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Acute pancreatitis is associated with a readmission rate ranging from 7 to 34%. Readmission rates are highest among biliary (4-37%) and alcohol-induced (2-60%) acute pancreatitis. Severe acute pancreatitis and necrotizing pancreatitis have readmission rates ranging from 20 to 75%. The most common causes of readmission include recurrent acute pancreatitis (17-45% of readmissions) and smoldering symptoms/local complications (17-38%). A number of risk scores reliably estimate risk of readmission in acute pancreatitis. Decreased rates of readmission were reported in patients that underwent same-admission cholecystectomy in biliary pancreatitis and alcohol cessation interventions in alcohol-induced pancreatitis. This review article discusses readmission in acute pancreatitis, including etiology, risk factors, and opportunities for improved patient care.

PDF file - 272K, CERT downregulation enhances HRG-induced ErbB2/ErbB3 signaling

Triple-negative breast cancers (TNBC) are especially refractory to treatment due to their negative hormone receptor and ErbB2/HER2 status. Therefore, the identification of cancer-associated deregulated signaling pathways is necessary to develop improved targeted therapies. Here, we show that expression of the ceramide transfer protein CERT is reduced in TNBCs. CERT transfers ceramide from the endoplasmic reticulum to the Golgi complex for conversion into sphingomyelin (SM). We provide evidence that by regulating cellular SM levels, CERT determines the signaling output of the EGF receptor (EGFR/ErbB1), which is upregulated in approximately 70% of TNBCs. CERT downregulation in breast cancer cells enhanced ErbB1 lateral mobility, ligand-induced autophosphorylation, internalization, and chemotaxis. Together, our findings provide a link between lipid metabolism at the Golgi with signaling at the plasma membrane, thereby implicating CERT loss in the progression of TNBCs. Cancer Res; 72(11); 2855–66. ©2012 AACR.

PDF file - 81K, Downregulation of SM synthases recapitulates the effects of CERT depletion on EGF-induced Akt activation and cell migration

PDF file - 155K, A second CERT-specific siRNA confirms the impact on EGF-induced Akt activation, cell migration and focal adhesion clustering

PDF file - 108K, Lipid analyses by MALDI-TOF mass spectrometry

PDF file - 121K, A second PLD2-specific siRNA confirms the effects on ErbB1 and Akt activation

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PDF file - 64K, CERT transcript levels in prostate cancer microarray datasets

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek’s disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek's disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of

Eine Echtzeit-Beobachtung der Aktivitat der Sauren Sphingomyelinase (ASM) ist notwendig zur Aufklarung ihrer Rolle in Lipid-Signaltransduktionsprozessen. Mithilfe von Phosphorodichloridat-Chemie gelang die Synthese von fluoreszierenden Phosphosphingolipiden mit der Fahigkeit zum FRET. Diese Sphingomyelin-Analoga sind Substrate fur die rekombinante humane ASM und ermoglichen die Verfolgung der ASM-Aktivitat mittels Fluoreszenzspektroskopie. Inkubation mit Zell-Lysaten von Wildtyp- und Knockout-Mausen zeigte eine ausschliesliche Spaltung durch ASM. Daruber hinaus nutzten wir systematisch die Umgebungsempfindlichkeit der Fluorophore, um signifikante Zuwachse in der Responsivitat zu erzielen. Die dabei angewendeten Konzepte konnten auch fur andere zukunftige Lipase-Sonden zum Einsatz kommen. Eine mikroskopische Abbildung der ASM-Aktivitat in lebenden Zellen wurde mittels Zweiphotonenanregung realisiert.

A fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.Abstract Figure

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

Circadian clocks are molecular timekeeping mechanisms that allow organisms to anticipate daily changes in their environment. The fundamental cellular basis of these clocks is delayed negative feedback gene regulation with PERIOD and CRYPTOCHROME containing protein complexes as main inhibitory elements. For a correct circadian period, it is essential that such clock protein complexes accumulate in the nucleus in a precisely timed manner, a mechanism that is poorly understood. We performed a systematic RNAi-mediated screen in human cells and identified 15 genes associated with the nucleo-cytoplasmic translocation machinery, whose expression is important for circadian clock dynamics. Among them was Transportin 1 (TNPO1), a non-classical nuclear import carrier, whose knockdown and knockout led to short circadian periods. TNPO1 was found in endogenous clock protein complexes and particularly binds to PER1 regulating its (but not PER2’s) nuclear localization. While PER1 is also transported to the nucleus by the classical, Importin β-mediated pathway, TNPO1 depletion slowed down PER1 nuclear import rate as revealed by fluorescence recovery after photobleaching (FRAP) experiments. In addition, we found that TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization. Together, our RNAi screen knocking down import carriers (but also export carriers) results in short and long circadian periods indicating that the regulatory pathways that control the timing of clock protein subcellular localization are far more complex than previously assumed. TNPO1 is one of the novel players essential for normal circadian periods and potentially for redox regulation of the clock., Author summary Circadian clocks are endogenous timekeeping mechanisms allowing organisms to anticipate daily changes in their environment. In mammals, the fundamental mechanism of these clocks is a delayed negative feedback loop, in which timely auto-repression of clock components is essential. This repression occurs at a transcriptional level and requires clock proteins to enter the nucleus in a precisely timed manner, a regulation that is little understood. We performed a systematic genetic screen for factors modulating subcellular localization in oscillating human cells and identified Transportin 1 (TNPO1) as a non-classical carrier protein required for a normal circadian period. The primary target of TNPO1 within the circadian clockwork is PERIOD1, whose nuclear shuttling is modulated by TNPO1. In addition, TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORFall1371encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacteriumAnabaenasp. PCC 7120. Here, ORFall1371was heterologously expressed inEscherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cationsin vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, withkcat = 48.2 s−1at pH 7.5 and 28 °C andKM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of anAnabaenasp. PCC 7120 mutant strain lackingall1371was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression ofall1371under combined nitrogen deprivation. All1371 might play a substantial role inAnabaenasp. PCC 7120 under these conditions.

Fur temporare Fliesgewasser wie die sommertrockenen Bache gibt es bundesweit noch kein biologisches Bewertungsverfahren zur Erfassung des okologischen Zustands. Innerhalb des Projektes dynaklim wurde ein solches Bewertungssystem entwickelt.

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Forster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.

Real-time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid-mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild-type and knock-out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two-photon-excitation microscopy.

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level.

Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity.

Summary 1. In recent years, river restoration science has been searching for biological indicators of improvement in the physical habitats of streams. To date, research has mainly focused on the use of fish and macroinvertebrates as indicators. Despite their importance in aquatic ecosystems, the response of macrophytes to habitat restoration has been rarely studied. 2. We investigated the macrophyte communities of 40 restored river reaches in the lowland and lower mountainous areas of Germany. Each restored reach was compared to an upstream, unrestored reach using a space-for-time-substitution approach. At each reach, a 100 m stretch was surveyed for submerged and emergent macrophytes, recording the quantity, abundance and growth form of each species. Additionally, microhabitat patterns (substrate, depth, current velocity) and channel parameters (mean and bankfull width, number of channel elements) were recorded. 3. Restored reaches had a significantly higher macrophyte cover, richness, diversity and number of growth forms. Macrophyte diversity and richness were both positively correlated with depth, current and substrate. 4. The analysis of growth forms showed that Lemnids, Helodids, Parvopotamids, Elodids, Peplids and Juncids are all significant indicators of restoration. These species all responded directly to the restoration measures either by highly increasing in abundance or by being present in the restored reaches and absent in the unrestored reaches. While the restored reaches of the lowland rivers were characterized by a high abundance of Peplids and Parvopotamids, the restored reaches of the mountain rivers showed a significantly higher presence and abundance of Lemnids and Helodids. 5. Three macrophyte species (Lemna minor, Persicaria hydropiper, Potamogeton crispus) were regarded as significant indicators of restoration. No species were found to be indicators of unrestored reaches. 6.Synthesis and applications. Macrophyte communities benefit from river restoration by showing increased cover, abundance and diversity. The main drivers of this enhancement are more natural and diverse substrates and an increased floodplain area in the restored reaches, as well as a greater variability of current and depth patterns. Monitoring of macrophytes could thus be an easy and cost-effective means to gauge the success of river restoration measures.

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content. (c) 2011 Elsevier Ireland Ltd. All rights reserved.

Aims Intracardiac electrograms (IEGMs) recorded by implantable cardioverter-defibrillators (ICDs) are essential for arrhythmia diagnosis and ICD therapy assessment. Short IEGM snapshots showing 3–10 s before arrhythmia detection were added to the Biotronik Home Monitoring system in 2005 as the first-generation IEGM Online. The RIONI study tested the primary hypothesis that experts’ ratings regarding the appropriateness of ICD therapy based on IEGM Online and on standard 30 s IEGM differ in

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor–acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor–acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

We developed a system for the assessment of ecological condition for rivers in the lower mountains and lowlands of the Hindu Kush-Himalayan region (Pakistan, India, Nepal, Bhutan, and Bangladesh). We used benthic invertebrates collected from 198 rivers, located in five different ecoregions and covering degradation gradients; samples were taken twice (pre-monsoon and post-monsoon) applying a multi-habitat sampling procedure. Out of 38 environmental parameters, we constructed complex principal component analysis (PCA) gradients, separately for the stressors organic pollution, eutrophication, floodplain land use, and hydromorphological degradation. Correlation analysis between invertebrate metrics and environmental parameters revealed those biological metrics that are most responsive to river deterioration. Redundant metrics were deleted, and the most robust metrics were selected. The range of the index values under reference conditions was defined, and a five-class river quality system was generated.

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.

Mice with a knockout (KO) of muscle LIM protein (MLP) exhibit many morphologic and clinical features of human cardiomyopathy. In humans, MLP-expression is downregulated both in ischemic and dilative cardiomyopathy. In this study, we investigated the effects of MLP on the electrophysiologic phenotype in vivo and on outward potassium currents.MLP-deficient (MLPKO) and wild-type (MLPWT) mice were subjected to long-term electrocardiogram (ECG) recording and in vivo electrophysiologic study. The whole-cell, patch-clamp technique was applied to measure voltage dependent outward K+ currents in isolated cardiomyocytes. Long-term ECG revealed a significant prolongation of RR mean (108 +/- 9 versus 99 +/- 5 ms), P (16 +/- 3 versus 14 +/- 1 ms), QRS (17 +/- 3 versus 13 +/- 1 ms), QT (68 +/- 8 versus 46 +/- 7 ms), QTc (66 +/- 6 versus 46 +/- 7 ms), JT (51 +/- 7 versus 34 +/- 7 ms), and JTc (49 +/- 5 versus 33 +/- 7 ms) in MLPKO versus MLPWT mice (P.05). During EP study, QT (80 +/- 8 versus 58 +/- 7 ms), QTc (61 +/- 6 versus 45 +/- 5 ms), JT (62 +/- 9 versus 43 +/- 6 ms), and JTc (47 +/- 5 versus 34 +/- 5 ms) were also significantly prolonged in MLPKO mice (P.05). Nonsustained VT was inducible in 9/16 MLPKO versus 2/15 MLPWT mice (P.05). Analysis of outward K+ currents in revealed a significantly reduced density of the slowly inactivating outward K+ current IK, slow in MLPKO mice (11 +/- 5 pA/pF versus 18 +/- 7 pA/pF; P.05).Mice with KO of MLP exhibit significant prolongation of atrial and ventricular conduction and an increased ventricular vulnerability. A reduction in repolarizing outward K+ currents may be responsible for these alterations.

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.

AIMS The purpose of this prospective, randomized, multicentre study was to investigate whether the incidence of ventricular tachyarrhythmia can be reduced in standard implantable cardioverter/defibrillator (ICD) patients by implanting a dual-chamber ICD capable of atrial therapy delivery. METHODS AND RESULTS A Jewel AF or GEM III AT ICD (Medtronic Inc., Minneapolis, MN, USA) was implanted in 122 patients (62.3 +/- 10.5 years; 84.4% male; coronary artery disease 71.3%; left ventricular ejection fraction 39.7 +/- 13.6%; secondary ICD indication 91%). Overall, 31.2% of the patients had paroxysmal atrial fibrillation (AF)/atrial tachycardia (AT) before ICD implantation (n = 38). Implantable cardioverter/defibrillator therapies for AT/AF were activated and de-activated every 3 months in a cross-over study design. The mean follow-up was 18.5 +/- 7.7 months (6.29 +/- 2.2 cross-over/patient). Overall, there were 684 episodes of ventricular tachyarrhythmias in 48.4% of patients (n = 59). In 33.6% of patients (n = 41), 532 supraventricular tachyarrhythmias occurred. Activation of ICD therapies for AT/AF did not result in a reduction of ventricular tachyarrhythmias (P = 0.92). Patients with monomorphic ventricular tachycardias (mVTs) as index arrhythmia for ICD implantation or inducible mVTs in the electrophysiological study had the highest probability of recurrences of ventricular tachyarrhythmias. CONCLUSION For patients with standard indications for ICD therapy and no indication for cardiac pacing, a dual-chamber ICD capable of atrial tachyarrhythmia treatment offers no benefit concerning a reduction of ventricular tachyarrhythmias.

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients’ behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbtS and dbtL) in Drosophila.

Aims Identification of risk factors for ventricular tachycardia/ventricular fibrillation (VT/VF) occurrence in patients with implantable cardioverter-defibrillators (ICD) is reasonable, because ICD patients with multiple risk factors might benefit from more aggressive anti-arrhythmic therapy for the prevention of arrhythmic events. Furthermore, in the era of prophylactic ICD therapy and limited healthcare resources, additional markers are needed for improved patient selection. Methods and results Thus, in Prospective Analysis of Risk Factor for Appropriate ICD Therapy (PROFIT), we prospectively analyzed the role of ejection fraction (EF), N-terminal probrain natriuretic peptide (NT-proBNP), New York Heart Association (NYHA) class, atrial fibrillation, and QRS-duration as independent predictors for VT/VF occurrence in 250 ICD patients. Kaplan–Meier analysis showed that EF , 40% (log-rank P ¼ 0.001), NT-proBNP levels higher than median (� 405 ng/L; log-rank P ¼ 0.04), QRS-duration � 150 ms (log-rank P ¼ 0.016), permanent atrial fibrillation (log-rank P ¼ 0.008), and higher NYHA class (log-rank P ¼ 0.029) were associated with VT/VF occurrence. By multivariate Cox regression analysis EF, QRS-duration and atrial fibrillation remained significantly associated with appropriate VT/VF therapy, whereas there was no relationship among NT-proBNP, NYHA class, and VT/VF occurrence. Stratifying patients according to the number of their independent risk factors (EF , 40%, AF, QRS-width � 150 ms) showed that patients with greater than or equal to two risk factors had a 100% 2-year risk of VT/VF occurrence, whereas patients with no or one risk factor had a 19.3 and 25% 2-year risk, respectively. Conclusions EF , 40%, permanent atrial fibrillation, and QRS � 150 ms are independent predictors for VT/VF occurrence in predominantly secondary prophylactic ICD patients. Combining all independent predictors, we developed a risk score for VT/VF occurrence identifying a subgroup of patients with two or more risk factors who had a 100% 2-year risk. Future studies will reveal if this risk score helps to identify ICD patients suitable for empirical anti-arrhythmic therapy and to improve patient selection for prophylactic ICD therapy.

Appropriate and inappropriate therapies of implantable cardioverter defibrillators (ICDs) have a major impact on morbidity and quality of life in ICD recipients. The recently introduced home monitoring of ICD devices is a promising new technique which remotely offers information about the status of the system. Stored intracardiac electrograms (IEGMs), which are essential for correct classification of appropriate and inappropriate ICD discharges, have until now not been available with ICD home monitoring on a day-by-day basis because of limitations of transferable data. We demonstrate the first compressed IEGMs daily transferable via home monitoring (IEGM-online). Validation of these electrograms will be performed in the Reliability of IEGM-Online Interpretation (RIONI) study. A total of 210 episodes of stored IEGMs will be collected by at least 12 European centres. The primary endpoint of this study is to investigate whether the IEGM-online based evaluation of the appropriateness of the ICDs therapeutic decision following episode detection is equivalent to the evaluation based on the complete ICD episode Holter extracted from the IEGM stored. The evaluation is independently done by an expert board of three experienced ICD investigators. The equivalence of the two methods is accepted if the evaluations yield a different conclusion for

Bei 20–30% der Patienten mit implantierbarem Kardioverter-Defibrillator (ICD) treten inadaquate ICD-Therapien aufgrund supraventrikularer Tachykardien auf. Zur Behandlung inadaquater ICD-Episoden stehen verschiedene Therapieansatze wie optimierte ICD-Programmierung, antiarrhythmische Begleitmedikation und Hochfrequenzablation zur Verfugung. Ein-Kammer-Detektionsalgorithmen sind geeignete Kriterien fur die Unterscheidung vor allem von Vorhofflimmern bzw. Sinustachykardien und Kammertachykardien. Zwei-Kammer-Detektionsalgorithmen erweitern das Spektrum der Ein-Kammer-Algorithmen durch die atriale Sensing-Information und konnen uber die Zahl und die Zuordnung atrialer zu ventrikularen Ereignissen die Unterscheidung von supraventrikularen und ventrikularen Tachykardien verbessern. Grosere kontrollierte, randomisierte Studien, die hinsichtlich inadaquater Episoden eine Uberlegenheit der Zwei-Kammer-ICDs uber Ein-Kammer-ICDs zeigen, fehlen allerdings. Moglicherweise profitieren Patienten mit langsamen Kammertachykardien, die aufgrund der niedrigen Detektionsfrequenz ein erhohtes Risiko fur inadaquate ICD-Therapien haben, von einem Zwei-Kammer-ICD. Hinsichtlich der medikamentosen Therapie zur Suppression inadaquater ICD-Therapien konnte die OPTIC-Studie zeigen, dass eine Therapie mit Sotalol einer konventionellen β-Blocker-Therapie uberlegen ist und die Kombination von Amiodaron und β-Blocker-Therapie am effektivsten ist. ICD-Patienten mit inadaquaten ICD-Therapien bei typischem Vorhofflattern profitieren von einer Radiofrequenzablation. Bei paroxysmalem Vorhofflimmern ist nach Ausschopfung einer medikamentosen Rezidivprophylaxe eine katheterinterventionelle Behandlung ebenfalls zu erwagen, die Effektivitat ist allerdings bei ICD-Patienten bisher nicht nachgewiesen. Bei ICD-Patienten mit chronisch permanentem Vorhofflimmern und inadaquaten ICD-Therapien ist nach maximaler frequenzmodulierender Therapie (β-Blocker, Digitalis und ggf. Amiodaron) im Einzelfall eine AV-Knoten-Ablation sinnvoll. Eine Umrustung auf einen biventrikularen ICD sollte allerdings prospektiv erwogen werden, um eine Verschlechterung der Herzinsuffizienz aufgrund der durch rechtsventrikulare Stimulation hervorgerufenen inter-/intraventrikularen Asynchronie zu vermeiden.