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1.  Two different network topologies yield bistability in models of mesoderm and anterior mesendoderm specification in amphibians 
Journal of Theoretical Biology  2014;353(100):67-77.
Understanding the Gene Regulatory Networks (GRNs) that underlie development is a major question for systems biology. The establishment of the germ layers is amongst the earliest events of development and has been characterised in numerous model systems. The establishment of the mesoderm is best characterised in the frog Xenopus laevis and has been well studied both experimentally and mathematically. However, the Xenopus network has significant differences from that in mouse and humans, including the presence of multiple copies of two key genes in the network, Mix and Nodal. The axolotl, a urodele amphibian, provides a model with all the benefits of amphibian embryology but crucially only a single Mix and Nodal gene required for the specification of the mesoderm. Remarkably, the number of genes within the network is not the only difference. The interaction between Mix and Brachyury, two transcription factors involved in the establishment of the endoderm and mesoderm respectively, is not conserved. While Mix represses Brachyury in Xenopus, it activates Brachyury in axolotl. Thus, whilst the topology of the networks in the two species differs, both are able to form mesoderm and endoderm in vivo. Based on current knowledge of the structure of the mesendoderm GRN we develop deterministic models that describe the time evolution of transcription factors in a single axolotl cell and compare numerical simulations with previous results from Xenopus. The models are shown to have stable steady states corresponding to mesoderm and anterior mesendoderm, with the in vitro model showing how the concentration of Activin can determine cell fate, while the in vivo model shows that β-catenin concentration can determine cell fate. Moreover, our analysis suggests that additional components must be important in the axolotl network in the specification of the full range of tissues.
Highlights
•We present models of mesendoderm specification in the urodele amphibian, the axolotl.•in vitro and in vivo models are simulated and compared with experimental data.•The model topology differs from that of the anuran amphibian, Xenopus laevis.•Steady states representing mesoderm and anterior mesendoderm are found in both models.•Both the axolotl and Xenopus topologies can account for similar qualitative data.
doi:10.1016/j.jtbi.2014.03.015
PMCID: PMC4029075  PMID: 24650939
Embryo development; Network motifs; Cell differentiation; Gene regulatory networks
2.  Case-control study of paternal occupation and social class with risk of childhood central nervous system tumours in Great Britain, 1962–2006 
British Journal of Cancer  2013;108(9):1907-1914.
Background:
Paternal occupational exposures have been proposed as a risk factor for childhood central nervous system (CNS) tumours. This study investigates possible associations between paternal occupational exposure and childhood CNS tumours in Great Britain.
Methods:
The National Registry of Childhood Tumours provided all cases of childhood CNS tumours born and diagnosed in Great Britain from 1962 to 2006. Controls without cancer were matched on sex, period of birth and birth registration sub-district. Fathers' occupations were assigned to one or more of 33 exposure groups. A measure of social class was also derived from father's occupation at the time of the child's birth.
Results:
Of 11 119 cases of CNS tumours, 5 722 (51%) were astrocytomas or other gliomas, 2 286 (21%) were embryonal and 985 (9%) were ependymomas. There was an increased risk for CNS tumours overall with exposure to animals, odds ratio (OR) 1.40 (95% confidence intervals (CIs) 1.01, 1.94) and, after adjustment for occupational social class (OSC), with exposure to lead, OR 1.18 (1.01, 1.39). Exposure to metal-working oil mists was associated with reduced risk of CNS tumours, both before and after adjustment for OSC, OR 0.87 (0.75, 0.99).
Risk of ependymomas was raised for exposure to solvents, OR 1.73 (1.02,2.92). For astrocytomas and other gliomas, risk was raised with high social contact, although this was only statistically significant before adjustment for OSC, OR 1.15 (1.01,1.31). Exposure to paints and metals appeared to reduce the risk of astrocytomas and embryonal tumours, respectively. However, as these results were the result of a number of statistical tests, it is possible they were generated by chance.
Higher social class was a risk factor for all CNS tumours, OR 0.97 (0.95, 0.99). This was driven by increased risk for higher social classes within the major subtype astrocytoma, OR 0.95 (0.91, 0.98).
Conclusion:
Our results provide little evidence that paternal occupation is a significant risk factor for childhood CNS tumours, either overall or for specific subtypes. However, these analyses suggest that OSC of the father may be associated with risk of some childhood CNS cancers.
doi:10.1038/bjc.2013.171
PMCID: PMC3658514  PMID: 23612452
childhood cancer; childhood CNS tumours; epidemiology; paternal occupation; social class
3.  Exploitation of interspecific diversity for monocot crop improvement 
Heredity  2013;110(5):475-483.
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.
doi:10.1038/hdy.2012.116
PMCID: PMC3630809  PMID: 23321705
Introgression mapping; comparative mapping; recombination; interspecific diversity; monocots; SNP markers
4.  Development and Implementation of Health and Wellness CBT for Individuals with Depression and HIV 
Cognitive and behavioral practice  2014;21(2):237-246.
Rates of depression are reported to be between 22–33% in adults with HIV, which is double that of the general population. Depression negatively affects treatment adherence and health outcomes of those with medical illnesses. Further, it has been shown in adults that reducing depression may improve both adherence and health outcomes. To address the issues of depression and non-adherence, Health and Wellness (H&W) Cognitive Behavioral Therapy (CBT) and medication management (MM) treatment strategies have been developed specifically for youth living with both HIV and depression. H&W CBT is based on other studies with uninfected youth and upon research on adults with HIV. H&W CBT uses problem-solving, motivational interviewing, and cognitive-behavioral strategies to decrease adherence obstacles and increase wellness. The intervention is delivered in 14 planned sessions over a 6-month period, with three different stages of CBT. This paper summarizes the feasibility and acceptability data from an open depression trial with 8 participants, 16–24 years of age, diagnosed with HIV and with a Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) diagnosis of depression, conducted at two treatment sites in the Adolescent Trials Network (ATN). Both therapists and subjects completed a Session Evaluation Form (SEF) after each session, and results were strongly favorable. Results from The Quick Inventory of Depressive Symptomatology-Clinician (QIDS-C) also showed noteworthy improvement in depression severity. A clinical case vignette illustrates treatment response. Further research will examine the use of H&W CBT in a larger trial of youth diagnosed with both HIV and depression.
doi:10.1016/j.cbpra.2013.07.003
PMCID: PMC4002170  PMID: 24795524
Cognitive Behavioral Therapy (CBT); Major Depressive Disorder (MDD); Human Immunodeficiency Virus (HIV); Adherence; Health
5.  Buckling of a growing tissue and the emergence of two-dimensional patterns☆ 
Mathematical Biosciences  2013;246(2):229-241.
Highlights
•We model the growth of gut epithelial cells cultured upon a deformable substrate.•Growth generates buckling instabilities, contributing to crypt formation in vivo.•Variations in mechanical properties have little effect on resulting configurations.•Configurations are controlled by growth patterns & interactions with strata below.
The process of biological growth and the associated generation of residual stress has previously been considered as a driving mechanism for tissue buckling and pattern selection in numerous areas of biology. Here, we develop a two-dimensional thin plate theory to simulate the growth of cultured intestinal epithelial cells on a deformable substrate, with the goal of elucidating how a tissue engineer might best recreate the regular array of invaginations (crypts of Lieberkühn) found in the wall of the mammalian intestine. We extend the standard von Kármán equations to incorporate inhomogeneity in the plate’s mechanical properties and surface stresses applied to the substrate by cell proliferation. We determine numerically the configurations of a homogeneous plate under uniform cell growth, and show how tethering to an underlying elastic foundation can be used to promote higher-order buckled configurations. We then examine the independent effects of localised softening of the substrate and spatial patterning of cellular growth, demonstrating that (within a two-dimensional framework, and contrary to the predictions of one-dimensional models) growth patterning constitutes a more viable mechanism for control of crypt distribution than does material inhomogeneity.
doi:10.1016/j.mbs.2013.09.008
PMCID: PMC3863975  PMID: 24128749
Buckling; Tissue growth; Pattern formation; von Kármán plate
6.  Case–control study of paternal occupation and childhood leukaemia in Great Britain, 1962–2006 
British Journal of Cancer  2012;107(9):1652-1659.
