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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.

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

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.

Development of an Assessment System to Evaluate the Ecological Status of Rivers in the Hindu Kush-Himalayan Region (ASSESS-HKH) was a 3-year research project funded by the European Union (Contract number: INCO-CT-2005-003659). This article provides an overview of this research project by summarising the objectives, the approaches and the main achievements. The main objective was to develop and apply a biological assessment system to evaluate the river’s ecological quality and to provide a scientific basis for the identification of sustainable water policy options and management strategies. The assessment tools were jointly developed by European partners, who provided their experience from recent research activities (STAR, AQEM) and Asian partners, who provided the knowledge about Asian river catchments and management necessities. The project was organised into eight work packages defining the time line for all phases, such as establishment of a stream typology and definition of reference conditions and stages of impairment classes for the rivers in the Asian countries, including a review of existing policies for water management. A specific part of the project was dedicated to increasing the overall poor knowledge of benthic invertebrates in the region and their value to the classification of the river’s ecological quality. All activities were accompanied by information events for local residents, universities and water managers. A total of 396 multi-habitat samples, from 115 rivers in five different ecoregions, were taken in two different seasons and accompanied by information on 95 parameters describing river and catchment characteristics. The benthic invertebrates in the samples were taxonomically identified based on keys generated within the project. Taxalists, with abundances per site, and field protocol information were entered into a specifically developed software tool. This dataset was the basis for developing ecological river assessment methods, called HKHscreening (Rapid Field Assessment), HKHbios (HKH Biotic Score) and HKHindex (Multimetric Index). Furthermore, a software tool (ECODAT) for using these methods was developed. The monitoring tools will serve citizens and scientists of the Hindu Kush-Himalayan region and will provide a scientific basis for policy recommendations, mitigation strategies, transnational water resource planning and sustainable ecosystem management. Additional outputs, including all sampling and laboratory protocols and project deliverables, together with the freely downloadable software, are available at the following website: http://www.assess-hkh.at.

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.

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.

The macrophyte surveys undertaken as part of the EU-funded STAR project are a unique resource allowing aquatic plant communities to be studied at a Pan-European scale (211 stream sites with macrophytes in 14 countries). Using this dataset, we examined the influence of organic pollution in relation to other environmental correlates of river plant community variation across Europe. We examined the relationships between several existing macrophyte metrics and nutrient enrichment, and we also explored the possibility of developing a pan-European macrophyte-based assessment system. We showed that trophic (nutrient) status is an important driver of aquatic plant communities in European rivers. We found that while most existing macrophyte metrics are useful, none can be applied at a pan-European scale in their current form. Our attempt to redesign the Mean Trophic Rank (MTR) index by the addition of further species, and the re-scoring of existing species, resulted in a considerable improvement in the relationship between MTR scores and nutrient variables. We conclude that an enlarged core group of macrophyte species can form part of an improved pan-European macrophyte-based bioassessment system, although regional modifications may be required to adequately describe the nutrient status of certain stream types.

Background— Major gender-based differences in the incidence of ventricular tachyarrhythmia after myocardial infarction have been shown in humans. Although the underlying mechanisms are unclear, earlier studies suggest that estrogen receptor–mediated effects play a major role in this process. Methods and Results— We examined the effect of estrogen receptor α (ERα) and estrogen receptor β (ERβ) on the electrophysiological phenotype in female mice with and without chronic anterior myocardial infarction. There was no significant difference in overall mortality, infarct size, and parameters of left ventricular remodeling when we compared infarcted ERα-deficient and ERβ-deficient mice with infarcted wild-type animals. In the 12-hour telemetric ECG recording 6 weeks after myocardial infarction, surface ECG parameters did not show significant differences in comparisons of ERα-deficient mice versus wild-type controls, infarcted versus noninfarcted ERα-deficient mice, and infarcted ERα-deficient versus infarcted wild-type mice. However, infarcted ERβ-deficient versus noninfarcted ERβ-deficient mice showed a significant prolongation of the QT (61±6 versus 48±8 ms; P P P P P P P P P I to ) in ERβ-deficient mice ( P Conclusions— Estrogen receptor β deficiency results in prolonged ventricular repolarization and decreased ventricular automaticity in female mice with chronic myocardial infarction.

To elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade.

The influenza virus hemagglutinin (HA) is known to play an essential role in virus infection. It is not only involved in cell receptor binding and uptake by endocytosis, but moreover, after internalization and trafficking to early and late endosomes, induces membrane fusion which leads to the release of the viral genome into the cell. It has been shown for several influenza strains that the pH of fusion of HA can be shifted by modulating its stability which results in altered infectivity of the virus. However, the underlying molecular mechanisms that lead to an altered pH dependence of membrane fusion are still unknown.Using experimental approaches and Molecular Dynamics studies we demonstrate that the introduction of charges in the vicinity of certain His residues can change its protonation state, thereby influencing the pH dependant conformational change of the HA protein. Histidines at key structural locations were identified and neighboring residues were mutated in the HA of highly (HP) or low pathogenic (LP) H5N1 and X31. Fusion activity and stability of recombinant proteins and resulting virus particles were assessed by fluorescence dequenching.First results showed that charges, especially at position 216, in the vicinity of His184 have a significant effect on the stability of the HA1 ectodomain in both H3 and H5 HA. Interestingly, Arginine at position 216 is naturally present in the H5 HP whereas the H5 LP has a Glutamate at this position. We could show that the exchange of these charges in both variants rescues the pH dependence of fusion of HP and LP, respectively. We suggest that influenza viruses adjust their pH of activation by the exchange of charged residues in the vicinity of conserved Histidines for optimized spread and stability.