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Made available in DSpace on 2021-06-25T10:24:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-23 In this study, a novel label-free electrochemical biosensor based on the zeolitic imidazole framework (ZIF-8) was developed for monitoring protein-protein interactions (PPIs). ZIF-8 was deposited on interdigitated electrodes and employed as a transducing material and simultaneously carried the thioredoxin-1 (Trx-1) protein, followed by the deposition of increased concentrations of the cytoplasmic domain of a disintegrin and metalloproteinase 17 (ADAM17cyto) known as the Trx-1 binding partner. Structural and morphological characterizations were used to validate and verify the formation of ZIF-8. The ZIF-8 crystals showed a rhombic dodecahedral structure with mainly exposed (011) facets, a mean particle size of 205 (±22) nm, and a ZIF-8 film thickness around 61 (±6) nm. The interaction between Trx-1 and ADAM17cyto proteins was analyzed through electrochemical impedance spectroscopy (EIS). The results indicate a linear and inverse relationship between the impedance responses at 0.1 Hz for ADAM17cyto concentrations from 50 nM to 8 μM, with a coefficient of variation from 1.0% to 11.4%. The proposed biosensor also displayed a significant selectivity and stability verified by using ADAM17cyto mutant and BSA as controls. As a proof-of-concept, we compared the results with a widely used type of PPI assay based on antibody recognition, the solid-phase binding assay, using the same proteins. The solid-phase binding assay was able to detect a significant binding only in ADAM17cyto concentrations above 0.5 μM, with a coefficient of variation varying from 5.4% to 27.5%. The results demonstrate that the developed biosensor was 10× more sensitive and reproducible than the conventional solid-phase binding assay. Furthermore, the developed electrochemical biosensor based on ZIF-8 provides a faster, label-free, and low-cost detection analysis, representing a novel strategy in detecting PPIs. Laboratório Nacional de Biociências (LNBio) Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) Laboratório Nacional de Nanotecnologia (LNNano) Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) Departamento de Biologia Molecular e Funcional Instituto de Biologia (IB) Universidade de Campinas (UNICAMP) Departamento de Físico-Química Instituto de Química (IQ) Universidade de Campinas (UNICAMP) Programa de Pós Graduação em Ciência e Tecnologia de Materiais (POSMAT) Universidade Estadual Paulista (UNESP) Programa de Pós Graduação em Ciência e Tecnologia de Materiais (POSMAT) Universidade Estadual Paulista (UNESP)

Background Ameloblastoma is a locally aggressive tumor, originated from odontogenic epithelium, and affects the jawbones with an elevated recurrence rate. The molecular mechanisms involved with the pathogenesis of this tumor remain undetermined. This review aimed to describe the current data regarding epigenetic alterations in ameloblastoma. Material and methods A systematized electronic search was performed in the English-language literature in three databases, combining the following keywords: ameloblastoma, epigenetic, methylation, noncoding RNA, histone acetylation. Results According to the gathered results of 11 studies in this review, epigenetic alterations could induce the development and progression of ameloblastoma. DNA methylation has been the most assessed mechanism in ameloblastomas. Conclusions Current literature data indicate that epigenetic events can be involved in the etiopathogenesis of ameloblastomas. Key words:Ameloblastoma, epigenetic, methylation, noncoding RNA, histone acetylation.

To integrate all the available data published in the English literature regarding the protein diagnostic and/or prognostic markers in salivary gland tumors identified by mass spectrometry (MS)-based discovery proteomics.An extensive search was carried out using MEDLINE/PubMed, EMBASE, Web of Science, and Scopus databases. Manual searching in Google Scholar and assessment of the reference list of the included articles also was performed. The risk of bias was assessed by the Joanna Briggs Institute Critical Appraisal tool for the specific type of study.A total of 1092 articles were initially retrieved within which 6 were used for data extraction, resulting in 145 cases of salivary gland tumors. The data was composted by eleven salivary gland tumor types. In total, 2136 proteins were detected by MS-based discovery proteomics in salivary gland tumors. Ninety-one proteins were proposed as potential diagnostic and/or prognostic markers. Of these, some have been identified in one or more studies, whereas fifteen were in common across studies and a total of seventy-six were non-repeat proteins.In summary, we compiled data about the proteomic profile of potential diagnostic and/or prognostic protein markers of the salivary gland tumors detected by MS-based discovery proteomics. The proteins ANXA1, ANXA5, CAPG, CRYAB, FGB, GNB2L1, IGHG1, PPIA, S100A9, and SOD1 were proposed as the most common potential diagnostic markers of salivary gland tumors.

