47 results on '"Patil AH"'
Search Results
2. Patterns of Unwanted Biological and Technical Expression Variation Among 49 Human Tissues.
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Nieuwenhuis TO, Giles HH, Arking JVA, Patil AH, Shi W, McCall MN, and Halushka MK
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- Humans, Organ Specificity, Cluster Analysis, Gene Expression Profiling
- Abstract
Tissue gene expression studies are impacted by biological and technical sources of variation, which can be broadly classified into wanted and unwanted variation. The latter, if not addressed, results in misleading biological conclusions. Methods have been proposed to reduce unwanted variation, such as normalization and batch correction. A more accurate understanding of all causes of variation could significantly improve the ability of these methods to remove unwanted variation while retaining variation corresponding to the biological question of interest. We used 17,282 samples from 49 human tissues in the Genotype-Tissue Expression data set (v8) to investigate patterns and causes of expression variation. Transcript expression was transformed to z-scores, and only the most variable 2% of transcripts were evaluated and clustered based on coexpression patterns. Clustered gene sets were assigned to different biological or technical causes based on histologic appearances and metadata elements. We identified 522 variable transcript clusters (median: 11 per tissue) among the samples. Of these, 63% were confidently explained, 16% were likely explained, 7% were low confidence explanations, and 14% had no clear cause. Histologic analysis annotated 46 clusters. Other common causes of variability included sex, sequencing contamination, immunoglobulin diversity, and compositional tissue differences. Less common biological causes included death interval (Hardy score), disease status, and age. Technical causes included blood draw timing and harvesting differences. Many of the causes of variation in bulk tissue expression were identifiable in the Tabula Sapiens data set of single-cell expression. This is among the largest explorations of the underlying sources of tissue expression variation. It uncovered expected and unexpected causes of variable gene expression and demonstrated the utility of matched histologic specimens. It further demonstrated the value of acquiring meaningful tissue harvesting metadata elements to use for improved normalization, batch correction, and analysis of both bulk and single-cell RNA-seq data., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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3. Lesions Misdiagnosed as Endodontic Pathosis: A Narrative Review.
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Patil AH, Singh S, Bedia AS, Sharma D, Sinha A, and Dahiya N
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In daily practice, clinicians come across certain radiographic abnormalities which may or may not be asymptomatic. This abstract discusses radiographic abnormalities encountered by clinicians in daily practice, some of which resemble endodontic lesions. Prompt attention is crucial as these lesions can be benign or malignant. The article emphasizes the importance of differential diagnosis for accurate identification of periapical pathosis., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Pharmacy and Bioallied Sciences.)
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- 2024
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4. Brooklyn plots to identify co-expression dysregulation in single cell sequencing.
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Patil AH, McCall MN, and Halushka MK
- Abstract
Altered open chromatin regions, impacting gene expression, is a feature of some human disorders. We discovered it is possible to detect global changes in genomically-related adjacent gene co-expression within single cell RNA sequencing (scRNA-seq) data. We built a software package to generate and test non-randomness using 'Brooklyn plots' to identify the percent of genes significantly co-expressed from the same chromosome in ∼10 MB intervals across the genome. These plots establish an expected low baseline of co-expression in scRNA-seq from most cell types, but, as seen in dilated cardiomyopathy cardiomyocytes, altered patterns of open chromatin appear. These may relate to larger regions of transcriptional bursting, observable in single cell, but not bulk datasets., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)
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- 2024
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5. Evaluation of Salivary Alkaline Phosphatase Levels in Passive Smokers of Different Age Groups.
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Mulla SA, Bedia AS, Nimmagadda HK, Bedia S, and Patil AH
- Abstract
Background The smoke inhaled by a nonsmoker from the smoldering end of a cigarette is referred to as passive smoke. The nicotine present in smoke is known to cause tissue damage and alter the enzymatic composition of the body. Alkaline phosphatase (ALP) is a group of intracellular hydrolytic enzymes known to partake in cellular metabolism. ALP levels are affected by smoking as well as passive smoking (PS) with a change in the pH of the oral cavity. The association of salivary alkaline phosphatase (S-ALP) levels in different age groups, gender, and times of exposure is not thoroughly explored yet, which was the primary aim of this study. Material and methods A total of 64 samples were collected from passive smokers and non-smokers. Unstimulated saliva (2-2.5 mL) was collected from each subject after obtaining their consent, followed by centrifuging and mixing with ALP reagent in a semi-autoanalyzer to obtain the S-ALP levels. Results Higher S-ALP levels were seen in passive smokers (34.70 IU/L) compared to healthy individuals (12 IU/L), which came to be statistically significant (p<0.01). S-ALP levels, when compared to different age groups and gender, were statistically insignificant (p>0.05). However, higher levels were seen in association with time of exposure in passive smokers where the data was statistically significant (p<0.01), suggesting tissue damage possibly due to oxidative stresses and tissue inflammation on continuous exposure for a minimum of 30-60 minutes daily as per our study. Conclusion Significantly high levels of S-ALP were found in passive smokers in comparison to non-smokers. This suggests that passive smoking has negative effects on the body tissues. Age, gender, and time of exposure of a non-smoker to tobacco smoke can lead to alterations in S-ALP levels. High levels of S-ALP were seen in individuals with prolonged exposure to tobacco smoke on a daily basis. Salivaomics can thus be used as a non-invasive, economical, and accurate alternative in tissue damage diagnosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Mulla et al.)
- Published
- 2023
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6. A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.
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Fromm B, Patil AH, and Halushka MK
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- Humans, Myocardium, Circulating MicroRNA, Myocarditis diagnosis, Myocarditis genetics
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- 2022
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7. A curated human cellular microRNAome based on 196 primary cell types.
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Patil AH, Baran A, Brehm ZP, McCall MN, and Halushka MK
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- Genome, Humans, Sequence Alignment, Sequence Analysis, RNA, MicroRNAs genetics, MicroRNAs metabolism, Software
- Abstract
Background: An incomplete picture of the expression distribution of microRNAs (miRNAs) across human cell types has long hindered our understanding of this important regulatory class of RNA. With the continued increase in available public small RNA sequencing datasets, there is an opportunity to more fully understand the general distribution of miRNAs at the cell level., Results: From the NCBI Sequence Read Archive, we obtained 6,054 human primary cell datasets and processed 4,184 of them through the miRge3.0 small RNA sequencing alignment software. This dataset was curated down, through shared miRNA expression patterns, to 2,077 samples from 196 unique cell types derived from 175 separate studies. Of 2,731 putative miRNAs listed in miRBase (v22.1), 2,452 (89.8%) were detected. Among reasonably expressed miRNAs, 108 were designated as cell specific/near specific, 59 as infrequent, 52 as frequent, 54 as near ubiquitous, and 50 as ubiquitous. The complexity of cellular microRNA expression estimates recapitulates tissue expression patterns and informs on the miRNA composition of plasma., Conclusions: This study represents the most complete reference, to date, of miRNA expression patterns by primary cell type. The data are available through the human cellular microRNAome track at the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgHubConnect) and an R/Bioconductor package (https://bioconductor.org/packages/microRNAome/)., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)
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- 2022
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8. Dissecting Plasmodium yoelii Pathobiology: Proteomic Approaches for Decoding Novel Translational and Post-Translational Modifications.
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Rex DAB, Patil AH, Modi PK, Kandiyil MK, Kasaragod S, Pinto SM, Tanneru N, Sijwali PS, and Prasad TSK
- Abstract
Malaria is a vector-borne disease. It is caused by Plasmodium parasites. Plasmodium yoelii is a rodent model parasite, primarily used for studying parasite development in liver cells and vectors. To better understand parasite biology, we carried out a high-throughput-based proteomic analysis of P. yoelii . From the same mass spectrometry (MS)/MS data set, we also captured several post-translational modified peptides by following a bioinformatics analysis without any prior enrichment. Further, we carried out a proteogenomic analysis, which resulted in improvements to some of the existing gene models along with the identification of several novel genes. Analysis of proteome and post-translational modifications (PTMs) together resulted in the identification of 3124 proteins. The identified PTMs were found to be enriched in mitochondrial metabolic pathways. Subsequent bioinformatics analysis provided an insight into proteins associated with metabolic regulatory mechanisms. Among these, the tricarboxylic acid (TCA) cycle and the isoprenoid synthesis pathway are found to be essential for parasite survival and drug resistance. The proteogenomic analysis discovered 43 novel protein-coding genes. The availability of an in-depth proteomic landscape of a malaria pathogen model will likely facilitate further molecular-level investigations on pre-erythrocytic stages of malaria., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
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- 2022
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9. Analysis of Stress Distribution on the Bone around an Implant Placed in the Anterior Maxilla Using Three Different Abutment Angulations by Means of Finite Element Analysis.
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Shende S, Jadhav A, Edake DN, Patil AH, Patil H, and Agrawal N
- Abstract
Aim: the aim of the study was to study the effect of stress distribution on the bone around an implant using angled abutments by means of finite element analysis in the anterior maxillary region., Materials and Methods: A three-dimensional (3D) model of the of patient's maxilla of right central incisor: tooth, bone, crown, implant, and abutment system were used in this study. The models were designed for three situations with straight abutment, i.e. 0°, 15°, and 20° angled abutment. The load of 178N was applied on the cingulum area of the prosthesis at an angle of 130° in relationship with the long axis of the implant. After applying the static loads on each model, the stress generated in the bone and the implant was recorded. The results will be analyzed by analysis of variance test., Results: The cortical and cancellous on Mises stresses in 20° abutment model were found to be maximum as compared to 15° abutment followed by 0° abutment. The stress was concentrated in the implant-abutment joint area. The overall stresses in 20° abutment model were found to more than 15° abutment followed by 0° abutment. The magnitude of stresses increased as the angulations increased., Conclusion: From the conclusions of this study, the stress is more multiplied in angled abutments, hence care needs to be taken when restoring implants using angled abutments, especially in patients with heavy masticatory load or when planning for cantilevering of restorations in these angled implant restoration., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Pharmacy and Bioallied Sciences.)
- Published
- 2021
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10. Correction: Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.
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Solanki HS, Raja R, Zhavoronkov A, Ozerov IV, Artemov AV, Advani J, Radhakrishnan A, Babu N, Puttamallesh VN, Syed N, Nanjappa V, Subbannayya T, Sahasrabuddhe NA, Patil AH, Prasad TSK, Gaykalova D, Chang X, Sathyendran R, Mathur PP, Rangarajan A, Sidransky D, Pandey A, Izumchenko E, Gowda H, and Chatterjee A
- Abstract
[This corrects the article DOI: 10.18632/oncoscience.395.].
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- 2021
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11. Mapping Post-Translational Modifications in Brain Regions in Alzheimer's Disease Using Proteomics Data Mining.
- Author
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Deolankar SC, Patil AH, Rex DAB, Subba P, Mahadevan A, and Prasad TSK
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- Brain, Data Mining, Humans, Protein Processing, Post-Translational, Proteomics, Alzheimer Disease genetics, Neurodegenerative Diseases
- Abstract
Alzheimer's disease (AD) is a leading cause of dementia and a neurodegenerative disease. Proteomics and post-translational modification (PTM) analyses offer new opportunities for a comprehensive understanding of pathophysiology of brain in AD. We report here multiple PTMs in patients with AD, harnessing publicly available proteomics data from nine brain regions and at three different Braak stages of disease progression. Specifically, we identified 7190 peptides with PTMs, corresponding to 2545 proteins from brain regions with intermediate tangles, and 6864 peptides with PTMs corresponding to 2465 proteins from brain regions with severe tangles. A total of 103 proteins with PTMs were expressed uniquely to intermediate tangles and severe tangles compared to no tangles. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggested the association of these proteins in AD progression through platelet activation. These modified proteins were also found to be enriched for the tricarboxylic acid (TCA) cycle, respiratory electron cycle, and detoxification of reactive oxygen species. The multi-PTM data reported here contribute to our understanding of the neurobiology of AD and highlight the prospects of omics systems science research in neurodegenerative diseases. The present study provides a region-wise classification for the proteins with PTMs along with their differential expression patterns, providing insights into the localization of these proteins upon modification. The catalog of multi-PTMs identified in the context of AD from different brain regions provides a unique platform for generating newer hypotheses in understanding the putative role of specific PTMs in AD pathogenesis.
- Published
- 2021
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12. miRge3.0: a comprehensive microRNA and tRF sequencing analysis pipeline.
- Author
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Patil AH and Halushka MK
- Abstract
MicroRNAs and tRFs are classes of small non-coding RNAs, known for their roles in translational regulation of genes. Advances in next-generation sequencing (NGS) have enabled high-throughput small RNA-seq studies, which require robust alignment pipelines. Our laboratory previously developed miRge and miRge2.0, as flexible tools to process sequencing data for annotation of miRNAs and other small-RNA species and further predict novel miRNAs using a support vector machine approach. Although miRge2.0 is a leading analysis tool in terms of speed with unique quantifying and annotation features, it has a few limitations. We present miRge3.0 that provides additional features along with compatibility to newer versions of Cutadapt and Python. The revisions of the tool include the ability to process Unique Molecular Identifiers (UMIs) to account for PCR duplicates while quantifying miRNAs in the datasets, correct erroneous single base substitutions in miRNAs with miREC and an accurate mirGFF3 formatted isomiR tool. miRge3.0 also has speed improvements benchmarked to miRge2.0, Chimira and sRNAbench. Finally, miRge3.0 output integrates into other packages for a streamlined analysis process and provides a cross-platform Graphical User Interface (GUI). In conclusion miRge3.0 is our third generation small RNA-seq aligner with improvements in speed, versatility and functionality over earlier iterations., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)
- Published
- 2021
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13. Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers.
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Verma RX, Kannan S, Lin BL, Fomchenko KM, Nieuwenhuis TO, Patil AH, Lukban C, Yang X, Fox-Talbot K, McCall MN, Kwon C, Kass DA, Rosenberg AZ, and Halushka MK
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- Animals, Cell Separation, Foot, Mice, Sequence Analysis, RNA, Muscle, Skeletal, Muscular Diseases
- Abstract
Background: Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type., Methods: We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers., Results: Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types., Conclusion: This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.
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- 2021
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14. Study of solvent variation on controlled synthesis of different nanostructured NiCo 2 O 4 thin films for supercapacitive application.
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Dhavale SB, Patil VL, Beknalkar SA, Teli AM, Patil AH, Patil AP, Shin JC, and Patil PS
- Abstract
The present investigation deals with controlled synthesis of nanostructured NiCo
2 O4 thin films directly on stainless steel substrates by facile and economical chemical bath deposition technique, without adding a surfactant or a binder. The consequences of different compositions of solvents on morphological and electrochemical properties have been studied systematically. We used different solvent composition as Double Distilled Water (DDW), DDW:Ethanol (1:1) and DDW: N, N dimethylformamide (1:1). The films have been named as NCO-W for DDW, NCO-WE for DDW: Ethanol (1:1) solvent and NCO-WD for DDW: N, N dimethylformamide (1:1) solvent. The morphologies of NiCo2 O4 thin films modify substantially with change in a solvent. NCO-W exhibited the spikes of Crossandra infundibuliformis like nanostructures. The NCO-WE favored the formation of uniformly distributed leaf-like nanostructure whereas NCO-WD showed randomly oriented nanoplates all over the surface area. The Electrochemical performance of these NiCo2 O4 thin films were studied using cyclic voltammetry, chronopotentiometry, and electrochemical impedance spectroscopy techniques. The NCO-W, NCO-WE and NCO-WD electrodes showed specific capacitance values of 271, 553 and 140 F/g respectively at the current density of 0.5 mA/cm2 and excellent capacitance retention of 90%, 91% and 80% after 2000 cycles for NCO-W, NCO-WE and NCO-WD samples respectively. This result reveals that NiCo2 O4 is a prominent electrode material for supercapacitor application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2021
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15. Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers.
