Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

Crude saliva of Amblyomma cajennense sensu stricto (Acari: Ixodidae) reduces locomotor activity and increases the hemocyte number in the females of Aedes aegypti (Diptera: Culicidae).

Aedes aegypti are vector insects of arboviruses such as dengue, Zika, and chikungunya. All available vector control methods have limited efficacy, highlighting the urgent need to find alternative ones. Evidence shows that arachnids like ticks are sources of biologically active compounds. Moreover, chemical modulation of the locomotor and immune systems of vector insects can be used to control arbovirus transmission. The present study evaluated the effectiveness of crude saliva of female Amblyomma cajennense sensu stricto (s.s.) ticks in reducing locomotor activity and inducing an immune response in Ae. aegypti females. Additionally, the study evaluated the protein constitution of tick saliva. For this purpose, the crude saliva obtained from several semi-engorged A. cajennense females was used. A volume of 0.2 nL of crude tick saliva was administered to mosquitoes by direct intrathoracic microinjection. The effect of the tick's saliva on the locomotor activity of the mosquito was observed using Flybox, a video-automated monitoring system, and the hemolymph hemocyte levels were quantified by reading slides under a light microscope. The protein concentration of the crude tick saliva was 1.27 µg/µL, and its electrophoretic profile indicates the presence of proteins with a molecular weight ranging between ∼17 and 95 kDa. Microplusins, ixodegrins, cystatin, actins, beta-actin, calponin, albumin, alpha-globulins, and hemoglobin were the main proteins identified by proteomics in the saliva of A. cajennense. The microinjected saliva had low toxicity for Ae. aegypti females and significantly reduced their locomotor activity, especially in the transition between the light and dark phases. The crude tick saliva did not change the period and rhythmicity of the circadian cycle. The tick saliva significantly increased the number of hemocytes two days after injection and reduced it after five days. These results suggest that further evaluation of the biological properties of tick saliva proteins against Ae. aegypti would be of interest.

Phenolic Profile and Antioxidant Properties in Extracts of Developing Açaí (Euterpe oleracea Mart.) Seeds.

We investigated changes in the phenolic profile and antioxidant properties in the extracts of developing seeds of açaí (Euterpe oleracea). Four developmental stages were evaluated, with earlier stages displaying higher antioxidant activity and polyphenols content, while mass spectrometry analysis identified procyanidins (PCs) as the major components of the extracts in all stages. B-type PCs varied from dimers to decamers, with A-type linkages in a smaller number. Extracted PCs decreased in average length from 20.5 to 10.1 along seed development. PC composition indicated that (-)-epicatechin corresponded to over 95% of extension units in all stages, while (+)-catechin presence as the starter unit increased from 42 to 78.8% during seed development. This variation was correlated to the abundance of key enzymes for PC biosynthesis during seed development. This study is the first to report PC content and composition variations during açaí seed development, which can contribute to studies on the plant's physiology and biotechnological applications.

Chitotriosidase 1 in the cerebrospinal fluid as a putative biomarker for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurodegenerative disease that affects motor, urinary, intestinal, and sensory functions. Typically, HAM/TSP is slowly progressive, but it may vary from limited motor disability after decades (very slow progression) to loss of motor function in a few years from disease onset (rapid). In this study, we aimed to identify prognostic biomarkers for HAM/TSP to support patient management. Thus, proteomic analysis of the cerebrospinal fluid (CSF) was performed with samples from HTLV-1 asymptomatic carriers (AC) (n=13) and HAM/TSP patients (n=21) with rapid, typical, and very slow progression using quantitative label-free liquid chromatography/tandem mass spectrometry. Enrichment analyses were also carried out to identify key biological processes associated with distinct neurological conditions in HTLV-1 infection. Candidate biomarkers were validated by ELISA in paired CSF and serum samples, and samples from HTLV-1-seronegative individuals (n=9) were used as controls. CSF analysis identified 602 proteins. Leukocyte/cell activation, immune response processes and neurodegeneration pathways were enriched in rapid progressors. Conversely, HTLV-1 AC and HAM/TSP patients with typical and very slow progression had enriched processes for nervous system development. Differential expression analysis showed that soluble vascular cell adhesion molecule 1 (sVCAM-1), chitotriosidase 1 (CHIT1), and cathepsin C (CTSC) were upregulated in HAM/TSP. However, only CHIT1 was significantly elevated after validation, particularly in HAM/TSP rapid progressors. In contrast, none of these biomarkers were altered in serum. Additionally, CSF CHIT1 levels in HAM/TSP patients positively correlated with the speed of HAM/TSP progression, defined as points in the IPEC-2 HAM/TSP disability scale per year of disease, and with CSF levels of phosphorylated neurofilament heavy chain, neopterin, CXCL5, CXCL10, and CXCL11. In conclusion, higher CSF levels of CHIT1 were associated with HAM/TSP rapid progression and correlated with other biomarkers of neuroinflammation and neurodegeneration. Therefore, we propose CHIT1 as an additional or alternative CSF biomarker to identify HAM/TSP patients with a worse prognosis.

Bacillus velezensis H2O-1 surfactin efficiently maintains its interfacial properties in extreme conditions found in post-salt and pre-salt oil reservoirs.

Biosurfactants are molecules with surfactant properties produced by microorganisms, and can be used in various industrial sectors, e.g., the oil industry. These molecules can be used in enhanced oil recovery (EOR) in the pre-salt and post-salt reservoirs, where conditions of temperature, pressure, and salinity are quite varied, requiring a study of the stability of these molecules under these conditions. Bacillus velezensis H2O-1 produces five different surfactin homologs with a fatty-acid chain ranging from C11 to C16 and with a high capacity to reduce surface (24.8 mN.m-1) and interfacial tensions (1.5 and 0.8 8 mN.m-1 using light, medium oil and n-hexadecane, respectively). The critical micellar concentration (CMC) was 38.7 mg.L-1. Inversion wettability tests were carried out under the salinity conditions found in the post-salt (35 g.L-1) and pre-salt (70 g.L-1) reservoirs, in which it was observed that the surfactin reversed 100 % of the wettability of the calcite impregnated with light and medium oil. Using a central composite rotatable design, we demonstrated that surfactin maintained its interfacial properties when subjected simultaneously to extreme conditions of pressure, temperature and salinity commonly found in the post-salt (70 °C, 70 g.L-1 and 27.58 MPa) and pre-salt (100 °C, 150 g.L-1 and 48.2 MPa) layers. The results presented here highlight the efficiency and stability of H2O-1 surfactin in environmental conditions found in pre-salt and post-salt oil reservoirs.

O-GlcNAcylation protein disruption by Thiamet G promotes changes on the GBM U87-MG cells secretome molecular signature.

Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon.

Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the A. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by MS and corroborates the idea that Wolbachia helps to block ZIKV infections in A. aegypti. The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in A. aegypti, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

Comparing intestinal versus diffuse gastric cancer using a PEFF-oriented proteomic pipeline.

Gastric cancer is the fifth most common malignant neoplasia and the third leading cause of cancer death worldwide. Mac-Cormick et al. recently showed the importance of considering the anatomical region of the tumor in proteomic gastric cancer studies; more differences were found between distinct anatomical regions than when comparing healthy versus diseased tissue. Thus, failing to consider the anatomical region could lead to differential proteins that are not disease specific. With this as motivation, we compared the proteomic profiles of intestinal and diffuse adenocarcinoma from the same anatomical region, the corpus. To achieve this, we used isobaric labeling (iTRAQ) of peptides, a 10-step HILIC fractionation, and reversed-phase nano-chromatography coupled online with a Q-Exactive Plus mass spectrometer. We updated PatternLab to take advantage of the new Comet-PEFF search engine that enables identifying post-translational modifications and mutations included in neXtProt's PSI Extended FASTA Format (PEFF) metadata. Our pipeline then uses a text-mining tool that automatically extracts PubMed IDs from the proteomic result metadata and drills down keywords from manuscripts related with the biological processes at hand. Our results disclose important proteins such as apolipoprotein B-100, S100 and 14-3-3 proteins, among many others, highlighting the different pathways enriched by each cancer type. SIGNIFICANCE: Gastric cancer is a heterogeneous and multifactorial disease responsible for a significant number of deaths every year. Despite the constant improvement of surgical techniques and multimodal treatments, survival rates are low, mostly due to limited diagnostic techniques and late symptoms. Intestinal and diffuse types of gastric cancer have distinct clinical and pathological characteristics; yet little is known about the molecular mechanisms regulating these two types of gastric tumors. Here we compared the proteomic profile of diffuse and intestinal types of gastric cancer from the same anatomical location, the corpus, from four male patients. This methodological design aimed to eliminate proteomic variations resulting from comparison of tumors from distinct anatomical regions. Our PEFF-tailored proteomic pipeline significantly increased the identifications as when compared to previous versions of PatternLab.

7-Ketocholesterol overcomes drug resistance in chronic myeloid leukemia cell lines beyond MDR1 mechanism.

