How to harvest the left internal mammary artery-a randomized controlled trial.

OBJECTIVES: It is uncertain whether Thunderbeat has a place in harvesting the left internal mammary artery (LIMA) and whether skeletonization is superior to pedicle-harvested LIMA. Some investigations have shown improved flowrates in the skeletonized graft. The aim of this study was to compare 3 groups of harvesting techniques: Pedicled, surgical skeletonized and skeletonized with Thunderbeat in terms of flow rates in the LIMA and postoperative in-hospital outcomes. METHODS: Patients undergoing coronary artery bypass grafting with the LIMA to the anterior descending artery were randomized to pedicled (n = 56), surgical skeletonized (n = 55) and skeletonized with Thunderbeat (n = 54). Main outcomes were blood flow and pulsatility index in the graft. RESULTS: No statistical difference between groups regarding flow in LIMA or pulsatility index. Similarly, no difference in postoperative bleeding or days of hospitalization. The duration of harvesting was faster for the pedicled technique compared with surgical skeletonized and skeletonized with Thunderbeat [mean total min: pedicled 20.2 min standard deviation (SD) ± 5.4; surgical skeletonized 28.6 min SD ± 8.7; skeletonized with Thunderbeat 28.3 min SD ± 9.11, P < 0.001]. No grafts discarded due to faulty harvesting and there was no graft failure within hospital stay. CONCLUSIONS: We found no difference between the harvesting methods except for a significantly faster harvesting time with the pedicled technique. However, non-touch skeletonized LIMA harvesting with Thunderbeat seems to be an effective alternative to traditional surgical skeletonized LIMA. The future will reveal whether patency is harvesting dependent. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05562908.

Site-directed genotype screening for elimination of antinutritional saponins in quinoa seeds identifies TSARL1 as a master controller of saponin biosynthesis selectively in seeds.

Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.

Biosynthesis and biotechnological production of the anti-obesity agent celastrol.

Obesity is a major health risk still lacking effective pharmacological treatment. A potent anti-obesity agent, celastrol, has been identified in the roots of Tripterygium wilfordii. However, an efficient synthetic method is required to better explore its biological utility. Here we elucidate the 11 missing steps for the celastrol biosynthetic route to enable its de novo biosynthesis in yeast. First, we reveal the cytochrome P450 enzymes that catalyse the four oxidation steps that produce the key intermediate celastrogenic acid. Subsequently, we show that non-enzymatic decarboxylation-triggered activation of celastrogenic acid leads to a cascade of tandem catechol oxidation-driven double-bond extension events that generate the characteristic quinone methide moiety of celastrol. Using this acquired knowledge, we have developed a method for producing celastrol starting from table sugar. This work highlights the effectiveness of combining plant biochemistry with metabolic engineering and chemistry for the scalable synthesis of complex specialized metabolites.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

The saponin bomb: a nucleolar-localized ß-glucosidase hydrolyzes triterpene saponins in Medicago truncatula.

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen or herbivore attack. Via co-expression analysis with public transcriptomes, we determined that the model legume Medicago truncatula has evolved a two-component system composed of a ß-glucosidase, denominated G1, and triterpene saponins, which are physically separated from each other in intact cells. G1 expression is root-specific, stress-inducible, and coregulated with that of the genes encoding the triterpene saponin biosynthetic enzymes. However, the G1 protein is stored in the nucleolus and is released and united with its typically vacuolar-stored substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates. We dub this two-component system the saponin bomb, in analogy with the mustard oil and cyanide bombs, commonly used to describe the renowned ß-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots.

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated.

A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination.

Eukaryotic cells use G-protein coupled receptors to sense diverse signals, ranging from chemical compounds to light. Here, we exploit the remarkable sensing capacity of G-protein coupled receptors to construct yeast-based biosensors for real-life applications. To establish proof-of-concept, we focus on cannabinoids because of their neuromodulatory and immunomodulatory activities. We construct a CB2 receptor-based biosensor, optimize it to achieve high sensitivity and dynamic range, and prove its effectiveness in three applications of increasing difficulty. First, we screen a compound library to discover agonists and antagonists. Second, we analyze 54 plants to discover a new phytocannabinoid, dugesialactone. Finally, we develop a robust portable device, analyze body-fluid samples, and confidently detect designer drugs like JWH-018. These examples demonstrate the potential of yeast-based biosensors to enable diverse applications that can be implemented by non-specialists. Taking advantage of the extensive sensing repertoire of G-protein coupled receptors, this technology can be extended to detect numerous compounds.

Reciprocal mutations of two multifunctional ß-amyrin synthases from Barbarea vulgaris shift α/ß-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), ß-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce ß-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of ß-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Cascading Effects of Root Microbial Symbiosis on the Development and Metabolome of the Insect Herbivore Manduca sexta L.

Root mutualistic microbes can modulate the production of plant secondary metabolites affecting plant-herbivore interactions. Still, the main mechanisms underlying the impact of root mutualists on herbivore performance remain ambiguous. In particular, little is known about how changes in the plant metabolome induced by root mutualists affect the insect metabolome and post-larval development. By using bioassays with tomato plants (Solanum lycopersicum), we analyzed the impact of the arbuscular mycorrhizal fungus Rhizophagus irregularis and the growth-promoting fungus Trichoderma harzianum on the plant interaction with the specialist insect herbivore Manduca sexta. We found that root colonization by the mutualistic microbes impaired insect development, including metamorphosis. By using untargeted metabolomics, we found that root colonization by the mutualistic microbes altered the secondary metabolism of tomato shoots, leading to enhanced levels of steroidal glycoalkaloids. Untargeted metabolomics further revealed that root colonization by the mutualists affected the metabolome of the herbivore, leading to an enhanced accumulation of steroidal glycoalkaloids and altered patterns of fatty acid amides and carnitine-derived metabolites. Our results indicate that the changes in the shoot metabolome triggered by root mutualistic microbes can cascade up altering the metabolome of the insects feeding on the colonized plants, thus affecting the insect development.

An Independent Evolutionary Origin for Insect Deterrent Cucurbitacins in Iberis amara.

Pieris rapae and Phyllotreta nemorum are Brassicaceae specialists, but do not feed on Iberis amara spp. that contain cucurbitacins. The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Using de novo assembled transcriptomics from I. amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase (IaCPQ). In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyze the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the draft genome assembly of I. amara showed that the IaCPQ was localized to the same scaffold together with CYP708A15 but spanning over 100 kb, this contrasts with the highly organized cucurbitacin gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This study thus provides new insight into the mechanism of recurrent evolution and diversification of a plant defensive chemical.

Phenotypic plasticity in chemical defence of butterflies allows usage of diverse host plants.

Host plant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keep most herbivores at bay, but not the larvae of Heliconius butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus and H. melpomene rosina have remarkable plasticity in their chemical defences. When feeding on Passiflora species with cyanogenic compounds that they can readily sequester, both species downregulate the biosynthesis of these compounds. By contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a fitness cost for the more specialist H. m. rosina, as adult size and weight for this species negatively correlate with biosynthesis levels, but not for the more generalist H. c. chioneus. By contrast, H. m rosina has increased performance when sequestration is possible on its specialized host plant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential hosts within the Passiflora genus, while maintaining their chemical defences.

Trigger team activation in the emergency department at a tertiary university hospital.

INTRODUCTION The aim of this study was to describe the diversity in the incidence, diagnosis and prognosis of patients met by the trigger teams for trauma, STEMI, stroke and medical emergency patients (MEP). METHODS This was a single-centre, retrospective cohort study of all patients admitted to Odense University Hospital (OUH) from November 2012 to September 2015 with trauma, STEMI, stroke or MEP trigger team activation on arrival. OUH is a tertiary referral centre for patients with severe trauma, STEMI (directly referred to the catheterisation laboratory), stroke and MEP. RESULTS A total of 8,075 trigger team activations were recorded, a median of eight calls per day (range: 1-18), covering 16.7% trauma calls, 28.3% STEMI calls, 19.7% stroke calls and 35.3% MEP calls. This corresponds to 160/100,000 person years (py) trauma calls, 65/100,000 py STEMI calls, 73/100,000 py stroke calls and 339/100,000 py MEP calls. Seven-day mortality was 10% (95% confidence interval (CI): 9-12%) for patients with trauma calls, 6% (95% CI: 5-7%) for STEMI calls, 3% (95% CI: 3-4%) for stroke calls and 16% (95% CI: 15-17%) for MEP calls. Patients from trauma, STEMI and stroke calls were discharged with a diagnosis within 3-5 International Classification of Diseases (version 10) main coding areas, whereas patients from MEP calls had discharge diagnoses within 13 main coding areas. CONCLUSION Patients with MEP calls are more frequent, have a more diverse aetiology and a higher mortality than patients in the other trigger teams. A need exists for further guidelines and research regarding MEP with a view to reducing the high mortality rate among MEP in the future. FUNDING Annmarie Lassen was funded by an unrestricted grant from the philanthropic fund the Tryg Foundation given to the University of Southern Denmark. TRIAL REGISTRATION The study was approved by the Danish Health Authority (Record no. 3-3013-1385/1) and the Danish Data Protection Agency (Record no. 2013-41-2435).

Plant triterpenoids with bond-missing skeletons: biogenesis, distribution and bioactivity.

