High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology

Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint™ technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags (“sample barcoding”) with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion

Sequence-Based Genotyping for Marker Discovery and Co-Dominant Scoring in Germplasm and Populations

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike

A New Direction to Athletic Performance: Understanding the Acute and Longitudinal Responses to Backward Running

Backward running (BR) is a form of locomotion that occurs in short bursts during many overground field and court sports. It has also traditionally been used in clinical settings as a method to rehabilitate lower body injuries. Comparisons between BR and forward running (FR) have led to the discovery that both may be generated by the same neural circuitry. Comparisons of the acute responses to FR reveal that BR is characterised by a smaller ratio of braking to propulsive forces, increased step frequency, decreased step length, increased muscle activity and reliance on isometric and concentric muscle actions. These biomechanical differences have been critical in informing recent scientific explorations which have discovered that BR can be used as a method for reducing injury and improving a variety of physical attributes deemed advantageous to sports performance. This includes improved lower body strength and power, decreased injury prevalence and improvements in change of direction performance following BR training. The current findings from research help improve our understanding of BR biomechanics and provide evidence which supports BR as a useful method to improve athlete performance. However, further acute and longitudinal research is needed to better understand the utility of BR in athletic performance programs

Leucocyte filtration of residual heart-lung machine blood in children undergoing congenital heart surgery

Cardiopulmonary bypass (CPB) leads to a generalized inflammatory reaction, resulting in increased postoperative leucocyte counts and decreased pulmonary function. In adults, removal of leucocytes from the residual heart - lung machine blood after CPB improved postoperative oxygenation. In children, however, the clinical effects of leucocyte filtration of the residual heart - lung machine blood are unknown. Therefore, we measured postoperative leucocyte counts and arterial blood oxygenation in children undergoing congenital cardiac surgery in a randomized prospective study. Anaesthesia and CPB were standardized. After CPB, the residual heart - lung machine blood was collected as usual. In a group of 25 children, this blood was filtered with a leucocyte depletion filter before transfusion. A control group of 25 children received this blood unfiltered. We found that the postoperative leucocyte counts were significantly lower in the filter group than in the control group ( p = 0.02, repeated measurements ANOVA). This difference reached a maximum on the second postoperative day (12.9 x 10(9)/L filter versus 15.9 x 10(9)/L control, p = 0.02, Student's t-test). Values for the arterial blood oxygenation on the first postoperative day were not different between the two groups (15.5 +/- 1 kPa filter versus 14.6 +/- 1.3 kPa control, p = 0.57, Student's t-test). We conclude that leucocyte filtration of the residual heart - lung machine blood reduced systemic leucocyte counts, but did not improve arterial blood oxygenation in children after congenital heart surgery

Efficiency and safety of leukocyte filtration during cardiopulmonary bypass for cardiac surgery

Background. Leukocyte filtration of systemic blood during cardiopulmonary bypass surgery to reduce post-operative morbidity has not yet been established because of the enormous leukocyte release from the third space. This study was designed to examine the efficiency and safety of leukocyte filtration by a new prototype large capacity leukocyte filter. Patients and methods. Patients undergoing cardiopulmonary bypass surgery were prospectively divided into two groups: a leukocyte removal group (n = 11) receiving leukocyte filtration during cardiopulmonary bypass and a control group (n = 20) with no filtration. The filtration efficiency was indicated by electronic leukocyte counts before and after filtration and the clinical efficiency to reduce post-operative morbidity was indicated by PaO2. Safety was indicated by monitoring the filtration pressure and leukocyte release products across the filter, as well as by examining the postfiltration filter by light and electron microscopy. Results. On an average, 75% of all entering leukocytes were removed by the filter. The post-operative PaO2, showed a tendency to improve after filtration. During filtration, the pressure across the filter material increased in five cases, accompanied by an increase in post-filter plasma hemoglobin and beta-glucuronidase. Within these filters accumulations of fibrin network with many trapped leukocytes were discovered microscopically. Conclusions. The filter was efficient in filtering leukocytes, but the filtration efficiency slowed at the end of filtration. Furthermore, the patients' post-operative parameters showed a tendency to improve after filtration. However, flow obstruction by means of clotting seems to be an important issue of safety involved in the filtration of large numbers of leukocytes for cardiopulmonary surgical patients. (C) 1999 Elsevier Science Ltd. All rights reserved

Multilink stent promotes less platelet and leukocyte adhesion than a traditional stainless steel stent: An in vitro experimental study

Background: Platelet and Leukocyte deposition onto metallic struts can be a crucial factor in the outcome of a coronary stenting procedure. By means of an in vitro, closed-loop circulation model, me aimed to assess blood-stent interaction patterns for a new stainless steel stent (MultiLink, Guidant Nederland BV, Nieuwegein, the Netherlands), Methods: The effect of MultiLink (n=20) on blood cells and blood activation was studied by biochemical assays, Platelet and leukocyte adhesion to MultiLink were studied by immunofluorocytometric assays (anti-GpIIIa [CD 61] and anti-CD11b labeled antibodies, respectively), and by scanning electron microscopy. MultiLink was compared with empty circuits (n=20) and to the Palmaz Schatz stent (n=20), Experiments mere performed both in the presence and in the absence of an antiplatelet agent (15 mug/mL of indomethacin). Results: No significant effect on blood cells and blood activation was demonstrated for MultiLink. Antiplatelet treatment significantly reduced platelet adhesion to Multilink (from 3.78+/-1.28 to 2.23+/-0.57x10(6) count per second [cps]/stent) but not to the Palmaz Schatz stent (from 4.11+/-0.31 to 5.02+/-1.29 x 10(6) cps/stent)(P=0.011), Leukocyte adhesion to MultiLink was significantly less than adhesion to the Palmaz Schatz stent (7.95+/-1.59 vs. 9.16+/-1.36x10(6) cps/ stent, respectively; P=0.016), regardless of the presence of antiplatelet treatment. Conclusions: When compared with a traditional stainless steel stent, MultiLink seems to have features of improved hemocompatibility, and single antiplatelet treatment is proposed as the treatment of choice to prevent platelet deposition