jSplice: a high-performance method for accurate prediction of alternative splicing events and its application to large-scale renal cancer transcriptome data.

MOTIVATION: Alternative splicing represents a prime mechanism of post-transcriptional gene regulation whose misregulation is associated with a broad range of human diseases. Despite the vast availability of transcriptome data from different cell types and diseases, bioinformatics-based surveys of alternative splicing patterns remain a major challenge due to limited availability of analytical tools that combine high accuracy and rapidity. RESULTS: We describe here a novel junction-centric method, jSplice, that enables de novo extraction of alternative splicing events from RNA-sequencing data with high accuracy, reliability and speed. Application to clear cell renal carcinoma (ccRCC) cell lines and 65 ccRCC patients revealed experimentally validatable alternative splicing changes and signatures able to prognosticate ccRCC outcome. In the aggregate, our results propose jSplice as a key analytic tool for the derivation of cell context-dependent alternative splicing patterns from large-scale RNA-sequencing datasets. AVAILABILITY AND IMPLEMENTATION: jSplice is a standalone Python application freely available at http://www.mhs.biol.ethz.ch/research/krek/jsplice CONTACT: wilhelm.krek@biol.ethz.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An actin-regulated importin α/ß-dependent extended bipartite NLS directs nuclear import of MRTF-A.

Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)α/ß-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Impα-Impß heterodimer requires both NLS basic elements, and is dependent on the Impα major and minor binding pockets. Binding of the Impα-Impß heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.

Validation of Becton Dickinson Barricor™ tubes for use with Abbott Alinity™ and Siemens Atellica® highly sensitive cardiac troponin I measuring systems.

BACKGROUND: BD Barricor™ tubes have been proposed to decrease laboratory turnaround time (TAT). We analytically validated and then clinically verified these tubes for use with Abbott Alinity™ and Siemens Atellica® highly sensitive cardiac troponin I (hs-cTnI) assays. METHODS: hs-cTnI measurements were undertaken in paired Barricor™ and in-use PSTII™ tubes on both systems. 359 matched samples with hs-cTnI levels between 3 and 15,000 ng/L (Atellica® values) were used to assess the hemolysis rate and make method comparisons. 599 paired patient samples were collected on emergency department (ED) admission to compare the performance of the rapid acute myocardial infarction (AMI) rule-out strategy based on hs-cTnI concentrations lower than recommended thresholds (<4 ng/L Alinity™; <5 ng/L Atellica®) when different tubes and systems were employed. RESULTS: No between-tube differences in hemolysis rate were seen when free hemoglobin concentrations in plasma samples were ≥0.25 g/L, even if PSTII™ showed a significant increase of hemolysis rate vs. Barricor™ (31% vs. 22%, p=0.007) when a lower cut-off for hemolysis (≥0.11 g/L) was employed on the Atellica® detection system. The alternate use of these tubes did not influence the hs-cTnI results obtained from either of the two assays, which remained markedly biased (~40%) irrespective of the tube used. The expected optimal ability of very low hs-cTnI values on ED admission for ruling out AMI was confirmed by using both systems regardless of the tube type. CONCLUSIONS: Barricor™ and PSTII™ tubes can provide analytically equivalent hs-cTnI results when used on either Alinity™ or Atellica® hs-cTnI assays.

Loss of PBRM1 expression is associated with renal cell carcinoma progression.

Although von Hippel-Lindau (VHL) tumor suppressor gene alterations dominate the genetic landscape of clear cell renal cell carcinoma (ccRCC), recent studies have identified new ccRCC genes, including SETD2, KDM6A, KDM5C, BAP1 and PBRM1. Strikingly, all these genes fall into a category of histone/chromatin regulators. Polybromo-1 (PBRM1) is the second most frequently mutated gene after VHL; however, the clinical relevance of its loss in ccRCC has not yet been reported. Here, we analyzed the expression of PBRM1, the product encoded by PBRM1, in ccRCC cell lines and in more than 300 RCC tumor samples. The data were correlated with clinicopathological parameters and VHL mutation status. We found that a significant number of ccRCC cancer cell lines lack detectable PBRM1 expression. Loss of PBRM1 was predominant in the clear cell subtype of RCC (~ 70%) and correlated with advanced tumor stage (p < 0.0001), low differentiation grade (p = 0.0002) and worse patient outcome (p = 0.025), but not with the VHL mutation status. Our results indicate a critical role for PBRM1 in the suppression of ccRCC progression. Moreover, the results suggest that functional inactivation of PBRM1 in the context of pVHL loss-of-function may represent a key event in facilitating the development of key aspects of an aggressive tumor behavior. Given the role of PBRM1 in chromatin modification, the gene expression pathways disrupted by the inactivation of this protein may lead to new treatment strategies for ccRCC.

An assessment of heart rate and blood pressure asymmetry in the diagnosis of vasovagal syncope in females.

Introduction: Heart Rate Asymmetry (HRA) describes a phenomenon of differences between accelerations and decelerations in human heart rate. Methods used for HRA assessment can be further implemented in the evaluation of asymmetry in blood pressure variations (Blood Pressure Asymmetry-BPA). Methods: We have analyzed retrospectively the series of heartbeat intervals extracted from ECG and beat-to-beat blood pressure signals from 16 vasovagal patients (age: 32.1 ± 13.3; BMI: 21.6 ± 3.8; all female) and 19 healthy subjects (age: 34.6 ± 7.6; BMI: 22.1 ± 3.4; all female) who have undergone tilt test (70°). Asymmetry was evaluated with Poincaré plot-based methods for 5 min recordings from supine and tilt stages of the test. The analyzed biosignals were heart rate (RR), diastolic (dBP) and systolic Blood Pressure (sBP) and Pulse Pressure (PP). In the paper we explored the differences between healthy and vasovagal women. Results: The changes of HRA indicators between supine and tilt were observed only in the control group (Porta Index p = 0.026 and Guzik Index p = 0.005). No significant differences in beat-to-beat variability (i.e. spread of points across the line of identity in Poincaré plot-SD1) of dBP was noted between supine and tilt in the vasovagal group (p = 0.433 in comparison to p = 0.014 in healthy females). Moreover, in vasovagal patients the PP was significantly different (supine: 41.47; tilt: 39.27 mmHg) comparing to healthy subjects (supine: 35.87; tilt: 33.50 mmHg) in supine (p = 0.019) and in tilt (p = 0.014). Discussion: Analysis of HRA and BPA represents a promising method for the evaluation of cardiovascular response to orthostatic stressors, however currently it is difficult to determine a subject's underlying health condition based only on these parameters.

Heart Rate Asymmetry Analysis During Head-Up Tilt Test in Healthy Men.

The purpose of this study is to assess the cardiovascular system response to orthostatic stress in a group of 133 healthy men using heart rate asymmetry (HRA) methods. HRA is a feature of variability in human heart rate which is dependent upon external and internal body conditions. The initial phases of head-up tilt test (HUTT), namely, supine and tilt, were chosen as the external body affecting factors. Various calculation methods of HRA, such as Porta's index (PI), Guzik's index (GI), and its variance based components, were used to assess the heart rate variability (HRV) and its asymmetry. We compared 5-min ECG recordings from both supine and tilt phases of HUT test. Short-term HRA was observed in 54.1% of men in supine phase and 65.4% of men in tilt phase. The study revealed significant increase of GI (from 0.50 to 0.52, p < 0.001) in the tilt phase as well as significant changes in HRV descriptors between HUTT phases. Our results showed that the variability of human heart rate and its asymmetry are sensitive to orthostatic stress. The study of short-term HRA is a potential additional tool to increase sensitivity in conditions where HUTT is a diagnostic tool, such as vasovagal syncope.

Identification of Protor as a novel Rictor-binding component of mTOR complex-2.

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.

ALR and liver regeneration.

Liver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical, infectious and toxic injury. The extraordinary ability of liver to regenerate is the effect of cross-talk between growth factors, cytokines, matrix components and many other factors. In this review we present recent findings and existing information about mechanisms that regulate liver growth, paying attention to augmenter of liver regeneration.