Divya Shrivastava

Antioxidant activity and DNA damage inhibition in vitro by a methanolic extract of Carissa carandas (Apocynaceae) leaves

Abstract The aim of this study was to determine the antioxidant and DNA damage inhibition potential of a methanolic extract of Carissa carandas leaves. The extract had significant (p < 0.05), dose-dependent DPPH radical scavenging activity (median inhibitory concentration, 73.1 μg/ml), total antioxidant activity, H2O2 scavenging activity (median inhibitory concentration, 84.03 μg/ml) and reducing power activity. The extract also completely protected pBR322 plasmid DNA from free radical-mediated oxidative stress in a DNA damage inhibition assay. The antioxidant and DNA damage inhibition properties of C. carandas can be attributed to a high content of phenolic compounds (84.0 mg gallic acid equivalents/g dry weight of extract), estimated in the Folin–Ciocalteau assay. The high antioxidant and DNA damage inhibiting potential of C. carandas could be used to develop antioxidant compounds for therapeutic applications.

LncRNAs and immunity: watchdogs for host pathogen interactions

Immune responses combat various infectious agents by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. The polygenic responses to these external stimuli are temporally and coordinately regulated. Specific lncRNAs are induced to modulate innate and adaptive immune responses which can function through various target interactions like RNA-DNA, RNA-RNA, and RNA-protein interaction and hence affect the immunogenic regulation at various stages of gene expression. LncRNA are found to be present in various immune cells like monocytes, macrophages, dendritic cells, neutrophils, T cells and B cells. They have been shown to be involved in many biological processes, including the regulation of the expression of genes, the dosage compensation and genomics imprinting, but the knowledge how lncRNAs are regulated and how they alter cell differentiation/function is still obscure. Further dysregulation of lncRNA has been seen in many diseases, but as yet very less research has been carried out to understand the role of lncRNAs in regulation during host-pathogens interactions. In this review, we summarize the functional developments and mechanism of action of lncRNAs, in immunity and defense of host against pathogens.

Rapid detection of extensively drug-resistant (XDR-TB) strains from multidrug-resistant tuberculosis (MDR-TB) cases isolated from smear-negative pulmonary samples in an Intermediate Reference Laboratory in India

BACKGROUND Direct sputum smear microscopy is commonly used for diagnosing tuberculosis (TB). The objectives of the study were first, to determine the recovery of Mycobacterium tuberculosis in smear-negative sputum samples through liquid culture (using MGIT 960) and solid culture (using LJ slant) and second, to screen multidrug-resistant isolates through line probe assay and further third, to identify XDR isolates through MGIT second-line DST from these positive MDR cultures in Delhi region. METHODS In this study, the sample size was 717 (sputum smear AFB negative and culture positive for M. tuberculosis complex by both solid and liquid culture methods) MDRTB suspects who were enrolled from January 2014 to December 2014 at the Intermediate Reference Laboratory in New Delhi Tuberculosis Centre, New Delhi. Rapid line probe assay was performed on all culture-positive samples, which were direct smear-negative specimens, and LPA-confirmed MDR samples were tested on MGIT 960 second-line DST for identification of XDR strains. RESULTS An overall increase in the culture positivity (9.4%) among these smear-negative cases shows a good sign of recovery from M. tuberculosis infection in these samples. 717 (9.4%) positive cultures (MGIT+LJ) were subjected to line probe assay. Out of these 717 cultures, 9 (1.2%) were confirmed as NTM, 50 (7%) were MDR, 4 (0.6%) were mono-rifampicin resistant and 654 (91.2%) cultures were sensitive to both drugs Rif and Inh, respectively. Out of these 54 (50 MDR +4 mono-RIF resistant) cultures as screened by LPA, 1 (1.8%) was XDR, 10 (18.6%) were mono-ofloxacin resistant and 1 (1.8%) was mono-Kanamycin resistant. Sensitivity to both drugs KAN and OFX was seen in 42 (77.8%) cultures. CONCLUSIONS Since the bacterial load in direct smear-negative suspected MDR samples is less, it is important to recover mycobacteria by rapid liquid culture method in such samples. Initial screening for MDRTB is to be done in such cases by performing rapid molecular genotypic drug susceptibility test such as LPA. Baseline second-line DST is also done to rule out the XDR cases among them for rapid and better management of XDRTB patients.

Genetic polymorphism of rare mutations in Mycobacterium tuberculosis-infected patients in Delhi

Background: There is a wide variation in existing Mycobacterium tuberculosis strains across the globe, and false results in line probe assay (LPA) can occur due to the presence of unique genetic mutations in different settings. Objectives: An attempt was made to observe uncommon mutations in multidrug-resistant tuberculosis (MDR-TB) strains and determination of genetic diversity by spoligotyping and to study the treatment outcome in patients with uncommon mutations. Materials and Methods: Band pattern analysis of LPA strips was performed as per manufacturers instructions. DNA sequencing was performed to confirm the presence of uncommon mutations in Intermediate Reference Laboratory in Delhi state. Results: Four uncommon mutations were recognized along with 12 unique spoligotype patterns which serve as an update to worldwide databases. The noteworthy presence of a spoligotype previously rarely seen in India was the SIT53/T1 pattern. Central Asian (CAS) spoligotype was highly associated with MDR followed by Beijing type. During follow-up, the treatment outcomes of cases showing uncommon mutations were considered as cured, after completion of their treatment. Conclusion: The rifampicin resistance appears to be an effective marker of MDR-TB. The presence of uncommon mutations confirms genetic polymorphism that may require treatment targeted at both drug-resistant and drug-susceptible phenotypes for the better management of patients with MDR-TB.

Band pattern analysis of mutations in rifampicin resistance strain of Mycobacterium tuberculosis by Line Probe assay in patients from Delhi, India

BACKGROUND The GenoType MTBDRplus, a commercial Line Probe Assay (LPA) kit from Hain Lifescience, Germany, is endorsed by Indias RNTCP Program for diagnosis of DRTB cases among smear-positive sputum samples. Although the LPA has been studied in several laboratories, there is a wide variation in existing M. tuberculosis strains across the globe, and false results can occur due to the presence of unique genetic mutations in different settings. AIM AND OBJECTIVE An attempt was made to carry out band pattern analysis using LPA and also to observe uncommon mutations in MDR strains. MATERIALS AND METHODS Sputum samples were collected from MDR suspects and transported to intermediate reference laboratory (IRL) at New Delhi Tuberculosis Centre in Delhi. Sputum decontamination, DNA extraction, amplification, hybridization, and band pattern analysis of Line Probe assay strips was performed as per manufacturers instructions. RESULTS Among the 3000 samples with interpretable LPA strips, rifampicin drug resistance with or without isoniazid was observed in 600 samples. The most common mutation detected by LPA in the rpoB gene was Ser516Leu (29.0%). Novel mutations reported in this study include mutation from CAG (Gin) to CAT (His) at codon 517, AGC (Ser)-AGG (Arg) at codon 512, ACA (Thr) to GCA (Ala) at codon 526, TTG (Leu)-CTG (Leu)s at codon 524. CONCLUSION High frequencies of uncommon mutations in rpoB gene by LPA were observed, highlighting possibility of those in-silico detected mutations that may not impart phenotypic resistance further.

Metabolomic and Biotechnological approaches to determine therapeutic potential of Withania somnifera (L.) Dunal: A Review

BACKGROUND Withania somnifera, a high value medicinal plant is a major source of pharmaceutically important active compounds withanolides. Withania somnifera has been used in ayurveda as health restorative and anabolic agent besides having anti-arthritic, antidepressant, anti-microbial, anti-inflammatory, anti-diabetic, anti-stress, neuroprotective and cardio-protective activities. HYPOTHESIS/PURPOSE The mining of the compound(s) of interest offers opportunity to identify desired attributes in the therapeutic area of interest. Metabolomic has become an important tool in the field of pharmacological and functional genomics of medicinal plants. The analysis supports the information regarding differential outline of the gene expression for increasing important withanolides viz. withanolide A and withaferin A in W. somnifera. STUDY DESIGN The bioinformatics and biotechnological approaches viz. tissue culture, genetic transformation, genomic, transcriptomic, proteomic, gene mining and metabolomic studies have opened new windows about engineering of withanolide production. METHODS Target and network analysis for maximum therapeutic potential of Withania somnifera have been determined by employing Genemania software for finding interactions among various human genes that are being affected by active constituents. RESULTS Some of the major bioactive compounds of Withania somnifera have been discussed on protein-protein, protein-DNA and genetic interactions with respect to gene and protein expression data, protein domains, metabolic profiling, root organ culture, genetic transformation and phenotypic screening profiles CONCLUSION: The implementation of latest bioinformatic tools in combination with biotechnological techniques for breeding platforms are important in conservation of medicinal plant species in danger. The current review is based on molecular and in vitro methodologies employed in W. somnifera for accepting their importance in the improvement of this valuable medicinal species.

Nanodentistry: novel approaches

Abstract Nanodentistry has evolved as a new science of nanotechnology that helps in diagnosing, treating, preventing oral and dental disease, and improving dental health by using nanomaterials. These nanomaterials include clusters of atoms, grains, fibers, films, crystals, and nanoholes. The orientation of these nanomaterials can be in one dimension, for example, nanofilms and foils; two-dimensional, for example, carbon nanotubes or three-dimensional, for example, quantum dots; nanoshells, and dendrimers. The characteristic nanoscale pattern present on the surface and within the nanomaterials determines their dedicated functionalities. These nanostructures have wide applications in oral diagnosis and therapeutics, which can reduce dosage and side effects of drugs. Dentifrices with hydroxyapetite crystals provide protective coating on tooth enamel and restores surface of damaged teeth. Currently, dental materials have been replaced by nature-analog, surface-engineered dental implants, and anisotropic tooth restorations. Remineralization of damaged teeth has been achieved by using “sensitive” toothpastes containing nanoparticles. Nanostructure Ti products are used in dental implants because of high biocompatibility especially Grade 4 titanium. It has been shown that fabricating a nanostructured titanium implant can regulate the macrophage inflammatory response and improve the performance of the implant. The unique property of dendrimers and nanoshells make them promising tools to be used as therapeutics for oral cancer treatment. Graphene/zinc oxide nanocomposite (GZNC) acts as an effective coating agent for dental implants by inhibiting Streptococcus mutans , which is the primary agent in dental caries. A significant reduction in the formation of oral biofilms was found in the presence of GZNC. This chapter focuses on the role of nanostructures to keep teeth and oral tissues healthy and functioning. The challenges faced by this technology for the betterment of human health have also been discussed.

