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Simple Summary: In this study, we carried out global proteomic profiling of esophageal squamous cell carcinoma and adjacent non-neoplastic esophageal tissue to identify putative biomarkers and targets. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. We also identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by 3q26 region, a known hotspot region in ESCC. Synoviolin 1 (SYVN1), a protein involved in endoplasmic reticulum (ER)-associated degradation and other ER stress response proteins were overexpressed in ESCC. SYVN1 could be a potential therapeutic target in ESCC. Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC. [ABSTRACT FROM AUTHOR]

Background: Tobacco consumption in smoking and non‐smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. Methods: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco—crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture‐based assays and mass spectrometry‐based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. Results: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle‐mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. Conclusions: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking. [ABSTRACT FROM AUTHOR]

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is known for its aggressiveness associated with poor prognosis. The molecular mechanisms underlying the invasion and metastasis are still poorly understood. An improved understanding of these mechanisms shall precede the development of new diagnostic tools and targeted therapies. We report an integrated approach using bioinformatics to predict candidate genes, coupled with proteomics and immunohistochemistry for validating their presence and involvement in OSCC pathways heralding invasion and metastasis. Four genes POSTN, TNC, CAV1 and FSCN1 were identified. A protein–protein interaction network analysis teamed with pathway analysis led us to propose the role of the identified genes in invasion and metastasis in OSCC. Further analyses of archived FFPE blocks of various grades of oral cancer was carried out using TMT-based mass spectrometry and immunohistochemistry. Results of this study expressed a strong communiqué and interrelationship between these candidate genes. This study emphasizes the significance of a molecular biomarker panel as a diagnostic tool and its correlation with the invasion and metastatic pathway of OSCC. An insight into the probable association of CAF's and these biomarkers in the evolution and malignant transformation of OSCC further magnifies the molecular-biological spectrum of OSCC tumour microenvironment. [ABSTRACT FROM AUTHOR]

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients.Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA).Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy.Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use. [ABSTRACT FROM AUTHOR]

The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm is incompletely understood. Aberrant protein expression may drive structural alterations of vasculature found in IA. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. To unravel differential protein expression in three clinical subgroups of IA patients: (1) unruptured aneurysm, (2) ruptured aneurysm without vasospasm, (3) ruptured aneurysm who developed vasospasm, we performed untargeted quantitative proteomic analysis of aneurysm tissue and serum samples from three subgroups of IA patients and control subjects. Candidate molecules were then validated in a larger cohort of patients using enzyme-linked immunosorbent assay. A total of 937 and 294 proteins were identified from aneurysm tissue and serum samples, respectively. Several proteins that are known to maintain structural integrity of vasculature were found to be dysregulated in the context of aneurysm. ORM1, a glycoprotein, was significantly upregulated in both tissue and serum samples of unruptured aneurysm patients. We employed a larger cohort of subjects (n = 26) and validated ORM1 as a potential biomarker for screening of unruptured aneurysms. Samples from ruptured aneurysms with vasospasm showed significant upregulation of MMP9, a protease, compared with ruptured aneurysms without vasospasm. We validated MMP9 as a potential biomarker for vasospasm in a larger cohort (n = 52). This study reports the first global proteomic analysis of the entire clinical spectrum of IA. Furthermore, this study suggests ORM1 and MMP9 as potential biomarkers for unruptured aneurysm and cerebral vasospasm, respectively. [ABSTRACT FROM AUTHOR]

