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1.  Rare Copy Number Variants 
Archives of general psychiatry  2010;67(4):318-327.
Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date.
To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia.
A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray.
The Wellcome Trust Case Control Consortium.
There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents.
Main Outcome Measures
Overall load of CNVs and presence of rare CNVs.
The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder.
Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder.
PMCID: PMC4476027  PMID: 20368508
2.  Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and cryoEM modeling 
Some capsid proteins built on the ubiquitous HK97-fold have accessory domains that impart specific functions. Bacteriophage P22 coat protein has a unique inserted I-domain. Two prior I-domain models from sub-nanometer cryoEM reconstructions differed substantially. Therefore, the NMR structure of the I-domain was determined, which also was used to improve cryoEM models of coat protein. The I-domain has an anti-parallel 6-stranded β-barrel fold, previously not observed in HK97-fold accessory domains. The D-loop, which is dynamic both in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. A newly described S-loop is important for capsid size determination, likely through intra-subunit interactions. Ten of eighteen coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.
PMCID: PMC4068711  PMID: 24836025
3.  Mother reports of maternal support following child sexual abuse: Preliminary psychometric data on the Maternal Self-report Support Questionnaire (MSSQ) 
Child abuse & neglect  2010;34(10):784-792.
Maternal support is an important factor in predicting outcomes following disclosure of child sexual abuse; however, definition of the construct has been unclear and existing measures of maternal support are utilized inconsistently and have limited psychometric data. The purpose of this study was to develop a reliable and valid mother-report measure for assessing maternal support following the disclosure of child sexual abuse.
Data from 2 very similar samples of mother-child pairs seeking forensic evaluation following the discovery of child sexual abuse were combined, resulting in a final sample of 246.
Exploratory factor analysis resulted in two reliable 7-item factors labeled “Emotional Support” and “Blame/Doubt, ” each of which had acceptable internal consistency. Analyses with a child-report measure of general maternal support the construct validity of the MSSQ. Concurrent validity analyses revealed unique relations with maternal ratings of child behavior problems and case characteristic data.
The study resulted in the development of a brief, easily scored self-report measure of maternal support with reasonable preliminary psychometric properties that could easily be utilized in other studies of sexually abused children.
Practice Implications
Adoption of this promising measure in future research will reduce the lack of cross-study measurement comparability that has characterized the maternal support literature to date, increase the feasibility of expanding upon current literature on maternal support, and may produce important information leading to clinical and theoretical innovation.
PMCID: PMC4443432  PMID: 20850181
4.  Holidays, celebrations, and commiserations: measuring drinking during feasting and fasting to improve national and individual estimates of alcohol consumption 
BMC Medicine  2015;13:113.
Accurate measures of alcohol consumption are critical in assessing health harms caused by alcohol. In many countries, there are large discrepancies between survey-based measures of consumption and those based on alcohol sales. In England, surveys measuring typical alcohol consumption account for only around 60% of alcohol sold. Here, using a national survey, we measure both typical drinking and atypical/special occasion drinking (i.e., feasting and fasting) in order to develop more complete measures of alcohol consumption.
A national random probability telephone survey was implemented (May 2013 to April 2014). Inclusion criteria were resident in England and aged 16 years or over. Respondents (n = 6,085) provided information on typical drinking (amounts per day, drinking frequency) and changes in consumption associated with routine atypical days (e.g., Friday nights) and special dinking periods (e.g., holidays) and events (e.g., weddings). Generalized linear modelling was used to identify additional alcohol consumption associated with atypical/special occasion drinking by age, sex, and typical drinking level.
Accounting for atypical/special occasion drinking added more than 120 million UK units of alcohol/week (~12 million bottles of wine) to population alcohol consumption in England. The greatest impact was seen among 25- to 34-year-olds with the highest typical consumption, where atypical/special occasions added approximately 18 units/week (144 g) for both sexes. Those reporting the lowest typical consumption (≤1 unit/week) showed large relative increases in consumption (209.3%) with most drinking associated with special occasions. In some demographics, adjusting for special occasions resulted in overall reductions in annual consumption (e.g., females, 65 to 74 years in the highest typical drinking category).
