Solvated Crystalline Forms of Nevirapine: Thermoanalytical and Spectroscopic Studies

The study is aimed at exploring the utility of thermoanalytical methods in the solid-state characterization of various crystalline forms of nevirapine. The different forms obtained by recrystallization of nevirapine from various solvents were identified using differential scanning calorimetry and thermogravimetric analysis (TGA). The appearance of desolvation peak accompanied by weight loss in TGA indicated the formation of solvates: hemi-ethanolate (Form I), hemi-acetonitrilate (Form II), hemi-chloroformate (Form III), hemi-THF solvate (Form IV), mixed hemi-ethanolate hemi-hydrate (Form V), and hemi-toluenate (Form VI). The higher desolvation temperatures of all the solvates except toluenate than their respective boiling point indicate tighter binding of solvent. Emphasis has been laid on the determination of heat capacity and heat of solution utilizing microreaction calorimeter to further distinguish the various forms. The enthalpy of solution (ΔH _sol), an indirect measure of the lattice energy of a solid, was well correlated with the crystallinity of all the solid forms obtained. The magnitude of ΔH _sol was found to be −14.14 kJ/mol for Form I and −2.83 kJ/mol for Form V in phosphate buffer of pH 2, exhibiting maximum ease of molecular release from the lattice in Form I. The heat capacity for solvation (ΔC _p) was found to be positive, providing information about the state of solvent molecules in the host lattice. The solubility and dissolution rate of the forms were also found to be in agreement with their enthalpy of solution. Form (I), being the most exothermic, was found to be the most soluble of all the forms.     

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase alpha-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein-protein contacts during the entire initiation process.     

Molecular Pathogenesis of Wilson Disease Among Indians: A Perspective on Mutation Spectrum in ATP7B gene, Prevalent Defects, Clinical Heterogeneity and Implication Towards Diagnosis

Aims We aim to identify the molecular defects in the ATP7B, the causal gene for Wilson disease (WD), in eastern Indian patients and attempt to assess the overall mutation spectrum in India for detection of mutant allele for diagnostic purposes. Methods Patients from 109 unrelated families and their first-degree relatives comprising 400 individuals were enrolled in this study as part of an ongoing project. Genomic DNA was prepared from the peripheral blood of Indian WD patients. PCR was done to amplify the exons and flanking regions of the WD gene followed by sequencing, to identify the nucleotide variants. Results In addition to previous reports, we recently identified eight mutations including three novel (c.3412 + 1G > A, c.1771 G > A, c.3091 A > G) variants, and identified patients with variable phenotype despite similar mutation background suggesting potential role of modifier locus. Conclusions So far we have identified 17 mutations in eastern India including five common mutations that account for 44% of patients. Comparative study on WD mutations between different regions of India suggests high genetic heterogeneity and the absence of a single or a limited number of common founder mutations. Genotype–phenotype correlation revealed that no particular phenotype could be assigned to a particular mutation and even same set of mutations in different patients showed different phenotypes.     

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFκB activation. The activation of NFκB by acrolein was induced as a result of degradation of IκBα over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.     

Decolorization and partial degradation of monoazo dyes in sequential fixed-film anaerobic batch reactor (SFABR)

Decolorization of two monoazo dyes, acid orange 6 (AO6) and acid orange 7 (AO7), were studied in sequential fixed-film anaerobic batch reactor (SFABR) with varying dye concentrations and 500 mg/L glucose as the co-substrate. More than 90% dye decolorization could be achieved, even at 300 mg/L, with both AO6 and AO7 and dye decolorization rates were 168 mg/L/d and 176 mg/L/d, respectively. COD removals with these two monoazo dyes were significantly different, as 75% and 35% decrease were observed with AO6 and AO7, respectively. UV-visible spectral as well as HPLC analysis of SFABR treated effluent showed the accumulation of 4-aminobenzenesulfonate (4-ABS) from AO6 and AO7. Aminoresorcinol (AR) formed from AO6 decolorization could not be detected at the end of SFABR cycle. This along with high COD removal indicated its further degradation. Formation of pink coloration on exposure to air indicated the presence of 1-amino-2-naphthol (AN) in AO7 fed reactor effluent. Thus both 4-ABS and AN were resistant to further degradation under anaerobic conditions. Presence of nitrate did not decrease the observed decolorization at the end of 24h SFABR cycle, although initial rate was decreased. This indicates the suitability of SFABR configuration for the treatment of azo-dye containing wastewaters in the presence of nitrate.     

