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1.  Selective disruption of Rb-Raf-1 kinase interaction inhibits pancreatic adenocarcinoma growth irrespective of gemcitabine sensitivity 
Molecular cancer therapeutics  2013;12(12):10.1158/1535-7163.MCT-12-0719.
Inactivation of the Retinoblastoma tumor suppressor protein, (Rb), is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n=95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (p=0.0272), which correlated with reduced overall patient survival (p=0.0186). To define the anti-tumor effects of RRD-251 (50μM), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were performed on gemcitabine sensitive and resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (p< 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50mpk) alone or in combination with gemcitabine (250mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (p<0.01). Combination of RRD-251 with gemcitabine demonstrated cooperative effect on tumor growth (p< 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer.
doi:10.1158/1535-7163.MCT-12-0719
PMCID: PMC3858536  PMID: 24107447
Pancreatic cancer; Retinoblastoma tumor suppressor protein; Rb; Raf-1 kinase; RRD-251
2.  Evaluation of Protein Dihedral Angle Prediction Methods 
PLoS ONE  2014;9(8):e105667.
Tertiary structure prediction of a protein from its amino acid sequence is one of the major challenges in the field of bioinformatics. Hierarchical approach is one of the persuasive techniques used for predicting protein tertiary structure, especially in the absence of homologous protein structures. In hierarchical approach, intermediate states are predicted like secondary structure, dihedral angles, Cα-Cα distance bounds, etc. These intermediate states are used to restraint the protein backbone and assist its correct folding. In the recent years, several methods have been developed for predicting dihedral angles of a protein, but it is difficult to conclude which method is better than others. In this study, we benchmarked the performance of dihedral prediction methods ANGLOR and SPINE X on various datasets, including independent datasets. TANGLE dihedral prediction method was not benchmarked (due to unavailability of its standalone) and was compared with SPINE X and ANGLOR on only ANGLOR dataset on which TANGLE has reported its results. It was observed that SPINE X performed better than ANGLOR and TANGLE, especially in case of prediction of dihedral angles of glycine and proline residues. The analysis suggested that angle shifting was the foremost reason of better performance of SPINE X. We further evaluated the performance of the methods on independent ccPDB30 dataset and observed that SPINE X performed better than ANGLOR.
doi:10.1371/journal.pone.0105667
PMCID: PMC4148315  PMID: 25166857
3.  ParaPep: a web resource for experimentally validated antiparasitic peptide sequences and their structures 
ParaPep is a repository of antiparasitic peptides, which provides comprehensive information related to experimentally validated antiparasitic peptide sequences and their structures. The data were collected and compiled from published research papers, patents and from various databases. The current release of ParaPep holds 863 entries among which 519 are unique peptides. In addition to peptides having natural amino acids, ParaPep also consists of peptides having d-amino acids and chemically modified residues. In ParaPep, most of the peptides have been evaluated for growth inhibition of various species of Plasmodium, Leishmania and Trypanosoma. We have provided comprehensive information about these peptides that include peptide sequence, chemical modifications, stereochemistry, antiparasitic activity, origin, nature of peptide, assay types, type of parasite, mode of action and hemolytic activity. Structures of peptides consisting of natural, as well as modified amino acids have been determined using state-of-the-art software, PEPstr. To facilitate users, various user-friendly web tools, for data fetching, analysis and browsing, have been integrated. We hope that ParaPep will be advantageous in designing therapeutic peptides against parasitic diseases.
Database URL: http://crdd.osdd.net/raghava/parapep/
doi:10.1093/database/bau051
PMCID: PMC4054663  PMID: 24923818
5.  Neuroligins Provide Molecular Links Between Syndromic and Non-Syndromic Autism 
Science signaling  2013;6(283):re4.
