Jagat Kumar Roy

Rab proteins: The key regulators of intracellular vesicle transport

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.

Nanoparticle-Induced Controlled Biodegradation and Its Mechanism in Poly(ε-caprolactone)

Poly(epsilon-caprolactone) (PCL)/layered silicate nanocomposites have been prepared via solution route. Two different organically modified nanoclays were used to compare the variation in properties based on organic modifications. The nanostructures, as observed from wide-angle X-ray diffraction and transmission electron microscopy, indicate intercalated and partially exfoliated hybrids depending on the nature of organic modification in nanoclay. The nanohybrids exhibit significant improvement in thermal and mechanical properties of the matrix as compared to neat polymer. The nanoclays act as nucleating agent for the crystallization of PCL. The biodegradability of pure PCL and its nanocomposites have been studied under controlled conditions in enzyme, pure microorganism (fungi), compost, Ganges water, and alkaline buffer solution. The rate of biodegradation of PCL has enhanced dramatically in nanohybrids and depends strongly on the media used. Scanning confocal, electron, and atomic force microscopes have used to demarcate the nature of biodegradation of pristine PCL and its nanocomposites. The change in biodegradation is rationalized in terms of the crystallization behavior and organic modification in nanoclays of the nanohybrids vis-a-vis the neat polymer. The extent of compatibility was measured quantitatively through the interaction parameter for two different nanoclays to compare and establish the reason for variation in their properties in nanohybrids. A biodegradation mechanism has been revealed for PCL and its nanocomposites through enzyme activity in varying pH environment.

Nanostructure controlled anti-cancer drug delivery using poly(ε-caprolactone) based nanohybrids

We have focused on the generation of various nanostructures of poly(e-caprolactone) (PCL) using surface modified layered silicate. The improved and diverse mechanical, thermal and surface properties have been explored depending upon the nanostructure of the nanohybrids. The incorporation of drug into those nanohybrids further alters the nanostructure and subsequent properties. The rate of biodegradation has been studied in detail, with plausible mechanisms in different enzyme media being suggested, their specificity and the tunability of the biodegradation rate was demonstrated, followed by their optimization. The scaffolds of PCL and its nanohybrids with and without drugs have been prepared through electrospinning to control the dimensions of the nanofibers and their controlled degradation. The in-depth studies of the biocompatibility in terms of cell adhesion, genotoxicity and hemocompatibility have been performed to verify the suitability of the nanohybrids for potential biomedical applications. The biocompatibility of the nanohybrids at the gene level has been tested by the subcellular localization of an important regulator of pro-apoptotic signalling cascade, HIPK2 in human epithelial cells, demonstrating the attuned nature of the particles under study within the biological system. The blood compatibilities of the pure PCL and its nanohybrids were studied by platelet aggregation, platelet adhesion, and in vitro hemolysis assay, elucidating the excellent hemocompatibility of the novel nanohybrids. Biocompatible and hemocompatible nanohybrids have been testified for drug delivery and show sustained and controlled release of anti-cancer drugs (dexamethasone) in the presence of two dimensional disc-like nanoparticles. Hence, the developed nanohybrids are a potential biomaterial, suitable for tissue engineering and drug delivery.

Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster.

Abstract The genes engrailed (en), hedgehog (hh), wingless (wg) and decapentaplegic (dpp) have been shown to play vital organising roles in the development and differentiation of thoracic imaginal discs. We have analysed the roles of these genes in organising the development and differentiation of the genital discs, which are bilaterally symmetrical and possess different primordia, namely, the male and female genital primordia and an anal primordium. Our results suggest that the organising activity of en in genital discs programs the normal development and differentiation of the genital disc by regulating the expression of hh. Hh in turn induces wg and dpp, the genes whose products act as secondary signalling molecules. Moreover, the complementary patterns of wg and dpp expression are essential for the bilateral symmetry and are maintained by mutual repression.

Rab11 is required for embryonic nervous system development in Drosophila

In eukaryotes, membrane trafficking is regulated by the small monomeric GTPases of Rab protein family. Rab11, an evolutionary conserved, ubiquitously expressed subfamily of the Rab GTPases, has been implicated in the regulation of vesicular trafficking through the recycling of endosomes. To dissect out the role of this protein during embryonic nervous system development, we have studied the expression pattern of Rab11 in the ventral nerve cord during neuronal differentiation in the Drosophila embryo. When the dominant-negative or constitutively-active mutant DRab11 proteins are expressed in neurons, or when homozygous mutant Rab11 embryos are analyzed, defects are found in the developing central nervous system, along with disorganization and misrouting of embryonic axons. Our results provide the first in vivo evidence that Rab11 is involved in the development of the nervous system during Drosophila embryogenesis.

