Publications by authors named "Balvinder Singh"

41 Publications

Structurally disordered C-terminal residues of GTP cyclohydrolase II are essential for its enzymatic activity.

J Biomol Struct Dyn 2021 May 25:1-14. Epub 2021 May 25.

CSIR-Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Chandigarh, India.

GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20-25C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from (GCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of GCHII in complex with GTP, Mg, and Zn were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190 position from the unstructured C-terminal region of GCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1926326DOI Listing
May 2021

Role of Cys-298 in specific recognition of glutathione by aldose reductase.

J Biomol Struct Dyn 2021 Feb 25:1-9. Epub 2021 Feb 25.

Department cum National Centre for Human Genome Studies & Research, Pharmacy Extention Block, Panjab University, Chandigarh, India.

Aldose reductase (AR) is an NADPH-dependent oxidoreductase that is well-studied for its role in Diabetes Mellitus. Glutathione conjugated aldehydes are efficiently catalysed by AR. We have employed molecular dynamics simulations to investigate the dynamics of a glutathione analog, γ-glutamyl-S-(1,2-di-carboxyethyl)-cysteinyl-glycine (DCEG), into the binding pocket of AR. Study revealed that backbone nitrogens of Ala-299 and Leu-300 form a tiny pocket gated by thiol group of Cys-298. The glycine moiety of DCEG was able to displace the thiol group of Cys-298 to make hydrogen bond interactions with backbone of Ala-299, Leu-300, and Leu-301. This study provides the details of the dynamic interactions of DCEG in the binding pocket of AR, and shall aid in the design/discovery of differential inhibitors against AR.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1891138DOI Listing
February 2021

Contentious Issue in Recurrent COVID-19 Infection: Reactivation or Reinfection.

Turk Thorac J 2020 Nov 1;21(6):463-466. Epub 2020 Nov 1.

Department of Pulmonary, Critical Care and Sleep Medicine, VMMC and Safdarjung Hospital, New Delhi, India.

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http://dx.doi.org/10.5152/TurkThoracJ.2020.20164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752104PMC
November 2020

Deciphering the Structural Enigma of HLA Class-II Binding Peptides for Enhanced Immunoinformatics-based Prediction of Vaccine Epitopes.

J Proteome Res 2020 11 26;19(11):4655-4669. Epub 2020 Oct 26.

Immunology Laboratory, CSIR-Institute of Microbial Technology, Chandigarh 160036, India.

Vaccines remain the most efficacious means to avoid and eliminate morbid diseases associated with high morbidity and mortality. Clinical trials indicate the gaining impetus of peptide vaccines against diseases for which an effective treatment still remains obscure. CD4 T-cell-based peptide vaccines involve immunization with antigenic determinants from pathogens or neoplastic cells that possess the ability to elicit a robust T helper cell response, which subsequently activates other arms of the immune system. The available predictors of human leukocyte antigen II (HLA-II) binding peptides are sequence-based techniques, which ostensibly have balanced sensitivity and specificity. Structural analysis and understanding of the cognate peptide and HLA-II interactions are essential to empirically derive a successful peptide vaccine. However, the availability of structure-based epitope prediction algorithms is inadequate compared with sequence-based prediction methods. The present study is an attempt to understand the structural aspects of HLA-II binders by analyzing the Protein Data Bank (PDB) complexes of pHLA-II. Furthermore, we mimic the peptide exchange mechanism and demonstrate the structural implication of an acidic environment on HLA-II binders. Finally, we discuss a structure-guided approach to decipher potential HLA-II binders within an antigenic protein. This strategy may accurately predict the peptide epitopes and thus aid in designing successful peptide vaccines.
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http://dx.doi.org/10.1021/acs.jproteome.0c00405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640962PMC
November 2020

Tobacco use and other aspects related to smoking among school-going adolescents aged 13-15 years in Malaysia: Analysis of three cross-sectional nationally representative surveys in 2003, 2009 and 2016.

Tob Induc Dis 2020 17;18:80. Epub 2020 Sep 17.

School of Pharmacy, Monash University Malaysia, Subang Jaya, Malaysia.

Introduction: Periodic surveys on tobacco use patterns and other aspects of tobacco use among school-going adolescents in Malaysia provide information on the effectiveness of anti-smoking measures implemented. However, such information is limited in Malaysia. We investigated the prevalence of smoking and other related aspects among middle-secondary school students in Malaysia from the years 2003-2016 to fill this gap.

Methods: We analyzed data from the Global Youth Tobacco Survey (GYTS) 2003, GYTS 2009, and the Tobacco and Electronic Cigarette Survey among Malaysia Adolescents (TECMA) 2016. The surveys employed multistage sampling to select representative samples of adolescents attending secondary school in Malaysia. Data were collected using a pre-validated self-administered anonymous questionnaire adopted from the GYTS.

Results: Between 2003 and 2016, major changes occurred in which there were reductions in the prevalence of ever smoking, current smoking, and susceptibility to smoking. Reductions were also observed in exposure to SHS in public places and in the home. The proportion of school-going adolescents who support a ban on smoking in public places increased between 2013 to 2016, and there was a significant reduction in the proportion of respondents that were offered 'free' cigarettes by tobacco company representatives. However, there was no difference in the proportion of adolescents who initiated smoking before the age of 10 years and current smokers seeking advice to quit smoking across the time period.

Conclusions: Our study indicates that the smoking policies and measures have been effective in reducing smoking prevalence, secondhand smoke exposure, and access to cigarettes, among school-going adolescents in Malaysia. However, measures to reduce smoking initiation and increase smoking cessation need to be strengthened to reduce the burden of smoking-related diseases in Malaysia in the long-term.
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http://dx.doi.org/10.18332/tid/127231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528268PMC
September 2020

Clinical and epidemiologic profile of the initial COVID-19 patients at a tertiary care centre in India.

Monaldi Arch Chest Dis 2020 Apr 10;90(1). Epub 2020 Apr 10.

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COVID-19 has now become a pandemic. It has spread from Wuhan, China, in December 2019 to a large number of countries within three months. The objective of this work is to report the initial experience with epidemiologic and clinical features, as well as with the management of COVID-19 patients in India. This is a descriptive case series of the first 21 COVID-19 infected patients confirmed with polymerase chain reaction (PCR) and admitted to a tertiary care centre in India from 01.02.2020 to 19.03.2020. Clinical, laboratory, and radiologic data were collected, including age, sex, nationality, travel history, symptoms, duration of stay, and comorbidities. The mean age of the population was 40.3 years with a male preponderance. Thirteen (62%) patients had recent travel history outside India in the previous 30 days, two thirds of whom had travelled to Italy. The most common symptoms were fever and cough (42.9%) followed by sore throat, headache and breathlessness. Vital and laboratory parameters were preserved in all patients and none of them required ventilatory support. Among the first 21 patients diagnosed with COVID-19 infection in India, the typical clinical presentation consisted in a mild upper respiratory tract infection predominantly affecting the young male population. One patient required supplemental oxygen. All patients recovered with no residual symptoms.   *The Safdarjung Hospital COVID 2019 working group: Nitesh Gupta, Sumita Agrawal, Pranav Ish, Suruchi Mishra, Rajni Gaind, Ganapathy Usha, Balvinder Singh, Manas Kamal Sen, Shibdas Chakrabarti (Consultant and Head, Pulmonary Medicine); NK Gupta (Professor, Pulmonary medicine); Dipak Bhattacharya (Consultant, Pulmonary medicine); Rohit Kumar (Assistant Professor, Pulmonary Medicine); Siddharth R. Yadav (Assistant Professor, Pulmonary Medicine); Rushika Saksena (Specialist, Microbiology); Rojaleen Das (Assistant Professor, Microbiology); Vikramjeet Dutta (Assistant Professor, Microbiology); Anupam Kr Anveshi (Senior Resident, Microbiology); Santvana Kohli (Assistant Professor, Anaesthesiology); Naveen KV (Assistant Professor,  Anaesthesiology); Amandeep Jaswal (Assistant Professor, Anaesthesiology).
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http://dx.doi.org/10.4081/monaldi.2020.1294DOI Listing
April 2020

HSPMdb: a computational repository of heat shock protein modulators.

Database (Oxford) 2020 01;2020

Council of Scientific and Industrial Research-Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India.

