Tear Proteomics in Children and Adolescents with Type 1 Diabetes: A Promising Approach to Biomarker Identification of Diabetes Pathogenesis and Complications.

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.

Data-Independent Acquisition Mass Spectrometry Analysis of FFPE Rectal Cancer Samples Offers In-Depth Proteomics Characterization of the Response to Neoadjuvant Chemoradiotherapy.

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.

Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer.

Prostate cancer (PCa) is one of the leading causes of death in men worldwide. The molecular features, associated with the onset and progression of the disease, are under vigorous investigation. Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources for large-scale studies; however, their application in proteomics is limited due to protein cross-linking. In this study, the adjustment of a protocol for the proteomic analysis of FFPE tissues was performed which was followed by a pilot application on FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data for the investigation of PCa. For the optimization, FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization methods (beads, sonication, boiling) and buffers (SDS based and urea-thiourea based). The proteome extraction efficacy was then evaluated based on protein identifications and reproducibility using SDS electrophoresis and high resolution LC-MS/MS analysis. Comparison between the FFPE and matched fresh frozen (FF) tissues, using an optimized protocol involving protein extraction with an SDS-based buffer following beads homogenization and boiling, showed a substantial overlap in protein identifications with a strong correlation in relative abundances (rs = 0.819, p < 0.001). Next, FFPE tissues (3 sections, 15 µm each per sample) from 10 patients with PCa corresponding to tumor (GS = 6 or GS ≥ 8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis. Proteins significantly deregulated between PCa GS ≥ 8 and PCa GS = 6 represented extracellular matrix organization, gluconeogenesis, and phosphorylation pathways. Proteins deregulated between cancerous and adjacent benign tissues, reflected increased translation, peptide synthesis, and protein metabolism in the former, which is consistent with the literature. In conclusion, the results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material.

Proteome-based classification of Nonmuscle Invasive Bladder Cancer.

DNA/RNA-based classification of bladder cancer (BC) supports the existence of multiple molecular subtypes, while investigations at the protein level are scarce. Here, we aimed to investigate if Nonmuscle Invasive Bladder Cancer (NMIBC) can be stratified to biologically meaningful groups based on the proteome. Tissue specimens from 117 patients at primary diagnosis (98 with NMIBC and 19 with MIBC), were processed for high-resolution proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteomics output was subjected to unsupervised consensus clustering, principal component analysis (PCA) and investigation of subtype-specific features, pathways, and gene sets. NMIBC patients were optimally stratified to three NMIBC proteomic subtypes (NPS), differing in size, clinicopathologic and molecular backgrounds: NPS1 (mostly high stage/grade/risk samples) was the smallest in size (17/98) and overexpressed proteins reflective of an immune/inflammatory phenotype, involved in cell proliferation, unfolded protein response and DNA damage response, whereas NPS2 (mixed stage/grade/risk composition) presented with an infiltrated/mesenchymal profile. NPS3 was rich in luminal/differentiation markers, in line with its pathological composition (mostly low stage/grade/risk samples). PCA revealed a close proximity of NPS1 and conversely, remoteness of NPS3 to the proteome of MIBC. Proteins distinguishing these two extreme subtypes were also found to consistently differ at the mRNA levels between high and low-risk subtypes of the UROMOL and LUND cohorts. Collectively, our study identifies three proteomic NMIBC subtypes and following a cross-omics validation in two independent cohorts, shortlists molecular features meriting further investigation for their biomarker or potentially therapeutic value.

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease.

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.

Proteomics in cardiovascular disease: recent progress and clinical implication and implementation.

INTRODUCTION: Although multiple efforts have been initiated to shed light into the molecular mechanisms underlying cardiovascular disease, it still remains one of the major causes of death worldwide. Proteomic approaches are unequivocally powerful tools that may provide deeper understanding into the molecular mechanisms associated with cardiovascular disease and improve its management. Areas covered: Cardiovascular proteomics is an emerging field and significant progress has been made during the past few years with the aim of defining novel candidate biomarkers and obtaining insight into molecular pathophysiology. To summarize the recent progress in the field, a literature search was conducted in PubMed and Web of Science. As a result, 704 studies from PubMed and 320 studies from Web of Science were retrieved. Findings from original research articles using proteomics technologies for the discovery of biomarkers for cardiovascular disease in human are summarized in this review. Expert commentary: Proteins associated with cardiovascular disease represent pathways in inflammation, wound healing and coagulation, proteolysis and extracellular matrix organization, handling of cholesterol and LDL. Future research in the field should target to increase proteome coverage as well as integrate proteomics with other omics data to facilitate both drug development as well as clinical implementation of findings.

Effect of Heme Oxygenase-1 Deficiency on Glomerular Proteomics.

BACKGROUND: The cytoprotective effect of heme oxygenase (HO)-1 in various forms of renal glomerular injury is established. However, little is known on the role of HO-1 in preserving glomerular structural/functional integrity in the absence of injury. The present study addressed this question in HO-1-deficient rats. METHODS: HO-1-deficient rats were generated using zinc finger nuclease-mediated HO-1 gene (Hmox1) disruption and studied. Glomeruli were isolated from HO-1-deficient (Hmox1-/-) rats and their wild type (WT) littermates for proteomic analysis. RESULTS: Glomerular lesions were characterized and differentially expressed proteins important for preserving integrity of the glomerular filtration barrier were identified. HO-1-deficient (Hmox1-/-) rats developed albuminuria with decreased glomerular filtration rate. In albuminuric rats, there were lesions resembling focal and segmental glomerulosclerosis (FSGS). Western blot analysis of the integral slit diaphragm proteins, nephrin and podocin revealed a significant decrease in nephrin, with no change in podocin. Proteomic analysis of glomerular protein lysates from Hmox1-/- and WT rats revealed differential expression of proteins previously linked with FSGS pathogenesis. Specifically, α-actinin-4, actin related protein 3, cytokeratins and novel candidates including transgelin-2 and lamins. Bioinformatic analysis predicted the upregulation of pathways implicated in platelet aggregation and fibrin clot formation. CONCLUSION: HO-1 is a putative regulator of proteins important in preserving glomerular structural stability and integrity, and in minimizing the activity of proinflammatory pathways.

Investigation of the human-gut-kidney axis by fecal proteomics, highlights molecular mechanisms affected in CKD.

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.

Pilot proteomic study of locally advanced rectal cancer before and after neoadjuvant chemoradiotherapy indicates high metabolic activity in non-responders' tumor tissue.

PURPOSE: In the search for candidate predictive biomarkers to evaluate response to neoadjuvant chemoradiotherapy (nCRT) in rectal cancer, only a few studies report proteomic profiles of tumor tissue before and after nCRT. The aim of our study was to determine differentially expressed proteins between responders and non-responders before and after the therapy in order to identify candidate molecules for prediction and follow-up of response to nCRT. EXPERIMENTAL DESIGN: The study has included tissue sections of rectal tumor and non-tumor mucosa from five responders and five non-responders taken before and after nCRT from patients with locally advanced rectal cancer. Extracted proteins were analyzed by LC-MS/MS analysis followed by a set of bioinformatics analyses. RESULT: Proteomics analysis provided a mean of approximately 1050 protein identifications per sample. A comparison of proteomic profiles between responders and non-responders has identified 18 differentially expressed proteins. Pathway analysis demonstrated high metabolic activity in non-responders' tumors before nCRT, indicating the presence of intrinsic chemoradioresistance in these subjects. Two proteins associated with poor prognosis in colorectal cancer, ADAM10 and CAD, were identified as candidate predictive biomarkers as they were present in non-responders only. CONCLUSIONS AND CLINICAL RELEVANCE: Shortlisted proteins from our study should be further validated as candidate biomarkers for response to routinely applied nCRT protocols.

Ferroptosis-protective membrane domains in quiescence.

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.

Plasma Proteomics in Healthy Subjects with Differences in Tissue Glucocorticoid Sensitivity Identifies A Novel Proteomic Signature.

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.

Proteomic Analysis of Prostate Cancer FFPE Samples Reveals Markers of Disease Progression and Aggressiveness.

Prostate cancer (PCa) is the second most common cancer in men. Diagnosis and risk assessment are widely based on serum Prostate Specific Antigen (PSA) and biopsy, which might not represent the exact degree of PCa risk. Towards the discovery of biomarkers for better patient stratification, we performed proteomic analysis of Formalin Fixed Paraffin Embedded (FFPE) prostate tissue specimens using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Comparative analysis of 86 PCa samples among grade groups 1-5 identified 301 significantly altered proteins. Additional analysis based on biochemical recurrence (BCR; BCR+ n = 14, BCR- n = 51) revealed 197 significantly altered proteins that indicate disease persistence. Filtering the overlapping proteins of these analyses, seven proteins (NPM1, UQCRH, HSPA9, MRPL3, VCAN, SERBP1, HSPE1) had increased expression in advanced grades and in BCR+/BCR- and may play a critical role in PCa aggressiveness. Notably, all seven proteins were significantly associated with progression in Prostate Cancer Transcriptome Atles (PCTA) and NPM1NPM1, UQCRH, and VCAN were further validated in The Cancer Genome Atlas (TCGA), where they were upregulated in BCR+/BCR-. UQCRH levels were also associated with poorer 5-year survival. Our study provides valuable insights into the key regulators of PCa progression and aggressiveness. The seven selected proteins could be used for the development of risk assessment tools.

Urine peptidome analysis in cardiorenal syndrome reflects molecular processes.

The cardiorenal syndrome (CRS) is defined as the confluence of heart-kidney dysfunction. This study investigates the molecular differences at the level of the urinary peptidome between CRS patients and controls and their association to disease pathophysiology. The urinary peptidome of CRS patients (n = 353) was matched for age and sex with controls (n = 356) at a 1:1 ratio. Changes in the CRS peptidome versus controls were identified after applying the Mann-Whitney test, followed by correction for multiple testing. Proteasix tool was applied to investigate predicted proteases involved in CRS-associated peptide generation. Overall, 559 differentially excreted urinary peptides were associated with CRS patients. Of these, 193 peptides were specifically found in CRS when comparing with heart failure and chronic kidney disease urinary peptide profiles. Proteasix predicted 18 proteases involved in > 1% of proteolytic cleavage events including multiple forms of MMPs, proprotein convertases, cathepsins and kallikrein 4. Forty-four percent of the cleavage events were produced by 3 proteases including MMP13, MMP9 and MMP2. Pathway enrichment analysis supported that ECM-related pathways, fibrosis and inflammation were represented. Collectively, our study describes the changes in urinary peptides of CRS patients and potential proteases involved in their generation, laying the basis for further validation.

A Novel Pipeline for Drug Repurposing for Bladder Cancer Based on Patients' Omics Signatures.

Multi-omics signatures of patients with bladder cancer (BC) can guide the identification of known de-risked therapeutic compounds through drug repurposing, an approach not extensively explored yet. In this study, we target drug repurposing in the context of BC, driven by tissue omics signatures. To identify compounds that can reverse aggressive high-risk Non-Muscle Invasive BC (NMIBC) to less aggressive low-risk molecular subtypes, the next generation Connectivity Map (CMap) was employed using as input previously published proteomics and transcriptomics respective signatures. Among the identified compounds, the ATP-competitive inhibitor of mTOR, WYE-354, showed a consistently very high score for reversing the aggressive BC molecular signatures. WYE-354 impact was assessed in a panel of eight multi-origin BC cell lines and included impaired colony growth and proliferation rate without any impact on apoptosis. Overall, with this study we introduce a promising pipeline for the repurposing of drugs for BC treatment, based on patients' omics signatures.

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma.

Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5-0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies.

High resolution analysis of the intracellular proteome of cervical cancer cell lines unveils novel regulators of cervical carcinogenesis.

Cervical cancer remains the fourth most common and most lethal type of cancer in women, despite the applied regular screening and prevention strategies, while the available treatment schemes still pose a threat to fertility. Substantial understanding of the underlying mechanisms and development of novel diagnostic, prognostic and therapeutic approaches are critical steps for improving cervical cancer management. Towards this goal, a comparative proteomic analysis was conducted between three cervical cancer cell lines (HeLa: HPV18+, SiHa: HPV16+, C33A: HPV­) and normal cervical keratinocytes (HCK1T). The total cell extract of each cell line was analyzed by liquid chromatography coupled to tandem mass spectrometry (LC­MS/MS). Differential expression analysis revealed 919, 826 and 1,370 deregulated proteins in the comparisons of HeLa, SiHa and C33A with HCK1T cell lines, respectively. Pathway enrichment analysis of the differentially expressed proteins highlighted common cancer characteristics such as high metabolic demands and increased cell turnover, confirming the validity of the proteomic results. Extensive literature mining of the consistently differentially expressed proteins that resulted from the three comparisons was performed leading to a shortlist of 21 proteins that are potentially involved in cervical malignancy. The criteria for this shortlisting were the association of the proteins with various types of cancer, while there is no study as yet associating their expression to cervical cancer. Moreover, the expression trend of two of the shortlisted proteins was validated using western blot analysis. The proteomic datasets generated in this study can be utilized to enrich the current knowledge on cervical cancer pathology and unveil key molecular mechanisms of carcinogenesis. In conclusion, the shortlist of consistently deregulated proteins between cervical cancer cell lines and normal cervical keratinocytes can be used for validation in clinical samples and in functional investigation experiments that could ultimately lead to the discovery of novel disease biomarkers and drug targets.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease.

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

Diagnostic and Prognostic Performance of Secreted Protein Acidic and Rich in Cysteine (SPARC) Assay for Detecting Primary and Recurrent Urinary Bladder Cancer.

PURPOSE: To evaluate the diagnostic and prognostic performance of Secreted Protein Acidic and Rich in Cysteine (SPARC) in detecting urinary bladder cancer (UBC). METHODS: The Integrated Study on Bladder Cancer (n = 571; mean age:69.4 ± 12.2 years) evaluates cross-sectionally SPARC diagnostic performance in primary (n = 264) and recurrent (n = 307) UBC. SPARC prognostic performance is evaluated in a nested cohort (n = 250) prospectively followed for 80 months to detect tumor relapse, recurrence and/or progression. Baseline urine samples are analyzed blindly using a commercially available SPARC ELISA assay, characterized for its analytical performance according to clinical test regulatory requirements (R&D Manufactures Inc.). RESULTS: While higher mean SPARC levels are detected in primary (p = 0.008) and recurrent (p < 0.0001) UBC, the assay has limited diagnostic performance (AUC:0.593; 95% CI:0.524-0.663). SPARC positive patients undergoing disease monitoring are more likely to develop tumor relapse (age and gender Adj. HR:1.52; 95% CI:1.04-2.22) and progression (Adj. HR:1.83; 95% CI:1.02-3.27). However, prognostic performance is affected by hematuria. CONCLUSIONS: SPARC diagnostic performance for detecting UBC appears insufficient for clinical implementation. In patients undergoing disease monitoring, SPARC is a promising prognostic marker for tumor relapse and/or progression, but is affected by hematuria.

Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment.

Although cardiovascular disease (CVD) is the leading cause of morbimortality worldwide, promising new drug candidates are lacking. We compared the arterial high-resolution proteome of patients with advanced versus early-stage CVD to predict, from a library of small bioactive molecules, drug candidates able to reverse this disease signature. Of the approximately 4000 identified proteins, 100 proteins were upregulated and 52 were downregulated in advanced-stage CVD. Arachidonyl trifluoromethyl ketone (AACOCF3), a cytosolic phospholipase A2 (cPLA2) inhibitor was predicted as the top drug able to reverse the advanced-stage CVD signature. Vascular cPLA2 expression was increased in patients with advanced-stage CVD. Treatment with AACOCF3 significantly reduced vascular calcification in a cholecalciferol-overload mouse model and inhibited osteoinductive signaling in vivo and in vitro in human aortic smooth muscle cells. In conclusion, using a systems biology approach, we have identified a potentially new compound that prevented typical vascular calcification in CVD in vivo. Apart from the clear effect of this approach in CVD, such strategy should also be able to generate novel drug candidates in other complex diseases.

Novel structural approaches concerning HPV proteins: Insight into targeted therapies for cervical cancer (Review).

Cervical cancer incidence is tightly linked to HPV infection, and particularly virus types 16 and 18 cause the majority of cases presenting with pre-cancerous stages of cervical intraepithelial neoplasia (CIN). Structural and functional information concerning HPV proteins can offer novel insight into the mechanism(s) of cancer progression in the cervical epithelium. Recently, novel structural determinants of the interactions of viral proteins with their targets in keratinocytes have been elucidated. These exciting findings open the way for the development of targeted anti-oncogenic therapies, and may eventually allow the introduction of novel approaches for a rational cervical cancer treatment.