The protective role of HSP27 in ocular diseases.

Heat shock proteins (HSPs) are stress-induced proteins that are important constituents of the cell's defense system. The activity of HSPs enhances when the cell undergoes undesirable environmental conditions like stress. The protective roles of HSPs are due to their molecular chaperone and anti-apoptotic functions. HSPs have a central role in the eye, and their malfunction has been associated with the manifestation of ocular diseases. Heat shock protein 27 (HSP27, HSPB1) is present in various ocular tissues, and it has been found to protect the eye from disease states such as retinoblastoma, uveal melanoma, glaucoma, and cataract. But some recent studies have shown the destructive role of HSP27 on retinal ganglionic cells. Thus, this article summarizes the role of heat shock protein 27 in eye and ocular diseases and will focus on the expression, regulation, and function of HSP27 in ocular complications.

Silk Fibroin Microparticle Scaffold for Use in Bone Void Filling: Safety and Efficacy Studies.

Silk fibroin (SF) is a natural biocompatible protein polymer extracted from cocoons of silkworm Bombyx mori. SF can be processed into a variety of different forms and shapes that can be used as scaffolds to support bone regeneration. Three-dimensional (3D) SF scaffolds have shown promise in bone-void-filling applications. In in vitro studies, it has been demonstrated that a microparticle-based SF (M-RSF) scaffold promotes the differentiation of stem cells into an osteoblastic lineage. The expression of differentiation markers was also significantly higher for M-RSF scaffolds as compared to other SF scaffolds and commercial ceramic scaffolds. In this work, we have evaluated the in vitro and in vivo biocompatibility of M-RSF scaffolds as per the ISO 10993 guidelines in a Good Laboratory Practice (GLP)-certified facility. The cytotoxicity, immunogenicity, genotoxicity, systemic toxicity, and implantation studies confirmed that the M-RSF scaffold is biocompatible. Further, the performance of the M-RSF scaffold to support bone formation was evaluated in in vivo bone implantation studies in a rabbit model. Calcium sulfate (CaSO4) scaffolds were chosen as reference material for this study as they are one of the preferred materials for bone-void-filling applications. M-RSF scaffold implantation sites showed a higher number of osteoblast and osteoclast cells as compared to CaSO4 implantation sites indicating active bone remodeling. The number density of osteocytes was double for M-RSF scaffold implantation sites, and these M-RSF scaffold implantation sites were characterized by enhanced collagen deposition, pointing toward a finer quality of the new bone formed. Moreover, the M-RSF scaffold implantation sites had a negligible incidence of secondary fractures as compared to the CaSO4 implantation sites (∼50% sites with secondary fracture), implying a reduction in postsurgical complications. Thus, the study demonstrates that the M-RSF scaffold is nontoxic for bone-void-filling applications and facilitates superior healing of fracture defects as compared to commercial calcium-based bone void fillers.

Silk fibroin and ceramic scaffolds: Comparative in vitro studies for bone regeneration.

Synthetic bone void fillers based on calcium ceramics are used to fill cavities in the bone and promote bone regeneration. More recently, silk fibroin (SF), a protein polymer obtained from Bombyx mori silkworm, has emerged as a promising material in bone void filling. In this work, we have compared the safety and efficacy of two types of silk fibroin-based bone void fillers with currently used and commercially available ceramic bone void fillers (based on calcium sulphate, beta tricalcium phosphate, and beta tricalcium phosphate with hydroxyapatite). Further, we have also evaluated these two types of SF scaffolds, which have strikingly different structural attributes. The biocompatibility of these scaffolds was comparable as assessed by cytotoxicity assay, cellular adhesion assay, and immunogenic assay. Ability of the scaffolds to support differentiation of human mesenchymal stem cells (hMSCs) into an osteoblastic lineage was also evaluated in an in vitro differentiation experiment using reverse transcriptase polymerase chain reaction analysis. These results revealed that cells cultured on SF scaffolds exhibit higher expression of early to late markers such as Runx2, BMPs, collagen, osterix, osteopontin, and osteocalcin as compared with ceramic-based scaffolds. This observation was further validated by studying the expression of alkaline phosphatase and calcium deposition. We also show that scaffolds made from same material of SF, but characterized by very different pore architectures, have diverse outcome in stem cell differentiation.

Silk fibroin micro-particle scaffolds with superior compression modulus and slow bioresorption for effective bone regeneration.

Silk fibroin (SF), a natural polymer produced by Bombyx mori silkworms, has been extensively explored to prepare porous scaffolds for tissue engineering applications. Here, we demonstrate, a scaffold made of SF, which exhibits compression modulus comparable to natural cancellous bone while retaining the appropriate porosities and interconnected pore architecture. The scaffolds also exhibit high resistance to in-vitro proteolytic degradation due to the dominant beta sheet conformation of the SF protein. Additionally, the scaffolds are prepared using a simple method of microparticle aggregation. We also demonstrate, for the first time, a method to prepare SF micro-particles using a Hexafluoroisopropanol-Methanol solvent-coagulant combination. SF microparticles obtained using this method are monodisperse, spherical, non-porous and extremely crystalline. These micro-particles have been further aggregated together to form a 3D scaffold. The aggregation is achieved by random packing of these microparticles and fusing them together using a dilute SF solution. Preliminary in-vitro cell culture and in-vivo implantation studies demonstrate that the scaffolds are biocompatible and they exhibit the appropriate early markers, making them promising candidates for bone regeneration.

Expression of multidrug resistance proteins in retinoblastoma.

AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Glutathione S-transferase (GSTM1, GSTT1) polymorphisms and JOAG susceptibility: A case control study and meta-analysis in glaucoma.

PURPOSE: Glutathione S transferase (GST) polymorphisms have been considered risk factors for the development of glaucoma. The aim of the present study was to investigate the association of glutathione S-transferase GSTT1 and GSTM1 genotypes with juvenile open-angle glaucoma (JOAG) in Indian patients. METHODS: A case-control study was performed to investigate the associations of GSTM1 and GSTT1 in juvenile open-angle glaucoma. The genotype of GSTM1 and GSTT1 were determined in 73 juvenile open-angle glaucoma patients, and 70 controls matched by age and sex by polymerase chain reaction method. We also performed a meta-analysis of sixteen published studies on GSTM1 and GSTT1 and evaluated the association between the GSTM1 and GSTT1 polymorphisms and glaucoma (JOAG & POAG). Published literature from PubMed and other databases were retrieved. All studies evaluating the association between GSTM1 and GSTT1 polymorphisms and glaucoma (JOAG & POAG) risk were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using random- or fixed-effects model. RESULTS: In the present study, we observed there is no association of GSTM1 (OR=0.680; 95% CI=0.323-1.433; p=0.311) or GSTT1 (OR=0.698; 95% CI=0.307-1.586; p=0.391) with JOAG. In the present meta-analysis, significantly increased glaucoma (JOAG & POAG) risk was found among subjects carrying GSTM1 null genotype (OR=1.177; 95% CI=1.028-1.348; p=0.018) but not among subjects carrying GSTT1 deletion genotype (OR=1.186; 95% CI=0.992-1.417; p=0.061). CONCLUSIONS: The present case-control study found that GSTM1 and GSTT1 polymorphism are not associated with JOAG risk in North Indian population. The present meta-analysis suggested that there might be a significant association of GSTM1 null genotype with glaucoma (JOAG & POAG) risk. To the best of our knowledge, this is the first study in the world to investigate role of GSTM1 and GSTT1 polymorphisms with JOAG susceptibility. Given the limited sample size, the associations between GST polymorphism and glaucoma risk needs further investigation.

Epigenetic regulation of human retinoblastoma.

Retinoblastoma is a rare type of eye cancer of the retina that commonly occurs in early childhood and mostly affects the children before the age of 5. It occurs due to the mutations in the retinoblastoma gene (RB1) which inactivates both alleles of the RB1. RB1 was first identified as a tumor suppressor gene, which regulates cell cycle components and associated with retinoblastoma. Previously, genetic alteration was known as the major cause of its occurrence, but later, it is revealed that besides genetic changes, epigenetic changes also play a significant role in the disease. Initiation and progression of retinoblastoma could be due to independent or combined genetic and epigenetic events. Remarkable work has been done in understanding retinoblastoma pathogenesis in terms of genetic alterations, but not much in the context of epigenetic modification. Epigenetic modifications that silence tumor suppressor genes and activate oncogenes include DNA methylation, chromatin remodeling, histone modification and noncoding RNA-mediated gene silencing. Epigenetic changes can lead to altered gene function and transform normal cell into tumor cells. This review focuses on important epigenetic alteration which occurs in retinoblastoma and its current state of knowledge. The critical role of epigenetic regulation in retinoblastoma is now an emerging area, and better understanding of epigenetic changes in retinoblastoma will open the door for future therapy and diagnosis.

Association between VEGF polymorphisms (-460 T/C and +936 C/T) and retinopathy of prematurity risk: A meta-analysis.

PURPOSE: Vascular endothelial growth factor (VEGF) contributes to the development of retinopathy of prematurity (ROP). A number of studies investigated the association of ROP with VEGF -460 T/C and +936 C/T polymorphisms but the results were conflicting. In order to derive a more precise estimation of the associations, we performed a meta-analysis of the relationship between VEGF -460 T/C and +936 C/T polymorphisms with ROP in all published studies. METHODS: A literature search was performed systematically using electronic databases. Published literature from PubMed and other databases was retrieved. The odds ratio (OR) with 95% confidence interval (CI) was used to estimate the pooled effect. Each -460 T/C and +936 C/T polymorphism included four case-control studies including case/control 249/308 and 179/250 respectively. RESULTS: Through literature search, we found that both VEGF -460 T/C and +936 C/T polymorphisms were not associated with ROP risk at allelic, co-dominant, dominant and recessive models. CONCLUSIONS: This meta-analysis suggests that the VEGF -460 T/C and +936 C/T polymorphism might contribute to genetic susceptibility of ROP. The association between VEGF -460 T/C and +936 C/T polymorphism and ROP risk awaits further investigation.

Vascular endothelial growth factor (VEGF-634G/C) polymorphism and retinopathy of prematurity: a meta-analysis.

PURPOSE: Vascular endothelial growth factor polymorphism (VEGF-634G/C, rs 2010963) has been considered a risk factor for the development of retinopathy of prematurity (ROP). However, the results remain controversial. Therefore, the aim of the present meta-analysis was to determine the association between VEGF-634G/C polymorphism and ROP risk. METHODS: Published literature from PubMed and other databases were retrieved. All studies evaluating the association between VEGF-634G/C polymorphism and ROP risk were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using random or fixed effects model. A total of six case-control studies including 355 cases and 471 controls were included. RESULTS: By pooling all the studies, we found that VEGF-634G/C polymorphism was not associated with ROP risk at co-dominant and allele levels and no association was also found in dominant and recessive models. While stratifying on ethnicity level no association was observed in Caucasian and Asian population. DISCUSSION: This meta-analysis suggests that VEGF-634G/C polymorphism may not be associated with ROP risk, the association between single VEGF-634G/C polymorphism and ROP risk awaits further investigation.

Physical characterization, magnetic measurements, REE geochemistry and biomonitoring of dust load accumulated during a protracted winter fog period and their implications.

