Molecular docking analysis of Clostridium perfringens beta toxin model with potential inhibitors from the ZINC database

Beta toxin from Clostridium perfringens after being secreted in gut is capable of causing necrotic enteritis in humans and several other animal species and does not respond to routinely used antibiotics. Therefore, there is a need to design an effective inhibitor for the Clostridium perfringens beta toxin (CPB) using cutting edge drug discovery technologies. Hence, potential CPB inhibitors were identified using computer aided screening of compounds from the ZINC database. Further, we document the molecular docking analysis of Clostridium perfringens beta toxin model (that revealed 4 binding pockets, A-D) with the identified potential inhibitors. We show that ZINC291192 [N-[(1-methylindol-3-yl) methyl eneamino]-7,10-dioxabicyclo[4.4.0]deca-2,4,11-triene-8- carboxamide] has optimal binding features with calculated binding energy of -10.38 kcal/mol and inhibition constant of 24.76 nM for further consideration.     

Sustained release implants of chloroquine phosphate for possible use in chemoprophylaxis of malaria

Implants of chloroquine phosphate (CQP) using biodegradable polymer, gelatin (G) and cross-linked gelatin (CLG) were prepared and evaluated to assess their physicochemical properties and in vitro release profile. The mechanism and kinetics of release were studied to correlate the release phenomenon with the formulation parameters. Out of many batches of the implants investigated, the implant prepared with 20% gelatin at 2:1 drug polymer ratio, 10% crosslinking agent and 2% plasticizer (Batch J) was found to provide optimum release behavior conforming to the requirements of a long term implant for a week. In vivo studies conducted on albino rats showed consistent therapeutic blood level over a period of 7 days. Mean residence time (MRT) of the drug released in the body, calculated as the ratio of the area under the first moment curve (AUMC) to area under concentration time curve (AUC) was 72 hr for implant against 2.42 hr for subcutaneous injection.     

BRCA2 deficiency in mice leads to meiotic impairment and infertility

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

Decomposition patterns of unprocessed and processed lignocellulosics in a freshwater fish pond

In an attempt to recycle the lignocellulosic wastes like Eichhornia crassipes, Salvinia cucullata and rice (Oryza sativa) straw as manurial inputs in freshwater fish pond ecosystem, a decomposition experiment was carried out in litter bags in an oligotrophic freshwater fish pond environment, with the above mentioned three substrates in unprocessed and microbially processed forms. The loss rates, associated microbial groups, oxygen consumption patterns and other related parameters like carbon, nitrogen, phosphorus, cellulose, hemicellulose and lignin were analysed. The mean daily dry matter loss rates (unprocessed: 10.44>6.97>1.97 and processed: 11.03>8.21>3.67) and oxygen uptake rate (unprocessed: 0.675>0.571>0.568 mg O₂ g^–1 h^–1 and processed: 0.592>0.424>0.407 mg O₂ g^–1 h^–1) in raw and processed substrates were in the sequence Eichhornia > rice straw > Salvinia. The oxygen consumption pattern almost covariated with variations in temperature of pond water, daily dry matter loss rates and fungal counts on substrates. During the decay, the percentage of N and P increased whereas that of C decreased, resulting in lowering of C/N and C/P ratios of the substrates. The structural polymeric fractions like cellulose and hemicellulose decreased along with dry matter whereas the lignin content increased after an initial decrease due to loss of other structural carbohydrates resulting in apparent per cent gain of lignin. A higher number of different heterotrophic bacterial groups was observed in the processed substrates as compared to their raw counterparts. However, cellulolytic bacterial numbers were found to fluctuate through the study period. The fungal load was found to be decreasing gradually as the decay progressed. In this study, bacteria were found to be the prominent microbial group responsible for the decay. The nitrogen-fixing, phosphatase-producing and phosphorus-solubilising bacterial groups were observed to play an important role in lowering the C/N and C/P ratios of the decomposing substrates during decay.     

Diagnostic Yield of Universal Urine Toxicology Screening in an Unselected Cohort of Stroke Patients

Background

Illicit drug use increases the risk of cerebrovascular events by a variety of mechanisms. A recent report suggested that universal urine toxicology (UTox) screening of patients with stroke may be warranted. We aimed to evaluate the diagnostic yield of urine drug screening among unselected patients admitted with acute stroke or transient ischemic attack (TIA).

Methods

Using a single-center prospective study design, we evaluated consecutive patients with acute ischemic stroke, TIA, intracerebral hemorrhage (ICH), or subarachnoid hemorrhage (SAH) over one year. Urine samples were collected within 48 hours of admission and analyzed for common classes of abused drugs. Prevalence of positive UTox screening was determined. We evaluated whether baseline demographics and clinical factors were associated with UTox results.

Results

Of 483 eligible patients (acute ischemic stroke 66.4%; TIA 18.8%; ICH 7.7%; SAH 7.0%), 414 (85.7%) completed UTox screening. The mean (standard deviation) age was 65.1 (15.6) years, 52.7% were male, and 64.3% were Caucasian. Twenty-two (4.6%) patients had positive screening—cannabinoids were detected in 13 cases (3.1%), cocaine in 5 cases (1.2%), amphetamines in 1 case, and phencyclidine in 1 case. The highest yield (14.1%) was observed in patients < 60 years old with history of tobacco use while it was < 5% in the remaining subgroups (p<0.01).

Conclusions

Consistent with current guidelines, a selective approach to UTox screening should be pursued in acute stroke evaluation. The highest diagnostic yield is likely to be for cannabinoids and cocaine testing in younger patients with a history of concurrent tobacco use.     

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) lumen triggers the opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca²⁺ store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.