Excessive Supraventricular Ectopic Activity Is Indicative of Paroxysmal Atrial Fibrillation in Patients with Cerebral Ischemia

Background

Detecting paroxysmal atrial fibrillation (PAF) in patients with cerebral ischemia is challenging. Frequent premature atrial complexes (PAC/h) and the longest supraventricular run on 24-h-Holter (SV-run_{24 h}), summarised as excessive supraventricular ectopic activity (ESVEA), may help selecting patients for extended ECG-monitoring, especially in combination with echocardiographic marker LAVI/a’ (left atrial volume index/late diastolic tissue Doppler velocity).

Methods

Retrospective analysis from the prospective monocentric observational trial Find-AF (ISRCTN-46104198). Patients with acute stroke or TIA were enrolled at the University Hospital Göttingen, Germany. Those with sinus rhythm at presentation received 7-day Holter-monitoring. ESVEA was quantified in one 24-hour interval free from PAF. Echocardiographic parameters were assessed prospectively.

Results

PAF was detected in 23/208 patients (11.1%). The median was 4 [IQR 1; 22] for PAC/h and 5 [IQR 0; 9] for SV-run_{24 h}. PAF was more prevalent in patients with ESVEA: 19.6% vs. 2.8% for PAC/h >4 vs. ≤4 (p<0.001); 17.0% vs. 4.9% for SV-run_{24 h} >5 vs. ≤5 beats (p = 0.003). Patients with PAF showed more supraventricular ectopic activity: 29 PAC/h [IQR 9; 143] vs. 4 PAC/h [1]; [14] and longest SV-run_{24 h} = 10 [5]; [21] vs. 0 [0; 8] beats (both p<0.001). Both markers discriminated between the PAF- and the Non-PAF-group (area under receiver-operator-characteristics-curve 0.763 [95% CI 0.667; 0.858] and 0.716 [0.600; 0.832]). In multivariate analyses log(PAC/h) and log(SV-run_{24 h}) were independently indicative of PAF. In Patients with PAC/h ≤4 and normal LAVI/a’ PAF was excluded, whereas those with PAC/h >4 and abnormal LAVI/a’ showed high PAF-rates.

Conclusions

ESVEA discriminated PAF from non-PAF beyond clinical factors including LAVI/a’ in patients with cerebral ischemia. Normal LAVI/a’+PAC/h ≤4 ruled out PAF, while prevalence was high in those with abnormal LAVI/a’+PAC/h >4.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.     

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity,Leading to the Under Replication of DNA

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.     

Role of ascorbate in lung cellular toxicity mediated by light-exposed parenteral nutrition solution

Neonatal lung injury has been induced experimentally by infusion of multivitamin-containing light-exposed parenteral nutrition (PN) solutions. The objective was to explore the role of ascorbate in toxic effects of light-exposed PN on primary cultured foetal rat lung epithelial cells. Hydroperoxides were measured in 3% amino acid solutions at baseline, immediately after addition of either multivitamins or ascorbate alone (400 μg/mL) and again after a 24-h period of exposure to (or protection from) ambient light. Cellular toxicity was assessed by [C(14)]adenine release. Multivitamins or ascorbate alone increased hydroperoxides in PN, which was attenuated by light protection. Light-exposed PN containing multivitamins was more toxic to cells than baseline or light-protected PN. Exposure to ascorbate at concentrations both lower (< 5 μg/mL) and higher (> 1000 μg/mL) than normally contained in PN-induced oxidant-mediated cell death, as indicated by protective effects of hydroperoxide and hydroxyl radical scavengers. This study concludes that ascorbate generates toxic amounts of peroxide in PN solutions. The types and physiological importance of hydroperoxides induced by pro-oxidant effects of ascorbate require further evaluation in vivo.     

Early chloroplastic alterations analysed by optical coherence tomography during a harpin-induced hypersensitive response

The hypersensitive response has been mostly studied by molecular and biochemical methods after sample destruction. The development of imaging techniques allows the monitoring of physiological changes before any signs of cell death. Here, we follow the early steps of a hypersensitive-like response induced by the bacterial elicitor harpin in Nicotiana sp. We describe cytological modifications after inoculation of the harpin protein, using confocal fluorescence microscopy (CFM) and optical coherence tomography (OCT), an interferometric-based microscopy. The changes detected by CFM occurred 5 h after harpin infiltration and corresponded to a redistribution of the chloroplasts from the upper to the inner regions of the palisade mesophyll cells which could be related to a perturbation in the microtubule network. Using OCT, we were able to detect a decrease in chloroplast backscattered signal as early as 30 min after harpin infiltration. A simple physical model, which accounted for the structure and distribution of thylakoid membranes, suggested that this loss of scattering could be associated with a modification in the refractive index of the thylakoid membranes. Our OCT observations were correlated with a decrease in photosynthesis, emphasizing changes in chloroplast structure as one of the earliest hallmarks of plant hypersensitive cell death.     

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.