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Search Results: 1 - 10 of 150012 matches for " Vineet K. Singh "
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High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus
Vineet K. Singh
International Journal of Microbiology , 2014, DOI: 10.1155/2014/615965
Abstract: Staphylococcus aureus is a major human and animal pathogen. Autolysins regulate the growth, turnover, cell lysis, biofilm formation, and the pathogenicity of S. aureus. Atl is the major autolysin in S. aureus. The biochemical and structural studies of staphylococcal Atl have been limited due to difficulty in cloning, high level overexpression, and purification of this protein. This study describes successful cloning, high level over-expression, and purification of two forms of fully functional Atl proteins. These pure proteins can be used to study the functional and structural properties of this important protein. 1. Introduction Staphylococcus aureus is an aggressive pathogen that is responsible for a wide array of diseases from mild skin infections to life-threatening conditions such as bacteremia, pneumonia, and endocarditis [1–4]. The emergence of multidrug resistance in S. aureus is an enormous public health concern and there is an immediate need for additional and alternative therapeutic targets for infections caused by this bacterium [5]. Autolysins are enzymes that degrade the peptidoglycan cell wall layer and are called peptidoglycan hydrolases. They represent a diverse group of enzymes and appear to have redundant roles and more than one function [6, 7]. These enzymes include N-acetylmuramidase, N-acetyl glucosaminidase, N-acetylmuramyl-L-alanine amidase, and endopeptidase [8–11]. While amidases break the bonds between the glycan strand and the stem peptides, the glycosidases are responsible for the cleavage of the glycan strands [7]. Cellular levels and activities of autolysins are believed to be intricately regulated and are proposed to play key roles in bacterial cell wall metabolism, daughter-cell separation, and antibiotic mediated cell lysis [2, 10]. Bacteria may secrete these autolysins and cause lysis of other bacteria that compete with S. aureus for nutrients [12]. The peptidoglycan hydrolases are important in bacterial pathogenicity [13, 14]. These enzymes modulate muropeptide release which in turn activates host innate immune responses [13]. There is plenty of evidence to suggest that most S. aureus autolysins result from the processing of full-length Atl [9, 15]. The full length Atl is an ~137?kDa protein with two catalytically active domains [15–18]. A 63.3?kDa N-terminal domain possesses amidase activity while a 53.6?kDa N-terminal domain possesses the glucosaminidase activity [15–18]. Many investigators [16, 17, 19] in the past have reported difficulty in cloning the full length S. aureus atl gene. A difficulty in overexpression
Expression of Four Methionine Sulfoxide Reductases in Staphylococcus aureus
Kuldeep Singh,Vineet K. Singh
International Journal of Microbiology , 2012, DOI: 10.1155/2012/719594
Abstract: Staphylococcus aureus possesses three MsrA enzymes (MsrA1, MsrA2, MsrA3) that reduce the S-epimer of methionine sulfoxide (MetO) and an MsrB enzyme that reduces R-MetO. The four msr genes are expressed from three different promoters. The msrA1/msrB genes are coexpressed. To determine the expression pattern of msr genes, three independent reporter strains were constructed where msr promoter was cloned in front of a promoterless lacZ and the resulting construct was integrated in the chromosome. Using these strains, it was determined that the msrA1/B expression is significantly higher in S. aureus compared to msrA2 or msrA3. Expression of msrA1/B was highest during stationary phase growth, but the expression of msrA2 and msrA3 was highest during the early to midexponential growth phase. Expression of msrA1/B was induced by oxacillin and the expression of msrA3 was upregulated by salt. Expression of msrA2 remained unchanged under all tested conditions.
