A transgenic strain of the model nematode Caenorhabditis elegans in which bioluminescence reports on relative, whole-organism ATP levels was used to test an environmentally-relevant mixture of pollutants extracted from processed sewage sludge. Changes in bioluminescence, following exposure to sewage sludge extract, were used to assess relative ATP levels and overall metabolic health. Reproductive function and longevity were also monitored. A short (up to 8 h) sublethal exposure of L4 larval stage worms to sewage sludge extract had a concentration-dependent, detrimental effect on energy status, with bioluminescence decreasing to 50–60% of the solvent control (1% DMSO). Following longer exposure (22–24 h), the energy status of the nematodes showed recovery as assessed by bioluminescence. Continuous exposure to sewage sludge extract from the L4 stage resulted in a shorter median lifespan relative to that of solvent or medium control animals, but only in the presence of 400–600 µM 5-fluoro-2′-deoxyuridine (FUdR), which was incorporated to inhibit reproduction. This indicated that FUdR increased lifespan, and that the effect was counteracted by SSE. Exposure to sewage sludge extract from the L1 stage led to slower growth and a delayed onset of egg laying. When L1 exposed nematodes reached the reproductive stage, no effect on egg laying rate or egg number in the uterus was observed. DMSO itself (1%) had a significant inhibitory effect on growth and development of C. elegans exposed from the L1 stage and on reproduction when exposed from the L4 stage. Results demonstrate subtle adverse effects on C. elegans of a complex mixture of environmental pollutants that are present, individually, in very low concentrations and indicate that our biosensor of energy status is a novel, sensitive, rapid, quantitative, whole-organism test system which is suitable for high throughput risk assessment of complex pollutant mixtures.

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Clinical success of in utero transplantation (IUT) using allogeneic hematopoietic stem cells (HSCs) has been limited to fetuses that lack an immune response to allogeneic cells due to severe immunological defects, and where transplanted genetically normal cells have a proliferative or survival advantage. Amniotic fluid (AF) is an autologous source of stem cells with hematopoietic potential that could be used to treat congenital blood disorders. We compared the ability of congenic and allogeneic mouse AF stem cells (AFSC) to engraft the hematopoietic system of time-mated C57BL/6J mice (E13.5). At 4 and 16 weeks of age, multilineage donor engraftment was higher in congenic versus allogeneic animals. In vitro mixed lymphocyte reaction confirmed an immune response in the allogeneic group with higher CD4 and CD8 cell counts and increased proliferation of stimulated lymphocytes. IUT with congenic cells resulted in 100% of donor animals having chimerism of around 8% and successful hematopoietic long-term engraftment in immune-competent mice when compared with IUT with allogeneic cells. AFSCs may be useful for autologous cell/gene therapy approaches in fetuses diagnosed with congenital hematopoietic disorders.

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.Chronic kidney disease (CKD), defined as reduced kidney function and/or evidence of kidney damage, is a major public health problem throughout the world. Major health problems around the globe, with consistent prevalence rates of 10-13% have been reported (depending on reference group and stage) 1,2 . Disease management is characterized by excessive financial costs (with expenses associated with CKD treatment, exceeding 100 B € in total, annually in Europe) 2,3 . Common risk factors for CKD include ageing of the population and increased rates of diabetes and hypertension 2,4 . Of note, patients with CKD have an overall 30-fold increased risk for suffering from Cardio Vascular Disease (CVD) complications, this being the main cause of CKD-associated deaths. Specifically, ~45% of patients with CKD stage 4-5 die from CVD 5 and risk of CVD increases with CKD severity, which is already significantly higher in early CKD compared to non-CKD 6,7 . Early identification of CKD and addressing modifiable risk factors is recommended, as it can reduce the risk of kidney failure and CVD by up to 50% 8 . Early detection or prediction of complications enable early intervention, thus could increase the chances for higher treatment efficacy 9-11 . CKD diagnosis is currently based on the detection of reduced estimated glomerular filtration rate (eGFR) and/or albuminuria, as indicators of renal dysfunction 12,13 . However, these markers have substantial suitable number of k-nearest neighbors was conducted iteratively as ...

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.

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Chronic kidney disease (CKD) is characterized by the loss of kidney function. The molecular mechanisms underlying the development and progression of CKD are still not fully understood. Among others, the urinary peptidome has been extensively studied, with several urinary peptides effectively detecting disease progression. However, their link to proteolytic events has not been made yet. This study aimed to predict the proteases involved in the generation of CKD-associated urinary excreted peptides in a well-matched (for age, sex, lack of heart disease) case-control study. The urinary peptide profiles from CKD (n = 241) and controls (n = 240) were compared and statistically analyzed. The in-silico analysis of the involved proteases was performed using Proteasix and proteases activity was predicted based on the abundance changes of the associated peptides. Predictions were cross-correlated to transcriptomics datasets by using the Nephroseq database. Information on the respective protease inhibitors was also retrieved from the MEROPS database. Totally, 303 urinary peptides were significantly associated with CKD. Among the most frequently observed were fragments of collagen types I, II and III, uromodulin, albumin and beta-2-microglobulin. Proteasix predicted 16 proteases involved in their generation. Through investigating CKD-associated transcriptomics datasets, several proteases are highlighted including members of matrix metalloproteinases (MMP7, MMP14) and serine proteases (PCSK5); laying the foundation for further studies towards elucidating their role in CKD pathophysiology.

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