Genome-wide QTL and bulked transcriptomic analysis reveals new candidate genes for the control of tuber carotenoid content in potato (Solanum tuberosum L.).

KEY MESSAGE: Genome-wide QTL analysis of potato tuber carotenoid content was investigated in populations of Solanum tuberosum Group Phureja that segregate for flesh colour, revealing a novel major QTL on chromosome 9. The carotenoid content of edible plant storage organs is a key nutritional and quality trait. Although the structural genes that encode the biosynthetic enzymes are well characterised, much less is known about the factors that determine overall storage organ content. In this study, genome-wide QTL mapping, in concert with an efficient 'genetical genomics' analysis using bulked samples, has been employed to investigate the genetic architecture of potato tuber carotenoid content. Two diploid populations of Solanum tuberosum Group Phureja were genotyped (AFLP, SSR and DArT markers) and analysed for their tuber carotenoid content over two growing seasons. Common to both populations were QTL that explained relatively small proportions of the variation in constituent carotenoids and a major QTL on chromosome 3 explaining up to 71 % of the variation in carotenoid content. In one of the populations (01H15), a second major carotenoid QTL was identified on chromosome 9, explaining up to 20 % of the phenotypic variation. Whereas the major chromosome 3 QTL was likely to be due to an allele of a gene encoding ß-carotene hydroxylase, no known carotenoid biosynthetic genes are located in the vicinity of the chromosome 9 QTL. A unique expression profiling strategy using phenotypically distinct bulks comprised individuals with similar carotenoid content provided further support for the QTL mapping to chromosome 9. This study shows the potential of using the potato genome sequence to link genetic maps to data arising from eQTL approaches to enhance the discovery of candidate genes underlying QTLs.

Aß42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes.

Aß (amyloid-ß peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aß, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aß42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aß. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aß binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aß, but not preformed fibrillar Aß. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aß and this may be mediated by mechanical damage to the membranes by Aß aggregation. This membrane disruption may play a key role in the mechanism of Aß-related cell toxicity in AD.

Why fibrin biomechanical properties matter for hemostasis and thrombosis.

Polymeric fibrin displays unique structural and biomechanical properties that contribute to its essential role of generating blood clots that stem bleeds. The aim of this review is to discuss how the fibrin clot is formed, how protofibrils make up individual fibrin fibers, what the relationship is between the molecular structure and fibrin biomechanical properties, and how fibrin biomechanical properties relate to the risk of thromboembolic disease. Fibrin polymerization is driven by different types of bonds, including knob-hole interactions displaying catch-slip characteristics, and covalent crosslinking of fibrin polypeptides by activated factor XIII. Key biophysical properties of fibrin polymer are its visco-elasticity, extensibility and resistance to rupture. The internal packing of protofibrils within fibers changes fibrin biomechanical behavior. There are several methods to analyze fibrin biomechanical properties at different scales, including AFM force spectroscopy, magnetic or optical tweezers and rheometry, amongst others. Clinically, fibrin biomechanical characteristics are key for the prevention of thromboembolic disorders such as pulmonary embolism. Future studies are needed to address unanswered questions regarding internal molecular structure of the fibrin polymer, the structural and molecular basis of its remarkable mechanical properties and the relationship of fibrin biomechanical characteristics with thromboembolism in patients with deep vein thrombosis and ischemic stroke.

Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds.

Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.

Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae.

Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.

Impact of Conventional and Integrated Management Systems on the Water-Soluble Vitamin Content in Potatoes, Field Beans, and Cereals.

The reduction of the environmental footprint of crop production without compromising crop yield and their nutritional value is a key goal for improving the sustainability of agriculture. In 2009, the Balruddery Farm Platform was established at The James Hutton Institute as a long-term experimental platform for cross-disciplinary research of crops using two agricultural ecosystems. Crops representative of UK agriculture were grown under conventional and integrated management systems and analyzed for their water-soluble vitamin content. Integrated management, when compared with the conventional system, had only minor effects on water-soluble vitamin content, where significantly higher differences were seen for the conventional management practice on the levels of thiamine in field beans (p < 0.01), Spring barley (p < 0.05), and Winter wheat (p < 0.05), and for nicotinic acid in Spring barley (p < 0.05). However, for all crops, variety and year differences were of greater importance. These results indicate that the integrated management system described in this study does not significantly affect the water-soluble vitamin content of the crops analyzed here.

Effect of rioprostil on aspirin-induced gastrointestinal mucosal changes in normal volunteers.

Rioprostil, a 15-deoxy-16-methyl prostaglandin E1, was evaluated for its effect on aspirin-induced gastrointestinal mucosal changes in normal volunteers. Fifty-six normal male volunteers were evaluated by endoscopy in a double-blind, placebo-controlled study. Aspirin was given at a dose of 975 mg four times per day. Rioprostil was given at doses of 60, 120, and 300 micrograms four times per day. Rioprostil, at both antisecretory and subantisecretory doses, prevented or reduced aspirin-induced injury. Increased stool frequency was the most common side effect and appeared to be a dose-related effect of rioprostil occurring at only antisecretory doses.

Metacognition in motor learning.

Research on judgments of verbal learning has demonstrated that participants' judgments are unreliable and often overconfident. The authors studied judgments of perceptual-motor learning. Participants learned 3 keystroke patterns on the number pad of a computer, each requiring that a different sequence of keys be struck in a different total movement time. Practice trials on each pattern were either blocked or randomly interleaved with trials on the other patterns, and each participant was asked, periodically, to predict his or her performance on a 24-hr test. Consistent with earlier findings, blocked practice enhanced acquisition but harmed retention. Participants, though, predicted better performance given blocked practice. These results augment research on judgments of verbal learning and suggest that humans, at their peril, interpret current ease of access to a perceptual-motor skill as a valid index of learning.

Virtual fluoroscopy: computer-assisted fluoroscopic navigation.

STUDY DESIGN: In vitro accuracy assessment of a novel virtual fluoroscopy system. OBJECTIVES: To investigate a new technology combining image-guided surgery with C-arm fluoroscopy. SUMMARY OF BACKGROUND DATA: Fluoroscopy is a useful and familiar technology to all musculoskeletal surgeons. Its limitations include radiation exposure to the patient and operating team and the need to reposition the fluoroscope repeatedly to obtain surgical guidance in multiple planes. METHODS: Fluoroscopic images of the lumbar spine of an intact, unembalmed cadaver were obtained, calibrated, and saved to an ). A was used for the sequential insertion of a light-emitting diode-fitted probe into the pedicles of L1-S1 bilaterally. The trajectory of a "virtual tool" corresponding to the tracked tool was overlaid onto the saved fluoroscopic views in real time. Live fluoroscopic images of the inserted pedicle probe were then obtained. Distances between the tips of the virtual and fluoroscopically displayed probes were quantified using the image-guided computer's measurement tool. Trajectory angle differences were measured using a standard goniometer and printed copies of the workstation computer display. The surgeon's radiation exposure was measured using thermolucent dosimeter rings. RESULTS: Excellent correlation between the virtual fluoroscopic images and live fluoroscopy was observed. Mean probe tip error was 0.97 +/- 0.40 mm. Mean trajectory angle difference between the virtual and fluoroscopically displayed probes was 2.7 degrees +/- 0.6 degrees. The thermolucent dosimeter rings measured no detectable radiation exposure for the surgeon. CONCLUSIONS: Virtual fluoroscopy offers several advantages over conventional fluoroscopy while providing acceptable targeting accuracy. It enables a single C-arm to provide real-time, multiplanar procedural guidance. It also dramatically reduces radiation exposure to the patient and surgical team by eliminating the need for repetitive fluoroscopic imaging for tool placement.

Accuracy requirements for image-guided spinal pedicle screw placement.

STUDY DESIGN: Accuracy requirement analysis for image-guided pedicle screw placement. OBJECTIVES: To derive theoretical accuracy requirements for image-guided spinal pedicle screw placement. SUMMARY OF BACKGROUND DATA: Underlying causes of inaccuracy in image-guided surgical systems and methods for quantifying this inaccuracy have been studied. However, accuracy requirements for specific spinal surgical procedures have not been delineated. In particular, the accuracy requirements for image-guided spinal pedicle screw placement have not been previously reported. METHODS: A geometric model was developed relating spinal pedicle anatomy to accuracy requirements for image-guided surgery. This model was used to derive error tolerances for pedicle screw placement when using clinically relevant screw diameters in the cervical (3.5 mm), thoracic (5.0 mm), and thoracolumbar spine (6.5 mm). The error tolerances were represented as the permissible rotational and translational deviations from the ideal screw trajectory that would avoid pedicle wall perforation. The relevant dimensions of the pedicle model were extracted from existing morphometric data. RESULTS: As anticipated, accuracy requirements were greatest at spinal levels where the relevant screw diameter approximated the dimensions of the pedicle. These requirements were highest for T5, followed in descending order by T4, T7, T6, T3, T12, L1, T8, T11, C4, L2, C3, T10, C5, T2, T9, C6, L3, C2, T1, C7, L4, and L5. Maximum permissible translational/rotational error tolerances ranged from 0.0 mm/0.0 degrees at T5 to 3.8 mm/12.7 degrees at L5. CONCLUSIONS: These results, obtained by mathematical analysis, demonstrate that extremely high accuracy is necessary to place pedicle screws at certain levels of the spine without perforating the pedicle wall. These accuracy requirements exceed the accuracy of current image-guided surgical systems, based on clinical utility errors reported in the literature. In actual use, however, these systems have been shown to improve the accuracy of pedicle screw placement. This dichotomy indicates that other factors, such as the surgeon's visual and tactile feedback, may be operative.

Progression of arterial disease in patients with healed venous ulcers.

A report of a study set up to discover whether patients with healed venous ulcers were receiving adequate investigation after recurrent ulceration.

Approaches to venous leg ulcer care within the community: compression, pinch skin grafts and simple venous surgery.

The traditional approach to leg ulcer care is both expensive and achieves poor outcomes. The outcome of treatment for patients with venous ulceration has been improved using a more scientific and research-based approach to patient assessment, adequate compression bandaging, use of other procedures such as pinch skin grafting and simple venous surgery, and the delivery of these innovations in patient care to the community as a whole. Multi-layer compression bandaging is superior to standard (one-layer) bandaging because it can easily be adapted to a wide range of ankle circumferences and leg sizes to provide sustained, graduated compression. Pinch skin grafting is cost-effective, accelerates healing, and, following adequate training, may be performed by specialist nurses in the community. Simple venous surgery may be offered under local anaesthesia when only the superficial veins are incompetent; however, deep venous surgery has yet to prove itself in clinical practice. Implementation of dedicated community leg ulcer clinics using these research-based findings will result in a substantially reduced prevalence of venous leg ulceration.

Community leg ulcer clinics: a comparative study in two health authorities.

OBJECTIVE: To compare the outcome and cost of care for leg ulcers in community leg ulcer clinics in Stockport District Health authority with Trafford District Health Authority as a control. DESIGN: Detailed cost and efficacy studies conducted prospectively over a three month period in both districts both before and one year after the introduction of five leg ulcer clinics in Stockport. SETTING: Two large district health authorities of broad socioeconomic mix and total population of 540,000. PATIENTS: All patients receiving treatment for an active leg ulcer, irrespective of the profession or location of their carer. MAIN OUTCOME MEASURES: The proportion of ulcerated limbs completely healed within three months and total cost of leg ulcer care. RESULTS: The introduction of community clinics in Stockport improved healing of leg ulcers from 66/252 (26%) in 1993 to 99/233 (42%) in 1994 (P < 0.001) compared with in Trafford, where 47/203 (23%) healed in 1993 and only 43/213 (20%) in 1994. This improved result in Stockport was achieved while the annual expenditure on care of leg ulcers was reduced from 409,991 pounds to only 253,371 pounds. In the same year the cost of leg ulcer care in Trafford increased from 556,039 pounds to 673,318 pounds. CONCLUSION: In the first year after the introduction of community clinics, before most patients in Stockport had access to these clinics, healing of leg ulcers was already improved whereas costs were reduced.

Direct characterization of fluid lipid assemblies on mercury in electric fields.

