Wheat TaNACα18 functions as a positive regulator of high-temperature adaptive responses and improves cell defense machinery.

Global wheat production amounted to >780 MMT during 2022-2023 whose market size are valued at >$128 billion. Wheat is highly susceptible to high-temperature stress (HTS) throughout the life cycle and its yield declines 5-7% with the rise in each degree of temperature. Previously, we reported an array of HTS-response markers from a resilient wheat cv. Unnat Halna and described their putative role in heat acclimation. To complement our previous results and identify the key determinants of thermotolerance, here we examined the cytoplasmic proteome of a sensitive cv. PBW343. The HTS-triggered metabolite reprograming highlighted how proteostasis defects influence the formation of an integrated stress-adaptive response. The proteomic analysis identified several promising HTS-responsive proteins, including a NACα18 protein, designated TaNACα18, whose role in thermotolerance remains unknown. Dual localization of TaNACα18 suggests its crucial functions in the cytoplasm and nucleus. The homodimerization of TaNACα18 anticipated its function as a transcriptional coactivator. The complementation of TaNACα18 in yeast and overexpression in wheat demonstrated its role in thermotolerance across the kingdom. Altogether, our results suggest that TaNACα18 imparts tolerance through tight regulation of gene expression, cell wall remodeling and activation of cell defense responses.

Protein kinase PfPK2 mediated signalling is critical for host erythrocyte invasion by malaria parasite.

Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum. We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.

PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.

A signaling network map of Lipoxin (LXA4): an anti-inflammatory molecule.

Lipoxins (LXs) are a class of endogenous bioactive lipid mediators that are involved in the regulation of inflammation. They exert immunomodulatory effects by regulating the behaviour of various immune cells, including neutrophils, macrophages, and T and B cells, by promoting the clearance of apoptotic neutrophils. This helps to dampen inflammation and promote tissue repair. LXs regulate the expression of many inflammatory genes by modulating the levels of transcription factors, such as nuclear factor κB (NF-κB), activator protein-1 (AP-1), nerve growth factor-regulated factor 1A binding protein 1 (NGF), and peroxisome proliferator activated receptor γ (PPAR-γ), which are elevated in various diseases, such as respiratory tract diseases, renal diseases, cancer, neurodegenerative diseases, and viral infections. Lipoxin-mediated signaling is involved in chronic inflammation, cancer, diabetes-associated kidney disease, lung injury, liver injury, endometriosis, respiratory tract diseases, neurodegenerative diseases, chronic cerebral hypoperfusion, and retinal degeneration. In this study, we systematically investigated the intricate network of lipoxin signaling by analyzing the relevant literature. The resulting map comprised 467 molecules categorized as activation/inhibition, enzyme catalysis, gene and protein expression, molecular associations, and translocation events. This map serves as a valuable resource for understanding the complexity of lipoxin signaling and its impact on various cellular functions.

Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii.

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.

Mining proteomics data to extract post-translational modifications associated with gastric cancer.

Gastric cancers are highly heterogeneous, deep-seated tumours associated with late diagnosis and poor prognosis. Post-translational modifications (PTMs) of proteins are known to be well-associated with oncogenesis and metastasis in most cancers. Several enzymes which drive PTMs have also been used as theranostics in cancers of the breast, ovary, prostate and bladder. However, there is limited data on PTMs in gastric cancers. Considering that experimental protocols for simultaneous analysis of multiple PTMs are being explored, a data-driven approach involving reanalysis of mass spectrometry-derived data is useful in cataloguing altered PTMs. We subjected publicly available mass spectrometry data on gastric cancer to an iterative searching strategy for fetching PTMs including phosphorylation, acetylation, citrullination, methylation and crotonylation. These PTMs were catalogued and further analyzed for their functional enrichment through motif analysis. This value-added approach delivered identification of 21,710 unique modification sites on 16,364 modified peptides. Interestingly, we observed 278 peptides corresponding to 184 proteins to be differentially abundant. Using bioinformatics approaches, we observed that majority of these altered PTMs/proteins belonged to cytoskeletal and extracellular matrix proteins, which are known to be perturbed in gastric cancer. The dataset derived by this mutiPTM investigation can provide leads to further investigate the potential role of altered PTMs in gastric cancer management.

6-Methylcoumarin rescues bacterial quorum sensing induced ribosome-inactivating stress in Caenorhabditis elegans.

INTRODUCTION: Bacterial pathogenicity has for long posed severe effects on patient care. Pseudomonas aeruginosa is a common cause of hospital-acquired infections and nosocomial illnesses. It is known to infect the host by colonizing through quorum sensing and the production of exotoxins. METHODS: The current effort is an analysis of proteomic alterations caused by P. aeruginosa PAO1 to study the effects of quorum sensing inhibitor 6-Methylcoumarin on PAO1 infectivity in the Caenorhabditis elegans model. RESULTS: Through tandem mass tag-based quantitative proteomics approaches, 229 proteins were found to be differentially regulated in infection and upon inhibition. Among these, 34 proteins were found to be dysregulated in both infection and quorum-sensing inhibition conditions. Along with the dysregulation of proteins involved in host-pathogen interaction, PAO1 was found to induce ribosome-inactivating stress accompanied by the downregulating mitochondrial proteins. This in turn caused dysregulation of apoptosis. The expression of multiple proteins involved in ribosome biogenesis and structure, oxidative phosphorylation, and mitochondrial enzymes were altered due to infection. This mechanism, adapted by PAO1 to survive in the host, was inhibited by 6-Methylcoumarin by rescuing the downregulation of ribosomal and mitochondrial proteins. CONCLUSIONS: Taken together, the data reflect the molecular alterations due to quorum sensing and the usefulness of inhibitors in controlling pathogenesis.

A comprehensive review on current understanding of bradykinin in COVID-19 and inflammatory diseases.

Bradykinin, a member of the kallikrein-kinin system (KKS), is a potent, short-lived vasoactive peptide that acts as a vasodilator and an inflammatory mediator in a number of signaling mechanisms. Bradykinin induced signaling is mediated through kinin B1 (BDKRB1) and B2 (BDKRB2) transmembrane receptors coupled with different subunits of G proteins (Gαi/Gα0, Gαq and Gß1γ2). The bradykinin-mediated signaling mechanism activates excessive pro-inflammatory cytokines, including IL-6, IL-1ß, IL-8 and IL-2. Upregulation of these cytokines has implications in a wide range of clinical conditions such as inflammation leading to fibrosis, cardiovascular diseases, and most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In SARS-CoV-2 infection, bradykinin is found to be at raised levels and is reported to trigger a diverse array of symptoms. All of this brings bradykinin to the core point as a molecule of immense therapeutic value. Our understanding of its involvement in various pathways has expanded with time. Therefore, there is a need to look at the overall picture that emerges from the developments made by deciphering the bradykinin mediated signaling mechanisms involved in the pathological conditions. It will help devise strategies for developing better treatment modalities in the implicated diseases. This review summarizes the current state of knowledge on bradykinin mediated signaling in the diverse conditions described above, with a marked emphasis on the therapeutic potential of targeting the bradykinin receptor.

Comprehensive Proteomic Analysis of Brucella melitensis ATCC23457 Strain Reveals Metabolic Adaptations in Response to Nutrient Stress.

