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Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets., 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 © 2024 Elsevier Inc. All rights reserved.)

Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

In recent years, proteogenomics and ribosome profiling studies have identified a large number of proteins encoded by noncoding regions in the human genome. They are encoded by small open reading frames (sORFs) in the untranslated regions (UTRs) of mRNAs and long non-coding RNAs (lncRNAs). These sORF encoded proteins (SEPs) are often <150AA and show poor evolutionary conservation. A subset of them have been functionally characterized and shown to play an important role in fundamental biological processes including cardiac and muscle function, DNA repair, embryonic development and various human diseases. How many novel protein-coding regions exist in the human genome and what fraction of them are functionally important remains a mystery. In this review, we discuss current progress in unraveling SEPs, approaches used for their identification, their limitations and reliability of these identifications. We also discuss functionally characterized SEPs and their involvement in various biological processes and diseases. Lastly, we provide insights into their distinctive features compared to canonical proteins and challenges associated with annotating these in protein reference databases., 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 © 2023. Published by Elsevier Inc.)

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tyrosine phosphorylation on proteins is an important posttranslational modification that regulates various processes in cells. Mass spectrometry-based phosphotyrosine profiling can reveal tyrosine kinase signaling activity in cells. Using quantitative proteomics strategies such as stable isotope labeling with amino acids in cell culture (SILAC) allows comparison of tyrosine kinase signaling activity across two to -three different conditions. In this book chapter, we discuss the reagents required and a step-by-step protocol to carry out phosphotyrosine profiling using SILAC., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

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.

Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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.

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7., Competing Interests: The authors have declared that no competing interests exist.

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.

Virulence-associated protein B and C toxin-antitoxin (TA) systems are widespread in prokaryotes, but their precise role in physiology is poorly understood. We have functionally characterized the VapBC22 TA system from Mycobacterium tuberculosis . Transcriptome analysis revealed that overexpression of VapC22 toxin in M. tuberculosis results in reduced levels of metabolic enzymes and increased levels of ribosomal proteins. Proteomics studies showed reduced expression of virulence-associated proteins and increased levels of cognate antitoxin, VapB22 in the Δ vapC22 mutant strain. Furthermore, both the Δ vapC22 mutant and VapB22 overexpression strains of M. tuberculosis were susceptible to killing upon exposure to oxidative stress and showed attenuated growth in guinea pigs and mice. Host transcriptome analysis suggests upregulation of the transcripts involved in innate immune responses and tissue remodeling in mice infected with the Δ vapC22 mutant strain. Together, we demonstrate that the VapBC22 TA system belongs to a key regulatory network and is essential for M. tuberculosis pathogenesis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Purpose : Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods : We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo . Results : We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions : Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

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.

Therapy resistance and recurrence in Glioblastoma is due to the presence of residual radiation resistant cells. However, because of their inaccessibility from patient biopsies, the molecular mechanisms driving their survival remain unexplored. Residual Radiation Resistant (RR) and Relapse (R) cells were captured using cellular radiation resistant model generated from patient derived primary cultures and cell lines. iTRAQ based quantitative proteomics was performed to identify pathways unique to RR cells followed by in vitro and in vivo experiments showing their role in radio-resistance. 2720 proteins were identified across Parent (P), RR and R population with 824 and 874 differential proteins in RR and R cells. Unsupervised clustering showed proteasome pathway as the most significantly deregulated pathway in RR cells. Concordantly, the RR cells displayed enhanced expression and activity of proteasome subunits, which triggered NFkB signalling. Pharmacological inhibition of proteasome activity led to impeded NFkB transcriptional activity, radio-sensitization of RR cells in vitro , and significantly reduced capacity to form orthotopic tumours in vivo . We demonstrate that combination of proteasome inhibitor with radio-therapy abolish the inaccessible residual resistant cells thereby preventing GBM recurrence. Furthermore, we identified first proteomic signature of RR cells that can be exploited for GBM therapeutics., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Cigarette smoke and chewing tobacco are well known risk factors associated with ESCC. However, molecular mechanisms associated with development of ESCC among smokers and chewers are poorly understood. MicroRNAs play an important role in regulating physiological and disease processes including esophageal cancer., Objective and Methods: In this study, we developed an in vitro model by treating non-neoplastic Het- 1A esophageal cell line with cigarette smoke and chewing tobacco. We carried out miRNA sequencing on Illumina HiSeq 2500 platform and compared miRNA expression pattern across cigarette smoke and chewing tobacco treated Het-1A cells with untreated cells., Results: We identified and quantified 433 miRNAs in both smoke exposed and chewing tobacco treated cells, of which 13 miRNAs showed significantly altered expression in cigarette smoke exposed cells while 25 miRNAs showed significantly altered expression in chewing tobacco treated cells. In addition, we predicted novel miRNAs from these data-sets. We evaluated miRNAs that showed selective or context dependent expression pattern in cigarette smoke exposed or chewing tobacco treated cells., Conclusion: In this study, we have comprehensively mapped miRNA expression pattern in response to cigarette smoke and chewing tobacco in Het-1A cells. We identified miRNAs that show altered expression in these cell models., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine 73 (head-domain) and Serine 431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1 T14,Y15 , EIF4EBP1 T46,T50 , EIF4B S422 , AKT1S1 T246,S247 , CTTN1 T401,S405,Y421 , and CAP1 S307/309 in K8-S73A/D mutant and CTTN1 T401,S405,Y421 , BUB1B S1043 , and CARHSP1 S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYC S176 , ZYX S344 , and PNN S692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

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.

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.

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.

A facile synthesis based on the addition of ascorbic acid to a mixture of Na2 PdCl4, K2 PtCl6, and Pluronic P123 results in highly branched core-shell nanoparticles (NPs) with a micro-mesoporous dandelion-like morphology comprising Pd core and Pt shell. The slow reduction kinetics associated with the use of ascorbic acid as a weak reductant and suitable Pd/Pt atomic ratio (1:1) play a principal role in the formation mechanism of such branched Pd@Pt core-shell NPs, which differs from the traditional seed-mediated growth. The catalyst efficiently achieves the reduction of a variety of olefins in good to excellent yields. Importantly, higher catalytic efficiency of dandelion-like Pd@Pt core-shell NPs was observed for the olefin reduction than commercially available Pt black, Pd NPs, and physically admixed Pt black and Pd NPs. This superior catalytic behavior is not only due to larger surface area and synergistic effects but also to the unique micro-mesoporous structure with significant contribution of mesopores with sizes of several tens of nanometers., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Superhydrophobic/superoleophilic composites HFGO@ZIF-8 have been prepared from highly fluorinated graphene oxide (HFGO) and the nanocrystalline zeolite imidazole framework ZIF-8. The structure-directing and coordination-modulating properties of HFGO allow for the selective nucleation of ZIF-8 nanoparticles at the graphene surface oxygen functionalities. This results in localized nucleation and size-controlled ZIF-8 nanocrystals intercalated in between HFGO layers. The composite microstructure features fluoride groups bonded at the graphene. Self-assembly of a unique micro-mesoporous architecture is achieved, where the micropores originate from ZIF-8 nanocrystals, while the functionalized mesopores arise from randomly organized HFGO layers separated by ZIF-8 nanopillars. The hybrid material displays an exceptional high water contact angle of 162° and low oil contact angle of 0° and thus reveals very high sorption selectivity, fast kinetics, and good absorbencies for nonpolar/polar organic solvents and oils from water. Accordingly, Sponge@HFGO@ZIF-8 composites are successfully utilized for oil-water separation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Annotation of protein coding genes in sequenced genomes has been routinely carried out using gene prediction programs guided by available transcript data. The advent of mass spectrometry has enabled the identification of proteins in a high-throughput manner. In addition to searching proteins annotated in public databases, mass spectrometry data can also be searched against conceptually translated genome as well as transcriptome to identify novel protein coding regions. This proteogenomics approach has resulted in the identification of novel protein coding regions in both prokaryotic and eukaryotic genomes. These studies have also revealed that some of the annotated noncoding RNAs and pseudogenes code for proteins. This approach is likely to become a part of most genome annotation workflows in the future. Here we describe a general methodology and approach that can be used for proteogenomics.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with poor prognosis. Here, we carried out liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based untargeted metabolomic analysis of ESCC serum samples. Statistical analysis resulted in the identification of 652 significantly dysregulated molecular features in serum from ESCC patients as compared to the healthy subjects. Phosphatidylcholines were identified as a major class of dysregulated metabolites in this study suggesting potential perturbation of phosphocholine metabolism in ESCC. By using a targeted MS/MS approach both in positive and negative mode, we were able to characterize and confirm the structure of seven metabolites. Our study describes a quantitative LC-MS approach for characterizing dysregulated lipid metabolism in ESCC., Biological Significance: Altered metabolism is a hallmark of cancer. We carried out (LC-MS)-based untargeted metabolomic profiling of serum from esophageal squamous cell carcinoma (ESCC) patients to characterize dysregulated metabolites. Phosphatidylcholine metabolism was found to be significantly altered in ESCC. Our study illustrates the use of mass spectrometry-based metabolomic analysis to characterize molecular alterations associated with ESCC. This article is part of a Special Issue entitled: Proteomics in India., (Copyright © 2015. Published by Elsevier B.V.)

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.

Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation., (© 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The in situ synthesis of air-stable zero-valent iron nanoparticles (NZVI) embedded in cellulose fibers leads to the assembly of highly reactive magnetic filter papers. These engineered materials display a wide range of applications in the treatment of wastewater and drinking water, including chromium removal, phenol degradation, environmental bioremediation, and catalysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cationic quaternized carbon dots (QCDs) and anionic graphene oxide sheets (GO) are combined via non-covalent interactions following a self-assembly pathway to form highly biocompatible and fluorescent hybrid materials. These hybrids act as selective probes with controlled labelling of the cell nucleus or cytoplasm depending on the QCD loading.

Chromosome-centric human proteome project (C-HPP) is a global initiative to comprehensively characterize proteins encoded by genes across all human chromosomes by teams focusing on individual chromosomes. Here, we report mass spectrometry-based identification and characterization of proteins encoded by genes on chromosome 12. Our study is based on proteomic profiling of 30 different histologically normal human tissues and cell types using high-resolution mass spectrometry. In our analysis, we identified 1,535 proteins encoded by 836 genes on human chromosome 12. This includes 89 genes that are designated as "missing proteins" by "neXtProt" as they did not have any prior evidence either by mass spectrometry or by antibody-based detection methods. We identified several variant peptides that reflected coding SNPs annotated in dbSNP database. We also confirmed the start sites of ∼200 proteins by identifying protein N-terminal acetylated peptides. We also identified alternative start sites for 11 proteins that were not annotated in public databases until now. Most importantly, we identified 12 novel protein coding regions on chromosome 12 using our proteogenomics strategy. All of the 12 regions have been annotated as pseudogenes in public databases. This study demonstrates that there is scope for significantly improving annotation of protein coding genes in the human genome using mass-spectrometry-derived data. Individual efforts as part of C-HPP initiative should significantly contribute toward enriching human protein annotation. The data have been deposited to ProteomeXchange with identifier PXD000561.

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.

The aqueous dispersion of graphene or reduced graphene oxide (RGO) is very much important to realize the full potential of these materials in many fields. Herein we present a simple route to prepare highly water dispersible aminoclay-RGO (AC-RGO) hybrids by the in situ condensation of aminoclay over graphene oxide (GO) followed by reduction with hydrazine hydrate. The resultant hybrids are stable in aqueous media even at concentrations up to 7.5 mg RGO per mL. To the best of our knowledge this is the highest concentration of an aqueous dispersion of RGO. Significantly, the hybrids are amphiphilic in nature and show simultaneous adsorption of Cytochrome C through hydrophobic interaction and DNA through electrostatic interaction. This strategy opens up new possibilities for the prospect of RGO in catalysis and biomedical applications.

The 21 cm background from the epoch of reionization is a promising cosmological probe: line-of-sight velocity fluctuations distort redshift, so brightness fluctuations in Fourier space depend upon angle, which linear theory shows can separate cosmological from astrophysical information. Nonlinear fluctuations in ionization, density, and velocity change this, however. The validity and accuracy of the separation scheme are tested here for the first time, by detailed reionization simulations. The scheme works reasonably well early in reionization (≲40% ionized), but not late (≳80% ionized).

Herein, we report a facile route for the synthesis of foam-like porous oxides using carbon spheres and polyvinylpyrrolidone (PVP) as sacrificial templates. The as-prepared porous structures were characterized by XRD, FESEM, TEM and BET methods. These foam-like macroporous oxides are formed through the fusion of oxide nanoparticles around the gas bubbles liberated during the combustion of composites and show good BET surface areas.

Aminoclay supported copper nanoparticles are effective in promoting [3+2] cycloaddition of azides with terminal alkynes to produce the corresponding 1,2,3-triazoles in excellent yields. The copper nanoparticles are highly reactive in water and can be recycled for four cycles with consistent activity.

Highly pure and solution processable white-light-emitting hybrids are presented. These soft-hybrids are designed by an organic-inorganic supramolecular co-assembly in water. White-light emission is achieved by partial energy transfer (ET) between donor and acceptor molecules anchored on the inorganic component. The unique and remarkable processability feature of these hybrids is demonstrated by painting/writing onto large glass and flexible plastic substrates., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work demonstrates the use of amino functionalized Mg-phyllosilicate clay/Nafion nanocomposite film embedded with Pt nanoparticles (Pt/AC/N) for catalyzing oxygen reduction reaction (ORR) in sulphuric acid medium. Pt/AC/N nanocomposite films were surface characterized using transmission electron microscope. Cyclic and linear scan voltammetry studies were carried out under hydrodynamic conditions taking rotating-ring disc electrode (RRDE) as the working electrode. The effects of clay content, Pt mass loading, electrode rotation rate, and temperature on the ORR kinetics were studied. The Tafel slopes were found to vary between 118 and 126 mV dec(-1) indicating a good ORR kinetics. The exchange current density values calculated after mass transfer correction ranged from 5.8×10(-7) to 2.4×10(-6) A cm(-2). From the RRDE disc currents, Koutecky-Levich plots were constructed and the ORR mechanism was found to follow a four electron path with minimum H(2)O(2) formation of ∼1.6%. The effect of temperature on ORR kinetics was found at 25, 40, and 50°C. The energy of activation calculated to be 7.68 kJ mol(-1) and comparable to the standard Pt/C catalyzed ORR systems., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A novel Pt nanoparticle (Pt NP) embedded aminoclay/Nafion (Pt/AC/N) nanocomposite catalyst film was prepared for oxygen reduction reaction by sol-gel method. The prepared nanocomposite films were surface characterized using XRD and TEM and thermal stability was studied by TGA. The prepared film has firmly bound Pt NP and could exhibit an improved electro-reduction activity compared to vulcan carbon/Nafion supported Pt NP (Pt/VC/N). Moreover, the Pt/AC/N film possessed good stability in the acidic environment. The limiting current density of the Pt/AC/N film with 35.4 μg/cm(2) of Pt loading was found to be 4.2 mA/cm(2), which is 30% higher than that of the Pt/VC/N. The maximum H2O2 intermediate formation was found to be ∼1.6% and the reaction found to follow a four electron transfer mechanism. Accelerated durability test for 2000 potential cycles showed that ca. 78% of initial limiting current was retained. The results are encouraging for possible use of the Pt/AC/N as the free-standing electrocatalyst layer for polymer electrolyte membrane fuel cells.