Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP

The Peroxisomal Targeting Signal 3 (PTS3) of the Budding Yeast Acyl-CoA Oxidase Is a Signal Patch

The specificity of import of peroxisomal matrix proteins is dependent on the targeting signals encoded within their amino acid sequences. Two known import signals, peroxisomal targeting signal 1 (PTS1), positioned at the C-termini and PTS2 located close to N-termini of these proteins are recognized by the Pex5p and Pex7p receptors, respectively. However, in several yeast species, including Saccharomyces cerevisiae, proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined. An example of such a protein is S. cerevisiae acyl-CoA oxidase (AOx) encoded by the POX1 gene. While it is known that its import is driven by its interaction with the N-terminal segment of Pex5p, which is separate from its C-terminal PTS1-recognizing tetratricopeptide domain, to date, no AOx polypeptide region has been implicated as critical for this interaction, and thus would constitute the long-sought PTS3 signal. Using random mutagenesis combined with a two-hybrid screen, we identified single amino acid residues within the AOx polypeptide that are crucial for this interaction and for the peroxisomal import of this protein. Interestingly, while scattered throughout the primary sequence, these amino acids come close to each other within two domains of the folded AOx. Although the role of one or both of these regions as the PTS3 signal is not finally proven, our data indicate that the signal guiding AOx into peroxisomal matrix is not a linear sequence but a signal patch

Early and Long-Term Results of Unprotected Left Main Coronary Artery Stenting The LE MANS (Left Main Coronary Artery Stenting) Registry

ObjectivesThe aim of the study was to evaluate early and late outcomes after percutaneous coronary intervention (PCI) of unprotected left main coronary artery disease (ULMCA) and to compare bare-metal stent (BMS) and drug-eluting stent (DES) subgroups.BackgroundPCI is an increasingly utilized method of revascularization in patients with ULMCA.MethodsThis multicenter prospective registry included 252 patients after ULMCA stenting enrolled between March 1997 and February 2008. Non–ST-segment elevation acute coronary syndrome was diagnosed in 58% of patients; ST-segment elevation myocardial infarction cases were excluded. Drug-eluting stents were implanted in 36.2% of patients.ResultsMajor adverse cardiovascular and cerebral events (MACCE) occurred in 12 (4.8%) patients during the 30-day period, which included 4 (1.5%) deaths. After 12 months there were 17 (12.1%) angiographically confirmed cases of restenosis. During long-term follow-up (1 to 11 years, mean 3.8 years) there were 64 (25.4%) MACCE and 35 (13.9%) deaths. The 5- and 10-year survival rates were 78.1% and 68.9%, respectively. Despite differences in demographical and clinical data in favor of BMS patients, unmatched analysis showed a significantly lower MACCE rate in DES patients (25.9% vs. 14.9%, p = 0.039). This difference was strengthened after propensity score matching. The DES lowered both mortality and MACCE for distal ULMCA lesions when compared with BMS. Ejection fraction <50% was the only independent risk factor influencing long-term survival.ConclusionsStenting of ULMCA is feasible and offers good long-term outcome. Implantation of DES for ULMCA decreased the risk of long-term MACCE, and particularly improved survival in patients with distal ULMCA disease

Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington’s disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart

No evidence of enhanced oxidant production in blood obtained from patients with obstructive sleep apnea

<p>Abstract</p> <p>Background</p> <p>Obstructive sleep apnea syndrome (OSAS) is a recognized risk factor for cardiovascular morbidity and mortality, perhaps due to causative exacerbations of systemic oxidative stress. Putative oxidative stress related to numerous episodes of intermittent hypoxia, may be an oxidants chief driving force in OSAS patients.</p> <p>Methods</p> <p>We assessed the resting and n-formyl-methionyl-leucyl-phenylalanine (fMLP)- induced whole blood chemiluminescence (as a measure of oxidant production by polymorphonuclear leukocytes and monocytes), ferric reducing ability of plasma (FRAP) and H₂O₂generation in the whole blood of 27 untreated OSAS patients, 22 subjects after a night of CPAP therapy and 11 controls without OSAS. All of them were matched to age, BMI (body mass index) and smoking habits. All parameters were measured before and after polysomnography-controlled sleep, individual results were obtained as a mean from duplicated experiments.</p> <p>Results</p> <p>No significant differences were distinguished between evening and morning blood chemiluminescence, H₂O₂activity and FRAP within and between all three study groups.</p> <p>For instance patients with untreated OSAS had similar morning and evening resting whole blood chemiluminescence (2.3 +/- 2.2 vs. 2.4 +/- 2.2 [aU·10^-4phagocytes]), total light emission after stimulation with fMLP (1790 +/- 1371 vs. 1939 +/- 1532 [aU·s·10^-4phagocytes]), as well as FRAP after 3 min. plasma incubation (602 +/- 202 vs. 671 +/- 221 [uM]). Although, in the subgroup of 11 patients with severe OSAS (apnea/hypopnea index 58 +/- 18/h and oxygen desaturation index 55 +/- 19/h), the morning vs. evening resting chemiluminescence and total light emission after stimulation with fMLP observed a propensity to elevate 2.5 +/- 2.7 vs. 1.9 +/- 1.8 [aU·10^-4phagocytes] and 1778 +/- 1442 vs. 1503 +/- 1391 [aU·s·10^-4phagocytes], respectively, these did not attain statistical significance (p > 0.05).</p> <p>Conclusion</p> <p>Our investigation exposed no evidence in the overproduction of oxidants via circulating phagocytes, once considered a culprit in the oxidative stress of OSAS patients.</p

Exercise capacity in children with isolated congenital complete atrioventricular block: does pacing make a difference?

