Residues at the interface between zinc binding and winged helix domains of human RECQ1 play a significant role in DNA strand annealing activity.

RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a ß-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the ß-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2-Zn-WH domains harbouring mutations on the ß-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.

Biochemical Characterization of VapC46 Toxin from Mycobacterium tuberculosis.

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin-antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.

Acoustic cavitation assisted destratified clay tactoid reinforced in situ elastomer-mimetic semi-IPN hydrogel for catalytic and bactericidal application.

Ultrasonicaion is non-chemical process where acoustic waves have been targeted to aqueous medium dispersed precursor materials. In situ synthesis of silver nanoparticles anchored in hydrogel matrix has been opted via ~20 kHz frequency assisted (bath sonication) synthesis having the ultrasonication power intensity (UPI) of ~106 J/m2. Power intensity is inversely proportional to the surface area of the clay tactoids. The hydrogel have been prepared by in situ 20 kHz assisted sonochemical destratification of laponite clay tactoids which could be terminologically stated as 'top-down method'. Silver nanoparticles (AgNPs) have been deposited in the surfaces of the porous matrix of hydrogel via 'soak and irradiate' method. Soaking of silver ions into the gel matrix is welcomed due to their efficient stabilization and fast transformation towards AgNPs. AgNPs played the key role in catalytic reduction and bactericidal activity. Moreover, the prepared hydrogel has enough robust to withstand cyclic stress, uniaxial stress and oscillatory stress which have been extensively justified by the physico-mechanical characterizations. The gel supported catalyst showed first order reaction kinetics and less time consuming period during reduction of 4-nitrophenol as a model pollutant.

Converting waste Allium sativum peel to nitrogen and sulphur co-doped photoluminescence carbon dots for solar conversion, cell labeling, and photobleaching diligences: A path from discarded waste to value-added products.

Proper waste utilization in order to promote value added product is a promising scientific practice in recent era. Inspiring from the recurring trend, we propose a single step oxidative pyrolysis derived fluorescent carbon dots (C-dots) from Allium sativum peel, which is a natural, nontoxic, and waste raw material. Because of its excellent optical properties, and photostability this C-dots have been used in versatile area of applications. Due to its immediate water dispersing character, C-dots reinforced Poly(acrylic acid) (PAA) films revealed improvement in uniaxial stretching behavior and can be used as transparent sunlight conversion film. The nanocomposite film has been tested against rigorous simulated sunlight which proved almost identical sunlight conversion behavior with no photo-bleachable character which is definitely added an extra quality of transparent polymer films. Moreover, the C-dots dispersion has been used as in vitro biomarker for living cells owing to its ease in solubility, biocompatibility, non-cytotoxicity and bright fluorescence even in subcutaneous environment. For this case, adipose derived mesenchymal stem cells (ADMSCs) have been chosen and injected to rabbit ear skin to perform two-photon imaging experiment. The present work opens a new avenue towards the large-scale synthesis of bio-waste based fluorescent C-dots, paving the way for their versatile applications.

Biocompatible carbon dots derived from κ-carrageenan and phenyl boronic acid for dual modality sensing platform of sugar and its anti-diabetic drug release behavior.

Detection of sugar by enzymatic assay has been suffering from costly, time-taking, instable and denaturation of glucose oxidase. Recently, chemosensors that have affinity towards boronate became the hot topic in the domain of monosaccharide detection. In this work, a novel strategy was addressed to fabricate carbon dots (C-dots) from linear sulfated polysaccharides κ- carrageenan and phenyl boronic acid for nonenzymatic monosaccharide (glucose) detection. The boronic acid group anchored C-dots surface can form assembly by covalently bonded with the cis-diol moiety of the glucose which caused fluorescence quenching of the C-dots. The inert surface nature of the luminescent C-dots enables them to sense as low as 1.7 µM glucose without the interference of other biomolecules. The proposed sensing system was successfully applied for assay of glucose in blood serum. Interestingly, these C-dots were used as a nano vehicle for delivery of anti-diabetic drug Metformin. Good biocompatibility results were found with MTT and hemolysis assay. Owing to its simplicity and effectiveness, the as-prepared C-dots offered great promise for blood sugar diagnosis and treatment.

Elucidation of the mechanism of disulfide exchange between staphylococcal thioredoxin2 and thioredoxin reductase2: A structural insight.

The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.

Zinc and nitrogen ornamented bluish white luminescent carbon dots for engrossing bacteriostatic activity and Fenton based bio-sensor.

Carbon dots with heteroatom co-doping associated with consummate luminescence features are of acute interest in diverse applications such as biomolecule markers, chemical sensing, photovoltaic, and trace element detection. Herein, we demonstrate a straightforward, highly efficient hydrothermal dehydration technique to synthesize zinc and nitrogen co-doped multifunctional carbon dots (N, Zn-CDs) with superior quantum yield (50.8%). The luminescence property of the carbon dots can be tuned by regulating precursor ratio and surface oxidation states in the carbon dots. A unique attribution of the as-prepared carbon dots is the high monodispersity and robust excitation-independent emission behavior that is stable in enormously reactive environment and over a wide range of pH. These N, Zn-CDs unveils captivating bacteriostatic activity against gram-negative bacteria Escherichia coli. Furthermore, the excellent luminescence properties of these carbon dots were applied as a platform of sensitive biosensor for the detection of hydrogen peroxide. Under optimized conditions, these N, Zn-CDs reveals high sensitivity over a broad range of concentrations with an ultra-low limit of detection (LOD) indicating their pronounced prospective as a fluorescent probe for chemical sensing. Overall, the experimental outcomes propose that these zero-dimensional nano-dots could be developed as bacteriostatic agents to control and prevent the persistence and spreading of bacterial infections and as a fluorescent probe for hydrogen peroxide detection.

Waste chimney oil to nanolights: A low cost chemosensor for tracer metal detection in practical field and its polymer composite for multidimensional activity.

Proper waste disposal from household and restaurants is becoming an important and recurring waste-management concern. Herein, a method of upcycling of waste kitchen chimney oil has been adopted to prepare fluorescent multifunctional carbon quantum dots. These nanodots showed superior biocompatibility, excellent optical properties, water solubility and high yield. Preparation of C-dots from highly abundant carbon source of waste refusals is highly effective in commercial aspect as well as in reducing the immense environmental pollution. The C-dots showed quasi-spherical size obtained from high resolution transmission electron microscopy (HRTEM) having an abundance of 1-4 nm in size. The ease of water dispersibility of the nanodots is a mere reflection of their surface polarity which has been supported by Fourier transformed infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). In the field of practical acceptability, the C-dots have been experimented to sense Fe3+ ion in a wide range of concentration (1 nM to 600 µM) with a detection limit of 0.18 nM which can be termed as 'tracer metal chemosensor'. Moreover, the prepared carbon dots were also tested against inter-cellular Fe3+ ion sensing probe. Lastly, we also fabricate the biopolymer­carbon dots composite for fluorescent marker ink and light emitting polymer film.

Sonochemical green reduction to prepare Ag nanoparticles decorated graphene sheets for catalytic performance and antibacterial application.

The emerging popularity and wide acceptance of green chemistry and environmentally benign/ecofriendly approaches have comprehensively considered for catalyst synthesis methods. Natural resource derived carbogenic quantum dots has been used in assistance with ultrasonic shock wave to graphene oxide (GO) aqueous dispersion in order to prepare reduced graphene oxide decorated with silver nanoparticles following the 'top-down' method. The total reduction process is done without using any toxic external reducing agents and any surfactants or stabilizers, thus it can be accepted as green method. Sonochemical destratification of the GO layers provides green attributes due to scalable, non-hazardous and relatively fast reduction to enhance surface area of the GO. Arresting the silver nanoparticles onto basal planes of graphene oxide can act as an efficient solid state support catalyst for fast reduction of toxic nitro aryls. Besides this work also reports bactericidal feature exhibited by the catalyst. Thus a dual functioning nanomaterial has been successfully developed which can be a suitable alternative for reductive forthcoming specialty/multifunctional membrane and other high-end medicinal or industrial applications.

