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The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry, identifying 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue. We defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. These data reveal tumour matrisome signatures in human NSCLC.Abstract Figure

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.

Ocean acidification is impacting marine life all over the world. Understanding how species can cope with the changes in seawater carbonate chemistry represents a challenging issue. We addressed this topic using underwater CO2 vents that naturally acidify some marine areas off the island of Ischia. In the most acidified area of the vents, having a mean pH value of 6.7, comparable to far-future predicted acidification scenarios (by 2300), the biomass is dominated by the brown alga Sargassum vulgare. The novelty of the present study is the characterization of the S. vulgare proteome together with metabolite analyses to identify the key proteins, metabolites, and pathways affected by ocean acidification. A total of 367 and 387 proteins were identified in populations grown at pH that approximates the current global average (8.1) and acidified sites, respectively. Analysis of their relative abundance revealed that 304 proteins are present in samples from both sites: 111 proteins are either higher or exclusively present under acidified conditions, whereas 120 proteins are either lower or present only under control conditions. Functionally, under acidification, a decrease in proteins related to translation and post-translational processes and an increase of proteins involved in photosynthesis, glycolysis, oxidation-reduction processes, and protein folding were observed. In addition, small-molecule metabolism was affected, leading to a decrease of some fatty acids and antioxidant compounds under acidification. Overall, the results obtained by proteins and metabolites analyses, integrated with previous transcriptomic, physiological, and biochemical studies, allowed us to delineate the molecular strategies adopted by S. vulgare to grow in future acidified environments, including an increase of proteins involved in energetic metabolism, oxidation-reduction processes, and protein folding at the expense of proteins involved in translation and post-translational processes.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

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Background: IgA vasculitis (IgAV) is the most common form of childhood vasculitis. A better understanding of its pathophysiology is required to identify new potential biomarkers and treatment targets. Objective: to assess the underlying molecular mechanisms in the pathogenesis of IgAV using an untargeted proteomics approach. Methods: Thirty-seven IgAV patients and five healthy controls were enrolled. Plasma samples were collected on the day of diagnosis before any treatment was initiated. We used nano-liquid chromatography–tandem mass spectrometry (nLC–MS/MS) to investigate the alterations in plasma proteomic profiles. For the bioinformatics analyses, databases including Uniprot, PANTHER, KEGG, Reactome, Cytoscape, and IntAct were used. Results: Among the 418 proteins identified in the nLC–MS/MS analysis, 20 had significantly different expressions in IgAV patients. Among them, 15 were upregulated and 5 were downregulated. According to the KEGG pathway and function classification analysis, complement and coagulation cascades were the most enriched pathways. GO analyses showed that the differentially expressed proteins were mainly involved in defense/immunity proteins and the metabolite interconversion enzyme family. We also investigated molecular interactions in the identified 20 proteins of IgAV patients. We extracted 493 interactions from the IntAct database for the 20 proteins and used Cytoscape for the network analyses. Conclusion: Our results clearly suggest the role of the lectin and alternate complement pathways in IgAV. The proteins defined in the pathways of cell adhesion may serve as biomarkers. Further functional studies may lead the way to better understanding of the disease and new therapeutic options for IgAV treatment.

The demand for jellyfish is growing worldwide, especially due to their high nutraceutical value. In this study, the extraction and characterization of crude gelatin from the brown cannonball jellyfish (Stomolophus meleagris), which is periodically found in large volumes on the American Pacific coasts, were carried out. The crude gelatin obtained by alkaline treatment, with subsequent heat and dialysis treatment, showed an ability to quench free radicals (via ABTS and ORAC methods), and protect human cells against oxidative damage (through inhibition of hemolysis by AAPH), and they protected against mutations caused by aflatoxin B1 in the Salmonella enterica Typhimurium TA100 strain. Furthermore, it was established that these extracts were innocuous for eukaryotic cells (genotoxicity assay). The amino acid profiles indicate a high concentration of glycine and proline, as well as charged amino acids. Electrophoretic, FT-IR, and 1H-NMR studies indicated that one of the main proteins present in this crude gelatin is collagen. The presence of collagen and other proteins was identified by proteomic studies. Alkaline crude gelatin from brown jellyfish could be considered as potential candidates to be evaluated as antioxidant agents in foods in future research.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Tesis doctoral defendida en la Facultad de Ciencias Biológicas de la Universidad Complutense de Madrid, [EN] Cone snails belong to the family Conidae and constitute a highly diversified monophyletic group (>900 species), which is generally found inhabiting the intertidal of tropical and subtropical areas worldwide. In addition to their high species diversity, cones are characterized by having a highly specialized venom apparatus for producing and injecting toxins (conotoxins) with a neurotoxic effect. These are used to hunt prey and to defend against predators or competitors. Conotoxins are usually short peptides, rich in cysteines and are often modified post-translationally in their native state. It is considered that cone snails are capable of producing highly variable venom cocktails at different hierarchical levels (individual, population, interspecific) and that venom composition could be linked to different diets, generally grouped into three large groups: worms (vermivores), fish (piscivores) and snails (molluscivores). Cone snail venom has a huge interest in evolutionary studies as well as for its potential as novel drug source. In order to study cone snail venom, the first step is to determine its exact composition by cataloguing the different conotoxins, hormones, and other functional peptides. Currently, it is possible to use high-throughput sequencing techniques to obtain the venom duct transcriptome i.e., all the transcripts expressed in this tissue and their level of expression. On the other hand, proteomics is able to precisely determine the peptide components of the venom that will target receptors in the prey or predator. The current availability of second generation high-throughput sequencing techniques is allowing the cataloguing and analysis of venoms of an increasing number of cone snails. The advent of third-generation long-read sequencing along with techniques that make use of chromatin conformation structure are allowing the generation of chromosome-level assemblies from non-model organisms. The availability of such high-quality genomes for venomous organisms represents a milestone that together with transcriptomics and proteomics (i.e., venomics) opens the possibility for studying the origin and evolution of venom from an integrative viewpoint. In this doctoral thesis a multidisciplinary venomic approach was used to compare 1) the diversity of conotoxins associated to different diets; 2) venom expression levels between Atlantic and Indo-Pacific species with convergent piscivorous diets; 3) venom expression levels between cryptic sister species living in sympatry and 4) to evaluate the genomic basis of conotoxin diversity by generating and analyzing a chromosome-scale assembly of a cone snail genome. Comparative approaches used new transcriptomes from venom ducts of three vermivorous species (Lautoconus ventricosus, Virroconus ebraeus and Virroconus judaeus) and a piscivorous species (Pionoconus magus). In addition, the venom duct proteomes of V. ebraeus and V. judaeus were determined. With the new generated data, the diversity of conotoxins in all the mentioned species was catalogued, leading to the discovery of seven new conotoxin superfamilies of yet unknown molecular targets. It was possible to identify differences in expression between the two piscivorous species from the Indo-Pacific (P. magus) and the Atlantic (Chelyconus ermineus). Comparative transcriptomic analyses between V. ebraeus and V. judaeus revealed significant differences in conotoxins expression levels, also between proteomes, between these two cryptic sister species, which could be associated with specific dietary adaptations. Finally, the genome of L. ventricosus was sequenced using long CLR PacBio reads and chromatin conformation capture techniques (Hi-C and Chicago). The genome assembly had a final size of 3.59 Gb mainly distributed into 35 major scaffolds representing pseudochromosomes. The genome, along with the venom duct transcriptomes, were analyzed to infer the genomic basis of conotoxin gene diversity. A close to one-to-one relationship between genes and transcripts was observed and conotoxin genes were found to be scattered across almost all pseudochromosomes. In addition, the new cone snail genome was compared to that of the caenogastropod Pomacea canaliculata and an ancient whole genome duplication could be confirmed, which could have had an important role both in the diversification of the group as well as in that of conotoxins., La presente Tesis Doctoral ha sido financiada por: Convocatoria 2017 de las ayudas para contratos predoctorales para la formación de doctores del Subprograma Estatal de Formación del Ministerio de Economía, Industria y Competitividad con código BES-2017–081195. Los proyectos del Ministerio de Ciencia e Innovación CGL2016-75255-C2-1-P [AEI/FEDER, UE y PID2019-103947GB-C22/AEI/10.13039/501100011033.

Candida albicans is the main causal pathogen of fungal infections in human beings. Although diverse anti-C. albicans drugs have been explored, the drug resistance and side effects of these drugs are intensifying. Thus, it is urgent to explore new anti-C. albicans compounds from natural products. In this study, we identified trichoderma acid (TA), a compound from Trichoderma spirale with a strong inhibitory effect on C. albicans. Transcriptomic and iTRAQ-based proteomic analyses of TA-treated C. albicans in combination with scanning electronic microscopy and reactive oxygen species (ROS) detection were performed to investigate the potential targets of TA. The most significant differentially expressed genes and proteins after TA treatment were verified through Western blot analysis. Our results revealed that mitochondrial membrane potential, endoplasmic reticulum, ribosomes in the mitochondria, and cell walls were disrupted in TA-treated C. albicans, leading to the accumulation of ROS. The impaired enzymatic activities of superoxide dismutase further contributed to the increase in ROS concentration. The high concentration of ROS led to DNA damage and cell skeleton destruction. The expression levels of Rho-related GTP-binding protein RhoE (RND3), asparagine synthetase (ASNS), glutathione S-transferase, and heat shock protein 70 were significantly up-regulated in response to apoptosis and toxin stimulation. These findings suggest that RND3, ASNS, and supereoxide dismutase 5 are the potential targets of TA, as further demonstrated through Western blot analysis. The combination of transcriptomic, proteomic, and cellular analyses would provide clues for the anti-C. albicans mechanism of TA and the defensive response mechanism of C. albicans. TA is thus recognized as a promising new anti-C. albicans leading compound that alleviates the hazard of C. albicans infection in human beings.

Fibromuscular dysplasia (FMD) is a non-atherosclerotic vascular disease that may involve medium-sized muscular arteries throughout the body. The majority of FMD patients are women. Although a variety of genetic, mechanical, and hormonal factors play a role in the pathogenesis of FMD, overall, its cause remains poorly understood. It is probable that the pathogenesis of FMD is linked to a combination of genetic and environmental factors. Extensive studies have correlated the arterial lesions of FMD to histopathological findings of arterial fibrosis, cellular hyperplasia, and distortion of the abnormal architecture of the arterial wall. More recently, the vascular phenotype of lesions associated with FMD has been expanded to include arterial aneurysms, dissections, and tortuosity. However, in the absence of a string-of-beads or focal stenosis, these lesions do not suffice to establish the diagnosis. While FMD most commonly involves renal and cerebrovascular arteries, involvement of most arteries throughout the body has been reported. Increasing evidence highlights that FMD is a systemic arterial disease and that subclinical alterations can be found in non-affected arterial segments. Recent significant progress in FMD-related research has led to improve our understanding of the disease's clinical manifestations, natural history, epidemiology, and genetics. Ongoing work continues to focus on FMD genetics and proteomics, physiological effects of FMD on cardiovascular structure and function, and novel imaging modalities and blood-based biomarkers that can be used to identify subclinical FMD. It is also hoped that the next decade will bring the development of multi-centred and potentially international clinical trials to provide comparative effectiveness data to inform the optimal management of patients with FMD.

Fibromuscular dysplasia (FMD) is a non-atherosclerotic vascular disease that may involve medium-sized muscular arteries throughout the body. The majority of FMD patients are women. Although a variety of genetic, mechanical, and hormonal factors play a role in the pathogenesis of FMD, overall, its cause remains poorly understood. It is probable that the pathogenesis of FMD is linked to a combination of genetic and environmental factors. Extensive studies have correlated the arterial lesions of FMD to histopathological findings of arterial fibrosis, cellular hyperplasia, and distortion of the abnormal architecture of the arterial wall. More recently, the vascular phenotype of lesions associated with FMD has been expanded to include arterial aneurysms, dissections, and tortuosity. However, in the absence of a string-of-beads or focal stenosis, these lesions do not suffice to establish the diagnosis. While FMD most commonly involves renal and cerebrovascular arteries, involvement of most arteries throughout the body has been reported. Increasing evidence highlights that FMD is a systemic arterial disease and that subclinical alterations can be found in non-affected arterial segments. Recent significant progress in FMD-related research has led to improve our understanding of the disease’s clinical manifestations, natural history, epidemiology, and genetics. Ongoing work continues to focus on FMD genetics and proteomics, physiological effects of FMD on cardiovascular structure and function, and novel imaging modalities and blood-based biomarkers that can be used to identify subclinical FMD. It is also hoped that the next decade will bring the development of multi-centred and potentially international clinical trials to provide comparative effectiveness data to inform the optimal management of patients with FMD.

The soil-borne necrotrophic fungus Rhizoctonia solani is one of destructive fungi causing severe yield losses in various important crops. However, the host defense mechanisms against the invasion of this pathogen are poorly understood. In this study, we employed an iTRAQ-based quantitative proteomic approach to investigate host proteins responsive to R. solani using the resistant rice cultivar YSBR1. As a whole, we identified 319 differentially accumulated proteins (DAPs) after inoculation of rice plants with R. solani. Functional categorization analysis indicates that these DAPs cover a broad range of functions. Notably, a substantial portion of the DAPs are involved in cell redox homeostasis, carbohydrate metabolism, and phenylpropanoid biosynthesis, or belong to pathogenesis-related proteins, indicating that these processes/proteins play important roles in host defense against R. solani. Interestingly, all of the DAPs involved in photosynthesis and chlorophyll biosynthetic processes, and part of the DAPs involved in phenylpropanoid biosynthesis, show reduced accumulation after R. solani infection, suggesting that R. solani probably inhibits host photosynthetic system and phenylpropanoid biosynthesis to facilitate infection and colonization. In conclusion, our results provide both valuable resources and new insights into the molecular mechanisms underlying rice and R. solani interaction.

Exposure to extended periods of darkness is a common source of abiotic stress that significantly affects plant growth and development. To understand how Nicotiana benthamiana responds to dark stress, the proteomes and metabolomes of leaves treated with darkness were studied. In total, 5763 proteins and 165 primary metabolites were identified following dark treatment. Additionally, the expression of autophagy‐related gene (ATG) proteins was transiently upregulated. Weighted gene coexpression network analysis (WGCNA) was utilized to find the protein modules associated with the response to dark stress. A total of four coexpression modules were obtained. The results indicated that heat‐shock protein (HSP70), SnRK1‐interacting protein 1, 2A phosphatase‐associated protein of 46 kDa (Tap46), and glutamate dehydrogenase (GDH) might play crucial roles in N. benthamiana’s response to dark stress. Furthermore, a protein–protein interaction (PPI) network was constructed and top‐degreed proteins were predicted to identify potential key factors in the response to dark stress. These proteins include isopropylmalate isomerase (IPMI), eukaryotic elongation factor 5A (ELF5A), and ribosomal protein 5A (RPS5A). Finally, metabolic analysis suggested that some amino acids and sugars were involved in the dark‐responsive pathways. Thus, these results provide a new avenue for understanding the defensive mechanism against dark stress at the protein and metabolic levels in N. benthamiana., Exposure to prolonged periods of darkness can affect plant growth and development. In this study, the response of Nicotiana benthamiana to dark stress was investigated by analyzing changes to the proteome and metabolome across 13 time points. A number of hub proteins were filtered through weighted gene coexpression network analysis to identify potential key proteins involved in the dark stress response.

Sorghum is a cereal with potential economic and nutritional properties. It has gained headway in the international market because of its nutritional content which is characterized for many bioactive compounds with antioxidant characteristics, and also, because it is gluten free. This work evaluated the proteomic profile of sorghum grains and its nutritional composition and functional profile after exposure to 7 different treatments (control, grind, dry heat, bursting, wet cooking with and without water and wet cooking in pressure). They were analyzed for chemical composition, protein profile, total phenolic compounds, anthocyanin content and antioxidant activity. The dry heat preserves the protein content, phenolic compounds, anthocyanins and presents between 94% and 95% of radical scavenging activity. Heat treatments that use the pressure promote the natural hydrolysis of proteins. Bursting treatment resulted in 45.6% of proteins and peptides in the range of 3.7; 5.93; 8.9 and 14 kDa. Wet cooking in pressure (SPC) showed a similar behavior, with 26.8% being the abundance of 14 and 14.3 kDa proteins and 25.3% of the peptides with less than 10 kDa, making up 52.1% of protein content. This hydrolysis promoted an important percentage of peptides and low molecular mass proteins which can have bioactive profile and improve healthy.

Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm analysis (CASA), while protein composition of the seminal plasma was studied using UHPLC-MS/MS. Specific proteins were more abundant in samples with poorer percentages of total motility, average path velocity and circular velocity, and were: Secreted phosphoprotein 1, Fructose-bisphosphate aldolase (p = 1,95E-09; q = 0.0005) and Malate dehydrogenase 1 (p = 1,41E-11; q = 0.002), to the contrary samples with better straight-line velocity values were enriched in Glutathione peroxidase (p=0.00013; q=0.04) and Triosephosphate isomerase (p=0.00015; q=0.04).

Soil salinization poses a huge challenge to the development of agriculture and seriously decreases crop yield and quality. In recent years, grafting has become one of the key agronomic techniques used to enhance plant abiotic stress tolerance. In this study, we found that watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] grafted onto bottle gourd (Lagenaria siceraria Standl.) significantly enhanced salt tolerance. Transcriptome analysis revealed that a total of 8462 differentially expressed genes (DEGs) were identified, and the number of up- and down-regulated genes were 3207 and 5255, respectively. The DEGs in the bottle gourd rootstock-grafted plants were mainly involved in carbon metabolism, photosynthesis, and plant hormone signal transduction. Furthermore, proteome analysis identified 28 differently expressed proteins (DEPs) in bottle gourd rootstock-grafted plants under salt stress. These DEPs were closely associated with amino acid and protein synthesis, photosynthesis, mitochondrial metabolism and carbon metabolism, and stress defense. Combined transcriptome and proteome analyses showed that salt stress-responded genes in bottle gourd rootstock-grafted watermelon seedlings were mainly involved in plant hormone signal transduction, photosynthesis, and amino acid synthesis pathways.

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)–Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI–MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS–MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

Complete proteomic datasets are available in the PRIDE partner repository (Perez-Riverol et al., 2019) under identification numbers: PXD030822 and 10.6019/PXD030822 for HepaRG cells, PXD031045 and 10.6019/PXD031045 for HepaRG culture media, PXD030887 and 10.6019/PXD030887 for PHHs, and PXD030892 and 10.6019/PXD030892 for PRHs. Metabolomic data are available in the MetaboLights repository (Haug et al., 2020) under the identification number: www.ebi.ac.uk/metabolights/MTBLS4063.; International audience; The biological effects of the pesticide and mitochondrial complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by combining proteomics and metabolomics. Both cell culture media and cellular lysates were analyzed in dose-response and kinetic experiments on the HepaRG cell line. Responses were compared with those obtained on primary human and rat hepatocytes. A multitude of phase I and II metabolites (>80) mainly common to HepaRG cells and primary hepatocytes and an increase in metabolization enzymes were observed. Synthesis of mitochondrion and oxidative phosphorylation complex constituents, fatty acid oxidation, and cellular uptake of lipids were induced to compensate for complex I inhibition and the decrease in ATP intracellular contents caused by TEBU. Secretion of the 20 S circulating proteasome and overall inhibition of acute inflammation followed by IL-6 secretion in later stages were observed in HepaRG cells. These effects were associated with a decrease in STAT1 and STAT3 transcription factor abundances, but with different kinetics. Based on identified TEBU targets, docking experiments, and nuclear receptor reporter assays, we concluded that liver cell response to TEBU is mediated by its interaction with the PPARγ transcription factor.

21 p.-9 fig.-1 tab., Leuconostoc lactis is found in vegetables, fruits, and meat and is used by the food industry in the preparation of dairy products, wines, and sugars. We have previously demonstrated that the dextransucrase of Lc. lactis (DsrLL) AV1n produces a high-molecular-weight dextran from sucrose, indicating its potential use as a dextran-forming starter culture. We have also shown that this bacterium was able to produce 10-fold higher levels of dextran at 20°C than at 37°C, at the former temperature accompanied by an increase in dsrLL gene expression. However, the general physiological response of Lc. lactis AV1n to cold temperature in the presence of sucrose, leading to increased production of dextran, has not been yet investigated. Therefore, we have used a quantitative proteomics approach to investigate the cold temperature-induced changes in the proteomic profile of this strain in comparison to its proteomic response at 37°C. In total, 337 proteins were found to be differentially expressed at the applied significance criteria (adjusted p-value ≤ 0.05, FDR 5%, and with a fold-change ≥ 1.5 or ≤ 0.67) with 204 proteins overexpressed, among which 13% were involved in protein as well as cell wall, and envelope component biosynthesis including DsrLL. Proteins implicated in cold stress were expressed at a high level at 20°C and possibly play a role in the upregulation of DsrLL, allowing the efficient synthesis of the protein essential for its adaptation to cold. Post-transcriptional regulation of DsrLL expression also seems to take place through the interplay of exonucleases and endonucleases overexpressed at 20°C, which would influence the half-life of the dsrLL transcript. Furthermore, the mechanism of cold resistance of Lc. lactis AV1n seems to be also based on energy saving through a decrease in growth rate mediated by a decrease in carbohydrate metabolism and its orientation toward the production pathways for storage molecules. Thus, this better understanding of the responses to low temperature and mechanisms for environmental adaptation of Lc. lactis could be exploited for industrial use of strains belonging to this species., This study was supported by the Spanish Ministry of Science, Innovation and Universities (grant no. RTI2018-097114-B-I00) and the Tunisian Ministry of Higher Education and Scientific Research.

Isoprene-emitting plants are better protected against thermal and oxidative stresses, which is a desirable trait in a climate-changing (drier and warmer) world. Here we compared the ecophysiological performances of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual environmental conditions (400 ppm of CO2 and 28 °C of average daily temperature) and in a future climate scenario (600 ppm of CO2 and 32 °C of average daily temperature). Furthermore, we intended to complement the present knowledge on the mechanisms involved in isoprene-induced resistance to water deficit stress by examining the proteome of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual climate. Isoprene emitters maintained higher photosynthesis and electron transport rates under moderate stress in future climate conditions. However, physiological resistance to water stress in the isoprene-emitting plants was not as marked as expected in actual climate conditions, perhaps because the stress developed rapidly. In actual climate, isoprene emission capacity affected the tobacco proteomic profile, in particular by upregulating proteins associated with stress protection. Our results strengthen the hypothesis that isoprene biosynthesis is related to metabolic changes at the gene and protein levels involved in the activation of general stress defensive mechanisms of plants.

Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1β cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.

Hormones are integral molecules in the human body, controlling most body systems and maintaining homeostasis in the body. They function as signal molecules for communication between cells, and are often secreted by one tissue with another as target. Protein hormones make up the majority of the hormones in the body, are mostly transported in the bloodstream, and bind to receptors on the cell surface since they can not diffuse through cell membranes. Hormones and their associated proteins are linked to many diseases, and could therefore have a lot of potential as biomarkers. With the use of the company Olink’s Proximity Extension Assay (PEA) technology the concentrations of human proteins in blood can be measured. These proteins can be combined into different sets, called panels, to measure biomarkers related to specific diseases or conditions. This project aimed to identify the protein hormones of the human body, and investigate their associated proteins and the pathways and potential diseases they were related to. Suggestions of new panels based on disease areas that were found to be particularly related to protein hormones were also to be put forward. Databases such as UniProt, Reactome, String, Signor, Olink Insight, and the Human Protein Atlas were used to investigate protein hormones and their associated proteins. The result of the research was compiled into three different panels: the overall Protein Hormone panel, the Wellness panel, and the Diabetes panel. These panels were based on the disease areas commonly found associated with protein hormones, and that were deemed to be of interest to Olink and to the research community. The research also resulted in a table detailing all found protein hormones with additional info on pathways and diseases, normal concentrations of the proteins in blood or plasma, and the associations between the hormones and other proteins. The financial viability and marketability of the suggested panels were also investigated in a business case. It was determined that the development of the new panels had potential to be of significant importance to research efforts, healthcare providers, and individual patients. While there are some risks associated with developing and marketing new products, it was concluded that the economical potential of the panels make them an opportunity worth considering for Olink.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection leads to a wide range of clinical manifestations and determines the need for personalized and precision medicine. To better understand the biological determinants of this heterogeneity, we explored the plasma proteome of 43 COVID-19 patients with different outcomes by an untargeted liquid chromatography-mass spectrometry approach. The comparison between asymptomatic or pauci-symptomatic subjects (MILDs), and hospitalised patients in need of oxygen support therapy (SEVEREs) highlighted 29 proteins emerged as differentially expressed: 12 overexpressed in MILDs and 17 in SEVEREs. Moreover, a supervised analysis based on a decision-tree recognised three proteins (Fetuin-A, Ig lambda-2chain-C-region, Vitronectin) that are able to robustly discriminate between the two classes independently from the infection stage. In silico functional annotation of the 29 deregulated proteins pinpointed several functions possibly related to the severity; no pathway was associated exclusively to MILDs, while several only to SEVEREs, and some associated to both MILDs and SEVEREs; SARS-CoV-2 signalling pathway was significantly enriched by proteins up-expressed in SEVEREs (SAA1/2, CRP, HP, LRG1) and in MILDs (GSN, HRG). In conclusion, our analysis could provide key information for ‘proteomically’ defining possible upstream mechanisms and mediators triggering or limiting the domino effect of the immune-related response and characterizing severe exacerbations.

