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1.  The modulatory effect of cell–cell contact on the tumourigenic potential of pre-malignant epithelial cells: a computational exploration 
Malignant development cannot be attributed alone to genetic changes in a single cell, but occurs as a result of the complex interplay between the failure of cellular regulation mechanisms and the presence of a permissive microenvironment. Although E-cadherin is classified as a ‘metastasis suppressor’ owing to its role in intercellular adhesion, the observation that it may be downregulated at a premalignant stage is indicative of additional roles in neoplastic development. We have used an agent-based computational model to explore the emergent behaviour resulting from the interaction of single and subpopulations of E-cadherin-compromised cells with unaffected normal epithelial cells within a monolayer environment. We have extended this to investigate the importance of local tissue perturbations in the form of scratch-wounding, or ablation of randomly-dispersed normal cells, on the growth of a single cell exhibiting E-cadherin loss. Our results suggest that the microenvironment with respect to localized cell density and normal/E-cadherin-compromised neighbours is crucial in determining whether an abnormal individual cell proliferates or remains dormant within the monolayer. These predictions raise important questions relating to the propensity for individual mutations to give rise to disease, and future experimental exploration of these will enhance our understanding of a complex, multifactorial pathological process.
PMCID: PMC3565802  PMID: 23097504
agent-based computational model; epithelial cells; E-cadherin; intercellular adhesion
2.  Body checking behaviors in men 
Body image  2009;6(3):10.1016/j.bodyim.2009.05.001.
Males have been facing increasing pressure from the media to attain a lean, muscular physique, and are at risk for body dissatisfaction, disturbed eating and exercise behaviors, and abuse of appearance- and performance-enhancing drugs (APEDs). The aim of the current study was to examine the relationship between body checking and mood, symptoms of muscle dysmorphia, importance of shape and weight, and APED use in undergraduate males. Body checking in males was correlated with weight and shape concern, symptoms of muscle dysmorphia, depression, negative affect, and APED use. Body checking predicted APED use and uniquely accounted for the largest amount of variance in Muscle Dysmorphic Disorder Inventory (MDDI) scores (16%). Findings support the view that body checking is an important construct in male body image, muscle dysmorphia, and body change strategies and suggest a need for further research.
PMCID: PMC3838885  PMID: 19482568
Body checking; Muscularity; Body image; Body dissatisfaction; Muscle dysmorphia; Anabolic-androgenic steroids
3.  Advances in quality control: mouse embryo morphokinetics are sensitive markers of in vitro stress 
Human Reproduction (Oxford, England)  2013;28(7):1776-1782.
Can time-lapse analysis of cell division timings [morphokinetics (MK)] in mouse embryos detect toxins at concentrations that do not affect blastocyst formation?
An MK algorithm enhances assay sensitivity while providing results 24–48 h sooner than the traditional mouse embryo assay (MEA).
Current quality control testing methodology is sensitive but further improvements are needed to assure optimal culture conditions. MKs of embryo development may detect small variations in culture conditions.
Cross sectional—control versus treatment. Mouse embryo development kinetics of 466 embryos were analyzed according to exposure to various concentrations of toxins and toxic mineral oil.
Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CH) (0, 2, 4, 6 and 8 µM) and Triton X-100 (TX-100; 0, 0.0008, 0.0012, 0.0016 and 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 min for 120 h in seven focal planes. End-points were timing and pattern of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 h.
BR was not affected for embryos cultured in the three lowest concentrations of CH and the four lowest concentrations of TX-100. In contrast, a unique MK model detected all concentrations tested (P < 0.05). The MK model identified toxicity in two lots of toxic mineral oil that did not affect BR (P < 0.05).
A limited number of toxins were used so that the results may not apply to all potential embryo toxins. A larger sample size may also demonstrate other statistically significant developmental kinetic parameters.
MKs in mouse embryos are a sensitive and efficient method for quality control testing of in vitro culture conditions. BR, the end-point of traditional quality control assays, did not detect sublethal concentrations of toxins in the culture milieu in our study. This study demonstrates that temporal variation at key developmental stages reflects the quality of the culture environment. An MEA that incorporates MK will provide enhanced sensitivity and faster turn-around times.
The study was supported by Mayo Clinic Department of Obstetrics and Gynecology Small Grant Program. The authors have no competing interests to declare.
PMCID: PMC3685335  PMID: 23595971
time-lapse imaging; morphokinetics; embryo culture; quality control; toxicity
4.  Differential Effects of the CRF-R1 Antagonist GSK876008 on Fear-Potentiated, Light- and CRF-Enhanced Startle Suggest Preferential Involvement in Sustained versus Phasic Threat Responses 
The amplitude of the acoustic startle response is increased when elicited in the presence of brief cues that predict shock (fear-potentiated startle) and also when elicited during sustained exposure to bright light (light-enhanced startle). Although both effects are thought to reflect fear or anxiety, their neuroanatomical substrates differ. Whereas fear-potentiated startle is disrupted by reversible inactivation of the central nucleus of the amygdala (CeA) but not the closely related bed nucleus of the stria terminalis (BNST), light-enhanced startle is disrupted by BNST inactivation but not by CeA inactivation. Intra-ventricular infusions of corticotropin releasing factor (CRF) also increase startle (CRF-enhanced startle) and this effect is mediated by CRF receptors within the BNST, with no involvement of the CeA. Together, these observations suggest that CeA- and BNST-dependent fear and anxiety may be differentially sensitive to CRF receptor blockade. We tested this by orally administering the novel, potent, and selective CRF-R1 antagonist GSK876008 to rats prior to CRF-enhanced, light-enhanced, or fear-potentiated startle testing. GSK876008 disrupted CRF-enhanced startle with a linear dose-response curve, and light-enhanced startle with a U-shaped dose-response curve, but did not disrupt fear-potentiated startle to a visual stimulus at any dose tested, and even augmented the response in some animals. GSK876008 also disrupted shock-related ‘baseline’ startles increases, which may have reflected context conditioning (shown elsewhere to also be BNST-dependent). Overall, these results suggest that short-duration CeA-dependent threat responses can be pharmacologically dissociated from longer-duration BNST-dependent responses in terms of their sensitivity to CRF1 receptor antagonists.
PMCID: PMC3586210  PMID: 19078950
fear; anxiety; startle; amygdala; bed nucleus of the stria terminalis; corticotropin releasing factor
5.  Paediatric community-acquired septic shock: results from the REPEM network study 
European Journal of Pediatrics  2013;172(5):667-674.
Introduction and purpose of the study
With this study we aimed to describe a “true world” picture of severe paediatric ‘community-acquired’ septic shock and establish the feasibility of a future prospective trial on early goal-directed therapy in children. During a 6-month to 1-year retrospective screening period in 16 emergency departments (ED) in 12 different countries, all children with severe sepsis and signs of decreased perfusion were included.
A 270,461 paediatric ED consultations were screened, and 176 cases were identified. Significant comorbidity was present in 35.8 % of these cases. Intensive care admission was deemed necessary in 65.7 %, mechanical ventilation in 25.9 % and vasoactive medications in 42.9 %. The median amount of fluid given in the first 6 h was 30 ml/kg. The overall mortality in this sample was 4.5 %. Only 1.2 % of the survivors showed a substantial decrease in Paediatric Overall Performance Category (POPC). ‘Severe’ outcome (death or a decrease ≥2 in POPC) was significantly related (p < 0.01) to: any desaturation below 90 %, the amount of fluid given in the first 6 h, the need for and length of mechanical ventilation or vasoactive support, the use of dobutamine and a higher lactate or lower base excess but not to any variables of predisposition, infection or host response (as in the PIRO (Predisposition, Infection, Response, Organ dysfunction) concept).
The outcome in our sample was very good. Many children received treatment early in their disease course, so avoiding subsequent intensive care. While certain variables predispose children to become septic and shocked, in our sample, only measures of organ dysfunction and concomitant treatment proved to be significantly related with outcome. We argue why future studies should rather be large multinational prospective observational trials and not necessarily randomised controlled trials.
PMCID: PMC3631515  PMID: 23354787
Paediatric; Child; Emergency medicine; Sepsis; Shock; Outcome
6.  Role of Subchondral Bone during Early-stage Experimental TMJ Osteoarthritis 
Journal of Dental Research  2011;90(11):1331-1338.
Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease that affects both cartilage and subchondral bone. We used microarray to identify changes in gene expression levels in the TMJ during early stages of the disease, using an established TMJ OA genetic mouse model deficient in 2 extracellular matrix proteins, biglycan and fibromodulin (bgn-/0fmod-/-). Differential gene expression analysis was performed with RNA extracted from 3-week-old WT and bgn-/0fmod-/- TMJs with an intact cartilage/subchondral bone interface. In total, 22 genes were differentially expressed in bgn-/0fmod-/- TMJs, including 5 genes involved in osteoclast activity/differentiation. The number of TRAP-positive cells were three-fold higher in bgn-/0fmod-/- TMJs than in WT. Quantitative RT-PCR showed up-regulation of RANKL and OPG, with a 128% increase in RANKL/OPG ratio in bgn-/0fmod-/- TMJs. Histology and immunohistochemistry revealed tissue disorganization and reduced type I collagen in bgn-/0fmod-/- TMJ subchondral bone. Early changes in gene expression and tissue defects in young bgn-/0fmod-/- TMJ subchondral bone are likely attributed to increased osteoclast activity. Analysis of these data shows that biglycan and fibromodulin are critical for TMJ subchondral bone integrity and reveal a potential role for TMJ subchondral bone turnover during the initial early stages of TMJ OA disease in this model.
PMCID: PMC3188464  PMID: 21917603
temporomandibular disorders; osteoclasts; matrix biology; bone biology osteoarthritis; cartilage
7.  Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance 
British Journal of Cancer  2011;105(4):575-585.
Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes.
A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays.
Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype.
Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.
PMCID: PMC3170957  PMID: 21712824
germ cell tumour; yolk sac tumour; germinoma; methylation; paediatric
8.  Consequences of Aberrant Insulin Regulation in the Brain: Can Treating Diabetes be Effective for Alzheimer’s Disease 
Current Neuropharmacology  2011;9(4):693-705.
There is an urgent need for new ways to treat Alzheimer’s disease (AD), the most common cause of dementia in the elderly. Current therapies are modestly effective at treating the symptoms, and do not significantly alter the course of the disease. Over the years, a range of epidemiological and experimental studies have demonstrated interactions between diabetes mellitus and AD. As both diseases are leading causes of morbidity and mortality in the elderly and are frequent co-morbid conditions, it has raised the possibility that treating diabetes might be effective in slowing AD. This is currently being attempted with drugs such as the insulin sensitizer rosiglitazone. These two diseases share many clinical and biochemical features, such as elevated oxidative stress, vascular dysfunction, amyloidogenesis and impaired glucose metabolism suggesting common pathogenic mechanisms. The main thrust of this review will be to explore the evidence from a pathological point of view to determine whether diabetes can cause or exacerbate AD. This was supported by a number of animal models of AD that have been shown to have enhanced pathology when diabetic conditions were induced. The one drawback in linking diabetes and insulin to AD has been the postmortem studies of diabetic brains demonstrating that AD pathology was not increased; in fact decreased pathology has often been reported. In addition, diabetes induces its own distinct features of neuropathology different from AD. There are common pathological features to be considered including vascular abnormalities, a major feature arising from diabetes; there is increasing evidence that vascular abnormalities can contribute to AD. The most important common mechanism between insulin-resistant (type II) diabetes and AD could be impaired insulin signaling; a form of toxic amyloid can damage neuronal insulin receptors and affect insulin signaling and cell survival. It has even been suggested that AD could be considered as “type 3 diabetes” since insulin can be produced in brain. Another common feature of diabetes and AD are increased advanced glycation endproduct-modified proteins are found in diabetes and in the AD brain; the receptor for advanced glycation endproducts plays a prominent role in both diseases. In addition, a major role for insulin degrading enzyme in the degradation of Aβ peptide has been identified. Although clinical trials of certain types of diabetic medications for treatment of AD have been conducted, further understanding the common pathological processes of diabetes and AD are needed to determine whether these diseases share common therapeutic targets.
PMCID: PMC3263463  PMID: 22654727
Alzheimer’s disease; pathology; neurodegeneration; glucose metabolism; amyloid beta.
9.  Sample Quality Control Within Various Next Generation Sequencing Workflows Using the New Agilent 2200 TapeStation 
This study evaluates the performance of the 2200 TapeStation System in various Next Generation Sequencing (NGS) workflows. Numerous sample types were checked for quality at different stages of NGS protocols including pre- and post-shearing, post adaptor ligation as well as pre-hybridisation and post-hybridisation within the SureSelect target enrichment workflow. The data shows that this new automated electrophoresis system provides qualitative information that enables informed decision making in all downstream steps. By providing a range of ScreenTape consumables with standard and high sensitivities along with a tailored analysis package, the system is able to QC gDNA, fragmented DNA, whole genome libraries and target enriched libraries, presenting descriptive analyses at each stage for multiple sequencer protocols. The data described here demonstrates that the 2200 TapeStation System has the range, sensitivity, precision and accuracy to meticulously QC samples within the NGS workflow.
PMCID: PMC3630620
10.  Control of HIF-1α and vascular signaling in fetal lung involves cross talk between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching periodicity clock 
Lung development requires coordinated signaling between airway and vascular growth, but the link between these processes remains unclear. Mammalian target of rapamycin complex-1 (mTORC1) can amplify hypoxia-inducible factor-1α (HIF-1α) vasculogenic activity through an NH2-terminal mTOR binding (TOS) motif. We hypothesized that this mechanism coordinates vasculogenesis with the fibroblast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of airway branching. First, we tested if the HIF-1α TOS motif participated in epithelial-mesenchymal vascular signaling. mTORC1 activation by insulin significantly amplified HIF-1α activity at fetal Po2 (23 mmHg) in human bronchial epithelium (16HBE14o-) and induced vascular traits (Flk1, sprouting) in cocultured human embryonic lung mesenchyme (HEL-12469). This enhanced activation of HIF-1α by mTORC1 was abolished on expression of a HIF-1α (F99A) TOS-mutant and also suppressed vascular differentiation of HEL-12469 cocultures. Next, we determined if vasculogenesis in fetal lung involved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 pathway. Fetal airway epithelium displayed distinct mTORC1 activity in situ, and its hyperactivation by TSC1−/− knockout induced widespread VEGF expression and disaggregation of Tie2-positive vascular bundles. FGF-10-coated beads grafted into fetal lung explants from Tie2-LacZ transgenic mice induced localized vascular differentiation in the peripheral mesenchyme. In rat fetal distal lung epithelial (FDLE) cells cultured at fetal Po2, FGF-10 induced mTORC1 and amplified HIF-1α activity and VEGF secretion without induction of ERK1/2. This was accompanied by the formation of a complex between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor, TSC2, which abolished GTPase activity directed against Rheb, the G protein inducer of mTORC1. Thus, mTORC1 links HIF-1α-driven vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching periodicity regulator.
PMCID: PMC2957420  PMID: 20622121
lung development; epithelium; mesenchyme; hypoxia; rheb; tuberous sclerosis complex
11.  Hybrid Mice as Genetic Models of High Alcohol Consumption 
Behavior genetics  2009;40(1):93-110.
We showed that F1 hybrid genotypes may provide a broader variety of ethanol drinking phenotypes than the inbred progenitor strains used to create the hybrids (Blednov et al. in Alcohol Clin Exp Res 29:1949–1958–2005). To extend this work, we characterized alcohol consumption as well as intake of other tastants (saccharin, quinine and sodium chloride) in five inbred strains of mice (FVB, SJL, B6, BUB, NZB) and in their reciprocal F1 hybrids with B6 (FVBxB6; B6xFVB; NZBxB6; B6xNZB; BUBxB6; B6xBUB; SJLxB6; B6xSJL). We also compared ethanol intake in these mice for several concentrations before and after two periods of abstinence. F1 hybrid mice derived from the crosses of B6 and FVB and also B6 and SJL drank higher levels of ethanol than their progenitor strains, demonstrating overdominance for two-bottle choice drinking test. The B6 and NZB hybrid showed additivity in two-bottle choice drinking, whereas the hybrid of B6 and BUB demonstrated full or complete dominance. Genealogical origin, as well as non-alcohol taste preferences (sodium chloride), predicted ethanol consumption. Mice derived from the crosses of B6 and FVB showed high sustained alcohol preference and the B6 and NZB hybrids showed reduced alcohol preference after periods of abstinence. These new genetic models offer some advantages over inbred strains because they provide high, sustained, alcohol intake, and should allow mapping of loci important for the genetic architecture of these traits.
PMCID: PMC3038337  PMID: 19798565
Alcohol intake; Inbred strains; F1 hybrid; Tastes; Overdominance
12.  The state of research into children with cancer across Europe: new policies for a new decade 
ecancermedicalscience  2011;5:210.
