Comparative Molecular Dynamics Simulation of Hepatitis C Virus NS3/4A Protease (Genotypes 1b, 3a and 4a) Predicts Conformational Instability of the Catalytic Triad in Drug Resistant Strains

The protease domain of the Hepatitis C Virus (HCV) nonstructural protein 3 (NS3) has been targeted for inhibition by several direct-acting antiviral drugs. This approach has had marked success to treat infections caused by HCV genotype 1 predominant in the USA, Europe, and Japan. However, genotypes 3 and 4, dominant in developing countries, are resistant to a number of these drugs and little progress has been made towards understanding the structural basis of their drug resistivity. We have previously developed a 4D computational methodology, based on 3D structure modeling and molecular dynamics simulation, to analyze the active sites of the NS3 proteases of HCV-1b and 4a in relation to their catalytic activity and drug susceptibility. Here, we improved the methodology, extended the analysis to include genotype 3a (predominant in South Asia including Pakistan), and compared the results of the three genotypes (1b, 3a and 4a). The 4D analyses of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) indicate conformational instability of the catalytic site in HCV-3a and 4a compared to that of HCV-1b NS3 protease. The divergence is gradual and genotype-dependent, with HCV-1b being the most stable, HCV-4a being the most unstable and HCV-3a representing an intermediate state. These results suggest that the structural dynamics behavior, more than the rigid structure, could be related to the altered catalytic activity and drug susceptibility seen in NS3 proteases of HCV-3a and 4a.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

Selective inhibition of cardiac cyclic nucleotide phosphodiesterases by doxorubicin and daunorubicin

Doxorubicin and daunorubicin, the anthracycline antitumor agents, were evaluated for their in vitro and in vivo effect on phosphodiesterase (PDE) activity in mouse tissues. Doxorubicin at a concentration of 10(-4)M inhibited cardiac c-AMP (adenosine 3',5', monophosphate) PDE activity 50% of the control whereas in lungs and spleen, the activity was inhibited only 20%. On the contrary no effect was seen in kidney and liver. In addition, cardiac c-GMP (guanosine 3',5' monophosphate) PDE appeared less sensitive to doxorubicin than c-AMP PDE though inhibition in heart was more pronounced than in any other tissue. It appears that daunorubicin inhibits c-AMP PDE activity in heart markedly less than doxorubicin. Kinetic studies indicate that both inhibitions of c-AMP and c-GMP PDE by doxorubicin were non-competitive with substrate. Intravenous administration of 20 and 30 mg/kg of free doxorubicin to CDF1 mice resulted in 33 and 39% decreases in cardiac c-AMP PDE activity respectively by 72 hrs. In contrast, similar intravenous injections of same doses of doxorubicin entrapped in cardiolipin liposomes had no effect on c-AMP PDE activity in any tissues. These studies demonstrate the relative selectivity of the cardiac cyclic nucleotide PDE inhibitory effect of doxorubicin suggesting that this inhibition might be one aspect of the mechanism of anthracycline-induced cardiotoxicity.     

Ethanol production by nitrogen-deficient yeast cells immobilized in a hollow-fiber membrane bioreactor

Summary Cells ofSaccharomyces cerevisiae ATCC 4126, immobilized within the macroporous walls of asymmetric hollow-fiber membranes, were alternately perfused with 10% glucose complex medium and with 10% glucose defined medium which was deficient in nitrogen. Using complex growth medium, ethanol productivities during the initial 10 h of culture attained a maximum level of 133 g/l-h based on the total fiber volume (3% ethanol). Productivities during nitrogen deficiency stabilized at 10 g/l-h (0.5 ethanol). In subsequent growth phases, ethanol production rates increased to levels 40–70% of initial growth-phase values, but the ability to regenerate the fermentation activity decreased with culture age. During nitrogen deficiency, the fermentation efficiency declined with a concomitant reduction in the total protein concentration of immobilized cells within the hollow-fiber membranes. The molar ratio of acetaldehyde to ethanol increased seven-fold during nitrogen deficiency, indicating that the overall decline in glycolytic activity was accompanied by preferential reduction in alcohol dehydrogenase activity. The molar ratio of glycerol to ethanol increased two-fold during nitrogen deficiency, and large lipid-like droplets accumulated within the nitrogen-deficient cells. In addition to these findings, we conclude that current hollow-fiber membrane reactors should be limited to cell cultures having low growth rates, low O₂ requirements, and low CO₂ production rates.     

Strand breakage in DNA by silicic acid

The alkaline unwinding assay has been used to demonstrate the formation of single-strand breaks in DNA on treatment with silicic acid. Double-stranded DNA, containing no single-strand breaks, when incubated with increasing concentrations of silicic acid, showed the formation of an increasing number of strand breaks per molecule. Experiments on reduction of silicic acid-treated DNA with NaBH4 suggested the possibility of creation of apurinic or apyrimidinic sites. The significance of silicic acid interaction with cellular DNA during asbestos exposure is discussed.     

Evaluation of Biken test for detection of Salmonella enterotoxin.

Biken test by using antiserum to purified Salmonella enterotoxin (SE) was standardized and carried out to screen salmonellae for their enterotoxigenicity. As many as 101 strains of Salmonella belonging to 15 different serotypes isolated from foods of animal origin were subjected to Biken test. Of these, 68 (67.32%) were found seropositive. The test correlated with the rabbit ligated ileal loop (RLIL) test completely for the detection of enterotoxin producing salmonellae with 24 test strains. However, 5 of the 13 strains which were negative in the RLIL test, yielded positive results with the Biken test.     

