Combinatorial reshaping of a lipase structure for thermostability: Additive role of surface stabilizing single point mutations

Thermostable lipases are of high priority for industrial applications. In the present study, targeted improvement of the thermostability of a lipase from metagenomic origin was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. A variant (LipR5) was generated after combination of two thermostabilizing mutations (R214C & N355K). Thermostability of the variant enzyme was analyzed by half-life measurement and circular dichroism (CD). To assess whether catalytic properties were affected by mutation, the optimal reaction conditions were determined. The protein LipR5, displayed optimum activity at 50 °C and pH 8.0. It showed two fold enhancement in thermostability (at 60 °C) as compared to LipR3 (R214C) and nearly 168 fold enhancement as compared to parent enzyme (LipR1). Circular dichroism and fluorescence study suggest that the protein structure had become more rigid and stable to denaturation. Study of 3D model suggested that Lys355 was involved in formation of a Hydrogen bond with OE1 of Glu284. Lys355 was also making salt bridge with OE2 of Glu284.     

CAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants

The availability of a simple, reproducible and cost-effective molecular marker is a prerequisite for plant genetic analysis. We have developed a novel promoter-targeted marker, CAAT box- derived polymorphism (CBDP) using the nucleotide sequence of CAAT box of plant promoters. CBDP, like random amplified polymorphic DNA (RAPD), uses single primer in polymerase chain reaction (PCR) for generating markers. However unlike RAPD, the CBDP primers are 18 nucleotides long and consist of a central CCAAT nucleotides core flanked by the filler sequence towards the 5′ end and di- or trinucleotides towards the 3′ end. In this study, a small set of 25 CBDP primer was designed and initially tested in a representative set of eight cultivars of jute for generation of polymorphic markers. Further, to achieve high reproducibility, a touchdown PCR was employed with an annealing temperature of 50ºC. All the CBDP primers generated polymorphic markers in jute cultivars, and an UPGMA dendrogram based on Jaccard’s similarity grouped them into two clusters represented by Corchorus capsularis and C. olitorius, respectively. Interestingly, such grouping of jute cultivars was consistent with genetic relationships established earlier for these cultivars using other DNA markers. Moreover, these CBDP primers also generated polymorphic markers in representative sets of cotton (Gossypium species) and linseed (Linum usitatissimum ) cultivars. Given the high success rate of CBDP primers in generating markers in the tested species and advantages like ease in marker development and assay with reproducible profiles, they could potentially be exploited in other species as well for assessing genetic diversity, cultivar identification, construction of linkage map and marker- assisted selection.     

Dendrimer,Liposomes, Carbon Nanotubes and PLGA Nanoparticles: One Platform Assessment of Drug Delivery Potential

Liposomes (LIP), nanoparticles (NP), dendrimers (DEN), and carbon nanotubes (CNTs), represent eminent classes of drug delivery devices. A study was carried out herewith by employing docetaxel (DTX) as model drug to assess their comparative drug delivery potentials. Under optimized conditions, highest entrapment of DTX was observed in CNT-based formulation (DTX-CNTs, 74.70 ± 4.9%) followed by nanoparticles (DTX-NP, 62.34 ± 1.5%), liposome (49.2 ± 1.51%), and dendrimers (28.26 ± 1.74%). All the formulations were found to be of nanometric size. In vitro release studies were carried out in PBS (pH 7.0 and 4.0), wherein all the formulations showed biphasic release pattern. Cytotoxicity assay in human cervical cancer SiHa cells inferred lowest IC₅₀ value of 1,235.09 ± 41.93 nM with DTX-CNTs, followed by DTX-DEN, DTX-LIP, DTX-NP with IC₅₀ values of 1,571.22 ± 151.27, 1,653.98 ± 72.89, 1,922.75 ± 75.15 nM, respectively. Plain DTX showed higher hemolytic toxicity of 22.48 ± 0.94%, however loading of DTX inside nanocarriers drastically reduced its hemolytic toxicity (DTX-DEN, 17.22 ± 0.48%; DTX-LIP, 4.13 ± 0.19%; DTX-NP, 6.43 ± 0.44%; DTX-CNTs, 14.87 ± 1.69%).KEY WORDS: carbon nanotubes, dendrimer, drug delivery, liposomes, nanoparticles, nanotechnology     