High prevalence of oncogenic HPV-16 in cervical smears of asymptomatic women of eastern Uttar Pradesh, India: A population-based study

In developing countries like India, occurrence of Human papillomavirus (HPV) in cervical cancer as well as in the asymptomatic population was observed to be very high. Studies on HPV prevalence have been conducted in different parts of the country but no data were available from the eastern region of Uttar Pradesh (UP). The present study aimed to determine the status of HPV prevalence and its association with different socio-demographic factors in this population. Prevalence of HPV was investigated in a total of 2424 cervical scrape samples of asymptomatic women. Primer sets from L1 consensus region of viral genome were used to detect the presence of HPV, and the positive samples were genotyped by sequencing. Univariate binary logistic regression analysis was used to evaluate association of socio-demographic factors with HPV. 9.9% of the clinically asymptomatic women were found to be infected with HPV comprising 26 different genotypes. Among HPV-positive women, 80.8% showed single infection, while 15.4% harboured multiple infections. HPV-16 (63.7%) was the most prevalent, followed by HPV-31 (6.7%), HPV-6 (5.4%), HPV-81 (4.6%) and HPV-33 (4.2%). Significant association of HPV with non-vegetarian diet (P < 0.05) and rural residential areas (P < 0.01) were observed. High prevalence of HPV-16 in asymptomatic women of this population, a frequency comparable to invasive cervical cancers, highlights an urgent need for a therapeutic HPV vaccine covering HPV-16 and other high-risk types to provide protection against the disease.

Tuned biodegradation using poly(hydroxybutyrate-co-valerate) nanobiohybrids: Emerging biomaterials for tissue engineering and drug delivery

Our studies focus on controlled and tuned biodegradation, mechanism of biodegradation, biocompatibility, genotoxicity and controlled and sustained drug release performance of poly(hydroxybutyrate-co-valerate) nanohybrids. Nanohybrids were prepared using layered silicates with two different surface modifiers through a solution route. Biodegradation has been investigated in compost, microorganisms (pseudomonas stutzeri) and pure enzymes, showing both enhanced and suppressed rates as compared to that of the pure polymer. Two nanoclays that increased the strong filler dependency phenomena were used to control the biodegradation rate. Complete biodegradation mechanism has been revealed, which indicates that the pH of the medium dictates the rate of biodegradation by activating the enzyme responsible for degradation. Biodegradation has also been controlled by optimizing the processing conditions of the specimens. Biocompatibility of the nanohybrids has been tested at the intra-cellular level by sub-cellular localization of HIPK2 protein, in addition to the typical cell adherence on the nanohybrid films demonstrating that there is no induced genotoxic stress either from the polymer or from the nanoparticles. Biocompatible nanohybrids have been used for drug delivery, showing sustained and controlled release of drugs in presence of nanoparticles. Hence, the developed nanohybrids illustrate potential biomaterials for tissue engineering and drug delivery.

Effects of Hoechst 33258 on condensation patterns of hetero- and euchromatin in mitotic and interphase nuclei of Drosophila nasuta

Abstract Embryonic and third instar larval brain cells of D. nasuta were cultured in vitro in the presence of Hoechst 33258 (H) and H + 5-bromodeoxyuridine (BUdR) for periods varying from 2 to 24 h at 24 °C. Air-dried chromosome preparations were made with and without hypotonic pretreatment and stained with Giemsa. Metaphase chromosomes from H-treated (2 h) embryonic preparations show typical inhibition of condensation of the A-T-rich heterochromatin as in mouse. Presence of BUdR with H causes inhibition of condensation in fewer embryonic metaphase cells, but in the affected metaphases the degree of inhibition is more severe. In larval brains, however, even a 24 h H or H + BUdR treatment does not cause any significant inhibition of heterochromatin condensation. It is suggested that the differences in H effect on metaphase chromosomes of embryos and larval brains is related to differences in chromosome organization in the two cell types. Exposure of H-treated embryonic as well as larval brain cells to a hypotonic salt solution prior to fixation causes a ‘supercondensation’ of the heterochromatic chromocentre in most interphase nuclei. Presence of BUdR along with H reduces the frequency of cells showing such ‘supercondensed’ chromocentre. The euchromatin region in H-treated interphase nuclei is, on the other hand, slightly more diffuse than in control nuclei. Apparently, H-binding to DNA affects the nucleoprotein organization in hetero- and euchromatic regions of interphase nuclei in specific ways.

Rab11 is essential for fertility in Drosophila.

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P‐insertion line, Rab11mo, was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11mo males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4‐Rab11/+; Rab11mo/Rab11mo egg chambers, confirming the requirement of Rab11 in these events.

Lessons learnt from human papillomavirus (HPV) vaccination in 45 low- and middle-income countries.

Objective To synthesise lessons learnt and determinants of success from human papillomavirus (HPV) vaccine demonstration projects and national programmes in low- and middle-income countries (LAMICs). Methods Interviews were conducted with 56 key informants. A systematic literature review identified 2936 abstracts from five databases; after screening 61 full texts were included. Unpublished literature, including evaluation reports, was solicited from country representatives; 188 documents were received. A data extraction tool and interview topic guide outlining key areas of inquiry were informed by World Health Organization guidelines for new vaccine introduction. Results were synthesised thematically. Results Data were analysed from 12 national programmes and 66 demonstration projects in 46 countries. Among demonstration projects, 30 were supported by the GARDASIL® Access Program, 20 by Gavi, four by PATH and 12 by other means. School-based vaccine delivery supplemented with health facility-based delivery for out-of-school girls attained high coverage. There were limited data on facility-only strategies and little evaluation of strategies to reach out-of-school girls. Early engagement of teachers as partners in social mobilisation, consent, vaccination day coordination, follow-up of non-completers and adverse events was considered invaluable. Micro-planning using school/ facility registers most effectively enumerated target populations; other estimates proved inaccurate, leading to vaccine under- or over-estimation. Refresher training on adverse events and safe injection procedures was usually necessary. Conclusion Considerable experience in HPV vaccine delivery in LAMICs is available. Lessons are generally consistent across countries and dissemination of these could improve HPV vaccine introduction.