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular “handshake” between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity., Mayaro virus (MAYV) is an emerging arbovirus in Central and South America that is transmitted by mosquitoes and causes arthritogenic disease. Here, the authors present the 4.4 Å resolution cryo-EM structure of MAYV and describe specific features of the virus, which could be exploited for the design of MAYV-specific diagnostics and therapeutics.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.

This communication discusses the current challenges of oral mucositis (OM) management during the pandemic COVID-19 outbreak and reflects about an extraoral photobiomodulation protocol as an optimal alternative for preventing and treating OM in advanced cancer patients while minimizing the risk of infection by avoiding intraoral manipulation.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging because changes can occur simultaneously at protease, their inhibitor, and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics, and peptidase predictions for studying proteolytic events in the saliva of 79 patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph-node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (area under the receiver operating characteristic curve > 0.85). In addition, endopeptidases and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from public repositories, reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis were further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by selected reaction monitoring–mass spectrometry, while minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of patients with OSCC and lymph-node metastasis and, at least in part, is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis., Graphical Abstract, Highlights • Proteolysis is accentuated in the saliva of patients with OSCC and lymph-node metastasis. • Differential saliva peptidome outperformed proteomics in patient classification. • Proteases implicated in differential proteolysis correlated with prognostic factors. • Reduced levels of protease inhibitors disturb the proteolytic balance in the saliva., In Brief Mass spectrometry–based peptidomics has been applied to the saliva of patients with oral squamous cell carcinoma, enabling the discovery of signatures correlated with poor prognostic factors, including lymph-node metastasis. Sequence analysis of differential peptides allowed the prediction of proteases implicated in endogenous proteolysis and associated with prognosis in head and neck cancers. The results of combined saliva peptidomics and proteomics revealed that the accentuated proteolysis in patients with oral squamous cell carcinoma and lymph-node metastasis concurs with reduced levels of protease inhibitors in saliva.

Osteopontin (OPN) is a calcium-binding glycol-phosphoprotein present in many physiologic and pathological processes. This protein can control bone cell adhesion, osteoclastic activity, and bone matrix mineralization. However, its participation in pathological processes such as atherosclerosis, sarcoidosis, tuberculosis, and cancer have been described. Some studies have shown that OPN may participate in the development and progression of oral cancer. Although the role of OPN in oral cancer is not fully understood, some studies have suggested that this protein may induce malignant phenotype of cells by activation of PI3K/AKT/mTOR pathway, which favors cell proliferation, invasion, metastasis, angiogenesis, and failure of treatment. This review discusses the possible mechanism of involvement of OPN in oral cancer and its potential clinical application in diagnosis and prognosis.

Background and objectives Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. Methods Protein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. Results A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. Conclusions Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. Significance This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

Cancer cells generally recruit and influence non-malignant immune cells to support the tumor growth. Classical Hodgkin lymphoma (cHL) is a good example because the affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (H-RS) cells, which are supported by a massive infiltrate of lymphocytes, fibroblasts, and innate immune cells. The transmembrane receptor CD30, which is selectively expressed on the H-RS cells, plays an important role, not only in cell stimulation and intercellular communication but also in tumor diagnosis and targeted tumor therapy. Different protein processing pathways influence its functionality. Depending on the conditions, the receptor is internalized or released. The release of CD30 occurs either as an intact molecule, embedded in the membrane of extracellular vesicles (EVs), or as a cleaved soluble ectodomain (sCD30). CD30 cleavage is predominantly catalyzed by ADAM10. The enzyme is catalytically active in cells as well as in EVs and gradually releases sCD30. Because the circulation contains no CD30+ donor cells, this mechanism explains that the cleaved ectodomain represents the predominant form of CD30 in the plasma of cHL patients. CD30 processing might influence the impact of CD30 antibody-drug conjugates, such as Brentuximab Vedotin (BV). Whereas, ADAM10-degraded CD30 impedes the BV efficacy, tumor-derived EVs load bystander cells with CD30 and generate new targets among supporter cells. This crossfire effect might contribute to the enormous clinical impact of BV, whereas the ADAM10-dependent cleavage to the mild systemic off-target effects of the treatment with BV.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has to cope with reactive oxygen and nitrogen species during its life cycle in order to ensure its survival and infection. The parasite detoxifies these species through a series of pathways centered on trypanothione that depend on glutathione or low molecular mass dithiol proteins such as tryparedoxins. These proteins transfer reducing equivalents to peroxidases, including mitochondrial and cytosolic peroxiredoxins, TcMPx and TcCPx, respectively. In T. cruzi two tryparedoxins have been identified, TXNI and TXNII with different intracellular locations. TXNI is a cytosolic protein while TXNII due to a C-terminal hydrophobic tail is anchored in the outer membrane of the mitochondrion, endoplasmic reticulum and glycosomes. TXNs have been suggested to be involved in a majority of biological processes ranging from redox mechanisms to protein translation. Herein, a comparison of the TXNII interactomes under physiological and oxidative stress conditions was examined. Under physiological conditions, apart from the proteins with unknown biological process annotation, the majority of the identified proteins are related to cell redox homeostasis and biosynthetic processes, while under oxidative stress conditions, are involved in stress response, cell redox homeostasis, arginine biosynthesis and microtubule based process. Interestingly, although TXNII interacts with both peroxiredoxins under physiological conditions, upon oxidative stress, TcMPx interaction prevails. The relevance of the interactions is discussed opening a new perspective of TXNII functions. Fil: Dias, L.. Universidade Estadual de Campinas; Brasil Fil: Peloso, E.F.. Universidade Estadual de Campinas; Brasil Fil: Leme, A.F.P.. Associaçáo Brasileira de Informática; Fil: Carnielli, C.M.. Associaçáo Brasileira de Informática; Fil: Pereira, C.N.. Universidade Estadual de Campinas; Brasil Fil: Werneck, C.C.. Universidade Estadual de Campinas; Brasil Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Gadelha, F.R.. Universidade Estadual de Campinas; Brasil

Head and neck cancer is globally challenging due to the resistance to therapy and aggressive behavior leading to high rates of mortality. Recent findings show that the tumor microenvironment plays a role in the maintenance and progression of many solid tumors, including head and neck cancer. The mechanisms involved in the modulation and regulation of the tumor microenvironment remain poorly understood. Increasing evidence suggests that epigenetic events can modulate the crosstalk between neoplastic and non-neoplastic cells during tumor progression. In this review, we explore the current understanding of the involvement of epigenetic events in the modulation of the tumor microenvironment and its impact on head and neck cancer behavior. We also explore the latest therapeutic strategies that use epigenetic-modulating drugs to manage tumor growth and progression.

Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with functions varying from the regulation of neuronal development and intracellular transport mechanisms to transcription regulation. One of its interactors is retinoic acid receptor (RAR), which is activated by retinoic acid and controls many target genes and physiological process. Based on previous evidence suggesting a possible nuclear role for FEZ1, we wanted to deepen our understanding of this function by addressing the FEZ1-RAR interaction. We performed in vitro binding experiments and assessed the interface of interaction between both proteins. We found that FEZ1-RAR interacted with a similar magnitude as RAR to its responsive element DR5 and that the interaction occurred in the coiled-coil region of FEZ1 and in the ligand-binding domain of RAR. Furthermore, cellular experiments were performed in order to confirm the interaction and screen for induced target genes from an 86-gene panel. The analysis of gene expression showed that only in the presence of retinoic acid did FEZ1 induce hoxb4 gene expression. This finding is consistent with data from the literature showing the hoxb4 gene functionally involved in development and acute myeloid leukemia, as is FEZ1.

