Prevalence and impact of sarcopenia in individuals with heart failure with reduced ejection fraction (the SARC-HF study): A prospective observational study protocol.

Sarcopenia, a clinical syndrome primarily associated with reduced muscle mass in the elderly, has a negative impact on quality of life and survival. It can occur secondarily to other diseases such as heart failure (HF), a complex clinical syndrome with high morbidity and mortality. The simultaneous occurrence of these two conditions can worsen the prognosis of their carriers, especially in the most severe cases of HF, as in patients with reduced left ventricular ejection fraction (LVEF). However, due to the heterogeneous diagnostic criteria for sarcopenia, estimates of its prevalence present a wide variation, leading to new criteria having been recently proposed for its diagnosis, emphasizing muscle strength and function rather than skeletal muscle mass. The primary objective of this study is to evaluate the prevalence of sarcopenia and/or dynapenia in individuals with HF with reduced LVEF according to the most recent criteria, and compare the gene and protein expression of those patients with and without sarcopenia. The secondary objectives are to evaluate the association of sarcopenia and/or dynapenia with the risk of clinical events and death, quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength. The participants will answer questionnaires to evaluate sarcopenia and quality of life, and will undergo the following tests: handgrip strength, gait speed, dual-energy X-ray absorptiometry, respiratory muscle strength, cardiopulmonary exercise, as well as genomic and proteomic analysis, and dosage of N-terminal pro-B-type natriuretic peptide and growth differentiation factor-15. An association between sarcopenia and/or dynapenia with unfavorable clinical evolution is expected to be found, in addition to reduced quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength.

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.

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques.

Lumbar disc degeneration (LDD) and low back pain (LBP) are two conditions that are closely related. Several studies have shown Cutibacterium acnes colonization of degenerated discs, but whether and how these finding correlates with LBP is unknown. A prospective study was planned to identify molecules present in lumbar intervertebral discs (LLIVD) colonized by C. acnes in patients with LDD and LBP and correlate them with their clinical, radiological, and demographic profiles. The clinical manifestations, risk factors, and demographic characteristics of participants undergoing surgical microdiscectomy will be tracked. Samples will be isolated and pathogens found in LLIVD will be characterized phenotypically and genotypically. Whole genome sequencing (WGS) of isolated species will be used to phylotype and detect genes associated with virulence, resistance, and oxidative stress. Multiomic analyses of LLIVD colonized and non-colonized will be carried out to explain not only the pathogen's role in LDD, but also its involvement in the pathophysiology of LBP. This study was approved by the Institutional Review Board (CAAE 50077521.0.0000.5258). All patients who agree to participate in the study will sign an informed consent form. Regardless of the study's findings, the results will be published in a peer-reviewed medical journal. Trials registration number NCT05090553; pre-results.

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.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Extracellular Vesicles from Bothrops jararaca Venom Are Diverse in Structure and Protein Composition and Interact with Mammalian Cells.

Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.

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.

Plasma metabolome study reveals metabolic changes induced by pharmacological castration and testosterone supplementation in healthy young men.

Testosterone is a hormone that plays a key role in carbohydrate, fat, and protein metabolism. Testosterone deficiency is associated with multiple comorbidities, e.g., metabolic syndrome and type 2 diabetes. Despite its importance in many metabolic pathways, the mechanisms by which it controls metabolism are not fully understood. The present study investigated the short-term metabolic changes of pharmacologically induced castration and, subsequently, testosterone supplementation in healthy young males. Thirty subjects were submitted to testosterone depletion (TD) followed by testosterone supplementation (TS). Plasma samples were collected three times corresponding to basal, low, and restored testosterone levels. An untargeted metabolomics study was performed by liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to monitor the metabolic changes induced by the altered hormone levels. Our results demonstrated that TD was associated with major metabolic changes partially restored by TS. Carnitine and amino acid metabolism were the metabolic pathways most impacted by variations in testosterone. Furthermore, our results also indicated that LH and FSH might strongly alter the plasma levels of indoles and lipids, especially glycerophospholipids and sphingolipids. Our results demonstrated major metabolic changes induced by low testosterone that may be important for understanding the mechanisms behind the association of testosterone deficiency and its comorbidities.

Pectinases Secretion by Saccharomyces cerevisiae: Optimization in Solid-State Fermentation and Identification by a Shotgun Proteomics Approach.

A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL-1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin-lyase and pectate-lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL-1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin-lyases, one pectate-lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.

Interspecies Isobaric Labeling-Based Quantitative Proteomics Reveals Protein Changes in the Ovary of Aedes aegypti Coinfected With ZIKV and Wolbachia.

Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits such as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of coinfections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3913 proteins identified, were also able to quantify 1044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary. Furthermore, from a total of 480 mosquito proteins modulated in our study, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, coinfection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects mainly were related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganism's infection.

Enzymes in the time of COVID-19: An overview about the effects in the human body, enzyme market, and perspectives for new drugs.