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Fomchenko KM, Walsh EM, Yang X, Verma RX, Lin BL, Nieuwenhuis TO, Patil AH, Fox-Talbot K, McCall MN, Kass DA, Rosenberg AZ, and Halushka MK
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- Humans, Muscle Fibers, Fast-Twitch, Muscle, Skeletal, Proteomics, Muscle Fibers, Slow-Twitch, Muscular Diseases
- Abstract
Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.
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- 2021
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16. Plant Omics: Metabolomics and Network Pharmacology of Liquorice, Indian Ayurvedic Medicine Yashtimadhu.
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Karthikkeyan G, Pervaje R, Subbannayya Y, Patil AH, Modi PK, and Prasad TSK
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- Computational Biology methods, Metabolome, Plant Extracts chemistry, Plant Extracts metabolism, Plant Extracts pharmacology, Glycyrrhiza metabolism, Metabolomics methods, Plants metabolism, Plants, Medicinal metabolism
- Abstract
Plant omics is an emerging field of systems science and offers the prospects of evidence-based evaluation of traditional herbal medicines in human diseases. To this end, the powdered root of Yashtimadhu ( Glycyrrhiza glabra L.), commonly known as liquorice, is frequently used in Indian Ayurvedic medicine with an eye to neuroprotection but its target proteins, mechanisms of action, and metabolites remain to be determined. Using a metabolomics and network pharmacology approach, we identified 98,097 spectra from positive and negative polarities that matched to ∼1600 known metabolites. These metabolites belong to terpenoids, alkaloids, and flavonoids, including both novel and previously reported active metabolites such as glycyrrhizin, glabridin, liquiritin, and other terpenoid saponins. Novel metabolites were also identified such as quercetin glucosides, coumarin derivatives, beta-carotene, and asiatic acid, which were previously not reported in relation to liquorice. Metabolite-protein interaction-based network pharmacology analyses enriched 107 human proteins, which included dopamine, serotonin, and acetylcholine neurotransmitter receptors among other regulatory proteins. Pathway analysis highlighted the regulation of signaling kinases, growth factor receptors, cell cycle, and inflammatory pathways. In vitro validation confirmed the regulation of cell cycle, MAPK1/3, PI3K/AKT pathways by liquorice. The present data-driven, metabolomics and network pharmacology study paves the way for further translational clinical research on neuropharmacology of liquorice and other traditional medicines.
- Published
- 2020
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17. CusVarDB: A tool for building customized sample-specific variant protein database from next-generation sequencing datasets.
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Kasaragod S, Mohanty V, Tyagi A, Behera SK, Patil AH, Pinto SM, Prasad TSK, Modi PK, and Gowda H
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- Humans, Proteomics, Computational Biology, Databases, Protein, High-Throughput Nucleotide Sequencing, Software
- Abstract
Cancer genome sequencing studies have revealed a number of variants in coding regions of several genes. Some of these coding variants play an important role in activating specific pathways that drive proliferation. Coding variants present on cancer cell surfaces by the major histocompatibility complex serve as neo-antigens and result in immune activation. The success of immune therapy in patients is attributed to neo-antigen load on cancer cell surfaces. However, which coding variants are expressed at the protein level can't be predicted based on genomic data. Complementing genomic data with proteomic data can potentially reveal coding variants that are expressed at the protein level. However, identification of variant peptides using mass spectrometry data is still a challenging task due to the lack of an appropriate tool that integrates genomic and proteomic data analysis pipelines. To overcome this problem, and for the ease of the biologists, we have developed a graphical user interface (GUI)-based tool called CusVarDB. We integrated variant calling pipeline to generate sample-specific variant protein database from next-generation sequencing datasets. We validated the tool with triple negative breast cancer cell line datasets and identified 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38 datasets, respectively., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Kasaragod S et al.)
- Published
- 2020
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18. CusVarDB: A tool for building customized sample-specific variant protein database from next-generation sequencing datasets.
- Author
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Kasaragod S, Mohanty V, Tyagi A, Behera SK, Patil AH, Pinto SM, Prasad TSK, Modi PK, and Gowda H
- Subjects
- Humans, Proteomics, Computational Biology, Databases, Protein, High-Throughput Nucleotide Sequencing, Software
- Abstract
Cancer genome sequencing studies have revealed a number of variants in coding regions of several genes. Some of these coding variants play an important role in activating specific pathways that drive proliferation. Coding variants present on cancer cell surfaces by the major histocompatibility complex serve as neo-antigens and result in immune activation. The success of immune therapy in patients is attributed to neo-antigen load on cancer cell surfaces. However, which coding variants are expressed at the protein level can't be predicted based on genomic data. Complementing genomic data with proteomic data can potentially reveal coding variants that are expressed at the protein level. However, identification of variant peptides using mass spectrometry data is still a challenging task due to the lack of an appropriate tool that integrates genomic and proteomic data analysis pipelines. To overcome this problem, and for the ease of the biologists, we have developed a graphical user interface (GUI)-based tool called CusVarDB. We integrated variant calling pipeline to generate sample-specific variant protein database from next-generation sequencing datasets. We validated the tool with triple negative breast cancer cell line datasets and identified 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38 datasets, respectively., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Kasaragod S et al.)
- Published
- 2020
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19. MAP2K1 is a potential therapeutic target in erlotinib resistant head and neck squamous cell carcinoma.
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Jain AP, Patel K, Pinto S, Radhakrishnan A, Nanjappa V, Kumar M, Raja R, Patil AH, Kumari A, Manoharan M, Karunakaran C, Murugan S, Keshava Prasad TS, Chang X, Mathur PP, Kumar P, Gupta R, Gupta R, Khanna-Gupta A, Sidransky D, Chatterjee A, and Gowda H
- Subjects
- Cell Line, Tumor, Datasets as Topic, Drug Delivery Systems, Drug Resistance, Neoplasm genetics, Epithelial-Mesenchymal Transition, Genomics, Humans, Metabolic Networks and Pathways, Phenotype, Proteomics, Squamous Cell Carcinoma of Head and Neck enzymology, Whole Genome Sequencing, Antineoplastic Agents therapeutic use, Erlotinib Hydrochloride therapeutic use, MAP Kinase Kinase 1 antagonists & inhibitors, Protein Kinase Inhibitors therapeutic use, Squamous Cell Carcinoma of Head and Neck drug therapy
- Abstract
Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.
- Published
- 2019
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20. Omics data-driven analysis identifies laminin-integrin-mediated signaling pathway as a determinant for cell differentiation in oral squamous cell carcinoma.
- Author
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Kulkarni S, Abdulla R, Jose M, Adyanthaya S, B Rex DA, Patil AH, Pinto SM, and Subbannayya Y
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- Adult, Biomarkers, Tumor genetics, Carcinoma, Squamous Cell pathology, Cohort Studies, Computational Biology, Databases, Protein, Head and Neck Neoplasms genetics, Head and Neck Neoplasms pathology, Humans, Immunohistochemistry, Integrins metabolism, Laminin metabolism, Middle Aged, Proof of Concept Study, Carcinoma, Squamous Cell genetics, Cell Differentiation, Data Mining, Integrins genetics, Laminin genetics, Signal Transduction
- Abstract
Background: In recent years, high-throughput omics technologies have been widely used globally to identify potential biomarkers and therapeutic targets in various cancers. However, apart from large consortiums such as The Cancer Genome Atlas, limited attempts have been made to mine existing datasets pertaining to cancers., Methods and Results: In the current study, we used an omics data analysis approach wherein publicly available protein expression data were integrated to identify functionally important proteins that revealed consistent dysregulated expression in head and neck squamous cell carcinomas. Our analysis revealed members of the integrin family of proteins to be consistently altered in expression across disparate datasets. Additionally, through association evidence and network analysis, we also identified members of the laminin family to be significantly altered in head and neck cancers. Members of both integrin and laminin families are known to be involved in cell-extracellular matrix adhesion and have been implicated in tumor metastatic processes in several cancers. To this end, we carried out immunohistochemical analyses to validate the findings in a cohort (n = 50) of oral cancer cases. Laminin-111 expression (composed of LAMA1, LAMB1, and LAMC1) was found to correlate with cell differentiation in oral cancer, showing a gradual decrease from well differentiated to poorly differentiated cases., Conclusion: This study serves as a proof-of-principle for the mining of multiple omics datasets coupled with selection of functionally important group of molecules to provide novel insights into tumorigenesis and cancer progression.