BACKGROUND: Shisha smoking has been associated with multiple diseases including oral cancer. However, a mechanistic study to investigate alteration of secreted proteins in oral cells due to shisha smoking is lacking. OBJECTIVES: Elucidation of differentially secreted proteins by immortalized human normal oral keratinocytes (OKF6/TERT1) upon chronic exposure to shisha. METHODS: OKF6/TERT1 was chronically treated with 0.5% shisha extract for 8 months. Conditioned media from shisha treated (OKF6/TERT1-Shisha) and untreated (OKF6/TERT1-Parental) cells were subjected to TMT-based quantitative proteomic analysis. Bioinformatics analysis of differentially secreted proteins was carried out using SignalP, SecretomeP and TMHMM. Immunoblot validation of selected proteins was carried out to confirm the proteomics results. RESULTS: Proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha secretome resulted in the identification of 1,598 proteins, of which 218 proteins were found to be differentially secreted (⩾ 1.5-fold; p -value ⩽ 0.05) in shisha treated cells. Bioinformatics analysis using prediction tools showed secretory potential of differentially secreted proteins identified in OKF6/TERT1-Shisha. Western blotting validated the expression of AKR1C2, HSPH1 and MMP9 in OKF6/TERT1-Shisha secretome in agreement with proteomic data. CONCLUSION: This study serves as a useful resource to understand the effect of chronic shisha smoking on the milieu of secreted proteins of oral cells. In vivo studies are warranted to supplement our in vitro data to elucidate the role of these proteins as early diagnostic biomarkers for oral carcinogenesis among shisha smokers. [ABSTRACT FROM AUTHOR]

Shisha (water pipe) smoking is falsely believed to be a hazard-free habit and has become a major public health concern. Studies have reported shisha smoking to be associated with oral lesions, as well as carcinomas of the lung, esophagus, bladder, and pancreas. A deeper understanding of the underlying molecular mechanisms would contribute to identification of biomarkers for targeted public health screening, therapeutic innovation, and better prognosis of associated diseases. In this study, we have established an in vitro chronic cellular model of shisha-exposed oral keratinocytes to study the effect of shisha on oral cells. Normal nontransformed, immortalized oral keratinocytes were chronically exposed to shisha extract for 8 months. This resulted in significant increase in cellular proliferation and cell invasion in shisha-exposed cells compared to the parental cells. Quantitative proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha cells resulted in the identification of 5515 proteins. Forty-three differentially expressed proteins were found to be common across all conditions. Bioinformatic analysis of the dysregulated proteins identified in the proteomic study revealed dysregulation of interferon pathway, upregulation of proteins involved in cell growth, and downregulation of immune processes. The present findings reveal that chronic exposure of normal oral keratinocytes to shisha leads to cellular transformation and dysregulation of immune response. To the best of our knowledge, this is the first report that has developed a model of oral keratinocytes chronically exposed to shisha and identified proteomic alterations associated with shisha exposure. However, further research is required to evaluate the health burden of shisha smoking. [ABSTRACT FROM AUTHOR]

Highlights • Chronic exposure to diesel particulate extract/ its vapor cause global proteomic alterations in primary skin keratinocytes. • DPE/DPE vapor alter proteins involved in maintaining skin integrity in primary skin keratinocytes. • DPE vapor affects proteins associated with OXPHOS and cell migration in skin keratinocytes. • Similar effects are observed upon DPE/DPE vapor treatment in 2D and 3D skin model. • Vitamin E partially restores altered proteins in DPE/vapor exposed skin keratinocytes. Abstract Background Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress. Objective To investigate global proteomic alterations in diesel particulate extract (DPE)/its vapor exposed skin keratinocytes. Methods We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/DPE vapor on primary skin keratinocytes. Results We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p ≤ 0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/DPE vapor. Conclusions Our study highlights distinct adverse effects of chronic exposure to DPE/DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leptospira , the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira . The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions. [ABSTRACT FROM AUTHOR]

The importance of olfaction to human health and disease is often underappreciated. Olfactory dysfunction has been reported in association with a host of common complex diseases, including neurological diseases such as Alzheimer's disease and Parkinson's disease. For health, olfaction or the sense of smell is also important for most mammals, for optimal engagement with their environment. Indeed, animals have developed sophisticated olfactory systems to detect and interpret the rich information presented to them to assist in day-to-day activities such as locating food sources, differentiating food from poisons, identifying mates, promoting reproduction, avoiding predators, and averting death. In this context, the olfactory bulb is a vital component of the olfactory system receiving sensory information from the axons of the olfactory receptor neurons located in the nasal cavity and the first place that processes the olfactory information. We report in this study original observations on the human olfactory bulb proteome in healthy subjects, using a high-resolution mass spectrometry-based proteomic approach. We identified 7750 nonredundant proteins from human olfactory bulbs. Bioinformatics analysis of these proteins showed their involvement in biological processes associated with signal transduction, metabolism, transport, and olfaction. These new observations provide a crucial baseline molecular profile of the human olfactory bulb proteome, and should assist the future discovery of biomarker proteins and novel diagnostics associated with diseases characterized by olfactory dysfunction. [ABSTRACT FROM AUTHOR]

Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ophthalmology and visual health research have received relatively limited attention from the personalized medicine community, but this trend is rapidly changing. Postgenomics technologies such as proteomics are being utilized to establish a baseline biological variation map of the human eye and related tissues. In this context, the choroid is the vascular layer situated between the outer sclera and the inner retina. The choroidal circulation serves the photoreceptors and retinal pigment epithelium (RPE). The RPE is a layer of cuboidal epithelial cells adjacent to the neurosensory retina and maintains the outer limit of the blood-retina barrier. Abnormal changes in choroid-RPE layers have been associated with age-related macular degeneration. We report here the proteome of the healthy human choroid-RPE complex, using reverse phase liquid chromatography and mass spectrometry-based proteomics. A total of 5309 nonredundant proteins were identified. Functional analysis of the identified proteins further pointed to molecular targets related to protein metabolism, regulation of nucleic acid metabolism, transport, cell growth, and/or maintenance and immune response. The top canonical pathways in which the choroid proteins participated were integrin signaling, mitochondrial dysfunction, regulation of eIF4 and p70S6K signaling, and clathrin-mediated endocytosis signaling. This study illustrates the largest number of proteins identified in human choroid-RPE complex to date and might serve as a valuable resource for future investigations and biomarker discovery in support of postgenomics ophthalmology and precision medicine. [ABSTRACT FROM AUTHOR]

Cigarette smoking has been associated with multiple negative effects on human skin. Long-term physiological effects of cigarette smoke are through chronic and not acute exposure. Molecular alterations due to chronic exposure to cigarette smoke remain unclear. Primary human skin keratinocytes chronically exposed to cigarette smoke condensate (CSC) showed a decreased wound-healing capacity with an increased expression of NRF2 and MMP9. Using quantitative proteomics, we identified 4728 proteins, of which 105 proteins were overexpressed (≥2-fold) and 41 proteins were downregulated (≤2-fold) in primary skin keratinocytes chronically exposed to CSC. We observed an alteration in the expression of several proteins involved in maintenance of epithelial barrier integrity, including keratin 80 (5.3 fold, p value 2.5 · 10-7), cystatin A (3.6-fold, p value 3.2 · 10-3), and periplakin (2.4-fold, p value 1.2 · 10-8). Increased expression of proteins associated with skin hydration, including caspase 14 (2.2-fold, p value 4.7 · 10-2) and filaggrin (3.6-fold, p value 5.4 · 10-7), was also observed. In addition, we report differential expression of several proteins, including adipogenesis regulatory factor (2.5-fold, p value 1.3 · 10-3) and histone H1.0 (2.5-fold, p value 6.3 · 10-3) that have not been reported earlier. Bioinformatics analyses demonstrated that proteins differentially expressed in response to CSC are largely related to oxidative stress, maintenance of skin integrity, and anti-inflammatory responses. Importantly, treatment with vitamin E, a widely used antioxidant, could partially rescue adverse effects of CSC exposure in primary skin keratinocytes. The utility of antioxidant-based new dermatological formulations in delaying or preventing skin aging and oxidative damages caused by chronic cigarette smoke exposure warrants further clinical investigations and multi-omics research. [ABSTRACT FROM AUTHOR]

Background: Retinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma. Methods: We used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors. Results: We identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies. Conclusions: In the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma. [ABSTRACT FROM AUTHOR]

