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1.  The Skeleton Forming Proteome of an Early Branching Metazoan: A Molecular Survey of the Biomineralization Components Employed by the Coralline Sponge Vaceletia Sp. 
PLoS ONE  2015;10(11):e0140100.
The ability to construct a mineralized skeleton was a major innovation for the Metazoa during their evolution in the late Precambrian/early Cambrian. Porifera (sponges) hold an informative position for efforts aimed at unraveling the origins of this ability because they are widely regarded to be the earliest branching metazoans, and are among the first multi-cellular animals to display the ability to biomineralize in the fossil record. Very few biomineralization associated proteins have been identified in sponges so far, with no transcriptome or proteome scale surveys yet available. In order to understand what genetic repertoire may have been present in the last common ancestor of the Metazoa (LCAM), and that may have contributed to the evolution of the ability to biocalcify, we have studied the skeletal proteome of the coralline demosponge Vaceletia sp. and compare this to other metazoan biomineralizing proteomes. We bring some spatial resolution to this analysis by dividing Vaceletia’s aragonitic calcium carbonate skeleton into “head” and “stalk” regions. With our approach we were able to identify 40 proteins from both the head and stalk regions, with many of these sharing some similarity to previously identified gene products from other organisms. Among these proteins are known biomineralization compounds, such as carbonic anhydrase, spherulin, extracellular matrix proteins and very acidic proteins. This report provides the first proteome scale analysis of a calcified poriferan skeletal proteome, and its composition clearly demonstrates that the LCAM contributed several key enzymes and matrix proteins to its descendants that supported the metazoan ability to biocalcify. However, lineage specific evolution is also likely to have contributed significantly to the ability of disparate metazoan lineages to biocalcify.
doi:10.1371/journal.pone.0140100
PMCID: PMC4633127  PMID: 26536128
2.  A conserved set of maternal genes? Insights from a molluscan transcriptome 
The early animal embryo is entirely reliant on maternal gene products for a ‘jump-start’ that transforms a transcriptionally inactive embryo into a fully functioning zygote. Despite extensive work on model species, it has not been possible to perform a comprehensive comparison of maternally-provisioned transcripts across the Bilateria because of the absence of a suitable dataset from the Lophotrochozoa. As part of an ongoing effort to identify the maternal gene that determines left-right asymmetry in snails, we have generated transcriptome data from 1 to 2-cell and ~32-cell pond snail (Lymnaea stagnalis) embryos. Here, we compare these data to maternal transcript datasets from other bilaterian metazoan groups, including representatives of the Ecydysozoa and Deuterostomia. We found that between 5 and 10% of all L. stagnalis maternal transcripts (~300-400 genes) are also present in the equivalent arthropod (Drosophila melanogaster), nematode (Caenorhabditis elegans), urochordate (Ciona intestinalis) and chordate (Homo sapiens, Mus musculus, Danio rerio) datasets. While the majority of these conserved maternal transcripts (“COMATs”) have housekeeping gene functions, they are a non-random subset of all housekeeping genes, with an overrepresentation of functions associated with nucleotide binding, protein degradation and activities associated with the cell cycle. We conclude that a conserved set of maternal transcripts and their associated functions may be a necessary starting point of early development in the Bilateria. For the wider community interested in discovering conservation of gene expression in early bilaterian development, the list of putative COMATs may be useful resource.
doi:10.1387/ijdb.140121ad
PMCID: PMC4594767  PMID: 25690965
maternal to zygotic transition; mollusk; MBT; MZT; Spiralia
3.  The NULevel trial of a scalable, technology-assisted weight loss maintenance intervention for obese adults after clinically significant weight loss: study protocol for a randomised controlled trial 
Trials  2015;16:421.
Background
Effective weight loss interventions are widely available but, after weight loss, most individuals regain weight. This article describes the protocol for the NULevel trial evaluating the effectiveness and cost-effectiveness of a systematically developed, inexpensive, scalable, technology-assisted, behavioural intervention for weight loss maintenance (WLM) in obese adults after initial weight loss.
Methods/Design
A 12-month single-centre, two-armed parallel group, participant randomised controlled superiority trial is underway, recruiting a total of 288 previously obese adults after weight loss of ≥5 % within the previous 12 months. Participants are randomly assigned to intervention or control arms, with a 1:1 allocation, stratified by sex and percentage of body weight lost (<10 % vs ≥10 %). Change in weight (kg) from baseline to 12 months is the primary outcome. Weight, other anthropometric variables and 7-day physical activity (assessed via accelerometer) measures are taken at 0 and 12 months. Questionnaires at 0, 6 and 12 months assess psychological process variables, health service use and participant costs. Participants in the intervention arm initially attend an individual face-to-face WLM consultation with an intervention facilitator and then use a mobile internet platform to self-monitor and report their diet, daily activity (via pedometer) and weight through daily weighing on wirelessly connected scales. Automated feedback via mobile phone, tailored to participants’ weight regain and goal progress is provided. Participants in the control arm receive quarterly newsletters (via links embedded in text messages) and wirelessly connected scales. Qualitative process evaluation interviews are conducted with a subsample of up to 40 randomly chosen participants. Acceptability and feasibility of procedures, cost-effectiveness, and relationships among socioeconomic variables and WLM will also be assessed.
Discussion
It is hypothesised that participants allocated to the intervention arm will show significantly lower levels of weight regain from baseline than those in the control arm. To date, this is the first WLM trial using remote real-time weight monitoring and mobile internet platforms to deliver a flexible, efficient and scalable intervention, tailored to the individual. This trial addresses a key research need and has the potential to make a vital contribution to the evidence base to inform future WLM policy and provision.
Trial registration
http://www.isrctn.com/ISRCTN14657176 (registration date 20 March 2014).
doi:10.1186/s13063-015-0931-7
PMCID: PMC4580361  PMID: 26395774
Behaviour; Randomised controlled trial; Clinical protocol; Obesity; Overweight; Weight loss; Weight loss maintenance
5.  Evaluation of Serum Carnitine Levels for Pediatric Patients Receiving Carnitine-Free and Carnitine-Supplemented Parenteral Nutrition 
Hospital Pharmacy  2014;49(6):549-553.
Purpose:
Carnitine is a carrier molecule transporting long-chain fatty acids (LCFAs) into the mitochondria for fatty acid β-oxidation. The purpose of this study is to evaluate the role of carnitine supplementation in parenteral nutrition (PN) within the pediatric population. Our goal was to determine a weight range for which empiric carnitine supplementation is justified and to determine a weight range at which a carnitine level should first be drawn to confirm a deficiency prior to supplementation. Secondarily, we tried to determine a relationship among carnitine deficiency, hypoglycemia, and hypertriglyceridemia.
Methods:
This was a retrospective observational study to evaluate 2 groups of pediatric patients (weighing 0.68 kg to 60 kg) who were NPO and receiving PN. The first group of patients (n = 454) received carnitine supplementation (15 mg/kg/day) upon initiation of PN. The second group (n = 299) did not receive carnitine supplementation until they were determined to have a carnitine deficiency.
Results:
The data indicated that 82% of the patients weighing less than 5 kg were deficient. Patients weighing more than 5 kg had serum carnitine levels within the normal range. Therefore, patients receiving PN and weighing less than 5 kg should be supplemented with carnitine. Comparison of triglyceride, glucose, and carnitine showed no statistically significant difference (P = .1936).
Conclusion:
Patients weighing more than 5 kg should have serum carnitine levels drawn within 7 days to determine whether supplementation is needed. There is no statistical correlation among carnitine deficiency, hypoglycemia, and hypertriglyceridemia.
doi:10.1310/hpj4906-549
PMCID: PMC4062735  PMID: 24958973
carnitine supplementation; infants; neonates; parenteral nutrition; TPN
6.  Detection of Pathogenic Bacteria During Rhinovirus Infection is Associated with Increased Respiratory Symptoms and Exacerbations of Asthma 
Background
Detection of either viral or bacterial pathogens is associated with wheezing in children, however the influence of both bacteria and virus on illness symptoms has not been described.
Objective
We evaluated bacterial detection during peak RV season in children with and without asthma to determine if an association exists between bacterial infection and the severity of RV illnesses.
Methods
308 children (166 with asthma, 142 without asthma) ages 4–12 years provided five consecutive weekly nasal samples during September, and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were performed on all nasal samples.
Results
Detection rates were 53%, 17% and 11% for H. influenzae, S. pneumoniae and M. catarrhalis, respectively, with detection of RV increasing the risk of detecting bacteria within the same sample (OR 2.0, 95% CI 1.4–2.7, p<0.0001) or the following week (OR 1.6 (1.1–2.4), p=0.02). In the absence of RV, S. pneumoniae was associated with increased cold symptoms (mean 2.7 (95% CI 2.0–3.5) vs. 1.8 (1.5–2.2), p=0.006) and moderate asthma exacerbations (18% (12%–27%) vs. 9.2% (6.7%–12%), p=0.006). In the presence of RV, S. pneumoniae was associated with increased moderate asthma exacerbations (22% (16%–29%) vs. 15% (11%–20%), p=0.01). Furthermore, M. catarrhalis detected alongside RV increased the likelihood of experiencing cold and/or asthma symptoms compared to isolated detection of RV (OR 2.0 (1.0–4.1), p=0.04). Regardless of RV status, H. influenzae was not associated with respiratory symptoms.
Conclusion
RV infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M. catarrhalis and S. pneumoniae contribute to the severity of respiratory illnesses, including exacerbations of asthma.
doi:10.1016/j.jaci.2014.02.030
PMCID: PMC4047978  PMID: 24698319
Rhinovirus; bacteria; asthma
8.  The Magellania venosa Biomineralizing Proteome: A Window into Brachiopod Shell Evolution 
Genome Biology and Evolution  2015;7(5):1349-1362.
Brachiopods are a lineage of invertebrates well known for the breadth and depth of their fossil record. Although the quality of this fossil record attracts the attention of paleontologists, geochemists, and paleoclimatologists, modern day brachiopods are also of interest to evolutionary biologists due to their potential to address a variety of questions ranging from developmental biology to biomineralization. The brachiopod shell is a composite material primarily composed of either calcite or calcium phosphate in close association with proteins and polysaccharides which give these composite structures their material properties. The information content of these biomolecules, sequestered within the shell during its construction, has the potential to inform hypotheses focused on describing how brachiopod shell formation evolved. Here, using high throughput proteomic approaches and next generation sequencing, we have surveyed and characterized the first shell-proteome and shell-forming transcriptome of any brachiopod, the South American Magellania venosa (Rhynchonelliformea: Terebratulida). We find that the seven most abundant proteins present in the shell are unique to M. venosa, but that these proteins display biochemical features found in other metazoan biomineralization proteins. We can also detect some M. venosa proteins that display significant sequence similarity to other metazoan biomineralization proteins, suggesting that some elements of the brachiopod shell-forming proteome are deeply evolutionarily conserved. We also employed a variety of preparation methods to isolate shell proteins and find that in comparison to the shells of other spiralian invertebrates (such as mollusks) the shell ultrastructure of M. venosa may explain the effects these preparation strategies have on our results.
doi:10.1093/gbe/evv074
PMCID: PMC4453069  PMID: 25912046
Brachiopoda; biomineralization; proteome; transcriptome; evolution; Magellania
9.  Early Life Viral Infections and the Development of Asthma – A Target for Asthma Prevention? 
Purpose of the Review
To discuss recent insights into the relationships between viral respiratory infections and asthma inception in the context of a long-term goal of moving towards prevention strategies for childhood asthma.
Recent Findings
There is strong evidence for respiratory syncytial virus (RSV) and human rhinovirus (RV) wheezing illnesses as important risk factors for asthma inception. The mechanisms underlying these relationships have been an intense area of study. Novel approaches for the prevention of virus infections and/or lessening the severity of associated illnesses are at various stages of development, and are important potential tools in efforts aimed at primary and secondary prevention of asthma.
Summary
Viral respiratory infections in early life are a major source of morbidity and critical in the development of asthma. Mechanisms by which these infections lead to asthma inception in susceptible individuals are emerging. Further, there are promising potential interventions currently available that should be tested in clinical trials. The goal of prevention of disease inception is clearly on the horizon.
doi:10.1097/ACI.0000000000000047
PMCID: PMC4083742  PMID: 24569522
rhinovirus; RSV; asthma; prevention; children
10.  Asthma: NHLBI Workshop on the Primary Prevention of Chronic Lung Diseases 
Annals of the American Thoracic Society  2014;11(Suppl 3):S139-S145.
Asthma is a common disease with enormous public health costs, and its primary prevention is an ambitious and important goal. Understanding of how host and environmental factors interact to cause asthma is incomplete, but persistent questions about mechanisms should not stop clinical research efforts aimed at reducing the prevalence of childhood asthma. Achieving the goal of primary prevention of asthma will involve integrated and parallel sets of research activities in which mechanism-oriented studies of asthma inception proceed alongside clinical intervention studies to test biologically plausible prevention ideas. For example, continued research is needed, particularly in young children, to uncover biomarkers that identify asthma risk and provide potential targets of intervention, and to improve understanding of the role of microbial factors in asthma risk and disease initiation. In terms of clinical trials that could be initiated now or in the near future, we recommend three interventions for testing: (1) preventing asthma through prophylaxis against respiratory syncytial virus and human rhinovirus infections of the airway; (2) immune modulation, using prebiotics, probiotics, and bacterial lysates; and (3) prevention of allergen sensitization and allergic inflammation, using anti-IgE. These interventions should be tested while other, more universal prevention measures that may promote lung health are also investigated. These potential universal lung health measures include prevention of preterm delivery; reduced exposure of the fetus and young infant to environmental pollutants, including tobacco smoke; prevention of maternal and child obesity; and management of psychosocial stress.
doi:10.1513/AnnalsATS.201312-448LD
PMCID: PMC4112503  PMID: 24754822
asthma; primary prevention; allergy; respiratory syncytial virus; rhinovirus
11.  An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis 
Background
The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded, labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic stage within a species.
Results
Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal.
Conclusions
This method that we present here will be of value to investigators employing L. stagnalis as a model for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0068-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0068-7
PMCID: PMC4379745  PMID: 25879449
Whole mount in situ hybridisation; Mollusc; Lymnaea stagnalis; Gene expression; Evolution; Development
12.  Emerging Issues in Pediatric Asthma: Gaps in EPR-3 Guidelines for Infants and Children 
There are many complexities to the treatment of infants and children with recurrent wheezing and asthma. NHLBI’s Expert Panel Report-3 (EPR3), published in 2007, provides guidance to clinicians who care for infants and children with asthma. Since that time, many important clinical trials have further informed the evidence base available to clinicians. In this manuscript, new approaches to long-term therapy, intermittent fixed-dose and dynamic dose therapies, and emerging therapies for asthma are reviewed. Further, additional gaps in guideline-base care and areas for future research are discussed.
doi:10.1007/s11882-014-0477-x
PMCID: PMC4330969  PMID: 25269401
Asthma; Wheezing; Children; Guideline; Infants; Inhaled Corticosteroids; Long Acting Beta Agonists; Anticholinergics; Biologics; Action Plan; Yellow Zone; Dynamic dose; Fixed dose
13.  Ethnic-Specific Associations of Rare and Low Frequency DNA Sequence Variants with Asthma 
Nature communications  2015;6:5965.
Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (<1%) and low frequency (1–5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls, and 590 case-parent trios representing European Americans, African Americans/African Caribbeans, and Latinos. Our study reveals one low frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample (P=4.31×10−6; OR=1.25; MAF=1.21%) and two genes harboring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12-21 asthma locus in the Latino and combined samples (P=7.81×10−8 and 4.09×10−8, respectively) and MTHFR in the African ancestry sample (P=1.72×10−6). Our results suggest that associations with rare and low frequency variants are ethnic specific and not likely to explain a significant proportion of the “missing heritability” of asthma.
doi:10.1038/ncomms6965
PMCID: PMC4309441  PMID: 25591454
14.  Ethnic-specific associations of rare and low-frequency DNA sequence variants with asthma 
Nature Communications  2015;6:5965.
Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (<1%) and low-frequency (1–5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls and 590 case–parent trios representing European Americans, African Americans/African Caribbeans and Latinos. Our study reveals one low-frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample (P=4.31 × 10−6; OR=1.25; MAF=1.21%) and two genes harbouring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12–21 asthma locus in the Latino and combined samples (P=7.81 × 10−8 and 4.09 × 10−8, respectively) and MTHFR in the African ancestry sample (P=1.72 × 10−6). Our results suggest that associations with rare and low-frequency variants are ethnic specific and not likely to explain a significant proportion of the ‘missing heritability’ of asthma.
Common variants account for only a small amount of the heritable risk for developing asthma. Using a meta-analysis approach, Igartua et al. identify one low-frequency missense mutation and two genes with functional variants that are associated with asthma, but only in specific ethnic groups.
doi:10.1038/ncomms6965
PMCID: PMC4309441  PMID: 25591454
15.  Asthma Across the Ages: Knowledge Gaps in Childhood Asthma Prepared for the 2014 theme issue in the Journal of Allergy and Clinical Immunology 
The Eunice Kennedy Shriver National Institute of Child Health and Human Development convened an Asthma Group in response to the Best Pharmaceuticals for Children Act (BPCA). The overall goal of the BPCA Program is to improve pediatric therapeutics through preclinical and clinical drug trials that lead to drug labeling changes.
While significant advances have been made in the understanding and management of asthma in adults with appropriately labeled medications, less information is available on the management of asthma in children. Indeed, many medications are inadequately labeled in children. In general, the younger the child, the less information there is available to guide clinicians. Since asthma often begins in early childhood, it is incumbent upon us to continue to address the primary questions raised in this review and carefully evaluate medications used to manage asthma in children.
Meanwhile, continued efforts should be made in defining effective strategies that reduce the risk of exacerbations. If the areas of defined need are addressed in the coming years, namely prevention of exacerbations and progression of disease, as well as primary intervention, we will see continuing reduction in asthma mortality and morbidity along with improved quality of life for children with asthma.
doi:10.1016/j.jaci.2013.10.018
PMCID: PMC3925634  PMID: 24290281
Asthma; asthma natural history; asthma progression; asthma biomarkers; childhood asthma; asthma pharmacotherapy
16.  The clinical characteristics of patients with glaucoma presenting to Botswana healthcare facilities: an observational study 
BMJ Open  2014;4(12):e005965.
Objective
This study aimed to establish the clinical characteristics of patients with glaucoma attending eye care facilities in Botswana, and management of glaucoma among patients who received care in these facilities. The study also aimed to calculate the number of new diagnoses of glaucoma within the glaucoma service.
Design
A prospective, hospital-based, observational study.
Setting
A multicentre study was undertaken in government-run eye departments in Botswana from June to August 2012.
Participants
All patients with a diagnosis of glaucoma attending clinics at seven study sites were invited to participate.
Outcome measures
Examination findings, diagnosis and management were extracted from individual patient-held medical charts. Sociodemographic characteristics, patient knowledge and understanding of glaucoma were assessed through face-to-face interviews. In addition, details of outpatient attendances for 2011 were collected from 21 government-run hospitals.
Results
The majority of the 366 patients interviewed had a diagnosis of primary glaucoma (86.6%). The diagnoses were mainly made by ophthalmologists (48.6%) and ophthalmic nurses (44.0%). Many patients (38.5%) had been symptomatic for over 6 months before visiting an eye clinic. The mean presenting intraocular pressure was 28.2 mm Hg (SD 11.9 mm Hg). Most follow-up patients (79.2%) had not received surgery, however, many (89.5%) would accept surgery. Only 11.5% of participants had heard of glaucoma prior to diagnosis. Many participants (35.9%) did not understand glaucoma after being diagnosed. The majority (94.9%) of living first-degree relatives had never been examined. The number of newly diagnosed glaucoma cases for 2011 in the south of the country was 14.1/100 000; 95% CI (12.0 to 16.5), in the north it was 16.2/100 000; 95% CI (13.8 to 19.0).
Conclusions
Glaucoma is a significant burden that presents challenges to ophthalmic services in Botswana. Many patients have limited understanding of the condition and poor access to services. There is a need to develop a treatment infrastructure to include safe surgery and a reliable supply of effective medication.
doi:10.1136/bmjopen-2014-005965
PMCID: PMC4256541  PMID: 25472657
EPIDEMIOLOGY
18.  Laparoscopic versus open colorectal resection for cancer and polyps: a cost-effectiveness study 
Background
Available evidence that compares outcomes from laparoscopic and open surgery for colorectal cancer shows no difference in disease free or survival time, or in health-related quality of life outcomes, but does not capture the short term benefits of laparoscopic methods in the early postoperative period.
Aim
To explore the cost-effectiveness of laparoscopic colorectal surgery, compared to open methods, using quality of life data gathered in the first 6 weeks after surgery.
Methods
Participants were recruited in 2006–2007 in a district general hospital in the south of England; those with a diagnosis of cancer or polyps were included in the analysis. Quality of life data were collected using EQ-5D, on alternate days after surgery for 4 weeks. Costs per patient, from a National Health Service perspective (in British pounds, 2006) comprised the sum of operative, hospital, and community costs. Missing data were filled using multiple imputation methods. The difference in mean quality adjusted life years and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets. The probability that laparoscopic surgery is cost-effective compared to open surgery for a given societal willingness-to-pay threshold is illustrated using a cost-effectiveness acceptability curve.
Results
The sample comprised 68 laparoscopic and 27 open surgery patients. At 28 days, the incremental cost per quality adjusted life year gained from laparoscopic surgery was £12,375. At a societal willingness-to-pay of £30,000, the probability that laparoscopic surgery is cost-effective, exceeds 65% (at £20,000 ≈60%). In sensitivity analyses, laparoscopic surgery remained cost-effective compared to open surgery, provided it results in a saving ≥£699 in hospital bed days and takes no more than 8 minutes longer to perform.
Conclusion
The study provides formal evidence of the cost-effectiveness of laparoscopic approaches and supports current guidelines that promote use of laparoscopy where suitably trained surgeons are available.
doi:10.2147/CEOR.S66247
PMCID: PMC4186576  PMID: 25298736
colorectal cancer; laparoscopy; cost-effectiveness; QALYs
19.  Whole-Genome Sequencing of Individuals from a Founder Population Identifies Candidate Genes for Asthma 
PLoS ONE  2014;9(8):e104396.
Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS.
doi:10.1371/journal.pone.0104396
PMCID: PMC4130548  PMID: 25116239
20.  Maternal microchimerism protects against the development of asthma 
Background
Maternal asthma and child’s sex are among the most significant and reproducible risk factors for the development of asthma. Although the mechanisms for these effects are unknown, they likely involve non-classical genetic mechanisms. One such mechanism could involve the transfer and persistence of maternal cells to her offspring, a common occurrence known as maternal microchimerism (MMc). MMc has been associated with many autoimmune diseases, but has not been investigated for a role in asthma or allergic disease.
Objective
We hypothesized that some of the observed risks for asthma may be due to different rates of transmission or persistence of maternal cells to children of mothers with asthma compared to children of mothers without asthma, or to sons compared to daughters. We further hypothesized that rates of MMc differ between children with and without asthma.
Methods
We tested these hypotheses in 317 subjects from three independent cohorts using a real-time quantitative PCR assay to detect a non-inherited HLA allele in the child.
Results
MMc was detected in 20.5% of subjects (range 16.8% – 27.1% in the three cohorts). We observed lower rates of asthma among MMc positive subjects compared to MMc negative subjects (odds ratio [OR] 0.38, 95% CI 0.19, 0.79; P=0.029). Neither maternal asthma nor sex of the child was a significant predictor of MMc in the child (P = 0.81 and 0.15, respectively).
Conclusions
Our results suggest for the first time that MMc may protect against the development of asthma.
doi:10.1016/j.jaci.2012.12.1575
PMCID: PMC3700564  PMID: 23434286
Microchimerism; maternal; asthma
21.  Do oral corticosteroids reduce the severity of acute lower respiratory tract illnesses in preschool children with recurrent wheezing? 
The Journal of allergy and clinical immunology  2013;131(6):10.1016/j.jaci.2013.01.034.
Background
Oral corticosteroids (OCSs) are recommended for severe wheezing episodes in children. However, limited evidence supports this intervention in preschool children with outpatient wheezing illnesses.
Objective
We sought to investigate whether OCSs reduce symptom scores during acute lower respiratory tract illnesses (LRTIs) in preschool children with recurrent wheeze
Methods
We performed post hoc and replication analyses in 2 outpatient cohorts of children aged 1 to 5 years with episodic wheezing participating in clinical trials. We compared symptom scores during LRTIs that were or were not treated with OCSs, adjusting for differences in disease and episode severity covariates.We stratified episodes by severity by using a propensity model. The primary outcome was the area under the curve (AUC) of total symptom scores among the more severe episodes.
Results
Two hundred fifteen participants from the Acute Intervention Management Strategies trial experienced 798 acute LRTIs, 112 of which were defined as severe based on propensity scores. The AUCs of total symptom scores did not differ between the episodes that were (n = 70) and were not (n = 42) treated with OCSs (P = .46) nor was there an OCS treatment effect on individual symptom scores. Similar analyses of the Maintenance Versus Intermittent Inhaled Corticosteroids in Wheezing Toddlers trial, involving 278 participants with 133 severe LRTIs, confirmed the above findings (P =.46 for AUC of total symptoms score comparison).
Conclusion
In 2 separate cohorts of preschool children with episodic wheezing, OCS treatment during clinically significant LRTIs did not reduce symptom severity during acute LRTIs, despite asthma controller medication use during most episodes. These findings need to be confirmed in a prospective randomized controlled trial.
doi:10.1016/j.jaci.2013.01.034
PMCID: PMC3810170  PMID: 23498594
Oral corticosteroids; episodic wheezing; preschool children
22.  Comparison of N-acetylmethionine reactivity between oxaliplatin and an oxaliplatin derivative with chiral (S,S) amine nitrogen atoms 
Inorganica chimica acta  2013;401:64-69.
We have synthesized an oxaliplatin derivative using N,N′-dimethyl-1,2-diaminocyclohexane (Me2dach) as the diamine ligand. The complex (S,R,R,S)-Pt(Me2dach)(oxalate), where S,R,R,S represents the chiralities at N,C,C,N, respectively, was prepared and characterized by 1H NMR spectroscopy, COSY, NOESY, and HMQC. Oxaliplatin reacts with N-acetylmethionine (N-AcMet) to form [Pt(dach)(N-AcMet-S)2] and [Pt(dach)(N-AcMet-S,N)], with the former favored at higher molar ratios of N-AcMet. In contrast, Pt(Me2dach)(oxalate) reacts to form [Pt(Me2dach)(N-AcMet-S,O)]+ even in the presence of excess N-AcMet. Molecular mechanics calculations are consistent with significant steric clashes in models of [Pt(Me2dach)(N-AcMet-S)2]. When N-AcMet was reacted with an excess of each platinum complex, the rate of N-AcMet decrease was very similar for both complexes. Thus, the methyl groups at the nitrogen atoms had little to no effect on the addition of the sulfur atom of a single N-acetylmethionine, but they prevented chelation of the amide nitrogen or coordination of a second N-acetylmethionine residue.
doi:10.1016/j.ica.2013.03.008
PMCID: PMC3635081  PMID: 23626375
Nuclear magnetic resonance; platinum; oxaliplatin; amino acids; methionine
23.  Infections and Their Role in Childhood Asthma Inception 
Summary
The association of early onset wheezing with common viral and bacterial infections has raised significant interest in the role of infections in childhood asthma inception. This article serves to review these relationships among infections, host factors, and asthma inception in childhood.
doi:10.1111/pai.12147
PMCID: PMC3977202  PMID: 24236893
inception; asthma; infection; wheezing; virus; childhood; genetics
24.  Gene Splicing of an Invertebrate Beta Subunit (LCavβ) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels 
PLoS ONE  2014;9(4):e92941.
The accessory beta subunit (Cavβ) of calcium channels first appear in the same genome as Cav1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Cavβ subunits (β1, β2, β3, β4) which associate with four Cav1 channel isoforms (Cav1.1 to Cav1.4) and three Cav2 channel isoforms (Cav2.1 to Cav2.3). Here we assess the fundamentally-shared features of the Cavβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCav1, LCav2, and LCavβ). Invertebrate Cavβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Cav2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCavβ subunit. LCavβ will also slow the inactivation kinetics of LCav3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Cavβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCavβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Cav2.2 and Cavβ3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCav2 nor LCavβ) appears to be compatible with this observed property.
doi:10.1371/journal.pone.0092941
PMCID: PMC3972191  PMID: 24690951
25.  Characterization of the pigmented shell-forming proteome of the common grove snail Cepaea nemoralis 
BMC Genomics  2014;15:249.
Background
With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail, Cepaea nemoralis, has served as a model for evolutionary biologists and population geneticists for decades. Surprisingly, the molecular mechanisms by which C. nemoralis generates this pigmented shelled diversity, and the degree of evolutionary conservation present between molluscan shell-forming proteomes, remain unknown.
Results
Here, using next generation sequencing and high throughput proteomics, we identify and characterize the major proteinaceous components of the C. nemoralis shell, the first shell-proteome for a pulmonate mollusc. The recent availability of several marine molluscan shell-proteomes, and the dataset we report here, allow us to identify 59 evolutionarily conserved and novel shell-forming proteins. While the C. nemoralis dataset is dominated by proteins that share little to no similarity with proteins in public databases, almost half of it shares similarity with proteins present in other molluscan shells. In addition, we could not find any indication that a protein (or class of proteins) is directly associated with shell pigmentation in C. nemoralis. This is in contrast to the only other partially characterized molluscan-shell pigmentation mechanism employed by the tropical abalone Haliotis asinina.
Conclusions
The unique pulmonate shell-forming proteome that we report here reveals an abundance of both mollusc-specific and pulmonate-specific proteins, suggesting that novel coding sequences, and/or the extensive divergence of these sequences from ancestral sequences, supported the innovation of new shell types within the Conchifera. In addition, we report here the first evidence that molluscs use independently evolved mechanisms to pigment their shells. This proteome provides a solid foundation from which further studies aimed at the functional characterization of these shell-forming proteins can be conducted.
doi:10.1186/1471-2164-15-249
PMCID: PMC4023409  PMID: 24684722
Biomineralization; Calcification; Mollusc; Pulmonate; Pigment; Shell; Protein; Evolution; Cepaea nemoralis

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