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The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator. Live-cell recordings have been an important tool for studying circadian rhythms. Here the authors use CRISPR gene editing mediated knock-in to fluorescently tag Per2 and Cry1, and study cellular circadian dynamics of these two clock proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The study introduces an approach to obtaining information about the preferences of benthic invertebrates for substrate and current velocity in a region with little prior knowledge of benthic invertebrates. These preferences are then used for river assessment. Substrate-specific sampling of 271 reference sites was conducted in lower mountainous and lowland areas of the Hindu Kush-Himalaya region. Statistical analysis revealed significant preferences for substrate type and current velocity for 50 taxa of Ephemeroptera, Plecoptera, Trichoptera, Coleoptera, Diptera, Odonata, Mollusca, and Oligochaeta. A 20-point system was developed to assign scores for substrate and current preferences. Scores from seven taxa of Ephemeroptera and Trichoptera revealed low ecological potential in response to habitat alteration. These data were used to develop four preference metrics. The Lithal metric is composed of 34 taxa with significant preferences for stony substrates (fine gravel to bedrock size). The Lithophile metric contains 21 taxa with strong statistical links to stony substrates, which were also found on other substrates. The Lithobiont metric consists of 13 taxa exclusively found on stones. The Lotic metric consists of 11 taxa with significant preferences for moderate-to-fast current velocities. Multi-habitat sampling was conducted at 181 sites reflecting a hydromorphological gradient. The Mann–Whitney U test and box-and-whisker plots were applied to test the relationship of the new metrics to hydromorphological stress. Of the four new metrics, the Lithal, Lithophile, and Lotic were able to detect impacts of hydromorphological degradation. [ABSTRACT FROM AUTHOR]

Within the ASSESS-HKH project (Development of an Assessment System to Evaluate the Ecological Status of Rivers in the Hindu Kush-Himalayan (HKH) region—a research project funded by the European Union; contract number: INCO-CT-2005-003659) a benthic invertebrate-based scoring system (HKHbios; Hindu Kush-Himalayan biotic score) was developed. The development was based on multi-habitat samples from 198 sampling sites located in five ecoregions and five Asian countries (Bangladesh, Bhutan, India, Nepal and Pakistan) taken in two different seasons (pre- and post-monsoon). Environmental and biological screening data were used to select macro-invertebrates as indicators for the ecological river quality. Taxa scores were assigned based on the range and distribution patterns of taxa amongst different degrees of impact and on available autecological information. In total, 199 taxa were scored for the HKHbios, which is calculated a weighted average score per taxon (ASPT). The range of the index values under different degrees of stress was evaluated and a five-class quality assessment system was generated for each ecoregion. Correlation analysis between the HKHbios, 38 selected environmental parameters and complex PCA gradients were used to test the response of the HKHbios to different kinds of impact. [ABSTRACT FROM AUTHOR]

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH. [ABSTRACT FROM AUTHOR]

Introduction: An increasing number of genetically altered mice with specific molecular cardiac defects are being assessed by electrophysiological studies and ECG monitoring. This approach should allow for the identification of critical genes involved in the arrhythmogenesis in myocardial infarction. Therefore it was the aim of this study to establish a standard for the in-vivo electrophysiological characteristics in the mouse model of chronic anterior myocardial infarction. Methods and Results: Using a minimized, invasive, in-vivo electrophysiological study, surface ECG parameters, sinus node function, atrial, atrio-ventricular and ventricular conduction and ventricular repolarization, and enhanced vulnerability to atrial and ventricular arrhythmia were studied in 20 wild-type C57BL/6 mice either under control or 11 weeks after large anterior myocardial infarction induced by ligation of the left anterior descending coronary artery. Telemetric ECG recording was performed in the same animals at baseline unrestrained, conscious condition to study surface ECG parameters, heart rate variability and the prevalence of supraventricular and ventricular arrhythmia. During electrophysiological study, infarcted mice showed an 81% increase of the angle of the QRS axis (p < 0.001) and a prolongation of the P wave by 23% (p = 0.01), the QRS complex by 39% (p = 0.001), the QT interval by 23% (p<0.05), the QTc interval by 30% (p < 0.005) and the JTc interval by 31% (p < 0.05) in comparison to control animals. Furthermore, there was a prolongation of the atrio-ventricular interval by 28% (p < 0.0005) and the atrio-ventricular functional refractory period by 26% in infarcted animals (p < 0.05), and inducibility of ventricular tachycardia in 4 of 6 infarcted versus in none of control animals (0 < 0.01). During telemetric ECG recording, there was a marked increase in ventricular ectopic activity in infarcted mice in comparison to controls (p < 0.05). Heart rate and time- and frequency-domain of heart rate variability were not significantly different in both groups (p > 0.05, respectively). Conclusions: The mouse model of chronic anterior myocardial infarction is associated with significant atrial and ventricular conduction disturbances and vulnerability to ventricular arrhythmia and thus may provide a highly valuable tool to study molecular determinants of arrhythmogenesis in myocardial infarction. [ABSTRACT FROM AUTHOR]

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We describe a rare case of an inappropriate defibrillator discharge during a sinus tachycardia with single fast sensed beats fulfilling the ventricular fibrillation rate criterion, activating the “combined count criterion,” although the prevalent sinus tachycardia was inside a ventricular tachycardia rate zone, programmed to monitor only mode. The “combined count criterion” is a safety feature that ensures defibrillation therapy in case of a tachyarrhythmia inside the ventricular tachycardia detection zone, that is temporarily crossing the ventricular fibrillation detection rate. This case reports shows that there is a low but unpredictable risk of device therapy even in a zone programmed to monitor only mode. This phenomenon should be considered when a monitor only zone is installed for revelation of slower tachyarrhythmias. [ABSTRACT FROM AUTHOR]

We have studied the kinetics of low pH-induced fusion between influenza virus A/PR 8/34 and human erythrocyte membranes in suspension by using an assay based on fluorescence dequenching (FDQ) of the lipophilic dye octadecylrhodamine B chloride (R 18). As shown previously (Clague et al. 1991) the onset of FDQ is preceded by a characteristic lag time ( t) following pH reduction. Whereas t represents only a subpopulation of fusing viruses with the shortest delay time we suggest here that a representative mean lag time µ of virus-cell fusion can be deduced from the R 18-assay. Kinetics of FDQ reflects the cumulative distribution function of lag times τ of single fusion events with the mean value µ. We show that t obtained from the onset of FDQ does not always reflect the fusion behaviour of the whole population of fusing viruses. While both lag times, t and µ exhibit a similar temperature dependence we found a significantly different dependence of both delay times on virus inactivation by low pH-pretreatment. We conclude that the mean lag time µ appears to be a more appropriate parameter describing the kinetics of virus-cell fusion. The analysis of delay times offers a new approach to test the validity of different kinetic models of HA-mediated fusion and to gain valuable information about HA-mediated fusion. The analysis confirms that the inactivation process proceeds via steps of the formation of the fusion pore. Although the increase of lag times can be explained by a depletion of fusion competent HA's, our data suggest that intermediate structures of HA along the inactivation pathway can still transform into a fusion site. [ABSTRACT FROM AUTHOR]

Binding of the fluorophore 1,1′-bis(4-anili-no) naphthalene-5,5′-disulfonic acid (bis-ANS) to influenza virus A/PR 8/34 is strongly enhanced at low pH. Binding is accompanied by a significant increase in fluorescence intensity. The binding and the fluorescence increase are associated with the low-pH induced conformational change of the viral spike protein, hemagglutinin, exposing hydrophobic binding sites. The data indicate that in addition to the hydrophobic N-terminus of HA2 other hydrophobic sequences of the HA ectodomain become accessible to bis-ANS at low pH. It is shown that the time course of the fluorescence increase of bis-ANS at low pH is determined by the conformational change of HA. The application of this assay for continuously monitoring the kinetics of the structural alteration in HA is discussed and its relevance for elucidating the temporal relationship between the conformational change of HA and virus-membrane fusion is outlined. [ABSTRACT FROM AUTHOR]