Overcoming childhood cancers is critically dependent on the state of research. Understanding how, with whom and what the research community is doing with childhood cancers is essential for ensuring the evidence-based policies at national and European level to support children, their families and researchers. As part of the European Union funded EUROCANCERCOMS project to study and integrate cancer communications across Europe, we have carried out new research into the state of research in childhood cancers. We are very grateful for all the support we have received from colleagues in the European paediatric oncology community, and in particular from Edel Fitzgerald and Samira Essiaf from the SIOP Europe office. This report and the evidence-based policies that arise from it come at a important junction for Europe and its Member States. They provide a timely reminder that research into childhood cancers is critical and needs sustainable long-term support.
PMCID: PMC3223943  PMID: 22276053
13.  Development and Validation of a Male Specific Body Checking Questionnaire 
Body checking may be an important behavioral consequence of body image disturbance. Despite the importance of body checking, few measurements of this construct exist, particularly for males. This study describes the development and validation of the Male Body Checking Questionnaire (MBCQ).
Convergent and divergent validity, factor structure, and reliability were tested in three separate samples of men and women.
Factor analyses suggested a reliable four-factor structure with evidence of a higher order global checking factor for men, but not women. The MBCQ demonstrated good concurrent and divergent validity. Short-term test-retest reliability was good with high internal consistency across time.
Interpretation of psychometrics and recommendations for subsequent research are discussed. The MBCQ is likely to be an appropriate tool for investigating body image-based pathology in males.
PMCID: PMC2892168  PMID: 19247988
Male Body Checking Questionnaire; confirmatory factor analysis; psychometrics; body image disturbance; muscle dysmorphia; gender differences; exploratory factor analysis
14.  Selective Participation of the Bed Nucleus of the Stria Terminalis and CRF in Sustained Anxiety-Like versus Phasic Fear-Like Responses 
The medial division of the central nucleus of the amygdala (CeAM) and the lateral division of the bed nucleus of the stria terminalis (BNSTL) are closely related. Both receive projections from the basolateral amygdala (BLA) and both project to brain areas that mediate fear-influenced behaviors. In contrast to CeAM however, initial attempts to implicate the BNST in conditioned fear responses were largely unsuccessful. More recent studies have shown that the BNST does participate in some types of anxiety and stress responses. Here, we review evidence suggesting that the CeAM and BNSTL are functionally complementary, with CeAM mediating short- but not long-duration threat responses (i.e., phasic fear) and BNSTL mediating long- but not short-duration responses (sustained fear or ‘anxiety’). We also review findings implicating the stress-related peptide corticotropin-releasing factor (CRF) in sustained but not phasic threat responses, and attempt to integrate these findings into a neural circuit model which accounts for these and related observations.
PMCID: PMC2783512  PMID: 19595731
anxiety; startle; amygdala; bed nucleus of the stria terminalis; corticotropin releasing factor
15.  Management of septic arthritis: a systematic review 
Annals of the Rheumatic Diseases  2007;66(4):440-445.
Objective: To evaluate the existing evidence on the diagnosis and management of septic arthritis in native joints.
Design: Systematic review.
Data sources: Cochrane Library, Medline, Embase, National Electronic Library for Health, reference lists, national experts.
Review methods: Systematic review of the literature with evaluation of the methodological quality of the selected papers using defined criteria set out by the Clinical Effectiveness and Evaluation Unit of the Royal College of Physicians.
Results: 3291 citations were initially identified. Of these, 189 full text articles were identified for potential selection. Following review of these full text articles, 80 articles were found to fulfil the inclusion criteria and were included in the final list. Conclusions were drawn on the diagnosis, investigation and management of septic arthritis.
Discussion: Little good quality evidence exists to guide the diagnosis and management of septic arthritis. Overall, no investigation is more reliable in the diagnosis of septic arthritis than the opinion of an experienced doctor. Aspiration and culture of synovial fluid is crucial to the diagnosis, but measurement of cell count is unhelpful. Antibiotics are clearly required for a prolonged period, but there are no data to indicate by which route or for how long. Key unanswered questions remain surrounding the medical and surgical management of the infected joint.
PMCID: PMC1856038  PMID: 17223664
16.  Etanercept 50 mg once weekly is as effective as 25 mg twice weekly in patients with ankylosing spondylitis 
Annals of the Rheumatic Diseases  2006;65(12):1572-1577.
To compare the efficacy, pharmacokinetics and safety of etanercept 50 mg once weekly with 25 mg twice weekly and placebo in patients with ankylosing spondylitis.
A 12‐week, double‐blind, placebo‐controlled study compared the effects of etanercept 50 mg once weekly, etanercept 25 mg twice weekly and placebo in 356 patients with active ankylosing spondylitis (3:3:1 randomisation, respectively). The primary end point was the proportion of patients achieving a response at week 12 based on the Assessment in Ankylosing Spondylitis Working Group criteria (ASAS 20). The pharmacokinetics of etanercept 50 mg once weekly and 25 mg twice weekly were analysed.
Baseline characteristics and disease activity were similar among the three groups: etanercept 50 mg once weekly, etanercept 25 mg twice weekly and placebo. The percentage of patients discontinuing therapy was 9.0%, 9.3% and 13.7% for the three respective groups. ASAS 20 response at 12 weeks was achieved by 74.2% of patients with etanercept 50 mg once weekly and 71.3% of those with etanercept 25 mg twice weekly, both significantly higher than the percentage of patients taking placebo (37.3%, p<0.001). Percentages of patients with ASAS 5/6 response (70.3%, 72.0% and 27.5%, respectively; p<0.001) and those with ASAS 40 response (58.1%, 53.3% and 21.6%, respectively; p<0.001) followed a similar pattern. Significant improvement (p<0.05) was seen in measures of disease activity, back pain, morning stiffness and C reactive protein levels as early as 2 weeks. Serum etanercept exposure was similar between the etanercept groups. Incidence of treatment‐emergent adverse events, including infections, was similar among all three groups, and no unexpected safety issues were identified.
Patients with ankylosing spondylitis can expect a comparable significant improvement in clinical outcomes with similar safety when treated with etanercept 50 mg once weekly or with 25 mg twice weekly.
PMCID: PMC1798458  PMID: 16968715
17.  Challenges to implementation of sepsis guidelines 
Critical Care  2009;13(Suppl 4):P29.
PMCID: PMC2776202
18.  Molecular Ecological Analysis of Fecal Bacterial Populations from Term Infants Fed Formula Supplemented with Selected Blends of Prebiotics▿  
Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n = 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n = 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n = 27] or 8 g/liter [PGL8 group; n = 25]). A parallel breast-fed group (BF group) (n = 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.
PMCID: PMC2643592  PMID: 19088307
19.  How do clinical features help identify paediatric patients with fractures following blunt wrist trauma? 
Emergency Medicine Journal : EMJ  2006;23(5):354-357.
Wrist injuries are a common presentation to the emergency department (ED). There are no validated decision rules to help clinicians evaluate paediatric wrist trauma. This study aimed to identify which clinical features are diagnostically useful in deciding the need for a wrist radiograph, and then to develop a clinical decision rule.
This prospective cohort study was carried out in the ED of Sheffield Children's Hospital. Eligible patients were recruited if presenting within 72 hours following blunt wrist trauma. A standardised data collection form was completed for all patients. The outcome measure was the presence or absence of a fracture. Univariate analysis was performed with the χ2 test. Associated variables (p<0.2) were entered into a multivariate model. Classification and regression tree (CART) analysis was used to derive the clinical decision rule.
In total, 227 patients were recruited and 106 children were diagnosed with fractures (47%). Of 10 clinical features analysed, six were found by univariate analysis to be associated with a fracture. CART analysis identified the presence of radial tenderness, focal swelling, or an abnormal supination/pronation as the best discriminatory features. Cross fold validation of this decision rule had a sensitivity of 99.1% (95% confidence interval 94.8% to 100%) and a specificity of 24.0% (17.2% to 32.3%). The radiography rate would be 87%.
Radial tenderness, focal swelling, and abnormal supination/pronation are associated with wrist fractures in children. The clinical decision rule derived from these features had a high sensitivity, but low specificity, and would not substantially alter our current radiography rate. The potential for a clinical decision rule for paediatric wrist trauma appears limited.
PMCID: PMC2564082  PMID: 16627835
wrist trauma; paediatrics; decision making; radiographs
20.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
PMCID: PMC2656934  PMID: 18843368
21.  Mid cavity hypertrophic cardiomyopathy 
Heart  2005;91(12):1522.
PMCID: PMC1769218  PMID: 16287729
Images in cardiology
22.  Regulatory Roles of CD1d-Restricted NKT Cells in the Induction of Toxic Shock-Like Syndrome in an Animal Model of Fatal Ehrlichiosis▿  
Infection and Immunity  2008;76(4):1434-1444.
CD1d-restricted NKT cells are key players in host defense against various microbial infections. Using a murine model of fatal ehrlichiosis, we investigated the role of CD1d-restricted NKT cells in induction of toxic shock-like syndrome caused by gram-negative, lipopolysaccharide-lacking, monocytotropic Ehrlichia. Our previous studies showed that intraperitoneal infection of wild-type (WT) mice with virulent Ehrlichia (Ixodes ovatus Ehrlichia [IOE]) results in CD8+ T-cell-mediated fatal toxic shock-like syndrome marked by apoptosis of CD4+ T cells, a weak CD4+ Th1 response, overproduction of tumor necrosis factor alpha and interleukin-10, and severe liver injury. Although CD1d−/− mice succumbed to high-dose IOE infection similar to WT mice, they did not develop signs of toxic shock, as shown by elevated bacterial burdens, low serum levels of tumor necrosis factor, normal serum levels of liver enzymes, and the presence of few apoptotic hepatic cells. An absence of NKT cells restored the percentages and absolute numbers of CD4+ and CD8+ T cells and CD11b+ cells in the spleen compared to WT mice and was also associated with decreased expression of Fas on splenic CD4+ lymphocytes and granzyme B in hepatic CD8+ lymphocytes. Furthermore, our data show that NKT cells promote apoptosis of macrophages and up-regulation of the costimulatory molecule CD40 on antigen-presenting cells, including dendritic cells, B cells, and macrophages, which may contribute to the induction of pathogenic T-cell responses. In conclusion, our data suggest that NKT cells mediate Ehrlichia-induced T-cell-mediated toxic shock-like syndrome, most likely via cognate and noncognate interactions with antigen-presenting cells.
PMCID: PMC2292873  PMID: 18212072
23.  Molecular Changes in the Polymerase Genes (PA and PB1) Associated with High Pathogenicity of H5N1 Influenza Virus in Mallard Ducks▿  
Journal of Virology  2007;81(16):8515-8524.
The highly pathogenic (HP) influenza viruses H5 and H7 are usually nonpathogenic in mallard ducks. However, the currently circulating HP H5N1 viruses acquired a different phenotype and are able to cause mortality in mallards. To establish the molecular basis of this phenotype, we cloned the human A/Vietnam/1203/04 (H5N1) influenza virus isolate that is highly pathogenic in ferrets, mice, and mallards and found it to be a heterogeneous mixture. Large-plaque isolates were highly pathogenic to ducks, mice, and ferrets, whereas small-plaque isolates were nonpathogenic in these species. Sequence analysis of the entire genome revealed that the small-plaque and the large-plaque isolates differed in the coding of five amino acids. There were two differences in the hemagglutinin (HA) gene (K52T and A544V), one in the PA gene (T515A), and two in the PB1 gene (K207R and Y436H). We inserted the amino acid changes into the wild-type reverse genetic virus construct to assess their effects on pathogenicity in vivo. The HA gene mutations and the PB1 gene K207R mutation did not alter the HP phenotype of the large-plaque virus, whereas constructs with the PA (T515A) and PB1 (Y436H) gene mutations were nonpathogenic in orally inoculated ducks. The PB1 (Y436H) construct was not efficiently transmitted in ducks, whereas the PA (T515A) construct replicated as well as the wild-type virus did and was transmitted efficiently. These results show that the PA and PB1 genes of HP H5N1 influenza viruses are associated with lethality in ducks. The mechanisms of lethality and the perpetuation of this lethal phenotype in ducks in nature remain to be determined.
PMCID: PMC1951362  PMID: 17553873
24.  The Genome of the Obligately Intracellular Bacterium Ehrlichia canis Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies†  
Journal of Bacteriology  2006;188(11):4015-4023.
Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, α-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).
PMCID: PMC1482910  PMID: 16707693
25.  Popliteus tendon rupture: a case report and review of the literature 
PMCID: PMC1724686  PMID: 12893726

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