Using an Isolated Rat Kidney Model to Identify Kidney Origin Proteins in Urine

<div><p>The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.</p></div

Evaluation of reporting quality for observational studies using routinely collected health data in pharmacovigilance

<p><b>Objectives</b>: To appraise the reporting quality of studies which concerned linezolid-related thrombocytopenia referring to REporting of studies Conducted using Observational Routinely collected health Data (RECORD) statement.</p> <p><b>Methods</b>: Medline, Embase, Cochrane library and clinicaltrial.gov were searched for observational studies concerning linezolid-related thrombocytopenia using routinely collected health data from 2000 to 2017. Two reviewers screened potential eligible articles and extracted data independently. Finally, reporting quality assessment was performed by two senior researchers using RECORD statement.</p> <p><b>Results</b>: Of 25 included studies, 11 (44.0%) mentioned the type of data in the title and/or abstract. In 38 items derived from RECORD statement, the median number of items reported in the included studies was 22 (inter-quartile range 18–27). Inadequate reporting issues were discovered in the following aspects: validation studies of the codes or algorithms, study size estimation, quantitative variables, subgroup statistical methods, missing data, follow-up/matching or sampling strategy, sensitivity analysis and cleaning methods, funding and role of funders and accessibility of protocol, raw data.</p> <p><b>Conclusion</b>: This study provides the evidence that the reporting quality of post-marketing safety evaluation studies conducted using routinely collected health data was often insufficient. Future stakeholders are encouraged to endorse the RECORD guidelines in pharmacovigilance.</p

SDS-PAGE analysis of perfusion-driven urine.

<p>(A) The proteins from the perfusion-driven urine with oxygen supplementation were resolved and compared with the proteins present in rat plasma and rat urine. Lane p1, p2, and p3 represents proteins acquired from the first, second, and third ten-minute intervals of the perfusion respectively (B) The proteins from the perfusion-driven urine without oxygen supplementation were resolved. Lane p4, p5, p6, and p7 represents proteins acquired from the first, second, third, and fourth ten-minute intervals of the perfusion respectively.</p

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by <i>in vitro</i> ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated <i>in vitro</i>, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed <i>in vivo</i>. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by <i>in vitro</i> ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated <i>in vitro</i>, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed <i>in vivo</i>. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale