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DystrophicThus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed.This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacentThis report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

Micro ribonucleic acids (miRNAs) are small non-coding RNA molecules that control gene expression by translational inhibition. They have been identified to play a role in a multitude of physiological and pathophysiological cellular processes amongst others in the heart. Due to their ability to be released into the blood as well as their stability in body fluids, they appear suitable as biomarkers. This review discusses the role of selected miRNA that currently emerge as biomarkers for coronary artery disease, their potential to discriminate between different diseases, as well as how they might be used as predictive tools for cardiac events or disease outcome. Furthermore, we propose procedural steps of miRNA analysis, to allow better comparison between studies in the future.

New insights into molecular genetics and pathomechanisms in epidermolysis bullosa (EB), methodological and technological advances in molecular biology as well as designated funding initiatives and facilitated approval procedures for orphan drugs have boosted translational research perspectives for this devastating disease. This is echoed by the increasing number of clinical trials assessing innovative molecular therapies in the field of EB. Despite remarkable progress, gene-corrective modalities, aimed at sustained or permanent restoration of functional protein expression, still await broad clinical availability. This also reflects the methodological and technological shortcomings of current strategies, including the translatability of certain methodologies beyond preclinical models as well as the safe, specific, efficient, feasible, sustained and cost-effective delivery of therapeutic/corrective information to target cells. This review gives an updated overview on status, prospects, challenges and limitations of current gene-targeted therapies.

Background: Highly viscous semen reduces sperm motility significantly and can contribute to infertility. When processing semen samples, few techniques exist to induce liquefaction in case of seminal hyperviscosity such as different washing steps and mechanical treatment. The use of alpha-chymotrypsin seems controversial due to possible negative effects on fertilisation rates after in vitro fertilization (IVF). The main objective of this study was to examine the influence of mild alpha-chymotrypsin treatment of semen samples on the fertilisation rate after artificial reproductive treatment (ART).Methods: The fertilization rate of 52 ART cycles was examined following IVF using a low dose of alpha-chymotrypsin to induce liquefaction of highly viscous semen and was compared to a control group of 88 ART cycles.Results: There was no significant difference in the fertilization rates of alpha-chymotrypsin treated semen samples compared to the control group; pregnancy rates were unaffected.Conclusions: The use of mild alpha-chymotrypsin treatment of semen samples in case of hyperviscosity does not appear to impact negatively on the fertilization rates after ART and could be regarded as an additional method to induce liquefaction of highly viscous semen samples in IVF.

Aims Exercise is a trigger for acute coronary events especially in the untrained. Identifying subjects at risk remains a challenge. We set out to assess whether a distinct pattern of micro ribonucleic acids (miRNAs) expressed in response to an acute bout of all-out exercise might exist that would allow discrimination between health and disease. Methods Twenty healthy subjects and 20 patients with coronary artery disease (CAD) performed an all-out cycle ergometry. Total RNA was extracted from blood drawn before and after exercise. Each blood sample was analysed for 187 target miRNAs by quantitative reverse transcription polymerase chain reaction. Results At baseline, 18 miRNAs allowed discrimination between healthy subjects and CAD patients. In response to an acute all-out exercise in healthy subjects 51 miRNAs and in CAD patients 60 miRNAs were significantly modulated (all p Conclusion In this most comprehensive analysis of exercise effects on circulating miRNAs to date we demonstrate for the first time that a distinct combination of miRNAs together with variables of exercise capacity allow robust discrimination between healthy subjects and CAD patients. We postulate that miRNAs may eventually serve as biomarkers to identify patients with CAD and possibly even those at risk of exercise-induced cardiac events.

Summary This short review article summarizes what is known clinically and biochemically about the seven human NADPH oxidases. Emphasis is put on the connection between mutations in the catalytic and regulatory subunits of Nox2, the phagocyte defense enzyme, with syndromes like chronic granulomatous disease, as well as a number of chronic inflammatory diseases. These arise paradoxically from a lack of reactive oxygen species production needed as second messengers for immune regulation. Both Nox2 and the six other human NADPH oxidases display signaling functions in addition to the functions of these enzymes in specialized biochemical reactions, for instance, synthesis of the hormone thyroxine. NADPH oxidases are also needed by Saccharomyces cerevisiae cells for the regulation of the actin cytoskeleton in times of stress or developmental changes, such as pseudohyphae formation. The article shows that in certain cancer cells Nox4 is also involved in the re-structuring of the actin cytoskeleton, which is required for cell mobility and therefore for metastasis.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Mitochondria are responsible for a series of metabolic functions. Superoxide leakage from the respiratory chain and the resulting cascade of reactive oxygen species-induced damage, as well as mitochondrial metabolism in programmed cell death, have been intensively studied during ageing in single-cellular and higher organisms. Changes in mitochondrial physiology and metabolism resulting in ROS are thus considered to be hallmarks of ageing. In this review, we address 'other' metabolic activities of mitochondria, carbon metabolism (the TCA cycle and related underground metabolism), the synthesis of Fe/S clusters and the metabolic consequences of mitophagy. These important mitochondrial activities are hitherto less well-studied in the context of cellular and organismic ageing. In budding yeast, they strongly influence replicative, chronological and hibernating lifespan, connecting the diverse ageing phenotypes studied in this single-cellular model organism. Moreover, there is evidence that similar processes equally contribute to ageing of higher organisms as well. In this scenario, increasing loss of metabolic integrity would be one driving force that contributes to the ageing process. Understanding mitochondrial metabolism may thus be required for achieving a unifying theory of eukaryotic ageing.