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1.  Multimeric Assembly of Host-Pathogen Adhesion Complexes Involved in Apicomplexan Invasion 
PLoS Pathogens  2014;10(6):e1004120.
PMCID: PMC4055764  PMID: 24945143
3.  Genetic Variants in FBN-1 and Risk for Thoracic Aortic Aneurysm and Dissection 
PLoS ONE  2014;9(4):e91437.
A recent genome wide association study (GWAS) by LeMaire et al. found that two single nucleotide polymorphisms (SNPs), rs2118181 and rs10519177 in the FBN-1 gene (encoding Fibrillin-1), were associated with thoracic aortic dissection (TAD), non-dissecting thoracic aortic aneurysm (TAA), and thoracic aortic aneurysm or dissection (TAAD); the largest effect was observed for the association of rs2118181 with TAD. We investigated whether rs2118181 and rs10519177 were associated with TAD, TAA, and TAAD in the Yale study.
The genotypes of rs2118181 and rs10519177 were determined for participants in the Yale study: 637 TAAD cases (140 TAD, 497 TAA) and 275 controls from the United States, Hungary, and Greece. The association of the genotypes with TAD, TAA and TAAD were assessed using logistic regression models adjusted for sex, age, study center and hypertension.
Results and Conclusions
In the Yale study, rs2118181 was associated with TAD: compared with non-carriers, carriers of the risk allele had an unadjusted odds ratio for TAD of 1.80 (95% CI 1.15–2.80) and they had odds ratio for TAD of 1.87 (95% CI 1.09–3.20) after adjusting for sex, age, study center and hypertension. We did not find significant differences in aortic size, a potential confounder for TAD, between rs2118181 risk variant carriers and non-carriers: mean aortic size was 5.56 (95% CI: 5.37–5.73) for risk variant carriers (CC+CT) and was 5.48 (95% CI: 5.36–5.61) for noncarriers (TT) (p = 0.56). rs2118181 was not associated with TAA or TAAD. rs10519177 was not associated with TAD, TAA, or TAAD in the Yale study. Thus, the Yale study provided further support for the association of the FBN-1 rs2118181SNP with TAD.
PMCID: PMC3990573  PMID: 24743685
4.  The Cellular Basis for Biocide-Induced Fluorescein Hyperfluorescence in Mammalian Cell Culture 
PLoS ONE  2014;9(1):e84427.
Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting ‘staining’. Although localized intensely stained regions of the cornea frequently occur after exposure to ‘adverse’ clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells.
We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining ‘hyperfluorescent’ cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4°C.
We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli.
PMCID: PMC3904830  PMID: 24489650
5.  Structural and Functional Basis for Inhibition of Erythrocyte Invasion by Antibodies that Target Plasmodium falciparum EBA-175 
PLoS Pathogens  2013;9(5):e1003390.
Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.
Author Summary
Malaria is a devastating parasitic disease that kills one million people annually. The parasites invade and multiply within red blood cells, leading to the clinical symptoms of malaria. Therefore, preventing red blood cell, entry through vaccines is an attractive approach to controlling the disease. Although widespread efforts to develop a vaccine by identifying and combining critical parasite blood-stage proteins are underway, a protective vaccine for malaria has proved challenging. This is in part because, while parasite proteins have the ability to elicit antibodies that prevent red blood cell invasion, these antibodies are a small proportion compared to the total collection of ineffective antibodies produced. We show an antibody that prevents red blood cell invasion targets regions of the critical parasite protein PfEBA-175 required for red blood cell engagement. We also show that an antibody that does not prevent red blood cell invasion recognizes a region far removed from important functional segments of PfEBA-175. Our work demonstrates that identifying the regions targeted by antibodies, and the mechanisms by which antibodies that prevent invasion function, should drive future vaccine development and studies measuring the effectiveness of current vaccine combinations.
PMCID: PMC3662668  PMID: 23717209
6.  Treatment outcome in adults with chronic fatigue syndrome: a prospective study in England based on the CFS/ME National Outcomes Database 
Background: Chronic fatigue syndrome (CFS) is relatively common and disabling. Over 8000 patients attend adult services each year, yet little is known about the outcome of patients attending NHS services.
Aim: Investigate the outcome of patients with CFS and what factors predict outcome.