Background:
Paternal occupational exposures have been proposed as a risk factor for childhood leukaemia. This study investigates possible associations between paternal occupational exposure and childhood leukaemia in Great Britain.
Methods:
The National Registry of Childhood Tumours provided all cases of childhood leukaemia born and diagnosed in Great Britain between 1962 and 2006. Controls were matched on sex, period of birth and birth registration subdistrict. Fathers' occupations were assigned to 1 or more of 33 exposure groups. Social class was derived from father's occupation at the time of the child's birth.
Results:
A total of 16 764 cases of childhood leukaemia were ascertained. One exposure group, paternal social contact, was associated with total childhood leukaemia (odds ratio 1.14, 1.05–1.23); this association remained significant when adjusted for social class. The subtypes lymphoid leukaemia (LL) and acute myeloid leukaemia showed increased risk with paternal exposure to social contact before adjustment for social class. Risk of other leukaemias was significantly increased by exposure to electromagnetic fields, persisting after adjustment for social class. For total leukaemia, the risks for exposure to lead and exhaust fumes were significantly <1. Occupationally derived social class was associated with risk of LL, with the risk being increased in the higher social classes.
Conclusion:
Our results showed some support for a positive association between childhood leukaemia risk and paternal occupation involving social contact. Additionally, LL risk increased with higher paternal occupational social class.
doi:10.1038/bjc.2012.359
PMCID: PMC3493752  PMID: 22968649
childhood cancer; childhood leukaemia; epidemiology; paternal occupation; social class
8.  The willingness of patients presenting with advanced glaucoma to participate in a trial comparing primary medical vs primary surgical treatment 
Eye  2011;26(2):300-306.
Aim
The aim of this study is to examine the attitudes of patients, who presented with advanced glaucoma in at least one eye, to participation in a randomised prospective trial comparing primary medical treatment with primary surgical treatment for advanced glaucoma.
Methods
Patients who had presented with advanced glaucoma (>15 dB loss mean deviation on Humphrey visual field testing) in at least one eye were asked to participate. Five focus groups comprising of 4–8 patients and consisting of 29 patients in total were undertaken. The group interviews were conducted by two experienced qualitative researchers, an ophthalmic clinician was present to clarify technical issues. The focus group discussions were taped and transcribed in full, and analysed through a process of familiarisation, open (inductive) coding, theme generation, theme refinement, and thematic mapping.
Results
Three overarching themes were identified: (1) the extent of patients' knowledge, (2) anxieties about surgery, and (3) concerns about compromised care due to trial involvement; these themes were further classified into eight sub-themes.
Conclusions
Patients' willingness to participate in randomised clinical studies is significantly connected to their level of comprehension and insight about the medical condition, its treatment, and the research process; misunderstandings about any of these aspects may act as a significant barrier to trial recruitment. Recruitment rates for future randomised trials may be enhanced by ensuring that patients have full and accurate information about the treatment alternatives, and that uncertainty exists for best patient outcomes between treatment options, and reassuring potential participants that the research process, in particular randomisation, will not compromise clinical care.
doi:10.1038/eye.2011.279
PMCID: PMC3272192  PMID: 22094298
advanced glaucoma; randomised controlled trial; patient barriers; qualitative analysis
9.  Linear polyalkylamines as fingerprinting agents in capillary electrophoresis of low molecular weight heparins and glycosaminoglycans 
Electrophoresis  2011;32(21):3070-3077.
