Adeno-associated virus-mediated gene transfer

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system.     

The biology of triploid fish

This review deals with major areas of triploidy research in fish. It includes not only methods for induction and detection of triploidy but also the impact of triploidy on morphology, anatomy, growth, haematology, energetics, behaviour, endocrinology and gonads in various species of fish, studied so far. The future prospects of research on triploid fish are discussed inviting researchers with diverse areas of interest in fish biology.     

A finite element framework for studying the mechanical response of macromolecules: application to the gating of the mechanosensitive channel MscL

The gating pathways of mechanosensitive channels of large conductance (MscL) in two bacteria (Mycobacterium tuberculosis and Escherichia coli) are studied using the finite element method. The phenomenological model treats transmembrane helices as elastic rods and the lipid membrane as an elastic sheet of finite thickness; the model is inspired by the crystal structure of MscL. The interactions between various continuum components are derived from molecular-mechanics energy calculations using the CHARMM all-atom force field. Both bacterial MscLs open fully upon in-plane tension in the membrane and the variation of pore diameter with membrane tension is found to be essentially linear. The estimated gating tension is close to the experimental value. The structural variations along the gating pathway are consistent with previous analyses based on structural models with experimental constraints and biased atomistic molecular-dynamics simulations. Upon membrane bending, neither MscL opens substantially, although there is notable and nonmonotonic variation in the pore radius. This emphasizes that the gating behavior of MscL depends critically on the form of the mechanical perturbation and reinforces the idea that the crucial gating parameter is lateral tension in the membrane rather than the curvature of the membrane. Compared to popular all-atom-based techniques such as targeted or steered molecular-dynamics simulations, the finite element method-based continuum-mechanics framework offers a unique alternative to bridge detailed intermolecular interactions and biological processes occurring at large spatial scales and long timescales. It is envisioned that such a hierarchical multiscale framework will find great value in the study of a variety of biological processes involving complex mechanical deformations such as muscle contraction and mechanotransduction.     

Soluble fms-Like tyrosine kinase 1 (sFlt1), endoglin and placental growth factor (PlGF) in preeclampsia among high risk pregnancies

Background

Differences in circulating concentrations of antiangiogenic factors sFlt1 and soluble endoglin (sEng) and the pro-angiogenic growth factor PlGF are reported to precede the onset of preeclampsia weeks to months in low-risk pregnant women. The objective of this study was to investigate whether similar changes can be detected in pregnant women at high-risk to develop the syndrome.

Methods

This study is a secondary analysis of the NICHD MFMU trial of aspirin to prevent preeclampsia in high-risk pregnancies. Serum samples were available from 194 women with pre-existing diabetes, 313 with chronic hypertension, 234 with multifetal gestation, and 252 with a history of preeclampsia in a previous pregnancy. Samples collected across pregnancy were analyzed in a blinded fashion for sFlt1, sEng and PlGF.

Results

The odds of developing preeclampsia were significantly increased among women with multiple fetuses for each 2-fold elevation in sFlt1, sEng and the ratio of angiogenic factors (e.g. OR 2.18, 95% CI 1.46-3.32), and significantly decreased for each 2-fold elevation in circulating PlGF (OR 0.50, 95% CI 0.30-0.82) between 7 and 26 weeks'' gestation. Cross-sectional analysis of the angiogenic factors across gestation showed significant differences during the third trimester in women who develop preeclampsia compared with appropriate controls in all high-risk groups. However, when data were examined in relation to the gestational week when preeclampsia was diagnosed only sFlt1 was significantly higher 2 to 5 weeks before the clinical onset of preeclampsia and only in women with previous preeclampsia.

Conclusions

The pattern of elevated concentrations of sFlt1 and sEng, and low PlGF in high-risk pregnant subjects who develop preeclampsia is similar to that reported in low-risk pregnant women. However, differences in these factors among high-risk women who do and do not develop preeclampsia are modest, and do not appear to be clinically useful predictors in these high-risk pregnant women.     

Molecular modeling studies on the ribosome

In the absence of a high resolution crystal structure for the ribosome, numerous research groups are carrying out low resolution structural studies using neutron diffraction, electron microscopy, fluorescence energy transfer, chemical crosslinking, chemical footprinting studies, and other methods. We have developed a computer-based refinement method for incorporating these data into low resolution three-dimensional models. The method is based on a molecular mechanics approach, with proteins represented by spherical particles of suitable diameter and the ribosomal RNA represented by a string of spherical pseudoatoms, one for each nucleotide. Experimental data are used to derive constraints that are introduced through a special force field (potential function). Models are refined by simulated annealing. Since every term in the force field is quadratic, any model that satisfies all of the input data has an energy of zero; higher energies indicate residual unsatisfied constraints. The residual energy provides a quantitative statement of model quality and can be used to identify conflicts in the experimental data. The method has been applied to the refinement of a low resolution model for the 30S subunit (the small subunit) of theE. coli ribosome. Since this is a very underdetermined system, the range of acceptable models has also been explored. This provides an estimate of the resolution of the structure, which is about 15 Å overall, with the uncertainty in position of individual nucleotides ranging from about 5 Å to 50 Å.     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Biology of <Emphasis Type="Italic">B. sorokiniana</Emphasis> (syn. <Emphasis Type="Italic">Cochliobolus sativus</Emphasis>) in genomics era

Bipolaris sorokiniana (Sacc.) Shoemaker is a hemi-biotrophic fungal pathogen, which is an anamorph (teleomorph Cochlibolus sativus). It causes spot blotch, root rot and leaf spot diseases in a number of cereals including wheat, barley and other small grain cereals. In the genomics era, the fungus has been subjected to a variety of studies using molecular approaches. Correct chromosome number was determined and molecular karyotypes were prepared using contour-clamped homogeneous electric field. Molecular maps were prepared using markers like RFLPs, SSRs, RAPDs and SNPs. For this purpose, segregating progenies derived from crosses between diverse isolates of the pathogen were used. Whole genome sequencing (WGS) data was collected not only for B. sorokiniana isolates, but also for several species of Cochliobolus. Genes involved in secondary metabolism and virulence were identified from genome sequences. The WGS data has also been utilized for comparative genomics giving useful information about evolutionary trends. A brief account of this information is presented in this review.     

Probiotic Potential of Autochthonous Bacteria Isolated from the Gastrointestinal Tract of Four Freshwater Teleosts

In this study, a total of 121 bacterial strains were isolated from the gastrointestinal tract of four teleostean species, namely striped snakehead (Channa striatus), striped dwarf catfish (Mystus vittatus), orangefin labeo (Labeo calbasu) and mrigal carp (Cirrhinus mrigala), among which 8 isolates showed promising antibacterial activity against four potential fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Pseudomonas fluorescens and were non-hemolytic. The isolates were further screened in response to fish bile tolerance and extracellular digestive enzyme activity. Two bacterial strains MVF1 and MVH7 showed highest tolerance and extracellular enzymes activities, and selected for further studies. Antagonistic activity of these two isolates was further confirmed by in vitro growth inhibition assay against four selected fish pathogens in liquid medium. Finally, these two bacterial strains MVF1 and MVH7 were selected as potential probiotic candidates and thus identification by partial 16S rRNA gene sequence analysis. The bacterial isolates MVF1 and MVH7 were identified as two strains of Bacillus sp.