Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.     

Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K_m for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.     

DNA demethylation in erythroleukaemia cells

Despite a fall in the proportion of CGs methylated, evidence has not been obtained for significant demethylation of prelabelled DNA when mouse erythroleukaemia cells are induced to differentiate. There is, however, a delay in the methylation of the DNA that is synthesised in the early period of induction, leading to its undermethylation by 30-50% and this may be a contributory cause of the observed fall in CG methylation.     

Improved Glucose Control and Reduced Body Weight in Rodents with Dual Mechanism of Action Peptide Hybrids

Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lep^ob/Lep ^ob mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (Hb_A1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.     

Parents, predators, parasites, and the evolution of eggshell colour in open nesting birds

The colourful surface of birds’ eggshells varies dramatically between species, but the selective pressures driving this variation remain poorly understood. We used a large comparative dataset to test several hypotheses proposed to explain the evolution of eggshell colouration. We tested the hypothesis that predation pressure might select for cryptic eggshells by examining the relationship between predation rate and egg colouration. We found that predation rates were positively related to eggshell brightness. The blackmail hypothesis suggests that females lay colourful eggshells to coerce males into providing additional care during incubation to keep colourful eggs covered. According to this hypothesis, conspicuous eggs should be found in situations with high risk of visual detection from predators or brood parasites. In support of this hypothesis, proportional blue-green chroma was positively related to parasitism risk, and eggs with higher proportional blue-green chroma or higher ultraviolet chroma received higher combined parental nest attendance during the incubation period. The sexual signalling hypothesis states that blue-green colour indicates female quality; however, we did not find that blue-green eggshell colour was greater in species where males participate in any form of parental care, and relative male provisioning was unrelated to blue-green eggshell chroma. We found some support for the hypothesis that brood parasitism may select for high inter-clutch variation in eggshell colour to facilitate egg recognition. In our dataset, parasitism risk was negatively related to inter-clutch repeatability of blue-green chroma. Our study highlights the diversity of selection pressures acting on the evolution of eggshell colour in birds and provides suggestions for novel areas of future key research direction.     

Nitrogen dynamics following field application of biochar in a temperate North American maize-based production system

Background and aims

Biochar additions to tropical soils have been shown to reduce N leaching and increase N use efficiency. No studies exist verifying reduced N leaching in field experiments on temperate agricultural soils or identifying the mechanism for N retention.

Methods

Biochar derived from maize stover was applied to a maize cropping system in central New York State at rates of 0, 1, 3, 12, and 30 t?ha^-1 in 2007. Secondary N fertilizer was added at 100, 90, 70, and 50 % of the recommended rate (108 kg N ha^-1). Nitrogen fertilizer enriched with ¹⁵?N was applied in 2009 to the 0 and 12 t?ha^-1 of biochar at 100 and 50 % secondary N application.

Results

Maize yield and plant N uptake did not change with biochar additions (p?>?0.05; n?=?3). Less N (by 82 %; p?<?0.05) was lost after biochar application through leaching only at 100 %?N fertilization. The reason for an observed 140 % greater retention of applied ¹⁵?N in the topsoil may have been the incorporation of added ¹⁵?N into microbial biomass which increased approximately three-fold which warrants further research. The low leaching of applied fertilizer ¹⁵?N (0.42 % of applied N; p?<?0.05) and comparatively high recovery of applied ¹⁵?N in the soil (39 %) after biochar additions after one cropping season may also indicate greater overall N retention through lower gaseous or erosion N losses with biochar.

Conclusions

Addition of biochar to fertile soil in a temperate climate did not improve crop growth or N use efficiency, but increased retention of fertilizer N in the topsoil.     

The Impact of the West Africa Ebola Outbreak on Obstetric Health Care in Sierra Leone

Background

As Sierra Leone celebrates the end of the Ebola Virus Disease (EVD) outbreak, we can begin to fully grasp its impact on already weak health systems. The EVD outbreak in West Africa forced many hospitals to close down or reduce their activity, either to prevent nosocomial transmission or because of staff shortages. The aim of this study is to assess the potential impact of EVD on nationwide access to obstetric care in Sierra Leone.

Methods and Findings

Community health officers collected weekly data between January 2014—May 2015 on in-hospital deliveries and caesarean sections (C-sections) from all open facilities (public, private for-profit and private non-profit sectors) offering emergency obstetrics in Sierra Leone. This was compared to official data of EVD cases per district. Logistic and Poisson regression analyses were used to compute risk and rate estimates. Nationwide, the number of in-hospital deliveries and C-sections decreased by over 20% during the EVD outbreak. The decline occurred early on in the EVD outbreak and was mainly attributable to the closing of private not-for-profit hospitals rather than government facilities. Due to difficulties in collecting data in the midst of an epidemic, limitations of this study include some missing data points.

Conclusions

Both the number of in-hospital deliveries and C-sections substantially declined shortly after the onset of the EVD outbreak. Since access to emergency obstetric care, like C-sections, is associated with decreased maternal mortality, many women are likely to have died due to the reduced access to appropriate care during childbirth. Future research on indirect health effects of health system breakdown should ideally be nationwide and continue also into the recovery phase. It is also important to understand the mechanisms behind the deterioration so that important health services can be reestablished.