Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes.

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS, and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1, and SOS1), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.

Expression Levels of PIWI-interacting RNA, piR-823, Are Deregulated in Tumor Tissue, Blood Serum and Urine of Patients with Renal Cell Carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3% of adult malignancies. P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of naturally occurring, short non-coding RNAs involved in silencing of transposable elements and in sequence-specific chromatin modifications. There were preliminary data published indicating that piR-823 expression is deregulated in circulating tumor cells and tumor tissue in gastric and kidney cancer, respectively. PATIENTS AND METHODS: In our study, we analyzed piR-823 levels in 588 biological specimens: tumor tissue (N=153), adjacent renal parenchyma (N=121), blood serum (N=178) and urine (N=20) of patients undergoing nephrectomy for RCC; and in blood serum (N=101) and urine (N=15) of matched healthy controls. Expression levels of piR-823 were determined in all biological specimens by quantitative real-time polymerase chain reaction, compared in patients and controls, and correlated with clinicopathological features of RCC. RESULTS: We identified a significant down-regulation of piR-823 in tumor tissue [p<0.0001, area under the curve (AUC)=0.7945]. On the contrary in blood serum and urine, the expression of piR-823 was significantly higher in patients with RCC compared to healthy individuals (p=0.0005, AUC=0.6264 and p=0.0157, AUC=0.7433, respectively). We further observed higher levels of piR-823 in tumor tissue to be associated with shorter disease-free survival of patients (p=0.0186) and a trend for higher piR-823 levels in serum to be associated with advanced clinical stages of RCC (p=0.0691). There were no other significant associations of piR-823 levels in any type of biological specimen with clinicopathological features of RCC. CONCLUSION: piR-823 is down-regulated in tumor tissue, but positively correlated with worse outcome, indicating its complex role in RCC pathogenesis. In blood serum, piR-823 is up-regulated, but with unsatisfactory analytical performance. Preliminary data indicate the promising diagnostic utility of urinary piR-823 in patients with RCC.

MiR-429 is linked to metastasis and poor prognosis in renal cell carcinoma by affecting epithelial-mesenchymal transition.

MicroRNAs (miRNAs) have been proven to be important oncogenes and tumor suppressors in wide range of cancers, including renal cell carcinoma (RCC). In our study, we evaluated miRNA-429 as potential diagnostic/prognostic biomarker in 172 clear cell RCC patients and as a potential regulator of epithelial-mesenchymal transition (EMT) in vitro. We demonstrated that miR-429 is down-regulated in tumor tissue samples (P < 0.0001) and is significantly associated with cancer metastasis (P < 0.0001), shorter disease-free (P = 0.0105), and overall survival (P = 0.0020). In addition, ectopic expression of miR-429 in 786-0 RCC cells followed by TGF-ß treatment led to increase in the levels of E-cadherin expression (P < 0.0001) and suppression of cellular migration (P < 0.0001) in comparison to TGF-ß-treated controls. Taken together, our findings suggest that miR-429 may serve as promising diagnostic and prognostic biomarker in RCC patients. We further suggest that miR-429 has a capacity to inhibit loss of E-cadherin in RCC cells undergoing EMT and consequently attenuate their motility.

Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

Urinary microRNAs as a new class of noninvasive biomarkers in oncology, nephrology, and cardiology.

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. In the last decade, number of evidences showing miRNAs contribution to the regulation of apoptosis, cellular proliferation, differentiation, and other important cellular processes is constantly growing. Specific miRNA expression signatures have been identified in variety of human cancers as well as pathologies of cardiovascular and urinary systems. Our chapter focuses on the potential of urinary miRNAs to serve as biomarkers in uro-oncology, nephrology, and cardiology. We discuss in detail recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in medicine. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in urologic cancers, nephrology, and cardiology.

Urine microRNAs as potential noninvasive biomarkers in urologic cancers.

Micro-ribonucleic acids (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The ability of miRNAs to inhibit translation of oncogenes and tumor suppressors implies that they may be involved in carcinogenesis. Our review focuses on the potential of urinary miRNAs to serve as biomarkers of urologic cancers. We discuss in detail the recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in urologic cancer. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in bladder, prostate, and kidney cancers.