Bluetongue Virus VP1 Polymerase Activity In Vitro: Template Dependency, Dinucleotide Priming and Cap Dependency

BACKGROUND: Bluetongue virus (BTV) protein, VP1, is known to possess an intrinsic polymerase function, unlike rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with the polymerases of other members of the Reoviridae family, BTV VP1 has not been characterized in detail. METHODS AND FINDINGS: Using an in vitro polymerase assay system, we demonstrated that BTV VP1 could synthesize the ten dsRNAs simultaneously from BTV core-derived ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus core-derived ssRNA templates that possess no sequence similarity with BTV. In contrast, dsRNAs were not synthesized from non-viral ssRNA templates by VP1, unless they were fused with specific BTV sequences. Further, we showed that synthesis of dsRNAs from capped ssRNA templates was significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last base of the dinucleotide complemented the 3' -terminal nucleotide of the ssRNA template. CONCLUSIONS: We showed that the polymerase activity was stimulated by two different factors: cap structure, likely due to allosteric effect, and dinucleotides due to priming. Our results also suggested the possible presence of cis-acting elements shared by ssRNAs in the members of family Reoviridae

Haulage Methods in Different Areas of Nepal and the Health Condition of the Porters in Kathmandu

The complicated geographical features of Nepal make transport of goods difficult. People have to depend on human power even today, especially porters who use a number of different transport styles. The objectives of this study were to document (1) the characteristics of haulage methods in relation to the geographical conditions, and (2) the diurnal activities and health of porters in Kathmandu. The observed methods used by the porters in Nepal to carry loads were divided into four main classes: (1) on the top of the head, (2) by handcart, (3) in baskets on a yoke across the shoulders, and (4) on the back using a tumpline. The method of carrying a load on the back with a tumpline was most commonly observed, although this style might cause damage to the spinal vertebrae. The study of the diurnal activities and health condition of porters was limited to those in Kathmandu. For this purpose seven porters were interviewed orally. The results can be summarised as follows: (1) luggage of about 60kg to 110kg could be carried, (2) six of the seven porters habitually smoked tobacco and drank alcohol, (3) many porters wore cloth tightly twisted around their waist, and (4) complaints of severe neck pain were not made, but all porters complained of knee and/or back pains. The cervical and lumbar vertebrae of these porters were examined by roentgenological analysis, but no abnormal changes were observed

Pandemic restrictions in 2020 highlight the significance of non-road NOx sources in central London

Fluxes of nitrogen oxides (NOxCombining double low lineNO+NO2) and carbon dioxide (CO2) were measured using eddy covariance at the British Telecommunications (BT) Tower in central London during the coronavirus pandemic. Comparing fluxes to those measured in 2017 prior to the pandemic restrictions and the introduction of the Ultra-Low Emissions Zone (ULEZ) highlighted a 73 % reduction in NOx emissions between the two periods but only a 20 % reduction in CO2 emissions and a 32 % reduction in traffic load. Use of a footprint model and the London Atmospheric Emissions Inventory (LAEI) identified transport and heat and power generation to be the two dominant sources of NOx and CO2 but with significantly different relative contributions for each species. Application of external constraints on NOx and CO2 emissions allowed the reductions in the different sources to be untangled, identifying that transport NOx emissions had reduced by >73 % since 2017. This was attributed in part to the success of air quality policy in central London but crucially due to the substantial reduction in congestion that resulted from pandemic-reduced mobility. Spatial mapping of the fluxes suggests that central London was dominated by point source heat and power generation emissions during the period of reduced mobility. This will have important implications on future air quality policy for NO2 which, until now, has been primarily focused on the emissions from diesel exhausts

Micrometeorological flux measurements of aerosol and gases above Beijing

Air pollution is estimated to cause 1.6 million premature deaths in China every year and in the winter 2016/17 Beijing had to issue health alerts and put in place ad hoc limitations on industrial and vehicular activity. Much of this pollution is attributed to emissions from industrial processes and in particular coal combustion. By contrast, the diffuse pollutant sources within the city are less well understood. This includes, e.g., emissions from the Beijing traffic fleet, the sewage system, food preparation, solid fuel combustion in the streets and small industrial processes. Within the framework of a major UK-Chinese collaboration to study air pollution and its impact on human health in Beijing, we therefore measured fluxes of a large range of pollutants from a height of 102 m on the 325 m meteorological tower at the Institute of Atmospheric Physics. Several instruments were mounted at 102 m: fluxes of CO2 and H2O were measured with an infrared gas analyser (LiCOR 7500) and fluxes of ozone with a combination of a relative fast-response ozone analyser (ROFI) and a 2B absolute O3 instrument. Total particle number fluxes were measured with a condensation particle counter (TSI CPC 3785), and size-segregated fluxes over the size range 0.06 to 20 μm with a combination of an optical Ultrafine High Sensitivity Aerosol Spectrometer (UHSAS) and an Aerodynamic Particle Sizer Spectrometer (TSI APS3321). Ammonia (NH3) fluxes were measured for the first time above the urban environment using an Aerodyne compact quantum cascade laser (QCL). In addition, composition resolved aerosol fluxes were measured with an Aerodyne Aerosol Mass Spectrometer (HR-ToF-AMS), operated in a measurement container at the bottom of the tower, which subsampled from a 120 m long copper tube (15 mm OD). The analysis so far suggests that, due to often low wind speeds, fluxes were at times de-coupled from the surface. Fluxes normalised by CO2, a tracer for the amount of fossil fuel consumed, should be less sensitive to transport effects. However, not only fluxes, but also these CO2-ratioed fluxes are highly variable in both space and time, indicating a complex mix of sources, which will be further investigated. The organic aerosol fluxes were the largest we have recorded to date at any urban measurement site. Nitrate, sulphate, chloride and ammonium all showed emissions that followed a similar diurnal cycle as the organic aerosol. Much of this aerosol is likely to have been formed by chemistry below the measurement height, but it nevertheless indicates significant sources of the precursor gases within the footprint. Comparing the measured fluxes of gas-phase NH3 and aerosol NH+4, at 102 m the aerosol phase makes a significant contribution to the reduced nitrogen emission

