Morphological and genetic characterisation of the root system architecture of selected barley recombinant chromosome substitution lines using an integrated phenotyping approach.

Discoveries on the genetics of resource acquisition efficiency are limited by the ability to measure plant roots in sufficient number and with adequate genotypic variability. This paper presents a root phenotyping study that explores ways to combine live imaging and computer algorithms for model-based extraction of root growth parameters. The study is based on a subset of barley Recombinant Chromosome Substitution Lines (RCSLs) and a combinatorial approach was designed for fast identification of the regions of the genome that contribute the most to variations in root system architecture (RSA). Results showed there was a strong genotypic variation in root growth parameters within the set of genotypes studied. The chromosomal regions associated with primary root growth differed from the regions of the genome associated with changes in lateral root growth. The concepts presented here are discussed in the context of identifying root QTL and its potential to assist breeding for novel crops with improved root systems.

Binding Force Dynamics of Streptococcus mutans-glucosyltransferase B to Candida albicans.

Candida albicans cells are often detected with Streptococcus mutans in plaque biofilms from children affected with early childhood caries. The coadhesion between these 2 organisms appears to be largely mediated by the S. mutans-derived exoenzyme glucosyltransferase B (GtfB); GtfB readily binds to C. albicans cells in an active form, producing glucans locally that provide enhanced binding sites for S. mutans. However, knowledge is limited about the mechanisms by which the bacterial exoenzyme binds to and functions on the fungal surface to promote this unique cross-kingdom interaction. In this study, we use atomic force microscopy to understand the strength and binding dynamics modulating GtfB-C. albicans adhesive interactions in situ. Single-molecule force spectroscopy with GtfB-functionalized atomic force microscopy tips demonstrated that the enzyme binds with remarkable strength to the C. albicans cell surface (~2 nN) and showed a low dissociation rate, suggesting a highly stable bond. Strikingly, the binding strength of GtfB to the C. albicans surface was ~2.5-fold higher and the binding stability, ~20 times higher, as compared with the enzyme adhesion to S. mutans. Furthermore, adhesion force maps showed an intriguing pattern of GtfB binding. GtfB adhered heterogeneously on the surface of C. albicans, showing a higher frequency of adhesion failure but large sections of remarkably strong binding forces, suggesting the presence of GtfB binding domains unevenly distributed on the fungal surface. In contrast, GtfB bound uniformly across the S. mutans cell surface with less adhesion failure and a narrower range of binding forces (vs. the C. albicans surface). The data provide the first insights into the mechanisms underlying the adhesive and mechanical properties governing GtfB interactions with C. albicans. The strong and highly stable GtfB binding to C. albicans could explain, at least in part, why this bacterially derived exoenzyme effectively modulates this virulent cross-kingdom interaction.

Percutaneous Retroperitoneal Splenorenal Shunt for Symptomatic Portal Vein Thrombosis After Liver Transplantation.

Acute or recurrent bleeding from ectopic varices is a potentially life-threatening condition in rare patients with extrahepatic complete portal vein thrombosis (PVT) after liver transplantation (LT). In this setting, the role of interventional radiology is very limited and surgical shunts, in particular splenorenal shunts are usually used, despite the high associated mortality. We present the first reports of the clinical use of a new minimally invasive technique, percutaneous retroperitoneal splenorenal shunt (PRESS), in two LT recipients with life-threatening variceal hemorrhage secondary to PVT. Both patients had a successful PRESS using a transplenic approach with resolution of bleeding, avoiding the need for a potentially complicated laparotomy. The PRESS procedure is a useful addition to the interventional armamentarium that can be used in cases unsuitable for surgical shunt, and refractory to endoscopic management. In the future, this technique may be an alternative to surgical shunts as the standard procedure in patients with extra-hepatic PVT, just as the transjugular intrahepatic portosystemic shunt (TIPS) procedure has become for the management of portal hypertension in the absence of PVT. Longer-term follow-up will be needed to establish the long-term success of this procedure.

Exploitation of interspecific diversity for monocot crop improvement.

In many cultivated crop species there is limited genetic variation available for the development of new higher yielding varieties adapted to climate change and sustainable farming practises. The distant relatives of crop species provide a vast and largely untapped reservoir of genetic variation for a wide range of agronomically important traits that can be exploited by breeders for crop improvement. In this paper, in what we believe to be the largest introgression programme undertaken in the monocots, we describe the transfer of the entire genome of Festuca pratensis into Lolium perenne in overlapping chromosome segments. The L. perenne/F. pratensis introgressions were identified and characterised via 131 simple sequence repeats and 1612 SNPs anchored to the rice genome. Comparative analyses were undertaken to determine the syntenic relationship between L. perenne/F. pratensis and rice, wheat, barley, sorghum and Brachypodium distachyon. Analyses comparing recombination frequency and gene distribution indicated that a large proportion of the genes within the genome are located in the proximal regions of chromosomes which undergo low/very low frequencies of recombination. Thus, it is proposed that past breeding efforts to produce improved varieties have centred on the subset of genes located in the distal regions of chromosomes where recombination is highest. The use of alien introgression for crop improvement is important for meeting the challenges of global food supply and the monocots such as the forage grasses and cereals, together with recent technological advances in molecular biology, can help meet these challenges.

Who benefits most from guided self-help for binge eating? An investigation into the clinical features of completers and non-completers.

Guided self-help (GSH) is a recommended first step in treatment for bulimia nervosa (BN) and binge eating disorder (BED) (NICE, 2004). It remains unclear what makes some individuals more likely to respond to this form of treatment than others. Forty-eight patients participated in this study using a GSH programme for binge eating. Profiles of treatment completers and non-completers are compared, and reasons for non-completion explored. Completion of treatment was associated with significant improvements in mood, general functioning and on measures of dietary restraint, frequency of objective binge eating (OBE), laxative misuse, self-induced vomiting (SIV) and driven exercise. Improvements were maintained at follow-up. Treatment non-completers reported significantly higher pre-treatment levels of depression and weight concern, and lower levels of general health and vitality. Reasons for discontinuing treatment were related to perceptions of the GSH programme; practicalities of the programme; and readiness to change. Whilst GSH can be effective for a sub-group of patients, factors such as pre-morbid level of depression, degree of weight concern, perceptions of the programme, and readiness to change may increase the likelihood of non-completion.

Development and pilot of a group skills-and-support intervention for mothers of children with feeding problems.

Child feeding problems are often associated with parental factors which may influence and maintain difficulties. This paper reports the development, pilot and preliminary evaluation of a group intervention for mothers of children with feeding problems. Themes for the group were derived from a survey of parents and professionals. Three pilot interventions were conducted in order to make an assessment of the feasibility, acceptability and potential for achieving change in levels of maternal mood, parenting stress and concerns related to feeding. While single case analysis revealed little change in standardised measures of mood and parenting stress, participants valued the social and emotional support offered by the group and reported improvements in concerns and maladaptive behaviours related to feeding. An intervention which provides support and a sense of a shared experience appears to have beneficial effects for mothers of children with feeding problems and therefore, may offer a constructive means of supporting this population.

Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces.

Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.

Resistance to powdery mildew in Spanish barley landraces is controlled by different sets of quantitative trait loci.

Twenty-two landrace-derived inbred lines from the Spanish Barley Core Collection (SBCC) were found to display high levels of resistance to a panel of 27 isolates of the fungus Blumeria graminis that exhibit a wide variety of virulences. Among these lines, SBCC145 showed high overall resistance and a distinctive spectrum of resistance compared with the other lines. Against this background, the main goal of the present work was to investigate the genetic basis underlying such resistance using a doubled haploid population derived from a cross between SBCC145 and the elite spring cultivar Beatrix. The population was genotyped with the 1,536-SNP Illumina GoldenGate Oligonucleotide Pool Assay (Barley OPA-1 or BOPA1 for short), whereas phenotypic analysis was performed using two B. graminis isolates. A major quantitative trait locus (QTL) for resistance to both isolates was identified on the long arm of chromosome 6H (6HL) and accounted for ca. 60% of the phenotypic variance. Depending on the B. graminis isolate tested, three other minor QTLs were detected on chromosomes 2H and 7H, which explained less than 5% of the phenotypic variation each. In all cases, the alleles for resistance derived from the Spanish parent SBCC145. The position, the magnitude of the effect observed and the proportion of phenotypic variation accounted for by the QTL on 6HL suggest this is a newly identified locus for broad-based resistance to powdery mildew.

