Organocatalyzed Solvent Free and Efficient Synthesis of 2,4,5‐Trisubstituted Imidazoles as Potential Acetylcholinesterase Inhibitors for Alzheimer's Disease

The catalytic potential of pyridine‐2‐carboxlic acid has been evaluated for efficient, green and solvent free synthesis of 2,4,5‐trisubstituted imidazole derivatives 3a – 3m . The compounds 3a – 3m were synthesized by one pot condensation reaction of substituted aromatic aldehydes, benzil, and ammonium acetate in good to excellent yields (74–96 %). To explore the potential of these compounds against Alzheimer's disease, their inhibitory activities against acetylcholinesterase (AChE) were evaluated. In this series of compounds, compound 3m , bearing one ethoxy and a hydroxy group on the phenyl ring on 2,4,5‐trisubstituted imidazoles, proved to be a potent AChE inhibitor (102.56±0.14). Structure–activity relationship (SAR) of these compounds was developed. Molecular dockings were carried out for the compounds 3m , 3e , 3k , 3c , 3a , 3d , 3j , and 3f in order to further investigate the binding mechanism. The inhibitor molecule was molecularly docked with acetylcholinesterase to further study its binding mechanism. The amino group of the compound 3m forms an H‐bond with the oxygen atom of the residue (i. e., THR121) which has a bond length of 3.051 Å.     

SLC26A4 Mutations in Patients with Moderate to Severe Hearing Loss

Mutations in SLC26A4 cause either syndromic or nonsyndromic hearing loss. We identified a link between hearing loss and DFNB4 in 3 of the 50 families participating in this study. Sequencing analysis revealed two SLC26A4 mutations, p.V239D and p.S57X, in affected members of the 3 families. These mutations have been previously reported in deaf individuals from the subcontinent, all of whom manifested profound deafness. The patients investigated in our study exhibited moderate to severe hearing loss. Our results show that inactivating SLC26A4 mutations that cause profound deafness can also be involved in the etiology of moderate to severe hearing loss. The type of mutation cannot predict the severity of the hearing loss in all cases, and there may be additional epistatic interactions that could modify the phenotype.     

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo‐4, Propidium iodide, and di‐8‐Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin‐eosin, Masson's tri‐chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

Production of avian influenza virus vaccine using primary cell cultures generated from host organs

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2–10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.     

The impact of illicit drug use on spontaneous hepatitis C clearance: experience from a large cohort population study

Background and Aims

Acute hepatitis C infection usually ends in chronic infection, while in a minority of patients it is spontaneously cleared. The current population-based study is performed on a large cohort in Golestan province of Iran to examine the demographic correlates of Spontaneous Hepatitis C Clearance.

Methods

Serum samples used in this study had been stored in biorepository of Golestan Cohort Study. These samples were evaluated for anti hepatitis C Virus by third generation Enzyme-linked immunosorbent assay (ELISA). Subjects who tested positive were then invited and tested by Recombinant Immunoblot Assay (RIBA) and Ribonucleic Acid Polymerase Chain Reaction test (PCR). If tested positive for RIBA, subjects were recalled and the two tests were re-done after 6 months. Those subjects who again tested positive for RIBA but negative for PCR were marked as cases of spontaneous clearance.

Results

49,338 serum samples were evaluated. The prevalence of Chronic Hepatitis C Virus (CHCV) infection based on PCR results was 0.31%. Among those who had acquired hepatitis C, the rate of SC was 38%. In multivariate analysis, illicit drug use both Injecting Use (OR = 3.271, 95% CI: 1.784–6.000, p-value<0.001) and Non-Injecting Use (OR = 1.901, 95% CI: 1.068–3.386, p-value = 0.029) were significant correlates of CHCV infection versus SC.

Conclusions

Illicit drug use whether intravenous or non-intravenous is the only significant correlate of CHCV, for which several underlying mechanisms can be postulated including repeated contacts with hepatitis C antigen.     

Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: a novel and alternative approach to suppress quorum-sensing mechanism

The present study was focused on evaluating the potential of Emblica officinalis against cariogenic properties of Streptococcus mutans, a causative microorganism for caries. The effect of crude extract and ethanolic fraction from Emblica officinalis fruit was analysed against S. mutans. The sub-MIC concentrations of crude and ethanolic fraction of E. officinalis were evaluated for its cariogenic properties such as acid production, biofilm formation, cell-surface hydrophobicity, glucan production, sucrose-dependent and independent adherence. Its effect on biofilm architecture was also investigated with the help of confocal and scanning electron microscopy (SEM). Moreover, expression of genes involved in biofilm formation was also studied by quantitative RT- PCR. This study showed 50% reduction in adherence at concentrations 156 μg/ and 312.5 μg/ml of crude extract and ethanolic fraction respectively. However, the biofilm was reduced to 50% in the presence of crude extract (39.04 μg/ml) and ethanolic fraction (78.08 μg/ml). Furthermore, effective reduction was observed in the glucan synthesis and cell surface hydrophobicity. The qRT-PCR revealed significant suppression of the genes involved in its virulence. Confocal and scanning electron microscopy clearly depicted the obliteration of biofilm structure with reference to control. Hence, this study reveals the potential of E. officinalis fruit extracts as an alternative and complementary medicine for dental caries by inhibiting the virulence factors of Streptococcus mutans.     

pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.