- Published
- 2019
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21. Dissecting Alzheimer's Disease Molecular Substrates by Proteomics and Discovery of Novel Post-translational Modifications.
- Author
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Deolankar SC, Patil AH, Koyangana SG, Subbannayya Y, Prasad TSK, and Modi PK
- Subjects
- Alzheimer Disease etiology, Biomarkers, Computational Biology methods, Databases, Protein, Gene Ontology, Humans, Neurodegenerative Diseases etiology, Neurodegenerative Diseases metabolism, Peptides, Protein Processing, Post-Translational, Signal Transduction, Tandem Mass Spectrometry, Alzheimer Disease metabolism, Proteome, Proteomics methods
- Abstract
Alzheimer's disease (AD) is a common complex disease and a major public health burden in both developed and developing countries. Postgenomic technologies such as proteomics and intelligent mining of multi-omics Big Data offer new prospects for diagnostics and therapeutics innovation for AD. In this context, it is noteworthy that mass spectrometry (MS) data are often searched against proteomics databases to unravel the identity of protein biomarkers. In contrast, only a fraction of the MS data can be matched to known proteins, while a large portion of such raw data remains underutilized. Furthermore, the spectral data can be mined for multiple high-confidence post-translational modifications (PTMs) without a priori enrichment. Thus, AD research stands to gain by greater attention to the biological mechanisms regulated by PTMs. Protein modifications may serve as diagnostic biomarkers or as novel molecular targets for drug discovery. We report here novel PTMs discovered in relation to the AD from MS/MS-based proteomic datasets. Publicly available label-free proteomics data were searched for select PTMs using SEQUEST-HT. Only high-confidence PTMs were analyzed using bioinformatics analysis. We identified 4961 unique modified peptides corresponding to 1856 proteins from AD datasets. Of these, 52 proteins were known to be involved in Alzheimer's pathway. Importantly, 3164 PTMs reported in this study are novel in the context of AD. Furthermore, protein quantification revealed expression of 13 high-abundant secretary proteins across multiple studies, which can be potentially harnessed in the future to develop biomarkers. In summary, this study identifies novel PTMs which might help develop new insights on the molecular substrates of AD and thus inform future development of novel diagnostics and treatments for this highly prevalent disease.
- Published
- 2019
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22. Global Proteome Profiling Reveals Drug-Resistant Traits in Elizabethkingia meningoseptica : An Opportunistic Nosocomial Pathogen.
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Agrawal A, Ravikumar R, Varun CN, Kumar M, Chatterjee O, Advani J, Gopalakrishnan L, Nagaraj S, Mohanty V, Patil AH, Sreeramulu B, Malik A, Pinto SM, and Prasad TSK
- Subjects
- Anti-Bacterial Agents pharmacology, Humans, Microbial Sensitivity Tests, Chryseobacterium drug effects, Chryseobacterium metabolism, Communicable Diseases metabolism, Proteomics methods
- Abstract
Elizabethkingia meningoseptica is Gram-negative, rod-shaped opportunistic bacterial pathogen increasingly reported in hospital-acquired outbreaks. This bacterium is well known to thrive in the hospital environment. One of the leading causes of meningitis in pediatric and immune-compromised patients, E. meningoseptica has been noted as a "pathogen of interest" in the context of nosocomial diseases associated with device-related infections in particular. This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings. This study provides the global proteome of E. meningoseptica . The reference strain E. meningoseptica ATCC 13253 was used for proteomic analysis using high-resolution Fourier transform mass spectrometry. The study provided translational evidence for 2506 proteins of E. meningoseptica . We identified multiple metallo-β-lactamases, transcriptional regulators, and efflux transporter proteins associated with multidrug resistance. A protein Car D, which is an enzyme of the carbapenem synthesis pathway, was also discovered in E. meningoseptica . Further, the proteomics data were harnessed for refining the genome annotation. We discovered 39 novel protein-coding genes and corrected four existing translations using proteogenomic workflow. Novel translations reported in this study enhance the molecular data on this organism, thus improving current databases. We believe that the in-depth proteomic data presented in this study offer a platform for accelerated research on this pathogen. The identification of multiple proteins, particularly those involved in drug resistance, offers new future opportunities to design novel and specific antibiotics against infections caused by E. meningoseptica .
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- 2019
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23. PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40).
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Subbannayya T, Leal-Rojas P, Zhavoronkov A, Ozerov IV, Korzinkin M, Babu N, Radhakrishnan A, Chavan S, Raja R, Pinto SM, Patil AH, Barbhuiya MA, Kumar P, Guerrero-Preston R, Navani S, Tiwari PK, Kumar RV, Prasad TSK, Roa JC, Pandey A, Sidransky D, Gowda H, Izumchenko E, and Chatterjee A
- Abstract
Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.
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- 2019
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24. Role of protein kinase N2 (PKN2) in cigarette smoke-mediated oncogenic transformation of oral cells.
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Rajagopalan P, Nanjappa V, Patel K, Jain AP, Mangalaparthi KK, Patil AH, Nair B, Mathur PP, Keshava Prasad TS, Califano JA, Sidransky D, Gowda H, and Chatterjee A
- Abstract
Smoking is the leading cause of preventable death worldwide. Though cigarette smoke is an established cause of head and neck cancer (including oral cancer), molecular alterations associated with chronic cigarette smoke exposure are poorly studied. To understand the signaling alterations induced by chronic exposure to cigarette smoke, we developed a cell line model by exposing normal oral keratinocytes to cigarette smoke for a period of 12 months. Chronic exposure to cigarette smoke resulted in increased cellular proliferation and invasive ability of oral keratinocytes. Proteomic and phosphoproteomic analyses showed dysregulation of several proteins involved in cellular movement and cytoskeletal reorganization in smoke exposed cells. We observed overexpression and hyperphosphorylation of protein kinase N2 (PKN2) in smoke exposed cells as well as in a panel of head and neck cancer cell lines established from smokers. Silencing of PKN2 resulted in decreased colony formation, invasion and migration in both smoke exposed cells and head and neck cancer cell lines. Our results indicate that PKN2 plays an important role in oncogenic transformation of oral keratinocytes in response to cigarette smoke. The current study provides evidence that PKN2 can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients with a history of smoking.
- Published
- 2018
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25. Proteome data of Anopheles stephensi ovary using high-resolution mass spectrometry.
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Dey G, Mohanty AK, Kumar M, Sreenivasamurthy SK, Patil AH, Keshava Prasad TS, and Kumar A
- Abstract
This article contains data on the proteins expressed in the ovaries of Anopheles stephensi , a major vector of malaria in India. Data acquisition was performed using a high-resolution Orbitrap-Velos mass spectrometer. The acquired MS/MS data was searched against An. stephensi protein database comprising of 11,789 sequences. Overall, 4407 proteins were identified, functional analysis was performed for the identified proteins and a protein-protein interaction map predicted. The data provided here is also related to a published article - "Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes" (Prasad et al., 2017) [1].
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- 2018
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26. Dissecting Candida Pathobiology: Post-Translational Modifications on the Candida tropicalis Proteome.
