Purification, characterization and USAGE of thermotolerant laccase FROM Bacillus sp. FOR biodegradation of synthetic dyes

A Bacillus sp. isolated from sediments of distillery unit was found to overproduce laccase when cultured in a synthetic media containing 1mM CuSO₄ and 10% distillery spent wash as inducers along with 1% dextrose (w/v) and 0.1% tryptone (w/v) as additional carbon and nitrogen sources. The extracellular purified enzyme was highly thermostable with a calculated half-life of 23 min at 75°C. The optimal pH and temperature of the Bacillus sp. laccase were recorded to be 3.0 and 35°C, respectively. Sodium azide and solvents like methanol and acetonitrile completely inhibited enzyme activity. The average molecular weight of the purified enzyme as determined by SDS-PAGE and zymogam studies was around 70 kDa. Kinetic parameters were detected by using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate. At high ABTS concentrations (> 6 mM) a substrate inhibition phenomenon appeared and K _M (0.60 mM), V _max (983.00 U/min) values were determined. The polypeptide sequences showed significant similarity with Cudependent oxidoreductases through MALDI-TOF MS analysis. In addition, the crude Bacillus sp. laccase showed enormous potential for decolorization of various recalcitrant dyes. The apparent high stability of this enzyme makes it a good candidate for its possible application in biotechnology.     

Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

This paper presents an electrochemical immune sensor for label free detection of epidermal growth factor receptor (EGFR) by immobilizing anti-EGFR antibody (Anti-EGFRab) on dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) on gold (Au) electrode. Electrochemical studies show that increased surface concentration of redox moieties onto Anti-EGFRab/DTSP immuno-electrode leads to high electron transport and improved sensing performance. The antigen-antibody complex demonstrates a high association constant (5×10(12)L/mol) that results in high affinity of Anti-EGFRab to EGFR, confirming that the DTSP-SAM provides a conducive environment for anti-EGFR immobilization. The electrochemical response of EA/Anti-EGFRab/DTSP/Au electrode as a function of EGFR concentrations exhibits a linear range from 1pg/mL to 100ng/mL, a detection limit of 1pg/mL at a sensitivity of 2.02μAM(-1)at a regression coefficient of 0.99.     

Hexagonal Boron Nitride‐Based Electrolyte Composite for Li‐Ion Battery Operation from Room Temperature to 150 °C

Batteries for high temperature applications capable of withstanding over 60 °C are still dominated by primary cells. Conventional rechargeable energy storage technologies which have exceptional performance at ambient temperatures employ volatile electrolytes and soft separators, resulting in catastrophic failure under heat. A composite electrolyte/separator is reported that holds the key to extend the capability of Li‐ion batteries to high temperatures. A stoichiometric mixture of hexagonal boron nitride, piperidinium‐based ionic liquid, and a lithium salt is formulated, with ionic conductivity reaching 3 mS cm^?1, electrochemical stability up to 5 V and extended thermal stability. The composite is used in combination with conventional electrodes and demonstrates to be stable for over 600 cycles at 120 °C, with a total capacity fade of less than 3%. The ease of formulation along with superior thermal and electrochemical stability of this system extends the use of Li‐ion chemistries to applications beyond consumer electronics and electric vehicles.     

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin‐induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.     

AMPK-dependent phosphorylation of lipid droplet protein PLIN2 triggers its degradation by CMA

Lipids stored in lipid droplets are hydrolyzed via either cytosolic lipases or a selective form of macroautophagy known as lipophagy. We recently demonstrated that chaperone-mediated autophagy (CMA) is required for the initiation of lipolysis by either of these independent lipolytic pathways. CMA selectively degrades the lipid droplet proteins perilipins (PLIN) 2 and 3 from the lipid droplet surface, thus, facilitating the recruitment of cytosolic lipases and autophagy effector proteins to the lipid droplets. PLIN2 phosphorylation was observed upon induction of lipolysis, but the phosphorylating kinase and the relation of this phosphorylation with CMA of PLIN2 remained unknown. Here, we report that phosphorylation of PLIN2 is dependent on AMPK and occurs after the interaction of PLIN2 with the CMA chaperone HSPA8/Hsc70. Our results highlight a role for posttranslational modifications in priming proteins to be amenable for degradation by CMA.     

Desiccation stress induces developmental heterochrony in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Stressful environments are known to perturb developmental patterns in insects. In the purview of desiccation as a stressor, relatively little is known about the developmental consequences linked with desiccation tolerance. In this study, we have particularly focused on the exploration of the temporal profile of postembryonic development in response to desiccation exposure in Drosophila melanogaster and the associated trade-offs. We document a correlation between variations in 20-hydroxyecdysone levels and the altered timing of metamorphic events during the life cycle. Following desiccation, we observed an extension in the larval longevity whereas the duration of the pupal and adult stages was significantly shortened. Alternately, feeding of 20-hydroxyecdysone apparently led to the restoration of the normal temporal pattern of development in the desiccated group. In spite of the desiccation-responsive heterochronic shifts in development, the overall lifespan post recovery remained almost unaltered among the desiccated and undesiccated groups suggesting plasticity in developmental control. This observation reminisces ‘canalization-like’ phenomenon that buffers alterations in the overall lifespan. We thus identified a desiccation-responsive period in the lifespan of D. melanogaster during which variations in ecdysone levels are capable to alter the temporal course of development.     

Identification and evaluation of quercetin as a potential inhibitor of naphthoate synthase from Enterococcus faecalis

Enterococcus faecalis is a gram‐positive, rod‐shape bacteria responsible for around 65% to 80% of all enterococcal nosocomial infections. It is multidrug resistant (MDR) bacterium resistant to most of the first‐line antibiotics. Due to the emergence of MDR strains, there is an urgent need to find novel targets to develop new antibacterial drugs against E. faecalis. In this regard, we have identified naphthoate synthase (1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase, EC: 4.1.3.36; DHNS) as an anti‐E. faecalis target, as it is an essential enzyme for menaquinone (vitamin K₂) synthetic pathway in the bacterium. Thus, inhibiting naphtholate synthase may consequently inhibit the bacteria's growth. In this regard, we report here cloning, expression, purification, and preliminary structural studies of naphthoate synthase along with in silico modeling, molecular dynamic simulation of the model and docking studies of naphthoate synthase with quercetin, a plant alkaloid. Biochemical studies have indicated quercetin, a plant flavonoid as the potential lead compound to inhibit catalytic activity of EfDHNS. Quercetin binding has also been validated by spectrofluorimetric studies in order to confirm the bindings of the ligand compound with EfDHNS at ultralow concentrations. Reported studies may provide a base for structure‐based drug development of antimicrobial compounds against E. faecalis.     

Exploring protein-protein interactions using the site-identification by ligand competitive saturation methodology

Protein docking methods are powerful computational tools to study protein-protein interactions (PPI). While a significant number of docking algorithms have been developed, they are usually based on rigid protein models or with limited considerations of protein flexibility and the desolvation effect is rarely considered in docking energy functions, which may lower the accuracy of the predictions. To address these issues, we introduce a PPI energy function based on the site-identification by ligand competitive saturation (SILCS) framework and utilize the fast Fourier transform (FFT) correlation approach. The free energy content of the SILCS FragMaps represent an alternative to traditional energy grids and they can be efficiently utilized to guide FFT-based protein docking. Application of the approach to eight diverse test cases, including seven from Protein Docking Benchmark 5.0, showed the PPI prediction using SILCS approach (SILCS-PPI) to be competitive with several commonly used protein docking methods indicating that the method has the ability to both qualitatively and quantitatively inform the prediction of PPI. Results show the utility of the SILCS-PPI docking approach for determination of probability distributions of PPI interactions over the surface of both partner proteins, allowing for identification of alternate binding poses. Such binding poses are confirmed by experimental crystal contacts in our test cases. While more computationally demanding than available PPI docking technologies, we anticipate that the SILCS-PPI docking approach will offer an alternative methodology for improved evaluation of PPIs that could be used in a variety of fields from systems biology to excipient design for biologics-based drugs.