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Objective: The interplay of gut microbiota with the kidney system in chronic kidney disease (CKD), is characterized by increased concentrations of uric acid in the gut, which in turn, may increase bacterial uricase activity and may lead to the generation of uremic toxins. Nevertheless, knowledge on these underlying bidirectional molecular mechanisms is still limited. Methods: In this exploratory study, proteomic analysis was performed on fecal samples, targeting to investigate this largely unexplored biological material as a source of information reflecting the gut-kidney axis. Specifically, fecal suspension samples from patients with CKD1 (n = 12) and CKD4 (n = 17) were analysed by LC-MS/MS, using both the Human and Bacterial UniProt RefSeq reviewed databases. Results: This fecal proteomic analysis collectively identified 701 human and 1011 bacterial proteins of high confidence. Differential expression analysis (CKD4/CKD1) revealed significant changes in human proteins (n = 8, including proteins such as galectin-3-binding protein and prolactin-inducible protein), that were found to be associated with inflammation and CKD. The differential protein expression of pancreatic alpha-amylase further suggested plausible reduced saccharolytic fermentation in CKD4/CKD1. Significant changes in bacterial proteins (n = 9, such as glyceraldehyde-3-phosphate dehydrogenase and enolase), participating in various carbohydrate and metabolic pathways important for the synthesis of butyrate, in turn suggested differential butyrate synthesis in CKD4/CKD1. Further, targeted quantification of fecal pancreatic alpha-amylase and butyrate in the same fecal suspension samples, supported these hypotheses. Conclusion: Collectively, this exploratory fecal proteomic analysis highlighted changes in human and bacterial proteins reflecting inflammation and reduced saccharolytic fermentation in CKD4/CKD1, plausibly affecting the butyrate synthesis pathways in advanced stage kidney disease. Integrative multi-omics validation is planned.

The aim of the current study was to investigate the tear proteome in children and adolescents with type 1 diabetes (T1D) compared to healthy controls, and to identify differences in the tear proteome of children with T1D depending on different characteristics of the disease. Fifty-six children with T1D at least one year after diagnosis, aged 6–17 years old, and fifty-six healthy age- and sex-matched controls were enrolled in this cross-sectional study. The proteomic analysis was based on liquid chromatography tandem mass spectrometry (LC-MS/MS) enabling the identification and quantification of the protein content via Data-Independent Acquisition by Neural Networks (DIA-NN). Data are available via ProteomeXchange with the identifier PXD052994. In total, 3302 proteins were identified from tear samples. Two hundred thirty-nine tear proteins were differentially expressed in children with T1D compared to healthy controls. Most of them were involved in the immune response, tissue homeostasis and inflammation. The presence of diabetic ketoacidosis at diagnosis and the level of glycemic control of children with T1D influenced the tear proteome. Tear proteomics analysis revealed a different proteome pattern in children with T1D compared to healthy controls offering insights on deregulated biological processes underlying the pathogenesis of T1D. Differences within the T1D group could unravel biomarkers for early detection of long-term complications of T1D.

Background: Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNPs) have been suggested as risk, prognostic, and predictive factors for colorectal cancer in various populations, but have not been validated so far. The aim of this study was to examine the association of MTHFR C677T (rs1801133) and A1298C (rs1801131) single nucleotide polymorphisms with the risk of rectal cancer as well as the response to neoadjuvant chemoradiotherapy (nCRT) based on 5-Fluorouracil (5-FU)/leucovorin (LV) in the locally advanced setting.Patients and methods: This case-control study included 119 healthy controls and 97 patients with locally advanced rectal cancer (LARC). For MTHFR genotyping, restriction fragment length polymorphism analysis (PCR-RFLP) was employed.Results:In silico analysis highlighted that SNPs C677T and A1298T correlate with MTHFR gene expression, and that gene expression profile correlates with cancer risk and stage. Using dominant and recessive models, it was found that the MTHFR 677CC vs. 677CT+677TT have increased risk of cancer development (odds ratio (OR): 2.27; 95% confidence interval (CI): 1.30–3.95, p = 0.002) as well as 677CC+677CT compared to 677TT (OR: 4.18, 95% CI: 1.16–14.99, p = 0.014). MTHFR 1298AA also shown increased risk for cancer development compared to 1298AC+1298CC (OR:2.0, 95% CI: 1.20–3.59, p = 0.035) Statistical analysis of combined genotypes highlighted the protective role of CT/AC combined genotype (OR: 3.15 95% CI: 1.576–6.279, p = 0.002) while the CC/AA genotype showed an increased risk for rectal cancer development (OR: 2.499, 95% CI: 1.246–5.081, p = 0.016) The carriers of the 677C/1298A haplotype had the highest risk for developing rectal cancer (OR: 1.74; 95% CI: 1.198–2.530, p = 0.002) while the 677T/1298C haplotype seems to provide a protective effect. (OR: 0.44; 95%CI 0.248–0.795, p = 0.003). No significant association with response to chemoradiotherapy was found.Conclusion: Our data point to MTHFR 667C allele and 1298A alleles as low-penetrance risk factors for rectal cancer in our population. To the best of our knowledge, this is the first study of this type performed on the Slavic population in the Western Balkan, as various population-based factors might also be significant our findings can be used for future meta-analyses and the construction of genetic cancer risk prediction panels.

Summary: Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Introduction: The standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (nCRT). To select patients who would benefit the most from nCRT, there is a need for predictive biomarkers. The aim of this study was to evaluate the role of clinical, pathological, radiological, inflammation-related genetic, and hematological parameters in the prediction of post-nCRT response.Materials and methods:In silico analysis of published transcriptomics datasets was conducted to identify candidate genes, whose expression will be measured using quantitative Real Time PCR (qRT-PCR) in pretreatment formaline-fixed paraffin-embedded (FFPE) samples. In this study, 75 patients with LARC were prospectively included between June 2020—January 2022. Patients were assessed for tumor response in week 8 post-nCRT with pelvic MRI scan and rigid proctoscopy. For patients with a clinical complete response (cCR) and initially distant located tumor no immediate surgery was suggested (“watch and wait” approach). The response after surgery was assessed using histopathological tumor regression grading (TRG) categories from postoperative specimens by Mandard. Responders (R) were defined as patients with cCR without operative treatment, and those with TRG 1 and TRG 2 postoperative categories. Non-responders (NR) were patients classified as TRG 3-5.Results: Responders group comprised 35 patients (46.6%) and NR group 53.4% of patients. Analysis of published transcriptomics data identified genes that could predict response to treatment and their significance was assessed in our cohort by qRT-PCR. When comparison was made in the subgroup of patients who were operated (TRG1 vs. TRG4), the expression of IDO1 was significantly deregulated (p < 0.05). Among hematological parameters between R and NR a significant difference in the response was detected for neutrophil-to-monocyte ratio (NMR), initial basophil, eosinophil and monocyte counts (p < 0.01). According to MRI findings, non-responders more often presented with extramural vascular invasion (p < 0.05).Conclusion: Based on logistic regression model, factors associated with favorable response to nCRT were tumor morphology and hematological parameters which can be easily and routinely derived from initial laboratory results (NMR, eosinophil, basophil and monocyte counts) in a minimally invasive manner. Using various metrics, an aggregated score of the initial eosinophil, basophil, and monocyte counts demonstrated the best predictive performance.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses’ results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = −0.58 to −0.842 and range for IGHM rs = −0.6 to −0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

Abatacept (CTLA4-Ig)—a monoclonal antibody which restricts T cell activation—is an effective treatment for rheumatoid arthritis (RA). Nevertheless, only 50% of RA patients attain clinical responses, while predictors of response are rather limited. Herein, we aimed to investigate for early biomarkers of response to abatacept, based on a detailed immunological profiling of peripheral blood (PB) cells and serum proteins. We applied flow cytometry and proteomics analysis on PB immune cells and serum respectively, of RA patients starting abatacept as the first biologic agent. After 6 months of treatment, 34.5% of patients attained response. At baseline, Th1 and FoxP3+ T cell populations were positively correlated with tender joint counts (p-value = 0.047 and p-value = 0.022, respectively). Upon treatment, CTLA4-Ig effectively reduced the percentages of Th1 and Th17 only in responders (p-value = 0.0277 and p-value = 0.0042, respectively). Notably, baseline levels of Th1 and myeloid cell populations were significantly increased in PB of responders compared to non-responders (p-value = 0.009 and p-value = 0.03, respectively). Proteomics analysis revealed that several inflammatory mediators were present in serum of responders before therapy initiation and strikingly 10 amongst 303 serum proteins were associated with clinical responses. Finally, a composite index based on selected baseline cellular and proteomics’ analysis could predict response to abatacept with a high sensitivity (90%) and specificity (88.24%).

Locally advanced rectal cancer (LARC) presents a challenge in identifying molecular markers linked to the response to neoadjuvant chemoradiotherapy (nCRT). This study aimed to utilize a sensitive proteomic method, data-independent mass spectrometry (DIA-MS), to extensively analyze the LARC proteome, seeking individuals with favorable initial responses suitable for a watch-and-wait approach. This research addresses the unmet need to understand the response to treatment, potentially guiding personalized strategies for LARC patients. Post-treatment assessment included MRI scans and proctoscopy. This research involved 97 LARC patients treated with intense chemoradiotherapy, comprising radiation and chemotherapy. Out of 97 LARC included in this study, we selected 20 samples with the most different responses to nCRT for proteome profiling (responders vs. non-responders). This proteomic approach shows extensive proteome coverage in LARC samples. The analysis identified a significant number of proteins compared to a prior study. A total of 915 proteins exhibited differential expression between the two groups, with certain signaling pathways associated with response mechanisms, while top candidates had good predictive potential. Proteins encoded by genes SMPDL3A, PCTP, LGMN, SYNJ2, NHLRC3, GLB1, and RAB43 showed high predictive potential of unfavorable treatment outcome, while RPA2, SARNP, PCBP2, SF3B2, HNRNPF, RBBP4, MAGOHB, DUT, ERG28, and BUB3 were good predictive biomarkers of favorable treatment outcome. The identified proteins and related biological processes provide promising insights that could enhance the management and care of LARC patients.

Abstract Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed “ontο MHCI” trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

The collagen family contains 28 proteins, predominantly expressed in the extracellular matrix (ECM) and characterized by a triple-helix structure. Collagens undergo several maturation steps, including post-translational modifications (PTMs) and cross-linking. These proteins are associated with multiple diseases, the most pronounced of which are fibrosis and bone diseases. This review focuses on the most abundant ECM protein highly implicated in disease, type I collagen (collagen I), in particular on its predominant chain collagen type I alpha 1 (COLα1 (I)). An overview of the regulators of COLα1 (I) and COLα1 (I) interactors is presented. Manuscripts were retrieved searching PubMed, using specific keywords related to COLα1 (I). COL1A1 regulators at the epigenetic, transcriptional, post-transcriptional and post-translational levels include DNA Methyl Transferases (DNMTs), Tumour Growth Factor β (TGFβ), Terminal Nucleotidyltransferase 5A (TENT5A) and Bone Morphogenic Protein 1 (BMP1), respectively. COLα1 (I) interacts with a variety of cell receptors including integrinβ, Endo180 and Discoidin Domain Receptors (DDRs). Collectively, even though multiple factors have been identified in association to COLα1 (I) function, the implicated pathways frequently remain unclear, underscoring the need for a more spherical analysis considering all molecular levels simultaneously.