Typical drinking alone can be a poor proxy for actual alcohol consumption. Accounting for atypical/special occasion drinking fills 41.6% of the gap between surveyed consumption and national sales in England. These additional units are inevitably linked to increases in lifetime risk of alcohol-related disease and injury, particularly as special occasions often constitute heavy drinking episodes. Better population measures of celebratory, festival, and holiday drinking are required in national surveys in order to adequately measure both alcohol consumption and the health harms associated with special occasion drinking.
Electronic supplementary material
The online version of this article (doi:10.1186/s12916-015-0337-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4494693  PMID: 25998218
Abstinence; Alcohol; Binge drinking; Holidays; Sales; Surveys
5.  Relationship between alcohol-attributable disease and socioeconomic status, and the role of alcohol consumption in this relationship: a systematic review and meta-analysis 
BMC Public Health  2015;15:400.
Studies show that alcohol consumption appears to have a disproportionate impact on people of low socioeconomic status. Further exploration of the relationship between alcohol consumption, socioeconomic status and the development of chronic alcohol-attributable diseases is therefore important to inform the development of effective public health programmes.
We used systematic review methodology to identify published studies of the association between socioeconomic factors and mortality and morbidity for alcohol-attributable conditions. To attempt to quantify differences in the impact of alcohol consumption for each condition, stratified by SES, we (i) investigated the relationship between SES and risk of mortality or morbidity for each alcohol-attributable condition, and (ii) where, feasible explored alcohol consumption as a mediating or interacting variable in this relationship.
We identified differing relationships between a range of alcohol-attributable conditions and socioeconomic indicators. Pooled analyses showed that low, relative to high socioeconomic status, was associated with an increased risk of head and neck cancer and stroke, and in individual studies, with hypertension and liver disease. Conversely, risk of female breast cancer tended to be associated with higher socioeconomic status. These findings were attenuated but held when adjusted for a number of known risk factors and other potential confounding factors. A key finding was the lack of studies that have explored the interaction between alcohol-attributable disease, socioeconomic status and alcohol use.
Despite some limitations to our review, we have described relationships between socioeconomic status and a range of alcohol-attributable conditions, and explored the mediating and interacting effects of alcohol consumption where feasible. However, further research is needed to better characterise the relationship between socioeconomic status alcohol consumption and alcohol-attributable disease risk so as to gain a greater understanding of the mechanisms and pathways that influence the differential risk in harm between people of low and high socioeconomic status.
Electronic supplementary material
The online version of this article (doi:10.1186/s12889-015-1720-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4409704  PMID: 25928558
Alcohol; Socioeconomic status; Morbidity; Mortality; Systematic review
6.  The contrast sensitivity function of the praying mantis Sphodromantis lineola 
The detection of visual motion and its direction is a fundamental task faced by several visual systems. The motion detection system of insects has been widely studied with the majority of studies focussing on flies and bees. Here we characterize the contrast sensitivity of motion detection in the praying mantis Sphodromantis lineola, an ambush predator that stays stationary for long periods of time while preying on fast-moving prey. In this, its visual behaviour differs from previously studied insects and we might therefore expect its motion detection system to differ from theirs. To investigate the sensitivity of the mantis we analyzed its optomotor response in response to drifting gratings with different contrasts and spatio-temporal frequencies. We find that the contrast sensitivity of the mantis depends on the spatial and temporal frequencies present in the stimulus and is separably tuned to spatial and temporal frequency rather than specifically to object velocity. Our results also suggest that mantises are sensitive to a broad range of velocities, in which they differ from bees and are more similar to hoverflies. We discuss our results in relation to the contrast sensitivities of other insects and the visual ecology of the mantis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00359-015-1008-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4510923  PMID: 25894490
Praying mantis; Contrast sensitivity; Visual ecology; Separable tuning; Optomotor
7.  A genetic risk score combining 32 SNPs is associated with body mass index and improves obesity prediction in people with major depressive disorder 
BMC Medicine  2015;13:86.
Obesity is strongly associated with major depressive disorder (MDD) and various other diseases. Genome-wide association studies have identified multiple risk loci robustly associated with body mass index (BMI). In this study, we aimed to investigate whether a genetic risk score (GRS) combining multiple BMI risk loci might have utility in prediction of obesity in patients with MDD.
Linear and logistic regression models were conducted to predict BMI and obesity, respectively, in three independent large case–control studies of major depression (Radiant, GSK-Munich, PsyCoLaus). The analyses were first performed in the whole sample and then separately in depressed cases and controls. An unweighted GRS was calculated by summation of the number of risk alleles. A weighted GRS was calculated as the sum of risk alleles at each locus multiplied by their effect sizes. Receiver operating characteristic (ROC) analysis was used to compare the discriminatory ability of predictors of obesity.
In the discovery phase, a total of 2,521 participants (1,895 depressed patients and 626 controls) were included from the Radiant study. Both unweighted and weighted GRS were highly associated with BMI (P <0.001) but explained only a modest amount of variance. Adding ‘traditional’ risk factors to GRS significantly improved the predictive ability with the area under the curve (AUC) in the ROC analysis, increasing from 0.58 to 0.66 (95% CI, 0.62–0.68; χ2 = 27.68; P <0.0001). Although there was no formal evidence of interaction between depression status and GRS, there was further improvement in AUC in the ROC analysis when depression status was added to the model (AUC = 0.71; 95% CI, 0.68–0.73; χ2 = 28.64; P <0.0001). We further found that the GRS accounted for more variance of BMI in depressed patients than in healthy controls. Again, GRS discriminated obesity better in depressed patients compared to healthy controls. We later replicated these analyses in two independent samples (GSK-Munich and PsyCoLaus) and found similar results.
A GRS proved to be a highly significant predictor of obesity in people with MDD but accounted for only modest amount of variance. Nevertheless, as more risk loci are identified, combining a GRS approach with information on non-genetic risk factors could become a useful strategy in identifying MDD patients at higher risk of developing obesity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12916-015-0334-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4407390  PMID: 25903154
Body mass index; Genetic risk score; Major depressive disorder; Obesity
8.  Is There Any Association between PEEP and Upper Extremity DVT? 
Background. We hypothesized that positive end-exploratory pressure (PEEP) may promote venous stasis in the upper extremities and predispose to upper extremity deep vein thrombosis (UEDVT). Methods. We performed a retrospective case control study of medical intensive care unit patients who required mechanical ventilation (MV) for >72 hours and underwent duplex ultrasound of their upper veins for suspected DVT between January 2011 and December 2013. Results. UEDVT was found in 32 (28.5%) of 112 patients. Nineteen (67.8%) had a central venous catheter on the same side. The mean ± SD duration of MV was 13.2 ± 9.5 days. Average PEEP was 7.13 ± 2.97 cm H2O. Average PEEP was ≥10 cm H2O in 23 (20.5%) patients. Congestive heart failure (CHF) significantly increased the odds of UEDVT (OR 4.53, 95% CI 1.13–18.11; P = 0.03) whereas longer duration of MV (≥13 vs. <13 days) significantly reduced it (OR 0.29, 95% CI 0.11–0.8; P = 0.02). Morbid obesity showed a trend towards significance (OR 3.82, 95% CI 0.95–15.4; P = 0.06). Neither PEEP nor any of the other analyzed predictors was associated with UEDVT. Conclusions. There is no association between PEEP and UEDVT. CHF may predispose to UEDVT whereas the risk of UEDVT declines with longer duration of MV.
PMCID: PMC4398918  PMID: 25922762
9.  Developing an Automated Database for Monitoring Ultrasound- and Computed Tomography-Guided Procedure Complications and Diagnostic Yield 
Journal of Digital Imaging  2013;27(2):270-279.
Monitoring complications and diagnostic yield for image-guided procedures is an important component of maintaining high quality patient care promoted by professional societies in radiology and accreditation organizations such as the American College of Radiology (ACR) and Joint Commission. These outcome metrics can be used as part of a comprehensive quality assurance/quality improvement program to reduce variation in clinical practice, provide opportunities to engage in practice quality improvement, and contribute to developing national benchmarks and standards. The purpose of this article is to describe the development and successful implementation of an automated web-based software application to monitor procedural outcomes for US- and CT-guided procedures in an academic radiology department. The open source tools PHP: Hypertext Preprocessor (PHP) and MySQL were used to extract relevant procedural information from the Radiology Information System (RIS), auto-populate the procedure log database, and develop a user interface that generates real-time reports of complication rates and diagnostic yield by site and by operator. Utilizing structured radiology report templates resulted in significantly improved accuracy of information auto-populated from radiology reports, as well as greater compliance with manual data entry. An automated web-based procedure log database is an effective tool to reliably track complication rates and diagnostic yield for US- and CT-guided procedures performed in a radiology department.
PMCID: PMC3948931  PMID: 24146357
Procedures; Complications; Diagnostic yield; Tracking; Automated
10.  Breaking the In Vitro Alveolar Type II Cell Proliferation Barrier while Retaining Ion Transport Properties 
Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative “ATI-like” cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a “conditionally reprogrammed cell” phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and “ATI-like” mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air–liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases.
PMCID: PMC4068919  PMID: 24191670
proliferation; transdifferentiation; ion transport
11.  Distinct Features in Natural History and Outcomes of Acute Hepatitis C 
Supplemental Digital Content is available in the text.
Acute hepatitis C (AHCV) provides a diagnostic challenge with diverse clinical presentations.
This study was aimed to examine the clinical and demographic features as well as outcomes in AHCV patients identified from inpatient and outpatient hospital settings.
Patients with suspected AHCV were recruited from Philadelphia VA Medical Center, Hospital of University of Pennsylvania and Brooklyn VA Medical Center between 2000 and 2010. AHCV was diagnosed by acute serum alanine aminotransferase elevation with anti-hepatitis C virus (HCV) seroconversion, HCV-RNA fluctuations above 1 log, and/or recent high-risk exposure without prior HCV infection, excluding those with human immunodeficiency virus infection. Clinical and therapeutic outcomes were monitored for at least 6 months.
A total of 40 AHCV patients were enrolled with a median follow-up of 129 weeks. They were mostly men (68%) and whites (73%) with median age of 43 years, diverse risk factors (33% injection drugs, 20% health care–associated, 3% sexual, and 45% unknown), and wide variations in peak alanine aminotransferase (143 to 3435 U/L) and total bilirubin levels (0.4 to 19.3 mg/dL). Viremia resolved spontaneously in 23% and persisted without therapy in 27%, whereas 50% received interferon α-based therapy with 90% cure (18/20). Distinct clinical scenarios included: (1) wide viremic fluctuations >1 log (65%) and intermittent HCV-RNA negativity; (2) autoantibodies (25% antinuclear antibodies, 69% antismooth muscle antibodies) or autoimmune features; (3) delayed spontaneous viral clearance in 2 patients; (4) rapid cirrhosis progression in 2 patients.
AHCV is a heterogenous disease that requires careful monitoring. The lack of apparent risk factor in high proportion of patients and its diverse presentations warrant diagnostic vigilance.
PMCID: PMC4112167  PMID: 24457946
hepatitis C virus; acute hepatitis; RNA fluctuation; clinical presentation; autoantibody
12.  Plant-Pathogenic Oomycetes, Escherichia coli Strains, and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State 
Applied and Environmental Microbiology  2014;80(16):4814-4820.
In the United States, surface water is commonly used to irrigate a variety of produce crops and can harbor pathogens responsible for food-borne illnesses and plant diseases. Understanding when pathogens infest water sources is valuable information for produce growers to improve the food safety and production of these crops. In this study, prevalence data along with regression tree analyses were used to correlate water quality parameters (pH, temperature, turbidity), irrigation site properties (source, the presence of livestock or fowl nearby), and precipitation data to the presence and concentrations of Escherichia coli, Salmonella spp., and hymexazol-insensitive (HIS) oomycetes (Phytophthora and Pythium spp.) in New York State surface waters. A total of 123 samples from 18 sites across New York State were tested for E. coli and Salmonella spp., of which 33% and 43% were positive, respectively. Additionally, 210 samples from 38 sites were tested for HIS oomycetes, and 88% were found to be positive, with 10 species of Phytophthora and 11 species of Pythium being identified from the samples. Regression analysis found no strong correlations between water quality parameters, site factors, or precipitation to the presence or concentration of E. coli in irrigation sources. For Salmonella, precipitation (≤0.64 cm) 3 days before sampling was correlated to both presence and the highest counts. Analyses for oomycetes found creeks to have higher average counts than ponds, and higher turbidity levels were associated with higher oomycete counts. Overall, information gathered from this study can be used to better understand the food safety and plant pathogen risks of using surface water for irrigation.