Background We aimed to compare absolute plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) and growth differentiation factor 15 (GDF-15) obtained by a conventional immunoassay with the corresponding relative concentrations from a proximity extension assay (PEA) and compare the prognostic impact of the protein levels obtained from these assays. Methods We evaluated 437 patients with peripheral arterial disease (PAD) and a population-based cohort of 643 individuals without PAD. Correlations were calculated using Spearman’s rank correlation coefficients (rho). The discriminatory accuracy of the protein levels to predict future cardiovascular events was analyzed with Cox regression and presented as time-dependent areas under the receiver-operator-characteristic curves (tdAUCs). Results For NT-proBNP, the two assays correlated with rho 0.93 and 0.93 in the respective cohort. The PEA values leveled off at higher values in both cohorts. The corresponding correlations for GDF-15 were 0.91 and 0.89. At 5 years follow-up, the tdAUCs in the patient cohort were similar for NT-proBNP and GDF-15 regardless of assay used (0.65–0.66). The corresponding tdAUCs in the population-based cohort were between 0.72 and 0.77. Conclusion Except for the highest levels of NT-proBNP, we suggest that PEA data for NT-proBNP and GDF-15 reliably reflects absolute plasma levels and contains similar prognostic information.

Idiopathic membranous nephropathy (IMN) is a pathologically defined disorder of the glomerulus, primarily responsible for nephrotic syndromes (NS) in nondiabetic adults. The underlying molecular mechanisms are still not completely clarified. To explore possible molecular and functional signatures, an optimised mass spectrometry (MS) method based on next-generation data-independent acquisition combined with ion-mobility was applied to serum of patients affected by IMN (n = 15) or by other glomerulopathies (PN) (n = 15). The statistical comparison highlighted a panel of 57 de-regulated proteins with a significant increase in lipoprotein-related proteins (APOC1, APOB, APOA1, APOL1 and LCAT) and a substantial quantitative alteration of key serpins (including A4, D1, A7, A6, F2, F1 and 1) possibly associated with IMN or NS and podocyte stress. A critical dysregulation in metabolisms of lipids (e.g., VLDL assembly and clearance) likely to be related to known hyperlipidemia in IMN, along with involvement of non-classical complement pathways and a putative enrolment of ficolin-2 in sustaining the activation of the lectin-mediated complement system have been pinpointed. Moreover, mannose receptor CD206 (MRC1-down in IMN) and biotinidase (BTD-up in IMN) are able alone to accurately distinguish IMN vs. PN. To conclude, our work provides key proteomic insights into the IMN complexity, opening the way to an efficient stratification of MN patients.

Background: In Sweden around 1400 people are affected by head and neck cancer each year, and around 400 of these tumours are located in the mobile tongue (SCCOT). A major problem with these tumours is the high degree of relapse. In order to broaden our understanding of the group of squamous cell carcinoma of the head and neck (SCCHN) tumours we evaluated and compared the outcomes of panendoscopy with biopsy, ultrasonography with fine needle aspiration cytology (US-FNAC), and preoperative positron emission tomography/computed tomography (PET/CT) data from the same patients. As patients with SCCHN frequently have distant metastasis and locoregional recurrences, machine learning (ML) techniques were used to create classification models that accurately predict the likelihood of an early recurrence. Materials and methods: From patients suspected of having head and neck cancer between 2014–2016 results from PET/CT, panendoscopy with biopsy and US-FNAC were compared. Clinical, genomic, transcriptomic, and proteomic markers identifying recurrence risk were investigated. In blood samples taken from healthy individuals, data from proteins relevant to inflammation and/or tumor processes were evaluated. The SHapley Additive Explanations (SHAP) approach was used to determine the best ML algorithm for feature selection. AdaBoost, Artificial neural networks (ANNs), Decision Tree (DT), eXtreme Gradient Boosting (XGBoost), and Support Vector Machine (SVM) were used to create prediction models. Clinical data from patients were analyzed using statistical and ML techniques. Results: The concordance between results from PET/CT and panendoscopy with biopsy was 91.3%, and somewhat lower, 89.1%, for PET/CT and US-FNAC. The top contributors to classification with the ML approach were five mRNAs (PLAUR, DKK1, AXIN2, ANG, VEGFA), and 10 proteins (RAD50, 4E-BP1, MYH11, MAP2K1, BECN1, NF2, RAB25, ERRFI1, KDR, SERPINE1), using the extreme gradient boosting (XGBoost) method. The SHAP approach was used for feature selection. Using data from analysis of proteins in blood and interpretable ML showed that the Support Vector Machine (SVM) had the best performance with a balanced accuracy of 0.863, and a ROC-AUC of 0.924. The top three contributors to the SVM prediction model's performance were IL10, TNF Receptor Associated Factor 2 (TRAF2), and Kallikrein Related Peptidase 12 (KLK12). Recurrence was correlated with diabetes (p = 0.003), radiographic neck metastasis (p = 0.010), and T stage (p = 0.0012). A ML model got an accuracy rate of 71.2%. In the SCCOT group, diabetics predominated over non-diabetics, and also had lower recurrence rates and better survival (p = 0.012). Conclusion: Results show that the combination PET/CT is useful in diagnosis of SCCHN. It further emphasizes the use of ML to identify transcriptomic and proteomic factors that are significant in predicting risk of recurrence in patients with SCCHN. It provides a methodical strategy for early diagnosis of SCCOT before onset of clinical symptoms using multidimensional plasma protein profiling and interpretable ML. A model for predicting recurrence of SCCOT is provided by ML utilizing clinical data. As SCCOT patients with co-existing diabetes showed a better prognosis than non-diabetics, results suggest that individuals with SCCOT, regardless of diabetes status, may benefit from therapeutic management of glucose levels.

Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of “persister” cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. On the other hand, bacteria often have multiple molecular mechanisms to adapt to fluctuating environments. Understanding these mechanisms, and their redundancy, requires examinations in depth at the molecular level. This thesis describes a time- and cell state-selective proteome-labeling approach that enables researchers to investigate heterogeneous systems and molecular redundancy. In Chapter 1, we review the concept of bacterial persistence. The definition of bacterial persistence is introduced. Both the differences and connections between bacterial persistence and resistance are covered. In particular, we discuss research related to Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen found in many cystic fibrosis patients. State-of-the-art technologies to investigate bacterial persistence are discussed, and we conclude that advanced tools are needed to advance research on bacterial persistence further. In Chapter 2, we highlight the concept of bioorthogonal noncanonical amino acid tagging (BONCAT). BONCAT is a powerful tool developed in the Tirrell and Schuman laboratories allowing the incorporation of noncanonical amino acids (ncAA) into newly-synthesized proteins. We review established strategies for proteomics, especially cell-selective proteomics. We introduce the concept and mechanism of BONCAT and address the advantages of BONCAT in the investigation of phenotypic heterogeneity and bacterial persistence. In Chapter 3, we describe our work using BONCAT for understanding bacterial persistence. In particular, we investigated the process of persister resuscitation, as it is closely related to the reoccurrence of P. aeruginosa infections. The characteristics of the heterogeneity of persister cells during persister awakening were examined by survival assays and by ScanLag, an automated colony-based system allowing high-throughput acquisition of time-lapse images, quantification, and analysis of growth of bacterial colonies. Two BONCAT methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (Aha), which avoids extensive usage of antibiotic markers and allows direct integration with PA14 transposon insertion library, or with L-azidonorleucine (Anl), which has the advantage of specificity, as well as direct application in nutrition-rich medium. Through BONCAT enrichment experiments, we found proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were up-regulated in the regrowth phase. We further explored whether the up-regulation was a result of the modulation of HigB-HigA toxin-antitoxin system. In Chapter 4, we describe our work for understanding molecular redundancy. The chapter follows up on our observation of up-regulation of pyochelin-related proteins during persister regrowth. We discuss the hypothesis that pyochelin confers a growth advantage in persister cells subject to carbon-limited conditions. In addition, we discuss the potential role of Fur, a ferric uptake regulator, in bacterial persistence.