Pandemic restrictions in 2020 highlight the significance of non-road NOx sources in central London

Fluxes of nitrogen oxides (NOx = NO + NO2) and carbon dioxide (CO2) were measured using eddy covariance at the British Telecommunications (BT) Tower in central London during the coronavirus pandemic. Comparing fluxes to those measured in 2017 prior to the pandemic restrictions and the introduction of the Ultra-Low Emissions Zone (ULEZ) highlighted a 73 % reduction in NOx emissions between the two periods but only a 20 % reduction in CO2 emissions and a 32 % reduction in traffic load. Use of a footprint model and the London Atmospheric Emissions Inventory (LAEI) identified transport and heat and power generation to be the two dominant sources of NOx and CO2 but with significantly different relative contributions for each species. Application of external constraints on NOx and CO2 emissions allowed the reductions in the different sources to be untangled, identifying that transport NOx emissions had reduced by >73 % since 2017. This was attributed in part to the success of air quality policy in central London but crucially due to the substantial reduction in congestion that resulted from pandemic-reduced mobility. Spatial mapping of the fluxes suggests that central London was dominated by point source heat and power generation emissions during the period of reduced mobility. This will have important implications on future air quality policy for NO2 which, until now, has been primarily focused on the emissions from diesel exhausts

Relationship between the Duration of Stay in Japan of Malaysian Subjects and the Suppression of Sweat Gland Sensitivity by lontophoretically Applied Acetylcholine

Tropical African and Thai Subjects regulate core temperature with less amount of sweat against heat compared to temperate Japanese subjects. Reduced sweating in tropical subjects was attributed to suppression of both central and peripheral sudomotor mechanisms. The objective of the study is to compare the local sweating response activated by acetycholine (ACh) applied iontophoretically among the Malaysians (n=12) of varying duration of stay in Japan. Based on their length of stay, Malaysian subjects were divided into 2 groups, MS (n=6) with a duration of stay of 3 to 15 months and ML (n=6) with 27 to 60 months. ACh, the primary transmitter for sudomotor innervation, was iontophoretically administered on the forearm. Sweating response elicited directly (DIR) and indirectly via axon reflex (AXR) were evaluated by quantitative sudomotor axon reflex test. Although the sweat onset time was tend to reduce with longer duration of stay among Malaysian subjects, there was no significant difference among the two groups. AXR (1), sweat volume elicited by axon reflex for 0-5 min was 0.96ﾂｱ0.11 mg/cm2 in MS and 1.40ﾂｱ0.23 mg/cm2 in ML. AXR(2), that for 6-11 min, was 1.09ﾂｱ0.13 mg/cm2 and 1.45ﾂｱ0.20 mg/cm2. DIR, sweat volume directly induced by ACh for 6-11 min, was 3.40ﾂｱ0.18 mg/cm2 and 3.96ﾂｱ0. 24 mg/cm2 in MS and ML , respectively. A slight positive correlation between DIR and the duration of stay in Japan was observed in Malaysian subjects, though not significant (p=0.14). From these results, suppressed neuroglandular response to ACh was confirmed in Malaysians. It is suggested that long-term heat-acclimatization acquired in tropical subjects may decay after immigration to temperate area

Modelling chemistry in the nocturnal boundary layer above tropical rainforest and a generalised effective nocturnal ozone deposition velocity for sub-ppbv NOx conditions

Measurements of atmospheric composition have been made over a remote rainforest landscape. A box model has previously been demonstrated to model the observed daytime chemistry well. However the box model is unable to explain the nocturnal measurements of relatively high [NO] and [O3], but relatively low observed [NO2]. It is shown that a one-dimensional (1-D) column model with simple O3 -NOx chemistry and a simple representation of vertical transport is able to explain the observed nocturnal concentrations and predict the likely vertical profiles of these species in the nocturnal boundary layer (NBL). Concentrations of tracers carried over from the end of the night can affect the atmospheric chemistry of the following day. To ascertain the anomaly introduced by using the box model to represent the NBL, vertically-averaged NBL concentrations at the end of the night are compared between the 1-D model and the box model. It is found that, under low to medium [NOx] conditions (NOx <1 ppbv), a simple parametrisation can be used to modify the box model deposition velocity of ozone, in order to achieve good agreement between the box and 1-D models for these end-of-night concentrations of NOx and O3. This parametrisation would could also be used in global climate-chemistry models with limited vertical resolution near the surface. Box-model results for the following day differ significantly if this effective nocturnal deposition velocity for ozone is implemented; for instance, there is a 9% increase in the following day’s peak ozone concentration. However under medium to high [NOx] conditions (NOx > 1 ppbv), the effect on the chemistry due to the vertical distribution of the species means no box model can adequately represent chemistry in the NBL without modifying reaction rate constants

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays