Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.     

Insecticidal Activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and Other Lepidopteran Pests

Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.     

Conversion efficiency of bank vole prion protein in vitro is determined by residues 155 and 170, but does not correlate with the high susceptibility of bank voles to sheep scrapie in vivo

The misfolded infectious isoform of the prion protein (PrP(Sc)) is thought to replicate in an autocatalytic manner by converting the cellular form (PrP(C)) into its pathogenic folding variant. The similarity in the amino acid sequence of PrP(C) and PrP(Sc) influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrP(C) to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie. Different conversion efficiencies obtained with bank vole and mouse PrP(C) in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrP(C) in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrP(C) primary sequence is decisive for its convertibility to the strain-specific PrP(Sc) conformation.     

The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.     

Understanding Low-Acuity Visits to the Pediatric Emergency Department

Background

Canadian pediatric emergency department visits are increasing, with a disproportionate increase in low-acuity visits locally (33% of volume in 2008-09, 41% in 2011-12). We sought to understand: 1) presentation patterns and resource implications; 2) parents’ perceptions and motivations; and 3) alternate health care options considered prior to presenting with low-acuity problems.

Methods

We conducted a prospective cohort study at our tertiary pediatric emergency department serving two provinces to explore differences between patients with and without a primary care provider. During four, 2-week study periods over 1 year, parents of low-acuity visits received an anonymous survey. Presentation times, interventions, diagnoses and dispositions were captured on a data collection form linked to the survey by study number.

Results

Parents completed 2,443 surveys (74.1% response rate), with survey-data collection form pairs available for 2,146 visits. Overall, 89.7% of respondents had a primary care provider; 68% were family physicians. Surprisingly, 40% of visits occurred during weekday office hours and 27.3% occurred within 4 hours of symptom onset; 67.5% of those early presenters were for injuries. Few parents sought care from their primary care provider (25%), health information line (20.7%), or urgent care clinic (18.5%); 36% reported that they believed their child’s problem required the emergency department. Forty-five percent required only a history, physical exam and reassurance; only 11% required an intervention not available in an office setting. Patients without a primary care provider were significantly more likely to present during weekday office hours (p = 0.003), have longer symptom duration (p<0.001), and not know of other options (p = 0.001).

Conclusions

Many parents seek pediatric emergency department care for low-acuity problems despite their child having a primary care provider. Ensuring timely access to these providers may help reduce pediatric emergency department overuse. Educational initiatives should inform parents about low-acuity problems and where appropriate care can/should be accessed.     

Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL toward fucose and mannose

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.     

BDNF VAL66MET polymorphism and memory decline across the spectrum of Alzheimer's disease

The brain-derived neurotrophic factor (BDNF) Val66Met (rs6265) polymorphism has been shown to moderate the extent to which memory decline manifests in preclinical Alzheimer's disease (AD). To date, no study has examined the relationship between BDNF and memory in individuals across biologically confirmed AD clinical stages (i.e., Aβ+). We aimed to understand the effect of BDNF on episodic memory decline and clinical disease progression over 126 months in individuals with preclinical, prodromal and clinical AD. Participants enrolled in the Australian Imaging, Biomarkers and Lifestyle (AIBL) study who were Aβ + (according to positron emission tomography), and cognitively normal (CN; n = 238), classified as having mild cognitive impairment (MCI; n = 80), or AD (n = 66) were included in this study. Cognition was evaluated at 18 month intervals using an established episodic memory composite score over 126 months. We observed that in Aβ + CNs, Met66 was associated with greater memory decline with increasing age and were 1.5 times more likely to progress to MCI/AD over 126 months. In Aβ + MCIs, there was no effect of Met66 on memory decline or on disease progression to AD over 126 months. In Aβ + AD, Val66 homozygotes showed greater memory decline, while Met66 carriers performed at a constant and very impaired level. Our current results illustrate the importance of time and disease severity to clinicopathological models of the role of BDNF Val66Met in memory decline and AD clinical progression. Specifically, the effect of BDNF on memory decline is greatest in preclinical AD and reduces as AD clinical disease severity increases.     

Microsomal epoxide hydrolase polymorphisms and lung cancer risk: a quantitative review

To investigate the role of microsomal epoxide hydrolase (mEH) polymorphisms in the aetiology of lung cancer and to assess the interaction between mEH polymorphisms and smoking, we performed a meta-analysis of seven published studies, which included 2078 cases and 3081 controls, and a pooled analysis of eight studies (four published and four unpublished at that time) with a total of 986 cases and 1633 controls. The combined metaanalysis odds ratios (ORs) were 0.98 (95% confidence interval [CI] = 0.72-1.35) for polymorphism at amino acid 113 in exon 3 (His/His versus Tyr/Tyr genotype) and 1.00 (95% CI= 0.71-1.41) for polymorphism at amino acid 139 in exon 4 (Arg/Arg versus His/ His genotype). In the pooled analysis, we observed a significant decrease in lung cancer risk (OR = 0.70, 95% CI = 0.51-0.96) for exon 3 His/His genotype after adjustment for age, sex, smoking and centre. The protective effect of exon 3 polymorphism seems stronger for adenocarcinoma of the lung than for other histological types. The OR for high predicted mEH activity, compared with low activity, was 1.54 (95% CI = 0.77-3.07) in the meta analysis and 1.18 (95% CI = 0.92-1.52) in the pooled analysis. We did not find a consistent modification of the carcinogenic effect of smoking according to mEH polymorphism, although the risk of lung cancer decreased among never smokers with high mEH activity and among heavy smokers with the exon 3 His/His genotype. In conclusion, this study suggests a possible effect of mEH polymorphisms at exon 3 in modulating lung cancer. If present, this effect may vary among different populations, possibly because of interaction with genetic or environmental factors.     

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.     

Trainable High Resolution Melt Curve Machine Learning Classifier for Large-Scale Reliable Genotyping of Sequence Variants

High resolution melt (HRM) is gaining considerable popularity as a simple and robust method for genotyping sequence variants. However, accurate genotyping of an unknown sample for which a large number of possible variants may exist will require an automated HRM curve identification method capable of comparing unknowns against a large cohort of known sequence variants. Herein, we describe a new method for automated HRM curve classification based on machine learning methods and learned tolerance for reaction condition deviations. We tested this method in silico through multiple cross-validations using curves generated from 9 different simulated experimental conditions to classify 92 known serotypes of Streptococcus pneumoniae and demonstrated over 99% accuracy with 8 training curves per serotype. In vitro verification of the algorithm was tested using sequence variants of a cancer-related gene and demonstrated 100% accuracy with 3 training curves per sequence variant. The machine learning algorithm enabled reliable, scalable, and automated HRM genotyping analysis with broad potential clinical and epidemiological applications.