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Patil AH, Datta KK, Behera SK, Kasaragod S, Pinto SM, Koyangana SG, Mathur PP, Gowda H, Pandey A, and Prasad TSK
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- Candida genetics, Candida tropicalis genetics, Candida tropicalis metabolism, Phosphorylation, Protein Processing, Post-Translational, Candida metabolism, Proteome metabolism
- Abstract
Candida tropicalis belongs to the non-albicans group of Candida, and causes epidermal, mucosal, or systemic candidiasis in immunocompromised individuals. Although the prevalence of candidiasis has increased worldwide and non-albicans Candida (NAC) are becoming more significant, there are very few studies that focus on the NAC biology. Proteins and their post-translational modifications (PTMs) are an integral aspect in the pathobiology of such medically important fungi. Previously, we had reported the largest proteomic catalog of C. tropicalis. Notably, PTMs can be identified from proteomics data without a priori enrichment for a particular PTM, thus allowing broad-scale omics analyses. In this study, we developed the "PTM-Pro," a graphical user interface-based tool for identification and summary of high-confidence PTM sites based on statistical threshold of users' choice. We mined available proteomic data of C. tropicalis, and using PTM-Pro identified nearly 600 high-confidence PTM sites. The PTMs identified include phosphorylation of serine, threonine, and tyrosine; acetylation, crotonylation, methylation, and succinylation of lysine. These PTMs reside on biologically significant molecules, including histones, enzymes, and transcription factors. To our knowledge, this is the first report of PTMs in C. tropicalis and lays a foundation for future investigations of C. tropicalis PTMs. In addition, the PTM-Pro offers a graphical user interface tool for research on PTM sites in the field of proteomics.
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- 2018
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27. Integrated Multi-Omic Analysis of Mycobacterium tuberculosis H37Ra Redefines Virulence Attributes.
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Pinto SM, Verma R, Advani J, Chatterjee O, Patil AH, Kapoor S, Subbannayya Y, Raja R, Gandotra S, and Prasad TSK
- Abstract
H37Ra is a virulence attenuated strain of Mycobacterium tuberculosis widely employed as a model to investigate virulence mechanisms. Comparative high-throughput studies have earlier correlated its avirulence to the presence of specific mutations or absence of certain proteins. However, a recent sequencing study of H37Ra, has disproved several genomic differences earlier reported to be associated with virulence. This warrants further investigations on the H37Ra proteome as well. In this study, we carried out an integrated analysis of the genome, transcriptome, and proteome of H37Ra. In addition to confirming single nucleotide variations (SNVs) and insertion-deletions that were reported earlier, our study provides novel insights into the mutation spectrum in the promoter regions of 7 genes. We also provide transcriptional and proteomic evidence for 3,900 genes representing ~80% of the total predicted gene count including 408 proteins that have not been identified previously. We identified 9 genes whose coding potential was hitherto reported to be absent in H37Ra. These include 2 putative virulence factors belonging to ESAT-6 like family of proteins. Furthermore, proteogenomic analysis enabled us to identify 63 novel proteins coding genes and correct 25 existing gene models in H37Ra genome. A majority of these were found to be conserved in the virulent strain H37Rv as well as in other mycobacterial species suggesting that the differences in the virulent and avirulent strains of M. tuberculosis are not entirely dependent on the expression of certain proteins or their absence but may possibly be ascertained to functional changes.
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- 2018
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28. Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes.
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Bhat MY, Advani J, Rajagopalan P, Patel K, Nanjappa V, Solanki HS, Patil AH, Bhat FA, Mathur PP, Nair B, Prasad TSK, Califano JA, Sidransky D, Gowda H, and Chatterjee A
- Subjects
- Biomarkers, Computational Biology methods, Environmental Exposure adverse effects, Humans, Keratinocytes pathology, Phenotype, Gene Expression Regulation, Keratinocytes metabolism, MicroRNAs genetics, Mouth Mucosa cytology, Smoking, Tobacco, Smokeless
- Abstract
Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.
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- 2018
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29. Bovine Milk Comparative Proteome Analysis from Early, Mid, and Late Lactation in the Cattle Breed, Malnad Gidda (Bos indicus).
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Mol P, Kannegundla U, Dey G, Gopalakrishnan L, Dammalli M, Kumar M, Patil AH, Basavaraju M, Rao A, Ramesha KP, and Prasad TSK
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- Animals, Biomarkers, Breeding, Cattle, Computational Biology methods, Female, Gene Ontology, Lactation, Mammary Glands, Animal, Milk metabolism, Milk Proteins genetics, Signal Transduction, Milk chemistry, Milk Proteins metabolism, Proteome, Proteomics methods
- Abstract
Bovine milk is important for both veterinary medicine and human nutrition. Understanding the bovine milk proteome at different stages of lactation has therefore broad significance for integrative biology and clinical medicine as well. Indeed, different lactation stages have marked influence on the milk yield, milk constituents, and nourishment of the neonates. We performed a comparative proteome analysis of the bovine milk obtained at different stages of lactation from the Indian indigenous cattle Malnad Gidda (Bos indicus), a widely available breed. The milk differential proteome during the lactation stages in B. indicus has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins at early, mid, and late lactation stages, we identified a total of 564 proteins, out of which 403 proteins were found to be differentially abundant at different lactation stages. As is expected of any body fluid proteome, 51% of the proteins identified in the milk were found to have signal peptides. Gene ontology analyses were carried out to categorize proteins altered across different lactation stages based on biological process and molecular function, which enabled us to correlate their significance in each lactation stage. We also investigated the potential pathways enriched in different lactation stages using bioinformatics pathway analysis tools. To the best of our knowledge, this study represents the first and largest inventory of milk proteins identified to date for an Indian cattle breed. We believe that the current study broadly informs both veterinary omics research and the emerging field of nutriproteomics during lactation stages.
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- 2018
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30. Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.
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Solanki HS, Raja R, Zhavoronkov A, Ozerov IV, Artemov AV, Advani J, Radhakrishnan A, Babu N, Puttamallesh VN, Syed N, Nanjappa V, Subbannayya T, Sahasrabuddhe NA, Patil AH, Prasad TSK, Gaykalova D, Chang X, Sathyendran R, Mathur PP, Rangarajan A, Sidransky D, Pandey A, Izumchenko E, Gowda H, and Chatterjee A
- Abstract
EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no potential conflict of interest.
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- 2018
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31. Altered signaling associated with chronic arsenic exposure in human skin keratinocytes.
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Mir SA, Renuse S, Sathe G, Khan AA, Patil AH, Nanjappa V, Bhat FA, Prasad TSK, Giri AK, Chatterjee A, and Gowda H
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- Humans, Phosphoproteins metabolism, Phosphorylation drug effects, Signal Transduction drug effects, Titanium chemistry, Arsenic toxicity, Keratinocytes drug effects, Keratinocytes metabolism
- Abstract
Modulation of signaling pathways upon chronic arsenic exposure remains poorly studied. Here, we carried out SILAC-based quantitative phosphoproteomics analysis to dissect the signaling induced upon chronic arsenic exposure in human skin keratinocyte cell line, HaCaT. We identified 4171 unique phosphosites derived from 2000 proteins. We observed differential phosphorylation of 406 phosphosites (twofold) corresponding to 305 proteins. Several pathways involved in cytoskeleton maintenance and organization were found to be significantly enriched (p<0.05). Our data revealed altered phosphorylation of proteins associated with adherens junction remodeling and actin polymerization. Kinases such as protein kinase C iota type (PRKCI), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), tyrosine-protein kinase BAZ1B (BAZ1B) and STE20 like kinase (SLK) were found to be hyperphosphorylated. Our study provides novel insights into signaling perturbations associated with chronic arsenic exposure in human skin keratinocytes. All MS/MS data have been deposited to the ProteomeXchange with identifier PXD004868., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
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32. Toward the human cellular microRNAome.
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McCall MN, Kim MS, Adil M, Patil AH, Lu Y, Mitchell CJ, Leal-Rojas P, Xu J, Kumar M, Dawson VL, Dawson TM, Baras AS, Rosenberg AZ, Arking DE, Burns KH, Pandey A, and Halushka MK
- Subjects
- Adult, Cell Line, Transformed, Cell Line, Tumor, Humans, Male, Organ Specificity, MicroRNAs biosynthesis, MicroRNAs genetics, RNA Processing, Post-Transcriptional physiology
- Abstract
MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species., (© 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.)
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- 2017
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33. Mosquito-Borne Diseases and Omics: Tissue-Restricted Expression and Alternative Splicing Revealed by Transcriptome Profiling of Anopheles stephensi.
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Sreenivasamurthy SK, Madugundu AK, Patil AH, Dey G, Mohanty AK, Kumar M, Patel K, Wang C, Kumar A, Pandey A, and Prasad TSK
- Subjects
- Animals, Anopheles metabolism, Anopheles parasitology, Fat Body metabolism, Female, Gastrointestinal Tract metabolism, Gene Expression Profiling, Gene Library, Gene Ontology, Humans, India, Insect Vectors metabolism, Insect Vectors parasitology, Malaria, Falciparum parasitology, Malpighian Tubules metabolism, Molecular Sequence Annotation, Organ Specificity, Ovary metabolism, Plasmodium falciparum pathogenicity, Plasmodium falciparum physiology, Sequence Analysis, DNA, Alternative Splicing, Anopheles genetics, Genome, Insect, Insect Vectors genetics, Transcriptome
- Abstract
Malaria is one of the most debilitating mosquito-borne diseases with high global health burdens. While much of the research on malaria and mosquito-borne diseases is focused on Africa, Southeast Asia accounts for a sizable portion of the global burden of malaria. Moreover, about 50% of the Asian malaria incidence and deaths have been from India. A promising development in this context is that the completion of genome sequence of Anopheles stephensi, a major malaria vector in Asia, offers new opportunities for global health innovation, including the progress in deciphering the vectorial ability of this mosquito species at a molecular level. Moving forward, tissue-based expression profiling would be the next obvious step in understanding gene functions of An. stephensi. We report in this article, to the best of our knowledge, the first in-depth study on tissue-based transcriptomic profile of four important organs (midgut, Malpighian tubules, fat body, and ovary) of adult female An. stephensi mosquitoes. In all, we identified over 20,000 transcripts corresponding to more than 12,000 gene loci from these four tissues. We present and discuss the tissue-based expression profiles of majority of annotated transcripts in An. stephensi genome, and the dynamics of their alternative splicing in these tissues, in this study. The domain-based Gene Ontology analysis of the differentially expressed transcripts in each of the mosquito tissue indicated enrichment of transcripts with proteolytic activity in midgut; transporter activity in Malpighian tubules; cell cycle, DNA replication, and repair activities in ovaries; and oxidoreductase activities in fat body. Tissue-based study of transcript expression and gene functions markedly enhances our understanding of this important malaria vector, and in turn, offers rationales for further studies on vectorial ability and identification of novel molecular targets to intercept malaria transmission.
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- 2017
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34. Long-Term Cigarette Smoke Exposure and Changes in MiRNA Expression and Proteome in Non-Small-Cell Lung Cancer.
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Advani J, Subbannayya Y, Patel K, Khan AA, Patil AH, Jain AP, Solanki HS, Radhakrishnan A, Pinto SM, Sahasrabuddhe NA, Thomas JK, Mathur PP, Nair BG, Chang X, Prasad TSK, Sidransky D, Gowda H, and Chatterjee A
- Subjects
- Biomarkers analysis, Carcinoma, Non-Small-Cell Lung, Cell Line, Tumor, Gene Expression Regulation drug effects, Gene Expression Regulation genetics, Humans, Lung Neoplasms diagnosis, Cigarette Smoking adverse effects, Lung Neoplasms genetics, MicroRNAs metabolism
- Abstract
Chronic exposure to cigarette smoke markedly increases the risk for lung cancer. Regulation of gene expression at the post-transcriptional level by miRNAs influences a variety of cancer-related interactomes. Yet, relatively little is known on the effects of long-term cigarette smoke exposure on miRNA expression and gene regulation. NCI-H292 (H292) is a cell line sensitive to cigarette smoke with mucoepidermoid characteristics in culture. We report, in this study, original observations on long-term (12 months) cigarette smoke effects in the H292 cell line, using microarray-based miRNA expression profiling, and stable isotopic labeling with amino acids in cell culture-based quantitative proteomic analysis. We identified 112 upregulated and 147 downregulated miRNAs (by twofold) in cigarette smoke-treated H292 cells. The liquid chromatography-tandem mass spectrometry analysis identified 3,959 proteins, of which, 303 proteins were overexpressed and 112 proteins downregulated (by twofold). We observed 39 miRNA target pairs (proven targets) that were differentially expressed in response to chronic cigarette smoke exposure. Gene ontology analysis of the target proteins revealed enrichment of proteins in biological processes driving metabolism, cell communication, and nucleic acid metabolism. Pathway analysis revealed the enrichment of phagosome maturation, antigen presentation pathway, nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, and cholesterol biosynthesis pathways in cigarette smoke-exposed cells. In conclusion, this report makes an important contribution to knowledge on molecular changes in a lung cell line in response to long term cigarette smoke exposure. The findings might inform future strategies for drug target, biomarker and diagnostics innovation in lung cancer, and clinical oncology. These observations also call for further research on the extent to which continuing or stopping cigarette smoking in patients diagnosed with lung cancer translates into molecular and clinical outcomes.
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- 2017
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35. Differential signaling through p190 and p210 BCR-ABL fusion proteins revealed by interactome and phosphoproteome analysis.
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Cutler JA, Tahir R, Sreenivasamurthy SK, Mitchell C, Renuse S, Nirujogi RS, Patil AH, Heydarian M, Wong X, Wu X, Huang TC, Kim MS, Reddy KL, and Pandey A
- Subjects
- Cytoskeletal Proteins metabolism, Humans, Leukemia etiology, Phosphorylation, STAT Transcription Factors physiology, Fusion Proteins, bcr-abl physiology, Signal Transduction physiology
- Abstract
Two major types of leukemogenic BCR-ABL fusion proteins are p190
BCR-ABL and p210BCR-ABL . Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. We took an integrated approach by coupling protein-protein interaction analysis using biotinylation identification with global phosphorylation analysis to investigate the differences in signaling between these two fusion proteins. Our findings suggest that p190BCR-ABL and p210BCR-ABL differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210BCR-ABL , we observed an increased engagement of molecules active proximal to the membrane and in the case of p190BCR-ABL , an engagement of molecules of the cytoskeleton. These differences in signaling could underlie the distinct leukemogenic process induced by these two protein variants.- Published
- 2017
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36. Identification and characterization of a grain micronutrient-related OsFRO2 rice gene ortholog from micronutrient-rich little millet (Panicum sumatrense).
- Author
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Chandel G, Dubey M, Gupta S, Patil AH, and Rao AR
- Abstract
Minor millets are considered as nutrient-rich cereals having significant effect in improving human health. In this study, a rice ortholog of Ferric Chelate Reductase (FRO2) gene involved in plant metal uptake has been identified in iron-rich Little millet (LM) using PCR and next generation sequencing-based strategy. FRO2 gene-specific primers designed from rice genome amplified 2.7 Kb fragment in LM genotype RLM-37. Computational genomics analyses of the sequenced amplicon showed high level sequence similarity with rice OsFRO2 gene. The predicted gene structure showed the presence of 6 exons and 5 introns and its protein sequence was found to contain ferric reductase and NOX_Duox_Like_FAD_NADP domains. Further, 3D structure analysis of FCR-LM model protein (494 amino acids) shows that it has 18 helices, 10 beta sheets, 10 strands, 41 beta turn and 5 gamma turn with slight deviation from the FCR-Os structure. Besides, the structures of FCR-LM and FCR-Os were modelled followed by molecular dynamics simulations. The overall study revealed both sequence and structural similarity between the identified gene and OsFRO2. Thus, a putative ferric chelate reductase gene has been identified in LM paving the way for using this approach for identification of orthologs of other metal genes from millets. This also facilitates mining of effective alleles of known genes for improvement of staple crops like rice.
