Arsenic-induced health effects and genetic damage in keratotic individuals: involvement of p53 arginine variant and chromosomal aberrations in arsenic susceptibility

In West Bengal, India, more than 6 million people are exposed to arsenic through drinking water. Chronic arsenic exposure results in several multisystemic non-cancerous as well as cancerous effects in humans. Among non-cancerous effects, arsenic-specific skin lesions, conjunctivitis, peripheral neuropathy and respiratory diseases are prominent. One of the major consequences of chronic arsenic exposure is keratosis, the precancerous state of skin cancer. The tumor suppressor protein p53 consists of a polymorphism proline72arginine reported to be associated with various types of cancers. Previously we have reported that the p53 codon 72 arginine (Arg) homozygous genotype is associated with the development of arsenic-induced keratosis. In the present study we have investigated the distribution of health effects and chromosomal aberrations (CAs) in the individuals with keratosis. We have compared individuals with keratosis with those without arsenic-induced skin lesions but drinking similar level of arsenic-contaminated water. Attempts have also been made to find out the association of the p53 risk genotype with health effects and chromosomal aberrations. This study comprises of 349 unrelated exposed individuals (162 individuals with keratosis and 187 individuals without arsenic-specific skin lesions) from highly arsenic-affected districts of West Bengal, India. The results showed that health effects (i.e. peripheral neuropathy, conjunctivitis and respiratory illness) and chromosomal aberrations were significantly higher in the keratotic group compared to individuals with no skin lesions. Moreover, individuals with the arginine homozygous genotype showed increased levels of chromosomal aberrations compared to individuals with other genotypes; however, we did not find any significant association of the risk genotype with health effects. This study suggests that individuals with keratosis are more susceptible to arsenic-induced health effects and genetic damage and that the arginine variant of p53 can further influence the repair capacity of arsenic-exposed individuals, leading to increased accumulation of chromosomal aberrations.     

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.     

Development of an Ointment Formulation Using Hot-Melt Extrusion Technology

Ointments are generally prepared either by fusion or by levigation methods. The current study proposes the use of hot-melt extrusion (HME) processing for the preparation of a polyethylene glycol base ointment. Lidocaine was used as a model drug. A modified screw design was used in this process, and parameters such as feeding rate, barrel temperature, and screw speed were optimized to obtain a uniform product. The product characteristics were compared with an ointment of similar composition prepared by conventional fusion method. The rheological properties, drug release profile, and texture characteristics of the hot-melt extruded product were similar to the conventionally prepared product. This study demonstrates a novel application of the hot-melt extrusion process in the manufacturing of topical semi-solids.     

Ex Vivo Evidence for the Contribution of Hemodynamic Shear Stress Abnormalities to the Early Pathogenesis of Calcific Bicuspid Aortic Valve Disease

The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly and is frequently associated with calcific aortic valve disease (CAVD). The most prevalent type-I morphology, which results from left-/right-coronary cusp fusion, generates different hemodynamics than a tricuspid aortic valve (TAV). While valvular calcification has been linked to genetic and atherogenic predispositions, hemodynamic abnormalities are increasingly pointed as potential pathogenic contributors. In particular, the wall shear stress (WSS) produced by blood flow on the leaflets regulates homeostasis in the TAV. In contrast, WSS alterations cause valve dysfunction and disease. While such observations support the existence of synergies between valvular hemodynamics and biology, the role played by BAV WSS in valvular calcification remains unknown. The objective of this study was to isolate the acute effects of native BAV WSS abnormalities on CAVD pathogenesis. Porcine aortic valve leaflets were subjected ex vivo to the native WSS experienced by TAV and type-I BAV leaflets for 48 hours. Immunostaining, immunoblotting and zymography were performed to characterize endothelial activation, pro-inflammatory paracrine signaling, extracellular matrix remodeling and markers involved in valvular interstitial cell activation and osteogenesis. While TAV and non-coronary BAV leaflet WSS essentially maintained valvular homeostasis, fused BAV leaflet WSS promoted fibrosa endothelial activation, paracrine signaling (2.4-fold and 3.7-fold increase in BMP-4 and TGF-β1, respectively, relative to fresh controls), catabolic enzyme secretion (6.3-fold, 16.8-fold, 11.7-fold, 16.7-fold and 5.5-fold increase in MMP-2, MMP-9, cathepsin L, cathepsin S and TIMP-2, respectively) and activity (1.7-fold and 2.4-fold increase in MMP-2 and MMP-9 activity, respectively), and bone matrix synthesis (5-fold increase in osteocalcin). In contrast, BAV WSS did not significantly affect α-SMA and Runx2 expressions and TIMP/MMP ratio. This study demonstrates the key role played by BAV hemodynamic abnormalities in CAVD pathogenesis and suggests the dependence of BAV vulnerability to calcification on the local degree of WSS abnormality.     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

Controlled surface trap state photoluminescence from CdS QDs impregnated in poly(methyl methacrylate)

A method of microwave (MW) assisted synthesis was employed to prepare cadmium sulfide (CdS) quantum dots (QDs) in dimethylformamide in the presence of poly(methyl methacrylate) (PMMA). The MW irradiation was carried out for a fixed time of 20-30 s and the size of QDs varied from 2.9-5.5 nm. Before each irradiation the solution was cooled down to ambient temperature and the irradiation process was repeated six times. An increase in the intensity and red shift of the characteristic UV-vis absorption peak originating from CdS QDs were observed with repeated MW irradiation, suggesting that the amount of generated CdS QDs increased within the PMMA network and aggregated with repeated MW irradiation. MW irradiation could influence selectively the nucleation and growing rates of PMMA-CdS QDs systems. The broadness and large Stokes shift of the emission from Cd(2+)-rich PMMA-CdS QDs was due to the surface trap state photoluminescence. The recombination of shallow trapped electrons and shallow trapped holes has been considered as the primary source of the surface trap state photoluminescence in Cd(2+)-rich PMMA-CdS QDs. The photoluminescence lifetime was observed to be decreased sharply when the amount of QDs was less, showing the emission decay was dependent on the surface property of PMMA-CdS QDs. The origin of the longer lifetime was due to the involvement of surface trap states and dependent on the amount of CdS QDs present within PMMA and its environment. The effect of the concentration of Cd(2+), S(2-) and PMMA on the generation of CdS QDs within PMMA and the effect of repeated MW irradiation on the optical properties was studied and the results are discussed in this article.