Phospholipid monolayers on mercury (Hg) surfaces have received substantial and extensive scientific interest not only because of their use as a biomembrane model but also for their application as a successful toxicity-sensing element. The monolayers show characteristic and very reproducible phase transitions manifest as consecutive voltammetric peaks in response to applied transverse electric fields. Unfortunately, apart from the results of simulation studies, there is a lack of data on the lipid phase structures to help interpret these voltammetric peaks. In this paper we report on the direct measurement of the structural changes underlying the phase transitions of phospholipid layers of dioleoyl phosphatidylcholine (DOPC) at electrified Hg surfaces using atomic force microscopy force-distance techniques. These direct measurements enable a description of the following structural changes in fluid lipid assemblies on a liquid electrode within an increasing transverse electric field. At about -1.0 V (vs Ag/AgCl) a field-facilitated ingress of ions and water into the monolayer results in a phase transition to a structured 2D emulsion. This is followed by a further phase transition at more negative potentials involving the readsorption of bilayer patches. At stronger values of field the bilayer patches form semivesicles, which subsequently collapse to form a monolayer of uncertain composition at very negative potentials. The observation that a monolayer on Hg converts to a bilayer by increasing the applied potential has allowed techniques to be developed for preparing and characterizing a near-continuous DOPC bilayer on Hg in an applied potential window within -1.0 and -1.4 V (vs Ag/AgCl).

Roles of fibrin α- and γ-chain specific cross-linking by FXIIIa in fibrin structure and function.

Factor XIII is responsible for the cross-linking of fibrin γ-chains in the early stages of clot formation, whilst α-chain cross-linking occurs at a slower rate. Although γ- and α-chain cross-linking was previously shown to contribute to clot stiffness, the role of cross-linking of both chains in determining clot structure is currently unknown. Therefore, the aim of this study was to determine the role of individual α- and γ-chain cross-linking during clot formation, and its effects on clot structure. We made use of a recombinant fibrinogen (γQ398N/Q399N/K406R), which does not allow for γ-chain cross-linking. In the absence of cross-linking, intact D-D interface was shown to play a potential role in fibre appearance time, clot stiffness and elasticity. Cross-linking of the fibrin α-chain played a role in the thickening of the fibrin fibres over time, and decreased lysis rate in the absence of α2-antiplasmin. We also showed that α-chain cross-linking played a role in the timing of fibre appearance, straightening fibres, increasing clot stiffness and reducing clot deformation. Cross-linking of the γ-chain played a role in fibrin fibre appearance time and fibre density. Our results show that α- and γ-chain cross-linking play independent and specific roles in fibrin clot formation and structure.

Four-limb compartment syndrome associated with the systemic capillary leak syndrome.

Systemic capillary leak syndrome, or the Clarkson syndrome, is an extremely rare condition in which increased capillary permeability results in a massive shift of fluid into the extravascular space. This is followed rapidly by hypotensive shock, haemoconcentration, and, potentially, substantial oedema of the limbs resulting in an acute compartment syndrome. It is important for orthopaedic surgeons to be aware of this syndrome as our medical colleagues, who initially care for these patients, are less familiar with the diagnosis and the need for emergency management of the associated compartment syndrome should it develop. There have been fewer than 100 cases of this entity reported. This case report is the first to describe the subsequent development of a compartment syndrome in all four limbs. Clinical vigilance and continuous monitoring of intracompartmental pressure is necessary in these patients in order to help reduce limb-threatening complications.

L-Ascorbic acid accumulation in fruit of Ribes nigrum occurs by in situ biosynthesis via the L-galactose pathway.

Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high vitamin C (l-ascorbic acid, AsA) content. In the present study, a systematic analysis of the mechanism of fruit AsA accumulation was undertaken. AsA accumulation occurred during fruit expansion and was associated with high in situ biosynthetic capacity via the l-galactose pathway and low rates of turnover. Cessation of AsA accumulation was associated with reduced biosynthesis and increased turnover. Translocation of AsA from photosynthetic or vegetative tissues contributed little to fruit AsA accumulation. Manipulation of substrate availability by defoliation had no effect on fruit AsA concentration but significantly reduced fruit yields. Supply of the AsA precursor l-galactono-1,4-lactone to intact, attached fruit transiently increased fruit AsA concentration which rapidly returned to control levels after removal of the compound. These data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.