Chronic kidney disease (CKD) is prevalent in 10% of world’s adult population. The role of protein glycosylation in causal mechanisms of CKD progression is largely unknown. The aim of this study was to identify urinary O-linked glycopeptides in association to CKD for better characterization of CKD molecular manifestations. Urine samples from eight CKD and two healthy subjects were analyzed by CE-MS/MS and glycopeptides were identified by a specific software followed by manual inspection of the spectra. Distribution of the identified glycopeptides and their correlation with Age, eGFR and Albuminuria were evaluated in 3810 existing datasets. In total, 17 O-linked glycopeptides from 7 different proteins were identified, derived primarily from Insulin-like growth factor-II (IGF2). Glycosylation occurred at the surface exposed IGF2 Threonine 96 position. Three glycopeptides (DVStPPTVLPDNFPRYPVGKF, DVStPPTVLPDNFPRYPVG and DVStPPTVLPDNFPRYP) exhibited positive correlation with Age. The IGF2 glycopeptide (tPPTVLPDNFPRYP) showed a strong negative association with eGFR. These results suggest that with aging and deteriorating kidney function, alterations in IGF2 proteoforms take place, which may reflect changes in mature IGF2 protein. Further experiments corroborated this hypothesis as IGF2 increased plasma levels were observed in CKD patients. Protease predictions, considering also available transcriptomics data, suggest activation of cathepsin S with CKD, meriting further investigation.

IL-33, a nuclear alarmin released during cell death, exerts context-specific effects on adaptive and innate immune cells, eliciting potent inflammatory responses. We screened blood, skin, and kidney tissues from patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease driven by unabated type I IFN production, and found increased amounts of extracellular IL-33 complexed with neutrophil extracellular traps (NETs), correlating with severe, active disease. Using a combination of molecular, imaging, and proteomic approaches, we show that SLE neutrophils, activated by disease immunocomplexes, release IL-33–decorated NETs that stimulate robust IFN-α synthesis by plasmacytoid DCs in a manner dependent on the IL-33 receptor ST2L. IL33-silenced neutrophil-like cells cultured under lupus-inducing conditions generated NETs with diminished interferogenic effect. Importantly, NETs derived from patients with SLE are enriched in mature bioactive isoforms of IL-33 processed by the neutrophil proteases elastase and cathepsin G. Pharmacological inhibition of these proteases neutralized IL-33–dependent IFN-α production elicited by NETs. We believe these data demonstrate a novel role for cleaved IL-33 alarmin decorating NETs in human SLE, linking neutrophil activation, type I IFN production, and end-organ inflammation, with skin pathology mirroring that observed in the kidneys.

Tumor cells undergoing epithelial to mesenchymal transition (EMT) and immune cells in tumor microenvironment (TME) reciprocally influence each other. Immune cells, by supplying TME with bioactive molecules including cytokines, chemokines, enzymes, metabolites, and by physical interactions with tumor cells via their receptors, represent an important factor that affects EMT. Chronical inflammation in TME favorizes tumor growth and invasiveness and stimulates synthesis of EMT promoting transcription factors. Natural killer (NK) cells, owing to their unique ability to exert cytotoxic function independent of major histocompatibility (MHC)-mediated antigen presentation, play a significant role in the control of metastasis in colorectal cancer (CRC). Although, the cross-talk between immune cells and tumor cells in general favors the induction of EMT and inhibition of antitumor immune responses, there are some changes in the immunogenicity of tumor cells during EMT of CRC cells that increase their susceptibility to NK cell cytotoxic lysis. However, suppressive TME downmodulates the expression of activating NK cell receptors, decreases the expression of activating and increases the expression of inhibitory NK cell ligands on tumor cells, and impairs NK cell metabolism that altogether negatively affects the overall NK cell function. Furthermore, process of EMT is often associated with increased expression of programmed cell death ligand (PD-L) and expression of immune checkpoint molecules PD-1, TIGIT, and TIM3 on functionally exhausted NK cells in TME in CRC. In this review we discuss modalities of cross-talk between tumor cells and NK cells, with regard of EMT-driven changes.