Keynote Symposium

The developmental potency of pre-implant parthenogentic goat embryos were compared under two chemical activation protocols in three different culture media groups. The in vitro matured oocytes were chemically activated by two protocols viz. P1 (CB-CHX-6DMAP) and P2 (Ca-CHX-6DMAP). The activated oocytes under both the protocols were developed in three culture media, viz. modified synthetic oviductal fluid (mSOF), research vitro cleave medium (RVCL), and RVCL-Blast. While comparing the developmental potential of activated oocytes, it was observed that the oocytes activated under P2 protocol pooled over three culture media group producing significantly higher mean cleavage rate (43.2?±?0.9 vs 40.6?±?1.5), blastocyst development (16.4?±?1.1 vs 12.6?±?0.8), and blastomere count (120.7?±?4.7 vs 113.2?±?4.1) as compared to P1 protocol. The comparison of effect of culture media pooled over protocol groups revealed that the mean cleavage rate observed under RVCL-Blast (44.8?±?1.3) and RVCL (45.3?±?0.5) were significantly higher (P?≤?0.01) than mSOF (35.8?±?1.2). However, the mean blastocyst development observed under RVCL-Blast group (18.8?±?3.2) was significantly higher than RVCL (14.0?±?0.8) and mSOF (10.8?±?0.4). Similarly, the mean blastomere count under RVCL-Blast group (136.0?±?3.7) was significantly higher (P?≤?0.01) than RVCL (114.7?±?1.0) and mSOF (100.2?±?0.5) groups. The semiquantitative RT PCR analysis showed the expression of pro-apoptotic caspase 3 gene in P1 and anti-apoptotic Mcl-1 gene in P2. This study concludes that the activation protocol P2 and embryo cultured under RVCL-Blast group were optimum for chemical activation and culture medium, respectively.     

Imaging of ex vivo nonmelanoma skin cancers in the optical and terahertz spectral regions Optical and Terahertz skin cancers imaging

We tested the hypothesis that polarization sensitive optical and terahertz imaging may be combined for accurate nonmelanoma skin cancer (NMSC) delineation. Nine NMSC specimens were imaged. 513 μm and 440 nm wavelengths were used for terahertz and optical imaging, respectively. Histopathology was processed for evaluation. Terahertz reflectance of NMSC was quantified. Our results demonstrate that cross‐polarized terahertz images correctly identified location of the tumours, whereas cross‐polarized and polarization difference optical images accurately presented morphological features. Cross‐polarized terahertz images exhibited lower reflectivity values in cancer as compared to normal tissue. Combination of optical and terahertz imaging shows promise for intraoperative delineation of NMSC (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

rbcL,a potential candidate DNA barcode loci for aconites: conservation of himalayan aconites

Molecular Biology Reports - Aconitum heterophyllum Wall. ex Royle and Aconitum balfourii Stapf, are two highly important, threatened medicinal plants of the Indian Himalayan Region. Root-tubers of...     

Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione

We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl(-)) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I(sc)) and trans-epithelial resistance (R(t)). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I(sc) with an IC(50) of 3.2 +/- 1 mum (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I(sc). Conversely, BAY 41-2272, a sGC stimulator, increased I(sc) in a dose-response fashion. The GSNO increase of I(sc) was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mum) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I(sc). However, smaller concentrations of oxy-Mb (1-10 mum), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I(sc) partially. Oxy-Mb did not reverse the increase of I(sc) following addition of GSNO and cysteine (50 mum). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.     

Genome Sequence of the Alkaliphilic Bacterium Nitritalea halalkaliphila Type Strain LW7, Isolated from Lonar Lake,India

An alkaliphilic bacterium, Nitritalea halalkaliphila LW7, which belongs to the family Cyclobacteriacae in the phylum Bacteroidetes, was isolated from Lonar Lake in Maharastra, India. Here we announce the draft genome sequence of the type strain LW7, which contains 3,633,701 bp with a G+C content of 48.58%.