Rakesh Kumar Sharma - Academia.edu (original) (raw)

Papers by Rakesh Kumar Sharma

Journal of proteomics, Jan 5, 2015

Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gas... more Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular ...

Journal of Proteome Research, 2015

Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads to permanent phy... more Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads to permanent physical and neurological disabilities in adults worldwide. Proteins associated with stroke pathogenesis may appear in the serum of AIS patients due to blood−brain barrier dysfunction, thus permitting the development of blood-based biomarkers for early diagnosis of stroke. These biomarkers could perhaps be an adjunct to the existing imaging modalities and aid in better management and therapeutic intervention during the course of the disease. For this exploratory study, a combination of multiplexed isobaric tagging using iTRAQ reagents and high resolution tandem mass spectrometry was used to identify differentially expressed proteins in serum samples from AIS patients. The quantitative proteomic analysis of serum from both AIS and control subjects revealed 389 high confidence protein identifications and their relative levels. Among them, 60 proteins showed a ≥1.5-fold change in the AIS subjects. We verified the altered serum levels of candidate proteins such as vWF, ADAMTS13, S100A7, and DLG4 through ELISA, and the results also corroborate with the experimental findings. vWF and ADAMTS13 are key players that regulate blood hemostasis, and their altered concentration may contribute to endothelial dysfunction. S100A7 is a novel candidate protein identified in this study that is also known to mediate inflammation, endothelial proliferation, and angiogenesis. The current study provided a potential and novel biomarker panel that may in turn provide diagnostic aid to the existing imaging modalities for the rapid diagnosis of ischemic stroke a Patient and control results are expressed as mean (SD) for each parameter. Comparisons were made by unpaired t test. Values of p <0.05 are statistically significant.

OH (Presented by Dr. Sharma) INTRODUCTION AND OBJECTIVES: We developed a new measure of oxidative... more OH (Presented by Dr. Sharma) INTRODUCTION AND OBJECTIVES: We developed a new measure of oxidative stress called ROS-TAC score and also reduced 9 sperm characteristics into 2 semen scores. The purpose of this study was to determine if the ROS-TAC and semen scores can be used to quantify the semen quality in patients with various diagnoses of infertility.

OH (Presented by Dr. Gil-Guzman) Reactive oxygen species (ROS) have been implicated to play an im... more OH (Presented by Dr. Gil-Guzman) Reactive oxygen species (ROS) have been implicated to play an important role in male infertility. ROS measurement by chemiluminescence assay using luminol as the probe is a simple and accurate method. The current protocols for ROS measurement are unclear about the optimum time period when the measurement is to be done following the specimen production. In addition, the optimum sperm concentration for the chemiluminescence assay is not defined. A concentration of 20 X 10 6 used generally for measuring ROS cannot be obtained in oligospermic patients. The purpose of our study was to examine the effect of time (age of the sample), and sperm concentration on ROS generation.

The presence of high concentrations of leukocytes in semen has been associated with ROS-induced s... more The presence of high concentrations of leukocytes in semen has been associated with ROS-induced sperm damage, impaired sperm function, and male infertility. However, the mechanism(s) responsible for these effects are not clearly understood. Sperm DNA damage in ejaculated sperm has recently been reported as an indicator of alterations in the regulation of spermiogenesis and of poor pregnancy outcome in IVF. The purpose of this study was to determine the extent of DNA damage in subsets of human spermatozoa at different stages of maturation obtained from leukocytospermic and from leukocyte-free semen samples. Methods: Semen samples obtained from infertile patients with leukocytospermia (n =10) (group 1), patients with abnormal semen parameters and leukocyte-free samples (n = 16) (group 2), and healthy donors with leukocyte-free semen samples (n = 16) (group 3), were fractionated using a three-step discontinuous ISolate gradient (47%, 70%, and 90%). Each fraction was evaluated for sperm concentration, motility, morphology, and DNA structure by the sperm chromatin structure assay (COMP at ). Values are expressed as the mean ± standard deviation. Results: Percent recovery of immature germ cells (seminal plasma/47% interface) and immature sperm (47%/70% interface) was highest in group 1 and lowest in group 3 (P <0.001). The recovery of morphologically normal, mature sperm (90% ISolate) was highest in group 3 and lowest in group 1 (P <0.0001). DNA damage in mature sperm from groups 1, 2, and 3, as measured by COMP at , was 49 ± 23, 27.6 ± 17, and 9.8 ± 7.1, respectively. Differences between groups 1 and 3 were statistically significant (P <0.001). Although the mean COMP at value was higher in group 1 than in group 2, these differences did not reach statistical significance. No significant correlation was found between DNA damage in mature sperm and age of semen sample.