The winter fog in India is a recurrent phenomenon for more than a decade now affecting the entire Himalayan and sub-Himalayan regions covering an area of nearly 500,000 km(2). Every winter (December-January), the air and surface transports in cities of northern India (Amritsar, New Delhi, Agra, Gwalior, Kanpur, Lucknow, and Allahabad) are severely disrupted with visibility reduced to <50 m at times. Since dust particles are known to act as nuclei for the fog formation, this study is aimed to carry out physicochemical characterization of the dust particulates accumulated during a protracted fog period from one of the severely fog affected cities of north India (Allahabad; 25°27'33.40″N-81°52'45.47″E). The dust-loaded tree leaves belonging to Ficus bengalensis and Ficus religiosa from 50 different locations between January 24 and 31, 2010 are sampled and characterized. The mass of dust, color, grain shape, size, phase constituents, and mineral magnetic parameters, such as magnetic susceptibility, SIRM, χ fd%, and S-ratio, show minor variation and the regional influence outweighs local anthropogenic contributions. The dust compositions show fractionated rare earth element pattern with a pronounced negative Eu anomaly similar to upper continental crust and further suggesting their derivation from sources located in parts of north and central India.

Pseudomonas aeruginosa in cystic fibrosis: pyocyanin negative strains are associated with BPI-ANCA and progressive lung disease.

The clinical consequence of chronic Pseudomonas aeruginosa colonization in cystic fibrosis (CF) varies between individuals for unknown reasons. Auto-antibodies against bactericidal/permeability increasing protein (BPI-ANCA) are associated with poor prognosis in CF. We hypothesize that there is a correlation between the presence of BPI-ANCA, the properties of the colonizing bacteria and the clinical conditions of the host. We compared isolates of P. aeruginosa from BPI-ANCA positive CF patients who have deteriorating lung disease with BPI-ANCA negative CF patients who are in stable clinical conditions. Epithelial cells (A549) and isolated polymorphonuclear granulocytes (PMNs) were stimulated with the isolates and cell death was analyzed with flow cytometry. We found that the ANCA associated strains in most cases showed pyocyanin negative phenotypes. These strains also induced less inflammatory response than the non-ANCA associated strains as shown by apoptosis and necrosis of epithelial cells and neutrophils. Our results suggest that colonization with strains of P. aeruginosa that induce a weak inflammatory response is associated with unfavorable outcome in CF. We speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying PMNs in the airways, contributing to progression in CF lung disease.

Somatic hypermutation in peritoneal B1b cells.

Murine B1 cells have been shown to be able to switch to IgA in vitro. In agreement, we could demonstrate in the peritoneum of mice the presence of IgA producing B1 cells. Interestingly, enzyme-linked immunospot assays of lipopolysaccharide stimulated cultures revealed that only the B1b cell subpopulation contained high numbers of such cells while IgA producing B cells were rare amongst the B2 and B1a cell populations. This was confirmed by RT-PCR on sorted peritoneal B cell subpopulations. In addition, the variable regions associated with IgA of peritoneal B1b cells displayed extensive variation due to somatic hypermutation. In contrast, mutations were found only at low frequencies in VH regions associated with IgM of both B1 cell populations. Thus, peritoneal B1b cells display many similarities to B2 cells. This finding is consistent with the idea of a layered immune system in which peritoneal B1a and splenic follicular B2 cells appear at the two extremes and peritoneal B1b and B2 cells represent intermediates.

Loss of lambda2(315) transgene copy numbers influences the development of B1 cells.

Transgenic L2 mice contain high numbers of the lambda2(315) immunoglobulin L chain gene in their germ line. They are characterized by an almost complete block in B2 cell development and dominance of B1 cells in their periphery. This was attributed to high transgene expression. Here, we describe a variant of such mice (L2V), which has lost half of the transgene copies. This results in decreased transgene expression. Consequently, such mice display less severe isotype exclusion and an increase in B cells expressing endogenous kappa light chains. In addition, the B2 cell compartment is enlarged. Nevertheless, L2V mice exhibit phosphatidylcholine (PtC) binding B cells expressing lambda L chains as well as an unaltered number of B1a cells expressing the dominating specificity usually encountered in L2 mice. Since in L2V mice transgene integration and regulation is identical to L2 mice, the correlation of decreased transgene expression and increased presence of B2 cells strongly suggests that high transgene expression is decisive for development of B1 cells in L2 mice.

Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature.

Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.