Molecular insights into RNA-binding properties of Escherichia coli-expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

Green approach to photoluminescent carbon dots for imaging of gram-negative bacteria Escherichia coli.

Fluorescent carbon dots, zero-dimensional nanomaterials with surface ligands, have been studied extensively over the past few years in biolabelling or fluorescence-based live cell assays. In the past, synthetic organic dyes have been used as cell tracking materials, but they have severe limitations; fluorescent carbon dots may pave the way to biolabelling and cell imaging. In this work, green fluorescent carbon dots have been synthesized from a green source, gram, without any sort of covalent or ionic modifications. These gram-derived carbon dots are unique with respect to synthetic commercial cell-tracking dyes as they are non-toxic, cell internalization occurs quickly, and they have excellent bioconjugation with bacterial cells. Our aim is to establish these carbon dots in a biolabelling assay with its other physicochemical features like the tunable luminescence property, high degree of water solubility and low toxicity, towards various environments (wide range of pH, high ionic strength). Our study introduces a new perspective on the commercialization of carbon dots as a potential alternative to synthetic organic dyes for fluorescence-based cell-labelling assays.

A simplistic approach to green future with eco-friendly luminescent carbon dots and their application to fluorescent nano-sensor 'turn-off' probe for selective sensing of copper ions.

Zero-dimensional fluorescent nanoparticles having specificity as molecular probe appears to be strategically balanced fluorescent nano-probes. In this work, purified lemon extract and l-arginine have been thermally coupled for the extremely acute detection of Cu2+ in aqueous medium. The Cu2+ ions may be captured by the amino groups on the surface of the nano-sensor to form cupric ammine complex resulting in quenched fluorescence via an inner filter effect. Our proposed nano-probe is N-doped carbon dots (NCDs) which are efficiently selective as fluorescent chemosensor due to enormous binding affinity towards Cu2+ in a wide range of concentration (0.05-300µM) within a few minutes.

Protection of human γB-crystallin from UV-induced damage by epigallocatechin gallate: spectroscopic and docking studies.

The transparency of the human eye lens depends on the solubility and stability of the structural proteins of the eye lens, the crystallins. Although the mechanism of cataract formation is still unclear, it is believed to involve protein misfolding and/or aggregation of proteins due to the influence of several external factors such as ultraviolet (UV) radiation, low pH, temperature and exposure to chemical agents. In this article, we report the study of UV induced photo-damage (under oxidative stress) of recombinant human γB-crystallin in vitro in the presence of the major green tea polyphenol, (-)-epigallocatechin gallate (EGCG). We have shown that EGCG has the ability to protect human γB-crystallin from oxidative stress-induced photo-damage.

Reconstitution of the RNA-dependent RNA polymerase activity of Antheraea mylitta cypovirus in vitro using separately expressed different functional domains of the enzyme.

Antheraea mylitta cytoplasmic polyhedrosis virus is a segmented dsRNA virus of the family Reoviridae. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, Antheraea mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp have demonstrated that the purified domains interact in vitro. The central polymerase domain (PD) shows nucleotide binding properties, but neither the N-terminal domain (NTD) nor the C-terminal domain (CTD). Isolated PD does not exhibit RdRp activity but this activity can be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated PD has increased internal motions in comparison to when it is associated with the NTD and CTD. The motions of the separated PD may lead to the formation of a less accessible RNA template-binding channel and, thus, impair RdRp activity.

Complete catalytic cycle of cofactor-independent phosphoglycerate mutase involves a spring-loaded mechanism.