Glycosaminoglycan (GAG) analysis represents a challenging frontier despite the advent of many high resolution technologies because of their unparalleled structural complexity. We previously developed a resolving agent aided capillary electrophoretic approach for fingerprinting low molecular weight heparins (LMWHs) to profile their microscopic differences and assess batch-to-batch variability. In this work, we study the application of this approach for fingerprinting other GAGs and analyze the basis for the fingerprints observed in CE. Whereas the resolving agents, linear polyalkylamines, could resolve the broad featureless electropherogram of LMWH into a large number of distinct, highly reproducible peaks, longer GAGs such as chondroitin sulfate, dermatan sulfate and heparin responded in a highly individualistic manner. Full-length heparin interacted with linear polyalkylamines very strongly followed by dermatan sulfate, while chondroitin sulfate remained essentially unaffected. Oversulfated chondroitin sulfate could be easily identified from full-length heparin. Scatchard analysis of the binding profile of enoxaparin with three linear polyalkylamines displayed a biphasic binding profile suggesting two distinctly different types of interactions. Some LMWH chains were found to interact with linear polyalkylamines with affinities as high as 10 nM, while others displayed nearly 5000-fold weaker affinities. These observations provide fundamental insight into the basis for fingerprinting of LMWHs by linear polyalkylamine-based resolving agents, which could be utilized in the design of advanced resolving agents for compositional profiling, direct sequencing and chemoinformatics studies.
doi:10.1002/elps.201100175
PMCID: PMC3516877  PMID: 22002802
Capillary electrophoresis; Chondroitin sulfate; Dermatan sulfate; Fingerprinting; Glycosaminoglycans; Low molecular weight heparin; Oversulfated chondroitin sulfate; Polyalkylamines
10.  Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma 
British Journal of Cancer  2012;107(12):1987-1996.
Background:
Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).
Methods:
MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.
Results:
Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.
Conclusion:
We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.
doi:10.1038/bjc.2012.525
PMCID: PMC3516695  PMID: 23169280
myeloma; microRNAs; biomarkers; diagnostics; cleaved tRNA; serum miRNAs
11.  Differences in NMDA Receptor Expression During Human Development Determine the Response of Neurons to HIV-Tat-mediated Neurotoxicity 
Neurotoxicity research  2010;19(1):138-148.
HIV infection of the CNS can result in neurologic dysfunction in a significant number of infected individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV infection in neurons. Thus, neuronal damage and dropout are likely due to indirect effects of HIV infection of other CNS cells, through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivating protein tat. We and others demonstrated that tat induces apoptosis in differentiated mature human neurons. We now demonstrate that the high level of tat toxicity observed in human neurons involves specific developmental stages that correlate with N-Methyl-D-Aspartate receptor (NMDAR) expression, and that tat toxicity is also dependent upon the species being analyzed. Our results indicate that tat treatment of primary cultures of differentiated human neurons with significant amounts of NMDAR expression induces extensive apoptosis. In contrast, tat treatment induces only low levels of apoptosis in primary cultures of immature human neurons with low or minimal expression of NMDAR. In addition, tat treatment has minimal effect on rat hippocampal neurons in culture, despite their high expression of NMDAR. We propose that this difference may be due to low expression of the NR2A subunit. These findings are important for an understanding of the many differences among tissue culture systems and species used to study HIV-tat-mediated toxicity.
doi:10.1007/s12640-010-9150-x
PMCID: PMC3464008  PMID: 20094923
HIV-1; NeuroAIDS; Glutamate; NMDA; Dementia; HAND
12.  Role of Binding and Nucleoside Diphosphate Kinase A in the Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator by AMP-activated Protein Kinase* 
The Journal of Biological Chemistry  2012;287(40):33389-33400.
Background: AMPK phosphorylates CFTR and inhibits PKA-stimulated CFTR channel gating by unclear mechanisms.
Results: NDPK-A, AMPK, and CFTR exist in a membrane-associated complex. AMPK-CFTR binding and NDPK-A catalytic function are required for CFTR inhibition by AMPK.
Conclusion: NDPK-A plays an integral role in the regulation of CFTR by AMPK.
Significance: Targeting the AMPK-CFTR interaction and NDPK-A function could yield new therapeutic strategies for CF.
