Proteomic Insights of Cowpea Response to Combined Biotic and Abiotic Stresses.

The co-occurrence of biotic and abiotic stresses in agricultural areas severely affects crop performance and productivity. Drought is one of the most adverse environmental stresses, and its association with root-knot nematodes further limits the development of several economically important crops, such as cowpea. Plant responses to combined stresses are complex and require novel adaptive mechanisms through the induction of specific biotic and abiotic signaling pathways. Therefore, the present work aimed to identify proteins involved in the resistance of cowpea to nematode and drought stresses individually and combined. We used the genotype CE 31, which is resistant to the root-knot nematode Meloidogyne spp. And tolerant to drought. Three biological replicates of roots and shoots were submitted to protein extraction, and the peptides were evaluated by LC-MS/MS. Shotgun proteomics revealed 2345 proteins, of which 1040 were differentially abundant. Proteins involved in essential biological processes, such as transcriptional regulation, cell signaling, oxidative processes, and photosynthesis, were identified. However, the main defense strategies in cowpea against cross-stress are focused on the regulation of hormonal signaling, the intense production of pathogenesis-related proteins, and the downregulation of photosynthetic activity. These are key processes that can culminate in the adaptation of cowpea challenged by multiple stresses. Furthermore, the candidate proteins identified in this study will strongly contribute to cowpea genetic improvement programs.

Revealing Corynebacterium glutamicum proteoforms through top-down proteomics.

Corynebacterium glutamicum is a bacterium widely employed in the industrial production of amino acids as well as a broad range of other biotechnological products. The present study describes the characterization of C. glutamicum proteoforms, and their post-translational modifications (PTMs) employing top-down proteomics. Despite previous evidence of PTMs having roles in the regulation of C. glutamicum metabolism, this is the first top-down proteome analysis of this organism. We identified 1125 proteoforms from 273 proteins, with 60% of proteins presenting at least one mass shift, suggesting the presence of PTMs, including several acetylated, oxidized and formylated proteoforms. Furthermore, proteins relevant to amino acid production, protein secretion, and oxidative stress were identified with mass shifts suggesting the presence of uncharacterized PTMs and proteoforms that may affect biotechnologically relevant processes in this industrial workhorse. For instance, the membrane proteins mepB and SecG were identified as a cleaved and a formylated proteoform, respectively. While in the central metabolism, OdhI was identified as two proteoforms with potential biological relevance: a cleaved proteoform and a proteoform with PTMs corresponding to a 70 Da mass shift.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

NBS-LRR-WRKY genes and protease inhibitors (PIs) seem essential for cowpea resistance to root-knot nematode.

Cowpea (Vigna unguiculata L. Walp) is a legume of great economic importance, however it is highly affected by nematodes. The present work aimed to identify proteins and genes involved in nematode resistance by proteomic and transcriptomic analysis. Plants of a genotype resistant (CE31) to root-knot nematode (Meloidogyne spp.) were collected 12 days after inoculation with Meloidogyne incognita and the total proteins and RNA were extracted from the root samples. Shotgun proteomic analysis was performed using an Orbitrap Elite mass spectrometer and the construction and sequencing of cDNA libraries were carried out in a Hi-Seq 2000 sequencing system. The proteomic and transcriptomic analyses revealed key processes involved in cowpea defense and some interesting candidates were further analyzed by RT-qPCR. Proteins and genes involved in essential biological processes were differentially accumulated such as, regulation of transcription, cell wall stiffening and microtubule-based process. However, the main defense strategies of Vigna unguiculata seem to be focused on the interaction of NBS-LRR and WRKY genes for the activation of R genes, production of protease inhibitors and maintenance of actin cytoskeleton. These are key processes that can culminate in the suppression of giant cell formation and consequently in the development of Meloidogyne incognita. SIGNIFICANCE: In this study, we identified proteins and transcripts regulated in cowpea resistant to the nematode Meloidogyne spp. upon inoculation. The results revealed key candidate genes involved in the activation of R genes, the production of protease inhibitors and maintenance of the actin cytoskeleton. These processes might be essential for cowpea resistance, as they can impede nematode nutrition, giant cell formation and consequently the development of Meloidogyne incognita.

Mapping Salivary Proteases in Sjögren's Syndrome Patients Reveals Overexpression of Dipeptidyl Peptidase-4/CD26.

Sjögren's Syndrome (SS) is an autoimmune exocrinopathy characterized by the progressive damage of salivary and lacrimal glands associated with lymphocytic infiltration. Identifying new non-invasive biomarkers for SS diagnosis remains a challenge, and alterations in saliva composition reported in patients turn this fluid into a source of potential biomarkers. Among these, proteases are promising candidates since they are involved in several key physio-pathological processes. This study evaluated differentially expressed proteases in SS individuals' saliva using synthetic fluorogenic substrates, zymography, ELISA, and proteomic approaches. Here we reported, for the first time, increased activity of the serine protease dipeptidyl peptidase-4/CD26 (DPP4/CD26) in pSS saliva, the expression level of which was corroborated by ELISA assay. Gelatin zymograms showed that metalloproteinase proteolytic band profiles differed significantly in intensity between control and SS groups. Focusing on matrix metalloproteinase-9 (MMP9) expression, an increased tendency in pSS saliva (p = 0.0527) was observed compared to the control group. Samples of control, pSS, and sSS were analyzed by mass spectrometry to reveal a general panorama of proteases in saliva. Forty-eight protein groups of proteases were identified, among which were the serine proteases cathepsin G (CTSG), neutrophil elastase (ELANE), myeloblastin (PRTN3), MMP9 and several protease inhibitors. This work paves the way for proteases to be explored in the future as biomarkers, emphasizing DPP4 by its association in several autoimmune and inflammatory diseases. Besides its proteolytic role, DPP4/CD26 acts as a cell surface receptor, signal transduction mediator, adhesion and costimulatory protein involved in T lymphocytes activation.

iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia.

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.

In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

An Integrative Sialomic Analysis Reveals Molecules From Triatoma sordida (Hemiptera: Reduviidae).

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts' hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector's biology, the hematophagous behaviour, and the Triatominae subfamily's evolution.

Novel Possible Protein Targets in Neovascular Age-Related Macular Degeneration: A Pilot Study Experiment.

Age-related macular degeneration (AMD) is among the world's leading causes of blindness. In its neovascular form (nAMD), around 25% of patients present further anatomical and visual deterioration due to persistence of neovascular activity, despite gold-standard treatment protocols using intravitreal anti-VEGF medications. Thus, to comprehend, the molecular pathways that drive choroidal neoangiogenesis, associated with the vascular endothelial growth factor (VEGF), are important steps to elucidate the mechanistic events underneath the disease development. This is a pilot study, a prospective, translational experiment, in a real-life context aiming to evaluate the protein profiles of the aqueous humor of 15 patients divided into three groups: group 1, composed of patients with nAMD, who demonstrated a good response to anti-VEGF intravitreal injections during follow-up (good responsive); group 2, composed of patients with anti-VEGF-resistant nAMD, who demonstrated choroidal neovascularization activity during follow-up (poor/non-responsive); and group 3, composed of control patients without systemic diseases or signs of retinopathy. For proteomic characterization of the groups, mass spectrometry (label-free LC-MS/MS) was used. A total of 2,336 proteins were identified, of which 185 were distinctly regulated and allowed the differentiation of the clinical conditions analyzed. Among those, 39 proteins, including some novel ones, were analyzed as potential disease effectors through their pathophysiological implications in lipid metabolism, oxidative stress, complement system, inflammatory pathways, and angiogenesis. So, this study suggests the participation of other promising biomarkers in neovascular AMD, in addition to the known VEGF.

Proteomic Mapping of Multifunctional Complexes Within Triatomine Saliva.

Triatomines are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent of Chagas disease. This neglected tropical disease represents a global health issue as it is spreading worldwide. The saliva of Triatominae contains miscellaneous proteins crucial for blood feeding acquisition, counteracting host's hemostasis while performing vasodilatory, anti-platelet and anti-coagulant activities, besides modulating inflammation and immune responses. Since a set of biological processes are mediated by protein complexes, here, the sialocomplexomes (salivary protein complexes) of five species of Triatominae were studied to explore the protein-protein interaction networks. Salivary multiprotein complexes from Triatoma infestans, Triatoma dimidiata, Dipetalogaster maxima, Rhodnius prolixus, and Rhodnius neglectus were investigated by Blue-Native- polyacrylamide gel electrophoresis coupled with liquid chromatography tandem mass spectrometry. More than 70 protein groups, uncovering the landscape of the Triatominae salivary interactome, were revealed. Triabin, actin, thioredoxin peroxidase and an uncharacterized protein were identified in sialocomplexes of the five species, while hexamerin, heat shock protein and histone were identified in sialocomplexes of four species. Salivary proteins related to triatomine immunity as well as those required during blood feeding process such as apyrases, antigen 5, procalins, and nitrophorins compose different complexes. Furthermore, unique proteins for each triatomine species were revealed. This study represents the first Triatominae sialocomplexome reference to date and shows that the approach used is a reliable tool for the analysis of Triatominae salivary proteins assembled into complexes.

Paternal Resistance Training Modulates Calcaneal Tendon Proteome in the Offspring Exposed to High-Fat Diet.

The increase in high-energy dietary intakes is a well-known risk factor for many diseases, and can also negatively impact the tendon. Ancestral lifestyle can mitigate the metabolic harmful effects of offspring exposed to high-fat diet (HF). However, the influence of paternal exercise on molecular pathways associated to offspring tendon remodeling remains to be determined. We investigated the effects of 8 weeks of paternal resistance training (RT) on offspring tendon proteome exposed to standard diet or HF diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, three times per week, with 8-12 dynamic movements per climb in a stair climbing apparatus). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into four groups (five animals per group): offspring from sedentary fathers were exposed either to control diet (SFO-C), or to high-fat diet (SFO-HF); offspring from trained fathers were exposed to control diet (TFO-C) or to a high-fat diet (TFO-HF). The Nano-LC-MS/MS analysis revealed 383 regulated proteins among offspring groups. HF diet induced a decrease of abundance in tendon proteins related to extracellular matrix organization, transport, immune response and translation. On the other hand, the changes in the offspring tendon proteome in response to paternal RT were more pronounced when the offspring were exposed to HF diet, resulting in positive regulation of proteins essential for the maintenance of tendon integrity. Most of the modulated proteins are associated to biological pathways related to tendon protection and damage recovery, such as extracellular matrix organization and transport. The present study demonstrated that the father's lifestyle could be crucial for tendon homeostasis in the first generation. Our results provide important insights into the molecular mechanisms involved in paternal intergenerational effects and potential protective outcomes of paternal RT.

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

BACKGROUND: Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. METHODS: After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. RESULTS: After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. CONCLUSIONS: To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

Paternal Resistance Training Induced Modifications in the Left Ventricle Proteome Independent of Offspring Diet.

Ancestral obesogenic exposure is able to trigger harmful effects in the offspring left ventricle (LV) which could lead to cardiovascular diseases. However, the impact of the father's lifestyle on the offspring LV is largely unexplored. The aim of this study was to investigate the effects of 8 weeks of paternal resistance training (RT) on the offspring left ventricle (LV) proteome exposed to control or high-fat (HF) diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, 3 times per week with weights secured to the animals' tails). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into 4 groups (5 animals per group): offspring from sedentary fathers, exposed to control diet (SFO-C); offspring from trained fathers, exposed to control diet (TFO-C); offspring from sedentary fathers, exposed to high-fat diet (SFO-HF); and offspring from trained fathers, exposed to high-fat diet (TFO-HF). The LC-MS/MS analysis revealed 537 regulated proteins among groups. Offspring exposure to HF diet caused reduction in the abundance levels of proteins related to cell component organization, metabolic processes, and transport. Proteins related to antioxidant activity, transport, and transcription regulation were increased in TFO-C and TFO-HF as compared with the SFO-C and SFO-HF groups. Paternal RT demonstrated to be an important intervention capable of inducing significant effects on the LV proteome regardless of offspring diet due to the increase of proteins involved into LV homeostasis maintenance. This study contributes to a better understanding of the molecular aspects involved in transgenerational inheritance.

Mild hydrothermal pretreatment of sugarcane bagasse enhances the production of holocellulases by Aspergillus niger.

Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB saccharification. The combined use of PS and pretreatment liquor (PL), referred to as whole pretreated slurry (WPS), as carbon source induced a more balanced up-regulation of cellulases, hemicellulases and pectinases and resulted in the highest increase (4.8-fold) in the release of total reducing sugars from SCB. The use of PL as the sole carbon source induced the modulation of A. niger's secretome towards hemicellulose degradation. Mild pretreatment allowed the use of PL in downstream biological operations without the need for undesirable detoxification steps.

Metabolic Peculiarities of Paracoccidioides brasiliensis Dimorphism as Demonstrated by iTRAQ Labeling Proteomics.

Paracoccidioidomycosis (PCM), a systemic mycosis with a high incidence in Latin America, is caused by thermodimorphic fungi of the Paracoccidioides genus. The contact with host occurs by the inhalation of conidia or mycelial propagules which once reaching the pulmonary alveoli differentiate into yeast cells. This transition process is vital in the pathogenesis of PCM allowing the fungus survival in the host. Thus, the present work performed a comparative proteome analysis of mycelia, mycelia-to-yeast transition, and yeast cells of Paracoccidioides brasiliensis. For that, tryptic peptides were labeled with iTRAQ and identified by LC-MS/MS and computational data analysis, which allowed the identification of 312 proteins differentially expressed in different morphological stages. Data showed that P. brasiliensis yeast cells preferentially employ aerobic beta-oxidation and the tricarboxylic acid cycle accompanied by oxidative phosphorylation for ATP production, in comparison to mycelia and the transition from mycelia-to-yeast cells. Furthermore, yeast cells show a metabolic reprogramming in amino acid metabolism and in the induction of virulence determinants and heat shock proteins allowing adaptation to environmental conditions during the increase of the temperature. In opposite of that, the alcoholic fermentation found to P. lutzii, at least under laboratory conditions, is strongly favored in mycelium compared to yeast cells. Thereby, the data strongly support substantial metabolic differences among members of the Paracoccidioides complex, when comparing the saprobiotic mycelia and the yeast parasitic phases.

Stress and cell cycle regulation during somatic embryogenesis plays a key role in oil palm callus development.

Oil palm is an oleaginous plant of relevant economic importance since its fruits are rich in vegetable oil. These plants have a single apical meristem and the main method for vegetative propagation is somatic embryogenesis. The aim of this study was to identify differentially abundant proteins from oil palm genotypes contrasting in the capacity of embryogenic competence acquisition, using shotgun proteomics. Oil palm leaves were subjected to callus induction and the material was collected in biological triplicates at 14 and 90 days of callus induction. LC-MS/MS analysis was performed and revealed a total of 4695 proteins. Responsive and non-responsive genotypes were compared at 14 and 90 days of callus induction and 221 differentially abundant proteins were obtained. The data analysis revealed several proteins mainly related to energy metabolism, stress response and regulation of cell cycle, further analyzed by qRT-PCR, which seem important for embryogenic development. We suggest some of these proteins as key factors for the success of callus formation in oil palm including antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway. These proteins may also be potential biomarkers for the acquisition of embryogenic competence. SIGNIFICANCE: Antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway are key factors for the success of callus formation in oil palm. The proteins identified in this study may be potential biomarkers for embryogenic competence acquisition.

Proteome analysis of Phytomonas serpens, a phytoparasite of medical interest.

The protozoan Phytomonas serpens (class Kinetoplastea) is an important phytoparasite that has gained medical importance due to its similarities to Trypanosoma cruzi, the etiological agent of Chagas disease. The present work describes the first proteome analysis of P. serpens. The parasite was separated into cytosolic and high density organelle fractions, which, together with total cell extract, were subjected to LC-MS/MS analyses. Protein identification was conducted using a comprehensive database composed of genome sequences of other related kinetoplastids. A total of 1,540 protein groups were identified among the three sample fractions. Sequences from Phytomonas sp. in the database allowed the highest number of identifications, with T. cruzi and T. brucei the human pathogens providing the greatest contribution to the identifications. Based on the proteomics data obtained, we proposed a central metabolic map of P. serpens, which includes all enzymes of the citric acid cycle. Data also revealed a new range of proteins possibly responsible for immunological cross-reactivity between P. serpens and T. cruzi.

Exploring the molecular complexity of Triatoma dimidiata sialome.

Triatoma dimidiata, a Chagas disease vector widely distributed along Central America, has great capability for domestic adaptation as the majority of specimens caught inside human dwellings or in peridomestic areas fed human blood. Exploring the salivary compounds that overcome host haemostatic and immune responses is of great scientific interest. Here, we provide a deeper insight into its salivary gland molecules. We used high-throughput RNA sequencing to examine in depth the T. dimidiata salivary gland transcriptome. From >51 million reads assembled, 92.21% are related to putative secreted proteins. Lipocalin is the most abundant gene family, confirming it is an expanded family in Triatoma genus salivary repertoire. Other putatively secreted members include phosphatases, odorant binding protein, hemolysin, proteases, protease inhibitors, antigen-5 and antimicrobial peptides. This work expands the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI from 388 to 3815. Additionally, we complemented the salivary analysis through proteomics (available data via ProteomeXchange with identifier PXD008510), disclosing the set complexity of 119 secreted proteins and validating the transcriptomic results. Our large-scale approach enriches the pharmacologically active molecules database and improves our knowledge about the complexity of salivary compounds from haematophagous vectors and their biological interactions. SIGNIFICANCE: Several haematophagous triatomine species can transmit Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the reemergence of this disease, new drugs for its prevention and treatment are considered priorities. For this reason, the knowledge of vector saliva emerges as relevant biological finding, contributing to the design of different strategies for vector control and disease transmission. Here we report the transcriptomic and proteomic compositions of the salivary glands (sialome) of the reduviid bug Triatoma dimidiata, a relevant Chagas disease vector in Central America. Our results are robust and disclosed unprecedented insights into the notable diversity of its salivary glands content, revealing relevant anti-haemostatic salivary gene families. Our work expands almost ten times the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI. Moreover, using an integrated transcriptomic and proteomic approach, we showed a correlation pattern of transcription and translation processes for the main gene families found, an important contribution to the research of triatomine sialomes. Furthermore, data generated here reinforces the secreted proteins encountered can greatly contribute for haematophagic habit, Trypanosoma cruzi transmission and development of therapeutic agent studies.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation.

The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.