Residues Arg283, Arg285, and Ile287 in the Nucleotide Binding Pocket of Bovine Viral Diarrhea Virus NS5B RNA Polymerase Affect Catalysis and Fidelity

Residues Arg283, Arg285, and Ile287 are highly conserved amino acids in bovine viral diarrhea virus RNA polymerase (BVDV RdRp) and RdRps from related positive-strand RNA viruses. This motif is an important part of the binding pocket for the nascent RNA base pair during initiation and elongation. We found that replacement of the arginines with alanines or more conserved lysines or replacement of isoleucine with alanine or valine alters the ability of the mutant RdRps to incorporate ribonucleotides efficiently. The reduced RdRp activity stems from both decreased ribonucleotide binding and decreased catalytic efficiency in both primer-dependent and de novo initiation, as shown by kinetic studies. In line with other studies on flaviviral RdRps, our data suggest that Arg283 and Ile287 may be implicated in ribonucleotide binding and positioning of the template base in the active site. Arg285 appears to be involved directly in the selection of cognate nucleotide. The findings for Arg285 and Ile287 mutants also agree with similar data from picornavirus RdRps.     

Continuing decline of a keystone tree species in the Kruger National Park,South Africa

Sclerocarya birrea subsp. caffra (marula), a keystone large tree species in southern Africa and heavily utilized by people and herbivores, is declining at an unprecedented rate in the Kruger National Park (KNP), South Africa. Due to increasing concerns over large tree declines in the KNP, mirroring those in eastern Africa over the last 50 years, and local extinctions of marula populations in the north‐western KNP, adult trees in the southern KNP were monitored between 2001 and 2010. Annual mortality rates across all adult size classes ranged between 3.2 and 4.6%. Loss of adult marula trees in some areas over the last decade exceeded 25%. Rainfall had a strong influence on mortality rates temporally and spatially, but the distance to the nearest perennial river had little influence. Overall, marula populations continue to decline and further local extinctions are possible. Given the clearly unacceptable trends of decline, the next step is to utilize this information (i) to develop predictive models for the future dynamics of marula populations incorporating all drivers; (ii) to advocate active management of refugia within the park where marula populations are protected from elephant impacts; and in combination with (iii) continued monitoring.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Cardiomyopathy Mutations in the Tail of β-Cardiac Myosin Modify the Coiled-coil Structure and Affect Integration into Thick Filaments in Muscle Sarcomeres in Adult Cardiomyocytes

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301–1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Do foods imported into the UK have a greater environmental impact than the same foods produced within the UK?

Purpose

This study of seven foods assessed whether there are modes or locations of production that require significantly fewer inputs, and hence cause less pollution, than others. For example, would increasing imports of field-grown tomatoes from the Mediterranean reduce greenhouse gas (GHG) emissions by reducing the need for production in heated greenhouses in the UK, taking account of the additional transport emissions? Is meat production in the UK less polluting than the import of red meat from the southern hemisphere?

Methods

We carried out a life-cycle inventory for each commodity, which quantified flows relating to life-cycle assessment (LCA) impact categories: primary energy use, acidification, eutrophication, abiotic resource use, pesticide use, land occupation and ozone depletion. The system boundary included all production inputs up to arrival at the retail distribution centre (RDC). The allocation of production burdens for meat products was on the basis of economic value. We evaluated indicator foods from which it is possible to draw parallels for foods whose production follows a similar chain: tomatoes (greenhouse crops), strawberries (field-grown soft fruit), apples (stored for year-round supply or imported during spring and summer), potatoes (early season imports or long-stored UK produce), poultry and beef (imported from countries such as Brazil) and lamb (imported to balance domestic spring–autumn supply).

Results and discussion

Total pre-farm gate global warming potential (GWP) of potatoes and beef were less for UK production than for production in the alternative country. Up to delivery to the RDC, total GWP were less for UK potatoes, beef and apples than for production elsewhere. Production of tomatoes and strawberries in Spain, poultry in Brazil and lamb in New Zealand produced less GWP than in the UK despite emissions that took place during transport. For foods produced with only small burdens of GWP, such as apples and strawberries, the burden from transport may be a large proportion of the total. For foods with inherently large GWP per tonne, such as meat products, burdens arising from transport may only be a small proportion of the total.

Conclusions

When considering the GWP of food production, imports from countries where productivity is greater and/or where refrigerated storage requirement is less will lead to less total GWP than axiomatic preference for local produce. However, prioritising GWP may lead to increases in other environmental burdens, in particular leading to both greater demands on and decreasing quality of water resources.     

Risk Factors and Screening for Trypanosoma cruzi Infection of Dutch Blood Donors

Background

Blood donors unaware of Trypanosoma cruzi infection may donate infectious blood. Risk factors and the presence of T. cruzi antibodies in at-risk Dutch blood donors were studied to assess whether specific blood safety measures are warranted in the Netherlands.

Methodology

Birth in a country endemic for Chagas disease (CEC), having a mother born in a CEC, or having resided for at least six continuous months in a CEC were considered risk factors for T. cruzi infection. From March through September 2013, risk factor questions were asked to all donors who volunteered to donate blood or blood components. Serum samples were collected from donors reporting one or more risk factors, and screened for IgG antibodies to T. cruzi by EIA.

Results

Risk factors for T. cruzi infection were reported by 1,426 of 227,278 donors (0.6%). Testing 1,333 at-risk donors, none (0.0%; 95%, CI 0.0–0.4%) was seroreactive for IgG antibodies to T. cruzi. A total of 472 donors were born in a CEC; 553 donors reported their mother being born in a CEC; and 1,121 donors reported a long-term stay in a CEC. The vast majority of reported risk factors were related to Suriname and Brazil. Overall, the participants resided for 7,694 years in CECs, which equals 2.8 million overnight stays. Of those, 1.9 million nights were spent in Suriname.

Conclusions/Significance

Asymptomatic T. cruzi infection appears to be extremely rare among Dutch blood donors. Blood safety interventions to mitigate the risk of T. cruzi transmission by transfusion would be highly cost-ineffective in the Netherlands, and are thus not required.