Autism is a common and heritable neuropsychiatric disorder that can be categorized into two types: syndromic and non-syndromic, the former of which are associated with other neurological disorders or syndromes. Molecular and functional links between syndromic and non-syndromic autism genes were lacking until studies aimed at understanding role of trans-synaptic adhesion molecule neuroligin, which is associated with non-syndromic autism, provided important connections. Here, we integrate data from these studies into a model of how neuroligin functions to control synaptic connectivity in the central nervous system and how neuroligin dysfunction may participate in the pathophysiology of autism. Understanding the complex functional interactions between neuroligins and other autism-associated proteins at the synapse is crucial to understand the pathology of autism. This understanding might bring us closer to development of therapeutic approaches for autism.
doi:10.1126/scisignal.2004102
PMCID: PMC4000534  PMID: 23838185
6.  Health care in rural India: A lack between need and feed 
South Asian Journal of Cancer  2014;3(2):143-144.
doi:10.4103/2278-330X.130483
PMCID: PMC4014652  PMID: 24818117
7.  Innovative Technique of Vascular Repair in Intra-Operative IVC Rupture During Lumbar Microdiscectomy: A Case Report 
Archives of Trauma Research  2013;2(3):133-135.
Background
Major vascular injury during a spinal surgery is a rare but most dreaded complication.
Case Presentation
A 39 years old female undergoing microscopic lumbar discectomy suddenly developed severe hypotension on table. The procedure was abandoned and the patient turned supine. It was diagnosed to be a major vessel tear and the patient was taken up for immediate successful vascular repair. To best of our knowledge such a repair procedure has not been described in literature.
Conclusions
Majority of such vascular injuries are dealt with primary repair of the defect by a vascular surgeon; however in our case the rent was big and placed on the undersurface making it very difficult for the vascular surgeon to approach or repair it primarily.
doi:10.5812/atr.11005
PMCID: PMC3950917  PMID: 24693524
Microdiscectomy Complications; Vascular Injury; Inferior Vena Cava Rupture; Anastomosis; Vascular Bypass
8.  Hemolytik: a database of experimentally determined hemolytic and non-hemolytic peptides 
Nucleic Acids Research  2013;42(Database issue):D444-D449.
Hemolytik (http://crdd.osdd.net/raghava/hemolytik/) is a manually curated database of experimentally determined hemolytic and non-hemolytic peptides. Data were compiled from a large number of published research articles and various databases like Antimicrobial Peptide Database, Collection of Anti-microbial Peptides, Dragon Antimicrobial Peptide Database and Swiss-Prot. The current release of Hemolytik database contains ∼3000 entries that include ∼2000 unique peptides whose hemolytic activities were evaluated on erythrocytes isolated from as many as 17 different sources. Each entry in Hemolytik provides comprehensive information about a peptide, like its name, sequence, origin, reported function, property such as chirality, types (linear and cyclic), end modifications as well as details pertaining to its hemolytic activity. In addition, tertiary structure of each peptide has been predicted, and secondary structure states have been assigned. To facilitate the scientific community, a user-friendly interface has been developed with various tools for data searching and analysis. We hope, Hemolytik will be useful for researchers working in the field of designing therapeutic peptides.
doi:10.1093/nar/gkt1008
PMCID: PMC3964980  PMID: 24174543
9.  Historical perspective of Indian neurology 
Objective:
To chronicle the history of medicine and neurology in India with a focus on its establishment and evolution.
Background:
The history of neurology in India is divided into two periods: ancient and modern. The ancient period dates back to the mid-second millennium Before Christ (B.C.) during the creation of the Ayurvedic Indian system of Medicine, which detailed descriptions of neurological disorders called Vata Vyadhi. The early 20th century witnessed the birth of modern Indian medicine with the onset of formal physician training at the nation's first allopathic medical colleges located in Madras (1835), Calcutta (1835) and Mumbai (1848). Prior to India's independence from Britain in 1947, only 25 medical schools existed in the entire country. Today, there are over 355. In 1951, physicians across the field of neurology and neurosurgery united to create the Neurological Society of India (NSI). Four decades later in 1991, neurologists branched out to establish a separate organization called the Indian Academy of Neurology (IAN).
Design/Methods:
Information was gathered through literature review using PubMed, MD Consult, OVID, primary texts and research at various academic institutions in India.
Results:
Neurological disorders were first described in ancient India under Ayurveda. The transition to modern medicine occurred more recently through formal training at medical schools beginning in the 1930's. Early pioneers and founders of the NSI (1951) include Dr. Jacob Chandy, Dr. B Ramamurthi, Dr. S. T. Narasimhan and Dr. Baldev Singh. Later, Dr. J. S. Chopra, a prominent neurologist and visionary, recognized the need for primary centers of collaboration and subsequently established the IAN (1991). The future of Neurology in India is growing rapidly. Currently, there are 1100 practicing neurologists and more than 150 post-graduate trainees who join the ranks every year. As the number of neurologists rises across India, there is an increase in the amount of basic, clinical and epidemiological research being conducted across the country every day.
Conclusions:
The history of neurology in India roots back to its rich culture and tradition. Over time, there has been great structural and organizational evolution and the future of neurology in India appears to be bright. However, the number of neurologists and research in neurology needs to experience a significant growth in the future to ensure the best patient care.
doi:10.4103/0972-2327.120422
PMCID: PMC3841583  PMID: 24339562
Ayurveda; dementia; Epilepsy; history; stroke
10.  Effect of chlorine dioxide and sodium hypochlorite on the dissolution of human pulp tissue – An in vitro study 
Background
Organic tissue dissolution is an important property of an irrigant which aids in the success of root canal treatment. Recent studies have advocated the use of Chlorine dioxide as an endodontic irrigant. The aim of this study is to compare the dissolution efficacy of chlorine dioxide and sodium hypochlorite on human pulp tissue.
Methods
In this study, 2% Sodium hypochlorite, 5% Chlorine dioxide and isotonic saline solution (control) were used. Thirty human pulp tissue specimens were exposed to three test solutions (n = 10) for 30 min following which the loss of weight was compared from the original weight by using a digital analytical balance.
Results
Sodium hypochlorite was more efficient in dissolving human pulp tissue when compared to Chlorine dioxide. Isotonic saline solution failed to dissolve any of the specimens.
Conclusion
5% Chlorine dioxide is capable of dissolving human pulp tissue but sodium hypochlorite was more effective.
doi:10.1016/j.mjafi.2011.11.003
PMCID: PMC3862608  PMID: 24532904
Sodium hypochlorite; Chlorine dioxide; Pulp tissue dissolution
13.  Neuroprotective Role of a Novel Copper Chelator against Aβ42 Induced Neurotoxicity 
Alzheimer's disease (AD) is a progressive neurodegenerative disease and associated with the extracellular deposits of amyloid-β peptide in hippocampus region. Metal ions like Cu, Fe and Zn are known to associate with the amyloid beta (Aβ) at high concentration and interaction of these ions with soluble and aggregated forms of Aβ peptide help in development of AD. Here we showed Cu mediated neurotoxicity in the eye tissues of transgenic Drosophila expressing human amyloid β and its rescue through a novel Cu chelator. In this context, we have synthesised and characterized the compound L 2,6-Pyridinedicarboxylic acid, 2,6-bis[2-[(4-carboxyphenyl) methylene] hydrazide] by Mass spectra (MS) and Elemental analysis (EA). The Cu chelation potential of the compound L is tested in vivo in Drosophila. Oral administration of Copper to the transgenic larvae resulted in severe degeneration in eye tissues, which was rescued by the supplementation of compound L. The levels of anti-oxidant markers like SOD and MDA were measured in compound L treated flies and found a significant rescue (P < 0.001). Further rescue of the eye degeneration phenotypes as revealed by SEM affirm the role of copper in Aβ toxicity. Hence, use of compound L, an amidoamine derivative, could be a possible therapeutic measure for Aβ induced neurotoxicity.
doi:10.1155/2013/567128
PMCID: PMC3789492  PMID: 24159420
14.  An Evaluation of Mass Drug Administration Compliance Against Filariasis of Tikamgarh District of Madhya Pradesh-A Household-Based Community Study 
Background:
Mass drug administration (MDA) means once-in-a-year administration of diethyl carbamazine (DEC) tablet to all people (excluding children under 2 years, pregnant women and severely ill persons) in identified endemic areas. It aims at cessation of transmission of lymphatic filariasis.
Objective:
To study the coverage and compliance of MDA in Tikamgarh district during the campaign in April 2010.
Materials and Methods:
The activities under MDA involved administration of DEC tablets to eligible population from endemic area by health staff and Integrated Child Development Scheme (ICDS) functionaries referred as drug distributors (DD) make house-to-house visits on select dates in 2010. DEC was administered to all people (excluding children under 2 years, pregnant women and severely ill persons) with the instruction to ingest the tablet preferably on the spot.
Study Design:
Cross-sectional population based house-to-house visit.
Setting:
Urban and rural areas in Tikamgarh district identified as endemic for filariasis where MDA 2010 was undertaken.
Study Variables:
Exploratory - Rural and urban clusters of Tikamgarh district; Outcome - coverage, compliance, actual coverage, side effects.
Analysis:
Percentage and proportions.
Results:
Four clusters, each comprising 30 households from Tikamgarh endemic district, yielded an eligible population of 641. The coverage rate was 607 (94.6% of eligible) with variation across different areas. The compliance with drug ingestion was 89.9% with a gap of 10.1% to be targeted by intensive IEC. The effective coverage (85.2%) was just above the target (85%). Side effects of DEC were minimum, transient and drug-specific. Overall coverage was marginally better in rural areas. The causes of poor coverage and compliance have been discussed and relevant suggestions have been made.
doi:10.4103/2249-4863.117395
PMCID: PMC3894026  PMID: 24479075
Diethyl carbamazine; lymphatic filariasis; Mass drug administration
15.  βArrestin-1 and Mcl-1 Modulate Self-Renewal Growth of Cancer Stem-Like Side-Population Cells in Non-Small Cell Lung Cancer 
PLoS ONE  2013;8(2):e55982.
Side population (SP) cells have been reported to have properties of cancer stem-like cells (CSCs) in non-small cell lung carcinoma (NSCLC), yet their molecular features have not been fully elucidated. Here we show that, NSCLC-SP cells were enriched in G0/G­1 phase of cell cycle, had higher aldehyde dehydrogenase activity as well as higher clonogenic and self-renewing ability compared to main population (MP) cells. Interestingly, SP cells were also able to trans-differentiate into angiogenic tubules in vitro and were highly tumorigenic as compared to MP cells. SP-derived tumors demonstrated the intratumoral heterogeneity comprising of both SP and MP cells, suggesting the self-renewal and differentiation ability of SP cells are manifested in vivo as well. βArrestin-1 (βArr1) is involved in the progression of various cancers including NSCLCs and we find that depletion of βArr1 significantly blocked the SP phenotype; whereas depletion of βArr2 had relatively minor effects. Ectopic expression of βArr1 resulted in increased SP frequency and ABCG2 expression while abrogation of βArr1 expression suppressed the self-renewal growth and expansion of A549 cells. Anti-apoptotic protein Mcl-1 is known to be one of the key regulators of self-renewal of tissue stem cells and is thought to contribute to survival of NSCLC cells. Our experiments show that higher levels of Mcl-1 were expressed in SP cells compared to MP cells at both transcriptional and translational levels. In addition, Obatoclax, a pharmacological inhibitor of Mcl-1, could effectively prevent the self-renewal of both EGFR-inhibitor sensitive and resistant NSCLC cells. In conclusion, our findings suggest that βArr1 and Mcl-1 are involved in the self-renewal and expansion of NSCLC-CSCs and are potential targets for anti-cancer therapy.
doi:10.1371/journal.pone.0055982
PMCID: PMC3572139  PMID: 23418490
16.  Nicotine-mediated induction of E-selectin in aortic endothelial cells requires Src kinase and E2F1 transcriptional activity 
Smoking is highly correlated with enhanced likelihood of atherosclerosis by inducing endothelial dysfunction. In endothelial cells, various cell-adhesion molecules including E-selectin, are shown to be upregulated upon exposure to nicotine, the addictive component of tobacco smoke; however, the molecular mechanisms underlying this induction are poorly understood. Here we demonstrate that nicotine induced E-selectin transcription in human aortic endothelial cells (HAECs) could be significantly blocked by α7-nAChR subunit inhibitor, α-BT, Src-kinase inhibitor, PP2, or siRNAs against Src or β-Arrestin-1 (β-Arr1). Further, chromatin immunoprecipitations show that E-selectin is an E2F1 responsive gene and nicotine stimulation results in increased recruitment of E2F1 on E-selectin promoter. Inhibiting E2F1 activity using RRD-251, a disruptor of the Rb-Raf-1 kinase interaction, could significantly inhibit the nicotine induced recruitment of E2F1 to the E-selectin promoter as well as E-selectin expression. Interestingly, stimulation of HAECs with nicotine results in increased adhesion of U937 monocytic cells to HAECs and could be inhibited by pre-treatment with RRD-251. Similarly, depletion of E2F1 or Src using RNAi blocked the increased adhesion of monocytes to nicotine stimulated HAECs. These results suggest that nicotine stimulated adhesion of monocytes to endothelial cells is dependent on the activation of α7-nAChRs, β-Arr1 and cSrc regulated increase in E2F1-mediated transcription of E-selectin gene. Therefore, agents such as RRD-251 that can target activity of E2F1 may have potential therapeutic benefit against cigarette-smoke induced atherosclerosis.
doi:10.1016/j.bbrc.2011.12.127
PMCID: PMC3273677  PMID: 22240023
E-selectin; atherosclerosis; monocyte adhesion; RRD-251; β-arrestin-1; Src; Rb
17.  Comparative evaluation of 15% ethylenediamine tetra-acetic acid plus cetavlon and 5% chlorine dioxide in removal of smear layer: A scanning electron microscope study 
Aims:
The purpose of this study was to compare the efficacy of smear layer removal by 5% chlorine dioxide and 15% Ethylenediamine Tetra-Acetic Acid plus Cetavlon (EDTAC) from the human root canal dentin.
Materials and Methods
Fifty single rooted human mandibular anterior teeth were divided into two groups of 20 teeth each and control group of 10 teeth. The root canals were prepared till F3 protaper and initially irrigated with 2% Sodium hypochlorite followed by 1 min irrigation with 15% EDTAC or 5% Chlorine dioxide respectively. The control group was irrigated with saline. The teeth were longitudinally split and observed under Scanning electron microscope SEM (×2000).
Statistical Analysis Used:
The statistical analysis was done using General Linear Mixed Model.
Results:
At the coronal thirds, no statistically significant difference was found between 15% EDTAC and 5% Chlorine dioxide in removing smear layer. In the middle and apical third region 15% EDTAC showed better smear layer removal ability than 5% Chlorine dioxide.
Conclusion:
Final irrigation with 15% EDTAC is superior to 5% chlorine dioxide in removing smear layer in the middle and apical third of radicular dentin.
doi:10.4103/0976-237X.111624
PMCID: PMC3703697  PMID: 23853455
Chlorine dioxide; ethylenediamine tetra-acetic acid plus cetavlon; smear layer removal
18.  Percutaneous Transvenous Angioplasty of Left Innominate Vein Stenosis Following Right Side Permanent Pacemaker Implantation- A Left Femoral Vein to Left Axillary Vein Approach 
Central venous stenosis after the insertion of a permanent pacemaker is a well recognized complication. This late complication is encountered when there is a need to change the pacemaker lead or extract it. We describe a young male who had such a complication after many years after right side pacemaker implantation. The lesion was managed percutaneously leading to placement of a new lead from the left side.
PMCID: PMC3513405  PMID: 23233760
Percutaneous Transvenous Angioplasty; Innominate Vein Stenosis; Permanent Pacemaker Implantation
19.  Lupus Nephritis. A Retrospective Analysis of Clinical Presentation and Outcomes from a single center 
Background and Objectives
Lupus nephritis (LN) is an ominous complication of Systemic lupus erythematosus (SLE) and the risk factors for the disease progression are not very well characterized.
Design, Setting, Participants and measurements
In a retrospective study, we evaluated the mode of presentation and outcomes of 163 consecutive patients with biopsy proven LN, who presented to our center between January 1999 and September 2008. Using stepwise logistic regression analysis we assessed risk factors independently associated with response to treatment as well as to progression to end stage renal disease (ESRD) in proliferative LN (PLN).
Results
Ninety percent of our patients belonged to minority population. Among 122 patients with class III and IV LN (PLN), 76 patients received intravenous cyclophosphamide (IVC) and 38 mycophenolate for induction while 34 patients received IVC, and 63 mycophenolate for maintenance. Thirty six (30%) patients with PLN progressed to ESRD and 3 patients died over a mean follow-up of 37.5 months. On multivariate analysis, chronicity index (CI) (p=0.0007) and hypertension (p=0.042) positively correlated with progression to ESRD and death and CI was associated with increased probability of non-response to treatment (p=0.001). Additionally, mycophenolate as maintenance agent was associated with increased likelihood of sustained complete remission and partial remission [p=0.045].
Conclusions
In patients with LN, Hypertension and a high CI are independent risk factors for progression to ESRD or death. Furthermore, a high CI is associated with poor response and mycophenolate as a maintenance agent may improve the response to treatment.
doi:10.1097/MAJ.0b013e3182199214
PMCID: PMC3176993  PMID: 21681076
lupus nephritis; outcomes; lupus; SLE; LN
20.  Nicotine Induces Inhibitor of Differentiation-1 in a Src-dependent Pathway Promoting Metastasis and Chemoresistance in Pancreatic Adenocarcinoma1 
Neoplasia (New York, N.Y.)  2012;14(12):1102-1114.
Smoking is a significant risk factor for pancreatic cancer, but the molecular mechanisms by which tobacco smoke components promote the growth and progression of these cancers are not fully understood. While nicotine, the addictive component of tobacco smoke, is not a carcinogen, it has been shown to promote the growth of non-small cell lung and pancreatic cancers in a receptor-dependent fashion. Here, we show that stimulation of pancreatic cancer cells with nicotine concentrations that are within the range of human exposure results in activation of Src kinase, which facilitated the induction of the inhibitor of differentiation-1 (Id1) transcription factor. Depletion of Id1 prevented nicotine-mediated induction of proliferation and invasion of pancreatic cancer cells, indicating that it is a major mediator of nicotine function. Nicotine could promote the growth and metastasis of pancreatic cancers orthotopically implanted into SCID mice; in addition, cells stably expressing a short hairpin RNA for Id1 did not grow or metastasize in response to nicotine. Nicotine could also confer resistance to apoptosis induced by gemcitabine in pancreatic cancer cells in vitro and depletion of Src or Id1 rendered the cells sensitive to gemcitabine. Further, nicotine could effectively inhibit the chemotherapeutic effects of gemcitabine on pancreatic tumors xenografted into mice. Clinical analyses of resected pancreatic cancer specimens demonstrated a statistically significant correlation between Id1 expression and phospho-Src, tumor grade/differentiation, and worsening overall patient survival. These results demonstrate that exposure to tobacco smoke components might promote pancreatic cancer progression, metastasis, and chemoresistance and highlight the role of Id1 in these processes.
PMCID: PMC3540937  PMID: 23308043
21.  Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N 
PLoS ONE  2012;7(11):e49291.
The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.
doi:10.1371/journal.pone.0049291
PMCID: PMC3493545  PMID: 23145144
22.  Is serum retinol binding protein-4: A predictor for diabetes in genetically high risk population? 
Background:
Retinol binding protein-4 (BP-4) a new adipocytokine, specifically binds to retinol, through experimental studies, reported its link between obesity and insulin resistance (IR). But till date no studies are available on influence of genetic predisposition of diabetes on RBP-4 expression. Hence, we aimed to study the influence of genetic predisposition of diabetes on the serum RBP-4 and its role in development of IR and diabetes in genetically high risk population.
Materials and Methods:
Healthy non diabetic individuals (age 18 to 22) were grouped into Group I: Control (n = 81), whose parents are non diabetic, non hypertensive and does not have any family history of coronary heart diseases. Group II: (n = 157) with one of their parents diabetic and Group III: (n = 47) with both parents diabetic. In all the participants, we estimated fasting serum RBP-4, insulin and glucose. Homeostasis model for assessment-insulin resistance (HOMA-IR) and homeostasis model for assessment-beta cell dysfunction (HOMA-B) were calculated from fasting serum insulin and glucose levels.
Results:
In this study, we observed significantly higher RBP-4 levels 12.71 ± 2.3 in Group-II and 13.25 ± 2 in Group-III, respectively when compared to Group-I 11.4 ± 1.8 (P < 0.01). RBP-4 showed a significantly strong positive correlation with plasma insulin, glucose and HOMA-IR in genetically high risk population (group II and III) P < 0.01. Linear regression analysis revealed a strong positive association of RBP-4 with parental diabetes even after adjusting for BMI, age and sex (OR 1.53, 95% CI 1.089-1.40).
Conclusion:
Higher serum RBP-4 and its positive correlation with Insulin, glucose, and HOMA-IR in healthy non diabetic participants of genetically high risk population, indicating its role as predictor for the onset of diabetes in coming future.
PMCID: PMC3702081  PMID: 23833574
Adipocytokines; diabetes; insulin resistance; retinol binding protein-4
23.  EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer 
Molecular Cancer  2012;11:73.
Background
Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling.
Results
SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples.
Conclusions
Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.
doi:10.1186/1476-4598-11-73
PMCID: PMC3497614  PMID: 23009336
Cancer stem-like cells; Side-population cells; Self-renewal; EGFR; Sox2
24.  Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment 
BMC Molecular Biology  2012;13:28.
Background
GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription.
Results
The present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse β-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment.
Conclusion
We propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.
doi:10.1186/1471-2199-13-28
PMCID: PMC3459802  PMID: 22978699
SMAR1; Diabetes; GAD65; p53; Streptozotocin
25.  A Novel Five Gene Signature Derived from Stem-Like Side Population Cells Predicts Overall and Recurrence-Free Survival in NSCLC 
PLoS ONE  2012;7(8):e43589.
Gene expression profiling has been used to characterize prognosis in various cancers. Earlier studies had shown that side population cells isolated from Non-Small Cell Lung Cancer (NSCLC) cell lines exhibit cancer stem cell properties. In this study we apply a systems biology approach to gene expression profiling data from cancer stem like cells isolated from lung cancer cell lines to identify novel gene signatures that could predict prognosis. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stem like properties. Differentially expressed genes that were over or under-expressed at least two fold commonly in all 4 cell lines were identified. We found 354 were upregulated and 126 were downregulated in SP cells compared to MP cells; of these, 89 up and 62 downregulated genes (average 2 fold changes) were used for Principle Component Analysis (PCA) and MetaCore™ pathway analysis. The pathway analysis demonstrated representation of 4 up regulated genes (TOP2A, AURKB, BRRN1, CDK1) in chromosome condensation pathway and 1 down regulated gene FUS in chromosomal translocation. Microarray data was validated using qRT-PCR on the 5 selected genes and all showed robust correlation between microarray and qRT-PCR. Further, we analyzed two independent gene expression datasets that included 360 lung adenocarcinoma patients from NCI Director's Challenge Set for overall survival and 63 samples from Sungkyunkwan University (SKKU) for recurrence free survival. Kaplan-Meier and log-rank test analysis predicted poor survival of patients in both data sets. Our results suggest that genes involved in chromosome condensation are likely related with stem-like properties and might predict survival in lung adenocarcinoma. Our findings highlight a gene signature for effective identification of lung adenocarcinoma patients with poor prognosis and designing more aggressive therapies for such patients.
doi:10.1371/journal.pone.0043589
PMCID: PMC3430700  PMID: 22952714

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