Evidence of dengue and chikungunya virus co-infection and circulation of multiple dengue serotypes in a recent Indian outbreak.

In India, dengue endemic areas overlap with chikungunya-affected areas and both the viruses are transmitted by same vector, Aedes aegypti - thereby increasing opportunity of co-infection by both viruses. Present study was carried out to understand the DENV-CHIKV infection dynamics during recent outbreaks in eastern India (West Bengal state) and its implication on disease manifestations. Blood was collected from 326 symptomatic febrile patients. Patients' serum was subjected to serological diagnosis for presence of anti-dengue-IgM, anti-chikungunya-IgM antibodies and dengue-NS1 antigen by ELISA. Viral RNA was extracted, and presence of dengue virus (DENV) and chikungunya virus (CHIKV) genome, their viral load (VL), and serotype among infected patients' plasma was determined by real-time qRT-PCR. Statistical analysis was performed by using EPI INFO software. DENV and CHIKV were detected in 54% and 33% of symptomatic patients respectively, among whom 23% were harboring both viruses. WHO classified warning signs were detected among 64% DENV patients and 61% DENV-CHIKV double-infected patients. Patients with warning signs always had much higher DEN VL than those without warning signs. Hemorrhagic manifestation and abdominal pain was found in significantly higher frequency among patients with high dengue VL (>10,000 copies/ml). DENV2 was the most predominant serotype among monotypic dengue patients, whereas DENV2-DENV4 combination was most prevalent among patients infected with dual dengue serotypes. This study indicated that DENV-CHIKV double infection and high dengue VL contributed towards severe disease manifestations among infected patients. DENV2 and DENV2-DENV4 combination were the most prevalent serotype(s) found in current outbreak.

Application of comet assay in the study of DNA damage and recovery in rohu (Labeo rohita) fingerlings after an exposure to phorate, an organophosphate pesticide.

Labeo rohita (rohu) fingerlings were exposed to different concentrations (0.001, 0.002 and 0.01 ppm) of phorate, an organophosphate pesticide; samplings were done at 24, 48, 72 and 96 h. The study was carried out to evaluate tissue specific genotoxic effects produced by phorate, on three different tissue systems and to assess DNA repair response in fish. Results of tissue specific DNA damage experiments showed low baseline damage in blood cells followed by gill and liver cells in control individuals whereas more DNA breaks were found in liver followed by gill and blood cells of treated individuals. Concentrations-dependent DNA damage showed a strong, linear and positive relationship (r(2) = >0.7) in all three tissues. Clear time-related increase in DNA damage was observed for all tissues exposed to all concentrations except in liver cells at 0.01 ppm, where the DNA damage declined significantly after 72 h. For the assessment of DNA repair response, fingerlings were first exposed to 0.01 ppm of phorate for 72 h and then transferred to pesticide free water. Tissue chosen for the repair experiment was liver. Samplings were done at 0, 3, 6, 12 and 24 h after the release of 72 h pesticide treated fishes into pesticide free water. Fishes showed a reduction in DNA breaks from 3 h onwards in pesticide free water and at 24 h returned to control level damage. The results indicate that phorate is a potential genotoxicant, comet assay can be used in DNA damage and repair analysis, response to pollutants in multicellular animals is often tissue specific.

Influence of stabilizers on quality of sandesh from buffalo milk.

Buffalo milk standardized to solids-not-fat (SNF) to fat ratio of 1.4 was added separately with 0.1% (w/w) each of carrageenan, sodium alginate and carboxymethyl cellulose and then heated, cooled and coagulated to obtain chhana which was converted into sandesh by adding 1.5% (w/w) wheat flour and 25% (w/w) cane sugar followed by heating (40 min/kg chhana). The treated samples of sandesh were compared with control prepared similarly manner but without stabilizer. Addition of stabilizer decreased hardness, fracturability, adhesiveness, cohesiveness, gumminess and chewiness of sandesh and improved sensory body and texture, colour and appearance as well as overall acceptability of the product when compared with control. Textural and sensory properties of different samples of sandesh indicated that the product made by adding carrageenan proved best. Carrageenan at 0.1% produced better results in terms of textural and sensory profile of sandesh as compared to 0, 0.075 and 0.125% (w/w) of carrageenan.

Gradient of reduced glutathione in the tail of Hemidactylus leschenaultii (Sauria: Gekkonidae).

A proximo-distal gradient of reduced glutathione (GSH), a non enzymatic antioxidant was observed in the original tails of the lizard, H. leschenaultii. In the regenerating tails, a gradual increase in the level of GSH was noted with tail elongation. In the newly regenerated tails also the level of GSH remained higher in the proximal part than the corresponding distal parts.

Harmonia sedecimnotata (F.): Predatory potential, biology, life table, molecular characterization, and field evaluation against Aphis gossypii Glover.

The ladybird beetle, Harmonia sedecimnotata (F.) was studied in biology, life table, consumption rates, molecular characterization, and field evaluation. The net reproductive rate (R0), based on the age-stage and two-sex life table, was 43.2 eggs/individual. The female adults lived longer (68.1 d) than the male adults (62.9 d). The rate of consumption increased with progress in each stage of development. Compared to the other larval stages of the predator, the fourth stadium consumed most quantities of Aphis gossypii Glover nymphs (Hemiptera: Aphididae) (200.4). Both female (2214.6) and male (1792.4) consumed more prey (nymphs) than larvae. The net rate of consumption was 1458.92 nymphs of melon aphids. There was no variation in the sequences of the two nucleotides out of 583 bp, H sedecimnotata China (EU392410) and India (MG720024). Our investigations demonstrated that inoculative release of 30 or 40 or 50 adults per 100 m2 attained high reduction of aphids (>90%). Thus, it may be recommended the release rate of 40 adults per 100 m2 to suppress the eggplant aphid population. H. sedecimnotata is therefore one of the most promising biological control agents for cotton aphids that can be achieved for instant control through an inoculative release of adults.

Isolation and characterization of chromatin from Neurospora crassa.

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mmicro and a minimum at 238-239 mmicro. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120-150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.

Dechlorination of PCB in the presence of plant nitrate reductase.

The dechlorination of PCB, specifically the noncoplanar congener PCB 153, has been observed in the presence of a crude nitrate reductase extract from Medicago sativa leaves. These observations were further confirmed using a commercially available and pure nitrate reductase from Zea mays. The presence of nitrate reductase increased PCB 153 dechlorination. Then, the addition of molybdenum, the enzyme's cofactor, enhanced dechlorination of the environmental contaminant. The ability of plant nitrate reductase to dechlorinate PCB is a new observation.

Lingual lipase in cystic fibrosis. Quantitation of enzyme activity in the upper small intestine of patients with exocrine pancreatic insufficiency.

We have measured the level of lingual lipase activity in gastric and duodenal aspirates of five patients with cystic fibrosis (CF) and pancreatic insufficiency. Lingual lipase activity (measured in vitro by the hydrolysis of long-chain triglyceride, tri-[3H]olein, at pH 4.2 and expressed in nanomoles FFA released per milliliter aspirate per minute) and pH in gastric and duodenal aspirates were measured at 10-min intervals during a a 30-min basal period and at 15-min intervals during a 2-h period after the ingestion of a test meal. In gastric aspirates, lingual lipase activity decreased from basal levels of 200 +/- 34 nmol FFA released per milliliter per minute (similar to values reported previously in normal subjects (Hamosh M., H. L. Klaeveman, R. O. Wolf, and R. O. Scow, 1975, J. Clin. Invest., 55:908-913) to 79 +/- 15 nmol FFA/ml per min during the first postprandial hour and returned to basal levels during the second postprandial hour, (206 +/- 39 nmol FFA/ml per min). Duodenal aspirates, obtained during basal conditions, had lingual lipase activity similar to that in the stomach, 178 +/- 63 nmol FFA/ml per min. Enzyme activity levels were 56 +/- 14 and 113 +/- 29 during the first and second postprandial hours. Measurements of total lipase activity delivered to the ligament of Treitz showed that lingual lipase amounted to 91.22 +/- 4.06% of the total lipase activity in the upper small intestine during the 150-min study period. The basal and postprandial gastric pH levels in the five CF patients studied (3.2 +/- 0.44, 4.0 +/- 0.16, and 4.4 +/- 0.4 for basal and first and second postprandial hours, respectively) did not differ from previously reported values for normal subjects. The pH of duodenal aspirates was however significantly lower (P less than 0.001) in CF patients, both under basal conditions (5.0 +/- 0.26) and during the first and second postprandial hours (4.9 +/- 0.13 and 4.4 +/- 0.36, respectively), than in normal subjects. The low postprandial duodenal pH enables lingual lipase to act not only in the stomach but to continue the hydrolysis of dietary fat in the upper small intestine of CF patients. The data presented show that lingual lipase remains fully active in CF and accounts for greater than 90% of total lipase activity in the upper small intestine. We suggest that, because of low intestinal pH in CF, enzyme replacement therapy containing lingual lipase could improve fat absorption in CF patients to a greater extent than the pancreatic preparations now in use.

Ahaetulla nasuta anomala (Annandale, 1906) (Squamata: Colubridae), resurrected as a valid species with marked sexual dichromatism.

In this paper we resolve the taxonomic confusion related to Ahaetulla nasuta anomala (Annandale, 1906). On the basis of molecular and morphological data, we remove it from the synonymy of Ahaetulla nasuta (Lacépède, 1789) and reinstate it as a valid species-Ahaetulla anomala. This species is sexually dichromatic, males are green and females are brown in colour. Though the brown morph morphologically resembles Ahaetulla pulverulenta (Duméril, Bibron & Duméril, 1854) there are significant morphological and genetic differences between these two species. Additional information on taxonomy, natural history and distribution of the species is provided.

Microbial cellulases - Diversity & biotechnology with reference to mangrove environment: A review.

Cellulose is an abundant natural biopolymer on earth, found as a major constituent of plant cell wall in lignocellulosic form. Unlike other compounds cellulose is not easily soluble in water hence enzymatic conversion of cellulose has become a key technology for biodegradation of lignocellulosic materials. Microorganisms such as aerobic bacteria, fungi, yeast and actinomycetes produce cellulase that degrade cellulose by hydrolysing the ß-1, 4-glycosidic linkages of cellulose. In contrast to aerobic bacteria, anaerobic bacteria lack the ability to effectively penetrate into the cellulosic material which leads to the development of complexed cellulase systems called cellulosome. Among the different environments, the sediments of mangrove forests are suitable for exploring cellulose degrading microorganisms because of continuous input of cellulosic carbon in the form of litter which then acts as a substrate for decomposition by microbe. Understanding the importance of cellulase, the present article overviews the diversity of cellulolytic microbes from different mangrove environments around the world. The molecular mechanism related to cellulase gene regulation, expression and various biotechnological application of cellulase is also discussed.

Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.

Phosphorus is an essential element for all life forms. Phosphate solubilizing bacteria are capable of converting phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-37, was isolated from mangrove soil of the Mahanadi river delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Serratia sp. The maximum phosphate solubilizing activity of the strain was determined to be 44.84 µg/ml, accompanied by a decrease in pH of the growth medium from 7.0 to 3.15. During phosphate solubilization, various organic acids, such as malic acid (237 mg/l), lactic acid (599.5 mg/l) and acetic acid (5.0 mg/l) were also detected in the broth culture through HPLC analysis. Acid phosphatase activity was determined by performing p-nitrophenyl phosphate assay (pNPP) of the bacterial broth culture. Optimum acid phosphatase activity was observed at 48 h of incubation (76.808 U/ml), temperature of 45 °C (77.87 U/ml), an agitation rate of 100 rpm (80.40 U/ml), pH 5.0 (80.66 U/ml) and with glucose as a original carbon source (80.6 U/ml) and ammonium sulphate as a original nitrogen source (80.92 U/ml). Characterization of the partially purified acid phosphatase showed maximum activity at pH 5.0 (85.6 U/ml), temperature of 45 °C (97.87 U/ml) and substrate concentration of 2.5 mg/ml (92.7 U/ml). Hence the present phosphate solubilizing and acid phosphatase production activity of the bacterium may have probable use for future industrial, agricultural and biotechnological application.

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths.

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.

DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53.

Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors.

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds.

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila.

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.

Dietary fiber in pancreatic disease: effect of high fiber diet on fat malabsorption in pancreatic insufficiency and in vitro study of the interaction of dietary fiber with pancreatic enzymes.

The effect of high fiber diet on fat malabsorption was evaluated in twelve patients with exocrine pancreatic insufficiency secondary to chronic alcoholic pancreatitis. Additionally, the effect of dietary fiber on pancreatic enzymes was examined in vitro, employing different concentrations of cellulose, pectin, and wheat bran incubated with amylase, lipase, and trypsin. Ingestion of a high fiber diet was associated with a small but significant (p less than 0.01) increase in fecal weight and fecal fat excretion. All patients complained of increased abdominal flatulence with high fiber diet, however, no significant increase in frequency of bowel movements was noted. In vitro studies demonstrated reduction in pancreatic enzyme activity by increasing concentration of dietary fiber and its components. These data suggest that steatorrhea may be enhanced with the ingestion of high fiber diet in patients with exocrine pancreatic insufficiency on oral pancreatic enzyme therapy. Increase in fecal fat excretion may, in part, be related to reduction in the activity of pancreatic enzymes by the dietary fiber.

Dietary fiber supplementation: effect on exocrine pancreatic secretion in man.

The effect of a dietary fiber supplementation program (20 g/d) on exocrine pancreatic gland secretion was evaluated in six healthy male subjects who underwent quantitative assessment of pancreatic enzyme secretion both before and after 4 wk of dietary fiber supplementation. A duodenal perfusion technique was used to quantify the concentrations and output of pancreatic enzymes after ingestion of a standard test meal. Samples were aspirated from the ligament of Trietz and analyzed for pH, total protein, amylase, trypsin, and lipase activity. No significant changes were observed in duodenal flow rate pH, total protein, amylase, or trypsin concentrations and outputs after fiber supplementation. A marked increase in mean (+/- SEM) lipase concentration (U/mL) and output (kU/min) in both the resting and postprandial states was seen, reaching statistical significance (p less than 0.05) at 120 min postprandial. These data suggest that in man, a 4-wk dietary fiber supplementation program can modulate pancreatic lipase secretion.

Selenium and acute alcoholism.

Selenium status was investigated in nine inebriated alcoholic subjects by collecting serial samples of blood and urine during hospitalization for alcohol detoxification. The selenium content of various alcoholic beverages and samples of hospital diets was also determined. Mean plasma selenium level and mean urinary excretion of selenium were both significantly lower (p less than 0.01) in alcoholic subjects as compared to the control subjects at the time of admission. Furthermore, the daily dietary intake of selenium before hospitalization was estimated to be below the recommended safe and adequate range in the majority of the alcoholic subjects. The selenium content of various alcoholic beverages was determined to be very low (0.1 to 0.8 microgram/dl). These data suggest that selenium depletion does occur in alcoholic subjects most likely due to poor dietary intake. Selenium depletion in this group of patients is corrected by cessation of ethanol ingestion and adequate dietary intake without additional selenium supplementation.

Evaluation of vitamin A absorption by using oil-soluble and water-miscible vitamin A preparations in normal adults and in patients with gastrointestinal disease.

We evaluated vitamin A absorption in 50 healthy adults and 26 gastrointestinal-disease patients by measuring the postabsorptive response in plasma retinyl esters after oral doses of the vitamin. On 3 consecutive days, two physiologic-dose tests of 2000-2400 retinol equivalents (RE) and one pharmacologic-dose test (84,000 RE) were administered. The physiologic doses were given as an oil-soluble or a water-miscible preparation. In gastrointestinal-disease patients the physiologic-dose test was highly correlated with the pharmacologic-dose test for the oil-soluble preparation as determined by peak rise (r = 0.50, P less than 0.05) and area under the curve (r = 0.56, P less than 0.01), suggesting that the physiologic dose is valid for investigating vitamin A absorption. Intestinal-disease or resection patients absorbed preparations poorly. Pancreatic-disease patients absorbed the oil-soluble preparation poorly. Physiologic rather than pharmacologic doses of vitamin A can be used to study vitamin A absorption.

DNA sequences encoding enolase are remarkably conserved from yeast to mammals.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11), particularly isoform neuron-specific enolase (NSE), is primarily localized in neurons and neuroendocrine cells and is a cancer diagnostic marker for brain tumors. Homology of enolase-coding DNA sequences from human, dog, cow, rat, mouse, rabbit, chicken, and yeast cells was investigated using hybridization techniques, percent sequence divergence, and amino acid analysis. Because enolase is a significant enzyme of the glycolytic pathway, enolase-coding DNA sequences have been found in all organisms tested so far. The human enzyme was found to be more like those of monkey and dog in structure than to those of chicken and yeast. The implications of the existence of the genetic conservation of enolase-coding DNA sequences in understanding concerted evolution as well as post-transcriptional regulation during differentiation are discussed. This is the first report is which sequence divergence in the coding region for enolase has been determined in a variety of organisms.