A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was *50 lg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.

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Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the ␣ subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA 163-256 to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA 174 -316 ). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds ϳ100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink.

Hepatitis C virus infects over 15 million patients from India and 2.86 million from Brazil. Detection of anti-hepatitis C virus antibodies has limited sensitivity during acute phase: the pre-seroconversion window period. Hepatitis C virus-RNA detection techniques are used to overcome this shortfall, but are costly and unavailable widely in developing countries. Estimation of hepatitis C virus core-antigen, a protein with highly conserved sequence, by enzyme-immunoassays is an economic and simpler alternative to RNA detection. This study was conducted in Delhi, involving 300 acute and chronic liver disease patients, tested for anti-hepatitis C virus 3rd-generation ELISA, hepatitis C virus core-antigen-ELISA and hepatitis C virus-RNA reverse transcription-polymerase chain reaction. Among the acute patients, hepatitis C virus core-antigen assay could identify 13 out of 14 pre-seroconversion window period cases and 6 out of 8 seroconverted cases, with a pre-seroconversion window period sensitivity of 92.9% and specificity of 100%. In hepatitis C virus core-antigen-positive cases, the viral load was in the range of 4900 to 1.46×10(6)IU/mL, whereas in hepatitis C virus core-antigen-negative cases, the range of viral load was 100-4500IU/mL. The cost of the hepatitis C virus core-antigen-ELISA was estimated around 3-4 times lesser than the in-house reverse transcription-polymerase chain reaction and 9-10 times lesser than the United States Food and Drug Administration approved reverse transcription-polymerase chain reaction. With a good sensitivity and specificity in the acute phase of infection, hepatitis C virus core-antigen-ELISA can thus be a useful alternative in the developing nations.

A high‐performance liquid chromatography method is described for the detection of adulteration of milk with soymilk, based on separation of sugars on NH2 column and their detection by refractive index detector. Sugars were extracted with 20% acetonitrile in the presence of Carrez solutions and quantified. Recovery of added raffinose to soymilk was 96.3%. Owing to relative high concentration of lactose in milk or adulterated milk, lactose peak was very broad and spread to retention time corresponding to sucrose and raffinose. However, stachyose peak remained well separated. Presence of stachyose peak in milk can be used for the detection of adulteration of milk with soymilk and the method can detect upto 5% soymilk in milk.