Skin cancer, including malignant melanoma (MM) and keratinocyte carcinoma (KC), historically named non-melanoma skin cancers (NMSC), represents the most common type of cancer among the white skin population. Despite decades of clinical research, the incidence rate of melanoma is increasing globally. Therefore, a better understanding of disease pathogenesis and resistance mechanisms is considered vital to accomplish early diagnosis and satisfactory control. The “Omics” field has recently gained attention, as it can help in identifying and exploring metabolites and metabolic pathways that assist cancer cells in proliferation, which can be further utilized to improve the diagnosis and treatment of skin cancer. Although skin tissues contain diverse metabolic enzymes, it remains challenging to fully characterize these metabolites. Metabolomics is a powerful omics technique that allows us to measure and compare a vast array of metabolites in a biological sample. This technology enables us to study the dermal metabolic effects and get a clear explanation of the pathogenesis of skin diseases. The purpose of this literature review is to illustrate how metabolomics technology can be used to evaluate the metabolic profile of human skin cancer, using a variety of analytical platforms including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Data collection has not been based on any analytical method. Refereed/Peer-reviewed

The sympathetic nervous system (SNS), particularly through the β2 adrenergic receptor (β2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting β2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under β2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under β-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Fusarium crown rot (FCR), typically caused by Fusarium pseudograminearum, is a severe soil-borne disease that, in recent years, has become an emerging threat to Chinese wheat crops. For the first time in this study, we investigated and compared the proteomic characteristics of two Chinese wheat varieties (04 Zhong 36 and Xinmai 26) at 24, 48, and 72 h post-inoculation using label-free quantitative proteomic analysis. A total of 9 234 proteins were successfully quantified, of which 783 were differentially expressed after inoculation. These proteins were mainly involved in metabolic, single-organism, and cellular processes. Thirty-three proteins associated with defense, cell wall formation, photosynthesis, etc., showed consistently different expression between the two genotypes at multiple time points. In particular, chitinase, which degrades chitin in the fungal cell wall and limits fungal growth, was exclusively and consistently upregulated in 04 Zhong 36 across the three time points. Other proteins such as flavonoid O-methyltransferase, glycosyltransferase, and peroxidase were only upregulated in 04 Zhong 36, and proteins, including the berberine bridge enzyme and rubisco large subunit-binding protein, were specifically downregulated in Xinmai 26. The expression of transcripts encoding eight selected proteins through qRT-PCR analysis supported the proteomic profiles. Overall, the results of this study allow us to understand FCR resistance in wheat at the protein level. Some proteins and their corresponding genes may be useful resources for the genetic improvement of FCR resistance in wheat.

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a disease that causes serious problems to the global citrus industry. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has been used in human medicine to diagnose various diseases caused by both fungi and bacteria. In agriculture, this technique has potential for the diagnosis of diseases due to the low cost of large-scale analysis and quickness. This study showed that MALDI-TOF MS combined with chemometric analysis was effective for differentiating the macromolecule profile of orange leaves with canker lesions, healthy leaves, and leaves with phytotoxicity symptoms, proving that this technique may be used for the rapid diagnosis of citrus canker.

Coronary artery disease (CAD) is the disease with one of the highest rates of mortality and morbidity in the world. It is characterized by chest discomfort (angina) and associated with the following risk factors: age, sex, ethnicity, family history, stress, obesity, hypertension and diabetes. To improve diagnosis and mitigate the development of the disease, there is the need for rapid and effective screening, with dried blood spot being a possible alternative in the future. The dried blood spot (DBS) is a test characterized by the collection of a drop of blood from a prick on a patient's finger and dried on filter paper. This method has the advantages of requiring a small sample volume, being minimally invasive, not requiring a healthcare professional and being low cost. This dissertation is a proof of concept where, using a set of 10 samples (5 control and 5 CAD patients), we performed proteomic quantification of DBS samples through mass spectrometry, western blot validation of 5 potential biomarkers (AMBP, Apo-A1, Apo-E, BNP and Gal-3); and finally, a bibliometric analysis that relates coronary artery disease, dry blood spot and target proteins. Through a proteomics-based approach, 221 proteins were identified and quantified, 14 of which were identified and quantified for the first time in DBS. From western blot analysis, AMBP and BNP proteins showed no statistical differences between the control and CAD groups, Apo-A1, Apo-E and Gal-3 proteins showed higher levels in the control group compared to the CAD group. Bibliometric analysis showed the relationship between proteins, CAD and DBS. DBS proved to be an effective method for recognizing CAD, since the results allowed the validation of mass spectrometry and western blot techniques in dry blood spot for CAD. Bibliometric analysis confirmed the association of coronary artery disease, dry blood spot and proteins. As this study is the first to integrate a mass spectrometry analysis, western blot and bibliometric analysis with the DBS technique for coronary artery disease, further studies are needed in the future. A doença arterial coronária (CAD) é a principal causa de mortalidade e morbilidade no mundo. É caraterizada pelo desconforto no peito (angina) e tem como fatores de risco: a idade, o sexo, a etnia, o histórico familiar, o stress, a obesidade, a hipertensão e a diabetes. Para melhorar o diagnóstico e atenuar o desenvolvimento da doença, há necessidade de um rastreio rápido e eficaz, sendo o ‘dried blood spot’ uma possível alternativa no futuro. O ‘dried blood spot’ (DBS) é um teste caraterizado pela coleta de uma gota de sangue a partir de uma picada num dedo do paciente e seca em papel de filtro. Este método tem as vantagens de necessitar de um pequeno volume da amostra, ser minimamente invasivo, não necessitar de um profissional de saúde e ser de baixo custo. Esta dissertação é uma prova de conceito onde, analisando um total de 10 amostras (5 controlo e 5 pacientes CAD), procurou-se fazer a quantificação proteómica das amostras de DBS a partir da espetrometria de massa, a validação por western blotting de 5 potenciais biomarcadores (AMBP, Apo-A1, Apo-E, BNP e Gal-3); e por fim, uma análise bibliométrica que relaciona a doença arterial coronária, o ‘dry blood spot’ e as proteínas alvo. Através de uma abordagem proteómica, foram identificadas e quantificadas 221 proteínas, sendo 14 proteínas pela primeira vez identificadas e quantificadas no DBS. Da análise western blotting, as proteínas AMBP e BNP não apresentaram diferenças estatísticas entre os grupos controlo e CAD, as proteínas Apo-A1, Apo-E e Gal-3 exibiram níveis mais elevados no grupo controlo em comparação com o grupo CAD. A análise bibliométrica mostrou a relação entre as proteínas, a CAD e o DBS. O DBS mostrou-se um método eficaz no reconhecimento da CAD, uma vez que, os resultados permitiram a validação das técnicas de espetrometria de massa e western blot no dry blood spot para a CAD. A análise bibliométrica confirmou a associação da doença arterial coronária, o dry blood spot e as proteínas. Sendo este estudo o primeiro que integra uma análise de espetrometria de massa, western blot e análise bibliométrica com a técnica de DBS para a doença arterial coronária, são necessários mais estudos no futuro para corroborar os resultados. Mestrado em Biomedicina Molecular

The spinal cord is one of the most important parts of the central nervous system (CNS). However, the spinal cord can be subject to injury (SCI), ranging from contusion to severance, due to life events (motor vehicle or bicycle accidents, physical aggression, falls etc.). Unfortunately, no effective treatment is known to date. In this context, it is important to characterize the molecular mechanisms involved in this physiopathology. To this end, a spatio-temporal proteomic study on rats with a spinal cord injury (SCI) was carried out. This made it possible to highlight that the rostral region located upstream of the lesion synthesizes factors involved in neurogenesis; whereas at the lesion site and in the caudal region located downstream of the lesion the factors produced are inflammatory in nature. Surprisingly, this study also showed that the secretomes from organotypic cultures of these different segments of injured spinal cord contain Gamma immunoglobulins (IgG), of the isotypes IgG1, IgG2A, IgG2B and IgG2C. Surprisingly, these appear as early as 12 hours after injury. However, during a conventional adaptive response, the production of IgG by B lymphocytes requires 7 to 8 days. Although it is currently considered that only B lymphocytes can produce IgG, this early expression supports the idea that these IgGs could be produced by CNS resident cells. Thus, the aim of this thesis was to identify the source of these IgGs and to determine their role in SCI. In the course of this work, we have shown that Igs can be produced in the central nervous system by neurons and astrocytes. Indeed, using multi-omics analysis, we revealed the constitutive expression of IgG2C and IgG2B heavy chain in neurons and astrocytes respectively. When we focused on the IgG2B heavy chain in astrocytes, overexpression and invalidation studies showed a role for this IgG in maintaining astrocyte identity. These Igs were found to be peculiar in that they did not have a classical form, but rather an aberrant form. That is, independent variable and constant parts for both the IgG heavy chain and the Kappa light chain. We were able to show by functional studies that these Igs had a role in the conservation of the astrocytic phenotype and were involved in neurogenesis and neuritic regrowth. They can now be called “neural derivated Igs”. The current dogma is that Ig is produced by B lymphocytes (LB) during an immune response. Our work tends to show that B cells are no longer the only ones to produce these Igs. In conclusion, our work has identified a new class of non-B cell derived Igs, opening the door to research on their roles and targets in the nervous system under normal or pathophysiological conditions.; La moelle épinière est l'une des parties les plus importantes du système nerveux central (SNC). Cependant, celle-ci peut être sujette à des lésions allant de la contusion à la section, à la suite d'accidents de la route, d'agressions physiques, de chutes, etc. Malheureusement, aucun traitement efficace n'est réellement connu à ce jour. Dans ce contexte, il est important de caractériser les mécanismes moléculaires mis en jeu lors de cette physiopathologie. Pour cela, une étude protéomique spatio-temporelle sur des rats présentant une lésion de la moelle épinière (LME) a été réalisée. Celle-ci a permis de mettre en évidence que la région rostrale localisée en amont de la lésion synthétise des facteurs impliqués dans la neurogenèse ; alors que sur le site de lésion et la région caudale localisée en aval de la lésion, les facteurs produits sont de nature inflammatoire. De façon surprenante, cette étude a également montré que les sécrétomes issus des cultures organotypiques de ces différents segments de la LME contiennent des immunoglobulines Gamma (IgG), d'isotypes IgG1, IgG2A, IgG2B et IgG2C. Celles-ci apparaissent dès 12 h après lésion. Or, lors d'une réponse adaptative conventionnelle, la production d'IgG par les lymphocytes B nécessite 7 à 8 jours. Bien qu'actuellement, il soit considéré que seuls les lymphocytes B soient capables de produire des IgG, cette expression précoce soutient l'idée que ces Igs pourraient être produites par des cellules résidentes du SNC. Ainsi, le but de cette thèse a été d'identifier la source de ces Igs et de déterminer leur rôle lors d'une LME. Au cours de ce travail, nous avons montré que des Igs peuvent être produites au sein même du système nerveux central par les neurones et les astrocytes. En effet, à l'aide d'analyses multi-omiques, nous avons révélé l'expression constitutive des chaînes lourdes IgG2C et IgG2B dans les neurones et les astrocytes respectivement. Lorsque nous nous sommes intéressés à la chaîne lourde IgG2B dans les astrocytes, des études de surexpression et d'invalidation ont montré un rôle de cette IgG dans le maintien de l'identité des astrocytes. Ces Igs se sont révélées particulières, car elles ne présentent pas une forme classique, mais plutôt une forme aberrante. C'est-à-dire des parties variables et constantes aussi bien pour la chaîne lourde IgG que pour la chaîne légère Kappa qui sont produites de façon indépendante. Nous avons pu montrer par des études fonctionnelles que ces Igs présenteraient un rôle dans la conservation du phénotype astrocytaire et seraient impliquées dans la neurogénèse et la repousse neuritique. Ces nouvelles immunoglobulines peuvent être nommées les « Igs dérivées des cellules neurales ». Ces résultats renforcent les dernières recherches montrant que les Igs peuvent produire par d'autres cellules que les cellules B. En conclusion, l'ensemble de ces travaux a permis de mettre en évidence une nouvelle classe d'Igs issu de cellules non-B, ouvrant la porte à des recherches portant sur leurs rôles et leurs cibles au sein du SNC dans des conditions physiologiques normales ou physiopathologiques.

Background Cisplatin‐based chemotherapy is a therapeutic strategy against non‐small cell lung cancer (NSCLC). However, cancers relapse after chemotherapy due to a dormant state of residual cancer cells. Extracellular vesicles and particles (EVPs) are active carriers of proteins and nucleic acid. Here, we aimed to study the molecular alterations and proteomic characteristics of EPV in dormant and reactivated cancer cells induced by cisplatin. Methods We used a short‐term single dose of cisplatin to induce the dormant and reactivated cell status. We examined the gene expressional profiling and proteomic profiling of EVPs from dormant and reactivated cancer cells by RNA‐sequencing and LC–MS/MS. Results We found substantial changes in gene expression and protein level in EVP. The genes with higher expression in dormant cancer cells were lipid transporter‐ and lipid metabolic‐related genes. A total of 111 EVP proteins were upregulated in dormant cancer cells compared to those in control cells. Fifty differential expressed proteins (DEPs) were identified in EVPs from reactivated cancer cells compared to those in dormant cancer cells. Among the DEPs, we found that apolipoproteins such as APOA1 and APOE were significantly increased in dormant cancer cell‐derived EVPs. Integration of EVP proteomes with transcriptional profiles of cancer cells revealed that the proteomic profiling of EVP derived from cancer cells can reflect the cellular status of cancer cells, which showed an activated lipid metabolism in dormant state. Conclusion Lipoproteins enriched in EVPs reflect the activated lipid metabolism in dormant cancer cells and may provide potential biomarkers or therapeutic targets for cisplatin‐based therapy., Integration of EVP proteomes with transcriptional profiles revealed that the proteomic profiling of EVP derived from cancer cells can reflect the cellular status of cancer cells, which showed an activated lipid metabolism in dormant state induced by chemotherapy. It may provide potential biomarkers or therapeutic targets for cisplatin‐based therapy.

Objective: With the rapid development of proteomics sequencing and RNA sequencing technology, multi-omics analysis has become a current research hotspot. Our previous study indicated that Xinjiang brown cattle have better meat quality than Kazakh cattle. In this study, Xinjiang brown cattle and Kazakh cattle were used as the research objects.Methods: Proteome sequencing and RNA sequencing technology were used to analyze the proteome and transcriptome of the longissimus dorsi muscle of the two breeds of adult steers (n = 3).Results: In this project, 22,677 transcripts and 1,874 proteins were identified through quantitative analysis of the transcriptome and proteome. By comparing the identified transcriptome and proteome, we found that 1,737 genes were identified at both the transcriptome and proteome levels. The results of the study revealed 12 differentially expressed genes and proteins: troponin I1, crystallin alpha B, cysteine, and glycine rich protein 3, phosphotriesterase-related, myosin-binding protein H, glutathione s-transferase mu 3, myosin light chain 3, nidogen 2, dihydropyrimidinase like 2, glutamate-oxaloacetic transaminase 1, receptor accessory protein 5, and aspartoacylase. We performed functional enrichment of these differentially expressed genes and proteins. The Kyoto encyclopedia of genes and genomes results showed that these differentially expressed genes and proteins are enriched in the fatty acid degradation and histidine metabolism signaling pathways. We performed parallel reaction monitoring (PRM) verification of the differentially expressed proteins, and the PRM results were consistent with the sequencing results.Conclusion: Our study provided and identified the differentially expressed genes and proteins. In addition, identifying functional genes and proteins with important breeding value will provide genetic resources and technical support for the breeding and industrialization of new genetically modified beef cattle breeds.

Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

Le développement de thérapies efficaces pour la sclérose latérale amyotrophique (SLA) est urgent et est dépendant de la compréhension des mécanismes moléculaires sous-jacents à la pathologie. La délocalisation de TDP-43 du noyau vers le cytoplasme est un événement observé dans la majorité des cas de SLA et nous avons démontré que la déplétion nucléaire de TDP-43 induit l’accumulation d’une isoforme alternative de Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), nommé hnRNP A1B. La protéine hnRNP A1B diffère de hnRNP A1 par l’inclusion de l’exon 7B qui allonge son domaine intrinsèquement désordonné de 52 acides aminés. HNRNPA1 est muté dans certains cas de SLA familiale et la présence de mutations augmente les propriétés d’agrégation de la protéine. Par contre, la contribution des agrégats à la pathogenèse n’est pas totalement comprise dû au manque de connaissances sur les fonctions de hnRNP A1B. L’isoforme hnRNP A1 est quant à elle très bien étudiée et est un membre important du métabolisme de l’ARN. Étonnamment, on connaît très peu l’isoforme hnRNP A1B. Nous avons donc examiné l’expression spatiotemporelle et la fonction de hnRNP A1B pour comprendre sa contribution à la biologie cellulaire et aux pathologies. Dans la première partie, nous avons découvert que l’expression de la protéine hnRNP A1B est plus restreinte au système nerveux central (SNC) comparé à hnRNP A1. Dans le SNC, hnRNP A1B peut aussi former un dimère résistant au SDS. De plus, nous avons démontré que l’expression de hnRNP A1B devient progressivement restreinte aux neurones moteurs dans la corne ventrale de la moelle épinière. Contrairement à hnRNP A1 qui est exprimée globalement. Nous avons aussi découvert que hnRNP A1B est présente dans le noyau et le cytoplasme des neurones moteurs alors que la protéine hnRNP A1 est nucléaire. De plus, hnRNP A1B forme des puncta dans les neurites. Dans la seconde partie, nous avons caractérisé l’interactome de hnRNP A1B dans la moelle épinière. Nous avons identifié et validé l’interaction de hnRNP A1B avec des protéines motrices. Nous avons démontré que les granules de hnRNP A1B contenaient des ARN messagers (ARNm) et des protéines nouvellement synthétisées. Ce résultat soutient le rôle de hnRNP A1B comme adaptateur dans le transport neuronal et régulateur de la traduction locale. Nos résultats démontrent que les deux isoformes de HNRNPA1 sont différentiellement exprimées dans les tissus et ont des localisations cellulaires distinctes. Ces résultats suggèrent que les deux isoformes ont des fonctions cellulaires spécifiques. Ainsi, nous proposons que hnRNP A1B joue une fonction cytoplasmique neuronale dans le transport des ARNm, qui n’est pas partagé par hnRNP A1. Les isoformes de HNRNPA1 peuvent donc contribuer distinctement dans la progression des pathologies., The development of an effective treatment for amyotrophic lateral sclerosis (ALS) is urgent and is expected to be informed by a better understanding of the molecular mechanisms underlying the pathology. TDP-43 mislocalization from the nucleus to the cytoplasm is observed in the majority of ALS cases and we have discovered that TDP-43 nuclear depletion drives the accumulation of an alternatively spliced variant of heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), termed hnRNP A1B. hnRNP A1B differs by the inclusion of the 7B exon that elongates its intrinsically disorder region by 52 amino acids. HNRNPA1 is mutated in rare cases of familial ALS and these diseaseassociated mutations drive aggregation, but their contribution to the pathogenesis is not clearly understood due to the lack of information about hnRNP A1B functions. hnRNP A1 is well studied and is a major player in RNA metabolism, but surprisingly little is known about the spliced variant hnRNP A1B. We then aim to investigate the spatiotemporal regulation and function of hnRNP A1B to understand its contribution to physiology and pathology. In the first part, we have found that hnRNP A1B’s expression is more restricted to the central nervous system (CNS) compared to hnRNP A1, and that it can form an SDSresistant dimer enriched in the CNS. Also, hnRNP A1B expression becomes progressively restricted to motor neurons in the ventral horn of the spinal cord, compared to hnRNP A1 which is more broadly expressed. We also demonstrate that hnRNP A1B is present in the nucleus and in the soma of motoneurons compared to hnRNP A1 which is nuclear. In addition, hnRNP A1B forms puncta in neuronal processes. In the second part, we have characterized hnRNP A1B’s interactome in the spinal cord. We have identified and validated the interactions between hnRNP A1B and cytoskeletal motor proteins. We have demonstrated that hnRNP A1B granules in neuronal process contains messenger RNA (mRNA) and contains nascent proteins which support the role for hnRNP A1B as an RNA adaptor in mRNA transport and a regulator of local translation. Our results demonstrate that both isoforms of HNRNPA1 are differentially expressed across tissues and have distinct localization profiles, suggesting that the two isoforms may have specific subcellular functions. Those finding support that hnRNP A1B may have a cytosolic function in neurons, RNA transport, that is not shared with hnRNP A1. Indeed, HNRNPA1 isoforms can uniquely contribute to disease progression.

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

Zearalenone (ZEN), also known as the F-2 toxin, is a common contaminant in cereal crops and livestock products. This experiment aimed to reveal the changes in the proteomics of ZEN-induced intestinal damage in weaned piglets by tandem mass spectrometry tags. Sixteen weaned piglets either received a basal diet or a basal diet supplemented with 3.0 mg/kg ZEN in a 32 d study. The results showed that the serum levels of ZEN, α-zearalenol, and β-zearalenol were increased in weaned piglets exposed to ZEN (p < 0.05). Zearalenone exposure reduced apparent nutrient digestibility, increased intestinal permeability, and caused intestinal damage in weaned piglets. Meanwhile, a total of 174 differential proteins (DEPs) were identified between control and ZEN groups, with 60 up-regulated DEPs and 114 down-regulated DEPs (FC > 1.20 or

The aim of this study was to explore the effect of zearalenone (ZEA) exposure on uterine development in weaned gilts by quantitative proteome analysis with tandem mass spectrometry tags (TMT). A total of 16 healthy weaned gilts were randomly divided into control (basal diet) and ZEA3.0 treatments groups (basal diet supplemented with 3.0 mg/kg ZEA). Results showed that vulva size and uterine development index were increased (ip/iamp;lt; 0.05), whereas serum follicle stimulation hormone, luteinizing hormone and gonadotropin-releasing hormone were decreased in gilts fed the ZEA diet (ip/iamp;lt; 0.05). ZEA, α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) were detected in the uteri of gilts fed a 3.0 mg/kg ZEA diet (ip/iamp;lt; 0.05). The relative protein expression levels of creatine kinase M-type (CKM), atriopeptidase (MME) and myeloperoxidase (MPO) were up-regulated (ip/iamp;lt; 0.05), whereas aldehyde dehydrogenase 1 family member (ALDH1A2), secretogranin-1 (CHGB) and SURP and G-patch domain containing 1 (SUGP1) were down-regulated (ip/iamp;lt; 0.05) in the ZEA3.0 group by western blot, which indicated that the proteomics data were dependable. In addition, the functions of differentially expressed proteins (DEPs) mainly involved the cellular process, biological regulation and metabolic process in the biological process category. Some important signaling pathways were changed in the ZEA3.0 group, such as extracellular matrix (ECM)-receptor interaction, focal adhesion and the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway (ip/iamp;lt; 0.01). This study sheds new light on the molecular mechanism of ZEA in the uterine development of gilts.

Objective: Aberrant activity of androgen receptor (AR) is the primary cause underlying development and progression of prostate cancer (PCa) and castration-resistant PCa (CRPC). Androgen signaling regulates gene transcription and lipid metabolism, facilitating tumor growth and therapy resistance in early and advanced PCa. Although direct AR signaling inhibitors exist, AR expression and function can also be epigenetically regulated. Specifically, lysine (K)-specific demethylases (KDMs), which are often overexpressed in PCa and CRPC phenotypes, regulate the AR transcriptional program. Methods: We investigated LSD1/UTX inhibition, two KDMs, in PCa and CRPC using a multi-omics approach. We first performed a mitochondrial stress test to evaluate respiratory capacity after treatment with MC3324, a dual KDM-inhibitor, and then carried out lipidomic, proteomic, and metabolic analyses. We also investigated mechanical cellular properties with acoustic force spectroscopy. Results: MC3324 induced a global increase in H3K4me2 and H3K27me3 accompanied by significant growth arrest and apoptosis in androgen -responsive and-unresponsive PCa systems. LSD1/UTX inhibition downregulated AR at both transcriptional and non-transcriptional level, showing cancer selectivity, indicating its potential use in resistance to androgen deprivation therapy. Since MC3324 impaired metabolic activity, by modifying the protein and lipid content in PCa and CRPC cell lines. Epigenetic inhibition of LSD1/UTX disrupted mitochondrial ATP production and mediated lipid plasticity, which affected the phosphocholine class, an important structural element for the cell membrane in PCa and CRPC associated with changes in physical and mechanical properties of cancer cells. Conclusions: Our data suggest a network in which epigenetics, hormone signaling, metabolite availability, lipid content, and mechano-metabolic process are closely related. This network may be able to identify additional hotspots for pharmacological intervention and un-derscores the key role of KDM-mediated epigenetic modulation in PCa and CRPC.(c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Both high-fat diet (HFD) alone and high-fructose plus HFD (HFr/HFD) cause diet-induced non-alcoholic fatty liver disease in murine models. However, the mechanisms underlying their impacts on inducing different levels of liver injury are yet to be elucidated. This study employed a proteomic approach to elucidate further on this issue. Adult male C57BL/6J mice were allocated to the HFD or the HFr/HFD group. After feeding for 12 weeks, all mice were euthanized and samples were collected. The proteomic profiles in liver tissues were analyzed using liquid chromatography–tandem mass spectrometry followed by canonical pathway analysis. We demonstrated that the mitochondrial oxidative phosphorylation (OXPHOS) pathway was the most significantly downregulated canonical pathway in the HFr/HFD group when compared with the HFD group. Within the OXPHOS pathway, the HFr/HFD group demonstrated significant downregulation of complexes I and III and significant upregulation of complex IV when compared with the HFD group. Moreover, the HFr/HFD group had lower protein levels of NADH: ubiquinone oxidoreductase subunits S3, S6, A5, and A12 in complex I (p < 0.001, =0.03

Eubacterium limosum is a strict anaerobic bacterium, belonging to the group of acetogens. It’s interest lies in its ability to transform one carbon (C1) feedstock into butyrate, a C4 molecule, through the oldest metabolic pathway on earth, called the Wood-Ljungdahl Pathway (WLP).Nevertheless, yields and productivities must be improved to reach performances close to chemical processes. For this purpose, metabolic engineering strategies are needed but improving our knowledge on primary metabolism is of paramount importance to reach this goal.The work performed in this thesis project aimed to characterize, by a systems biology approach, the central metabolism of E. limosum B2 growing in chemostat cultures on methanol or on glucose as a carbon source.Following an adaptive laboratory evolution process, the wild strain was cultivated on synthetic methanol medium without yeast extract. The maximum growth rate for the evolved clone was improved by 2.72. In addition, we succeeded in substituting the cysteine of the synthetic medium, described as essential, with sodium thiosulfate. The wild type strain was unable to grow under these conditions, suggesting positive mutations in the evolved clone. The complete genome of the wild strain was first sequenced, de novo, followed by the genomes of the population on several generations and the isolated clone in order to identify and study the dynamics of genetic evolution. No mutation that could explain this change in phenotype was detected in the genes encoding enzymes of the primary metabolism. A proteomic study was carried out on the wild type and the adapted strain to clarify the adaptation mechanisms. Among overexpressed proteins measured in the adapted strain, four proteins involved in central metabolism, including two in the WLP and two in gluconeogenesis, as well as a protein complex linked to sulfur assimilation were identified. In addition, exploration of the methylome revealed a homologous recombination event on the type I restriction-modification system between the two strains. This event by changing the methylome of the evolved clone, could play a key role in the regulatory mechanism on synthetic methanol medium.The adapted strain was grown in chemostat cultures for the characterization of its central metabolism on methanol or glucose as a carbon source. The physiological parameters were4determined as well as all the input and output fluxes. These values were integrated into a in silico genome scale model to estimate the specific fluxes of each enzyme of primary metabolism. From these estimations, energy conversation models were developed for the two conditions studied. The absolute number of protein copies per cell was then determined and these data allowed the estimation of the in vivo turnover rates for each enzyme. This parameter was useful to compare enzyme activities and identify the most limiting ones.For the transcriptomic part, the development of a total mRNA extraction method was a major challenge. We developed a strategy for the absolute quantification of mRNA per cell reinforced by the addition of two mixes of external standards during the extraction process. The first was added after the cell lysis step and the second before ribodepletion. RNA-Seq sequencing showed satisfactory results with a good correlation between the number of reads obtained for each standard and their theoretical concentration, indicating a minimal loss of mRNA during the extraction process. The determination of the absolute mRNA content for each gene is now possible.By combining proteomics and transcriptomics data, the in vivo specific synthesis rate of each protein can be determined. Our work will improve the in silico model of E. limosum and allow the use of a rational metabolic engineering approach.; Eubacterium limosum est une bactérie anaérobie stricte, faisant partie du groupe des acétogènes. Son intérêt réside dans sa capacité à transformer des sources de carbone à un atome de carbone (C1) telle que le méthanol en butyrate, composé en C4 présentant un intérêt industriel. Néanmoins, les rendements et productivité doivent être améliorés pour se rapprocher des performances de l’industrie chimique. L’ingénierie métabolique des souches permettrait d’atteindre cet objectif mais pour cela il est nécessaire d’améliorer nos connaissances du métabolisme central.L’objectif principal de ce projet de thèse était de caractériser le métabolisme central de E. limosum B2 cultivé en chemostat sur méthanol ou sur glucose comme source de carbone, par une approche de biologie des systèmes.Suivant un processus d’adaptation évolutive en laboratoire, la souche sauvage a été adaptée milieu synthétique méthanol sans extrait de levure. Le taux de croissance maximal chez le clone isolé a été amélioré par 2,72. De plus, nous sommes parvenus à substituer la cystéine du milieu synthétique, décrite comme indispensable, par du thiosulfate de sodium. La souche sauvage s’est montrée incapable de croître dans ces conditions, suggérant l’apparition de mutations favorable chez le mutant. Le génome de la souche sauvage a d’abord été séquencé de novo, suivi des génomes de la population sur plusieurs générations et de trois clones isolés pour étudier la dynamique d’évolution génétique. Aucune mutation pouvant expliquer ce changement de phénotype n’a a été détectée dans le métabolisme central. Une étude protéomique a été réalisée sur la souche sauvage et adaptée afin d’éclaircir les mécanismes d’adaptations. Parmi les protéines surproduites chez la souche adaptée, deux dans le WLP et deux en gluconéogenèse, ainsi qu’un complexe lié à l’assimilation du soufre ont été identifiés. De plus, l’exploration du méthylome a révélé un évènement de recombinaison homologue au niveau du système de restriction-modification de type I entre les deux souches. Cette modification pourrait jouer un rôle clé dans le mécanisme de régulation sur milieu minéral méthanol.La souche adaptée a été cultivée en chémostat pour la caractérisation de son métabolisme central sur méthanol ou sur glucose comme seule source de carbone. Les paramètres physiologiques ont été déterminés et les flux d’entrée et de production ont été calculés. Ces valeurs ont été intégrées dans un modèle in silico à l’échelle du génome pour estimer les flux spécifiques de chaque enzyme du métabolisme primaire. De ces estimations, des modèles de conversation de l’énergie ont été élaborés pour les deux conditions étudiées. Le nombre absolu de copies de protéines par cellule a ensuite été déterminé puis ces données ont permis d’estimer la constante catalytique in vivo de chaque enzyme du métabolisme central. Ce paramètre nous a été utile pour comparer les activités des enzymes et identifier les plus limitantes.Pour la partie transcriptomique, le développement d’une méthode d’extraction totale des ARNm a été un défi majeur. Nous avons élaboré une stratégie de quantification absolue des ARNm par cellule renforcée par l’ajout de deux mélanges de standards externes lors du procédé d’extraction des ARNm. Le premier a été ajouté après l’étape de lyse cellulaire et le second avant la ribodéplétion. Les résultats de séquençage par RNA-Seq ont été satisfaisant, avec une bonne corrélation entre le nombre de « reads » obtenus pour chaque standard et leur concentration théorique, indiquant une perte minimale en ARNm durant le processus d’extraction. La détermination de la quantité absolue de chaque transcrits est désormais possible.En combinant les données de protéomique et de transcriptomique, la vitesse spécifique de traduction in vivo de chaque protéine pourra être déterminée. Nos travaux permettront d’améliorer le modèle in silico d'E. limosum et permettre l’utilisation d’une approche rationnelle d’ingénierie métabolique.

Salt stress is a major abiotic stress factor affecting crop production, and understanding of the response mechanisms of seed germination to salt stress can help to improve crop tolerance and yield. The differences in regulatory pathways during germination in different salt-tolerant barley seeds are not clear. Therefore, this study investigated the responses of different salt-tolerant barley seeds during germination to salt stress at the proteomic and metabolic levels. To do so, the proteomics and metabolomics of two barley seeds with different salt tolerances were comprehensively examined. Through comparative proteomic analysis, 778 differentially expressed proteins were identified, of which 335 were upregulated and 443 were downregulated. These proteins, were mainly involved in signal transduction, propanoate metabolism, phenylpropanoid biosynthesis, plant hormones and cell wall stress. In addition, a total of 187 salt-regulated metabolites were identified in this research, which were mainly related to ABC transporters, amino acid metabolism, carbohydrate metabolism and lipid metabolism; 72 were increased and 112 were decreased. Compared with salt-sensitive materials, salt-tolerant materials responded more positively to salt stress at the protein and metabolic levels. Taken together, these results suggest that salt-tolerant germplasm may enhance resilience by repairing intracellular structures, promoting lipid metabolism and increasing osmotic metabolites. These data not only provide new ideas for how seeds respond to salt stress but also provide new directions for studying the molecular mechanisms and the metabolic homeostasis of seeds in the early stages of germination under abiotic stresses.

3 pág. Departamento de Biotecnología, The role of nucleated red blood cells (RBCs) as cellular mediators of the immune response in fish has emerged as a hot research subject that has attracted fish immunologists’ attention during the past decade. Fish are the most primitive vertebrates, possessing many of the cell types and molecules found in higher vertebrates. However, the immune system of fish is peculiarly dissimilar to that of higher vertebrates. Immune response is still poorly understood in most fish species, particularly in species recently introduced to aquaculture. These facts have limited the development of strategies to combat infectious diseases so far. Concerning fish RBCs, a number of biological processes relevant to immunity had been described for them: (i) pathogen recognition; (ii) clearance of pathogens by means of binding microbial immune complexes and (iii) production of cytokines or specific signalling molecules in response to pathogens. This Special Issue aimed to gather new research findings and advances on the role of RBCs in mediating the immune response in fish, with the hope that this forum could foster further collaborations in this emerging area of research. In this Research Topic, the advances on the immune response of fish RBCs to viruses or to different antiviral prophylactics will be presented. It contains articles related to the RBCs immune response to RNA and DNA viruses, as well as to different types of prophylactics, such an antigen-based DNA vaccine or a nanosctructured recombinant antigenic protein., European Research Council fund to MO-V (ERC Starting Grant GA639249)