What´s in the "pure" prosody?

Detectors for accents and phrase boundaries have been developed which derive prosodic features from the speech signal and its fundamental frequency to support other modules of a speech understanding system in an early analysis stage, or in cases where no word hypotheses are available.The detectors underlying Gaussian distribution classifiers were trained with 50 minutes and tested with 30 minutes of spontaneous speech, yielding recognition rates of 74% for accents and 86% for phrase boundaries. Since this material was prosodically hand labelled, the question was, which labels for phrase boundaries and accentuation were only guided by syntactic or semantic knowledge, and which ones are really prosodically marked.Therefore a small test subset has been resynthesized in such a way that comprehensibility was lost, but the prosodic characteristics were kept. This subset has been re-labelled by 11listeners with nearly the same accuracy as the detectors

Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons

Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends

Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA double-strand breaks1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or AP sites)2. At single strand breaks, 5' terminal abasic sites are excised by pol β's 5'dRP lyase activity3,4,5,6: we show here in vitro and in cells that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'dRP/AP lyase activity (Figure 1a). Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping distinguish this pathway from otherwise robust alternate NHEJ pathways. Surprisingly, the NHEJ factor Ku can be identified as an effective 5'dRP/AP lyase. Similar to other lyases7, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff base covalent intermediate with the abasic site. We demonstrate using cell extracts that Ku is essential for efficient removal of AP sites near double-strand breaks and, consistent with this result, joining of such breaks is specifically reduced in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that processed ends; our data supports an unexpected direct role for Ku in end processing steps as well

Essential role for polymerase specialization in cellular nonhomologous end joining

Nonhomologous end joining (NHEJ) is a DNA double-strand break repair pathway required for development of the adaptive immune response, maintenance of cellular proliferative capacity, and the response to several commonly used cancer treatments. A major challenge faced by this pathway is that chromosome breaks can have dirty end structures, making them difficult to repair. We show here that two mammalian DNA polymerases have an unexpectedly pivotal role in helping resolve such ends. Each is proficient in different contexts and has a differing impact on repair fidelity. This work sheds light on how NHEJ has evolved to be flexible during repair and identifies two polymerases as critical for this process

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo

T wave abnormalities, high body mass index, current smoking and high lipoprotein (a) levels predict the development of major abnormal Q/QS patterns 20 years later. A population-based study

BACKGROUND: Most studies on risk factors for development of coronary heart disease (CHD) have been based on the clinical outcome of CHD. Our aim was to identify factors that could predict the development of ECG markers of CHD, such as abnormal Q/QS patterns, ST segment depression and T wave abnormalities, in 70-year-old men, irrespective of clinical outcome. METHODS: Predictors for development of different ECG abnormalities were identified in a population-based study using stepwise logistic regression. Anthropometrical and metabolic factors, ECG abnormalities and vital signs from a health survey of men at age 50 were related to ECG abnormalities identified in the same cohort 20 years later. RESULTS: At the age of 70, 9% had developed a major abnormal Q/QS pattern, but 63% of these subjects had not been previously hospitalized due to MI, while 57% with symptomatic MI between age 50 and 70 had no major Q/QS pattern at age 70. T wave abnormalities (Odds ratio 3.11, 95% CI 1.18–8.17), high lipoprotein (a) levels, high body mass index (BMI) and smoking were identified as significant independent predictors for the development of abnormal major Q/QS patterns. T wave abnormalities and high fasting glucose levels were significant independent predictors for the development of ST segment depression without abnormal Q/QS pattern. CONCLUSION: T wave abnormalities on resting ECG should be given special attention and correlated with clinical information. Risk factors for major Q/QS patterns need not be the same as traditional risk factors for clinically recognized CHD. High lipoprotein (a) levels may be a stronger risk factor for silent myocardial infarction (MI) compared to clinically recognized MI

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures, but it is unclear how the steps employed for resolution are determined. We sought to address this question by analyzing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85% to 6%. End-processing by nucleases and polymerases is increased to compensate, though paths with the fewest number of steps to generate a substrate suitable for ligation are favored. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening