Urinary CE-MS peptide marker pattern for detection of solid tumors

Urinary profiling datasets, previously acquired by capillary electrophoresis coupled to mass-spectrometry were investigated to identify a general urinary marker pattern for detection of solid tumors by targeting common systemic events associated with tumor-related inflammation. A total of 2,055 urinary profiles were analyzed, derived from a) a cancer group of patients (n = 969) with bladder, prostate, and pancreatic cancers, renal cell carcinoma, and cholangiocarcinoma and b) a control group of patients with benign diseases (n = 556), inflammatory diseases (n = 199) and healthy individuals (n = 331). Statistical analysis was conducted in a discovery set of 676 cancer cases and 744 controls. 193 peptides differing at statistically significant levels between cases and controls were selected and combined to a multi-dimensional marker pattern using support vector machine algorithms. Independent validation in a set of 635 patients (293 cancer cases and 342 controls) showed an AUC of 0.82. Inclusion of age as independent variable, significantly increased the AUC value to 0.85. Among the identified peptides were mucins, fibrinogen and collagen fragments. Further studies are planned to assess the pattern value to monitor patients for tumor recurrence. In this proof-of-concept study, a general tumor marker pattern was developed to detect cancer based on shared biomarkers, likely indicative of cancer-related features

Polymerization-incompetent uromodulin in the pregnant stroke-prone spontaneously hypertensive rat

The kidney is centrally involved in blood pressure regulation and undergoes extensive changes during pregnancy. Hypertension during pregnancy may result in an altered urinary peptidome that could be used to indicate new targets of therapeutic or diagnostic interest. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of maternal chronic hypertension. Capillary electrophoresis-mass spectrometry was conducted to interrogate the urinary peptidome in SHRSP and the control Wistar–Kyoto strain at three time points: prepregnancy and gestational days 12 and 18. The comparison within and between the Wistar–Kyoto and SHRSP peptidome at all time points detected 123 differentially expressed peptides (fold change >1.5; P<0.05). Sequencing of these peptides identified fragments of collagen α-chains, albumin, prothrombin, actin, serpin A3K, proepidermal growth factor, and uromodulin. Uromodulin peptides showed a pregnancy-specific alteration in SHRSP with a 7.8-fold (P<0.01) and 8.8-fold (P<0.05) increase at gestational days 12 and 18, respectively, relative to the Wistar–Kyoto. Further investigation revealed that these peptides belonged to the polymerization-inhibitory region of uromodulin. Two forms of uromodulin (polymerization competent and polymerization incompetent) were found in urine from both Wistar–Kyoto and SHRSP, where the polymerizationincompetent form was increased in a pregnancy-specific manner in SHRSP. Nifedipine-treated pregnant SHRSP showed only polymerization-competent uromodulin, indicating that calcium may be mechanistically involved in uromodulin polymerization. This study highlights, for the first time, a potential role of uromodulin and its polymerization in hypertensive pregnancy

Dual mTOR/PI3K inhibition limits PI3K-dependent pathways activated upon mTOR inhibition in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of kidney cysts leading to kidney failure in adulthood. Inhibition of mammalian target of rapamycin (mTOR) slows polycystic kidney disease (PKD) progression in animal models, but randomized controlled trials failed to prove efficacy of mTOR inhibitor treatment. Here, we demonstrate that treatment with mTOR inhibitors result in the removal of negative feedback loops and up-regulates pro-proliferative phosphatidylinositol 3-kinase (PI3K)-Akt and PI3K-extracellular signal-regulated kinase (ERK) signaling in rat and mouse PKD models. Dual mTOR/PI3K inhibition with NVP-BEZ235 abrogated these pro-proliferative signals and normalized kidney morphology and function by blocking proliferation and fibrosis. Our findings suggest that multi-target PI3K/mTOR inhibition may represent a potential treatment for ADPKD

Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice

Nephropathy is among the most frequent complications of diabetes and the leading cause of end-stage renal disease. Despite the success of novel drugs in animal models, the majority of the subsequent clinical trials employing those drugs targeting diabetic nephropathy failed. This lack of translational value may in part be due to an inadequate comparability of human disease and animal models that often capture only a few aspects of disease. Here we overcome this limitation by developing a multimolecular noninvasive humanized readout of diabetic nephropathy based on urinary peptidomics. The disease-modified urinary peptides of 2 type 2 diabetic nephropathy mouse models were identified and compared with previously validated urinary peptide markers of diabetic nephropathy in humans to generate a classifier composed of 21 ortholog peptides. This classifier predicted the response to disease and treatment with inhibitors of the renin-angiotensin system in mice. The humanized classifier was significantly correlated with glomerular lesions. Using a human type 2 diabetic validation cohort of 207 patients, the classifier also distinguished between patients with and without diabetic nephropathy, and their response to renin-angiotensin system inhibition. Thus, a combination of multiple molecular features common to both human and murine disease could provide a significant change in translational drug discovery research in type 2 diabetic nephropathy

Predictive performance and clinical application of COV50, a urinary proteomic biomarker in early COVID-19 infection: a prospective multicentre cohort study

Background: The SARS-CoV-2 pandemic is a worldwide challenge. The CRIT-CoV-U pilot study generated a urinary proteomic biomarker consisting of 50 peptides (COV50), which predicted death and disease progression from SARS-CoV-2. After the interim analysis presented for the German Government, here, we aimed to analyse the full dataset to consolidate the findings and propose potential clinical applications of this biomarker. Methods: CRIT-CoV-U was a prospective multicentre cohort study. In eight European countries (Austria, France, Germany, Greece, North Macedonia, Poland, Spain, and Sweden), 1012 adults with PCR-confirmed COVID-19 were followed up for death and progression along the 8-point WHO scale. Capillary electrophoresis coupled with mass spectrometry was used for urinary proteomic profiling. Statistical methods included logistic regression and receiver operating characteristic curve analysis with a comparison of the area under curve (AUC) between nested models. Hospitalisation costs were derived from the care facility corresponding with the Markov chain probability of reaching WHO scores ranging from 3 to 8 and flat-rate hospitalisation costs adjusted for the gross per capita domestic product of each country. Findings: From June 30 to Nov 19, 2020, 228 participants were recruited, and from April 30, 2020, to April 14, 2021, 784 participants were recruited, resulting in a total of 1012 participants. The entry WHO scores were 1-3 in 445 (44%) participants, 4-5 in 529 (52%) participants, and 6 in 38 (4%) participants; and of all participants, 119 died and 271 had disease progression. The odds ratio (OR) associated with COV50 in all 1012 participants for death was 2·44 (95% CI 2·05-2·92) unadjusted and 1·67 (1·34-2·07) when adjusted for sex, age, BMI, comorbidities, and baseline WHO score; and for disease progression, the OR was 1·79 (1·60-2·01) when unadjusted and 1·63 (1·41-1·91) when adjusted (p Interpretation: The urinary proteomic COV50 marker might be predictive of adverse COVID-19 outcomes. Even in people with mild-to-moderate PCR-confirmed infections (WHO scores 1-4), the 0·04 COV50 threshold justifies earlier drug treatment, thereby potentially reducing the number of days in hospital and associated costs. Funding: German Federal Ministry of Health

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

Comparison of urine and plasma peptidome indicates selectivity in renal peptide handling

Purpose: Urine is considered to be produced predominantly as a result of plasma filtration in the kidney. However, the origin of the native peptides present in urine has never been investigated in detail. Therefore, we aimed to obtain a first insight into the origin of urinary peptides based on a side‐by‐side comprehensive analysis of the plasma and urine peptidome. Methods: Twenty‐two matched urine and plasma samples were analyzed for their peptidome using capillary electrophoresis coupled to mass spectrometry (CE‐MS; for relative quantification) and CE‐ or LC coupled to tandem mass spectrometry (CE‐ or LC‐ MS/MS; for peptide identification). The overlap and association of abundance of the different peptides present in these two body fluids were evaluated. Results: We were able to identify 561 plasma and 1461 urinary endogenous peptides. Only 90 peptides were detectable in both urine and plasma. No significant correlation was found when comparing the abundance of these common peptides, with the exception of collagen fragments. This observation was also supported when comparing published peptidome data from both plasma and urine. Conclusions and clinical relevance: Most of the plasma peptides are not detectable in urine, possibly due to tubular reabsorption. The majority of urinary peptides may in fact originate in the kidney. The notable exception is collagen fragments, which indicates potential selective exclusion of these peptides from tubular reabsorption. Experimental verification of this hypothesis is warranted

1,8-Naphthalimides 3-substituted with imine or β-ketoenamine unit evaluated as compounds for organic electronics and cell imaging

In this paper, we describe both new as well as described in our previous works 1,8- naphthalimide derivatives substituted at the 3-C position with imine or -ketoenamine unitin order to demonstrate a broader scope of research enabling of analysis between the structureproperties relationship relevant to the application of these compounds in organic electronics and cellular imaging. Thermal, physicochemical, optical, electrochemical, electroluminescence, and biological properties of a series of derivatives containing the 1,8-naphthalimideunit were tested and compared. This allowed the determination of impact of substituents in the imide part (hexylamine, phenylethyl, benzyl, fluorobenzyl, methylbenzyl), type of bond (imine or ketoenamine) as well as the substituent on the naphthalene ring (2-hydroxyphenyl, 5-bromo-2- hydroxyphenyl, 3,5-diodo-2-hydroxyphenyl, pyrimidines) on their properties. Moreover, the properties in the aggregating state were tested in the MeOH/PBS system. Imines are susceptible to the hydrolysis process and aggregation-caused photoluminescence quenching(ACQ). In turn,-ketoenamine shown excited-state intramolecular proton transfer promoted by aggregation (AIEE). Our studies can be helpful in the further design of compounds containing the 1,8- naphthalimide structure for various applications