The rising pandemic caused by a coronavirus, resulted in a scientific quest to discover some effective treatments against its etiologic agent, the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). This research represented a significant scientific landmark and resulted in many medical advances. However, efforts to understand the viral mechanism of action and how the human body machinery is subverted during the infection are still ongoing. Herein, we contributed to this field with this compilation of the roles of both viral and human enzymes in the context of SARS-CoV-2 infection. In this sense, this overview reports that proteases are vital for the infection to take place: from SARS-CoV-2 perspective, the main protease (Mpro ) and papain-like protease (PLpro ) are highlighted; from the human body, angiotensin-converting enzyme-2, transmembrane serine protease-2, and cathepsins (CatB/L) are pointed out. In addition, the influence of the virus on other enzymes is reported as the JAK/STAT pathway and the levels of lipase, enzymes from the cholesterol metabolism pathway, amylase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase are also be disturbed in SARS-CoV-2 infection. Finally, this paper discusses the importance of detailed enzymatic studies for future treatments against SARS-CoV-2, and how some issues related to the syndrome treatment can create opportunities in the biotechnological market of enzymes and the development of new drugs.

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.

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

BACKGROUND: 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.

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.

Monitoring casbene synthase in Jatropha curcas tissues using targeted proteomics.

BACKGROUND: Casbene synthase (CS) is responsible for the first committed step in the biosynthesis of phorbol esters (PE) in the Euphorbiaceae. PE are abundant in the seeds of the biofuel crop Jatropha curcas and its toxicity precludes the use of the protein-rich cake obtained after oil extraction as an animal feed and the toxicity of the fumes derived from burning PE containing biofuel is also a matter of concern. This toxicity is a major hindrance to exploit the potential of this crop as a source of raw material to produce biodiesel. For this reason, the current research on J. curcas is mainly focused on the understanding of the biosynthesis and site of synthesis of PE, as an avenue for the development of genotypes unable to synthesize PE in its seeds. RESULTS: Here, we present targeted proteomics assays (SRM and PRM) to detect and quantify CS in leaves, endosperm, and roots of two J. curcas genotypes with contrasting levels of PE. These assays were based on the use of reference isotopic labeled synthetic peptides (ILSP) predicted from 12 gene models of CS from the J. curcas genome. CONCLUSION: Our targeted proteomics methods were able to detect and quantify, for the first time, CS gene products and demonstrate the distribution of CS isoforms only in roots from J. curcas genotypes with a high and low concentration of PE. These methods can be expanded to monitor CS, at the protein level, in different tissues and genotypes of J. curcas.

Assessing the effects of an acute exposure to worst-case concentration of Cry proteins on zebrafish using the embryotoxicity test and proteomics analysis.

Cry1C, Cry1F and Cry1Ab are insecticidal proteins from Bacillus thuringiensis (Bt) which are expressed in transgenic crops. Given the entry of these proteins into aquatic environments, it is relevant to evaluate their impacts on aquatic organisms. In this work, we sought to evaluate the effects of Cry1C, Cry1F and Cry1Ab on zebrafish embryos and larvae of a predicted worst-case scenario concentration of these proteins (set to 1.1 mg/L). For that, we coupled a traditional toxicity approach (the zebrafish embryotoxicity test and dosage of enzymatic biomarkers) to gel free proteomics analysis. At the concentration tested, these proteins did not cause adverse effects in the zebrafish early life stages, either by verifying phenotypic endpoints of toxicity or alterations in representative enzymatic biomarkers (catalase, glutathione-S-tranferase and lactate-dehydrogenase). At the molecular level, the Cry proteins tested lead to very small changes in the proteome of zebrafish larvae. In a global way, these proteins upregulated the expression of vitellogenins. Besides that, Cry1C e Cry1F deregulated heterogeneous nuclear ribonucleoproteins (Hnrnpa0l and Hnrnpaba, respectively), implicated in mRNA processing and gene regulation. Overall, these data lead to the conclusion that Cry1C, Cry1F and Cry1Ab proteins, even at a very high concentration, have limited effects in the early stages of zebrafish life.

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)

Evaluation of the effects of humic acids on maize root architecture by label-free proteomics analysis.

Humic substances have been widely used as plant growth promoters to improve the yield of agricultural crops. However, the mechanisms underlying this effect remain unclear. Root soluble protein profiles in plants 11 days after planting and cultivated with and without humic acids (HA, 50 mg CL-1), were analyzed using the label-free quantitative proteomic approach. Cultivation of maize with HA resulted in higher fresh weight of roots than in untreated plants (control). Plants treated with HA showed increased number, diameter and length of roots. In the proteomics analysis, differences were detected in the following categories: energy metabolism, cytoskeleton, cellular transport, conformation and degradation of proteins, and DNA replication. Thirty-four proteins were significantly more abundant in the seedlings treated with HA, whereas only nine proteins were abundant in the control. The effects on root architecture, such as the induction of lateral roots and biomass increase were accompanied by changes in the energy metabolism-associated proteins. The results show that the main effect of HA is protective, mainly associated with increased expression of the 2-cys peroxidase, putative VHS/GAT, and glutathione proteins. Indeed, these proteins had the highest fold-difference. Overall, these results improve our understanding of the molecular mechanisms of HA-promoted plant growth.