The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition.

Proteomic analysis of extracellular matrices (ECM) of dentoalveolar tissues can provide insights into developmental, pathological, and reparative processes. However, targeted dissection of mineralized tissues, dental cementum (DC), alveolar bone (AB), and dentin (DE), presents technical difficulties. We demonstrate an approach combining EDTA decalcification and laser capture microdissection (LCM), followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), to analyze proteome profiles of these tissues. Using the LCM-LC-MS/MS approach, a total of 243 proteins was identified from all tissues, 193 proteins in DC, 147 in AB, and 135 proteins DE. Ninety proteins (37% of total) were common to all tissues, whereas 52 proteins (21%) were overlapping in only two. Also, 101 (42%) proteins were exclusively detected in DC (60), AB (15), or DE (26). Identification in all tissues of expected ECM proteins including collagen alpha-1(I) chain (COL1A1), collagen alpha-1(XII) chain (COL12A1), biglycan (BGN), asporin (ASPN), lumican (LUM), and fibromodulin (FMOD), served to validate the approach. Principal component analysis (PCA) and hierarchical clustering identified a high degree of similarity in DC and AB proteomes, whereas DE presented a distinct dataset. Exclusively and differentially identified proteins were detected from all three tissues. The protein-protein interaction network (interactome) of DC was notable for its inclusion of several indicators of metabolic function (e.g. mitochondrial proteins, protein synthesis, and calcium transport), possibly reflecting cementocyte activity. The DE proteome included known and novel mineralization regulators, including matrix metalloproteinase 20 (MMP-20), 5' nucleotidase (NT5E), and secreted phosphoprotein 24 (SPP-24 or SPP-2). Application of the LCM-LC-MS/MS approach to dentoalveolar tissues would be of value in many experimental designs, including developmental studies of transgenic animals, investigation of treatment effects, and identification of novel regenerative factors.

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods.Several disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.

Lip and oral cancer are one of the most frequent types of cancers in the world. Cultural habits and ethnic variations may influence the epidemiologic profile of patients with this neoplasm. The GLOBOCAN project is updated annually with cancer estimates and statistics around the world. The aim of this review is to compare statistics of lip and oral cancer based on GLOBOCAN 2012 and 2018. The review was conducted using the online platform of data statistics available by International Agency for Research on Cancer. Data not available on the platform were added based on a preview of epidemiology articles about these neoplasms. In 2018, 54 464 more cases were diagnosed compared to GLOBOCAN 2012. Men are more affected than women in all countries, in both projects. One-half of patients died in both projects. Therefore, ethnic variations have great importance on the epidemiologic characterization of oral cancer because cultural habits that lead to more exposure to risk factors increase incidence and mortality of lip and oral cancer. Apoio Financeiro: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo.

Objective To determine the proteome profile of adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAc) and to identify a protein signature useful to differentiate both neoplasms. Study Design Ten cases of AdCC and 10 cases of PAc were retrospectively retrieved and submitted to laser microdissection for enrichment of neoplastic tissue. The samples were submitted to liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the proteomics data were analyzed using the MaxQuant software. MS/MS spectra were searched against the Human UniProt database, whereas statistical analyses were performed with Perseus software. Results The LC-MS/MS analysis identified 1957 proteins. Among these, 261 proteins were exclusively identified in AdCC and 106 proteins only in PAc. Clustering analysis of the 394 statistically significant proteins identified in both tumors showed a clear separation of the tumors. A total of 16 proteins were upregulated in each tumor, 6 in AdCC and 10 in PAc, and their cluster analysis also revealed clear differences between both neoplasms. Conclusions Global proteome can effectively discriminate AdCC and PAc, and the use of a protein signature may represent a useful diagnostic auxiliary to differentiate both tumors in difficult cases. Support: FAPESP 2015/16056-0 and FAPEMIG.