Item does not contain fulltextThe management of patients with isolated congenital complete atrioventricular block (CCAVB) has changed during the last decades. The current policy is to pace the majority of patients based on a variety of criteria, among which is limited exercise capacity. Data regarding exercise capacity in this population stems from previous publications reporting small case series of unpaced patients. Therefore, we have investigated the exercise capacity of a group of contemporary children with CCAVB. Sixteen children (mean age 11.5 +/- 4; seven boys, nine girls) with CCAVB were tested. In 13 patients, a median number of three pacemakers were implanted, whereas in three patients no pacemaker was given. All patients had an echocardiogram and completed a cardiopulmonary cycle exercise test. Exercise parameters were determined and compared with reference values obtained from healthy Dutch peers. The peak oxygen uptake/body mass was reduced to 34.4 +/- 9.5 ml kg(-1) min(-1) (79 +/- 24% of predicted) and the ventilatory threshold was reduced to 52 +/- 17% of peak oxygen uptake (78 +/- 21% of predicted), whereas the peak work load/body mass was 2.8 +/- 0.6 W/kg (91 +/- 24% of predicted), which was similar to controls. Importantly, 25% of the paced patients showed upper rate restriction by the pacemaker. In conclusion, children with CCAVB show a reduced peak oxygen uptake and ventilatory threshold, whereas they show normal peak work rates. This indicates that they generate more energy during exercise from anaerobic energy sources. Paced children with CCAVB do not perform better than unpaced children.1 april 201

Operating a full tungsten actively cooled tokamak: overview of WEST first phase of operation

WEST is an MA class superconducting, actively cooled, full tungsten (W) tokamak, designed to operate in long pulses up to 1000 s. In support of ITER operation and DEMO conceptual activities, key missions of WEST are: (i) qualification of high heat flux plasma-facing components in integrating both technological and physics aspects in relevant heat and particle exhaust conditions, particularly for the tungsten monoblocks foreseen in ITER divertor; (ii) integrated steady-state operation at high confinement, with a focus on power exhaust issues. During the phase 1 of operation (2017–2020), a set of actively cooled ITER-grade plasma facing unit prototypes was integrated into the inertially cooled W coated startup lower divertor. Up to 8.8 MW of RF power has been coupled to the plasma and divertor heat flux of up to 6 MW m−2 were reached. Long pulse operation was started, using the upper actively cooled divertor, with a discharge of about 1 min achieved. This paper gives an overview of the results achieved in phase 1. Perspectives for phase 2, operating with the full capability of the device with the complete ITER-grade actively cooled lower divertor, are also described

A Genomic Screen Revealing the Importance of Vesicular Trafficking Pathways in Genome Maintenance and Protection against Genotoxic Stress in Diploid Saccharomyces cerevisiae Cells

The ability to survive stressful conditions is important for every living cell. Certain stresses not only affect the current well-being of cells but may also have far-reaching consequences. Uncurbed oxidative stress can cause DNA damage and decrease cell survival and/or increase mutation rates, and certain substances that generate oxidative damage in the cell mainly act on DNA. Radiomimetic zeocin causes oxidative damage in DNA, predominantly by inducing single- or double-strand breaks. Such lesions can lead to chromosomal rearrangements, especially in diploid cells, in which the two sets of chromosomes facilitate excessive and deleterious recombination. In a global screen for zeocin-oversensitive mutants, we selected 133 genes whose deletion reduces the survival of zeocin-treated diploid Saccharomyces cerevisiae cells. The screen revealed numerous genes associated with stress responses, DNA repair genes, cell cycle progression genes, and chromatin remodeling genes. Notably, the screen also demonstrated the involvement of the vesicular trafficking system in cellular protection against DNA damage. The analyses indicated the importance of vesicular system integrity in various pathways of cellular protection from zeocin-dependent damage, including detoxification and a direct or transitional role in genome maintenance processes that remains unclear. The data showed that deleting genes involved in vesicular trafficking may lead to Rad52 focus accumulation and changes in total DNA content or even cell ploidy alterations, and such deletions may preclude proper DNA repair after zeocin treatment. We postulate that functional vesicular transport is crucial for sustaining an integral genome. We believe that the identification of numerous new genes implicated in genome restoration after genotoxic oxidative stress combined with the detected link between vesicular trafficking and genome integrity will reveal novel molecular processes involved in genome stability in diploid cells

Badania stanu nasmarowania łożysk tocznych

The research aimed to assess the lubrication condition of rolling bearings dismounted from previously operated passenger car alternators. The tests measured the vibrations and evaluated the technical condition of the bearings based on selected estimators of the vibration acceleration signal subjected to earlier band-pass filtration in the high-frequency range of 8-10kHz. Next, the bearings have been disassembled, allowing inspection of the lubricant condition for each measured bearings and the visual assessment of individual components. Based on the test results, it was observed that the mean value and standard deviation of considered features of vibration acceleration signals in the 8-10kHz band might be helpful in the classification of the lubrication condition.Celem badań była ocena stanu nasmarowania łożysk tocznych wymontowanych z wcześniej eksploatowanych alternatorów samochodów osobowych. Badania polegały na pomiarze drgań i ocenie stanu technicznego łożysk w oparciu o wybrane estymatory sygnału przyspieszeń drgań poddawanego wcześniejszej filtracji pasmowo przepustowej w zakresie wysokich częstotliwości 8-10kHz. Następnie łożyska zostały zdemontowane, co pozwoliło na zbadanie stanu smaru każdego łożyska i ocenę wizualną poszczególnych elementów. Na podstawie wyników badań zaobserwowano, że wartość średnia i odchylenie standardowe wybranych cech sygnałów przyspieszeń drgań w paśmie 8-10kHz mogą być przydatne w klasyfikacji stanu nasmarowania