Begomovirus and Associated Satellite Components Infecting Cluster Bean (Cyamopsis tetragonoloba) in Pakistan

Cluster bean (Cyamopsis tetragonoloba) is a legume that is grown widely on the Indian subcontinent. Leaf curl symptoms of cluster bean plants collected in the Punjab, Pakistan, were shown to be associated with the begomovirus Papaya leaf curl virus; the first time this virus has been identified infecting cluster bean in Pakistan. The virus was shown to be associated with Tomato leaf curl betasatellite. Additionally, some cluster bean plants were shown to also harbour Cotton leaf curl Multan alphasatellite. The significance of these findings is discussed.     

Sooty Falcon Falco concolor reproduction and population dynamics on the islands in the Sea of Oman

Knowledge of demographic parameters affecting population dynamics is critical to the formulation of effective conservation strategies. Sooty Falcon Falco concolor is a little‐studied, Near‐threatened species; estimates of global population size and trend for this species are uncertain. They lay eggs during mid‐summer and sometimes nest in colonies. This unusual breeding ecology suggests that demographic parameters driving their population growth rate may differ from those of most other falcons. We studied Sooty Falcon reproduction at breeding aggregations on Fahal Island and the Daymaniyat islands in the Sea of Oman during 2007–2014, modelled population growth and identified important life history parameters using elasticity analysis. The mean (± se) clutch and brood size was 2.83 ± 0.06 and 2.11 ± 0.07, respectively. Overall, 11.7% of nests failed between the egg and nestling stages, and the failure rate differed significantly between Fahal and the Daymaniyats, and across years. The mean proportion of eggs that hatched annually was 0.66 ± 0.02, and broods were significantly smaller on the Daymaniyats than on Fahal. Falcons on Fahal Island had a higher rate of hatching, a higher rate of nests that produced at least one chick, and produced more chicks per nest than on the Daymaniyats. We suggest that Fahal's proximity to the mainland gives breeding Sooty Falcons access to a more plentiful and stable source of food, especially during the period between arrival from the wintering grounds and the onset of the autumn migration of prey birds, resulting in the better reproductive rates for falcons on Fahal Island, relative to those on the Daymaniyat Islands. The annual asymptotic population growth rate (λ) was 0.87 (95% confidence interval (CI) 0.75–0.99), suggesting a declining population, although Sooty Falcons enjoyed a slightly higher population growth rate on Fahal than on the Daymaniyats. Because our study population is on the edge of the breeding range and is isolated from other breeding areas, measures to improve reproductive success of Sooty Falcons breeding on the islands in the Sea of Oman could be important for conservation of Sooty Falcons in Oman.     

Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we have developed thiolated gelatin nanoparticles that can release the payload in the highly reducing environment, such as in response to glutathione. Thiolated gelatin was synthesized by covalent modification of the primary amino groups of Type B gelatin using 2-iminothiolane (Traut's reagent). The degree of thiolation of the polymers ranged from 0 to 43.71 mmol of reduced sulfhydryl (SH) groups when the amount of 2-iminothiolane was increased up to 100 mg per gram of the biopolymer. Cytotoxicity evaluations carried out by the formazan (MTS) assay showed that the thiolated gelatin prepared with 20 mg and 40 mg of 2-iminothiolane (SHGel-20 and SHGel-40) per gram of gelatin had comparable cell viability profile to that of the unmodified gelatin. In vitro release studies of fluorescein isothiocyanate (FITC)-labeled dextran (mol wt. 70 000 Da), when encapsulated in gelatin and thiolated gelatin nanoparticles (150-250 nm in diameter), was found to be affected by the presence of glutathione (GSH) in the medium. The presence of GSH was found to enhance the release by about 40% in case of thiolated gelatin and about 20% in gelatin nanoparticles under similar conditions of temperature and GSH concentrations. Qualitative and quantitative analysis of transfection in NIH-3T3 murine fibroblast cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by fluorescence confocal microscopy and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 h. Quantitative results from FACS showed that the thiolated gelatin nanoparticles (SHGel-20) were significantly more effective in transfecting NIH-3T3 cells than other carrier systems examined. The results of this study show that thiolated gelatin nanoparticles would serve as a biocompatible intracellular delivery system that can release the payload in a highly reducing environment.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Epidemiology of severe trauma among status Aboriginal Canadians: a population-based study

Background

Aboriginal Canadians are considered to be at increased risk of major trauma. However, population-based studies characterizing the distribution, determinants and outcomes of major trauma in this group are lacking. We sought to measure the impact of ethnicity, as reflected by Aboriginal status, on the incidence of severe trauma and to broadly define the epidemiologic characteristics of severe trauma among status Aboriginal Canadians in a large health region.

Methods

This population-based, observational study involves all adults (people ≥ 16 years) resident in the Calgary Health Region between Apr. 1, 1999, and Mar. 31, 2002. Stratification of the population into status Aboriginal Canadians and the reference population was performed by Alberta Health and Wellness using an alternate premium arrangement field within the personal health care number. Injury incidence was determined by identifying all injuries with severity scores of 12 or greater in the Alberta Trauma Registry, regional corporate data and the Office of the Medical Examiner.

Results

Aboriginal Canadians were at much higher risk than the reference population in the Calgary Health Region of sustaining severe trauma (257.2 v. 68.8 per 100 000; relative risk [RR] 3.7, 95% confidence interval [CI] 3.0–4.6). Aboriginal Canadians were found to be at significantly increased risk of injuries resulting from motor vehicle crashes (RR 4.8, 95% CI 3.5–6.5), assault (RR 11.1, 95% CI 6.2–18.6) and traumatic suicide (RR 3.1, 95% CI 1.4–6.1). A trend toward higher median injury severity scores was observed among Aboriginal Canadians (21 v. 18, p = 0.09). Although the case-fatality rate among Aboriginal Canadians was less than half that in the reference population (14/93 [15%] v. 531/1686 [31%], p < 0.0001), population mortality was almost 2 times greater (RR = 1.8, 95% CI 1.0–3.0, p = 0.046).

Interpretation

Severe trauma disproportionately affects Aboriginal Canadians.In Canada, injury is the leading cause of death among people under the age of 45 and the leading cause of potential years of life lost.¹ Although difficult to quantify, the cost of injury was estimated to be at least $12.7 billion in 1998.² Trauma has been known, even in industrialized countries, to disproportionately affect the most marginalized members of society.³ Aboriginal Canadians are considered to be particularly at risk, and data showing alarming patterns of trauma mortality in this group are beginning to emerge. Unfortunately, the number of studies looking at injury risk among Aboriginal Canadians is small,⁴ and little attention has been paid to quantifying the risk of nonfatal injury. Better understanding of the nature of trauma risk and outcome among Aboriginal Canadians could lead to more effective prevention and treatment strategies.In this study, we used a population-based design in an attempt to quantify the impact of injury, both fatal and nonfatal, on the Aboriginal community in a large, heterogeneous Canadian region with over 1 million urban and rural inhabitants. We sought to measure the impact of ethnicity (defined by registered status within the definition of the Indian Act⁵) on the incidence of severe trauma and to broadly define the epidemiologic characteristics of severe trauma among status Aboriginal Canadians.     

Cytosolic NADH redox and thiol oxidation regulate pulmonary arterial force through ERK MAP kinase

An ERK MAP kinase-mediated contractile mechanism previously reported to be activated by peroxide and stretch in bovine coronary arteries is shown in this study to be present in endothelium-denuded bovine pulmonary arteries and subject to regulation by modulation of cytosolic NAD(H) redox through the lactate dehydrogenase reaction. Although our previous work identified an acute PO2-dependent peroxide-mediated relaxation of bovine pulmonary arteries on exposure to lactate, a 30-min treatment with 10 mM lactate enhanced ERK phosphorylation and increased force generation to 30 mM KCl. Hypoxia inhibited these responses to lactate. Increases in ERK phosphorylation and the enhancement of force generation by lactate and stretch are attenuated in the presence of inhibitors of Nox oxidase (0.1 mM apocynin) or ERK activation (10 microM PD-98059) and by 0.1 mM ebselen. Additionally, incubation of pulmonary arteries with 10 mM pyruvate lowered basal levels of ERK phosphorylation, and it inhibited both the ERK phosphorylation and the enhancement in force generation to 30 mM KCl caused by stretch. Treatment of pulmonary arteries with the thiol oxidant diamide (1 microM) elicited what appears to be a peroxide-independent increase in force and ERK phosphorylation that were both attenuated by PD-98059. Thus pulmonary arteries possess a peroxide-elicited contractile mechanism involving ERK MAP kinase, which is stimulated by stretch and regulated through the control of Nox oxidase activity by the availability of cytosolic NADH.     

Percutaneous permeation of enantiomers and racemates of chiral drugs and prediction of their flux ratios using thermal data: a pharmaceutical perspective

Albeit pharmacological, pharmacokinetic, and toxicological differences between enantiomeric pairs or between the pure enantiomers and racemate of chiral drugs are known to exist for decades, we are just beginning to realize that there are apparent differences between these species with respect to their percutaneous permeation as well. Such differences in permeation are likely to be enhanced when chiral drugs are formulated with chiral excipients, necessitating a careful assessment of the effect of formulation excipients on the permeation as well as the overall therapeutic outcomes. The in vitro transport data from the preclinical investigations, using laboratory animal models and/or in vitro cell culture systems, must be carefully validated in vivo as there are differences between these models and the human skin. Mathematical models such as MTMT that utilize the interdependence of certain physicochemical characteristics and percutaneous permeability have a predictive value in assessing the flux behavior of enantiomers and racemates.     

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle

Calcineurin mediates repression of plasma membrane Ca²⁺-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which ⁴⁵Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca²⁺ concentrations ([Ca²⁺]_i) increased twofold, and real-time RT-PCR and Western blot revealed a 40% decrease in PMCA4 expression levels from G₀ to G₁/S in the cell cycle, where PMCA4 constituted 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G₀ to G₁/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G₁/S. By contrast, Ca²⁺-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G₀ to G₁/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G₁/S-associated repression of PMCA4. These data show that G₁/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca²⁺]_i through alternate targets. calcineurin; c-Myb; plasma membrane Ca²⁺-ATPase-4; cell cycle     

High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane

We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations. The ability to cleave PDT-Bimane off the protein enables rapid determination of these parameters, and positions it as an ideal fluorophore for automated, high-throughput structural studies of protein folding, the detection of protein-protein interactions, and the monitoring of real-time conformational changes.