Does the Urinary Calcium/Citrate Ratio Add to the Diagnostic Workup of Children at Risk of Kidney Stones? A Cross-Sectional Study

The purpose of the study was to evaluate urinary citrate/creatinine (U Ci /U Cr ) and urinary calcium/citrate (U Ca /U Ci ) ratios for distinguishing stone formers (SF) from non-stone formers (NSF) in an at-risk population. This was a retrospective study that included all pediatric patients who underwent urinary citrate testing from April 2017 to March 2018. The urinary levels of citrate, calcium, sodium, potassium, creatinine, oxalate, urate, pH, and specific gravity (SG) were measured in our clinical laboratory. Diagnosis of kidney stones was obtained through chart review. A total of 97 patients were included (46 NSF and 51 SF). The U Ci /U Cr ratio was not significantly different between NSF and SF. Median U Ca /U Cr ratio was higher in SF (0.67) compared with NSF (0.21, p \u3c 0.0001). The median ratio of U Ca /U Ci was also higher in SF (1.30) than in NSF (0.65, p = 0.001). Oxalate, urate, pH, SG, and urinary sodium/potassium ratio did not differentiate between the SF and NSF. Positive correlation was seen between U Ca /U Cr and urinary sodium/creatinine U Na /U Cr (p \u3c 0.0001), as well as between U Ca /U Cr and U Ci /U Cr (p \u3c 0.0001). The study has demonstrated significantly higher U Ca /U Ci and U Ca /U Cr in SF compared with NSF, while the use of urinary oxalate, urate, pH, and SG did not differentiate between SF from NSF. We also confirmed a positive correlation between U Na /U Cr and U Ca /U Cr . While the utility of U Ca /U Cr is well established, our data suggest that U Ca /U Ci rather than U Ci /U Cr may be more predictive in the clinical setting when evaluating for nephrolithiasis

Large-scale screening of preferred interactions of human src homology-3 (SH3) domains using native target proteins as affinity ligands

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3-ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern

BACKGROUND. The role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients’ plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811–825, S-881–895, and N-156–170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSION. Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats. FUNDING. Ontario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/ Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit a-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein- protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions

Assay precision and risk of misclassification at rule-out cut-offs for high-sensitivity cardiac troponin

Clinical trials and guidelines support the use of very low high-sensitivity cardiac troponin (hs-cTn) results to rule-out a myocardial infarction (MI) ( 1) ). The International Federation of Clinical Chemistry and Laboratory Medicine Committee on Clinical Applications of Cardiac Biomarkers committee, through a modeling approach, suggests assays need to have a lower limit near 3 ng/L and an analytical variation of 10% below 7 ng/L if these low values are to perform consistently in practice ( 2) ). Our objectives for the present study were to assess: i) if any type of instrument or individual instrument could achieve a coefficient of variation (CV) of ≤10% at very low hs-cTn cut-offs (i.e., targets) recommended in clinical pathways; ii) the frequency of results at the hs-cTn target, above the target and below the target, with the latter group representing potential misclassification to the low risk group where the target level would in the intermediate risk range.<br/

In focus: perplexing increase of urinary stone disease in children, adolescent and young adult women and its economic impact

Background Urinary stone disease (USD) historically has affected older men, but studies suggest recent increases in women, leading to a near identical sex incidence ratio. USD incidence has doubled every 10 years, with disproportionate increases amongst children, adolescent, and young adult (AYA) women. USD stone composition in women is frequently apatite (calcium phosphate), which forms in a higher urine pH, low urinary citrate, and an abundance of urinary uric acid, while men produce more calcium oxalate stones. The reasons for this epidemiological trend are unknown. Methods This perspective presents the extent of USD with data from a Canadian Province and a North American institution, explanations for these findings and offers potential solutions to decrease this trend. We describe the economic impact of USD. Findings There was a significant increase of 46% in overall surgical interventions for USD in Ontario. The incidence rose from 47.0/100,000 in 2002 to 68.7/100,000 population in 2016. In a single United States institution, the overall USD annual unique patient count rose from 10,612 to 17,706 from 2015 to 2019, and the proportion of women with USD was much higher than expected. In the 10–17-year-old patients, 50.1% were girls; with 57.5% in the 18–34 age group and 53.6% in the 35–44 age group. The roles of obesity, diet, hormones, environmental factors, infections, and antibiotics, as well as the economic impact, are discussed. Interpretation We confirm the significant increase in USD among women. We offer potential explanations for this sex disparity, including microbiological and pathophysiological aspects. We also outline innovative solutions – that may require steps beyond typical preventive and treatment recommendations

How should we assess renal function in neonates and infants?

AIM: Review of current knowledge on assessing renal function in term and preterm neonates. METHODS: Literature review and analysis of own data. RESULTS: Prematurity, genetic, environmental and maternal factors may alter peak nephron endowment and life-long renal function. Nephrogenesis continues until 34-36 weeks of gestation, but it is altered with premature delivery. Variability of nephron endowment has a substantial impact on the clearance of renally excreted drugs. Postnatally, glomerular function rate (GFR) increases daily, doubles by two weeks, and slowly reaches full maturity at 18 months of age. Ideally, renal function biomarkers should be expressed as age-independent z-scores, and evidence suggests indexing these values to post-conceptual age rather than chronological age. Newborn and maternal serum creatinine correlate tightly for more than 72 hours after delivery, rendering this biomarker unsuitable for the assessment of neonatal renal function. Cystatin C does not cross the placenta and may be the preferred biomarker in the neonate. Here, we provide preliminary data on the natural evolution of the cystatin C eGFR in infancy. CONCLUSION: Cystatin C may be superior for GFR estimation in neonates, but the best approach to drug dosing of renally excreted drugs remains to be established