seasonal epidemic as well as global pandemic. This was evidenced by recent emergence and continued prevalent 2009 swine origin pandemic H1N1 Influenza A virus, provoking first true pandemic in the past 40 years. In the course of its evolution, the virus acquired many mutations and multiple unidentified molecular determinants are likely responsible for the ability of the 2009 H1N1 virus to cause increased disease severity in humans. Availability of limited data on complete genome hampers the continuous monitoring of this type of events. Outbreaks with considerable morbidity and mortality have been reported from all parts of the country.

Methods/Results

Considering a large number of clinical cases of infection complete genome based sequence characterization of Indian H1N1pdm virus and their phylogenetic analysis with respect to circulating global viruses was undertaken, to reveal the phylodynamic pattern of H1N1pdm virus in India from 2009–2011. The Clade VII was observed as a major circulating clade in phylogenetic analysis. Selection pressure analysis revealed 18 positively selected sites in major surface proteins of H1N1pdm virus.

Conclusions

This study clearly revealed that clade VII has been identified as recent circulating clade in India as well globally. Few clade VII specific well identified markers undergone positive selection during virus evolution. Continuous monitoring of the H1N1pdm virus is warranted to track of the virus evolution and further transmission. This study will serve as a baseline data for future surveillance and also for development of suitable therapeutics.     

Quick Diagnosis of Human Brain Meningitis Using Omp85 Gene Amplicon as a Genetic Marker

The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease.     

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H₂O₂) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H₂O₂ using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O₂) concentration of ventilation gas on lung rate of H₂O₂ release into the perfusate. Baseline lung rate of H₂O₂ release was 8.45?±?0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O₂ in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O₂-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H₂O₂, and that most of acute hyperoxia-enhanced lung rate of H₂O₂ release is from nonmitochondrial rather than mitochondrial sources.     

ZD6474 enhances paclitaxel antiproliferative and apoptotic effects in breast carcinoma cells

Chemotherapy employing paclitaxel and docetaxel is widely used for treating early‐stage breast cancer and metastasis, which is frequently associated with overexpression of epidermal growth factor receptor (EGFR) and resistance to apoptosis. ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR, inhibits cell proliferation of solid tumors, including breast. Phase III clinical trials using ZD6474 in non‐small cell lung carcinoma when combined with standard chemotherapy appear promising. In order to improve the antineoplastic activity of paclitaxel, we presently investigated the effects of ZD6474 in combination with paclitaxel in EGFR and VEGFR expressing human breast cancer cell lines MCF‐7 and MDA‐MB‐231. ZD6474 synergistically decreased cell viability when used in combination with paclitaxel. ZD6474 inhibited cyclin D1 and cyclin E expression and induced p53 expression when combined with paclitaxel. The combination of ZD6474 with paclitaxel versus either agent alone also more potently down‐regulated the antiapoptotic bcl‐2 protein, up‐regulated pro‐apoptotic signaling events involving expression of bax, activation of caspase‐3 and caspase‐7 proteins, and induced poly(ADP‐ribose) polymerase resulting in apoptosis. ZD6474 combined with paclitaxel inhibited anchorage‐independent colony formation and invasion of breast cancer cells in vitro as compared to either single agent, indicating a potential involvement of altered expression and reorganization of cytoskeletal proteins in combinatorial treated breast cancer cells. Collectively, our studies indicate that incorporating an anti‐EGFR plus VEGFR strategy (ZD6474) with chemotherapy (paclitaxel), where clinical studies of dose‐intensive paclitaxel therapy are currently in progress, may be more effective in treating patients with locally advanced or metastatic breast cancer than either approach alone. J. Cell. Physiol. 226: 375–384, 2011. © 2010 Wiley‐Liss, Inc.     

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.     

Poly-ethers from Winogradskyella poriferorum: Antifouling potential, time-course study of production and natural abundance

A sponge-associated bacterium, Winogradskyella poriferorum strain UST030701-295T was cultured up to 100 l for extraction of antifouling bioactive compounds. Five poly-ethers were isolated and partially characterized based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS); two of them showed inhibitory effects on biofilm formation of marine bacteria and larval settlement of macro-foulers but did not produce any adverse effects on the phenotypes of zebra fish embryos at a concentration of 5 μg ml⁻¹. The effect of culture duration on the production of the poly-ethers and the bioactivity of the relevant extracts was monitored over a period of 12 days. The total crude poly-ether production increased from day 2 to day 5 and the highest bioactivity was observed on day 3. The poly-ethers were found to be localized in the cellular fraction of the extracts, implying their natural occurrence. The potent bioactivity of these poly-ethers together with their high natural abundance in bacteria makes them promising candidates as ingredients in antifouling applications.