Reproductive seasonality in the Indian pygmy field mouse,Mus terricolor

The Indian pygmy field mouse, Mus terricolor, is a tiny, yet economically and ecologically important crop pest found throughout South-East Asia. There are no systematic reports exploring its reproductive physiology. We report the presence of distinct periods of annual reproductive activity and quiescence in M. terricolor. Body weight in males and females, relative weights of testis, epididymis and seminal vesicle in males, ovarian and uterine weight in females, gonadal histomorphic changes, testicular and ovarian cholesterol, sialic acid in epididymis, fructose in seminal vesicle, uterine protein content, melatonin in males and females, testosterone in males, estradiol, and progesterone in females were studied over a period of three years in both wild-caught and lab-acclimated mice. The number of Graafian follicles and corpora lutea, and plasma estradiol and progesterone, along with relative weights of ovary and uterus in females exhibited a peak in the months of October–January, compared to June. Based on histomorphic and hormonal status, the major reproductively active season is the winter (short-day breeding). There is a brief period of sub-maximal reproductive activity in April. M. terricolor is reproductively inactive in the summer, monsoons, and autumn. The results establish M. terricolor as a seasonal breeder in the field, with interesting implications for pest management.     

16SrRNA sequencing of Dye decolorizing bacteria isolated from Soil

Dye׳s residues in textile effluents are hazardous for humans and animals health. Such pollutants can be degraded into non-harmful molecules using biological approaches that are considered cheaper and ecologically safer. Isolated 15 bacterial cultures from soil that could be used in biological system were showed decolorization capacity for Acid Green dye (33.9% to 94.0%) using thin layer chromatography and broth culture method. The most promising cultures (AMC3) to decolorize Acid green Dye (94.6%) was re-coded as NSDSUAM for submitting at IMTECH, Chandigarh for sequencing. The 16SrRNA sequencing suggested that it can be a variant of Pseudomonas geniculata (99.85% identical similarity) with difference of 2 base pairs to reference strain Pseudomonas geniculata ATCC 19374(T). Thus present study proposed dye decolorizing efficiency of the isolated strain of Pseudomonas geniculata that was previously unnoticed. The sequence is deposited in NCBI GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"KP238100","term_id":"745856746","term_text":"KP238100"}}KP238100.     

Strand 6B deformation and residues exposure towards N-terminal end of helix B during proteinase inhibition by Serpins

Serine Protease inhibitors (Serpins) like antithrombin, antitrypsin, neuroserpin, antichymotrypsin, protein C-inhibitor and plasminogen activator inhibitor is involved in important biological functions like blood coagulation, fibrinolysis, inflammation, cell migration and complement activation. Serpins native state is metastable, which undergoes transformation to a more stable state during the process of protease inhibition. Serpins are prone to conformation defects, however little is known about the factors and mechanisms which promote its conformational change and misfolding. Helix B region in serpins is with several point mutations which result in pathological conditions due to polymerization. Helix B analysis for residue burial and cavity was undertaken to understand its role in serpin structure function. A structural overlap and an accessible surface area analysis showed the deformation of strand 6B and exposure of helix B at N-terminal end in cleaved conformation but not in the native and latent conformation of various inhibitory serpins. A cleaved polymer like conformation of antitrypsin also showed deformation of s6B and helix B exposure. Cavity analysis showed that helix B residues were part of the largest cavity in most of the serpins in the native state which increase in size during the transformation to cleaved and latent states. These data for the first time show the importance of strand 6B deformation and exposure of helix B in smooth insertion of the reactive center loop during serpin inhibition and indicate that helix B exposure due to variants may increase its polymer propensity. ABBREVIATIONS: serpin -serine protease inhibitors RCL -reactive center loop ASA -accessible surface area.