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.
doi:10.1074/jbc.M112.396036
PMCID: PMC3460441  PMID: 22869372
AMP-activated Kinase (AMPK); CFTR; Chloride Transport; Ion Channels; Oocyte; Patch Clamp Electrophysiology; Phosphorylation; Calu-3 Cells; Nm23; Nucleoside Diphosphate Kinase
13.  Phylogeography of microbial phototrophs in the dry valleys of the high Himalayas and Antarctica 
High-elevation valleys in dry areas of the Himalayas are among the most extreme, yet least explored environments on Earth. These barren, rocky valleys are subjected to year-round temperature fluctuations across the freezing point and very low availability of water and nutrients, causing previous workers to hypothesize that no photoautotrophic life (primary producers) exists in these locations. However, there has been no work using modern biogeochemical or culture-independent molecular methods to test the hypothesis that photoautotrophs are absent from high Himalayan soil systems. Here, we show that although microbial biomass levels are as low as those of the Dry Valleys of Antarctica, there are abundant microbial photoautotrophs, displaying unexpected phylogenetic diversity, in barren soils from just below the permanent ice line of the central Himalayas. Furthermore, we discovered that one of the dominant algal clades from the high Himalayas also contains the dominant algae in culture-independent surveys of both soil and ice samples from the Dry Valleys of Antarctica, revealing an unexpected link between these environmentally similar but geographically very distant systems. Phylogenetic and biogeographic analyses demonstrated that although this algal clade is globally distributed to other high-altitude and high-latitude soils, it shows significant genetic isolation by geographical distance patterns, indicating local adaptation and perhaps speciation in each region. Our results are the first to demonstrate the remarkable similarities of microbial life of arid soils of Antarctica and the high Himalayas. Our findings are a starting point for future comparative studies of the dry valleys of the Himalayas and Antarctica that will yield new insights into the cold and dry limits to life on Earth.
doi:10.1098/rspb.2010.1254
PMCID: PMC3030841  PMID: 20826485
cryophilic algae; cyanobacteria; cold deserts; dry valleys; subnival zone soils
14.  Translating HIV Interventions into Practice: Community-based Organizations’ Experiences with the Diffusion of Effective Behavioral Interventions (DEBIs) 
Social science & medicine (1982)  2010;71(10):1839-1846.
Efficacious behavioral interventions developed to address the spread of HIV/STIs are currently being disseminated in the USA through a national diffusion program (DEBI) spearheaded by the Centers for Disease Control and Prevention (CDC). Understanding how interventions are translated to real world settings is necessary to further scientific knowledge of this process and to facilitate future translation efforts in public health. Prior studies have begun to elucidate how agencies translate behavioral interventions into practice, but further work is needed. Guided by the ADAPT framework, we examined agencies’ assessment, preparation, and implementation of interventions. Our qualitative interview-based study focused on six community-based agencies in California (United States) funded to implement three group-level HIV interventions. Findings showed considerable variation in the extent to which agencies engaged in assessment and broad-based preparation and in the ease with which agencies implemented the interventions. The findings provide insight into the process that agencies undergo in the translation of effective behavioural interventions and illustrate how agencies can inform logic models that guide translation. We also identify relevant dimensions of existing models, including the ADAPT framework and Roger’s (1995 and Roger’s (2005) diffusion of innovations in organizations, that have value for agencies that are translating research to practice.
doi:10.1016/j.socscimed.2010.08.011
PMCID: PMC2994322  PMID: 20926169
USA; HIV/AIDS; translation research; evidence-based intervention; behavioral interventions; diffusion of innovations
15.  Axisymmetric indentation of curved elastic membranes by a convex rigid indenter 
Motivated by applications to seed germination, we consider the transverse deflection that results from the axisymmetric indentation of an elastic membrane by a rigid body. The elastic membrane is fixed around its boundary, with or without an initial pre-stretch, and may be initially curved prior to indentation. General indenter shapes are considered, and the load–indentation curves that result for a range of spheroidal tips are obtained for both flat and curved membranes. Wrinkling may occur when the membrane is initially curved, and a relaxed strain-energy function is used to calculate the deformed profile in this case. Applications to experiments designed to measure the mechanical properties of seed endosperms are discussed.