BackgroundDiseases of the anterior segment of the eye may present different mechanisms, intensity of symptoms, and impact on the patients’ quality of life and vision. The tear film is in direct contact with the ocular surface and cornea and can be easily accessed for sample collection, figuring as a promising source of potential biomarkers for diagnosis and treatment control. This study aimed to evaluate tear proteomic profile in 3 distinct ocular diseases: keratoconus (corneal ectasia), severe dry eye related to graft-versus-host-disease (tear film dysfunction and ocular inflammatory condition) and pterygium (conjunctival fibrovascular degenerative disease).MethodsTear samples were collected from patients of each condition and a control group. By using mass spectrometric analysis combined with statistics and bioinformatics tools, a detailed comparison of protein profile was performed.ResultsAfter Student’s t-test analyses comparing each condition to the control group, we found the following number of differentially expressed proteins: 7 in keratoconus group, 29 in pterygium group, and 79 in GVHD group. Following multivariate analyses, we also report potential candidates as biomarkers for each disease.ConclusionsWe demonstrated herein that mass spectrometry-based proteomics was able to indicate proteins that differentiate three distinct ocular conditions, which is a promising tool for the diagnosis of ocular diseases.

Established restorative protocols for patients after head and neck radiotherapy are lacking, increasing the failure rates of dental adhesive restorations.The purpose of this systematic review and meta-analysis was to analyze the evidence regarding the impact of head and neck radiotherapy on the longevity of dental adhesive restorations.A search was performed using PubMed, Scopus, and Embase in May 2018 (updated in November 2020). Data extraction was performed regarding the percentage of restoration failure among dental adhesive materials, including glass ionomer cements, resin-modified glass ionomer cements, and composite resins. Risk of bias was assessed by the meta-analysis of statistics assessment and review instrument (MAStARI). Confidence in cumulative evidence was evaluated by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) protocol.Four studies met the inclusion criteria. All included studies were classified as having a moderate risk of bias and reported results regarding class V restorations. Overall, composite resins presented lower failure rates at 2 years (30%) when compared with resin-modified glass ionomer (41%) and glass ionomer cements (57%). Meta-analysis showed that the risk of failure with glass ionomer cements was greater than with resin-modified glass ionomer cements (RR: 1.71, P.001). Composite resins presented lower risk of failure when compared with glass ionomer (RR: 2.29, P.001) and resin-modified glass ionomer cements (RR: 1.30, P=.03). Three studies reported results regarding fluoride compliance, which had a negative effect on the survival rates of glass ionomer and resin-modified glass ionomer cements and a positive effect on composite resin restorations.The results suggest that composite resin restorations associated with fluoride gel compliance seems to be the best alternative for restoring class V lesions in patients after head and neck radiotherapy. However, the results showed moderate certainty of evidence, which justifies the need for more randomized clinical trials regarding this subject.

International audience; Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen

Salivary gland tumors (SGTs) comprise a heterogeneous group of benign and malignant neoplasms that exhibit significant variability in their microscopic appearance, clinical presentation, and biological behavior. The etiologic factors are unknown; however, chromosomic translocation, secondary radiation, and chemotherapy can be associated with the development of SGT. It has been indicated that epigenetic alterations can be responsible for the development and progress of these neoplasms. The epigenetic mechanisms are defined as a set of DNA changes that do not alter the sequence of nucleotide bases but alter the expression of the proteins. These alterations have been studied in the SGT, and they were associated with the development and progress of these neoplasms and may influence on SGT prognosis. Hence, we critically review the currently available data on the participation of epigenetic events on salivary gland tumors.

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

In numerous types of cancer, the primary tumor site can show a correlation with disease behavior and survival outcomes. In salivary gland tumors (SGTs) this association remains controversial. This study assessed the association between primary sites of SGTs and prognosis. Studies from five databases were assessed and a meta-analysis was performed using studies that presented 95 % confidence interval (95 % CI), hazard ratio (HR) and survival analysis. Gathered information from 46,361 patients showed that site had a prognostic impact on SGTs. Tumors involving minor salivary glands showed worse overall survival (HR = 1.60; 95 % CI = 1.17-2.19; p = 0.003), disease-specific survival (HR=1.63; 95 % CI = 1.12-2.37; p = 0.01), and cause-specific survival (HR=2.10; 95 % CI = 1.72-2.55; p = 0.00001). Tumors from major salivary glands showed better recurrence-free survival (HR=2.31; 95 % CI = 1.77-3.02; p = 0.00001), and locoregional control of disease (HR=2.66; 95 % CI = 1.20-5.91; p = 0.02). Our results showed that the primary site of SGTs has an impact on patient prognosis.

Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell– and SCC-9 cell–derived EVs. A multi-omics integration identified 11 ‘hub proteins’ significantly decreased at the metastatic site compared with primary tumor–derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven ‘hub proteins’ in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC., Graphical Abstract, Highlights • Proteomic, miRNA, metabolomic, and lipidomic profiles were mapped in oral cancer EVs. • The molecular profile of EVs was associated with the lymph node metastatic phenotype. • A multi-omics integrative analysis revealed 11 highly connected ‘hub proteins.’ • ‘Hub proteins’ from EVs are candidates as prognostic markers in oral cancer., In Brief A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 ‘hub proteins’ that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer.

Only few studies on snake venoms were dedicated to deeply characterize the toxin secretion of animals from the Colubridae family, despite the fact that they represent the majority of snake diversity. As a consequence, some evolutionary trends observed in venom proteins that underpinned the evolutionary histories of snake toxins were based on data from a minor parcel of the clade. Here, we investigated the proteins of the totally unknown venom from Phalotris mertensi (Dipsadinae subfamily), in order to obtain a detailed profile of its toxins and to appreciate evolutionary tendencies occurring in colubrid venoms. By means of integrated omics and functional approaches, including RNAseq, Sanger sequencing, high-resolution proteomics, recombinant protein production, and enzymatic tests, we verified an active toxic secretion containing up to 21 types of proteins. A high content of Kunitz-type proteins and C-type lectins were observed, although several enzymatic components such as metalloproteinases and an L-amino acid oxidase were also present in the venom. Interestingly, an arguable venom component of other species was demonstrated as a true venom protein and named svLIPA (snake venom acid lipase). This finding indicates the importance of checking the actual protein occurrence across species before rejecting genes suggested to code for toxins, which are relevant for the discussion about the early evolution of reptile venoms. Moreover, trends in the evolution of some toxin classes, such as simplification of metalloproteinases and rearrangements of Kunitz and Wap domains, parallel similar phenomena observed in other venomous snake families and provide a broader picture of toxin evolution.

The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.

Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.

Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications.Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration.

// Hinrich P. Hansen 1 , Ahmad Trad 2 , Maria Dams 1 , Paola Zigrino 3 , Marcia Moss 4 , Maximilian Tator 1 , Gisela Schon 1 , Patricia C Grenzi 1 , Daniel Bachurski 1 , Bruno Aquino 5 , Horst Durkop 6 , Katrin S Reiners 1 , Michael von Bergwelt-Baildon 1 , Michael Hallek 1 , Joachim Grotzinger 2 , Andreas Engert 1 , Adriana F Paes Leme 5 , Elke Pogge von Strandmann 1 1 Department of Internal Medicine I, University of Cologne, Cologne, Germany 2 Department of Biochemistry, University Kiel, Kiel, Germany 3 Department of Dermatology, University of Cologne, Cologne, Germany 4 BioZyme Inc., Apex, North Carolina, USA 5 Brazilian Biosciences National Laboratory, LNBio, CNPEM, Campinas, Brazil 6 Pathodiagnostik Berlin, Berlin, Germany Correspondence to: Hinrich P. Hansen, e-mail: h.hansen@uni-koeln.de Keywords: Hodgkin lymphoma, extracellular vesicle, CD30, ADAM10, Brentuximab-Vedotin Received: January 11, 2016 Accepted: March 31, 2016 Published: April 20, 2016 ABSTRACT The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30 – /CD30L + mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30 + EVs.

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-beta 1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

Oral leukoplakia (OL) is a white lesion with high potential of recurrence and malignant transformation. The variable clinical and histopathological features of OL may potentially impact both treatment and prognosis. Current literature shows that post treatment rates of recurrence and malignant transformation vary widely. The use of surgical lasers have been proposed with the objective of improving outcomes. We performed a systematic review and a comprehensive meta-analysis dedicated to pooling the rates of recurrence and malignant transformation of OL lesions treated using the main types of surgical lasers available. Scopus, MEDLINE/PubMed, and Embase were searched electronically. A total of 36 articles met the inclusion criteria. Selected studies included OL lesions that were treated by evaporation or excision using Nd:YAG laser, Er:YAG laser, CO2 laser, KTP laser, or diode laser. The results of this systematic review and meta-analysis suggest that surgical laser excision of OL may decrease recurrence rates but have no effect on the malignant transformation of OL when compared with conventional treatments.

Proliferative verrucous leukoplakia (PVL) is a subtype of oral leukoplakia, which is not commonly associated with risk factors such as alcohol and smoking; it has high rates of recurrence after treatment and has a high risk of malignant transformation. This study described the clinical features of 34 patients with PVL (1991-2016). Of these, the vast majority was female (31%-91.17%) and the mean age was 69.08 years. Regarding risk factors, only 2 patients smoked and 5 consumed alcoholic beverages. The most frequently affected sites were the buccal mucosa and lateral border of the tongue. The average number of site affected with leukoplakia was 3.41 and the development of squamous cell carcinoma occurred in 7 of the patients (20%). PVL starts as an isolated lesion but develops important changes throughout the follow-up period. Therefore, constant clinical monitoring is necessary.

Objectives The aim of this study was to characterize the salivary proteomic profile of patients treated for head and neck cancers (HNCs) and correlate it with the risk of developing severe radiation-related oral mucositis (OM). Study Design Forty-one patients with oral squamous cell carcinoma (OSCC) who underwent adjuvant radiotherapy (RT) or chemoradiotherapy (CRT) were included. All patients were submitted to dental conditioning protocols prior to RT. OM and OM-related pain were evaluated daily during RT and graded according to the Common Toxicity Criteria for Adverse Events (National Cancer Institute, version 4.0, 2010) and the visual analogue scale (VAS). For the molecular analysis, whole saliva was collected immediately before RT and subjected to proteomic analysis by means of liquid chromatography–mass spectrometry (LTQ Orbitrap Velos MS; Thermo Fisher Scientific, Bremen, Germany) and label-free protein quantification. The results obtained from the targeted proteomic analysis were compared with OM clinical outcomes. Statistical analysis was performed by using Wilcoxon's test. Results Of the study patients, 73% presented with stage III/IV OSCC; 58% were submitted to CRT protocols, with a mean RT dose of 66 Gy. Most of the patients (66%) had visible tumor areas at the time of saliva collection; 43.9% had grade 2 and 31.7% had grade 3 as the highest OM grade during RT, with a mean highest reported VAS score of 3.53. For the target proteomics analysis, a total of 65 proteins were observed to be mostly related to biologic processes, such as immune responses, peptidase inhibitor activity, and the inflammatory system. The macrophage migration inhibitory factor (MIF HUMAN) was statistically significant when correlated with OM grade. MIF was observed in patients with grades 1 to 4 OM and showed higher abundance for OM grades 3 and 4 compared with grades 1 and 2 (P = .04). Conclusions The correlation of MIF with the severity of OM is compatible with the role of MIF as a proinflammatory protein. This seems to be the first study to describe this association; therefore, future prospective studies would be ideal to observe pre- and post-RT alterations in salivary MIF levels to validate this result and better understand the role of MIF in the pathogenesis of OM.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

The interaction of single-layer graphene oxide (SLGO) and multi-layered graphene oxide (MLGO) with a cell culture medium (i.e. DMEM) was studied by evaluating fetal bovine serum (FBS) protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions. SLGO and MLGO exhibited different colloidal behavior in the culture medium, which was visualized by cryogenic transmission electron microscopy in situ analysis. Exploring proteomics and bioinformatics tools, 394 and 290 proteins were identified on the SLGO and MLGO hard corona compositions, respectively. From this amount, 115 proteins were exclusively detected on the SLGO and merely 11 on MLGO. SLGO enriched FBS proteins involved in metabolic processes and signal transduction, while MLGO enriched proteins involved in cellular development/structure, and lipid transport/metabolic processes. Such a distinct corona profile is due to differences on surface chemistry, aggregation behavior and the surface area of GO materials. Hydrophilic interactions were found to play a greater role in protein adsorption by MLGO than SLGO. Our results point out implications for in vitro studies of graphene oxide materials concerning the effective dose delivered to cells and corona bioactivity. Finally, we demonstrated the importance of integrating conventional and modern techniques thoroughly to understand the GO-FBS complexes towards more precise, reliable and advanced in vitro nanotoxicity assessment.

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor−node−metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis., Oral cancer has region-specific histopathological and molecular characteristics, complicating its classification by the standard tumor-node-metastasis system. Here, the authors combine discovery and targeted proteomics with IHC to identify region-specific and saliva biomarkers for oral cancer prognosis.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.

BACKGROUND Physiological roles for the periodontal ligament (PDL) include tooth eruption and anchorage, force absorption, and provision of proprioceptive information. Despite the advances in understanding the biology of PDL cells, there is a lack of information regarding the molecular signature of deciduous (DecPDL) and permanent (PermPDL) PDL tissues. Thus, the present study was designed to characterize the membrane proteome of DecPDL and PermPDL cells. METHODS Primary PDL cells were obtained (n = 6) and a label-free quantitative proteome of cell membrane-enriched components was performed. Proteome findings were validated by quantitative polymerase chain reaction and Western blot assays in fresh human tissues (n = 8) and primary cell cultures (n = 6). In addition, confocal microscopy was used to verify the expression of target factors in the PDL cell cultures. RESULTS Comparative gene ontology enrichment analysis evidenced that most stickling differences involved "endomembrane system" (PICALM, STX4, and LRP10), "hydrolase activity" (NCSTN and XRCC6), "protein binding" (PICALM, STX4, GPNMB, VASP, extended-synaptotagmin 2 [ESYT2], and leucine-rich repeat containing 15 [LRRC15]), and "isomerase activity" (FKBP8). Data are available via ProteomeXchange with identifier PXD010226. At the transcript level, high PICALM in DecPDL and ESYT2 and LRRC15 in PermPDL were confirmed in fresh PDL tissues. Furthermore, Western blot analysis confirmed increased levels of PICALM, LRRC15, and ESYT2 in cells and/or fresh tissues, and confocal microscopy confirmed the trends for PICALM and LRRC15 expression in PDL cells. CONCLUSION We report the first comprehensive characterization of the membrane protein machinery of DecPDL and PermPDL cells, and together, we identified a distinct molecular signature for these cell populations, including unique proteins for DecPDL and PermPDL.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

To cope with toxic levels of H2S, the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens employ the bigR operon to oxidize H2S into sulfite. The bigR operon is regulated by the transcriptional repressor BigR and it encodes a bifunctional sulfur transferase (ST) and sulfur dioxygenase (SDO) enzyme, Blh, required for H2S oxidation and bacterial growth under hypoxia. However, how Blh operates to enhance bacterial survival under hypoxia and how BigR is deactivated to derepress operon transcription is unknown. Here, we show that the ST and SDO activities of Blh are in vitro coupled and necessary to oxidize sulfide into sulfite, and that Blh is critical to maintain the oxygen flux during A. tumefaciens respiration when oxygen becomes limited to cells. We also show that H2S and polysulfides inactivate BigR leading to operon transcription. Moreover, we show that sulfite, which is produced by Blh in the ST and SDO reactions, is toxic to Citrus sinensis and that X. fastidiosa-infected plants accumulate sulfite and higher transcript levels of sulfite detoxification enzymes, suggesting that they are under sulfite stress. These results indicate that BigR acts as a sulfide sensor in the H2S oxidation mechanism that allows pathogens to colonize plant tissues where oxygen is a limiting factor.

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.