The annual economic burden of visual disorders in the United States was estimated at $139 billion. Ophthalmology is therefore one of the salient application fields of postgenomics biotechnologies such as proteomics in the pursuit of global precision medicine. Interestingly, the protein composition of the human iris tissue still remains largely unexplored. In this context, the uveal tract constitutes the vascular middle coat of the eye and is formed by the choroid, ciliary body, and iris. The iris forms the anterior most part of the uvea. It is a thin muscular diaphragm with a central perforation called pupil. Inflammation of the uvea is termed uveitis and causes reduced vision or blindness. However, the pathogenesis of the spectrum of diseases causing uveitis is still not very well understood. We investigated the proteome of the iris tissue harvested from healthy donor eyes that were enucleated within 6 h of death using high-resolution Fourier transform mass spectrometry. A total of 4959 nonredundant proteins were identified in the human iris, which included proteins involved in signaling, cell communication, metabolism, immune response, and transport. This study is the first attempt to comprehensively profile the global proteome of the human iris tissue and, thus, offers the potential to facilitate biomedical research into pathological diseases of the uvea such as Behcet's disease, Vogt Koyonagi Harada's disease, and juvenile rheumatoid arthritis. Finally, we make a call to the broader visual health and ophthalmology community that proteomics offers a veritable prospect to obtain a systems scale, functional, and dynamic picture of the eye tissue in health and disease. This knowledge is ultimately pertinent for precision medicine diagnostics and therapeutics innovation to address the pressing needs of the 21st century visual health. [ABSTRACT FROM AUTHOR]

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (). [ABSTRACT FROM AUTHOR]

The debilitating disease kala-azar or visceral leishmaniasis is caused by the kinetoplastid protozoan parasite Leishmania donovani . The parasite is transmitted by the hematophagous sand fly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with the transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes . Understanding the molecular interaction of the sand fly and Leishmania , during the development of parasite within the sand fly gut is crucial to the understanding of the parasite life cycle. The complete genome sequences of sand flies ( Phlebotomus and Lutzomyia ) are currently not available and this hinders identification of proteins in the sand fly vector. The current study utilizes a three frame translated transcriptomic data of P. papatasi in the absence of genomic sequences to analyze the mass spectrometry data of P. papatasi cell line using a proteogenomic approach. Additionally, we have carried out the proteogenomic analysis of P. papatasi by comparative homology-based searches using related sequenced dipteran protein data. This study resulted in the identification of 1313 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Proteins present in human saliva offer an immense potential for clinical applications. However, exploring salivary proteome is technically challenged due to the presence of amylase and albumin in high abundance. In this study, we used four workflows to analyze human saliva from healthy individuals which involved depletion of abundant proteins using affinity-based separation methods followed by protein or peptide fractionation and high resolution mass spectrometry analysis. We identified a total of 1256 human salivary proteins, 292 of them being reported for the first time. All identifications were verified for any shared proteins/peptides from the salivary microbiome that may conflict with the human protein identifications. On integration of our results with the analyses reported earlier, we arrived at an updated human salivary proteome containing 3449 proteins, 808 of them have been reported as differentially expressed proteins in oral cancer tissues. The secretory nature of 598 of the 808 proteins has also been supported on the basis of the presence of signal sequence, transmembrane domain or association with exosomes. From this subset, we provide a priority list of 139 proteins along with their proteotypic peptides, which may serve as a reference for targeted investigations as secretory markers for clinical applications in oral malignancies. This article is part of a Special Issue entitled: Proteomics in India. [ABSTRACT FROM AUTHOR]

