Development of a Prediction Model for Short-Term Remission of Patients with Crohn's Disease Treated with Anti-TNF Drugs.

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

diaPASEF Proteomics and Feature Selection for the Description of Sputum Proteome Profiles in a Cohort of Different Subtypes of Lung Cancer Patients and Controls.

The high mortality, the presence of an initial asymptomatic stage and the fact that diagnosis in early stages reduces mortality justify the implementation of screening programs in the populations at risk of lung cancer. It is imperative to develop less aggressive methods that can complement existing diagnosis technologies. In this study, we aimed to identify lung cancer protein biomarkers and pathways affected in sputum samples, using the recently developed diaPASEF mass spectrometry (MS) acquisition mode. The sputum proteome of lung cancer cases and controls was analyzed through nano-HPLC-MS using the diaPASEF mode. For functional analysis, the results from differential expression analysis were further analyzed in the STRING platform, and feature selection was performed using sparse partial least squares discriminant analysis (sPLS-DA). Our results showed an activation of inflammation, with an alteration of pathways and processes related to acute-phase, complement, and immune responses. The resulting sPLS-DA model separated between case and control groups with high levels of sensitivity and specificity. In conclusion, we showed how new-generation proteomics can be used to detect potential biomarkers in sputum samples, and ultimately to discriminate patients from controls and even to help to differentiate between different cancer subtypes.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

SWATH-based proteomics reveals processes associated with immune evasion and metastasis in poor prognosis colorectal tumours.

Newly emerged proteomic methodologies, particularly data-independent acquisition (DIA) analysis-related approaches, would improve current gene expression-based classifications of colorectal cancer (CRC). Therefore, this study was aimed to identify protein expression signatures using SWATH-MS DIA and targeted data extraction, to aid in the classification of molecular subtypes of CRC and advance in the diagnosis and development of new drugs. For this purpose, 40 human CRC samples and 7 samples of healthy tissue were subjected to proteomic and bioinformatic analysis. The proteomic analysis identified three different molecular CRC subtypes: P1, P2 and P3. Significantly, P3 subtype showed high agreement with the mesenchymal/stem-like subtype defined by gene expression signatures and characterized by poor prognosis and survival. The P3 subtype was characterized by decreased expression of ribosomal proteins, the spliceosome, and histone deacetylase 2, as well as increased expression of osteopontin, SERPINA 1 and SERPINA 3, and proteins involved in wound healing, acute inflammation and complement pathway. This was also confirmed by immunodetection and gene expression analyses. Our results show that these tumours are characterized by altered expression of proteins involved in biological processes associated with immune evasion and metastasis, suggesting new therapeutic options in the treatment of this aggressive type of CRC.

A multiomic atlas of the aging hippocampus reveals molecular changes in response to environmental enrichment.

Aging involves the deterioration of organismal function, leading to the emergence of multiple pathologies. Environmental stimuli, including lifestyle, can influence the trajectory of this process and may be used as tools in the pursuit of healthy aging. To evaluate the role of epigenetic mechanisms in this context, we have generated bulk tissue and single cell multi-omic maps of the male mouse dorsal hippocampus in young and old animals exposed to environmental stimulation in the form of enriched environments. We present a molecular atlas of the aging process, highlighting two distinct axes, related to inflammation and to the dysregulation of mRNA metabolism, at the functional RNA and protein level. Additionally, we report the alteration of heterochromatin domains, including the loss of bivalent chromatin and the uncovering of a heterochromatin-switch phenomenon whereby constitutive heterochromatin loss is partially mitigated through gains in facultative heterochromatin. Notably, we observed the multi-omic reversal of a great number of aging-associated alterations in the context of environmental enrichment, which was particularly linked to glial and oligodendrocyte pathways. In conclusion, our work describes the epigenomic landscape of environmental stimulation in the context of aging and reveals how lifestyle intervention can lead to the multi-layered reversal of aging-associated decline.

Exploring the Anti-Inflammatory Effect of Inulin by Integrating Transcriptomic and Proteomic Analyses in a Murine Macrophage Cell Model.

Inulin is a natural polysaccharide classified as a soluble fiber with demonstrated prebiotic activity. Prebiotics can reduce intestinal and systemic inflammation through modulation of the gut microflora and their metabolites. Additionally, extensive research is illuminating the role of macrophages in the interaction between gut microbiota and many systemic inflammatory diseases. In this study, the anti-inflammatory properties of inulin were evaluated using a murine macrophage cell model (RAW 264.7) of inflammation, and the immunomodulatory mechanism was investigated using omics technologies. The cells underwent comprehensive transcriptomic and proteomic analyses to identify the mechanisms responsible for the observed anti-inflammatory phenotype. Functional analyses of these omics results revealed two potential mechanisms that may lead to an overall reduction in cytokine and chemokine transcription: the inhibition of the NF-κB signaling pathway, leading to the downregulation of proinflammatory factors such as COX2, and the promotion of the phase II defense protein Hmox1 via the Nrf2 pathway. This study provides promising targets for research on immune modulation by dietary fibers and offers new strategies for the design of functional ingredients, foods, and nutraceutical products, which could ultimately lead to personalized nutrition and improved consumer health.

Proteomic analysis of postprandial high-density lipoproteins in healthy subjects.

The relationship between the functionality and composition of high-density lipoproteins (HDL) is yet not fully studied, and little is known about the influence of the diet in HDL proteome. Therefore, the aim of this research was to elucidate the HDL proteome associated to postprandial hyperlipidemia. Male volunteers were recruited for an interventional study with high fatty acid-based meals. Blood samples were collected before the intake (baseline), and 2-3 (postprandial peak) and 5-6 (postprandial post peak) hours later. HDL were purified and the protein composition was quantified by LC-MS/MS. Statistical analysis was performed by lineal models (amica) and by ANOVA and multi-t-test of the different conditions (MetaboAnalyst). Additionally, a clustering of the expression profiles of each protein was done with coseq R package (RStudio). Initially, 320 proteins were identified but only 119 remained after the filtering. APOM, APOE, APOB, and APOA2, proteins previously identified in the HDL proteome, were the only proteins with a statistically significant altered expression in postprandial hyperlipidemia when compared to baseline (p values <0.05 and logFC >1). In conclusion, we have been able to describe several behaviors of the whole HDL proteome during the postprandial hyperlipidemic metabolism.

Food authentication of commercially-relevant shrimp and prawn species: from classical methods to Foodomics.

Although seafood species identification has traditionally relied on morphological analysis, sometimes this is difficult to apply for the differentiation among penaeid shrimps owing to their phenotypic similarities and to the frequent removal of external carapace during processing. The objective of this review is to provide an updated and extensive overview on the molecular methods for shrimp and prawn species authentication, in which several omics approaches based on protein and DNA analysis are described. DNA-based methods include the amplification by PCR of different genes, commonly the mitochondrial 16S ribosomal RNA and cytochrome oxidase I genes. A recently described method based on RFLP coupled to PCR turned out to be particularly interesting for species differentiation and origin identification. Protein analysis methods for the characterization and detection of species-specific peptides are also summarized, emphasizing some novel proteomics-based approaches, such as phyloproteomics, peptide fragmentation, and species-specific peptide detection by HPLC coupled to multiple reaction monitoring (MRM) MS, the latter representing the fastest method described to date for species authentication in food.

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis.

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.

Shotgun Proteomics for Food Microorganism Detection.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

Re-analysis of SARS-CoV-2-infected host cell proteomics time-course data by impact pathway analysis and network analysis: a potential link with inflammatory response.

Coronavirus disease 2019 (COVID-19), caused by an outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in Wuhan, China, has led to an unprecedented health and economic crisis worldwide. To develop treatments that can stop or lessen the symptoms and severity of SARS-CoV-2 infection, it is critical to understand how the virus behaves inside human cells, and so far studies in this area remain scarce. A recent study investigated translatome and proteome host cell changes induced in vitro by SARS-CoV-2. Here, we use the publicly available proteomics data from this study to re-analyze the in vitro cellular consequences of SARS-CoV-2 infection by impact pathways analysis and network analysis. Notably, proteins linked to the inflammatory response, but also proteins related to chromosome segregation during mitosis, were found to be altered in response to viral infection. Upregulation of inflammatory response proteins is in line with the propagation of inflammatory reaction and lung injury that is observed in advanced stages of COVID-19 patients and which worsens with age.

α-Linolenic and γ-linolenic acids exercise differential antitumor effects on HT-29 human colorectal cancer cells.

α-Linolenic acid (ALA, 18:3n-3) and γ-gamma linolenic acid (GLA, 18:3n-6) are polyunsaturated fatty acids (PUFA) that improve the human health. The present study focused on testing the in vitro antitumor actions of pure ALA and GLA on the HT-29 human colorectal cancer cell line. Cell viability was checked by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, cell membrane damage by the lactate dehydrogenase assay, apoptosis was tested by both caspase-3 activity trial and transmission electron microscopy images, and protein composition was analyzed by quantitative proteomics analysis. MTT test revealed IC50 values of 230 and 255 µM for ALA and GLA, respectively, at 72 h. After 24 h of incubation, both ALA and GLA induced apoptosis on HT-29 colorectal cancer cells according to the caspase-3 assay and microscopy images. SWATH/MS analysis evidenced that ALA significantly affected the mitochondrial protein import pathway and the citric acid cycle pathway, while GLA did not significantly affect any particular pathway. In summary, both ALA and GLA showed concentration-dependent inhibitory effects on HT-29 cells viability and induced cell death by apoptosis. ALA significantly affected cellular pathways, while GLA does not have specific actions on either pathway. Both n-3 and n-6 C18 PUFA are bioactive food components useful in the colorectal cancer prevention.

Mass spectrometry characterization of species-specific peptides from arginine kinase for the identification of commercially relevant shrimp species.

The identification of commercial shrimp species is a relevant issue to ensure correct labeling, maintain consumer confidence and enhance the knowledge of the captured species, benefiting both, fisheries and manufacturers. A proteomic approach, based on 2DE, tryptic in-gel digestion, MALDI-TOF MS, and ESI-MS/MS analyses, is proposed for the identification of shrimp species with commercial interest. MALDI-TOF peptide mass fingerprint from arginine kinase tryptic digests were used for the identification of seven commercial, closely related species of Decapoda shrimps. Further identification and characterization of these peptides was performed by CID on an ESI-IT instrument, database search and de novo sequence interpretation, paying special attention to differential, species-specific peptides. Fisheries and manufacturers may take advantage of this methodology as a tool for a rapid and effective seafood product identification and authentication, providing and guaranteeing the quality and safety of the foodstuffs to consumers.

Review of Recent DNA-Based Methods for Main Food-Authentication Topics.

Adulteration and mislabeling of food products and the commercial fraud derived, either intentionally or not, is a global source of economic fraud to consumers but also to all stakeholders involved in food production and distribution. Legislation has been enforced all over the world aimed at guaranteeing the authenticity of the food products all along the distribution chain, thereby avoiding food fraud and adulteration. Accordingly, there is a growing need for new analytical methods able to verify that all the ingredients included in a foodstuff match the qualities claimed by the manufacturer or distributor. In this sense, the improved performance of most recent DNA-based tools in term of sensitivity, multiplexing ability, high-throughput, and relatively low-cost give them a game-changing role in food-authenticity-related topics. Here, we provide a thorough and updated vision on the recently reported approaches that are applying these DNA-based tools to assess the authenticity of food components and products.

SWATH proteomic profiling of prostate cancer cells identifies NUSAP1 as a potential molecular target for Galiellalactone.

Galiellalactone (GL) is a fungal metabolite that presents antitumor and antiinflammatory activities in vitro and in vivo. Previous studies have shown that GL targets NF-κB and STAT3 pathways and induces G2/M cell cycle arrest in androgen-insensitive prostate cancer cells. In this study, we show that GL-induced cell cycle arrest is independent of the NF-κB and STAT3 pathways in DU145 and PC-3 cells, and also that GL did not affect cell cycling in androgen-sensitive prostate cancer cells such as LNCaP and 22Rv1 cells. In addition, we showed confluence resistance to GL in DU145 cells. Using a SWATH proteomic approach we identified a down-regulation of Nucleolar and spindle associated protein 1 (NUSAP1) under DU145 confluence. Moreover the expression of NUSAP1 in LNCaP cells is low compared to DU145 cells. The inhibition of NUSAP1 by siRNAs induced resistance to GL in DU145 cells, suggesting that NUSAP1 may be a target for GL and could be useful as a biomarker for the responsiveness of the antitumor activity of GL. Altogether, our finding shed light to the potential of GL to be developed as a novel treatment of castration resistance prostate cancer.

SWATH Differential Abundance Proteomics and Cellular Assays Show In Vitro Anticancer Activity of Arachidonic Acid- and Docosahexaenoic Acid-Based Monoacylglycerols in HT-29 Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH) - mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions.

Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection.

A typical clinical manifestation of growth hormone deficiency (GHD) is a short stature resulting from delayed growth, but GHD affects bone health, cardiovascular function and metabolic profile and therefore quality of life. Although early GH treatment during childhood has been shown to improve outcomes, no single biochemical parameter is currently available for the accurate diagnosis of GHD in children. There is hence a need for non-invasive biomarkers. In this study, the relative abundance of serum proteins from GHD children and healthy controls was measured by next-generation proteomics SWATH-MS technology. The data generated was analysed by machine-learning feature-selection algorithms in order to discover the minimum number of protein biomarkers that best discriminate between both groups. The analysis of serum proteins by a SWATH-MS approach yielded a useful method for discovering potential biomarkers of GHD in children. A total of 263 proteins were confidently detected and quantified in each sample. Pathway analysis indicated an effect on tissue/organ structure and morphogenesis. The top ten serum protein biomarker candidates were identified after applying feature-selection data analysis. The combination of three proteins - apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein - demonstrated the best classification performance for our data. In addition, the apolipoprotein group resulted in strong over-representation, thus highlighting these proteins as an additional promising biomarker panel. SIGNIFICANCE: Currently there is no single biochemical parameter available for the accurate diagnosis of growth hormone (GH) deficiency (GHD) in children. Simple GH measurements are not an option: because GH is released in a pulsatile action, its blood levels fluctuate throughout the day and remain nearly undetectable for most of that time. This makes measurements of GH in a single blood sample useless for assessing GH deficiency. Actually, the diagnosis of GHD includes a combination of direct and indirect non-accurate measurements, such as taking several body measurements, testing GH levels in multiple blood samples after provocative tests (GH peak <7.3ng/mL, using radioimmunoassay), and conducting magnetic resonance imaging (MRI), among others. Therefore, there is a need for simple, non-invasive, accurate and cost-effective biomarkers. Here we report a case-control study, where relative abundance of serum proteins were measured by next-generation proteomics SWATH-MS technology in 15 GHD children and 15healthy controls matched by age, sex, and not receiving any treatment. Data generated was analysed by machine learning feature selection algorithms. 263 proteins could be confidently detected and quantified on each sample. The top 10 serum protein biomarker candidates could be identified after applying a feature selection data analysis. The combination of three proteins, apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein, showed the best classification performance for our data. In addition, the fact that the pathway and GO analysis we performed pointed to the apolipoproteins as over-represented highlights this protein group as an additional promising biomarker panel for the diagnosis of GHD and for treatment evaluation.

Proteomics Study Reveals That Docosahexaenoic and Arachidonic Acids Exert Different In Vitro Anticancer Activities in Colorectal Cancer Cells.

Two polyunsaturated fatty acids, docosahexaenoic acid (DHA) and arachidonic acid (ARA), as well as derivatives, such as eicosanoids, regulate different activities, affecting transcription factors and, therefore, DNA transcription, being a critical step for the functioning of fatty-acid-derived signaling. This work has attempted to determine the in vitro anticancer activities of these molecules linked to the gene transcription regulation of HT-29 colorectal cancer cells. We applied the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test along with lactate dehydrogenase and caspase-3 assays; proteome changes were assessed by "sequential windowed acquisition of all theoretical mass spectra" quantitative proteomics, followed by pathway analysis, to determine the affected molecular mechanisms. In all assays, DHA inhibited cell proliferation of HT-29 cells to a higher extent than ARA and acted primarily by downregulating proteasome particles, while ARA presented a dramatic effect on all six DNA replication helicase particles. The results indicated that both DHA and ARA are potential chemopreventive agent candidates.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Review on proteomics for food authentication.

UNLABELLED: Consumers have the right to know what is in the food they are eating. Accordingly, European and global food regulations require that the provenance of the food can be guaranteed from farm to fork. Many different instrumental techniques have been proposed for food authentication. Although traditional methods are still being used, new approaches such as genomics, proteomics, and metabolomics are helping to complement existing methodologies for verifying the claims made about certain food products. During the last decade, proteomics (the large-scale analysis of proteins in a particular biological system at a particular time) has been applied to different research areas within food technology. Since proteins can be used as markers for many properties of a food, even indicating processes to which the food has been subjected, they can provide further evidence of the foods labeling claim. This review is a comprehensive and updated overview of the applications, drawbacks, advantages, and challenges of proteomics for food authentication in the assessment of the foods compliance with labeling regulations and policies. SIGNIFICANCE: This review paper provides a comprehensive and critical overview of the application of proteomics approaches to determine the authenticity of several food products updating the performances and current limitations of the applied techniques in both laboratory and industrial environments.