Development of a molecular cytogenetic recombination assay for barley.

Distal localisation of chiasmata is common to many cereals and grasses, which consigns many genes of the complement to recombination backwaters. Releasing this potential untapped genetic variation for use in advanced breeding programmes is an ambitious and technically demanding challenge, necessitating controlled shifts in the distribution of crossover events. As part of a collaborative programme to manipulate recombination in barley, we are developing a robust and reliable molecular cytogenetic assay for recombination in this species, which will be used to gauge the success of our forward and reverse genetic interventions. Single-locus bacterial artificial chromosome clones and rDNA probes identify the 7 somatic chromosomes of the complement. Meiocytes at pachytene of meiosis were embedded in polyacrylamide and hybridised in situ with centromere and telomere probes, followed by immunolocalisation of the synaptonemal complex-associated protein Asy1 which highlights the bivalents' axes. Optical sectioning, deconvolution and image analysis of the z-stacks of the nuclei allowed the disentanglement of each bivalent and the construction of an accurate meiotic ideogram. The landing of single-locus bacterial artificial chromosomes and the detection of late recombination proteins will complete the assay and provide a means of discerning subtle changes in recombination in this species.

Classification of eating disturbance in children and adolescents: proposed changes for the DSM-V.

Childhood and adolescence are critical periods of neural development and physical growth. The malnutrition and related medical complications resulting from eating disorders such as anorexia nervosa (AN), bulimia nervosa (BN) and eating disorder not otherwise specified may have more severe and potentially more protracted consequences during youth than during other age periods. The consensus opinion of an international workgroup of experts on the diagnosis and treatment of child and adolescent eating disorders is that (a) lower and more developmentally sensitive thresholds of symptom severity (e.g. lower frequency of purging behaviours, significant deviations from growth curves as indicators of clinical severity) be used as diagnostic boundaries for children and adolescents, (b) behavioural indicators of psychological features of eating disorders be considered even in the absence of direct self-report of such symptoms and (c) multiple informants (e.g. parents) be used to ascertain symptom profiles. Collectively, these recommendations will permit earlier identification and intervention to prevent the exacerbation of eating disorder symptoms.

Effects of ascertainment bias and marker number on estimations of barley diversity from high-throughput SNP genotype data.

The capability of molecular markers to provide information of genetic structure is influenced by their number and the way they are chosen. This study evaluates the effects of single nucleotide polymorphism (SNP) number and selection strategy on estimates of germplasm diversity and population structure for different types of barley germplasm, namely cultivar and landrace. One hundred and sixty-nine barley landraces from Syria and Jordan and 171 European barley cultivars were genotyped with 1536 SNPs. Different subsets of 384 and 96 SNPs were selected from the 1536 set, based on their ability to detect diversity in landraces or cultivated barley in addition to corresponding randomly chosen subsets. All SNP sets except the landrace-optimised subsets underestimated the diversity present in the landrace germplasm, and all subsets of SNP gave similar estimates for cultivar germplasm. All marker subsets gave qualitatively similar estimates of the population structure in both germplasm sets, but the 96 SNP sets showed much lower data resolution values than the larger SNP sets. From these data we deduce that pre-selecting markers for their diversity in a germplasm set is very worthwhile in terms of the quality of data obtained. Second, we suggest that a properly chosen 384 SNP subset gives a good combination of power and economy for germplasm characterization, whereas the rather modest gain from using 1536 SNPs does not justify the increased cost and 96 markers give unacceptably low performance. Lastly, we propose a specific 384 SNP subset as a standard genotyping tool for middle-eastern landrace barley.

Image correlation microscopy for uniform illumination.

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.

Primate maternal placental angiography.

BACKGROUND: In humans, it is known that blood flow is directed to the gravid uterus from two (right and left) pelvic uterine arteries. The extent of supply from the tubo-ovarian anastomosis (joining of the ovarian and uterine arteries) is unknown. The aim of this study was to delineate the arterial blood supply to the placenta via systematic angiography in normal pregnancies in a non-human primate, the baboon (Papio hamadryas). METHODS: The assessment of the distribution of blood supply with single-shot 3-vessel angiography (aorta, right and left common iliac arteries), allowed assessment of bilateral supply and possible ovarian supply (n=9). In 2-vessel pictures (aorta and left or right iliac), the contralateral supply was determined by subtraction of the ipsilateral supply from the total supply (n=7). The studies were all approved by the Institutional animal welfare committee and were conducted as part of a broader project investigating preeclampsia. RESULTS: The animals were 9 years of age and 140 days of gestation for the 3 vessel study and 154 days of gestation for the 2 vessel study. The angiograms were more likely to have cotyledons perfused by the left uterine artery (p=0.012) than the right. Overall, 55% of placentae had 5-44% of supply overlapping and 22% had 10-15% ovarian contribution to blood supply. DISCUSSION: This study demonstrates the variation in primate uteroplacental blood flow including the contribution of ovarian arteries and left and right collateralization. Similarity to human vascular anatomy strengthens the use of primate species as a model of human placentation.

Classification of child and adolescent eating disturbances. Workgroup for Classification of Eating Disorders in Children and Adolescents (WCEDCA).

OBJECTIVE: : The purpose of this article is to summarize major conceptual and clinical variables related to age-appropriate and developmentally appropriate classification of eating problems and disorders in children and adolescents. METHOD: A review of current classifications and related literature in child development is provided. Problems with current classification schemes are identified and discussed. RESULTS: Current classifications are inadequate to address the clinical and research needs of children and adolescents with eating disturbances and disorders. CONCLUSION: A range of possible changes in classification strategies for eating disorders in children and adolescents are described.

SFP genotyping from affymetrix arrays is robust but largely detects cis-acting expression regulators.

The recent development of Affymetrix chips designed from assembled EST sequences has spawned considerable interest in identifying single-feature polymorphisms (SFPs) from transcriptome data. SFPs are valuable genetic markers that potentially offer a physical link to the structural genes themselves. However, most current SFP prediction methodologies were developed for sequenced species although SFPs are particularly valuable for species with complex and unsequenced genomes. To establish the sensitivity and specificity of prediction, we explored four methods for identifying SFPs from experiments involving two tissues in two commercial barleys and their doubled-haploid progeny. The methods were compared in terms of numbers of SFPs predicted and their ability to identify known sequence polymorphisms in the features, to confirm existing SNP genotypes and to match existing maps and individual haplotypes. We identified >4000 separate SFPs that accurately predicted the SNP genotype of >98% of the doubled-haploid (DH) lines. They were highly enriched for features containing sequence polymorphisms but all methods uniformly identified a majority of SFPs ( approximately 64%) in features for which there was no sequence polymorphism while 5% mapped to different locations, indicating that "SFPs" mainly represent polymorphism in cis-acting regulators. All methods are efficient and robust at predicting markers for gene mapping.

A high density barley microsatellite consensus map with 775 SSR loci.

A microsatellite or simple sequence repeat (SSR) consensus map of barley was constructed by joining six independent genetic maps based on the mapping populations 'Igri x Franka', 'Steptoe x Morex', 'OWB(Rec) x OWB(Dom)', 'Lina x Canada Park', 'L94 x Vada' and 'SusPtrit x Vada'. Segregation data for microsatellite markers from different research groups including SCRI (Bmac, Bmag, EBmac, EBmag, HVGeneName, scsssr), IPK (GBM, GBMS), WUR (GBM), Virginia Polytechnic Institute (HVM), and MPI for Plant Breeding (HVGeneName), generated in above mapping populations, were used in the computer program RECORD to order the markers of the individual linkage data sets. Subsequently, a framework map was constructed for each chromosome by integrating the 496 "bridge markers" common to two or more individual maps with the help of the computer programme JoinMap 3.0. The final map was calculated by following a "neighbours" map approach. The integrated map contained 775 unique microsatellite loci, from 688 primer pairs, ranging from 93 (6H) to 132 (2H) and with an average of 111 markers per linkage group. The genomic DNA-derived SSR marker loci had a higher polymorphism information content value (average 0.61) as compared to the EST/gene-derived SSR loci (average 0.48). The consensus map spans 1,068 cM providing an average density of one SSR marker every 1.38 cM. Such a high-density consensus SSR map provides barley molecular breeding programmes with a better choice regarding the quality of markers and a higher probability of polymorphic markers in an important chromosomal interval. This map also offers the possibilities of thorough alignment for the (future) physical map and implementation in haplotype diversity studies of barley.

Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1.

Preeclampsia is a complication of pregnancy with significant morbidity and mortality for the mother and the fetus. Presumptions are made that placental hypoxia has a causative role in the clinical syndrome. Furthermore, soluble fms-like tyrosine kinase 1 (sFLT-1) has been shown to have a role in the maternal syndrome of preeclampsia. We investigated the relationship between uteroplacental ischemia (UPI), the maternal clinical syndrome of preeclampsia and sFLT-1 in non-human primates. The induction of UPI in a pregnant non-human primate resulted in the development of a clinical entity analogous to human preeclampsia. This was illustrated by the increase in blood pressure, development of proteinuria, and renal histological changes identical to human preeclampsia. A significant elevation in the placental and peripheral blood mononuclear cell sFLT-1 mRNA expression was noted, translating to a significant elevation in circulating sFLT-1. Thus, this sequence suggests that a pathogenic reduction in placental perfusion results in the development of the maternal syndrome of preeclampsia and an increase in circulating sFLT-1, which is derived both from placental and extra-placental sources.

Rheological analysis and measurement of neutrophil indentation.

Aspects of neutrophil mechanical behavior relevant to the formation of adhesive contacts were assessed by measuring the dependence of the contact area between the cell and a spherical substrate under controlled loading. Micropipettes were used to bring neutrophils into contact with spherical beads under known forces, and the corresponding contact area was measured over time. The neutrophil was modeled as a viscous liquid drop with a constant cortical tension. Both the equilibrium state and the dynamics of the approach to equilibrium were examined. The equilibrium contact area increased monotonically with force in a manner consistent with a cell cortical tension of 16-24 pN/microm. The dynamic response matched predictions based on a model of the cell as a growing drop using published values for the effective viscosity of the cell. The contact pressure between the cell and substrate at equilibrium is predicted to depend on the curvature of the contacting substrate, but to be independent of the impingement force. The approach to equilibrium was rapid, such that the time-averaged stress for a two-second impingement was within 20% of the equilibrium value. These results have implications for the role of mechanical force in the formation of adhesive contacts.

Neutrophil adhesive contact dependence on impingement force.

Neutrophil capture and recruitment from the circulation requires the formation of specific receptor/ligand bonds under hydrodynamic forces. In the present study we examine bond formation between beta2-integrins on neutrophils and immobilized ICAM-1 while using micropipettes to control the force of contact between the cell and substrate. Magnesium was used to induce the high affinity conformation of the integrins, and bond formation was assessed by measuring the probability of adhesion during repeated contacts. Increasing the impingement force caused an increase in the contact area and led to a proportional increase in adhesion probability (from approximately 20 to 50%) over the range of forces tested (50-350 pN). In addition, different-sized beads were used to change the force per unit area in the contact zone (contact stress). We conclude that for a given contact stress, the rate of bond formation increases linearly with contact area, but that increasing contact stress results in higher intrinsic rates of bond formation.

Long-term outcome of radiological-guided insertion of implanted central venous access port devices (CVAPD) for the delivery of chemotherapy in cancer patients: institutional experience and review of the literature.

Central venous access port devices (CVAPD) are necessary for delivery of prolonged infusional chemotherapy or in patients with poor peripheral venous access. Previous studies of Hickman catheters report complication rates in about 45% of patients. Our aim was to assess the early and late complication rate, and duration that the CVAPD remained functional, following insertion by interventional radiologists in patients with solid tumours. A prospective study was undertaken in 110 consecutive patients who had insertion of 111 subclavian CVAPD. The median age of patients was 57 years (range 17-83), 64 were females; 68 patients (61%) had gastrointestinal tumours and 25 (23%) had breast cancer. CVAPD were successfully implanted in all but one patient. There were four (4%) immediate major complications: thrombosis 2 and pneumothorax 2. Nine patients (8%) had bruising or pain. Four devices (4%) became infected. In total, 100 CVAPD (90%) were either removed as planned at the end of treatment (n=23) after a median 203 days, or remained in situ for a median of 237 days (7-1133). Premature removal occurred in eight patients due to infection (n=4), thrombosis (n=3) or faulty device (n=1). Four patients were lost to follow-up. Radiological insertion of CVAPD is safe and convenient with low rates of complications.