In the present study, a comprehensive proteomic analysis of Brucella melitensis (B. melitensis) strain ATCC23457 was carried out to investigate proteome alterations in response to in vitro-induced nutrient stress. Our analysis resulted in the identification of 2440 proteins, including 365 hypothetical proteins and 850 potentially secretory proteins representing ~77.8% of the B. melitensis proteome. Utilizing a proteogenomics approach, we provide translational evidence for eight novel putative protein-coding genes and confirmed the coding potential of 31 putatively annotated pseudogenes, thus refining the existing genome annotation. Further, using a label-free quantitative proteomic approach, new insights into the cellular processes governed by nutrient stress, including enrichment of amino acid metabolism (E), transcription (K), energy production and conversion (C), and biogenesis (J) processes were obtained. Pathway analysis revealed the enrichment of survival and homeostasis maintenance pathways, including type IV secretion system, nitrogen metabolism, and urease pathways in response to nutrient limitation. To conclude, our analysis demonstrates the utility of in-depth proteomic analysis in enabling improved annotation of the B. melitensis genome. Further, our results indicate that B. melitensis undergoes metabolic adaptations during nutrient stress similar to other Brucella. sp, and adapts itself for long-term persistence and survival.

Deciphering metabolomic alterations in seminal plasma of crossbred (Bos taurus X Bos indicus) bulls through comparative deep metabolomic analysis.

The incidence of sub-fertility is higher in crossbred bulls compared to zebu bulls. In the present study, we analysed the metabolomic profile of seminal plasma from crossbred and zebu bulls and uncovered differentially expressed metabolites between these two breeds. Using a high-throughput LC-MS/MS-based approach, we identified 990 and 1,002 metabolites in crossbred and zebu bull seminal plasma respectively. After excluding the exogenous metabolites, we found that 50 and 68 putative metabolites were unique to crossbred and zebu bull seminal plasma, respectively, whilst 87 metabolites were common to both. After data normalisation, 63 metabolites were found to be dysregulated between crossbred and zebu bull seminal plasma. Observed pathways included Linoleic acid metabolism (observed metabolite was phosphatidylcholine) in crossbred bull seminal plasma whereas inositol phosphate metabolism (observed metabolites were phosphatidylinositol-3,4,5-trisphosphate/inositol 1,3,4,5,6-pentakisphosphate/myo-inositol hexakisphosphate) was observed in zebu bull seminal plasma. Abundance of Tetradecanoyl-CoA was significantly higher, whilst abundance of Taurine was significantly lower in crossbred bull seminal plasma. In conclusion, the present study established the seminal plasma metabolomic profile in crossbred and zebu bulls and suggest that increased lipid peroxidation coupled with low concentrations of antioxidants in seminal plasma might be associated with high incidence of sub-fertility in crossbred bulls.

Molecular Profiling Associated with Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2)-Mediated Carcinogenesis in Gastric Cancer.

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. We showed previously that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), a serine-threonine kinase, is highly expressed in gastric cancer and leads to progression. In the present study, we identified the molecular networks involved in CAMKK2-mediated progression of gastric adenocarcinoma. Treatment of gastric cancer cell lines with a CAMKK2 inhibitor, STO-609, resulted in decreased cell migration, invasion, and colony-forming ability and a G1/S-phase arrest. In addition, tandem mass tag (TMT)-based quantitative proteomic analysis resulted in the identification of 7609 proteins, of which 219 proteins were found to be overexpressed and 718 downregulated (1.5-fold). Our data identified several key downregulated proteins involved in cell division and cell proliferation, which included DNA replication licensing factors, replication factor C, origin recognition complex, replication protein A and GINS, and mesenchymal markers, upon CAMKK2 inhibition. Immunoblotting and immunofluorescence results showed concordance with our mass spectroscopy data. Taken together, our study supports CAMKK2 as a novel therapeutic target in gastric cancer.

Hyperactivation of MEK/ERK pathway by Ca2+ /calmodulin-dependent protein kinase kinase 2 promotes cellular proliferation by activating cyclin-dependent kinases and minichromosome maintenance protein in gastric cancer cells.