- Published
- 2017
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37. Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.
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Verma R, Pinto SM, Patil AH, Advani J, Subba P, Kumar M, Sharma J, Dey G, Ravikumar R, Buggi S, Satishchandra P, Sharma K, Suar M, Tripathy SP, Chauhan DS, Gowda H, Pandey A, Gandotra S, and Prasad TS
- Subjects
- Humans, Mass Spectrometry, Mycobacterium tuberculosis pathogenicity, Phosphoproteins genetics, Phosphorylation genetics, Proteomics methods, Signal Transduction genetics, Tuberculosis genetics, Tuberculosis pathology, Mycobacterium tuberculosis genetics, Phosphoproteins biosynthesis, Proteome genetics, Tuberculosis microbiology
- Abstract
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.
- Published
- 2017
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38. Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.
- Author
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Prasad TS, Mohanty AK, Kumar M, Sreenivasamurthy SK, Dey G, Nirujogi RS, Pinto SM, Madugundu AK, Patil AH, Advani J, Manda SS, Gupta MK, Dwivedi SB, Kelkar DS, Hall B, Jiang X, Peery A, Rajagopalan P, Yelamanchi SD, Solanki HS, Raja R, Sathe GJ, Chavan S, Verma R, Patel KM, Jain AP, Syed N, Datta KK, Khan AA, Dammalli M, Jayaram S, Radhakrishnan A, Mitchell CJ, Na CH, Kumar N, Sinnis P, Sharakhov IV, Wang C, Gowda H, Tu Z, Kumar A, and Pandey A
- Subjects
- Animals, Anopheles genetics, Exons genetics, Gene Expression Profiling, Proteome genetics, Proteomics, Genome genetics, High-Throughput Nucleotide Sequencing methods, Molecular Sequence Annotation, Transcriptome genetics
- Abstract
Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes., (© 2017 Prasad et al.; Published by Cold Spring Harbor Laboratory Press.)
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- 2017
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39. Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells.
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Raja R, Sahasrabuddhe NA, Radhakrishnan A, Syed N, Solanki HS, Puttamallesh VN, Balaji SA, Nanjappa V, Datta KK, Babu N, Renuse S, Patil AH, Izumchenko E, Prasad TS, Chang X, Rangarajan A, Sidransky D, Pandey A, Gowda H, and Chatterjee A
- Subjects
- Animals, Carcinoma, Non-Small-Cell Lung drug therapy, Cell Line, Tumor, Cell Proliferation, Cell Survival, ErbB Receptors metabolism, Gene Silencing, Humans, Lung Neoplasms drug therapy, Male, Mice, Mice, Inbred NOD, Mice, SCID, Neoplasm Transplantation, Phosphorylation, Proteome, Pyrazoles pharmacology, Pyrroles pharmacology, RNA, Small Interfering metabolism, Signal Transduction, Tobacco Products, rac1 GTP-Binding Protein metabolism, Carcinoma, Non-Small-Cell Lung metabolism, Lung Neoplasms metabolism, Smoke adverse effects, p21-Activated Kinases metabolism
- Abstract
Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers., Competing Interests: The authors declare that they have no potential conflicts of interest.
- Published
- 2016
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40. Phosphotyrosine profiling of curcumin-induced signaling.
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Sathe G, Pinto SM, Syed N, Nanjappa V, Solanki HS, Renuse S, Chavan S, Khan AA, Patil AH, Nirujogi RS, Nair B, Mathur PP, Prasad TSK, Gowda H, and Chatterjee A
- Abstract
Background: Curcumin, derived from the rhizome Curcuma longa, is a natural anti-cancer agent and has been shown to inhibit proliferation and survival of tumor cells. Although the anti-cancer effects of curcumin are well established, detailed understanding of the signaling pathways altered by curcumin is still lacking. In this study, we carried out SILAC-based quantitative proteomic analysis of a HNSCC cell line (CAL 27) to investigate tyrosine signaling in response to curcumin., Results: Using high resolution Orbitrap Fusion Tribrid Fourier transform mass spectrometer, we identified 627 phosphotyrosine sites mapping to 359 proteins. We observed alterations in the level of phosphorylation of 304 sites corresponding to 197 proteins upon curcumin treatment. We report here for the first time, curcumin-induced alterations in the phosphorylation of several kinases including TNK2, FRK, AXL, MAPK12 and phosphatases such as PTPN6, PTPRK, and INPPL1 among others. Pathway analysis revealed that the proteins differentially phosphorylated in response to curcumin are known to be involved in focal adhesion kinase signaling and actin cytoskeleton reorganization., Conclusions: The study indicates that curcumin may regulate cellular processes such as proliferation and migration through perturbation of the focal adhesion kinase pathway. This is the first quantitative phosphoproteomics-based study demonstrating the signaling events that are altered in response to curcumin. Considering the importance of curcumin as an anti-cancer agent, this study will significantly improve the current knowledge of curcumin-mediated signaling in cancer.
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- 2016
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41. Proteogenomics of Candida tropicalis--An Opportunistic Pathogen with Importance for Global Health.
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Datta KK, Patil AH, Patel K, Dey G, Madugundu AK, Renuse S, Kaviyil JE, Sekhar R, Arunima A, Daswani B, Kaur I, Mohanty J, Sinha R, Jaiswal S, Sivapriya S, Sonnathi Y, Chattoo BB, Gowda H, Ravikumar R, and Prasad TS
- Subjects
- Candida tropicalis genetics, Culture Media, Conditioned, Mass Spectrometry, Candida tropicalis metabolism, Fungal Proteins genetics, Genome, Fungal, Global Health
- Abstract
The frequency of Candida infections is currently rising, and thus adversely impacting global health. The situation is exacerbated by azole resistance developed by fungal pathogens. Candida tropicalis is an opportunistic pathogen that causes candidiasis, for example, in immune-compromised individuals, cancer patients, and those who undergo organ transplantation. It is a member of the non-albicans group of Candida that are known to be azole-resistant, and is frequently seen in individuals being treated for cancers, HIV-infection, and those who underwent bone marrow transplantation. Although the genome of C. tropicalis was sequenced in 2009, the genome annotation has not been supported by experimental validation. In the present study, we have carried out proteomics profiling of C. tropicalis using high-resolution Fourier transform mass spectrometry. We identified 2743 proteins, thus mapping nearly 44% of the computationally predicted protein-coding genes with peptide level evidence. In addition to identifying 2591 proteins in the cell lysate of this yeast, we also analyzed the proteome of the conditioned media of C. tropicalis culture and identified several unique secreted proteins among a total of 780 proteins. By subjecting the mass spectrometry data derived from cell lysate and conditioned media to proteogenomic analysis, we identified 86 novel genes, 12 novel exons, and corrected 49 computationally-predicted gene models. To our knowledge, this is the first high-throughput proteomics study of C. tropicalis validating predicted protein coding genes and refining the current genome annotation. The findings may prove useful in future global health efforts to fight against Candida infections.
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- 2016
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42. Dysregulation of splicing proteins in head and neck squamous cell carcinoma.
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Radhakrishnan A, Nanjappa V, Raja R, Sathe G, Chavan S, Nirujogi RS, Patil AH, Solanki H, Renuse S, Sahasrabuddhe NA, Mathur PP, Prasad TS, Kumar P, Califano JA, Sidransky D, Pandey A, Gowda H, and Chatterjee A
- Subjects
- Alternative Splicing genetics, Biomarkers, Tumor genetics, Carcinoma, Squamous Cell pathology, Cell Line, Tumor, Cell Proliferation genetics, Gene Expression Regulation, Neoplastic, Head and Neck Neoplasms pathology, Humans, Neoplasm Proteins biosynthesis, Neoplasm Proteins genetics, Phosphorylation, Protein Serine-Threonine Kinases genetics, Proteomics, Squamous Cell Carcinoma of Head and Neck, Biomarkers, Tumor biosynthesis, Carcinogenesis genetics, Carcinoma, Squamous Cell genetics, Head and Neck Neoplasms genetics, Protein Serine-Threonine Kinases biosynthesis
- Abstract
Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.
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- 2016
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43. Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.
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Subbannayya T, Leal-Rojas P, Barbhuiya MA, Raja R, Renuse S, Sathe G, Pinto SM, Syed N, Nanjappa V, Patil AH, Garcia P, Sahasrabuddhe NA, Nair B, Guerrero-Preston R, Navani S, Tiwari PK, Santosh V, Sidransky D, Prasad TS, Gowda H, Roa JC, Pandey A, and Chatterjee A
- Subjects
- Biomarkers, Tumor genetics, Cell Line, Tumor, Cell Proliferation genetics, Cell Survival genetics, Early Detection of Cancer, Gallbladder Neoplasms diagnosis, Gallbladder Neoplasms pathology, Gene Expression Regulation, Neoplastic, Humans, Intramolecular Oxidoreductases genetics, Macrophage Migration-Inhibitory Factors genetics, Macrophages metabolism, Macrophages pathology, Neoplasm Proteins biosynthesis, Proteomics, Biomarkers, Tumor biosynthesis, Gallbladder Neoplasms genetics, Intramolecular Oxidoreductases biosynthesis, Macrophage Migration-Inhibitory Factors biosynthesis, Prognosis
- Abstract
Background: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer., Methods: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA., Results: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells., Conclusions: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.
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- 2015
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44. Quantitative phosphoproteomic analysis of IL-33-mediated signaling.
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Pinto SM, Nirujogi RS, Rojas PL, Patil AH, Manda SS, Subbannayya Y, Roa JC, Chatterjee A, Prasad TS, and Pandey A
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- Amino Acid Sequence, Animals, Cell Line, Macrophages chemistry, Mass Spectrometry, Mice, Molecular Sequence Data, Phosphopeptides analysis, Phosphopeptides immunology, Phosphoprotein Phosphatases analysis, Phosphoprotein Phosphatases immunology, Phosphorylation, Protein Interaction Maps, Protein Kinases analysis, Protein Kinases immunology, Proteomics, Interleukin-6 immunology, Macrophages immunology, Proteins analysis, Proteins immunology, Signal Transduction
- Abstract
Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
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45. Proteomic analysis of human vitreous humor.
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Murthy KR, Goel R, Subbannayya Y, Jacob HK, Murthy PR, Manda SS, Patil AH, Sharma R, Sahasrabuddhe NA, Parashar A, Nair BG, Krishna V, Prasad TK, Gowda H, and Pandey A
- Abstract
Background: The vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing metabolic nutrient requirements of the lens, coordinating eye growth and providing support to the retina. It is in close proximity to the retina and reflects many of the changes occurring in this tissue. The biochemical changes occurring in the vitreous could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. In this study, we investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry., Results: The vitreous humor was subjected to multiple fractionation techniques followed by LC-MS/MS analysis. We identified 1,205 proteins, 682 of which have not been described previously in the vitreous humor. Most proteins were localized to the extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). Classification based on molecular function showed that 27% had catalytic activity, 10% structural activity, 10% binding activity, 4% cell and 4% transporter activity. Categorization for biological processes showed 28% participate in metabolism, 20% in cell communication and 13% in cell growth. The data have been deposited to the ProteomeXchange with identifier PXD000957., Conclusion: This large catalog of vitreous proteins should facilitate biomedical research into pathological conditions of the eye including diabetic retinopathy, retinal detachment and cataract.
- Published
- 2014
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46. A draft map of the human proteome.
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Kim MS, Pinto SM, Getnet D, Nirujogi RS, Manda SS, Chaerkady R, Madugundu AK, Kelkar DS, Isserlin R, Jain S, Thomas JK, Muthusamy B, Leal-Rojas P, Kumar P, Sahasrabuddhe NA, Balakrishnan L, Advani J, George B, Renuse S, Selvan LD, Patil AH, Nanjappa V, Radhakrishnan A, Prasad S, Subbannayya T, Raju R, Kumar M, Sreenivasamurthy SK, Marimuthu A, Sathe GJ, Chavan S, Datta KK, Subbannayya Y, Sahu A, Yelamanchi SD, Jayaram S, Rajagopalan P, Sharma J, Murthy KR, Syed N, Goel R, Khan AA, Ahmad S, Dey G, Mudgal K, Chatterjee A, Huang TC, Zhong J, Wu X, Shaw PG, Freed D, Zahari MS, Mukherjee KK, Shankar S, Mahadevan A, Lam H, Mitchell CJ, Shankar SK, Satishchandra P, Schroeder JT, Sirdeshmukh R, Maitra A, Leach SD, Drake CG, Halushka MK, Prasad TS, Hruban RH, Kerr CL, Bader GD, Iacobuzio-Donahue CA, Gowda H, and Pandey A
- Subjects
- Adult, Cells, Cultured, Databases, Protein, Fetus metabolism, Fourier Analysis, Gene Expression Profiling, Genome, Human genetics, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Internet, Mass Spectrometry, Molecular Sequence Annotation, Open Reading Frames genetics, Organ Specificity, Protein Biosynthesis, Protein Isoforms analysis, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Sorting Signals, Protein Transport, Proteome analysis, Proteome chemistry, Proteome genetics, Pseudogenes genetics, RNA, Untranslated genetics, Reproducibility of Results, Untranslated Regions genetics, Proteome metabolism, Proteomics
- Abstract
The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.
- Published
- 2014
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47. Genome-wide Profiling of RNA splicing in prostate tumor from RNA-seq data using virtual microarrays.
- Author
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Srinivasan S, Patil AH, Verma M, Bingham JL, and Srivatsan R
- Abstract
Background: Second generation RNA sequencing technology (RNA-seq) offers the potential to interrogate genome-wide differential RNA splicing in cancer. However, since short RNA reads spanning spliced junctions cannot be mapped contiguously onto to the chromosomes, there is a need for methods to profile splicing from RNA-seq data. Before the invent of RNA-seq technologies, microarrays containing probe sequences representing exon-exon junctions of known genes have been used to hybridize cellular RNAs for measuring context-specific differential splicing. Here, we extend this approach to detect tumor-specific splicing in prostate cancer from a RNA-seq dataset., Method: A database, SPEventH, representing probe sequences of under a million non-redundant splice events in human is created with exon-exon junctions of optimized length for use as virtual microarray. SPEventH is used to map tens of millions of reads from matched tumor-normal samples from ten individuals with prostate cancer. Differential counts of reads mapped to each event from tumor and matched normal is used to identify statistically significant tumor-specific splice events in prostate., Results: We find sixty-one (61) splice events that are differentially expressed with a p-value of less than 0.0001 and a fold change of greater than 1.5 in prostate tumor compared to the respective matched normal samples. Interestingly, the only evidence, EST (BF372485), in the public database for one of the tumor-specific splice event joining one of the intron in KLK3 gene to an intron in KLK2, is also derived from prostate tumor-tissue. Also, the 765 events with a p-value of less than 0.001 is shown to cluster all twenty samples in a context-specific fashion with few exceptions stemming from low coverage of samples., Conclusions: We demonstrate that virtual microarray experiments using a non-redundant database of splice events in human is both efficient and sensitive way to profile genome-wide splicing in biological samples and to detect tumor-specific splicing signatures in datasets from RNA-seq technologies. The signature from the large number of splice events that could cluster tumor and matched-normal samples into two tight separate clusters, suggests that differential splicing is yet another RNA phenotype, alongside gene expression and SNPs, that can be exploited for tumor stratification.
- Published
- 2012
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