Highlights
► Axisymmetric indentation of elastic membranes. ► General axisymmetric indenter profiles considered. ► Initially curved elastic membranes, not just flat membranes. ► Strain-energy function important for large deformations.
doi:10.1016/j.ijnonlinmec.2011.04.030
PMCID: PMC3268561  PMID: 22298913
Indentation; Hyperelastic; Membrane; Deformation; Endosperm
16.  AMPK Agonists Ameliorate Sodium and Fluid Transport and Inflammation in Cystic Fibrosis Airway Epithelial Cells 
The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl− channel and epithelial Na+ channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (Isc), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2–5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent Isc in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red–dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03–1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.
doi:10.1165/rcmb.2009-0147OC
PMCID: PMC2891496  PMID: 19617399
metformin; cystic fibrosis transmembrane conductance regulator; ENaC; airway surface liquid; inflammation
17.  Sero-survey of rubella IgM antibodies among children in Jos, Nigeria 
Virology Journal  2011;8:244.
Sero-survey of rubella IgM antibodies was carried out among children aged 0-10 years in Jos, Nigeria. Blood samples were collected from the subjects and sera extracted. Of the 93(100%) assayed for the rubella IgM antibody, 42(45.2%) were seropositive for rubella IgM antibody while 51(54.8%) were seronegative. A breakdown of the seropositive subjects reveals that 14(15.1%) of the infected children were males while 28(30.1%) were females. Those subjects within the age groups of 1-2, 3-4 and 5-6 years had the highest prevalence of 8(8.6%) followed by those within the age groups of 7-8, 9-10 years with 7(7.5%). Blood transfusion as a risk factor did not show any significant influence on the status of the subjects. The demographic data of the mothers of the subjects were also linked with the seropositivity of the children.
doi:10.1186/1743-422X-8-244
PMCID: PMC3113311  PMID: 21595973
Sero-survey; rubella IgM antibodies; children; Nigeria
18.  Paternal occupation and neuroblastoma: a case–control study based on cancer registry data for Great Britain 1962–1999 
British Journal of Cancer  2010;102(3):615-619.
Background:
Neuroblastoma is the most common malignancy of infancy but little is known about the aetiological factors associated with the development of this tumour. A number of epidemiological studies have previously examined the risk associated with paternal occupational exposures but most have involved small numbers of cases. Here we present results from a large, population-based, case–control study of subjects diagnosed over a period of more than 30 years and recorded in the national registry of childhood tumours in Great Britain.
Methods:
A case–control study of paternal occupational data for 2920 cases of neuroblastoma, born and diagnosed in Great Britain between 1962 and 1999 and recorded in the National Registry of Childhood Tumours, and 2920 controls from the general population matched on sex, date of birth and birth registration district. Paternal occupations at birth, of the case or control child, were grouped by inferred exposure using an occupational exposure classification scheme. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CI), for each of the 32 paternal occupational exposure groups.
Results:
Only paternal occupational exposure to leather was statistically significantly associated with neuroblastoma, OR=5.00 (95% CI 1.07–46.93). However, this association became non-significant on correction for multiple testing.
Conclusion:
Our findings do not support the hypothesis that paternal occupational exposure is an important aetiological factor for neuroblastoma.
doi:10.1038/sj.bjc.6605504
PMCID: PMC2822941  PMID: 20068570
neuroblastoma; case–control study; paternal occupation
19.  Impact of epoxyeicosatrienoic acids in lung ischemia-reperfusion injury 
Objective
Epoxyeicosatrienoic acids (EETs) are protective in both myocardial and brain ischemia, variously attributed to activation of KATP channels or blockade of adhesion molecule upregulation. In this study we tested whether EETs would be protective in lung ischemia-reperfusion injury.
Methods
The filtration coefficient (Kf), a measure of endothelial permeability, and expression of the adhesion molecules VCAM and ICAM were measured after 45 min ischemia and 30 min reperfusion in isolated rat lungs.
Results
Kf increased significantly after ischemia-reperfusion alone vs time controls, an effect dependent upon extracellular Ca2+ though not on the EET-regulated channel TRPV4. Inhibition of endogenous EET degradation or administration of exogenous 11,12- or 14,-15-EET at reperfusion significantly limited the permeability response to ischemia-reperfusion. The beneficial effect of 11,12-EET was not prevented by blockade of KATP channels nor by blockade of TRPV4. Finally, 11,12-EET-dependent alteration in adhesion molecules expression is unlikely to explain its beneficial effect, since the expression of the adhesion molecules VCAM and ICAM in lung after ischemia-reperfusion was similar to that in controls.
Conclusion
EETs are beneficial in the setting of lung ischemia-reperfusion, when administered at reperfusion. However, further study will be needed to elucidate the mechanism of action.
doi:10.1111/j.1549-8719.2009.00013.x
PMCID: PMC2923642  PMID: 20163540
20.  Changes in MRI signal intensity during hypercapnic challenge under conscious and anesthetized conditions 
Magnetic resonance imaging  2003;21(9):995-1001.
Most functional magnetic resonance imaging (fMRI) studies in animals are conducted under anesthesia to minimize motion artifacts. However, methods and techniques have been developed recently for imaging fully conscious rats. Functional MRI studies on conscious animals report enhanced BOLD signal changes as compared to the anesthetized condition. In this study, rats were exposed to different concentrations of carbon dioxide (CO2) while conscious and anesthetized to test whether cerebrovascular reactivity may be contributing to these enhanced BOLD signal changes. Hypercapnia produced significantly greater increases in MRI signal intensity in fully conscious animals (6.7–13.3% changes) as when anesthetized with 1% isoflurane (3.2–4.9% changes). In addition, the response to hypercapnia was more immediate in the conscious condition (< 30s) with signal risetimes twice as fast as in the anesthetized state (60s). Both cortical and subcortical brain regions showed a robust, dose- dependent increase in MRI signal intensity with hypercapnic challenge while the animals were conscious but little or no change when anesthetized. Baseline variations in MRI signal were higher while animals were conscious but this was off set by greater signal intensity changes leading to a greater contrast-to-noise ratio, 13.1 in conscious animals, as compared to 8.0 in the anesthetized condition. In summary, cerebral vasculature appears to be more sensitive to hypercapnic challenge in the conscious condition resulting in enhanced T2* MRI signal intensity and the potential for better BOLD signal changes during functional imaging.
PMCID: PMC2962949  PMID: 14684202
Functional magnetic resonance imaging; Vascular reactivity; Bold signal intensity; Regional cerebral blood flow
21.  Frequency of bleb manipulations after trabeculectomy surgery 
Aim
To quantify the type and frequency of postoperative bleb manipulations undertaken after modern glaucoma surgery.
Methods
Bleb manipulations were recorded after trabeculectomy surgery on 119 consecutive patients with at least 1 year of follow‐up. The type of intervention and time after surgery were recorded. Statistical analysis identified success rates at various intraocular pressure (IOP) cut‐off definitions and identified factors that increased the risk for bleb manipulation.
Results
In all, 78.2% of trabeculectomies were followed by some form of bleb manipulation. Almost 49% of blebs underwent massage and a similar number required at least one suture removal, 31.1% required at least one 5‐fluorouracil (5‐FU) injection and 25.2% required at least one needling and 5‐FU injection. The median time to the first intervention for massage, suture removal, 5‐FU injection, and needling and 5‐FU injection were 1, 14, 14 and 43 days, respectively. IOP measurements were higher at all follow‐up time points in the intervention group. Failure to achieve specific IOP target pressures was also generally poorer in the 5‐FU, and needling and 5‐FU intervention groups.
Conclusions
Postoperative intervention is a frequent occurrence after modern glaucoma surgery. This requires intensive postoperative follow‐up and is a labour‐intensive undertaking. Despite interventions in our group of patients, IOP in the intervention group was always higher than in the group that required no intervention.
doi:10.1136/bjo.2006.109835
PMCID: PMC1955637  PMID: 17272388
22.  Stability of Cholesterol, 7-Ketocholesterol and β-Sitosterol during Saponification: Ramifications for Artifact Monitoring of Sterol Oxide Products 
Cholesterol has been used to monitor artifact generation. Stability differences among cholesterol oxide products (COPs) and cholesterol in thermal and alkaline conditions are theorized. Thus, use of cholesterol may be unsuitable for detection of artifacts generated from COPs. Stability of cholesterol was compared to that of 7-ketocholesterol (7-keto) and β-sitosterol (βS) under various thermal and alkaline saponification conditions: 1 M methanolic KOH for 18 h at 24 °C (1 M18hr24°C, Control), 18 h at 37 °C (1M18hr37°C), 3 h at 45 °C (1M3hr45°C), and 3.6 M methanolic KOH for 3 h at 24 °C (3.6M3hr24°C). Trends indicated that cholesterol in solution was more stable than 7-keto under all conditions. Compared to βS, cholesterol was more stable under all conditions except for 1M18hr37°C for which stabilities were similar. Compounds were more labile in heat than alkalinity. Poor recoveries of 7-keto during cold saponification with high alkalinity were attributed to alkaline instability. 7-Keto, less stable than cholesterol, should be used to monitor artifact generation during screening of various methods that include thermal and alkaline conditions. In a preliminary analysis of turkey meat, more 3,5-7-one was generated from spiking with cholesterol than with 7-keto.
doi:10.1007/s11746-010-1572-3
PMCID: PMC2923713  PMID: 20835297
Oxysterols; Phytosterols; Artifacts; Thermal and alkaline saponification
24.  A novel use of amniotic membrane in the management of tube exposure following glaucoma tube shunt surgery 
Aim
To report a novel technique using amniotic membrane to cover exposed glaucoma tube shunts.
Methods
A consecutive series of three cases that underwent drainage tube shunt surgery with the Ahmed valve for intractable glaucoma. All three patients developed exposure of the tube secondary to necrosis of the overlying bovine pericardial patch and conjunctiva. Repair of the defect was carried out with a double layer of amniotic membrane, the inner one acting as a graft and the outer as a patch. Autologous serum was used to promote epithelial growth.
Results
Successful lasting closure of the conjunctival defect was achieved in all cases.
Conclusion
Erosion of the drainage tube following shunt surgery is a potentially serious problem. It can be successfully managed using a double layer of amniotic membrane.
doi:10.1136/bjo.2005.084905
PMCID: PMC1856991  PMID: 16547316
amniotic membrane; glaucoma; tube shunt
25.  Development of a Genomic Microsatellite Library in Perennial Ryegrass (Lolium perenne) and its Use in Trait Mapping 
Annals of Botany  2008;101(6):845-853.
Background and Aims
Perennial ryegrass (Lolium perenne) is one of the key forage and amenity grasses throughout the world. In the UK it accounts for 70 % of all agricultural land use with an estimated farm gate value of £6 billion per annum. However, in terms of the genetic resources available, L. perenne has lagged behind other major crops in Poaceae. The aim of this project was therefore the construction of a microsatellite-enriched genomic library for L. perenne to increase the number of genetic markers available for both marker-assisted selection in breeding programmes and gene isolation.
Methods
Primers for 229 non-redundant microsatellite markers were designed and used to screen two L. perenne genotypes, one amenity and one forage. Of the 229 microsatellites, 95 were found to show polymorphism between amenity and forage genotypes. A selection of microsatellite primers was selected from these 95 and used to screen two mapping populations derived from intercrossing and backcrossing the two forage and amenity grass genotypes.
Key Results and Conclusions
The utility of the resulting genetic maps for analysis of the genetic control of target traits was demonstrated by the mapping of genes associated with heading date to linkage groups 4 and 7.
doi:10.1093/aob/mcn016
PMCID: PMC2710216  PMID: 18281692
Microsatellites; Lolium perenne; perennial ryegrass; trait mapping

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