Thottethodi Subrahmanya Keshava Prasad

Genomewide mRNA profiling of esophageal squamous cell carcinoma for identification of cancer biomarkers

Esophageal squamous cell carcinoma (ESCC) is a common cancer worldwide that has a poor survival rate among patients mainly because of lack of early markers to identify this cancer. Molecular mechanisms contributing to initiation and progression of esophageal squamous cell carcinoma are still poorly understood. Development of DNA microarrays technology allows high-throughput identification of gene expression profiles in cancers. In order to identify molecules as candidates for early diagnosis and/or therapeutic targets, we analyzed mRNA expression profiles of 20 surgically resected specimens of ESCC and compared them to their adjacent normal epithelium using whole genome DNA microarrays. We observed 119 genes significantly upregulated in ESCC samples as compared to the adjacent normal epithelium. The expression of two previously unreported overexpressed genes, ORAOV2 and FAP, was validated at the protein level by immunohistochemical labeling of tissue microarrays (TMAs) and archival tissue sections. Overexpression of ORAOV2 was observed in 116/118 (98%) of ESCC cases, while FAP overexpression was in 79/116 (68%) of cases. Osteopontin, which was identified in earlier studies, was observed to be upregulated in 114/118 (97%) cases. Overall, using this approach, we have identified a number of promising novel candidates that can be validated further for their potential to serve as biomarkers for ESCC.

SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome

The identification of secreted proteins that are differentially expressed between non-neoplastic and esophageal squamous cell carcinoma (ESCC) cells can provide potential biomarkers of ESCC. We used a SILAC-based quantitative proteomic approach to compare the secretome of ESCC cells with that of non-neoplastic esophageal squamous epithelial cells. Proteins were resolved by SDS-PAGE, and tandem mass spectrometry analysis (LC-MS/MS) of in-gel trypsin-digested peptides was carried out on a high-accuracy qTOF mass spectrometer. In total, we identified 441 proteins in the combined secretomes, including 120 proteins with >2-fold upregulation in the ESCC secretome vs. that of non-neoplastic esophageal squamous epithelial cells. In this study, several potential protein biomarkers previously known to be increased in ESCC including matrix metalloproteinase 1, transferrin receptor, and transforming growth factor beta-induced 68 kDa were identified as overexpressed in the ESCC-derived secretome. In addition, we identified several novel proteins that have not been previously reported to be associated with ESCC. Among the novel candidate proteins identified, protein disulfide isomerase family a member 3 (PDIA3), GDP dissociation inhibitor 2 (GDI2), and lectin galactoside binding soluble 3 binding protein (LGALS3BP) were further validated by immunoblot analysis and immunohistochemical labeling using tissue microarrays. This tissue microarray analysis showed overexpression of protein disulfide isomerase family a member 3, GDP dissociation inhibitor 2, and lectin galactoside binding soluble 3 binding protein in 93%, 93% and 87% of 137 ESCC cases, respectively. Hence, we conclude that these potential biomarkers are excellent candidates for further evaluation to test their role and efficacy in the early detection of ESCC.

Characterization of traumatic brain injury in human brains reveals distinct cellular and molecular changes in contusion and pericontusion

Traumatic brain injury (TBI) contributes to fatalities and neurological disabilities worldwide. While primary injury causes immediate damage, secondary events contribute to long‐term neurological defects. Contusions (Ct) are primary injuries correlated with poor clinical prognosis, and can expand leading to delayed neurological deterioration. Pericontusion (PC) (penumbra), the region surrounding Ct, can also expand with edema, increased intracranial pressure, ischemia, and poor clinical outcome. Analysis of Ct and PC can therefore assist in understanding the pathobiology of TBI and its management. This study on human TBI brains noted extensive neuronal, astroglial and inflammatory changes, alterations in mitochondrial, synaptic and oxidative markers, and associated proteomic profile, with distinct differences in Ct and PC. While Ct displayed petechial hemorrhages, thrombosis, inflammation, neuronal pyknosis, and astrogliosis, PC revealed edema, vacuolation of neuropil, axonal loss, and dystrophic changes. Proteomic analysis demonstrated altered immune response, synaptic, and mitochondrial dysfunction, among others, in Ct, while PC displayed altered regulation of neurogenesis and cytoskeletal architecture, among others. TBI brains displayed oxidative damage, glutathione depletion, mitochondrial dysfunction, and loss of synaptic proteins, with these changes being more profound in Ct. We suggest that analysis of markers specific to Ct and PC may be valuable in the evaluation of TBI pathobiology and therapeutics. We have characterized the primary injury in human traumatic brain injury (TBI). Contusions (Ct) – the injury core displayed hemorrhages, inflammation, and astrogliosis, while the surrounding pericontusion (PC) revealed edema, vacuolation, microglial activation, axonal loss, and dystrophy. Proteomic analysis demonstrated altered immune response, synaptic and mitochondrial dysfunction in Ct, and altered regulation of neurogenesis and cytoskeletal architecture in PC. Ct displayed more oxidative damage, mitochondrial, and synaptic dysfunction compared to PC.

Quantitative proteomics for identifying biomarkers for Rabies.

IntroductionRabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.MethodsIn this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.Results and conclusionWe identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.

Proteomic analysis of purified protein derivative of Mycobacterium tuberculosis.

BackgroundPurified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as “purified,” in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterization is likely to help in short-listing the relevant antigens required to prepare a less complex and more potent reagent for diagnostic purposes.ResultsProteomic analysis of Connaught Tuberculin 68 (PPD-CT68), a tuberculin preparation generated from M. tuberculosis, was carried out in this study. PPD-CT68 is the protein component of a commercially available tuberculin preparation, Tubersol, which is used for tuberculin skin testing. Using a high resolution LTQ-Orbitrap Velos mass spectrometer, we identified 265 different proteins. The identified proteins were compared with those identified from PPD M. bovis, PPD M. avium and PPD-S2 from previous mass spectrometry-based studies. In all, 142 proteins were found to be shared between PPD-CT68 and PPD-S2 preparations. Out of the 354 proteins from M. tuberculosis–derived PPDs (i.e. proteins in either PPD-CT68 or PPD-S2), 37 proteins were found to be shared with M. avium PPD and 80 were shared with M. bovis PPD. Alignment of PPD-CT68 proteins with proteins encoded by 24 lung infecting bacteria revealed a number of similar proteins (206 bacterial proteins shared epitopes with 47 PPD-CT68 proteins), which could potentially be involved in causing cross-reactivity. The data have been deposited to the ProteomeXchange with identifier PXD000377.ConclusionsProteomic and bioinformatics analysis of different PPD preparations revealed commonly and differentially represented proteins. This information could help in delineating the relevant antigens represented in various PPDs, which could further lead to development of a lesser complex and better defined skin test antigen with a higher specificity and sensitivity.

Proteogenomic analysis of pathogenic yeast Cryptococcus neoformans using high resolution mass spectrometry

BackgroundCryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported.ResultsWe performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing.ConclusionsProteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans.

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

Downregulation of cornulin in esophageal squamous cell carcinoma

Early events in the development of esophageal squamous cell carcinoma (ESCC) are poorly understood and many of the key molecules involved have not yet been identified. We previously used isobaric tags for a relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach to identify differentially expressed proteins in ESCC tissue as compared to the adjacent normal mucosa. Cornulin was identified as one of the major downregulated molecules in ESCC. Cornulin is a member of the S100 fused-type protein family, which has an EF-hand calcium binding motif and multiple tandem repeats of specific peptide motifs. Cornulin was 5-fold downregulated in ESCC as compared to normal epithelium mirroring our previous findings in a gene expression study of ESCC. In the present study, we performed immunohistochemical validation of cornulin (CRNN) in a larger set of patients with ESCC. Downregulation of cornulin was observed in 89% (n=239) of 266 different ESCC tissues arrayed on tissue microarrays (TMAs). Expression of cornulin was observed in the prickle and functional cell layers of normal esophageal mucosa, localized predominantly in the cytoplasm and perinuclear region. The large majority of ESCC cases had little or no expression of cornulin in the carcinoma or stroma. These findings suggest that cornulin is an important molecule in normal esophageal pathology and is likely lost during the conversion of normal to neoplastic epithelium.

Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function

Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX‐deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX‐SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis demonstrated down‐regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under‐expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases.

Overexpression of Periostin and Lumican in Esophageal Squamous Cell Carcinoma

To identify biomarkers for early detection for esophageal squamous cell carcinoma (ESCC), we previously carried out a genome-wide gene expression profiling study using an oligonucleotide microarray platform. This analysis led to identification of several transcripts that were significantly upregulated in ESCC compared to the adjacent normal epithelium. In the current study, we performed immunohistochemical analyses of protein products for two candidates genes identified from the DNA microarray analysis, periostin (POSTN) and lumican (LUM), using tissue microarrays. Increased expression of both periostin and lumican was observed in 100% of 137 different ESCC samples arrayed on tissue microarrays. Increased expression of periostin and lumican was observed in carcinoma as well as in stromal cell in the large majority of cases. These findings suggest that these candidates can be investigated in the sera of ESCC patients using ELISA or multiple reaction monitoring (MRM) type assays to further explore their utility as biomarkers.