Expression of Four Methionine Sulfoxide Reductases in Staphylococcus aureus
Kuldeep Singh,Vineet K. Singh
International Journal of Microbiology , 2012, DOI: 10.1155/2012/719594
Abstract: Staphylococcus aureus possesses three MsrA enzymes (MsrA1, MsrA2, MsrA3) that reduce the S-epimer of methionine sulfoxide (MetO) and an MsrB enzyme that reduces R-MetO. The four msr genes are expressed from three different promoters. The msrA1/msrB genes are coexpressed. To determine the expression pattern of msr genes, three independent reporter strains were constructed where msr promoter was cloned in front of a promoterless lacZ and the resulting construct was integrated in the chromosome. Using these strains, it was determined that the msrA1/B expression is significantly higher in S. aureus compared to msrA2 or msrA3. Expression of msrA1/B was highest during stationary phase growth, but the expression of msrA2 and msrA3 was highest during the early to midexponential growth phase. Expression of msrA1/B was induced by oxacillin and the expression of msrA3 was upregulated by salt. Expression of msrA2 remained unchanged under all tested conditions. 1. Introduction Staphylococcus aureus is a versatile and aggressive pathogen responsible for causing a wide array of diseases ranging from mild skin infections such as folliculitis and carbuncles to life-threatening conditions such as bacteremia, pneumonia, and endocarditis [1–3]. In response to S. aureus invasion, the host immune system recruits neutrophils and macrophages that trigger the release of highly reactive oxygen species such as hydrogen peroxide, hydroxyl radical, singlet oxygen, and hypochlorous acid. These highly reactive species lead to the oxidation of DNA, lipids, and proteins [4]. In proteins, oxidative damage usually leads to a loss of protein function that disturbs cellular processes and metabolism [5, 6]. Such oxidative damage includes oxidation of the sulfur atom of methionine producing methionine sulfoxide. Oxidation of methionine results in two diastereomic forms of MetO (R-MetO and S-MetO). These two stereoisomeric MetO products are reduced by two different kinds of Msr enzymes—MsrA and MsrB. MsrA specifically reduces S-MetO, whereas MsrB specifically reduces R-MetO [7–9]. The MsrA and MsrB proteins share no homology at the primary sequence or structural levels. Orthologs of msrA and msrB are present in most organisms [10, 11]. In bacterial species, the genetic organization of msrA and msrB shows great variation. In numerous cases, msrA and msrB are transcribed as independent units and are located in different regions of the chromosome [12]. However, in many bacterial species, these two genes are located adjacent to each other and are cotranscribed [12–14]. In a few cases like
Antioxidant Functions of Nitric Oxide Synthase in a Methicillin Sensitive Staphylococcus aureus
Manisha Vaish,Vineet K. Singh
International Journal of Microbiology , 2013, DOI: 10.1155/2013/312146
Abstract:
Antioxidant Functions of Nitric Oxide Synthase in a Methicillin Sensitive Staphylococcus aureus
Manisha Vaish,Vineet K. Singh
International Journal of Microbiology , 2013, DOI: 10.1155/2013/312146
Abstract: Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria including Staphylococcus aureus possess a gene encoding nitric oxide synthase (SaNOS) in their chromosome. In this study it was determined that under normal growth conditions, expression of SaNOS was highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion of SaNOS led to increased susceptibility of the mutant cells compared to wild-type S. aureus. While inhibition of SaNOS activity by the addition of L-NAME increased sensitivity of the wild-type S. aureus to oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of the SaNOS mutant. The SaNOS mutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-type S. aureus. 1. Introduction Staphylococcus aureus is a Gram-positive bacterial pathogen that colonizes anterior nares and mucosal surfaces in humans and is responsible for causing a wide array of diseases from mild skin infections to life-threatening conditions such as bacteremia, pneumonia, and endocarditis [1–4]. The emerging resistant strains of S. aureus exacerbate efforts to control or properly treat staphylococcal infections [5]. The host immune system responds to bacterial infections in a concerted manner to eliminate this pathogen. This involves recruitment of polymorphonuclear leukocytes and macrophages to the site of infection and ingestion of invading bacteria. Uptake of bacteria triggers oxygen-dependent and oxygen-independent microbicidal pathways in the phagocytic cells. The oxygen-dependent pathway generates superoxide anion ( ) that serves as a precursor for additional reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical, singlet oxygen, hypochlorous acid (HOCl), and peroxynitrite [6–9]. S. aureus utilizes various strategies to defend itself against host immune attack. It produces antioxidant enzymes such as superoxide dismutase that converts superoxide anion to H2O2, catalase that converts H2O2 to water and oxygen, and alkyl hydroperoxide reductases that detoxify H2O2, peroxynitrites and hydroperoxides [10, 11]. In addition to their ability to protect from host’s oxidants, S. aureus infections impose oxidative stress in a host [12]. During infection with a methicillin resistant S. aureus strain, host neutrophils respond by an increase in nitric oxide production [12]. Nitric oxide (NO) is a free radical synthesized by nitric
Continuous wavelet transform with the Shannon wavelet from the point of view of hyperbolic partial differential equations
Eugene B. Postnikov,Vineet K. Singh
Mathematics , 2015, DOI: 10.1007/s10476-015-0206-2
Abstract: We identify the result of the continuous wavelet transform with the difference of solutions of two hyperbolic partial differential equations, for which wavelet's shift and scale are considered as independent variables on 2D plane. The characteristic property, which follows from the introduced representation is is the fact that the transform's values inside the triangle defined by two characteristics (a=const, b=const) and crossecting them slopped line on a scale-shift plane (a,b) are completely and uniquely defined by the value of transform and its derivatives along the last mentioned line.
Utilization of case presentations in medical microbiology to enhance relevance of basic science for medical students
Neal R. Chamberlain,Melissa K. Stuart,Vineet K. Singh,Neil J. Sargentini
Medical Education Online , 2012, DOI: 10.3402/meo.v17i0.15943
Abstract: Background : Small-group case presentation exercises (CPs) were created to increase course relevance for medical students taking Medical Microbiology (MM) and Infectious Diseases (ID) Methods : Each student received a unique paper case and had 10 minutes to review patient history, physical exam data, and laboratory data. Students then had three minutes to orally present their case and defend why they ruled in or out each of the answer choices provided, followed by an additional three minutes to answer questions. Results : Exam scores differed significantly between students who received the traditional lecture-laboratory curriculum (Group I) and students who participated in the CPs (Group II). In MM, median unit exam and final exam scores for Group I students were 84.4% and 77.8%, compared to 86.0% and 82.2% for Group II students (P < 0.018; P < 0.001; Mann-Whitney Rank Sum Test). Median unit and final ID exam scores for Group I students were 84.0% and 80.0%, compared to 88.0% and 86.7% for Group II students (P < 0.001; P < 0.001). Conclusion : Students felt that the CPs improved their critical thinking and presentation skills and helped to prepare them as future physicians.
Primary Angiosarcoma of the Tongue: A Case Report  [PDF]
Vineet Chadha, Digvijay Singh Rawat, Beni Prasad
International Journal of Otolaryngology and Head & Neck Surgery (IJOHNS) , 2012, DOI: 10.4236/ijohns.2012.12011
Abstract: Sarcomas are rare tumors of head and neck region and constitute less than 1% of all cancers in this region. Primary angiosarcoma of the oral cavity is an extremely rare malignancy with few reported cases in literature till now. We present a case of a 50-year-old woman who was diagnosed as angiosarcoma after appropriate histopathological and immunohistochemical evaluation.
Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays
Martin J Hessner, Vineet K Singh, Xujing Wang, Shehnaz Khan, Michael R Tschannen, Thomas C Zahrt
BMC Genomics , 2004, DOI: 10.1186/1471-2164-5-12
Abstract: Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression).This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.Historically, the investigation of genetic alterations has focused on the study of single genes. In recent years, development of DNA microarrays has allowed researchers to study the complete genome of an organism and profile transcriptional expression patterns of up to tens of thousands of genes in a single experiment [1-3]. Consequently, this technology is already providing important insights into the biological properties of various organisms, making it a mainstream component of biomedical research [3-10].DNA microarrays are currently based on one o
Structure-Processing-Property Relationship of Poly(Glycolic Acid) for Drug Delivery Systems 1: Synthesis and Catalysis
Vineet Singh,Meena Tiwari
International Journal of Polymer Science , 2010, DOI: 10.1155/2010/652719
Abstract: Till date, market is augmented with a huge number of improved drug delivery systems. The success in this area is basically due to biodegradable polymers. Although conventional systems of drug delivery utilizing the natural and semisynthetic polymers so long but synthetic polymer gains success in the controlled drug delivery area due to better degradation profile and controlled network and functionality. The polyesters are the most studied class group due the susceptible ester linkage in their backbone. The Poly(glycolic Acid) (PGA), Poly(lactic acid) (PLA), and Polylactide-co-glycolide (PLGA) are the best profiled polyesters and are most widely used in marketed products. These polymers, however, still are having drawbacks which failed them to be used in platform technologies like matrix systems, microspheres, and nanospheres in some cases. The common problems arose with these polymers are entrapment inefficiency, inability to degrade and release drugs with required profile, and drug instability in the microenvironment of the polymers. These problems are forcing us to develop new polymers with improved physicochemical properties. The present review gave us an insight in the various structural elements of Poly(glycolic acid), polyester, with in depth study. The first part of the review focuses on the result of studies related to synthetic methodologies and catalysts being utilized to synthesize the polyesters. However the author will also focus on the effect of processing methodologies but due some constraints those are not included in the preview of this part of review. 1. Introduction Biodegradablepolymers can be efficiently utilized for various purposes such as drug delivery, orthopaedic, dental, and tissue engineering [1–7]. Such sophisticated applications usually require polymers with narrowly defined material properties. For a polymer to be used in drug delivery system, it is desired that it should degrade in prerequisite manner [8–12]. The rate of degradation (Hydrolytic and proteolytic degradation) of any polymer chiefly depends on its primary structure properties (Backbone and functionality characteristics), and secondary structural properties such as morphology, mechanical properties (tensile strength and modulus), the thermal properties (glass transition temperature , softening or melting point, degradation temperature) and the viscoelastic properties (storage and Loss moduli and tan?δ). The quality of these physicochemical properties depends on the structural features, such as backbone characteristics, functionalities, and crystal packing
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