PMCID: PMC4135776  PMID: 24878603
13.  Comorbid medical illness in bipolar disorder 
The British Journal of Psychiatry  2014;205(6):465-472.
Individuals with a mental health disorder appear to be at increased risk of medical illness.
To examine rates of medical illnesses in patients with bipolar disorder (n = 1720) and to examine the clinical course of the bipolar illness according to lifetime medical illness burden.
Participants recruited within the UK were asked about the lifetime occurrence of 20 medical illnesses, interviewed using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) and diagnosed according to DSM-IV criteria.
We found significantly increased rates of several medical illnesses in our bipolar sample. A high medical illness burden was associated with a history of anxiety disorder, rapid cycling mood episodes, suicide attempts and mood episodes with a typically acute onset.
Bipolar disorder is associated with high rates of medical illness. This comorbidity needs to be taken into account by services in order to improve outcomes for patients with bipolar disorder and also in research investigating the aetiology of affective disorder where shared biological pathways may play a role.
PMCID: PMC4248234  PMID: 25359927
14.  Antibacterial gene transfer across the tree of life 
eLife  2014;3:e04266.
Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group.
eLife digest
Living things inherit most of their genetic material from their parents, so genes tend to be passed on from one generation to the next—from ancestors to descendants. Sometimes, however, DNA is transferred from one organism to another by other means. These events, collectively called horizontal gene transfer, are fairly common in nature; genes have been passed between different species as well as between different groups of organisms. For example, genes that confer resistance to antibacterial drugs have transferred from one species of bacteria to another, and other genes have also ‘jumped’ from bacteria to plants or animals.
Now Metcalf et al. have studied a gene that first arose in bacteria and that encodes an enzyme called a lysozyme. This enzyme breaks down the outer casing of a bacterial cell: a step that is required for a bacterium to reproduce and divide in two. When Metcalf et al. searched for relatives of the lysozyme gene, they found copies in many other species of bacteria and revealed that this gene has been repeatedly transferred between different bacteria. Members of the lysozyme gene family have also ‘jumped out’ of bacteria and into other organisms at least four times. Metcalf et al. found related lysozyme genes in a plant, an insect, many species of fungi, and a single-celled microbe (called an archaeon) that lives at hot, deep-sea vents.
A gene family being spread this widely across the tree of life has not been seen before. Nevertheless, as DNA is a common biological language to all living things, it is likely that all the different species that have received a lysozyme gene might use it for similar purposes.
Metcalf et al. reveal that the lysozyme could be being used as an antibacterial molecule. The archaeon lysozyme can kill a broad range of bacteria; and when the gene was transferred into Escherichia coli bacteria, only the bacteria that mutated the lysozyme gene to render it useless were able to survive. Metcalf et al. also revealed that the archaeon microbe produces more of the enzyme if bacteria are present, which allows it to outcompete these bacteria.
These findings suggest that there may be a number of horizontally transferred genes that have antibacterial activity against a wide range of bacteria. Searching for these genes—particularly in the largely underexplored group of archaea—might reveal new sources for antibiotic drugs to treat bacterial infections.
PMCID: PMC4241558  PMID: 25422936
horizontal gene transfer; antibiotic; lysin; archaea; other
15.  Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers 
Journal of proteome research  2013;12(10):10.1021/pr400307u.
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions. 480 LC-MS/MS runs delivered >250 GB of data in two months. Several analysis algorithms were compared. At 1 % false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
PMCID: PMC3817719  PMID: 24004147
immunodepletion; Seppro; IgY14; iTRAQ; EMMOL normalization; TripleTOF; Orbitrap; Q Exactive
16.  Gene expression in whole lung and pulmonary macrophages reflects the dynamic pathology associated with airway surface dehydration 
BMC Genomics  2014;15(1):726.
Defects in airway mucosal defense, including decreased mucus clearance, contribute to the pathogenesis of human chronic obstructive pulmonary diseases. Scnn1b-Tg mice, which exhibit chronic airway surface dehydration from birth, can be used as a model to study the pathogenesis of muco-obstructive lung disease across developmental stages. To identify molecular signatures associated with obstructive lung disease in this model, gene expression analyses were performed on whole lung and purified lung macrophages collected from Scnn1b-Tg and wild-type (WT) littermates at four pathologically relevant time points. Macrophage gene expression at 6 weeks was evaluated in mice from a germ-free environment to understand the contribution of microbes to disease development.
Development- and disease-specific shifts in gene expression related to Scnn1b over-expression were revealed in longitudinal analyses. While the total number of transgene-related differentially expressed genes producing robust signals was relatively small in whole lung (n = 84), Gene Set Enrichment Analysis (GSEA) revealed significantly perturbed biological pathways and interactions between normal lung development and disease initiation/progression. Purified lung macrophages from Scnn1b-Tg mice exhibited numerous robust and dynamic gene expression changes. The expression levels of Classically-activated (M1) macrophage signatures were significantly altered at post-natal day (PND) 3 when Scnn1b-Tg mice lung exhibit spontaneous bacterial infections, while alternatively-activated (M2) macrophage signatures were more prominent by PND 42, producing a mixed M1-M2 activation profile. While differentially-regulated, inflammation-related genes were consistently identified in both tissues in Scnn1b-Tg mice, there was little overlap between tissues or across time, highlighting time- and tissue-specific responses. Macrophages purified from adult germ-free Scnn1b-Tg mice exhibited signatures remarkably similar to non-germ-free counterparts, indicating that the late-phase macrophage activation profile was not microbe-dependent.
Whole lung and pulmonary macrophages respond independently and dynamically to local stresses associated with airway mucus stasis. Disease-specific responses interact with normal developmental processes, influencing the final state of disease in this model. The robust signatures observed in Scnn1b-Tg lung macrophages highlight their critical role in disease pathogenesis. These studies emphasize the importance of region-, cell-type-, and time-dependent analyses to fully dissect the natural history of disease and the consequences of disease on normal lung development.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-726) contains supplementary material, which is available to authorized users.
PMCID: PMC4247008  PMID: 25204199
Scnn1b-Tg mice; Pulmonary macrophage activation; Inflammation; Mucus clearance defect; Gene expression profiling; Lung development; Airway surface liquid dehydration
17.  Adverse childhood experiences and associations with health-harming behaviours in young adults: surveys in eight eastern European countries 
To evaluate the association between adverse childhood experiences – e.g. abuse, neglect, domestic violence and parental separation, substance use, mental illness or incarceration – and the health of young adults in eight eastern European countries.
Between 2010 and 2013, adverse childhood experience surveys were undertaken in Albania, Latvia, Lithuania, Montenegro, Romania, the Russian Federation, The former Yugoslav Republic of Macedonia and Turkey. There were 10 696 respondents – 59.7% female – aged 18–25 years. Multivariate modelling was used to investigate the relationships between adverse childhood experiences and health-harming behaviours in early adulthood including substance use, physical inactivity and attempted suicide.
Over half of the respondents reported at least one adverse childhood experience. Having one adverse childhood experience increased the probability of having other adverse childhood experiences. The number of adverse childhood experiences was positively correlated with subsequent reports of health-harming behaviours. Compared with those who reported no adverse experiences, respondents who reported at least four adverse childhood experiences were at significantly increased risk of many health-harming behaviours, with odds ratios varying from 1.68 (95% confidence interval, CI: 1.32–2.15) – for physical inactivity – to 48.53 (95% CI: 31.98–76.65) – for attempted suicide. Modelling indicated that prevention of adverse childhood experiences would substantially reduce the occurrence of many health-harming behaviours within the study population.
Our results indicate that individuals who do not develop health-harming behaviours are more likely to have experienced safe, nurturing childhoods. Evidence-based programmes to improve parenting and support child development need large-scale deployment in eastern European.