There is a growing concern regarding the potential detrimental effects of sperm processing on spe... more There is a growing concern regarding the potential detrimental effects of sperm processing on sperm DNA integrity. We examined the extent of DNA damage in infertile patients with abnormal semen characteristics using two sperm preparation methods, the results were compared with a population of healthy controls. Methods: Semen samples were collected from 22 infertile men and 21 normal healthy male volunteers. These samples were processed by a 2-layer density gradient technique (ISolate) and the swim-up method. Sperm characteristics such as motility, production of reactive oxygen species (ROS) by chemiluminescence assay, and DNA damage by sperm chromatin structure assay (SCSA) were evaluated both before and after processing by each method as well as between the patients and donors. Values are expressed as median and 25%, 75% interquartile range. Results: Patient samples before sperm preparation showed poor motility (P <0.0001), abnormal ROS (P <0.02), and a high amount of DNA damage [25.9 (21.3, 45.4)] compared to controls [19.4 (13.6, 25.15); p <0.02]. Both sperm preparation techniques were equally effective in improving sperm motility (P <0.0001) and lowering ROS levels (P <0.04). Semen samples prepared by the swim-up technique had significantly lower amounts of DNA damage compared to those prepared by Isolate (P <0.02); as well as to original unprocessed semen samples (P <0.01). Conclusions: Semen samples processed by swim-up technique show minimal amounts of DNA damage compared to those prepared by density gradient media such as ISolate. Since DNA damage is linked to the potential risk of transmission of genetic anomalies to the offspring and according to the latest reports sperm DNA damage of >30% by SCSA technique is predictive of failure of fertilization by in vitro fertilization method. In view of our results, we conclude that laboratories using density gradient media in sperm preparation for assisted reproduction should monitor those patients who fail to fertilize in the presence of normal egg and semen quality. Perhaps in these patients, sperm chromatin damage may be an underlying cause of fertilization failure; and, therefore, these patients could be tested for sperm DNA damage and appropriately counseled about the poor outcome by IVF and offered alternate therapeutic options.

Semen analysis is the primary tool to assess the status of infertile men. However, a subset of me... more Semen analysis is the primary tool to assess the status of infertile men. However, a subset of men with normal semen parameters may still be infertile and are classified as idiopathic. Additional tests to determine the cause of infertility include measurement of reactive oxygen species (ROS) levels and sperm DNA damage. DNA damage in sperm has been recently reported as a very sensitive indicator of abnormal spermiogenesis and poor pregnancy outcome. In this study, we evaluated semen parameters, ROS production and DNA damage in healthy donors and in patients examined for infertility. Methods: Semen samples were collected from 90 infertile men and 34 normal healthy male volunteers. Samples were centrifuged and resuspended in PBS medium. ROS production was measured by the chemiluminescence assays in the resulting sperm suspensions. DNA damage was assessed by the sperm chromatin structure assay in neat semen and results expressed as percent COMP at values. Chi-square tests were used for statistical hypothesis testing. Results: Although samples with abnormal semen parameters were more likely to be associated with elevated ROS levels than samples with normal semen parameters (66% versus 37%; p=0.01), this relationship was not found between semen parameters and sperm DNA damage (28% versus 33%; p=0.65). Also, elevated ROS levels per se were not significantly associated with increased DNA damage (p=0.21). Of the 90 patients, 27 (30%) had normal concentration, motility, and WHO morphology levels. Among these patients with normal semen parameters, 17 (63%) had ROS levels >1 million cpm and COMP at values >30%. However, only 1 (4%) patient with normal semen parameters had these values. Also, of the 63 patients with at least one abnormal semen parameter, only 11 (17%) had both elevated ROS and COMP at . Of the 30 donor samples with normal semen parameters, 5 (20%) had ROS levels >1 million cpm, 2 (7%) had COMP at values >30%, and none had both. Conclusions: Normal standard semen parameters are not always indicative of normal sperm function. The results of this study indicate that significant DNA damage, as measured by the SCSA assay, can be found in sperm from samples with normal semen parameters. In contrast, ROS production by washed spermatozoa was correlated with abnormal semen parameters. Therefore, patients diagnosed with unexplained male infertility should be evaluated for sperm DNA damage.

Introduction and Objective: Recent studies have shown differential levels of apoptosis in subsets... more Introduction and Objective: Recent studies have shown differential levels of apoptosis in subsets of spermatozoa at different stages of maturation. Caspase activity (aCP), disruption of mitochondrial membrane potential (∆Ψm), and DNA fragmentation are integral parts of apoptosis. This study was designed to estimate the aCp, ∆Ψm, DNA fragmentation, and their interrelation in subsets of ejaculated human spermatozoa.

Introduction and Objective: Cryopreservation (CRP) of semen is associated with a significant dama... more Introduction and Objective: Cryopreservation (CRP) of semen is associated with a significant damage to spermatozoa and might be related to apoptosis. Caspase activity (aCp), disruption of mitochondrial membrane potential (∆Ψm), and DNA fragmentation are integral parts of apoptosis. This study aims at evaluating the differences in the levels of aCp, ∆Ψm and DNA fragmentation in the fresh and CRP sperm.

Introduction and Objectives: Semen characteristics measured in a semen analysis are often correla... more Introduction and Objectives: Semen characteristics measured in a semen analysis are often correlated, indicating that underlying measures of semen quality can be used to reduce the number of variables evaluated. The purpose of our study was to compare the semen quality and the seminal oxidative stress (ROS-TAC score) in men with idiopathic infertility with normal donors (controls), and to a known group of fertile and infertile men treated for their infertility. Methods: Principal component analysis was applied to nine semen characteristics (concentration, motility, morphology, and sperm motion characteristics assessed by computer assisted semen analyzer) to provide a standardized score in 36 men with idiopathic infertility and 19 controls attending our infertility clinic. Reactive oxygen species (ROS) production and total antioxidant capacity TAC) was measured by chemiluminescence assays. ROS-TAC score was formulated using principal components to predict fertility potential in these men. A logistic regression analysis comparing the fertile (n = 13) and infertile (n = 39) men (treated male factor cases) was used to provide estimates of fertility based on the ROS-TAC score. Results: Compared to controls, patients with idiopathic infertility had significantly lower sperm concentration (37.53 ± 6.89 vs. 69.4 ± 10.03; P <0.009), sperm motility (37.93 ± 3.36 vs. 55.5 ± 4.8; P <0.003), and normal morphology (30.39 ± 2.23 vs. 39.8 ± 3.2; P <0.02). The idiopathic group had lower semen quality scores (83.0 ± 14.5) than controls (100.0 ± 10.0; P <0.004). The ROS level was higher in men with idiopathic infertility (2.3 ± 0.21) compared to controls (1.3 ± 0.3; P = 0.006); whereas, the TAC level was lower in men with idiopathic infertility (1014.75 ± 79.22) compared to controls (1653 ± 115.29; P = 0.001). In addition, idiopathic infertility patients had significantly lower ROS-TAC scores (32.8 ± 14.2) than controls (50.0 ± 10.0; P<0.0001). An estimated 64% of men with idiopathic infertility will remain infertile during one-year follow-up based on logistic regression analysis. Conclusions: Patients with idiopathic infertility have lower scores of semen quality and ROS-TAC compared to controls. Sixty-four percent of men with idiopathic infertility tend to remain infertile within one year. Both semen score and ROS-TAC scores provide important information about the semen quality and fertilizing potential, this information may be used in the medical managmeent of infertile patients with idiopathic etiologies.

Introduction and Objective: Oxidative stress, as a result of elevated levels of reactive oxygen s... more Introduction and Objective: Oxidative stress, as a result of elevated levels of reactive oxygen species (ROS) and depressed levels of antioxidants is associated with male infertility. Increased levels of cytokines are also reported in the seminal plasma of infertile men. We assessed semen characteristics, levels of ROS, non-enzymatic total antioxidant capacity (TAC), a composite ROS-TAC score, and interleukin-6 (IL-6) in infertile patients with varicocele and fertile and infertile patients after a vasectomy reversal procedure and compared them to normal healthy men.

FEMS microbiology letters, Jan 10, 2015

Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. ... more Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. We report the draft genome sequence of T. rubrum var. raubitschekii from Delhi, India, isolated from a patient presenting symptoms of onychomycosis. The total estimated genome size of the clinical isolate is 25.2 MB containing 8265 predicted protein-coding sequences, 91 tRNA and 15 rRNA genes. Sequence analysis of the secreted subtilases, one of the major virulence factors in dermatophytes, clusters them into three subfamilies with distinct sequence features. The genome sequence is a step in understanding diversity of dermatophytes worldwide and will aid in identification of virulence factors and dissecting mechanisms of pathogenesis among them.

Journal of the American Chemical Society, Jan 17, 2010

Monosubstituted acyclic (E)-1,3-dienes undergo efficient hydrovinylation giving (Z)-3-alkylhexa-1... more Monosubstituted acyclic (E)-1,3-dienes undergo efficient hydrovinylation giving (Z)-3-alkylhexa-1,4-dienes upon treatment with catalytic amounts of bidentate phosphine-CoCl(2) complexes {[P~P](CoCl(2))} and Me(3)Al in an atmosphere of ethylene. The regioselectivity of the reaction (i.e., 1,4- or 1,2-addition) depends on the nature of the ligand and temperature at which the reaction is carried out. Complexes derived from (RR)-DIOP and (SS)-BDDP at -45 degrees C give very high enantioselectivities for several prototypical 1,3-dienes. In sharp contrast to the corresponding Ni(II)-catalyzed hydrovinylation, 1-aryl-substituted 1,3-dienes give almost exclusively achiral linear 1,4-addition products, unless the 2-position is also substituted. Solid-state structures of the precatalysts are presented.

Archives of neurology, 2011

Although traditionally regarded as spared, a range of oculomotor dysfunction has been recorded in... more Although traditionally regarded as spared, a range of oculomotor dysfunction has been recorded in patients with amyotrophic lateral sclerosis (ALS). Most frequent is ophthalmoparesis, particularly in patients with prolonged survival; however, pursuit, nystagmus, and saccadic impairments have also been reported. The apparent resistance to pathologic involvement of oculomotor (and sphincter) control pathways in most patients with ALS has prompted comparative study to establish the key pathways that underlie motor neuronal vulnerability, with the hope of generating novel therapeutic strategies. Developments in the assessment of oculomotor function, including portable eye-tracking devices, have revealed more subtle impairments in ALS in relation to phenotype, which can now be better understood through parallel elucidation of the normal cerebral oculomotor control network. Given the clinicopathologic overlap between ALS and some types of frontotemporal dementia, the study of oculomotor f...

Stem cells and cloning : advances and applications, 2012

Throughout their lifetime, an individual may sustain many injuries and recover spontaneously over... more Throughout their lifetime, an individual may sustain many injuries and recover spontaneously over a period of time, without even realizing the injury in the first place. Wound healing occurs due to a proliferation of stem cells capable of restoring the injured tissue. The ability of adult stem cells to repair tissue is dependent upon the intrinsic ability of tissues to proliferate. The amazing capacity of embryonic stem cells to give rise to virtually any type of tissue has intensified the search for similar cell lineage in adults to treat various diseases including cardiovascular diseases. The ability to convert adult stem cells into pluripotent cells that resemble embryonic cells, and to transplant those in the desired organ for regenerative therapy is very attractive, and may offer the possibility of treating harmful disease-causing mutations. The race is on to find the best cells for treatment of cardiovascular disease. There is a need for the ideal stem cell, delivery strategie...

Clinical pharmacology : advances and applications, 2010

The potential interaction between clopidogrel and proton pump inhibitors (PPI) in patients with a... more The potential interaction between clopidogrel and proton pump inhibitors (PPI) in patients with acute coronary syndrome (ACS) raises serious concerns for cardiologists. However, in patients on this combination of drugs, there is no conclusive evidence of an increase in adverse cardiovascular events. From pharmacologic and pharmacodynamic perspectives, there is a real interaction between clopidogrel and PPIs because of the competitive inhibition of CYP2C19 isoenzyme which is required for biotransformation of clopidogrel to its active metabolite. The consequent decrease in the availability of this active metabolite leads to attenuation of antiplatelet efficacy of clopidogrel. In several observational trials, it was shown that decreased antiplatelet effect of clopidogrel due to PPIs may translate into poor cardiovascular outcomes. However, an incomplete RCT (COGENT) and a post hoc analysis of two large trials (PRINCIPLE-TIMI 44 and TRITON-TIMI 38 trial) showed no significant adverse ca...

Fertility and Sterility, 2004

Objective(s)To measure levels of lipid peroxidation (LPO) and total antioxidant capacity (TAC) in... more Objective(s)To measure levels of lipid peroxidation (LPO) and total antioxidant capacity (TAC) in the follicular fluid from patients undergoing IVF and to examine the association among LPO and TAC and oocyte maturity, embryo quality, fertilization, cleavage, and pregnancy rates.