Cofactor-independent phosphoglycerate mutase (iPGM), an important enzyme in glycolysis and gluconeogenesis, catalyses the isomerization of 2- and 3-phosphoglycerates by an Mn(2+)-dependent phospho-transfer mechanism via a phospho-enzyme intermediate. Crystal structures of bi-domain iPGM from Staphylococcus aureus, together with substrate-bound forms, have revealed a new conformation of the enzyme, representing an intermediate state of domain movement. The substrate-binding site and the catalytic site are present in two distinct domains in the intermediate form. X-ray crystallography complemented by simulated dynamics has enabled delineation of the complete catalytic cycle, which includes binding of the substrate, followed by its positioning into the catalytic site, phospho-transfer and finally product release. The present work describes a novel mechanism of domain movement controlled by a hydrophobic patch that is exposed on domain closure and acts like a spring to keep the protein in open conformation. Domain closing occurs after substrate binding, and is essential for phospho-transfer, whereas the open conformation is a prerequisite for efficient substrate binding and product dissociation. A new model of catalysis has been proposed by correlating the hinge-bending motion with the phospho-transfer mechanism.

Expression, purification, crystallization and preliminary X-ray diffraction studies of phosphoglycerate mutase from Staphylococcus aureus NCTC8325.

Phosphoglycerate mutase (PGM) is a key enzyme in carbohydrate metabolism. It takes part in both glycolysis and gluconeogenesis. PGM from pathogenic Staphylococcus aureus (NCTC8325) was cloned in pQE30 expression vector overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from two different conditions, (i) 0.1 M HEPES pH 7.5, 20%(w/v) polyethylene glycol 10,000 and (ii) 0.2 M NaCl, 0.1 M bis-tris pH 6.5, 25%(w/v) polyethylene glycol 3350, at 25°C by the sitting-drop vapour-diffusion method. Crystals grown at pH 7.5 diffracted to 2.5 Šresolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 77.0, b = 86.11, c = 94.07 Å. Crystals from the second condition at pH 6.5 diffracted to 2.00 Šresolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 73.21, b = 81.75, c = 89.18 Å. X-ray diffraction data have been collected and processed to the maximum resolution to determine the structure of PGM. The structure has been solved by molecular replacement and structure refinement is now in progress.

Spectroscopic studies of the binding of Cu(II) complexes of oxicam NSAIDs to alternating G-C and homopolymeric G-C sequences.

Drugs belonging to the Non-steroidal anti-inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing them with a bioactive metal like copper, show an enhancement in their anti-cancer effects compared to the bare drugs, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that Cu(II)-NSAIDs can directly bind to the DNA backbone. The ability of the copper complexes of NSAIDs namely meloxicam and piroxicam to bind to the DNA backbone could be a possible molecular mechanism behind their enhanced anticancer effects. Elucidating base sequence specific interaction of Cu(II)-NSAIDs to the DNA will provide information on their possible binding sites in the genome sequence. In this work, we present how these complexes respond to differences in structure and hydration pattern of GC rich sequences. For this, binding studies of Cu(II) complexes of piroxicam [Cu(II)-(Px)2 (L)2] and meloxicam [Cu(II)-(Mx)2 (L)] with alternating GC (polydG-dC) and homopolymeric GC (polydG-polydC) sequences were carried out using a combination of spectroscopic techniques that include UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy. The Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC (Kb=11.5×10(3) M(-1)) to a weak binder of polydG-polydC (Kb=5.02×10(3) M(-1)), while Cu(II)-piroxicam changes from a strong binder of polydG-polydC (Kb=8.18×10(3) M(-1)) to a weak one of polydG-dC (Kb=2.18×10(3) M(-1)), point to the sensitivity of these complexes to changes in the backbone structures/hydration. Changes in the profiles of UV absorption band and CD difference spectra, upon complex binding to polynucleotides and the results of competitive binding assay using ethidium bromide (EtBr) fluorescence indicate different binding modes in each case.