PMCID: PMC4208567  PMID: 25378755
18.  UV Light Inactivation of Human and Plant Pathogens in Unfiltered Surface Irrigation Water 
Fruit and vegetable growers continually battle plant diseases and food safety concerns. Surface water is commonly used in the production of fruits and vegetables and can harbor both human- and plant-pathogenic microorganisms that can contaminate crops when used for irrigation or other agricultural purposes. Treatment methods for surface water are currently limited, and there is a need for suitable treatment options. A liquid-processing unit that uses UV light for the decontamination of turbid juices was analyzed for its efficacy in the treatment of surface waters contaminated with bacterial or oomycete pathogens, i.e., Escherichia coli, Salmonella enterica, Listeria monocytogenes, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, and Phytophthora capsici. Five-strain cocktails of each pathogen, containing approximately 108 or 109 CFU/liter for bacteria or 104 or 105 zoospores/liter for Ph. capsici, were inoculated into aliquots of two turbid surface water irrigation sources and processed with the UV unit. Pathogens were enumerated before and after treatment. In general, as the turbidity of the water source increased, the effectiveness of the UV treatment decreased, but in all cases, 99.9% or higher inactivation was achieved. Log reductions ranged from 10.0 to 6.1 and from 5.0 to 4.2 for bacterial pathogens and Ph. capsici, respectively.
PMCID: PMC3911201  PMID: 24242253
19.  Isolation and Characterization of the Herpes Simplex Virus 1 Terminase Complex 
Journal of Virology  2014;88(1):225-236.
During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.
PMCID: PMC3911699  PMID: 24155374
20.  Relationship between obesity and the risk of clinically significant depression: Mendelian randomisation study 
Obesity has been shown to be associated with depression and it has been suggested that higher body mass index (BMI) increases the risk of depression and other common mental disorders. However, the causal relationship remains unclear and Mendelian randomisation, a form of instrumental variable analysis, has recently been employed to attempt to resolve this issue.
To investigate whether higher BMI increases the risk of major depression.
Two instrumental variable analyses were conducted to test the causal relationship between obesity and major depression in RADIANT, a large case-control study of major depression. We used a single nucleotide polymorphism (SNP) in FTO and a genetic risk score (GRS) based on 32 SNPs with well-established associations with BMI.
Linear regression analysis, as expected, showed that individuals carrying more risk alleles of FTO or having higher score of GRS had a higher BMI. Probit regression suggested that higher BMI is associated with increased risk of major depression. However, our two instrumental variable analyses did not support a causal relationship between higher BMI and major depression (FTO genotype: coefficient –0.03, 95% CI –0.18 to 0.13, P = 0.73; GRS: coefficient –0.02, 95% CI –0.11 to 0.07, P = 0.62).
Our instrumental variable analyses did not support a causal relationship between higher BMI and major depression. The positive associations of higher BMI with major depression in probit regression analyses might be explained by reverse causality and/or residual confounding.
PMCID: PMC4076654  PMID: 24809401
21.  Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks 
As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys→Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.
PMCID: PMC3651811  PMID: 23483515
Antibody characterization; Protein structure; Fast photochemical oxidation of proteins (FPOP); Top-down; Ion mobility; Antibody mutants; IgG2
22.  Replication of bipolar disorder susceptibility alleles and identification of 2 novel genome-wide significant associations in a new bipolar disorder case-control sample 
Molecular psychiatry  2012;18(12):1302-1307.
We have conducted a genotyping study using a custom Illumina Infinium HD genotyping array, the ImmunoChip, in a new UK sample of 1,218 bipolar disorder cases and 2,913 controls that have not been used in any studies previously reported independently or in meta-analyses. The ImmunoChip was designed prior to the publication of the Psychiatric GWAS Consortium Bipolar Disorder Working Group (PGC-BD) meta-analysis data. As such 3106 SNPs with a P value less than 1×10−3 from the bipolar disorder meta-analysis by Ferreira et al., 2008 were genotyped. We report support for two of the three most strongly associated chromosomal regions in the Ferreira study, CACNA1C (rs1006737, p=4.09×10−4) and 15q14 (rs2172835, p=0.043) but not ANK3 (rs10994336, p=0.912). We have combined our ImmunoChip data (569 quasi-independent SNPs from the 3016 SNPs genotyped) with the recently published PGC-BD meta-analysis data, using either the PGC-BD combined discovery and replication data where available or just the discovery data where the SNP was not typed in a replication sample in PGC-BD. Our data provide support for two regions, at ODZ4 and CACNA1C, with prior evidence for genome-wide significant association in PGC-BD meta-analysis. In addition, the combined analysis shows two novel genome-wide significant associations. First, rs7296288 (P = 8.97 × 10−9, OR = 0.9), an intergenic polymorphism on chromosome 12 located between RHEBL1 and DHH. Secondly, rs3818253 (P = 3.88 × 10−8, OR = 1.16), an intronic SNP on chromosome 20q11.2 in the gene TRPC4AP which lies in a high linkage disequilibrium region along with the genes GSS and MYH7B.
PMCID: PMC3971368  PMID: 23070075
Bipolar disorder; genome-wide significant association; ImmunoChip; PGC-BD; rs7296288; rs3818253
23.  Strong genetic evidence for a selective influence of GABAA receptors on a component of the bipolar disorder phenotype 
Molecular psychiatry  2008;15(2):146-153.
Despite compelling evidence for a major genetic contribution to risk of bipolar mood disorder, conclusive evidence implicating specific genes or pathophysiological systems has proved elusive. In part this is likely to be related to the unknown validity of current phenotype definitions and consequent aetiological heterogeneity of samples. In the recent Wellcome Trust Case Control Consortium (WTCCC) genome-wide association analysis of bipolar disorder (1868 cases, 2938 controls) one of the most strongly associated polymorphisms lay within the gene encoding the GABAA receptor β1 subunit, GABRB1. Aiming to increase biological homogeneity, we sought the diagnostic subset that showed the strongest signal at this polymorphism and used this to test for independent evidence of association with other members of the GABAA receptor gene family. The index signal was significantly enriched in the 279 cases meeting Research Diagnostic Criteria for schizoaffective disorder, bipolar type (p=3.8×10−6). Independently, these cases showed strong evidence that variation in GABAA receptor genes influences risk for this phenotype (independent system-wide p=6.6×10−5) with association signals also at GABRA4, GABRB3, GABRA5 and GABRR1. Our findings have the potential to inform understanding of presentation, pathogenesis and nosology of bipolar disorders. Our method of phenotype refinement may be useful in studies of other complex psychiatric and non-psychiatric disorders.
PMCID: PMC3967096  PMID: 19078961
24.  Genome Wide Association Analysis of Copy Number Variation in Recurrent Depressive Disorder 
Molecular psychiatry  2011;18(2):183-189.
Large, rare copy number variants (CNV) have been implicated in a variety of psychiatric disorders, but the role of CNVs in recurrent depression is unclear. We performed a genome-wide analysis of large, rare CNVs in 3,106 cases of recurrent depression, 459 controls screened for lifetime-absence of psychiatric disorder and 5,619 unscreened controls from phase 2 of the Wellcome Trust Case Control Consortium (WTCCC2). We compared the frequency of cases with CNVs against the frequency observed in each control group, analysing CNVs over the whole genome, genic, intergenic, intronic and exonic regions. We found that deletion CNVs were associated with recurrent depression while duplications were not. The effect was significant when comparing cases to WTCCC2 controls (p=7.7×10−6, OR =1.25 (95% CI 1.13 - 1.37)) and to screened controls (p=5.6×10−4, OR=1.52 (95% CI 1.20 - 1.93). Further analysis showed that CNVs deleting protein coding regions were largely responsible for the association. Within an analysis of regions previously implicated in schizophrenia, we found an overall enrichment of CNVs in our cases when compared to screened controls (p=0.019). We observe an ordered increase of samples with deletion CNVs, with the lowest proportion seen in screened controls, the next highest in unscreened controls and the highest in cases. This may suggest that the absence of deletion CNVs, especially in genes, is associated with resilience to recurrent depression.
PMCID: PMC3939438  PMID: 22042228

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