Enhancement of Dissolution and Skin Permeability of Pentazocine by Proniosomes and Niosomal Gel

Proniosomes (PN) are the dry water-soluble carrier systems that may enhance the oral bioavailability, stability, and topical permeability of therapeutic agents. The low solubility and low oral bioavailability due to extensive first pass metabolism make Pentazocine as an ideal candidate for oral and topical sustained release delivery. The present study was aimed to formulate the PNs by quick slurry method that are converted to niosomes (liquid dispersion) by hydration, and subsequently formulated to semisolid niosomal gel. The PNs were found in spherical shape in the SEM and stable in the physicochemical and thermal analysis (FTIR, TGA, and XRD). The quick slurry method produced high recovery (>?80% yield) and better flow properties (θ?=?28.1–37.4°). After hydration, the niosomes exhibited desirable entrapment efficiency (44.45–76.23%), size (4.98–21.3 μm), and zeta potential (??9.81 to ??21.53 mV). The in vitro drug release (T_100%) was extended to more than three half-lives (2–4 h) and showed good fit to Fickian diffusion indicated by Korsmeyer-Peppas model (n?=?0.136–0.365 and R²?=?0.9747–0.9954). The permeation of niosomal gel was significantly enhanced across rabbit skin compared to the pure drug-derived gel. Therefore, the PNs are found promising candidates for oral as dissolution enhancement and sustained release for oral and topical delivery of pentazocine for the management of cancer pain.     

Do rivers influence fine-scale population genetic structure of tigers in the Sundarbans?

Global tiger Panthera tigris populations mostly survive within the geographically fragmented forest patches, thereby limited genetic exchange between isolated populations. Assessing the genetic status of these populations can reveal the effects of dispersal barriers and provide critical insights to guide future conservation actions. Using non-invasively collected biological samples, we investigated fine-scale genetic structure of tigers in the Sundarbans mangrove forests intersected by the complex river systems, and which holds one of the largest global tiger populations. We genotyped 52 tiger samples at 10 polymorphic microsatellite loci, and sequenced 33 of them for a total of 1263 base-pairs at four mitochondrial gene fragments. Microsatellite analyses exhibit a signature of fine-scale genetic structure, which might have been the consequence of limited tiger dispersal due to wide rivers across the Sundarbans. Similarly, mitochondrial data show a historic pattern of population isolation that might be due to wider rivers across the entire Sundarbans shared by Bangladesh and India. Given the intrinsic nature of the mangrove habitat embedded with numerous rivers, increased commercial traffic and human activities may further impede tiger dispersal across wide rivers, escalating further genetic isolation of the Sundarbans tigers.     

<Emphasis Type="Italic">Ab initio</Emphasis> calculations of ionic hydrocarbon compounds with heptacoordinate carbon

Ionic hydrocarbon compounds that contain hypercarbon atoms, which bond to five or more atoms, are important intermediates in chemical synthesis and may also find applications in hydrogen storage. Extensive investigations have identified hydrocarbon compounds that contain a five- or six-coordinated hypercarbon atom, such as the pentagonal-pyramidal hexamethylbenzene, C₆(CH₃)₆²⁺, in which a hexacoordinate carbon atom is involved. It remains challenging to search for further higher-coordinated carbon in ionic hydrocarbon compounds, such as seven- and eight-coordinated carbon. Here, we report ab initio density functional calculations that show a stable 3D hexagonal-pyramidal configuration of tropylium trication, (C₇H₇)³⁺, in which a heptacoordinate carbon atom is involved. We show that this tropylium trication is stable against deprotonation, dissociation, and structural deformation. In contrast, the pyramidal configurations of ionic C₈H₈ compounds, which would contain an octacoordinate carbon atom, are unstable. These results provide insights for developing new molecular structures containing hypercarbon atoms, which may have potential applications in chemical synthesis and in hydrogen storage.

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Graphical abstract Possible structural transformations of stable configurations of (C₇H₇)³⁺, which may result in the formation of the pyramidal structure that involves a heptacoordinate hypercarbon atom.

     

Short-term movements and strong dependence on figs of binturongs (<Emphasis Type="Italic">Arctictis binturong</Emphasis>) in Bornean rainforests

We evaluated short-term movements of three radio-collared binturongs in relation to food distribution in Bornean rainforests, in addition to the basic ecological information on their home-range size and diet. Mean 95% fixed kernel and 95% MCP home-range size were 4.24?±?0.79 km² and 1.54?±?0.89 km², respectively (mean ± SD). We recorded 13 fig Ficus species and four non-fig species as their foods. Fig trees accounted for 87.5% of the feeding sites of the three collared binturongs, and 87.9% including uncollared individuals. Our results suggested that binturongs’ short-term movements were strongly affected by food distribution, especially figs. They feed on various fig species and may remember the location and fruiting periods of fig trees. They may use the biggest fig species, F. punctata, as a fallback food when other foods are scarce. Although this is the first systematic study to describe movement and feeding habits of binturongs, further studies are needed to understand their ecology so that proper measures can be designed for their conservation.