Serum levels of angiogenic and anti-angiogenic factors: their prognostic relevance in locally advanced hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prototype tumor wherein angiogenesis plays a vital role in its progression. The role of VEGF, a major angiogenic factor in HCC is known; however, the role of anti-angiogenic factors simultaneously with the angiogenic factors has not been studied before. Hence, in this study, the serum levels of major angiogenic [Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 (Ang-2)] and anti-angiogenic (endostatin, angiostatin) factors were analyzed and correlated with clinico-radiological features and with outcome. A total of 150 patients (50 HCC, 50 cirrhosis and 50 chronic hepatitis) and 50 healthy controls were enrolled in this study. Serum levels of VEGF, Ang-2, endostatin, and angiostatin were estimated by enzyme-linked immunosorbent assay. HCC shows significantly elevated serum levels of angiogenic factors VEGF and Ang-2 and of anti-angiogenic factors endostatin and angiostatin. ROC curve analysis for serum VEGF yielded an optimal cut-off value of 225.14 pg/ml, with a sensitivity of 78 % and specificity of 84.7 % for a diagnosis of HCC and its distinction from other group. Using this value, the univariate and multivariate analysis revealed significantly poor outcome in patients with higher levels of serum VEGF (p = 0.009). Combinatorial analysis revealed that patients with higher levels of both angiogenic and anti-angiogenic factors showed poor outcome. Serum VEGF correlates with poor survival of HCC patients and, therefore, serves as a non-invasive biomarker of poor prognosis. Moreover, elevated levels of anti-angiogenic factors occur endogenously in HCC patients.     

Subcellular dynamics of variants of SG2NA in NIH3T3 fibroblasts

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)‐tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3β‐ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP‐tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP‐tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation‐dephosphorylation. The inhibition of GSK3β by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP‐tagged 35, 52, and 87 kDa, inhibition of GSK3β caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3β by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular‐signal‐regulated‐kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3β, and ERK. Taken together, the variants of SG2NA are modulated by the kinase‐phosphatase network in a similar but characteristic manner.     

Exploring the Zika Genome to Design a Potential Multiepitope Vaccine Using an Immunoinformatics Approach

International Journal of Peptide Research and Therapeutics - Zika is one of the most dreaded viruses which has left mankind crippled for over years. Current no vaccines for Zika are available in...     

Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome

Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.     

Fibrotic Remodeling of the Extracellular Matrix through a Novel (Engineered,Dual-Function) Antibody Reactive to a Cryptic Epitope on the N-Terminal 30 kDa Fragment of Fibronectin

Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached “RGD” sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis - migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.     

Histone deacetylases 9 and 10 are required for homologous recombination

We tested the role of histone deacetylases (HDACs) in the homologous recombination process. A tissue-culture based homology-directed repair assay was used in which repair of a double-stranded break by homologous recombination results in gene conversion of an inactive GFP allele to an active GFP gene. Our rationale was that hyperacetylation caused by HDAC inhibitor treatment would increase chromatin accessibility to repair factors, thereby increasing homologous recombination. Contrary to expectation, treatment of cells with the inhibitors significantly reduced homologous recombination activity. Using RNA interference to deplete each HDAC, we found that depletion of either HDAC9 or HDAC10 specifically inhibited homologous recombination. By assaying for sensitivity of cells to the interstrand cross-linker mitomycin C, we found that treatment of cells with HDAC inhibitors or depletion of HDAC9 or HDAC10 resulted in increased sensitivity to mitomycin C. Our data reveal an unanticipated function of HDAC9 and HDAC10 in the homologous recombination process.     

Immune response to different fractions of Taenia solium cyst fluid antigens in patients with neurocysticercosis

The immunopathogenesis of neurocysticercosis (NCC) largely remains unknown. We analyzed the immune response to different fractions of Taenia solium cyst fluid antigens in patients with NCC. Lymphocytes were separated from 48 patients with NCC-related active epilepsy and 30 healthy controls. T. solium (isolated from pig muscles) antigens (crude lysate, CL; cyst wall, CW and cyst fluid, CF) at 20 μg/well concentrations were used to stimulate the cells in a lymphocyte transformation test (LTT). Only CF antigen stimulated cell proliferation significantly greater than control (p < 0.001), hence cyst fluid antigens were further studied. The CF antigens were electro-blotted on nitrocellulose membrane (NC), cut at 0.5 cm distance and particulate antigens were prepared. A total of 12 fractions, designated F1 to F12 according to molecular weight were tested in-vitro for LTT. After 72 h of stimulation by the different fractions, Th1 (IL-1β, TNF-α, IL-2) and Th2 (IL-4, IL-10) cytokine responses were determined in culture supernatants by ELISA. Low molecular weight fractions F1 through F4 (Mol. wt. < 25 kDa) were found to be potent inducers of cytokines. Fractions F1, F3 and F4 induced the production of Th1 (IL-1β, TNF-α, IL-2), whereas F2 induced the production of Th2 (IL-4 and IL-10) cytokine. The study shows that the low molecular weight fractions of CF antigens are immuno-dominant. Most of these fractions (F1, F3, F4) induce strong Th1 immune response except F2 which induces Th2 response. Further studies are needed to identify the different antigens present in these fractions to determine the molecules responsible for the immune response.     

Characterization of alkalotolerant bacterial community by 16S rDNA-based denaturing gradient gel electrophoresis method for degradation of dibenzofuran in soil microcosm

An alkalotolerant bacterial community was developed by continuous enrichment in the chemostat in presence of dibenzofuran (DF) as sole carbon source. Six different types of bacterial isolates were cultured on nutrient broth agar plates together with six operational taxonomic units (OTUs) at pH 7.0 and pH 8.0 by 16S rDNA-DGGE method. However, isolates of microbial community was declined from three OTUs (pH 9.0) to two at pH 10.0 after enrichment in alkaline condition. Among the six isolates tested for degradation of DF, Pseudomonas sp. and Bacillus sp. the members of alkalotolerant bacterial community had better potency to degrade dibenzofuran. Alkalotolerant bacterial community introduced in soil microcosm for evaluation of survival of most suitable isolates and degradation of dioxin-like compound indicated more than 90% degradation of dibenzofuran after 45 days by the bacterial community enriched for 180 days in the chemostat at pH 10, however, microbial community was not competent to utilize even 50% DF after day 30, not enriched in the chemostat. The survival of competent bacteria monitored by DGGE method in soil microcosm indicated presence of two major alkalotolerant isolates for utilization of dibenzofuran, substantiated the results and significance of alkalotolerant bacteria for in situ bioremediation of dioxin-like compounds in the environment.