The N-terminus region of Drp1, a Rint1 family protein is essential for cell survival and its interaction with Rad50 protein in fission yeast S.pombe

BackgroundDefects in DNA repair pathway can lead to double-strand breaks leading to genomic instability. Earlier we have shown that S.pombe Drp1, a Rint1/Tip1 family protein is required for the recovery from DNA damage.MethodsVarious truncations of Drp1 protein were constructed and their role in DNA damage response and interaction with Rad50 protein has been studied by co-immunoprecipitation and pull-down assays.ResultsThe structural and functional analysis of Drp1 protein revealed that the N-terminus region of Drp1 is indispensable for the survival. The C-terminus truncation mutants, drp1C1Δ and drp1C2Δ exhibit temperature sensitive phenotype and are hypersensitive against DNA damaging agents with elevated level of Rad52-YFP foci at non-permissive temperature indicating the impairment for DNA damage repair pathway. The essential N-terminus region of Drp1 interacts with the C-terminus region of Rad50 and might be involved in influencing the MRN/X function. Small-angle X-ray (SAXS) analysis revealed three-domain like shapes in Drp1 protein while the C-terminus region of Rad50 exhibit unusual bulges. Computational docking studies revealed the amino acid residues at the C-terminus region of Rad50 that are involved in the interaction with the residues present at the N-terminal region of Drp1 indicating the importance of the N-terminal region of Drp1 protein.ConclusionsWe have identified the region of Drp1 and Rad50 proteins that are involved in the interaction and their role in the DNA damage response pathway has been analyzed.General significanceThe functional and structural aspects of fission yeast Drp1 protein and its interaction with Rad50 have been elucidated.     

Serum proteins among Dusads of Bihar

Horizontal starch gel electrophoresis has been employed for the detection of haptoglobin, transferrin and albumin phenotypes among 88 Dusads of Bihar. No variant of the haptoglobins or transferrins has been found in this sample, whereas one individual showed bisalbuminemia.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

Design,synthesis and biological evaluation of novel indole-xanthendione hybrids as selective estrogen receptor modulators

Ground breaking clinical therapeutic advances in the treatment of breast cancer (BC) is the introduction of selective estrogen receptor modulators (SERMs). We have expeditiously designed and synthesized indole-xanthendione hybrids by coalescing the indole nucleus with xanthendione. All the compounds were first screened for anti-proliferative activity, cytotoxicity and ER-α binding affinity by utilizing ER-α dominant T47D BC cell lines, PBMCs and ER-α competitor assay kit. From this study, two representative compounds 6e and 6f showing most promising activity were advanced for gene expression studies for targeting ER-α. Cell imaging experiment undoubtedly indicate that both the compounds were able to cross cellular bio membrane and accumulate thus instigating cytotoxicity. RT-PCR and Western blotting experiments further strengthened that both compounds altered the expression of mRNA and receptor protein of ER-α, thereby forestalling downstream transactivation and signalling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that indole moiety of the compounds 6e and 6f helps in the anchoring of the xanthendione moiety in the hydrophobic region of the cavity thus enabling the compound to bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these finding collectively imply that compound 6e and 6f represents a novel potent ER-α antagonist and in the development of SERMs for the management of BC.     

Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres

Late 8-cell blastomeres were harvested within the first 45 min after entering mitosis. Some mitotic cells were analysed within the ensuing 2 h for the organization of their surface in relation to their progress through mitosis. Whereas in most late interphase cells microvilli were restricted to a discrete polar region, in mitotic cells at all stages from early metaphase to immediately postcytokinesis microvilli were found to be present over more of the cell surface. Other mitotic cells were placed in nocodazole to arrest them in M-phase for up to 10 h. They were found to show an even more extensive distribution of microvilli over the whole surface, the longer periods of incubation yielding more extended coverage such that many cells no longer appeared to have any residual surface polarity. Removal from nocodazole at all time points from 1 to 10 h resulted in most cells completing mitosis to yield pairs of cells which, in most cases, resembled pairs derived from nonarrested blastomeres and in which a defined polar area of microvilli was restored. However, the percentage of differentiative divisions decreased after 6 h arrest. If, instead of removing cells from nocodazole, they were placed in both nocodazole and cytochalasin D (CCD) for periods of up to 3 h, most microvilli retracted to reveal a tight polar zone of CCD-resistant microvilli. This result suggests that a heterogeneity of cytocortical organization may still exist within the arrested mitotic cell. We propose a model to explain the origin of this heterogeneity of organization and its relationship to the generation of cell diversity.     

Cerebrospinal fluid ferritin and albumin index: potential candidates for scoring system to differentiate between bacterial and viral meningitis in children

Objectives: To evaluate the diagnostic role of cerebrospinal fluid (CSF) ferritin and albumin index (AI?=?CSF albumin/serum albumin?×?1000) in differentiating acute bacterial meningitis (ABM) from acute viral meningitis (AVM) in children.

Methods: The study included 42 cases each of ABM and AVM in pediatric age group. Receiver operating characteristic (ROC) analysis was carried out for CSF ferritin and AI. Binary logistic regression was also done.

Results: CSF ferritin and AI were found significantly higher in ABM compared to AVM. Model obtained using AI and CSF ferritin along with conventional criteria is better than existing models.     

Naturally Occurring Variants of the Dysglycemic Peptide Pancreastatin: DIFFERENTIAL POTENCIES FOR MULTIPLE CELLULAR FUNCTIONS AND STRUCTURE-FUNCTION CORRELATION*

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca²⁺ levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by ∼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in ∼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.     

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.     

Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity

Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and are associated with the accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about the cytotoxic effects of these porphyrin nanostructures. Previously, we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), to cause organelle-selective protein aggregation, which we posited to be a major mechanism by which fluorescent porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein-binding ability. UV–visible spectrophotometry showed pH-mediated reversible transformations of PP-IX nanostructures. Biochemical analysis showed that PP-IX nanostructure size modulated PP-IX-induced protein oxidation and protein aggregation. Furthermore, albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. PP-IX binding quenched albumin intrinsic fluorescence and oxidized His-91 residue to Asn/Asp, likely via a previously described photo-oxidation mechanism for other proteins. Extracellular albumin protected from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX nanostructures in the context of porphyrias and offers insights into potential novel therapeutic approaches.     

Redescription of Robustodorus megadorus with Molecular Characterization and Analysis of Its Phylogenetic Position within the Family Aphelenchoididae

Based on a new record of the rare species Robustodorus megadorus from Utah, the generic diagnosis was amended to include the following characters: a labial disc surrounded by six pore-like sensilla; the absence of a cephalic disc; a lobed cephalic region devoid of annulation; a hexagonal inner cuticular structure of the pouch surrounding the stylet cone; large stylet knobs, rounded in outline and somewhat flattened on their lateral margins; a large spermatheca with an occluded lumen and lacking sperm; the excretory pore located between the median bulb and nerve ring. The stylet orifice consists of an open, ventral, elongate slit or groove. These characters distinguish the genus from the closely related genus Aphelenchoides. A lectotype and paralectotypes were designated. Results of phylogenetic analyses of the 18S and D2-D3 of 28S rRNA gene sequences revealed that R. megadorus occupies a basal position within one of the two main clades of the subfamily Aphelenchoidinae and shares close relationships with a species group of the genus Aphelenchoides that includes A. blastophthorus, A. fragariae, A. saprophilus, A. xylocopae, and A. subtenuis. Several specimens in our collection of R. megadorus were infected with Pasteuria sp. as were some of the paralectotypes.