The aqueous humor is a colorless, transparent fluid that fills the anterior chamber of the eye. It plays an important role in maintaining the intraocular pressure and providing nourishment to the lens and cornea. The constitution of the aqueous humor is controlled by the blood-aqueous barrier. Though this ocular fluid has been extensively studied, its role in ocular physiology is still not completely understood. In this study, aqueous humor samples were collected from 250 patients undergoing cataract surgery, subjected to multiple fractionation strategies and analyzed on a Fourier transform LTQ-Orbitrap Velos mass spectrometer. In all, we identified 763 proteins, of which 386 have been identified for the first time in this study. Sorbitol dehydrogenase (SORD), filensin (BFSP1), and phakinin (BFSP2) are some of the proteins that have not been previously reported in the aqueous humor. Gene Ontology analysis revealed 35% of the identified proteins to be extracellular, with a majority of them involved in cell communication and signal transduction. This study comprehensively reports 386 novel proteins that have important potential as biomarker candidates for future research into personalized medicine and diagnostics aimed towards improving visual health. [ABSTRACT FROM AUTHOR]

Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes. [ABSTRACT FROM AUTHOR]

Dicer is a crucial RNase III enzyme in miRNA biogenesis pathway. Although numerous studies have been carried out to investigate the role of miRNAs and Dicer in the regulation of biological processes, few studies have examined proteomic alterations upon knockout of Dicer. We employed a Cre--loxP-based inducible knockout mouse system to investigate the proteome regulated by Dicer-dependent miRNAs. We utilized spiked liver lysates from metabolically labeled mice to quantify the subtle changes in the liver proteome upon deletion of Dicer. We identified 2137 proteins using high resolution tandem mass spectrometry analysis. The upregulated proteins included several enzymes involved in peroxisomal β-oxidation of fatty acids and a large majority of the upregulated proteins involved in lipid metabolism were known PPARα targets. MRM-based assays were carried out to confirm the upregulation of enzymes including peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, cytochrome P450 3A13, cytochrome P450 3A41 and myristoylated alanine-rich protein kinase C substrate. Further, miRNA-124 which is predicted to regulate expression of peroxisomal bifunctional enzyme was confirmed to be downregulated in the Dicer knockout mice. Our study demonstrates the strength of coupling knockout mouse models and quantitative proteomic strategies to investigate functions of individual proteins in vivo. [ABSTRACT FROM AUTHOR]

Anopheles gambiae has a well-adapted system for host localization, feeding, and mating behavior, which are all governed by neuronal processes in the brain. However, there are no published reports characterizing the brain proteome to elucidate neuronal signaling mechanisms in the vector. To this end, a large-scale mapping of the brain proteome of An. gambiae was carried out using high resolution tandem mass spectrometry, revealing a repertoire of >1800 proteins, of which 15% could not be assigned any function. A large proportion of the identified proteins were predicted to be involved in diverse biological processes including metabolism, transport, protein synthesis, and olfaction. This study also led to the identification of 10 GPCR classes of proteins, which could govern sensory pathways in mosquitoes. Proteins involved in metabolic and neural processes, chromatin modeling, and synaptic vesicle transport associated with neuronal transmission were predominantly expressed in the brain. Proteogenomic analysis expanded our findings with the identification of 15 novel genes and 71 cases of gene refinements, a subset of which were validated by RT-PCR and sequencing. Overall, our study offers valuable insights into the brain physiology of the vector that could possibly open avenues for intervention strategies for malaria in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: Most solid tumours, including melanoma (skin cancer), are riddled with areas lacking adequate oxygen supply due to insufficient vasculature. Cancer cells in these regions are resistant to therapies and contribute to cancer spread and poor treatment response in patients. Understanding the mechanisms by which cancer cells adapt to survive in such a hostile environment will provide novel avenues for treatment. In this study, we investigated mechanisms that melanoma cells use to adapt and survive in an oxygen-poor environment. We used four different melanoma cell lines and studied how protein levels and phosphorylation patterns on thousands of proteins change when the cells are exposed to poor oxygen conditions. This revealed potential mechanisms on which cancer cells are dependent for survival. These survival mechanisms can be potentially targeted to achieve durable response to therapy. We demonstrate this by targeting one such mechanism required for cancer cell survival. Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin–proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies. [ABSTRACT FROM AUTHOR]

Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer. [ABSTRACT FROM AUTHOR]