Molar incisor hypomineralization is a complex developmental enamel defect that affects the permanent dentition of children with significant functional and aesthetic implications. Saliva is an ideal diagnostic tool and ensures patients’ compliance by diminishing the discomfort especially in pediatric population. Lately, salivary proteome analysis has progressively evolved in various biomedical disciplines. As changes in saliva composition are associated with oral diseases, it is reasonable to assume that the saliva proteome of MIH-affected children might be altered compared to healthy children. This study analyzed the human and microbial salivary proteome in children with MIH in order to identify salivary markers indicative of the pathology. The conducted proteomic analysis generated a comprehensive dataset comprising a total of 1515 high confidence identifications and revealed a clear discrimination between the two groups. Statistical comparison identified 142 differentially expressed proteins, while the pathway analysis indicated deregulation of inflammation, immune response mechanisms, and defense response to bacteria in MIH patients. Bacterial proteome analysis showed a lower diversity for the microbial species, which highlights the dysbiotic environment established in the MIH pathology.

Background: The increased prevalence of cardiovascular disease (CVD) indicates a demand for novel therapeutic approaches. Proteome analysis of vascular tissues from animal models and humans with CVD could lead to the identification of novel druggable targets. Methods: LC-MS/MS analysis of thoracic aortas from three mouse models of non-diabetic and diabetic (streptozotocin (STZ)-induced) atherosclerosis followed by bioinformatics/pathway analysis was performed. Selected findings were confirmed by proteomics analysis of human vessels from patients with CVD as well as in vitro studies (migration, proliferation, angiogenesis assays) using endothelial (HUVEC) cells. Findings: Comparative tissue proteomics of low density lipoprotein receptor deficient (Ldlr−/−) and diabetic Ldlr−/− (Ldlr−/−STZ) with wild type (WT) animals led to the identification of 284 differentially expressed proteins in both models. Among them, 177 proteins were also differentially expressed in diabetic apolipoprotein E deficient (ApoE−/−STZ) mice, suggesting expression changes associated with atherosclerosis independent of the model used. These proteins recapitulated the hallmarks of atherosclerosis. Comparison of these findings with differentially expressed proteins in human vessels with CVD enabled shortlisting of six commonly dysregulated proteins. Among them, lysine-specific demethylase 5D (KDM5D) exhibited pronounced overexpression accompanied by a reduction in the protein levels of its substrate, the trimethylated lysine 4 of histone H3 (H3K4me3), in patients with CVD. Functional interference studies applying a KDM5 inhibitor on HUVEC reduced cell proliferation, migration and tube-forming ability in vitro. Interpretation: This high-throughput proteomics strategy identified KDM5 histone demethylases being potentially involved in CVD, possibly by affecting H3K4 methylation. Fund: [SysVasc, HEALTH-2013 603288], [ERA-CVD PROACT: ANR-17-ECVD-0006, 01KL1805], [FRM, DEQ20170336759]. Keywords: Cardiovascular disease, Atherosclerosis, Diabetes, Proteomics, KDM5, H3K4

Chronic kidney disease (CKD) is predominant in 10% of the world’s adult population, and is increasingly considered a silent epidemic. Gut microbiota plays an essential role in maintaining host energy homeostasis and gut epithelial integrity. Alterations in gut microbiota composition, functions and, specifically, production of metabolites causing uremic toxicity are often associated with CKD onset and progression. Here, we present the latest omics (transcriptomics, proteomics and metabolomics) studies that explore the connection between CKD and gut microbiome. A review of the available literature using PubMed was performed using the keywords “microb*”, “kidney”, “proteom”, “metabolom” and “transcript” for the last 10 years, yielding a total of 155 publications. Following selection of the relevant studies (focusing on microbiome in CKD), a predominance of metabolomics (n = 12) over transcriptomics (n = 1) and proteomics (n = 6) analyses was observed. A consensus arises supporting the idea that the uremic toxins produced in the gut cause oxidative stress, inflammation and fibrosis in the kidney leading to CKD. Collectively, findings include an observed enrichment of Eggerthella lenta, Enterobacteriaceae and Clostridium spp., and a depletion in Bacteroides eggerthii, Roseburia faecis and Prevotella spp. occurring in CKD models. Bacterial species involved in butyrate production, indole synthesis and mucin degradation were also related to CKD. Consequently, strong links between CKD and gut microbial dysbiosis suggest potential therapeutic strategies to prevent CKD progression and portray the gut as a promising therapeutic target.

Significant inter-individual variation in terms of susceptibility to several stress-related disorders, such as myocardial infarction and Alzheimer’s disease, and therapeutic response has been observed among healthy subjects. The molecular features responsible for this phenomenon have not been fully elucidated. Proteomics, in association with bioinformatics analysis, offer a comprehensive description of molecular phenotypes with clear links to human disease pathophysiology. The aim of this study was to conduct a comparative plasma proteomics analysis of glucocorticoid resistant and glucocorticoid sensitive healthy subjects and provide clues of the underlying physiological differences. For this purpose, 101 healthy volunteers were given a very low dose (0.25 mg) of dexamethasone at midnight, and were stratified into the 10% most glucocorticoid sensitive (S) (n = 11) and 10% most glucocorticoid resistant (R) (n = 11) according to the 08:00 h serum cortisol concentrations determined the following morning. One month following the very-low dose dexamethasone suppression test, DNA and plasma samples were collected from the 22 selected individuals. Sequencing analysis did not reveal any genetic defects in the human glucocorticoid receptor (NR3C1) gene. To investigate the proteomic profile of plasma samples, we used Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and found 110 up-regulated and 66 down-regulated proteins in the S compared to the R group. The majority of the up-regulated proteins in the S group were implicated in platelet activation. To predict response to cortisol prior to administration, a random forest classifier was developed by using the proteomics data in order to distinguish S from R individuals. Apolipoprotein A4 (APOA4) and gelsolin (GSN) were the most important variables in the classification, and warrant further investigation. Our results indicate that a proteomics signature may differentiate the S from the R healthy subjects, and may be useful in clinical practice. In addition, it may provide clues of the underlying molecular mechanisms of the chronic stress-related diseases, including myocardial infarction and Alzheimer’s disease.

Background: The absence of efficient inhibitors for diabetic kidney disease (DKD) progression reflects the gaps in our understanding of DKD molecular pathogenesis. Methods: A comprehensive proteomic analysis was performed on the glomeruli and kidney cortex of diabetic mice with the subsequent validation of findings in human biopsies and omics datasets, aiming to better understand the underlying molecular biology of early DKD development and progression. Results: LC–MS/MS was employed to analyze the kidney proteome of 2 DKD models: Ins2Akita (early and late DKD) and db/db mice (late DKD). The abundance of detected proteins was defined. Pathway analysis of differentially expressed proteins in the early and late DKD versus the respective controls predicted dysregulation in DKD hallmarks (peroxisomal lipid metabolism and β-oxidation), supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, the consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal and upregulated mitochondrial proteins. Tissue sections from 16 DKD patients were analyzed by IHC confirming our results. Conclusion: Our study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity, providing new perspectives and potential markers of diabetic kidney dysfunction.

Abstract Background Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. Methods LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Results Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. Conclusions This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD.

Saliva, an essential oral secretion involved in protecting the oral cavity’s hard and soft tissues, is readily available and straightforward to collect. Recent studies have analyzed the salivary proteome in children and adolescents with extensive carious lesions to identify diagnostic and prognostic biomarkers. The current study aimed to investigate saliva’s diagnostic ability through proteomics to detect the potential differential expression of proteins specific for the occurrence of carious lesions. For this study, we performed bioinformatics and functional analysis of proteomic datasets, previously examined by our group, from samples of adolescents with regulated and unregulated type 1 diabetes, as they compare with healthy controls. Among the differentially expressed proteins relevant to caries pathology, alpha-amylase 2B, beta-defensin 4A, BPI fold containing family B member 2, protein S100-A7, mucin 5B, statherin, salivary proline-rich protein 2, and interleukin 36 gamma were significantly downregulated in poorly-controlled patients compared to healthy subjects. In addition, significant biological pathways (defense response to the bacterium, beta-defensin activity, proline-rich protein activity, oxygen binding, calcium binding, and glycosylation) were deregulated in this comparison, highlighting specific molecular characteristics in the cariogenic process. This analysis contributes to a better understanding of the mechanisms involved in caries vulnerability in adolescents with unregulated diabetes.

Abstract IgA nephropathy (IgAN) is the most prevalent among primary glomerular diseases worldwide. Although our understanding of IgAN has advanced significantly, its underlying biology and potential drug targets are still unexplored. We investigated a combinatorial approach for the analysis of IgAN-relevant -omics data, aiming at identification of novel molecular signatures of the disease. Nine published urinary proteomics datasets were collected and the reported differentially expressed proteins in IgAN vs. healthy controls were integrated into known biological pathways. Proteins participating in these pathways were subjected to multi-step assessment, including investigation of IgAN transcriptomics datasets (Nephroseq database), their reported protein-protein interactions (STRING database), kidney tissue expression (Human Protein Atlas) and literature mining. Through this process, from an initial dataset of 232 proteins significantly associated with IgAN, 20 pathways were predicted, yielding 657 proteins for further analysis. Step-wise evaluation highlighted 20 proteins of possibly high relevance to IgAN and/or kidney disease. Experimental validation of 3 predicted relevant proteins, adenylyl cyclase-associated protein 1 (CAP1), SHC-transforming protein 1 (SHC1) and prolylcarboxypeptidase (PRCP) was performed by immunostaining of human kidney sections. Collectively, this study presents an integrative procedure for -omics data exploitation, giving rise to biologically relevant results.

The gold standard vascular substitutes, used in cardiovascular surgery, are the Dacron or expanded polytetrafluoroethylene (ePTFE)-derived grafts. However, major adverse reactions accompany their use. For this purpose, decellularized human umbilical arteries (hUAs) may be proven as a significant source for the development of small diameter conduits. The aim of this study was the evaluation of a decellularization protocol in hUAs. To study the effect of the decellularization to the hUAs, histological analysis was performed. Then, native and decellularized hUAs were biochemically and biomechanically evaluated. Finally, broad proteomic analysis was applied. Histological analysis revealed the successful decellularization of the hUAs. Furthermore, a great amount of DNA was removed from the decellularized hUAs. Biomechanical analysis revealed statistically significant differences in longitudinal direction only in maximum stress (p < 0.013) and strain (p < 0.001). On the contrary, all parameters tested for circumferential direction exhibited significant differences (p < 0.05). Proteomic analysis showed the preservation of the extracellular matrix and cytoskeletal proteins in both groups. Proteomic data are available via ProteomeXchange with identifier PXD020187. The above results indicated that hUAs were efficiently decellularized. The tissue function properties of these conduits were well retained, making them ideal candidates for the development of small diameter vascular grafts.

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease.