Chronic myeloid leukemia (CML) is a myeloproliferative disease with a characteristic BCR-ABL tyrosine kinase (TK) fusion protein. Despite the clinical efficacy accomplished by TKIs therapies, disease progression may affect patient response rate to these inhibitors due to a multitude of factors that could lead to development of a mechanism known as multidrug resistance (MDR). 7-Ketocholesterol (7KC) is an oxidized cholesterol derivative that has been extensively reported to cause cell death in a variety of cancer models. In this study, we showed the in vitro efficacy of 7KC against MDR leukemia cell line, Lucena. 7KC treatment induced reduction in cell viability, together with apoptosis-mediated cell death. Moreover, downregulation of MDR protein caused intracellular drug accumulation and 7KC co-incubation with either Daunorubicin or Vincristine reduced cell viability compared to the use of each drug alone. Additionally, quantitative label-free mass spectrometry-based protein quantification showed alteration of different molecular pathways involved in cell cycle arrest, induction of apoptosis and misfolded protein response. Conclusively, this study highlights the effect of 7KC as a sensitizing agent of multidrug resistance CML and elucidates its molecular mechanisms. SIGNIFICANCE: CML patients treated with tyrosine kinase inhibitors (TKIs) have showed a 5-year estimated overall survival of 89%, with cumulative complete cytogenetic response of 87%. However, development of drug resistance is a common feature of the disease progression. This study aimed at showing the effect of 7KC as a cytotoxic and sensitizing agent of multidrug resistance CML cell lines. The cellular and molecular basis of this compound were elucidated using a comprehensive strategy based on quantitative proteomic and cell biology assays. We showed that 7KC induced cell death and overcomes drug resistance in CML through mechanisms that go beyond the classical MDR1 pathways.

A proteomic approach to compare saliva from individuals with and without oral leukoplakia.

BACKGROUND: Oral leukoplakia is the most common potentially malignant disorder in the oral cavity and can precede carcinoma. This study aimed to identify possible oral leukoplakia salivary biomarkers. METHODS: Unstimulated saliva was collected from participants and protein concentration was determined. Proteins were then precipitated with cold acetone and separated using 2DE over a pH range of 3-10. Spot demarcation and matching were performed and protein identification was done through MS analysis. Oral leukoplakia tissues were submitted to immunohistochemistry analysis for keratin 10 (CK10). A complementary analysis of oral leukoplakias that were not included previously was performed in addition. RESULTS: 226±10 spots were identified in oral leukoplakia 2DE gels, and 262±12 spots were identified in volunteers. Twenty-two spots were highly abundant in oral leukoplakias or not detected in the control group, such as apolipoprotein A1, alpha amylase, cystatins, keratin 10, and lysozyme precursor. All were identified. All oral leukoplakia cases were immunopositive for CK10, mainly in the superficial epithelial layers. CONCLUSIONS: The 2DE salivary protein profiles of individuals with and without oral leukoplakia were observably different. CK10 appears to be an interesting protein and should be further studied in oral carcinogenesis. SIGNIFICANCE: MS-based proteomics enables large-scale analysis of proteins. Proteomics can provide detailed descriptions of proteomes of cells and tissues, including body fluids, and appears as a powerful tool to study human disorders. Saliva is readily accessible through non invasive collection and can mirror diverse disease states. Saliva from both diseased and healthy subjects can be analyzed through 2DE and differences between groups could be found. Routine immunohistochemistry analysis confirmed one of these findings, with CK10 being positive tissues from individuals with oral leukoplakia. Therefore, the present study allows insights into development of an important potential oral cancer precursor, named oral leukoplakia. However, the results can be extrapolated and tested in other precancer states, such as proliferative verrucous leukoplakia, patients at risk of oral cancer due to lifestyle behavior and/or cancer history in the family or even those who are under surveillance after a treated primary oral cancer.

iTRAQ-Based Shotgun Proteomics Approach for Relative Protein Quantification.

Shotgun proteomics has a key role in quantitative estimation of proteins from biological systems under different conditions, which is crucial in the understanding of their functional roles. Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry is based on pre-labeling of peptides with mass tags which allows the multiplex analysis of up to eight proteomes simultaneously. We describe here a detailed protocol for sample preparation and iTRAQ 4-plex labeling for relative quantification of multiple samples from human and plant tissues. We also present two strategies for peptide fractionation after the iTRAQ labeling protocol.

Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense.

A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50=67.0 µg mL⁻¹), Neurospora sp. (IC50=57.5 µg mL⁻¹) and Colletotrichum gloeosporioides (IC50=32.1 µg mL⁻¹). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids.

Proteomic analysis of urine in rats chronically exposed to fluoride.

Urine is an ideal source of materials to search for potential disease-related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2-DE-based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F(-)) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F(-) for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F(-) excretion. Urinary proteome profiles were examined using 2-DE and Colloidal Coomassie Brilliant Blue staining. A dose-response regarding F(-) intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F(-), control vs. 50 ppm F(-) and 5 ppm F(-) vs. 50 ppm F(-) groups, respectively. Two proteins regulated by androgens (androgen-regulated 20-KDa protein and α-2µ-globulin) and one related to detoxification (aflatoxin-B1-aldehyde-reductase) were identified by MALDI-TOF-TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F(-) toxicity, even in low doses.