Heat shock proteins (Hsp) are among highly conserved proteins across all domains of life. Though originally discovered as a cellular response to stress, these proteins are also involved in a wide range of cellular functions such as protein refolding, protein trafficking and cellular signalling. A large number of potential Hsp modulators are under clinical trials against various human diseases. As the number of modulators targeting Hsps is growing, there is a need to develop a comprehensive knowledge repository of these findings which is largely scattered. We have thus developed a web-accessible database, HSPMdb, which is a first of its kind manually curated repository of experimentally validated Hsp modulators (activators and inhibitors). The data was collected from 176 research articles and current version of HSPMdb holds 10 223 entries of compounds that are known to modulate activities of five major Hsps (Hsp100, Hsp90, Hsp70, Hsp60 and Hsp40) originated from 15 different organisms (i.e. human, yeast, bacteria, virus, mouse, rat, bovine, porcine, canine, chicken, Trypanosoma brucei and Plasmodium falciparum). HSPMdb provides comprehensive information on biological activities as well as the chemical properties of Hsp modulators. The biological activities of modulators are presented as enzymatic activity and cellular activity. Under the enzymatic activity field, parameters such as IC50, EC50, DC50, Ki and KD have been provided. In the cellular activity field, complete information on cellular activities (percentage cell growth inhibition, EC50 and GI50), type of cell viability assays and cell line used has been provided. One of the important features of HSPMdb is that it allows users to screen whether or not their compound of interest has any similarity with the previously known Hsp modulators. We anticipate that HSPMdb would become a valuable resource for the broader scientific community working in the area of chaperone biology and protein misfolding diseases. HSPMdb is freely accessible at http://bioinfo.imtech.res.in/bvs/hspmdb/index.php.
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http://dx.doi.org/10.1093/database/baaa003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043294PMC
January 2020

ImmtorLig_DB: repertoire of virtually screened small molecules against immune receptors to bolster host immunity.

Sci Rep 2019 02 28;9(1):3092. Epub 2019 Feb 28.

Indian Institute of Technology Ropar, Rupnagar, 140001, India.

Host directed therapies to boost immunity against infection are gaining considerable impetus following the observation that use of antibiotics has become a continuous source for the emergence of drug resistant strains of pathogens. Receptors expressed by the cells of immune system play a cardinal role in initiating sequence of events necessary to ameliorate many morbid conditions. Although, ligands for the immune receptors are available; but their use is limited due to complex structure, synthesis and cost-effectiveness. Virtual screening (VS) is an integral part of chemoinformatics and computer-aided drug design (CADD) and aims to streamline the process of drug discovery. ImmtorLig_DB is a repertoire of 5000 novel small molecules, screened from ZINC database and ranked using structure based virtual screening (SBVS) against 25 immune receptors which play a pivotal role in defending and initiating the activation of immune system. Consequently, in the current study, small molecules were screened by docking on the essential domains present on the receptors expressed by cells of immune system. The screened molecules exhibited efficacious binding to immune receptors, and indicated a possibility of discovering novel small molecules. Other features of ImmtorLig_DB include information about availability, clustering analysis, and estimation of absorption, distribution, metabolism, and excretion (ADME) properties of the screened small molecules. Structural comparisons indicate that predicted small molecules may be considered novel. Further, this repertoire is available via a searchable graphical user interface (GUI) through http://bioinfo.imtech.res.in/bvs/immtor/ .
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http://dx.doi.org/10.1038/s41598-018-36179-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395627PMC
February 2019

Morbid Sequences Suggest Molecular Mimicry between Microbial Peptides and Self-Antigens: A Possibility of Inciting Autoimmunity.

Front Microbiol 2017 9;8:1938. Epub 2017 Oct 9.

Immunology Laboratory, CSIR-Institute of Microbial Technology, Chandigarh, India.

Understanding etiology of autoimmune diseases has been a great challenge for designing drugs and vaccines. The pathophysiology of many autoimmune diseases may be attributed to molecular mimicry provoked by microbes. Molecular mimicry hypothesizes that a sequence homology between foreign and self-peptides leads to cross-activation of autoreactive T cells. Different microbial proteins are implicated in various autoimmune diseases, including multiple sclerosis, human type 1 diabetes, primary biliary cirrhosis and rheumatoid arthritis. It may be imperative to identify the microbial epitopes that initiate the activation of autoreactive T cells. Consequently, in the present study, we employed immunoinformatics tools to delineate homologous antigenic regions between microbes and human proteins at not only the sequence level but at the structural level too. Interestingly, many cross-reactive MHC class II binding epitopes were detected from an array of microbes. Further, these peptides possess a potential to skew immune response toward Th1-like patterns. The present study divulges many microbial target proteins, their putative MHC-binding epitopes, and predicted structures to establish the fact that both sequence and structure are two important aspects for understanding the relationship between molecular mimicry and autoimmune diseases. Such findings may enable us in designing potential immunotherapies to tolerize autoreactive T cells.
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http://dx.doi.org/10.3389/fmicb.2017.01938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640720PMC
October 2017

Eukaryotic-type serine/threonine kinase mediated phosphorylation at Thr perturbs mycobacterial guanylate kinase activity.

Biosci Rep 2017 Dec 15;37(6). Epub 2017 Nov 15.

CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India

Guanylate kinase is an essential and conserved enzyme in nucleotide biosynthetic pathway that transfers phosphoryl group of ATP to GMP for yielding GDP. Here, we report the phosphorylation of guanylate kinase from (mGmk) by eukaryotic-type Ser/Thr kinase, PknA. Mass spectrometric studies identified Thr and Thr as phosphorylatable residues in mGmk. To evaluate the significance of phosphorylation in these threonines, two point (T101A and T169A) and one double (T101A-T169A) mutants were generated. The kinase assay with these mutant proteins revealed the major contribution of Thr compared with Thr in the phosphorylation of mGmk. Kinetic analysis indicated that p-mGmk was deficient in its enzymatic activity compared with that of its un-phosphorylated counterpart. Surprisingly, its phosphoablated (T169A) as well as phosphomimic (T169E) variants exhibited decreased activity as was observed with p-mGmk. Structural analysis suggested that phosphorylation of Thr might affect its interaction with Arg, which is crucial for the functioning of mGmk. In fact, the R166A and R166K mutant proteins displayed a drastic decrease in enzymatic activity compared with that of the wild-type mGmk. Molecular dynamics (MD) studies of mGmk revealed that upon phosphorylation of Thr, the interactions of Arg/Arg with Glu, Asp and residues of the loop in GMP-binding domain are perturbed. Taken together, our results illuminate the mechanistic insights into phosphorylation-mediated modulation of the catalytic activity of mGmk.
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http://dx.doi.org/10.1042/BSR20171048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686395PMC
December 2017

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS.

Sci Rep 2017 01 12;7:40452. Epub 2017 Jan 12.

CSIR-Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh 160036, India.

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg is still elusive. Therefore, mechanistic role played by Mg in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg have shown entry of Mg from bulk solvent into active site. Presence of Mg in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a "lid" covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.
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http://dx.doi.org/10.1038/srep40452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228156PMC
January 2017

Management of complex femoral nonunion with monorail external fixator: A prospective study.

J Clin Orthop Trauma 2016 Oct-Dec;7(Suppl 2):191-200. Epub 2016 Jun 8.

Director Professor, Ex-Head of Department, Central Institute of Orthopedics, VMMC & Safdarjung Hospital, Newdelhi, India.

Aim: To evaluate 30 patients who underwent distraction osteogenesis with monorail external fixator for complex femoral nonunion.

Method: Complex femoral nonunion includes infective non-union, gap nonunion, and limb-length discrepancy secondary to traumatic bone loss, which needs specialized treatment to ensure the functional integrity of femoral bone. 30 patients, including 28 male and 2 female (aged 22-62 years) patients, underwent surgical debridement followed by bone transport with monorail fixator. The lengthening index, radiographic consolidation index, functional status, bone healing, and various problems, obstacles, and complications encountered during the treatment were assessed.

Results: Patients underwent a mean of 2.2 (range 1-4) surgeries before presentation. The mean bone defect after surgical debridement was 5.83 cm (range 2-16 cm). The mean treatment duration was 204.7 days (range 113-543 days). The mean lengthening index was 13.06 days/cm with range from 12 to 16 days/cm. Mean maturation index was 23.51 days/cm with range from 17 to 45.5 days/cm. In our study, bone result was excellent in 17, good in 9, fair in 3, and poor in 1 patient. In our study functional outcome is excellent in 9 [30%], good in 14 [46.67%], fair in 5, and poor in 2 patients. In our study, we encountered 34 problems, 17 obstacles, and 8 complications.

Conclusion: We concluded that monorail external fixator is an effective treatment option for complex nonunion femoral shaft fracture and its functional outcome is comparable with any other treatment options. Lack of complications and its effectiveness makes monorail external fixator the treatment of choice for complex nonunion femoral shaft.
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http://dx.doi.org/10.1016/j.jcot.2016.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5197217PMC
June 2016

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation.

J Biomol Struct Dyn 2017 May 4;35(6):1153-1167. Epub 2016 May 4.

a Bioinformatics Centre , Council of Scientific & Industrial Research - Institute of Microbial Technology , Sector 39A, Chandigarh , India.

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5'-(T/C)TAATTAA(T/A)(T/A)G-3' DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein-DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll-3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll-3A01-Al. In addition, this change in secondary structure of Cll does not allow it to form protein-protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al-3A01-Cll where Cll binds to Al-3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein-protein interactions while proposing likely mechanism of recognition in ternary complex, Al-Cll-DNA.
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http://dx.doi.org/10.1080/07391102.2016.1175967DOI Listing
May 2017

PEPstrMOD: structure prediction of peptides containing natural, non-natural and modified residues.

Biol Direct 2015 Dec 21;10:73. Epub 2015 Dec 21.

Bioinformatics Centre, CSIR-Institute of Microbial Technology, Sec 39-A, Chandigarh, 160036, India.

Background: In the past, many methods have been developed for peptide tertiary structure prediction but they are limited to peptides having natural amino acids. This study describes a method PEPstrMOD, which is an updated version of PEPstr, developed specifically for predicting the structure of peptides containing natural and non-natural/modified residues.

Results: PEPstrMOD integrates Forcefield_NCAA and Forcefield_PTM force field libraries to handle 147 non-natural residues and 32 types of post-translational modifications respectively by performing molecular dynamics using AMBER. AMBER was also used to handle other modifications like peptide cyclization, use of D-amino acids and capping of terminal residues. In addition, GROMACS was used to implement 210 non-natural side-chains in peptides using SwissSideChain force field library. We evaluated the performance of PEPstrMOD on three datasets generated from Protein Data Bank; i) ModPep dataset contains 501 non-natural peptides, ii) ModPep16, a subset of ModPep, and iii) CyclicPep contains 34 cyclic peptides. We achieved backbone Root Mean Square Deviation between the actual and predicted structure of peptides in the range of 3.81-4.05 Å.

Conclusions: In summary, the method PEPstrMOD has been developed that predicts the structure of modified peptide from the sequence/structure given as input. We validated the PEPstrMOD application using a dataset of peptides having non-natural/modified residues. PEPstrMOD offers unique advantages that allow the users to predict the structures of peptides having i) natural residues, ii) non-naturally modified residues, iii) terminal modifications, iv) post-translational modifications, v) D-amino acids, and also allows extended simulation of predicted peptides. This will help the researchers to have prior structural information of modified peptides to further design the peptides for desired therapeutic property. PEPstrMOD is freely available at http://osddlinux.osdd.net/raghava/pepstrmod/.
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http://dx.doi.org/10.1186/s13062-015-0103-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687368PMC
December 2015

Cosmetic arm lengthening with monorail fixator.

Chin J Traumatol 2015 ;18(3):170-4

Central Institute of Orthopaedics, Safdarjung Hospital, New Delhi 110029, India.

Upper limb length discrepancy is a rare occurrence. Humerus shortening may need specialized treatment to restore the functional and cosmetic status of upper limb. We report a case of humerus lengthening of 9 cm with a monorail external fixator and the result was observed during a 2-year follow-up. Humerus lengthening needs specialized focus as it is not only a cosmetic issue but also a functional demand. The monorail unilateral fixator is more functional and cosmetically acceptable, and thus becomes an effective treatment option.
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http://dx.doi.org/10.1016/j.cjtee.2015.10.001DOI Listing
January 2017

Identification of Critical Amino Acids Conferring Lethality in VopK, a Type III Effector Protein of Vibrio cholerae: Lessons from Yeast Model System.

PLoS One 2015 21;10(10):e0141038. Epub 2015 Oct 21.

Institute of Microbial Technology, Council of Scientific and Industrial Research, Molecular Biology Division, Chandigarh, India.

VopK, a type III effector protein, has been implicated in the pathogenesis of Vibrio cholerae strains belonging to diverse serogroups. Ectopic expression of this protein exhibits strong toxicity in yeast model system. In order to map critical residues in VopK, we scanned the primary sequence guided by available data on various toxins and effector proteins. Our in silico analysis of VopK indicated the presence of predicted MCF1-SHE (SHxxxE) serine peptidase domain at the C-terminus region of the protein. Substitution of each of the predicted catalytic triad residues namely Ser314, His353 and Glu357 with alanine resulted in recombinant VopK proteins varying in lethality as evaluated in yeast model system. We observed that replacement of glutamate357 to alanine causes complete loss in toxicity while substitutions of serine314 and histidine353 with alanine exhibited partial loss in toxicity without affecting the stability of variants. In addition, replacement of another conserved serine residue at position 354 (S354) within predicted S314H353E357 did not affect toxicity of VopK. In essence, combined in silico and site directed mutagenesis, we have identified critical amino acids contributing to the lethal activity of VopK in yeast model system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619451PMC
June 2016

Quantification of minerals and trace elements in raw caprine milk using flame atomic absorption spectrophotometry and flame photometry.

J Food Sci Technol 2015 Aug 31;52(8):5299-304. Epub 2014 Aug 31.

College Central Laboratory, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 125004 Haryana India.

This study reports minerals and trace elements quantification in raw caprine milk of Beetal breed, reared in Northern India and their feed, fodder & water using flame atomic absorption spectrophotometry and flame photometry. The mineral and trace elements' concentration in the milk was in the order: K > Ca > Na > Fe > Zn > Cu. The results showed that minerals concentration in caprine milk was lesser than reference values. But trace elements concentration (Fe and Zn) was higher than reference values. Multivariate statistical techniques, viz., Pearsons' correlation, Cluster analysis (CA) and Principal component analysis (PCA) were applied to analyze the interdependences within studied variables in caprine milk. Significantly positive correlations were observed between Fe - Zn, Zn - K, Ca - Na and Ca - pH. The results of correlation matrix were further supported by Cluster analysis and Principal component analysis as primary cluster pairs were found for Ca - pH, Ca - Na and Fe - Zn in the raw milk. No correlation was found between mineral & trace elements content of the milk and feed.
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http://dx.doi.org/10.1007/s13197-014-1538-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519456PMC
August 2015

Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.

Proteins 2015 10;83(10):1813-22

CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.

Although unglycosylated HuEpo is fully functional, it has very short serum half-life. However, the mechanism of in vivo clearance of human Epo (HuEpo) remains largely unknown. In this study, the relative importance of protease-sensitive sites of recombinant HuEpo (rHuEpo) has been investigated by analysis of structural data coupled with in vivo half-life measurements. Our results identify α3-α4 inter-helical loop region as a target site of lysosomal protease Cathepsin L. Consistent with previously-reported lysosomal degradation of HuEpo, these results for the first time identify cleavage sites of rHuEpo by specific lysosomal proteases. Furthermore, in agreement with the lowered exposure of the peptide backbone around the cleavage site, remarkably substitutions of residues with bulkier amino acids result in significantly improved in vivo stability. Together, these results have implications for the mechanism of in vivo clearance of the protein in humans.
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http://dx.doi.org/10.1002/prot.24865DOI Listing
October 2015

Rv2031c of Mycobacterium tuberculosis: a master regulator of Rv2028-Rv2031 (HspX) operon.

Front Microbiol 2015 27;6:351. Epub 2015 Apr 27.

Institute of Microbial Technology, Council of Scientific and Industrial Research Chandigarh, India.

Genes belonging to the same operon are transcribed as a single mRNA molecule in all prokaryotes. The genes of the same operon are presumed to be involved in similar metabolic and physiological processes. Hence, computational analysis of constituent proteins could provide important clues to the functional relationships within the operonic genes. This tends to be more fruitful in the case of Mycobacterium tuberculosis (Mtb), considering the number of hypothetical genes with unknown functions and interacting partners. Dramatic advances in the past decade have increased our knowledge of the mechanisms that tubercle bacilli employ to survive within the host. But the phenomenon of Mtb latency continues to baffle all. Rv2031c belonging to dormancy regulon of Mtb is predominantly expressed during latency, with myriad immunological roles. Thus we attempted to analyze the operon comprising Rv2031c protein to gain insights into its role during latency. In the current study, we have carried out computational analysis of proteins encoded by genes known to be a part of this operon. Our study includes phylogenetic analysis, modeling of protein 3D structures, and protein interaction network analysis. We describe the mechanistic role in the establishment of latency and regulation of DevS-DevR component system. Additionally, we have identified the probable role of these proteins in carbohydrate metabolism, erythromycin tolerance, and nucleotide synthesis. Hence, these proteins can modulate the metabolism of Mtb inside the host cells and can be important for its survival in latency. The functional characterization and interactome of this important operon can give insight into its role during latency along with the exploitation of constituent proteins as drug targets and vaccine candidates.
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http://dx.doi.org/10.3389/fmicb.2015.00351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410610PMC
May 2015

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

J Biomol Struct Dyn 2015 12;33(10):2069-82. Epub 2015 Jan 12.

a Bioinformatics Centre , CSIR-Institute of Microbial Technology , Sector 39A, Chandigarh , India.

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.
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http://dx.doi.org/10.1080/07391102.2014.995709DOI Listing
May 2016

The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.

Mol Genet Genomics 2014 Dec 5;289(6):1171-82. Epub 2014 Jul 5.

Molecular Biology and Microbial Physiology Division, Institute of Microbial Technology (Council of Scientific and Industrial Research), Sector 39A, Chandigarh, 160036, India.

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.
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http://dx.doi.org/10.1007/s00438-014-0878-5DOI Listing
December 2014

An unusual cause of radial nerve palsy.

Chin J Traumatol 2014 ;17(3):175-7

Central Institute of Orthopedics, VMMC and Safdarjung Hospital, New Delhi 110029, India.

Neurapraxia frequently occurs following traction injury to the nerve intraoperatively, leading to radial nerve palsy which usually recovers in 5-30 weeks. In our case, we had operated a distal one-third of humeral shaft fracture and fixed it with 4.5 mm limited contact dynamic compression plate. The distal neurovascular status of the limb was assessed postoperatively in the recovery room and was found to be intact and all the sensory-motor functions of the radial nerve were normal. On the second postoperative day, following the suction drain removal and dressing, patient developed immediate radial nerve palsy along with wrist drop. We reviewed the literature and found no obvious cause for the nerve palsy and concluded that it was due to traction injury to the radial nerve while removing the suction drain in negative pressure.
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November 2015

Insights into structural mechanisms of gating induced regulation of aquaporins.

Prog Biophys Mol Biol 2014 Apr 1;114(2):69-79. Epub 2014 Feb 1.

Bioinformatics Center, Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160036, India.

Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.
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http://dx.doi.org/10.1016/j.pbiomolbio.2014.01.002DOI Listing
April 2014

Heavy metals bioconcentration from soil to vegetables and assessment of health risk caused by their ingestion.

Biol Trace Elem Res 2014 Mar 25;157(3):256-65. Epub 2014 Jan 25.

Centre for Radio-Ecology, Guru Jambheshwar University of Science and Technology, Hisar, 125001, Haryana, India,

The present study was undertaken to assess the non-carcinogenic human health risk of heavy metals through the ingestion of locally grown and commonly used vegetables viz. Raphanus sativus (root vegetable), Daucus carota (root vegetable), Benincasa hispida (fruit vegetable) and Brassica campestris leaves (leafy vegetable) in a semi-urbanized area of Haryana state, India. Heavy metal quantification of soil and vegetable samples was done using flame atomic absorption spectrophotometer. Lead, cadmium and nickel concentration in vegetable samples varied in range of 0.12-6.54 mg kg(-1), 0.02-0.67 mg kg(-1) and <0.05-0.41 mg kg(-1), respectively. Cadmium and lead concentration in some vegetable samples exceeded maximum permissible limit given by World Health Organization/Food and Agriculture Organization and Indian standards. Much higher concentrations of Pb (40-190.5 mg kg(-1)), Cd (0.56-9.85 mg kg(-1)) and Ni (3.21-45.87 mg kg(-1)) were reported in corresponding vegetable fields' soils. Correlation analysis revealed the formation of three primary clusters, i.e. Cu-Cd, Cd-Pb and Ni-Zn in vegetable fields' soils further supported by cluster analysis and principal component analysis. Bioconcentration factor revealed that heavy metals' uptake was more by leafy vegetable than root and fruit vegetables. Hazard index of all the vegetables was less than unity; thus, the ingestion of these vegetables is unlikely to pose health risks to the target population.
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http://dx.doi.org/10.1007/s12011-014-9892-zDOI Listing
March 2014

Phosphorylation of Ser-180 of rat aquaporin-4 shows marginal affect on regulation of water permeability: molecular dynamics study.

J Biomol Struct Dyn 2014 Apr 7;32(4):555-66. Epub 2013 May 7.

a Bioinformatics Center , CSIR-Institute of Microbial Technology, Council of Scientific and Industrial Research , Sector 39A, Chandigarh , 160036 , India .

Water permeation through rat aquaporin-4 (rAQP4), predominantly found in mammalian brain is regulated by phosphorylation of Ser-180. The present study has been carried out to understand the structural mechanism of regulation of water permeability across the channel. Molecular dynamics (MD) simulations have been carried out to investigate the structural changes caused due to phosphorylation of Ser-180 in the tetrameric assembly of rAQP4 along with predicted C-terminal region (255-323). The interactions involving opposite charges are observed between cytoplasmic loops and the C-terminal region during MD simulations. This results in movement of C-terminal region of rAQP4 towards the cytoplasmic mouth of water channel. Despite this movement, there was a gap between C-terminal region and cytoplasmic mouth of the channel through which water molecules were able to gain entry into the channel. The interactions between C-terminus and loop D of neighboring monomers in a tetrameric assembly appear to prevent the complete closure of cytoplasmic mouth of the water channel. Further, the rates of water permeation through phosphorylated and unphosphorylated rAQP4 have also been compared. The simulation studies showed a continuous movement of water in a single file across pore of unphosphorylated as well as phosphorylated rAQP4.
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http://dx.doi.org/10.1080/07391102.2013.780981DOI Listing
April 2014

Molecular characterization and expression analysis of a novel cystatin-like gene in a hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758].

Fish Shellfish Immunol 2013 Feb 12;34(2):683-7. Epub 2012 Dec 12.

National Bureau of Fish Genetic Resources (ICAR), Lucknow, UP, India.

A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.
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http://dx.doi.org/10.1016/j.fsi.2012.11.018DOI Listing
February 2013

Insufficient (sub-native) helix content in soluble/solid aggregates of recombinant and engineered forms of IL-2 throws light on how aggregated IL-2 is biologically active.

Protein J 2012 Oct;31(7):529-43

Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.

Interleukin 2 (IL-2) is an extremely aggregation-prone, all-alpha helical cytokine. In its receptor-bound state, ~72 % of the polypeptide chain adopts helical structure and there is no beta sheet content whatsoever. In the past, recombinant IL-2 has been formulated and used therapeutically in humans, following production in E. coli. Therapeutic IL-2 consists entirely of functionally-active soluble aggregates with ~30 subunits per aggregate particle. Side-effects attributed to aggregation resulted in discontinuation of usage over a decade ago. Structurally, and biochemically, activity in IL-2 aggregates can potentially be explained in one of two ways : (a) individual IL-2 chains exist in sterically-accessible, receptor binding-competent (native) structures, allowing aggregates to bind directly to IL-2 receptors (IL-2R); alternatively, (b) IL-2 chains dissociate from aggregates, become free to adopt native structure, and then bind to IL-2R. We produced native IL-2 and numerous engineered forms in E. coli with the objective of obtaining insights into these possibilities. Each IL-2 variant was subjected to size exclusion chromatography, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). All forms produced and studied (including those with native IL-2 sequences) turned out to aggregate and also display less than ~50 % helix content as well as significant beta sheet content. No conditions were found that obviate aggregation. Aggregated IL-2 is thus insufficiently native-like to bind to IL-2R. Activity in aggregates thus probably owes to adoption of receptor binding-competent structures by chains that have already dissociated from aggregates.
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http://dx.doi.org/10.1007/s10930-012-9429-2DOI Listing
October 2012

A temperature and salt-tolerant L-glutaminase from gangotri region of uttarakhand himalaya: enzyme purification and characterization.

Appl Biochem Biotechnol 2012 Apr 26;166(7):1723-35. Epub 2012 Feb 26.

Department of Biochemistry, Sardar Bhagwan Singh PG Institute of Biomedical Sciences and Research, Balawala, Dehradun 248161, India.

Purification and characterization of halotolerant, thermostable alkaline L-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. L-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0-11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1-20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. L-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K(m) and V(max) values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived L-glutaminase reported so far.
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http://dx.doi.org/10.1007/s12010-012-9576-0DOI Listing
April 2012

A single-amino-acid substitution in the C terminus of PhoP determines DNA-binding specificity of the virulence-associated response regulator from Mycobacterium tuberculosis.

J Mol Biol 2010 May 2;398(5):647-56. Epub 2010 Apr 2.

Institute of Microbial Technology (CSIR), Sector-39A, Chandigarh-160036, India.

The Mycobacterium tuberculosis PhoP-PhoR two-component system is essential for virulence in animal models of tuberculosis. Genetic and biochemical studies indicate that PhoP regulates the expression of more than 110 genes in M. tuberculosis. The C-terminal effector domain of PhoP exhibits a winged helix-turn-helix motif with the molecular surfaces around the recognition helix (alpha 8) displaying strong positive electrostatic potential, suggesting its role in DNA binding and nucleotide sequence recognition. Here, the relative importance of interfacial alpha 8-DNA contacts has been tested through rational mutagenesis coupled with in vitro binding-affinity studies. Most PhoP mutants, each with a potential DNA contacting residue replaced with Ala, had significantly reduced DNA binding affinity. However, substitution of nonconserved Glu215 had a major effect on the specificity of recognition. Although lack of specificity does not necessarily correlate with gross change in the overall DNA binding properties of PhoP, structural superposition of the PhoP C-domain on the Escherichia coli PhoB C-domain-DNA complex suggests a base-specific interaction between Glu215 of PhoP and the ninth base of the DR1 repeat motif. Biochemical experiments corroborate these results, showing that DNA recognition specificity can be altered by as little as a single residue change of the protein or a single base change of the DNA. The results have implications for the mechanism of sequence-specific DNA binding by PhoP.
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http://dx.doi.org/10.1016/j.jmb.2010.03.056DOI Listing
May 2010
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