OBJECTIVES:Urinary Proteomics in Predicting Heart Transplantation Outcomes (uPROPHET; NCT03152422) aims: (i) to construct new multidimensional urinary proteomic (UP) classifiers that after heart transplantation (HTx) help in detecting graft vasculopathy, monitoring immune system activity and graft performance, and in adjusting immunosuppression; (ii) to sequence UP peptide fragments and to identify key proteins mediating HTx-related complications; (iii) to validate UP classifiers by demonstrating analogy between UP profiles and tissue proteomic signatures (TP) in diseased explanted hearts, to be compared with normal donor hearts; (iv) and to identify new drug targets. This article describes the uPROPHET database construction, follow-up strategies and baseline characteristics of the HTx patients. METHODS:HTx patients enrolled at the University Hospital Gasthuisberg (Leuven) collected mid-morning urine samples. Cardiac biopsies were obtained at HTx. UP and TP methods and the statistical work flow in pursuit of the research objectives are described in detail in the Data supplement. RESULTS:Of 352 participants in the UP study (24.4% women), 38.9%, 40.3%, 5.7% and 15.1% had ischemic, dilated, hypertrophic or other cardiomyopathy. The median interval between HTx and first UP assessment (baseline) was 7.8 years. At baseline, mean values were 56.5 years for age, 25.2 kg/m2 for body mass index, 142.3/84.8 mm Hg and 124.2/79.8 mm Hg for office and 24-h ambulatory systolic/diastolic pressure, and 58.6 mL/min/1.73 m2 for the estimated glomerular filtration rate. Of all patients, 37.2% and 6.5% had a history of mild (grade = 1B) or severe (grade ≥ 2) cellular rejection. Anti-body mediated rejection had occurred in 6.2% patients. The number of follow-up urine samples available for future analyses totals over 950. The TP study currently includes biopsies from 7 healthy donors and 15, 14, and 3 patients with ischemic, dilated, and hypertrophic cardiomyopathy. CONCLUSIONS:uPROPHET constitutes a solid resources for UP and TP research in the field of HTx and has the ambition to lay the foundation for the clinical application of UP in risk stratification in HTx patients.

Background: Recent findings have shown that the fibrin gel derived from cord blood units (CBUs) play a significant role in wound healing and tissue regeneration. The aim of this study was to standardize the fibrin gel production process in order to allow for its regular use. Methods: CBUs (n = 200) were assigned to 4 groups according to their initial volume. Then, a two-stage centrifugation protocol was applied in order to obtain platelet rich plasma (PRP). The concentration of platelets (PLTs), white blood cells (WBCs) and red blood cells (RBCs) were determined prior to and after the production process. In addition, targeted proteomic analysis using multiple reaction monitoring was performed. Finally, an appropriate volume of calcium gluconate was used in PRP for the production of fibrin gel. Results: The results of this study showed that high volume CBUs were characterized by greater recovery rates, concentration and number of PLTs compared to the low volume CBUs. Proteomic analysis revealed the presence of key proteins for regenerative medicine. Fibrin gel was successfully produced from CBUs of all groups. Conclusion: In this study, low volume CBUs could be an alternative source for the production of fibrin gel, which can be used in multiple regenerative medicine approaches.

ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms.

Background: Multipotent Mesenchymal Stromal Cells (MSCs) are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS). However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL) from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB) and cord blood (CB) PLs were determined using the Multiple Reaction Monitoring (MRM). Finally, the cell doubling time (CDT), tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.

Muscle invasive bladder carcinoma is a complex, multifactorial disease caused by disruptions and alterations of several molecular pathways that result in heterogeneous phenotypes and variable disease outcome. Combining this disparate knowledge may offer insights for deciphering relevant molecular processes regarding targeted therapeutic approaches guided by molecular signatures allowing improved phenotype profiling. The aim of the study is to characterize muscle invasive bladder carcinoma on a molecular level by incorporating scientific literature screening and signatures from omics profiling. Public domain omics signatures together with molecular features associated with muscle invasive bladder cancer were derived from literature mining to provide 286 unique protein-coding genes. These were integrated in a protein-interaction network to obtain a molecular functional map of the phenotype. This feature map educated on three novel disease-associated pathways with plausible involvement in bladder cancer, namely Regulation of actin cytoskeleton, Neurotrophin signalling pathway and Endocytosis. Systematic integration approaches allow to study the molecular context of individual features reported as associated with a clinical phenotype and could potentially help to improve the molecular mechanistic description of the disorder.

Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.