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An in vitro infection model system to study proteins expressed during interaction of mycobacterium with murine macrophages  [PDF]
Neelja Singhal, Prashant Sharma, Bhavnesh Kumar, Utpal Sengupta, Krishnamurthy Venkatesan, Deepa Bisht
Advances in Bioscience and Biotechnology (ABB) , 2010, DOI: 10.4236/abb.2010.13025
Abstract: Resurgence of mycobacterial diseases particularly tuberculosis has caused a renewed interest to unravel the strategies employed by mycobacteria for intracellular survival. In spite of advancement in mycobacterial research, our knowledge about genes and their corresponding functional proteins involved during the interaction of mycobacterium with host’s macrophages is fragmentary. This study pertains to development of a suitable in vitro model using murine macrophages and Mycobacterium bovis BCG to study proteins expressed during macrophage-myco bacterium interactions. Peritoneal macrophages from BALB/c mice were infected with M. bovis BCG and intracellular replication was assessed by {3H} thymi- dine uptake assay which was maximal when macrophage to mycobacterium ratio was 1:10. SDS-PAGE was employed to study the proteins expressed and selected proteins were subjected to mass spectrometry. Seven proteins found to be upregulated during macrophage-mycobacterium interaction were identified by MALDI-TOF. The results indicate that the present in vitro infection model was able to support the growth of M. bovis BCG in murine macrophages and is an ideal model to determine the pattern of functions of gene expression during the interaction of mycobacterium with macrophages. The differentially expressed proteins will help in understanding the mycobacterial molecular basis of adaptation to intracellular macrophage environment.
Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates
Neelja Singhal, Prashant Sharma, Manish Kumar, Beenu Joshi, Deepa Bisht
Proteome Science , 2012, DOI: 10.1186/1477-5956-10-14
Abstract: On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug
Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis
Prashant Sharma, Bhavnesh Kumar, Yash Gupta, Neelja Singhal, Vishwa Katoch, Krishnamurthy Venkatesan, Deepa Bisht
Proteome Science , 2010, DOI: 10.1186/1477-5956-8-59
Abstract: Two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was employed for analyzing the protein profiles. Homology and in silico characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG), I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. In silico docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.The computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug action other than established primary sites and hence may help in search of novel chemotherapeutic agents at these new sites as inhibitors.Tuberculosis is one of the most challenging infectious diseases. Globally, 9.2 million new cases and 1.7 million deaths occur due to this disease [1]. Its impact on public health is further aggravated by co-infection with human immunodeficiency virus, emergence of multi-drug resistant strains and reactivation of the dormant bacteria. Attempt for primary prevention using Bacillus Calmette Guerin (BCG) and other integral vaccines have generally been disappointing though some subunit vaccines are under trial [2]. The excessive emergence of drug resistant tuberculosis has stimulated interest in the under
A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis
Kumar, Gavish;Shankar, Hari;Bisht, Deepa;Sharma, Prashant;Singhal, Neelja;Katoch, V.M.;Joshi, Beenu;
Brazilian Journal of Microbiology , 2010, DOI: 10.1590/S1517-83822010000200005
Abstract: sample preparation for two-dimensional gel electrophoresis (2de) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of m. tuberculosis. high lipid content in mycobacteria limits the use of common methods as it can hinder the 2de run. this study highlights the significance of sds-tca procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.
Cation Distribution in Lithium Ferrite (LiFe5O8) Prepared via Aerosol Route  [PDF]
Sonal Singhal, Kailash Chandra
Journal of Electromagnetic Analysis and Applications (JEMAA) , 2010, DOI: 10.4236/jemaa.2010.21008
Abstract: Nano size lithium ferrite was prepared through aerosol route and characterized using TEM, XRD, magnetic measurements and Mössbauer spectroscopy. The particle size of as obtained samples were found to be ~10 nm through TEM, that increases upto ~80 nm on annealing at 1200 oC. The unit cell parameter ‘a’ calculated using XRD, confirms the formation of ?-LiFe5O8. Room temperature Mössbauer spectra of as obtained sample of all the ferrites exhibited broad doublet suggesting super paramagnetic nature. This doublet further resolved into two doublets and assigned to the surface region atoms and internal region atoms of the particles. The annealed samples (1200 oC) show broad sextets, which were fitted with two sextets indicating different local environment of both tetrahedrally and octahedrally coordinated Fe-cation. Cation distribution obtained from the X-ray, magnetic and Mössbauer data confirms that the three fifth of the iron atom goes in to the octahedral site.
Endovascular and Percutaneous Management of the Pseudoaneurysms  [PDF]
Sahu Chandradev, Singhal Ateesh
Open Journal of Radiology (OJRad) , 2014, DOI: 10.4236/ojrad.2014.43032

Introduction: Pseudoaneurysm is a dilatation of an artery with actual disruption of one or more layers of its wall, rather than just expansion of wall layers. Pseudoaneurysms are common vascular abnormalities. Computed Tomography (CT), Magnetic Resonance Imaging (MRI), sonography, and angiography all are valuable modalities in the imaging workup of pseudoaneurysms. Conventional angiography remains the gold standard for diagnosis. Therapeutic options have evolved in recent years from the traditional surgical option toward a less invasive approach which includes procedures such as Ultrasound (US)-guided compression, US-guided percutaneous thrombin injection, and endovascular management (embolization, stent-graft placement and endovascular coiling). Objective: In this article, we discuss pseudoaneurysms of various arteries, and available percutaneous and endovascular treatment options. Materials and Methods: Sixteen patients diagnosed with pseudoaneurysm involving visceral and peripheral arteries were included in this study. The initial diagnosis was made with CDUS, CECT, and CTA followed by catheter angiogram for therapeutic procedures. Results: All the 16 patients with pseudoaneurysms were successfully treated with endovascular and percutaneous embolization to achieve stasis. Conclusion: CT, MRI, sonography, and angiography may all be valuable in the imaging workup of pseudoaneurysms. The use of minimally invasive treatment has led to a marked decrease in the morbidity and mortality rates for pseudoaneurysms.

GIS Based Methodology for Groundwater Flow Estimation across the Boundary of the Study Areain Groundwater Flow Modeling  [PDF]
Vijai Singhal, Rohit Goyal
Journal of Water Resource and Protection (JWARP) , 2011, DOI: 10.4236/jwarp.2011.311092
Abstract: Pali district, Rajasthan, India has been facing severe pollution of groundwater due to release of untreated industrial effluent of textile industries into the Bandi River flowing through the Pali city. A groundwater flow and transport modeling exercise has been undertaken by MNIT, Jaipur, India to understand the groundwater flow regime and to study the different scenarios. In the modeling exercise partially penetrating ephemeral rivers have been taken as part of model boundaries wherever more appropriate boundaries were not available in the near vicinity. These boundaries have been considered as constant flow boundaries. Aim of this paper is to present a methodology to calculate the average flux through such boundaries from readily available data such as bore logs and groundwater levels. The study area boundary was divided in to several cross sections and average values of groundwater flow gradients normal to the boundary were calculated for different monsoon and non monsoon seasons for different years. The entire boundary was then regrouped into 8 boundary segments on the basis of average values of gradients for individual line segments and mean gradient values for these line segments were calculated. Values of ground level, bottom elevations of hydros-tratigraphic layers and average water depth were extracted for a number of points on these line segments from the respective layers and these values were used to calculate equivalent horizontal hydraulic conductivity of the multi-layered aquifer system at every point. The Darcy’s law was then used to calculate inflow/outflow per m length of the boundary at each point. The methodology presented here is simple and is based on the assumption that the groundwater level gradients do not change significantly for different seasons and amongst different years which has been validated in the present groundwater modeling study. The paper demonstrates a GIS based methodology to work out inflow/outflow across boundary of a study area in the cases where no flow boundaries in the vicinity of the study area cannot be identified.
Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications  [PDF]
Anjali Sharma, Barkha Singhal
Advances in Bioscience and Biotechnology (ABB) , 2019, DOI: 10.4236/abb.2019.103004
Abstract: The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.
Effect of Non-Homogeneity on Thermally Induced Vibration of Orthotropic Visco-Elastic Rectangular Plate of Linearly Varying Thickness  [PDF]
Arun Kumar Gupta, Pooja Singhal
Applied Mathematics (AM) , 2010, DOI: 10.4236/am.2010.14043
Abstract: The analysis presented here is to study the effect of non-homogeneity on thermally induced vibration of orthotropic visco-elastic rectangular plate of linearly varying thickness. Thermal vibrational behavior of non-homogeneous rectangular plates of variable thickness having clamped boundary conditions on all the four edges is studied. For non–homogeneity of the plate material, density is assumed to vary linearly in one direction. Using the method of separation of variables, the governing differential equation is solved. An approximate but quite convenient frequency equation is derived by using Rayleigh-Ritz technique with a two-term deflection function. Time period and deflection at different points for the first two modes of vibration are calculated for various values of temperature gradients, non- homogeneity constant, taper constant and aspect ratio. Comparison studies have been carried out with non-homogeneous visco-elastic rectangular plate to establish the accuracy and versatility.
Thermal Effect on Free Vibration of Non-Homogeneous Orthotropic Visco-Elastic Rectangular Plate of Parabolically Varying Thickness  [PDF]
Arun Kumar Gupta, Pooja Singhal
Applied Mathematics (AM) , 2010, DOI: 10.4236/am.2010.16060
Abstract: A simple model presented here is to study the thermal effect on vibration of non-homogeneous orthotropic visco-elastic rectangular plate of parabolically varying thickness having clamped boundary conditions on all the four edges. For non-homogeneity of the plate material, density is assumed to vary linearly in one direction. Using the separation of variables method, the governing differential equation has been solved for vibration of non-homogeneous orthotropic viscoelastic rectangular plate. An approximate frequency equation is derived by using Rayleigh-Ritz technique with a two-term deflection function. Results are calculated for time period and deflection at different points, for the first two modes of vibration, for various values of temperature gradients, non-homogeneity constant, taper constant and aspect ratio and shown by graphs.
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