Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

Summary We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from –150 to +150 mV. The data are consistent with the on-off mechanism proposed by E. B. George et al. (J. Membrane Biol.103:245–253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5–10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.ASW, Carnegie-Mellon University     

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.     

Role of polymorphic XRCC6 (Ku70)/XRCC7 (DNA-PKcs) genes towards susceptibility and prognosis of lung cancer patients undergoing platinum based doublet chemotherapy

The DNA repair genes XRCC6 and XRCC7 formed an integral part of double strand break repair (DSBR) pathway. The two genes are thought to play an important role in the repair of lethal double strand damage on DNA. Polymorphic DSBR genes are studied to effect genomic stability. We intend to explore the association of DSBR genes i.e. XRCC6 and XRCC7 with susceptibility and survival in North Indian lung cancer patients. DNA isolation and genotyping was done for 320 controls and 330 lung cancer cases enrolled in the study. Each and every lung cancer study subjects were made a telephonic call and were followed for their health after administration of chemotherapy. Statistical analysis for susceptibility was done using logistic regression analysis. Survival analysis was done using Kaplan–Meier followed by Cox-regression. Small cell lung cancer (SCLC) subtype posed an amplified risk towards lung cancer in case of XRCC7 6721G>T (OR?=?4.11, p?=?0.0040). Gene-environment interaction analysis revealed that non-smokers with heterozygous genotype (CG) in case of XRCC6 61C>G showed a strong protective effect (OR?=?0.38, p?=?0.01) towards lung cancer. Survival analysis revealed poor prognosis in case of XRCC6 61C>G SCLC subtype. XRCC6 and XRCC7 were not involved in overall susceptibility and survival. However, in case of XRCC7 6721G>T subjects with SCLC subtype showed an increased susceptibility while poor prognosis in case of XRCC6 61C>G.     

Simulation Based Investigation of Deleterious nsSNPs in ATXN2 Gene and Its Structural Consequence Toward Spinocerebellar Ataxia

Spinocerebellar degeneration, termed as ataxia is a neurological disorder of central nervous system, characterized by limb in‐coordination and a progressive gait. The patient also demonstrates specific symptoms of muscle weakness, slurring of speech, and decreased vibration senses. Expansion of polyglutamine trinucleotide (CAG) within ATXN2 gene with 35 or more repeats, results in spinocerebellar ataxia type‐2. Protein ataxin‐2 coded by ATXN2 gene has been reported to have a crucial role in translation of the genetic information through sequestering the histone acetyl transferases (HAT) resulting in a state of hypo‐acetylation. In the present study, we have evaluated the outcome for 122 non synonymous single nucleotide polymorphisms (nsSNPs) reported within ATXN2 gene through computational tools such as SIFT, PolyPhen 2.0, PANTHER, I‐mutant 2.0, Phd‐SNP, Pmut, MutPred. The apo and mutant (L305V and Q339L) form of structures for the ataxin‐2 protein were modeled for gaining insights toward 3D spatial arrangement. Further, molecular dynamics simulations and structural analysis were performed to observe the brunt of disease associated nsSNPs toward the strength and secondary properties of ataxin‐2 protein structure. Our results showed that, L305V is a highly deleterious and disease causing point substitution. Analysis based on RMSD, RMSF, Rg, SASA, number of hydrogen bonds (NH bonds), covariance matrix trace, projection analysis for eigen vector demonstrated a significant instability and conformation along with rise in mutant flexibility values in comparison to the apo form of ataxin‐2 protein. The study provides a blue print of computational methodologies to examine the ataxin‐blend SNPs. J. Cell. Biochem. 119: 499–510, 2018. © 2017 Wiley Periodicals, Inc.     

Microwave-assisted synthesis of novel 5-substituted benzylidene amino-2-butyl benzofuran-3-yl-4-methoxyphenyl methanones as antileishmanial and antioxidant agents

A series of 5-substitutedbenzylideneamino-2-butylbenzofuran-3-yl-4-methoxyphenyl methanones is synthesized and evaluated for antileishmanial and antioxidant activities. Compounds 4f (IC₅₀?=?52.0?±?0.09?µg/ml), 4h (IC₅₀?=?56.0?±?0.71?µg/ml) and 4l (IC₅₀?=?59.3?±?0.55?µg/ml) were shown significant antileishmanial when compared with standard sodium stibogluconate (IC₅₀?=?490.0?±?1.5?µg/ml). Antioxidant study revealed that compounds 4i (IC₅₀?=?2.44?±?0.47?µg/ml) and 4l (IC₅₀?=?3.69?±?0.44?µg/ml) have shown potent comparable activity when compared with standard ascorbic acid (IC₅₀?=?3.31?±?0.34?µg/ml). Molecular docking study was carried out which replicating results of biological activity in case of initial hits 4f and 4h suggesting that these compounds have a potential to become lead molecules in drug discovery process. In silico ADME study was performed for predicting pharmacokinetic profile of the synthesised antileishmanial agents and expressed good oral drug like behaviour.     

CRISPR/Cas9-mediated efficient editing in <Emphasis Type="Italic">phytoene desaturase</Emphasis> (<Emphasis Type="Italic">PDS</Emphasis>) demonstrates precise manipulation in banana cv. Rasthali genome

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1–5) or deletion (1–4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.     

Biological response of using municipal solid waste compost in agriculture as fertilizer supplement

Waste management and declining soil fertility are the two main issues experienced by all developing nations, like India. Nowadays, agricultural utilization of Municipal Solid Waste (MSW) is one of the most promising and cost effective options for managing solid waste. It is helpful in solving two current burning issues viz. soil fertility and MSW management. However, there is always a potential threat because MSW may contain pathogens and toxic pollutants. Therefore, much emphasis has been paid to composting of MSW in recent years. Application of compost from MSW in agricultural land helps in ameliorating the soil’s physico-chemical properties. Apart from that it also assists in improving biological response of cultivated land. Keeping the present situation in mind, this review critially discusses the current scenario, agricultural utilization of MSW compost, role of soil microbes and soil microbial response on municipal solid waste compost application.