Covering: up to December 2018 The polycyclic ABCD(E) framework of triterpenoids can miss a single endocyclic C-C bond as a result of a modification of the cyclization cascade that triggers their formation (interrupted- or diverted cascades), or can be the result of post-cyclization ring cleavage by late-stage oxidative modifications (seco-triterpenoids). Because of mechanistic and biogenetic differences, ring opening associated with loss of a skeletal fragment, as in nor-seco-triterpenoids (limonoids, quassinoids), will not be covered, nor will compounds where ring opening is part of a fragmentation cascade or of a multiple diversion from it. Even with these limitations, 342 bond-missing triterpenoids could be retrieved from the literature, with transversal distribution in the plant kingdom. Their structural diversity translates into a variety of biological targets, with dominance of potential applications in the realm of cancer, neuroprotection, and anti-infective therapy. In addition to the bioactivity and chemotaxonomic relevance of bond-missing triterpenoids, current knowledge on the genetic basis of interrupted- and diverted oxidosqualene cyclases will be summarized. This untapped source of enzymes could be useful to selectively modify triterpenoids by metabolic engineering, circumventing the bottlenecks of their isolation (poor yield or inadequate supply chain) to explore new areas of their chemical space.

Variation of chemical compounds in wild Heliconiini reveals ecological factors involved in the evolution of chemical defenses in mimetic butterflies.

Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide-releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well-studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.

Predictive Markers of Atrial Fibrillation in Patients with Transient Ischemic Attack.

BACKGROUND: Atrial fibrillation (AF) is a major cause of cardio-embolism in patients with stroke and transient ischemic attack (TIA). Insertable cardiac monitors (ICM) make long-term monitoring for AF possible, but limited health care resources make patient selection important. AF is associated with atherosclerosis and markers of this could potentially be used to guide AF monitoring. METHODS AND RESULTS: One-hundred fourteen TIA-patients without AF were thoroughly monitored for AF with ECG, 72-hour Holter monitoring and ICM with a median monitoring time of 2.2 years. Patients with AF (n = 18) were significantly older than patients without AF (age 71.1 versus 64.4 years, P = .008) but were otherwise similar in regards to comorbidities. AF patients had significantly thicker carotid intima-media and also more often presence of carotid plaques than patients without AF, but no difference was found after adjusting for age and sex. No difference in noncontrast cardiac CT calculated coronary artery calcium score was found between the 2 groups. Serum biomarkers did not differ between groups, except for brain natriuretic peptide (BNP), where patients with BNP in the upper tertile were more likely to have AF than patients with BNP in the lowest tertile, odds ratio 5.96 (95% confidence interval 1.04-34.07, P = .045). CONCLUSIONS: Carotid intima-media thickness and coronary artery calcium score were poor predictors of AF in patients with TIA. Apart from BNP, the examined biomarkers (hs-CRP, MR-proADM, c-TnI, copeptin) had no predictive value, but larger scale studies are needed to confirm these findings.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.

BACKGROUND: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. RESULTS: To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. CONCLUSION: Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

Statin use and peripheral nerve function-A prospective follow-up study.

PURPOSE: To examine the association between use of statins and risk of deterioration of peripheral nerve function. METHODS: We prospectively followed patients who initiated statin treatment and compared them with statin never-users (non-users). At the time of inclusion and at 1-year follow-up, participants underwent tests for peripheral nerve function (ie nerve conduction studies, quantitative sensory testing), skin biopsies and ratings of symptoms and signs of neuropathy. We selected five tests of nerve function and the intraepidermal nerve fibre density (IENFD) a priori as primary outcomes. We used linear regression to test for differences between statin users and non-users with Holm-Bonferroni-corrected statistical significance level of .05. RESULTS: Comparisons were based on 57 statin users and 46 non-users. Changes in nerve function test results during follow-up were not uniform with regard to direction and were statistically not significant with the exception of IENFD (change in IENFD: statin users 1 fibre/mm vs. non-statin users -2 fibres/mm; P-value = .006). None of the participants developed overt peripheral neuropathy. However, five statin users developed neuropathy-like symptoms and a post hoc analysis showed a significant decrease in vibration sensitivity compared to asymptomatic statin users. CONCLUSION: Statin use was not clearly associated with increased risk of deterioration of peripheral nerve function analysed at a group level. However, given the sample size limitations of our study and the findings of our post hoc analysis, we cannot preclude that peripheral nerve function may be affected in some individuals exposed to statins.

Contralateral Transfer of the Phenomenon of Repeated Bout Rate Enhancement in Unilateral Index Finger Tapping.

These hypotheses were tested: (1) Freely chosen frequency in unilateral index finger tapping is correlated between the two index fingers, and (2) A 3-min bout of unilateral index finger tapping followed by 10 min rest results in an increase of the freely chosen tapping frequency performed by the contralateral index finger in a second bout. Thirty-two adults participated. Freely chosen tapping frequencies from first bouts were 167.2 ± 79.0 and 161.5 ± 69.4 taps/min for the dominant and non-dominant hand, respectively (p=.434). These variables correlated (R=.86, p<.001). When bout one and two were performed with the dominant and non-dominant hand, respectively, the frequency increased by 8.1%±17.2% in bout two (p=.011). In opposite order, the frequency increased by 14.1%±17.5% (p<.001), which was not different from the ∼8% (p=.157).

The dynamics of cyanide defences in the life cycle of an aposematic butterfly: Biosynthesis versus sequestration.

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.