Poly-(3-hexylthiophene) self-assembled monolayer based cholesterol biosensor using surface plasmon resonance technique

Cholesterol oxidase (ChOx) has been covalently immobilized onto 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified poly-(3-hexylthiophene) (P3HT) self-assembled monolayer (SAM) onto gold coated glass plates. These ChOx/FNAB/P3HT/Au bio-electrodes have been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical impedance technique, cyclic voltammetric technique and atomic force microscopic (AFM) technique, respectively. The ChOx/FNAB/P3HT/Au bio-electrodes were utilized for the estimation of cholesterol concentration in standard solutions using surface plasmon resonance (SPR) technique. It is shown that this SPR biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with detection limit of 50 mg/dl, sensitivity of 1.0 4 m degrees /(mg dl), reusability of around 15 times and a shelf-life of about 10 weeks when stored at 4 degrees C.     

Nucleic acid sensor for insecticide detection

Nucleic acid sensor based on polyaniline (PANI) has been fabricated by covalently immobilizing double stranded calf thymus (dsCT) DNA onto perchlorate (ClO(-) (4))-doped PANI film deposited onto indium-tin-oxide (ITO) glass plate using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. These dsCT-DNA-PANI-ClO(4)/ITO and PANI-ClO(4)/ITO electrodes have been characterized using square wave voltammetry, electrochemical impedance, scanning electron microscopy (SEM) and Fourier-transform-infrared (FTIR) measurements. This disposable dsCT-DNA-PANI-ClO(4)/ITO bioelectrode, stable for about 4 months, can be used to detect cypermethrin (0.005 ppm) and trichlorfon (0.01 ppm) in 30 and 60 s, respectively.     

Bottlenecks drive temporal and spatial genetic changes in alpine caddisfly metapopulations

Background

Extinction and re-colonisation of local populations is common in ephemeral habitats such as temporary streams. In most cases, such population turnover leads to reduced genetic diversity within populations and increased genetic differentiation among populations due to stochastic founder events, genetic drift, and bottlenecks associated with re-colonisation. Here, we examined the spatio-temporal genetic structure of 8 alpine caddisfly populations inhabiting permanent and temporary streams from four valleys in two regions of the Swiss Alps in years before and after a major stream drying event, the European heat wave in summer 2003.

Results

We found that population turnover after 2003 led to a loss of allelic richness and gene diversity but not to significant changes in observed heterozygosity. Within all valleys, permanent and temporary streams in any given year were not differentiated, suggesting considerable gene flow and admixture between streams with differing hydroperiods. Large changes in allele frequencies after 2003 resulted in a substantial increase in genetic differentiation among valleys within one to two years (1-2 generations) driven primarily by drift and immigration. Signatures of genetic bottlenecks were detected in all 8 populations after 2003 using the M-ratio method, but in no populations when using a heterozygosity excess method, indicating differential sensitivity of bottleneck detection methods.

Conclusions

We conclude that genetic differentiation among A. uncatus populations changed markedly both temporally and spatially in response to the extreme climate event in 2003. Our results highlight the magnitude of temporal population genetic changes in response to extreme events. More specifically, our results show that extreme events can cause rapid genetic divergence in metapopulations. Further studies are needed to determine if recovery from this perturbation through gradual mixing of diverged populations by migration and gene flow leads to the pre-climate event state, or whether the observed changes represent a new genetic equilibrium.     

Thermostable hexameric form of Eis (Rv2416c) protein of M. tuberculosis plays an important role for enhanced intracellular survival within macrophages

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective T(H)1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ～75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ～50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.     

Streptomyces antibioticalis, a Novel Species from a Sanitary Landfill Soil

A novel isolate belonging to the genus Streptomyces, strain SL-4^T, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4^T were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4^T contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4^T and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357^T ({"type":"entrez-nucleotide","attrs":{"text":"DQ026672","term_id":"68137786","term_text":"DQ026672"}}DQ026672), S. albogriseolus NRRLB 1305^T ({"type":"entrez-nucleotide","attrs":{"text":"AJ494865","term_id":"21742835","term_text":"AJ494865"}}AJ494865), S viridodiastaticus NBRC 13106^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184317","term_id":"90960133","term_text":"AB184317"}}AB184317), S. caelestis NRRL 2418^T ({"type":"entrez-nucleotide","attrs":{"text":"X80824","term_id":"587528","term_text":"X80824"}}X80824), S. flavoviridis NBRC 12772^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184842","term_id":"90960663","term_text":"AB184842"}}AB184842), S. pilosus NBRC 12807^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184161","term_id":"90959977","term_text":"AB184161"}}AB184161) and S. longispororuber NBRC 13488^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184440","term_id":"90960256","term_text":"AB184440"}}AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA–DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4^T and its relatives S. atrovirens,S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4^T representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4^T (=CCM 7434^T=MTCC 8588^T).     

Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization

The growth conditions for chitinase production by Trichoderma asperellum UTP-16 in solid state fermentation was optimized using response surface methodology based on central composite design. The chitinase production was optimized, using one-factor at a time approach, with six independent variables (temperature, pH, NaCl, incubation period, nitrogen and carbon sources) and 3.31 Units per gram dry substrate (U gds⁻¹) exo-chitinase yield was obtained. A 21.15% increase was recorded in chitinase activity (4.01 U gds⁻¹) through surface response methodology, indicates that it is a powerful and rapid tool for optimization of physical and nutritional variables. Further, efficiency of crude enzyme was evaluated against phytopathogenic Fusarium spp. and a mycelial growth inhibition up to 3.5–6.5 mm was achieved in well diffusion assay. These results could be supplemented as basic information for the development of enzyme based formulation of T. asperellum UTP-16 and its use as a biocontrol agent.     

Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses

Recombinant adenoviruses containing a double-stranded DNA genome of 26–45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies.     

CoaSim: A flexible environment for simulating genetic data under coalescent models

Background  

Coalescent simulations are playing a large role in interpreting large scale intra-specific sequence or polymorphism surveys and for planning and evaluating association studies. Coalescent simulations of data sets under different models can be compared to the actual data to test the importance of different evolutionary factors and thus get insight into these.     

Effect of glucose on carbohydrate synthesis from alanine or lactate in hepatocytes from starved rats.

Euglena gracilis was found to contain a peroxidase that specifically require L-ascorbic acid as the natural electron donor in the cytosol. The presence of an oxidation-reduction system metabolizing L-ascorbic acid was demonstrated in Euglena cells. Oxidation of L-ascorbic acid by the peroxidase, and the absence of ascorbic acid oxidase activity, suggests that the system functions to remove H2O2 in E. gracilis, which lacks catalase.