Although CAMKK2 is overexpressed in several cancers, its role and relevant downstream signaling pathways in gastric cancer (GC) are poorly understood. Treatment of AGS GC cells with a CAMKK2 inhibitor, STO-609, resulted in decreased cell proliferation, cell migration, invasion, colony-forming ability, and G1/S-phase arrest. Quantitative phosphoproteomics in AGS cells with the CAMKK2 inhibitor led to the identification of 9603 unique phosphosites mapping to 3120 proteins. We observed decreased phosphorylation of 1101 phosphopeptides (1.5-fold) corresponding to 752 proteins upon CAMKK2 inhibition. Bioinformatics analysis of hypo-phosphorylated proteins revealed enrichment of MAPK1/MAPK3 signaling. Kinase enrichment analysis of hypo-phosphorylated proteins using the X2K Web tool identified ERK1, cyclin-dependant kinase 1 (CDK1), and CDK2 as downstream substrates of CAMKK2. Moreover, inhibition of CAMKK2 and MEK1 resulted in decreased phosphorylation of ERK1, CDK1, MCM2, and MCM3. Immunofluorescence results were in concordance with our mass spectroscopy data and Western blot analysis results. Taken together, our data reveal the essential role of CAMKK2 in the pathobiology of GC through the activation of the MEK/ERK1 signaling cascade.

Preliminary comparative deep metabolomic analysis of spermatozoa from zebu and crossbred cattle suggests associations between metabolites, sperm quality and fertility.

Poor semen quality and infertility/subfertility are more frequent in crossbred than zebu bulls. Using a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach, we established the preliminary metabolomic profile of crossbred and zebu bull spermatozoa (n=3 bulls each) and identified changes in sperm metabolomics between the two groups. In all, 1732 and 1240 metabolites were detected in zebu and crossbred bull spermatozoa respectively. After excluding exogenous metabolites, 115 and 87 metabolites were found to be unique to zebu and crossbred bull spermatozoa respectively whereas 71 metabolites were common to both. In the normalised data, 49 metabolites were found to be differentially expressed between zebu and crossbred bull spermatozoa. The significantly enriched (P<0.05) pathways in spermatozoa were taurine and hypotaurine metabolism (observed metabolites taurine and hypotaurine) in zebu and glycerophospholipid metabolism (observed metabolites phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine) in crossbred bulls. The abundance of nitroprusside (variable importance in projection (VIP) score >1.5) was downregulated, whereas that of l-cysteine, acetyl coenzyme A and 2'-deoxyribonucleoside 5'-diphosphate (VIP scores >1.0) was upregulated in crossbred bull spermatozoa. In conclusion, this study established the metabolomic profile of zebu and crossbred bull spermatozoa and suggests that aberrations in taurine, hypotaurine and glycerophospholipid metabolism may be associated with the higher incidence of infertility/subfertility in crossbred bulls.

Triton X-114 Fractionated Subcellular Proteome of Leptospira interrogans Shows Selective Enrichment of Pathogenic and Outer Membrane Proteins in the Detergent Fraction.

The Triton X-114-based solubilization and temperature-dependent phase separation of proteins is used for subcellular fractionation where, aqueous, detergent, and pellet fractions represents cytoplasmic, outer membrane (OM), and inner membrane proteins, respectively. Mass spectrometry-based proteomic analysis of Triton X-114 fractions of proteomic analysis of Leptospira interrogans identified 2957 unique proteins distributed across the fractions. The results are compared with bioinformatics predictions on their subcellular localization and pathogenic nature. Analysis of the distribution of proteins across the Triton X-114 fractions with the predicted characteristics is performed based on "number" of unique type of proteins, and "quantity" which represents the amount of unique protein. The highest number of predicted outer membrane proteins (OMPs) and pathogenic proteins are found in aqueous and pellet fractions, whereas detergent fraction representing the OM has the highest quantity of OMPs and pathogenic proteins though lower in number than the aqueous and pellet fractions. This leaves the possibility of an upsurge in pathogenic proteins and OMPs on the OM under pathogenic conditions suggesting their potential use to combat leptospirosis. Further, the Triton X-114 subcellular fractions are more correlated to enrichment of pathogenic proteins predicted by MP3 software than predicted localization.

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.

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.

Proteogenomic examination of esophageal squamous cell carcinoma (ESCC): new lines of inquiry.

Introduction: Esophageal squamous cell carcinoma (ESCC), a histopathologic subtype of esophageal cancer is a major cause of cancer-related morbidity and mortality worldwide. This is primarily because patients are diagnosed at an advanced stage by the time symptoms appear. The genomics and mass spectrometry-based proteomics continue to provide important leads toward biomarker discovery for ESCC. However, such leads are yet to be translated into clinical utilities. Areas covered: We gathered information pertaining to proteomics and proteogenomics efforts in ESCC from the literature search until 2020. An overview of omics approaches to discover the candidate biomarkers for ESCC were highlighted. We present a summary of recent investigations of alterations in the level of gene and protein expression observed in biological samples including body fluids, tissue/biopsy and in vitro-based models. Expert opinion: A large number of protein-based biomarkers and therapeutic targets are being used in cancer therapy. Several candidates are being developed as diagnostics and prognostics for the management of cancers. High-resolution proteomic and proteogenomic approaches offer an efficient way to identify additional candidate biomarkers for diagnosis, monitoring of disease progression, prediction of response to chemo and radiotherapy. Some of these biomarkers can also be developed as therapeutic targets.

The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal Insights into Cell Differentiation and Function.

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.

Dynamics of Dual Specificity Phosphatases and Their Interplay with Protein Kinases in Immune Signaling.

Dual specificity phosphatases (DUSPs) have a well-known role as regulators of the immune response through the modulation of mitogen-activated protein kinases (MAPKs). Yet the precise interplay between the various members of the DUSP family with protein kinases is not well understood. Recent multi-omics studies characterizing the transcriptomes and proteomes of immune cells have provided snapshots of molecular mechanisms underlying innate immune response in unprecedented detail. In this study, we focus on deciphering the interplay between members of the DUSP family with protein kinases in immune cells using publicly available omics datasets. Our analysis resulted in the identification of potential DUSP-mediated hub proteins including MAPK7, MAPK8, AURKA, and IGF1R. Furthermore, we analyzed the association of DUSP expression with TLR4 signaling and identified VEGF, FGFR, and SCF-KIT pathway modules to be regulated by the activation of TLR4 signaling. Finally, we identified several important kinases including LRRK2, MAPK8, and cyclin-dependent kinases as potential DUSP-mediated hubs in TLR4 signaling. The findings from this study have the potential to aid in the understanding of DUSP signaling in the context of innate immunity. Further, this will promote the development of therapeutic modalities for disorders with aberrant DUSP signaling.

Secretome analysis of diarrhea-inducing strains of Escherichia coli.

Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by Gram-negative bacteria. Enterohemorrhagic Escherichia coli, O157:H7, is the major pathogen often causing outbreaks. However, studies have reported that the significant outbreaks caused by non-O157:H7 E. coli strains, also known as "Big-Six" serogroup strains, are increasing. There is no systematic study describing differential secreted proteins from these non-O157:H7 E. coli strains. In this study, we carried out MS-based differential secretome analysis using tandem mass tags labeling strategy of non-O157:H7 E. coli strains, O103, O111, O121, O145, O26, and O45. We identified 1241 proteins, of which 565 proteins were predicted to be secreted. We also found that 68 proteins were enriched in type III secretion system and several of them were differentially expressed across the strains. Additionally, we identified several strain-specific secreted proteins that could be used for developing potential markers for the identification and strain-level differentiation. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli Big-Six serogroup and the several of these strain-specific secreted proteins can be further studied to develop potential markers for identification and strain-level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and detection and identification of bio threats in biodefense.

SILAC-based quantitative proteomic analysis reveals widespread molecular alterations in human skin keratinocytes upon chronic arsenic exposure.

Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin.