Project description:The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-β1 (M(IL-10+TGF-β1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-β1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-β1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-β1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.
Project description:COVID-19 is the most impacting global pandemic of all times with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-COV-2 infection. For this reason, it is urgent to carry out studies that help to clarify the molecular changes after SARS-COV-2 infection. In this work, we show a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. The differential proteome was narrowed down by PLS-DA tool and the enrichment analysis was performed with FunRich. In parallel OralInt was used to determine interspecies PPIs between humans and SARS-CoV-2. Five dysregulated biological processes were identified in COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. Interactomics analysis allowed the identification of new PPIs between the virus and the host, representing molecular aspects that could be important to understand this disease.
Project description:LC-MS/MS of in-solution trypsin digested parasite protein extracts was carried out to examine the proteome of MSO-treated Pf parasites. Proteins were extracted from the untreated and MSO-treated parasite pellets of two independent experiments (Exp1: T7_R Vs T8; Exp2: T3_R Vs T4_R). For each experiment, two different sets of untreated and MSO-treated cultures synchronized for ring stages and early trophozoites were used. MSO treatment was carried out at 50 M concentration for 12 h. A total number of 149 proteins associated with various metabolic and cellular functions, cytoadherence and host invasion, Hb degradation, etc., were downregulated in MSO-treated parasites. This also included important asparagine-rich proteins such as tRNA ligases, components of RNA processing and protein degradation pathways, lipocalin associated with hemozoin formation and antimalarial drug sensitivity, heat shock protein 110c essential for stabilizing the asparagine repeat-rich parasite proteins etc. Only ten proteins were found to be upregulated in MSO-treated Pf3D7 parasites. For downregulated proteins, proteins identified in both the untreated controls of two independent experiments and either undetectable or significantly downregulated (>1.5 fold) in MSO-treated Pf3D7 parasites were considered. For upregulated proteins, proteins significantly upregulated (>1.5 fold) in both the MSO-treated parasites of two independent experiments and/or undetectable in the untreated controls but detectable in the MSO-treated parasites were considered.
Project description:LC-MS/MS of in-solution trypsin digested FV protein extracts was carried out for the FVs isolated from PbWT and PbFCKO parasites. The FV preparations had signature FV proteins such as plasmepsin IV, berghepain, aminopeptidases, subunits of vacuolar-type H+ATPase (V-type H ATPase), together with parasitophorous vacuolar (PV) proteins including exported protein 1 (Exp1), Exp2, early transcribed membrane protein, PV1, PV5 (lipocalin) etc., and Rab GTPases associated with cytostome-FV trafficking. A total number of 251 and 201 proteins could be identified for WT (P1) and FCKO (P2) FVs, respectively, and 175 proteins were common between them suggesting an overall consistency in the preparations. The results obtained have suggested that the FV proteome in FCKO parasites is compromised. None of the subunits of V-type H+ATPase - a proton pump maintaining the acidic pH of FV could be detected in FCKO FVs indicating the lower abundance of these proteins. In addition, berghepain-2 - a cysteine protease involved in Hb degradation could not be detected in FCKO FVs.
Project description:Recent advances in genome-wide techniques allowed the identification of thousands of non-coding RNAs with various sizes in eukaryotes, some of which have further been shown to serve important functions in many biological processes. However, in model plant Arabidopsis, novel intermediate-sized ncRNAs (im-ncRNAs) (50~300nt) have very limited information. By using a modified isolation strategy combined with deep-sequencing technology, we identified 838 im-ncRNAs in Arabidopsis globally. More than half (58%) are new ncRNA species, mostly evolutionary divergent. Interestingly, annotated protein-coding genes with 5’-UTR derived novel im-ncRNAs tend to be highly expressed. For intergenic im-ncRNAs, their average abundances were comparable to mRNAs in seedlings, but subsets exhibited significantly lower expression in senescing leaves. Further, intergenic im-ncRNAs were regulated by similar genetic and epigenetic mechanisms as those of protein-coding genes, and some showed developmentally-regulated expression patterns. Large-scale reverse genetic screening showed that the down-regulation of a number of im-ncRNAs resulted in either obvious molecular changes or abnormal developmental phenotypes in vivo, indicating the functional importance of im-ncRNAs in plant growth and development. Together, our results demonstrate that novel Arabidopsis im-ncRNAs are developmentally-regulated and functional components discovered in the transcriptome. Genome-wide maps of Intermediate-size Non-coding RNAs in Arabidopsis
Project description:Recent research has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis can modulate the subsequent phases of the process, as well as the behaviour of the resulting plants ex vitro. In this sense, the development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental temperature changes. As a result, in this study, the proteome of radiata pine somatic embryos was studies by LC-MS after the application of high temperatures during induction of embryonal masses (23 °C, 8 weeks, control, 40 °C, 4 h, 60 °C, 5 min). Then, the content of specific soluble sugars and sugar alcohols by HPLC was analysed. Results confirmed a decreased initiation rate of embryonal masses under high temperatures and a slight increase in the production of somatic embryos, as observed in previous studies. Besides, heat provoked a long-term readjustment of the protein synthesis machinery, by regulating translation and the composition of ribosomes. The levels of proteins related to methylation and posttranscriptional regulation mechanisms were also altered under high temperatures, coupled with higher contents of heat shock proteins and chaperones, transmembrane transport proteins, enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol), and changes in the composition of cell-wall carbohydrates. These modifications occurred at the expense of the synthesis of molecules involved in oxidative stress responses, such as peroxidases, thiamine, phenolic compounds and some amino acids, by a decreased activity of the glycolytic pathway and reduced nitrogen assimilation.
Project description:The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenisation and protein extraction are difficulted by tissue calcification. Reproducibility of proteome studies is key when minding clinical translation of the findings. Thus, we aimed to optimise a protocol for aortic valve homogenisation and protein extraction, and to develop a standard operating procedure (SOP), which can be used by researchers to maximise protein yield, while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenisation with a Potter apparatus, with a more advanced bead-beating system. Once confirmed the superiority of the latter, we further optimised it by testing the effect of beads size, the number of homogenisation cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenisation cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2-mL O-ring tubes. As a proof-of-concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterised, resulting in the quantification of proteins across 6 orders of magnitude, and uncovering some putative proteins dysregulated by sex.
Project description:Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.
Project description:The global trend on (male)infertility is concerning and the unidentifiable causes in half of the cases demands a better understanding of the molecular, biochemical and metabolic mechanisms as well as the identification of external and internal factors operating behind it, that might help to explain this apparently unjustified infertility. This can only be achieved through a comprehensive analysis of the infertile men, in which external (lifestyle, occupational and environmental factors) and internal factors (psychological distress) should also be evaluated and considered, but also assessing sperm function beyond the routine seminal analysis. In this prospective cohort study, 79 sperm samples, from men who were male partners in couples seeking for infertility treatment, were collected at the Reproductive Medicine Unit (CHUC) from July 2018 to July 2022 and analyzed by SWATH-MS. Based on couples’ clinical data, seminal/hormonal analysis, and strict exclusion criteria, samples were categorized in the different groups, namely control (CTRL; 50 individuals), idiopathic infertile (ID; 19 patients) and unexplained infertile (UMI; 10 patients) men. In general, ID patients presented the worst sperm functional profile, while the UMI patients were observed to be similar to controls. These differences were also observed at the proteomics levels, which revealed 145 differentially expressed proteins (DEP) between the three groups, with more than 120 proteins being altered between ID and the other groups, and only 14 proteins were considered altered between CTRL and UMI.
Project description:Dioecy is an important sexual system wherein, male and female flowers are borne on separate unisexual plants. Knowledge of sex-related differences can enhance our understanding in molecular and developmental processes leading to unisexual flower development. Coccinia grandis is a dioecious species belonging to Cucurbitaceae, a family well-known for diverse sexual systems. Male and female plants of C. grandis have 22A+XY and 22A+XX chromosomes respectively. Previously, we have reported a gynomonoecious form (GyM) (22A+XX) of C. grandis bearing morphologically hermaphrodite flowers (GyM-H) and female flowers (GyM-F). Also, we showed that foliar spray of silver nitrate on female C. grandis plant induces development of morphologically hermaphrodite buds (Ag-H) despite the absence of Y chromosome. To identify sex-related differences, total protein from the flower buds of male, female, GyM-H and Ag-H of C. grandis at early and middle stages of development were analysed by a powerful label-free proteomics approach on ABSCIEX Triple TOF 5600 platform.