Contaminant sensors: nanotechnology-based contaminant sensors

The remarkable properties of nanomaterials, such as their unique electrical, optical, and chemical properties as compared to macromaterials, make them ideal candidates for sensor design. Nanosensors offer unique advantages such as high sensitivity and accuracy, quick response, direct localized detection, and enhanced sensing surface area, which can be easily functionalized. Detection of contaminants in the food industry not only ensures safe storage and transportation within the sell-by period, but also reduces potential health risks involved due to presence of pathogenic microorganisms and the associated toxins. Additionally, contaminant sensors can be used to detect banned dyes and adulterants, pharmacological residues, such as antibiotics and hormones, and allergens. Advancements in the field of food nanosensing including novel nanosensory approaches called e-nose and e-tongue, lab-on-chip nanotechnology-based pathogen sensors, surface-enhanced Raman scattering-based sensors and aptamer-based sensors are discussed along with their current and potential applications

Nanostructures as Antimicrobial Therapeutics

Abstract Developing countries encounter huge economic losses due to frequent epidemics of infectious diseases. The major contributing factor is the indiscriminate use of antibiotics leading to the emergence of resistant strains that are unaffected by existing antibiotics. Moreover, infectious diseases associated with the formation of biofilms composed of a large number of bacterial communities are not easily penetrable by antibiotics at normal doses. Possible routes to the generation of new antimicrobial agents include finding new targets for antimicrobial activity, combination compounds showing simultaneous action on more than one target, and new delivery mechanisms to improve target specificity. Nanomaterials have opened a new avenue as antimicrobial agents owing to their unique properties, such as surface chemistry (functionalizable structure), chemical stability, appropriate size, high surface area to mass ratio, high reactivity, stability, robustness, durability, biosafety, and biocompatibility. Nanoparticles can be utilized to either enhance antimicrobial activity of existing antibiotics, as physical agents that cause cell damage (photothermal activity), or for encapsulating antimicrobial agents for efficient drug delivery. Nanomaterials studied for their compatibility as alternative medicine include metal nanoparticles (gold and silver nanoparticles), metal oxide nanoparticles (oxides of magnesium, titanium, zinc, calcium, copper, aluminium, cesium), and organic nanoparticles (poly-ɛ-lysine, quaternary ammonium compounds, cationic quaternary polyelectrolytes, N-halamine compounds, polysiloxanes, benzoic acid, phenol, and parahydroxybenzoate esters). Inorganic nanoparticles are preferred over organic nanoparticles owing to their stability in harsh process conditions. The modes of action of these nanoparticles are variable, depending on the types of nanoparticles conjugated. These actions include damage to cell membrane by altering permeability due to generation of reactive oxygen species, release of ions that react with cell constituents, such as DNA, and sulfur-containing proteins that inhibit DNA replication and enzyme functions, respectively, contact killing by cationic surfaces or photothermal damage. Although all these nanoparticles have tremendous applications as antimicrobial agents, extensive toxicity testing is required prior to their usage in order to ascertain their effects in a biological system. This chapter will highlight the recent advancements in applications of these antimicrobial nanoparticles, their unique properties, and modes of action along with their biocompatibility issues.

Biodegradation and Decolorization of Azo Dye (Deep Red Glx) by Alkaliphilic Bacilluscereus Strain BPL Isolated from Textile Effluent Contaminant Soil

The Gram positive, spore forming bacterium was isolated from textile effluent contaminated soil and was examined for its ability to degrade azo dye (deep red glx). On analysis of 16S rRNA gene sequence, strain showed 99% sequence similarity with Bacillus cereus ATCC 14579 and belongs to group Firmicutes of domain Bacteria. The strain has been assigned name BPL and the gene sequence was deposited in Genbank with accession number KU510086. The Bacillus cereus strain BPL was able to decolorize deep red glx with efficiency of 78% in minimal salt medium (MSM) supplemented with dye at concentration of 0.5gl. Decolorization studies have shown that the strain was able to decolorize deep red glx in pH range of 4-9 at 37C with optimum pH of 9 and adaptability to decolorize dye in alkaline conditions. Other parameters such as nitrogen sources, carbon sources and osmotic concentration were also examined. When supplemented with nitrogen source (peptone, 5gl), carbon source (maltose, 5gl) and NaCl (10gl), the decolorization obtained were 85%, 87% and 82% respectively. The Bacillus cereus strain BPL was found to decolorize azo dye deep red glx and shows good possibilities for biodegradation and decolorization of textile effluents.