Design: Longitudinal patient cohort.
Methods: We used data from six CFS/ME (myalgic encephalomyelitis) specialist services to measure changes in fatigue (Chalder Fatigue Scale), physical function (SF-36), anxiety and depression (Hospital Anxiety and Depression Scale) and pain (visual analogue pain rating scale) between clinical assessment and 8–20 months of follow-up. We used multivariable linear regression to investigate baseline factors associated with outcomes at follow-up.
Results: Baseline data obtained at clinical assessment were available for 1643 patients, of whom 834 (51%) had complete follow-up data. There were improvements in fatigue [mean difference from assessment to outcome: −6.8; 95% confidence interval (CI) −7.4 to −6.2; P < 0.001]; physical function (4.4; 95% CI 3.0–5.8; P < 0.001), anxiety (−0.6; 95% CI −0.9 to −0.3; P < 0.001), depression (−1.6; 95% CI −1.9 to −1.4; P < 0.001) and pain (−5.3; 95% CI −7.0 to −3.6; P < 0.001). Worse fatigue, physical function and pain at clinical assessment predicted a worse outcome for fatigue at follow-up. Older age, increased pain and physical function at assessment were associated with poorer physical function at follow-up.
Conclusions: Patients who attend NHS specialist CFS/ME services can expect similar improvements in fatigue, anxiety and depression to participants receiving cognitive behavioural therapy and graded exercise therapy in a recent trial, but are likely to experience less improvement in physical function. Outcomes were predicted by fatigue, disability and pain at assessment.
PMCID: PMC3665909  PMID: 23538643
7.  Genome Annotation of Five Mycoplasma canis Strains 
Journal of Bacteriology  2012;194(15):4138-4139.
To understand its potential to cause invasive disease, the genome of Mycoplasma canis strain PG14T from a dog's throat was compared to those of isolates from the genital tract or brain of dogs. The average nucleotide identity between strain pairs is 98%, and their genome annotations are similar.
PMCID: PMC3416566  PMID: 22815452
8.  AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway 
Molecular Biology of the Cell  2012;23(18):3612-3623.
A GPCR ubiquitin-independent MVB/lysosomal sorting pathway is regulated by the adaptor protein complex-3 (AP-3) and ALIX, a noncanonical ESCRT component. AP-3 binds to a PAR1 C-tail–localized, tyrosine-based motif and mediates PAR1 lysosomal degradation. AP-3 also facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
PMCID: PMC3442409  PMID: 22833563
9.  Life expectancy of HIV-1-positive individuals approaches normal conditional on response to antiretroviral therapy: UK Collaborative HIV Cohort Study 
Life expectancies (LEs) of patients in UK Collaborative HIV Cohort (UK CHIC) stratified by CD4 count at start of antiretroviral therapy (ART) have been estimated [1] but not gains in years of life in response to ART. We estimated LE associated with attained CD4 count and viral suppression at different durations of ART. Patients in UK CHIC aged > 20 years who started ART in 2000 to 2008 (excluding person who injects drugs) were followed to end of 2010. All-cause mortality was ascertained from clinic notes and by linkage to national records. We used the nearest CD4 count before ART and the last in each of years 1 to 5 of ART and determined whether patients were virally suppressed (HIV-1 RNA < 400 copies/mL) in the past year for those remaining under follow-up. Poisson models were used to estimate mortality rates by sex, age, latest CD4 count (<200, 200 to 349,≥350) and viral suppression for each duration of ART. Abridged life tables were constructed from age-specific mortality rates to estimate LE for ages 20 to 85 years. Results are presented as the average number of years that will be lived after exact age 35 years. A total of 17,021 patients started ART from 2000 to 2008 of whom 708 (4.2%) died; 3956 (23%) were lost to study follow-up. There was no difference in mortality between those with attained CD4 350 to 499 and ≥ 500. On starting ART, male LE at exact age 35 was 36, 44 and 42 (female LE 38, 46 and 44) years for attained CD4 < 200, 200 to 349,≥350, respectively; after 5 years on ART, it was 22, 42 and 46 (female LE 27, 46 and 51) years, respectively. Only 17% of patients had CD4 ≥ 350 at ART start, compared with 78% of patients on ART for > 5 years. The difference in LE between suppressed versus unsuppressed patients was around 11 years. The figure shows that both CD4 count and viral suppression contribute to changes in LE. Male patients that increased their CD4 in the 1st year of ART from < 200 to 200–349 or ≥ 350 gained 6 and 11 years of LE to 42 and 48 years, respectively, with similar rises for women. Overall, LE was 4 years greater for those on ART for > 5 years compared with those starting ART. Individuals that attain viral suppression and a CD4 count > 350 within 1 year of ART start have a normal LE with 35-year olds estimated to live to over 80 years on average. LE in patients with CD4 count < 200 beyond 5 years on ART drops by 15 years. Estimated LE may be biased by under-ascertainment of deaths, missing CD4 measurements and extrapolation beyond available data.
PMCID: PMC3512455
10.  ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting 
The Journal of Cell Biology  2012;197(3):407-419.
A novel MVB/lysosomal sorting pathway for signaling receptors bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be broadly applicable to GPCRs containing YPXnL motifs.
The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.
PMCID: PMC3341166  PMID: 22547407
11.  Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer 
British Journal of Cancer  2011;104(11):1755-1761.
The activity of the protein kinase Akt is frequently dysregulated in cancer and is an important factor in the growth and survival of tumour cells. Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif. Phosphorylation of Ser473 has been extensively studied in tumour samples as a correlate for Akt activity, yet the phosphorylation of Thr308 or of downstream Akt substrates is rarely assessed.
The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates – PRAS40, TSC2 and TBC1D4 – in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).
Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.
The phosphorylation of Thr308 is a more reliable biomarker for the protein kinase activity of Akt in tumour samples than Ser473. Any evaluation of the link between Akt phosphorylation or activity in tumour samples and the prediction or prognosis of disease should, therefore, focus on measuring the phosphorylation of Akt on Thr308 and/or at least one downstream Akt substrate, rather than Akt Ser473 phosphorylation alone.
PMCID: PMC3111153  PMID: 21505451
protein kinase B; Akt; biomarker; tumour; phosphorylation
13.  Polymorphism in the Apolipoprotein(a) Gene, Plasma Lipoprotein(a), Cardiovascular Disease, and Low-dose Aspirin Therapy 
Atherosclerosis  2008;203(2):371-376.
A minor allele variant (rs3798220) of apolipoprotein(a) has been reported to be associated with elevated plasma lipoprotein(a) [Lp(a)] and increased cardiovascular risk. We investigated whether this allele was associated with elevated Lp(a) and cardiovascular risk in Women's Health Study, a randomized trial of low-dose aspirin, and whether aspirin reduced cardiovascular risk in minor allele carriers.
Methods and Results
Genotypes of rs3798220 were determined for 25,131 initially healthy Caucasian participants. Median Lp(a) levels at baseline were 10.0, 79.5, and 153.9 mg/dL for major allele homozygotes, minor allele heterozygotes, and minor allele homozygotes, respectively (P<0.0001). During the 9.9 years of follow-up, minor allele carriers (3.7%) in the placebo group had two-fold higher risk of major cardiovascular events than non-carriers (age-adjusted hazard ratio (HR) = 2.21, 95% CI 1.39−3.52). Among carriers, risk was reduced more than two-fold by aspirin: for aspirin compared with placebo the age-adjusted HR was 0.44 (95% CI: 0.20−0.94); risk was not significantly reduced among non-carriers (age-adjusted HR=0.91, 95% CI: 0.77−1.08). This interaction between carrier status and aspirin allocation was significant (P=0.048).
In the Women's Health Study, carriers of an apolipoprotein(a) variant had elevated Lp(a), doubled cardiovascular risk, and appeared to benefit more from aspirin than non-carriers.
PMCID: PMC2678922  PMID: 18775538
cardiovascular disease; aspirin; lipoproteins; genetics; Lp(a)
14.  Primary prevention of cardiovascular disease: a web‐based risk score for seven British black and minority ethnic groups 
Heart  2006;92(11):1595-1602.
To recalibrate an existing Framingham risk score to produce a web‐based tool for estimating the 10‐year risk of coronary heart disease (CHD) and cardiovascular disease (CVD) in seven British black and minority ethnic groups.
Risk prediction models were recalibrated against survey data on ethnic group risk factors and disease prevalence compared with the general population. Ethnic‐ and sex‐specific 10‐year risks of CHD and CVD, at the means of the risk factors for each ethnic group, were calculated from the product of the incidence rate in the general population and the prevalence ratios for each ethnic group.
Two community‐based surveys.
3778 men and 4544 women, aged 35–54, from the Health Surveys for England 1998 and 1999 and the Wandsworth Heart and Stroke Study.
Main outcome measures
10‐year risk of CHD and CVD.
10‐year risk of CHD and CVD for non‐smoking people aged 50 years with a systolic blood pressure of 130 mm Hg and a total cholesterol to high density lipoprotein cholesterol ratio of 4.2 was highest in men for those of Pakistani and Bangladeshi origin (CVD risk 12.6% and 12.8%, respectively). CHD risk in men with the same risk factor values was lowest in Caribbeans (2.8%) and CVD risk was lowest in Chinese (5.4%). Women of Pakistani origin were at highest risk and Chinese women at lowest risk for both outcomes with CVD risks of 6.6% and 1.2%, respectively. A web‐based risk calculator (ETHRISK) allows 10‐year risks to be estimated in routine primary care settings for relevant risk factor and ethnic group combinations.
In the absence of cohort studies in the UK that include significant numbers of black and minority ethnic groups, this risk score provides a pragmatic solution to including people from diverse ethnic backgrounds in the primary prevention of CVD.
PMCID: PMC1861244  PMID: 16762981
15.  Cardiovascular disease risk assessment in older women: can we improve on Framingham? British Women's Heart and Health prospective cohort study 
Heart  2006;92(10):1396-1401.
To develop a cardiovascular risk assessment tool that is feasible and easy to use in primary care (general practice (GP) model).
Prospective cohort study.
23 towns in the United Kingdom.
3582 women aged 60 to 79 years who were free of coronary heart disease (CHD) at entry into the British Women's Heart and Health Study.
Main outcome measures
Predictive performance of a GP model compared with the standard Framingham model for both CHD and cardiovascular disease (CVD).
The Framingham tool predicted CHD events over 5 years accurately (predicted 5.7%, observed 5.5%) but overpredicted CVD events (predicted 10.5%, observed 6.8%). In higher‐risk groups, Framingham overpredicted both CHD and CVD events and was poorly calibrated for this cohort. Including C‐reactive protein and fibrinogen with standard Framingham risk factors did not improve discrimination of the model. The GP model, which used age, systolic blood pressure, smoking habit and self‐rated health (all of which can be easily obtained in one surgery visit) performed as well as the Framingham risk tool: area under the receiver operating curve discrimination statistic was 0.66 (95% confidence interval (CI) 0.62 to 0.70) for CHD and 0.67 (95% CI 0.64 to 0.71) for CVD compared with 0.65 (95% CI 0.61 to 0.68) and 0.66 (95% CI 0.62 to 0.69) for the corresponding Framingham models.
An alternative risk assessment based on only a simple routine examination and a small number of pertinent questions may be more useful in the primary care setting. This model appears to perform well but needs to be tested in different populations.
PMCID: PMC1861043  PMID: 16547204
17.  Modulation of Lymphocyte Proliferation by Antioxidants in Chronic Beryllium Disease 
Rationale: Occupational exposure to beryllium (Be) can result in chronic granulomatous inflammation characterized by the presence of Be-specific CD4+ T cells. Studies show that oxidative stress plays a role in the pathogenesis of chronic inflammatory disorders.
Objectives: We hypothesized that Be-induced oxidative stress modulates the proliferation of Be-specific CD4+ T cells.
Methods: Thirty-three subjects with chronic beryllium disease (CBD), 15 subjects with beryllium sensitization, and 28 healthy normal control subjects were consecutively enrolled from the Occupational and Environmental Health Clinic of the National Jewish Medical and Research Center.
Measurements and Main Results: All studies were performed with Ficoll-Hypaque–isolated peripheral blood mononuclear cells from subsets of the study subjects. Decreased intracellular levels of the thiol antioxidants, glutathione and cysteine, were observed in peripheral blood mononuclear cells from subjects with beryllium sensitization and CBD, as compared with healthy control subjects. Beryllium stimulation decreased intracellular thiol antioxidants by more than 40%, accompanied by increased reactive oxygen species levels and the proliferation of Be-specific blood CD4+ T cells from subjects with CBD. Be-induced T-cell proliferation was inhibited by treatment with the thiol antioxidant N-acetylcysteine or the catalytic antioxidant manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP). MnTBAP treatment also inhibited T-cell proliferation in response to the unrelated, MHC class II–restricted antigen tetanus toxoid. Treatment of CBD blood lymphocytes, but not antigen-presenting cells, with MnTBAP decreased Be-induced T-cell proliferation by more than 40%.
Conclusions: Beryllium can mediate a thiol imbalance leading to oxidative stress that may modulate the proliferation and clonal expansion of Be-specific blood CD4+ T cells. These data suggest that Be-induced oxidative stress plays a role in the pathogenesis of granulomatous inflammation in CBD.
PMCID: PMC2361422  PMID: 18218990
T cells; reactive oxygen species; glutathione; N-acetylcysteine; oxidative stress
18.  Single nucleotide polymorphisms associated with coronary heart disease predict incident ischemic stroke in the Atherosclerosis Risk in Communities (ARIC) study 
Ischemic stroke and coronary heart disease (CHD) may share genetic factors contributing to a common etiology. This study investigates whether 51 single nucleotide polymorphisms (SNPs) associated with CHD in multiple antecedent studies are associated with incident ischemic stroke in the Atherosclerosis Risk in Communities (ARIC) study. From the multi-ethnic ARIC cohort of 14,215 individuals, 495 validated ischemic strokes were identified. Cox proportional hazards models, adjusted for age and gender, identified three SNPs in Whites and two SNPs in Blacks associated with incident stroke (p≤0.05). The rs11628722 polymorphism in SERPINA9 was associated with incident stroke in Whites and Blacks, even after taking into account traditional risk factors. The idea that ischemic stroke and CHD may share some common genetic factors, such as variation in SERPINA9, should be investigated in other studies.
PMCID: PMC2662496  PMID: 18799872
genetics; stroke; ischemic; myocardial infarction; polymorphism
19.  Association of Gene Variants with Incident Myocardial Infarction in the Cardiovascular Health Study 
We asked if single nucleotide polymorphisms (SNPs) that had been nominally associated with cardiovascular disease in antecedent studies were also associated with cardiovascular disease in a population–based prospective study of 4,522 individuals aged 65 or older.
Based on antecedent studies, we prespecified a risk allele and an inheritance model for each of 74 SNPs. We then tested the association of these SNPs with myocardial infarction (MI) in the Cardiovascular Health Study (CHS).
The prespecified risk alleles of 8 SNPs were nominally associated (1-sided P<0.05) with increased risk of MI in white CHS participants. The false discovery rate for these 8 was 0.43, suggesting that about 4 of these 8 are likely to be true positives. The 4 of these 8 SNPs that had the strongest evidence for association with cardiovascular disease prior to testing in CHS (association in 3 antecedent studies) were in KIF6 (CHS HR=1.29; 90%CI 1.1–1.52), VAMP8 (HR=1.2; 90%CI 1.02–1.41), TAS2R50 (HR=1.13; 90%CI 1–1.27), and LPA (HR=1.62; 90%CI 1.09–2.42).
Although most of the SNPs investigated were not associated with MI in CHS, evidence from this investigation combined with previous studies suggests that 4 of these SNPs are likely associated with MI.
PMCID: PMC2636623  PMID: 17975119
coronary disease; myocardial infarction; genetics; polymorphisms
20.  Early onset neonatal meningitis in Australia and New Zealand, 1992–2002 
Objectives: To study the epidemiology of early onset neonatal bacterial meningitis (EONBM) in Australasia.
Design: Prospective surveillance study, 1992–2002, in 20 neonatal units in Australia and New Zealand. EONBM was defined as meningitis occurring within 48 hours of delivery.
Results: There were 852 babies with early onset sepsis, of whom 78 (9.2%) had EONBM. The incidence of early onset group B streptococcal meningitis fell significantly from a peak of 0.24/1000 live births in 1993 to 0.03/1000 in 2002 (p trend = 0.002). There was no significant change over time in the incidence of Escherichia coli meningitis. The rate of EONBM in very low birthweight babies was 1.09/1000 compared with the rate in all infants of 0.11/1000. The overall rate of EONBM was 0.41/1000 in 1992 and 0.06 in 2001, but this trend was not significant (p trend = 0.07). Case-fatality rates for EONBM did not change significantly with time. Birth weight <1500 g (odds ratio (OR) 7.2 (95% confidence interval (CI) 4.8 to 10.9)) and Gram negative bacillary meningitis (OR 3.3 (95% CI 2.2 to 4.9)) were significant risk factors for mortality. Sixty two percent of the 129 babies who died from early onset sepsis or suspected sepsis did not have a lumbar puncture performed.
Conclusion: The incidence of early onset group B streptococcal meningitis has fallen, probably because of maternal intrapartum antibiotic prophylaxis, without a corresponding change in E coli meningitis. Gram negative bacillary meningitis still carries a worse prognosis than meningitis with a Gram positive organism.
PMCID: PMC1721922  PMID: 15878934
21.  Mitigation of indirect environmental effects of GM crops 
Currently, the UK has no procedure for the approval of novel agricultural practices that is based on environmental risk management principles. Here, we make a first application of the ‘bow-tie’ risk management approach in agriculture, for assessment of land use changes, in a case study of the introduction of genetically modified herbicide tolerant (GMHT) sugar beet. There are agronomic and economic benefits, but indirect environmental harm from increased weed control is a hazard. The Farm Scale Evaluation (FSE) trials demonstrated reduced broad-leaved weed biomass and seed production at the field scale. The simplest mitigation measure is to leave a proportion of rows unsprayed in each GMHT crop field. Our calculations, based on FSE data, show that a maximum of 2% of field area left unsprayed is required to mitigate weed seed production and 4% to mitigate weed biomass production. Tilled margin effects could simply be mitigated by increasing the margin width from 0.5 to 1.5 m. Such changes are cheap and simple to implement in farming practices. This case study demonstrates the usefulness of the bow-tie risk management approach and the transparency with which hazards can be addressed. If adopted generally, it would help to enable agriculture to adopt new practices with due environmental precaution.
PMCID: PMC2176167  PMID: 17439853
genetically modified crops; environmental risk assessment; sugar beet; mitigation; biodiversity
22.  Beryllium-Induced TNF-α Production Is Transcription-Dependent in Chronic Beryllium Disease 
Beryllium (Be)-antigen presentation to Be-specific CD4+ T cells from the lungs of patients with chronic beryllium disease (CBD) results in T cell proliferation and TNF-α secretion. We tested the hypothesis that Be-induced, CBD bronchoalveolar lavage (BAL) T cell, transcription-dependent, TNF-α secretion was accompanied by specific transcription factor upregulation. After 6 h of Be stimulation, CBD BAL cells produced a median of 883 pg/ml TNF-α (range, 608–1,275 pg/ml) versus 198 pg/ml (range, 116–245 pg/ml) by unstimulated cells. After 12 h CBD BAL cells produced a median of 2,963 pg/ml (range, 99–9,424 pg/ml) TNF-α versus 55 pg/ml (range, 0–454) by unstimulated cells. Using real-time RT-PCR, Be-stimulated TNF-α production at 6 h was preceded by a 5-fold increase in TNF-α pre-mRNA copy number:β-actin copy number (Be median ratio 0.21; unstimulated median ratio 0.04). The median ratio of mature TNF-α mRNA:β-actin mRNA was upregulated 1.4-fold (Be median ratio 0.17; unstimulated median ratio 0.12). Be exposure in the presence of the transcription inhibitor pentoxifylline (PTX) decreased CBD BAL cell TNF-α pre-mRNA levels > 60%, whereas treatment with the mRNA splicing inhibitor 2-aminopurine (2AP) decreased levels 40% relative to Be exposure alone. PTX treatment decreased mature TNF-α mRNA levels 50% while 2AP decreased levels > 80%, relative to Be exposure alone. Beryllium exposure specifically upregulated transcription factors AP-1 and NF-κB. The data suggest that Be exposure induces transcription-dependent TNF-α production, potentially due to upregulation of specific transcription factors.
PMCID: PMC2176111  PMID: 16980557
granuloma; T lymphocytes; cytokines; gene regulation; lung
23.  Effects of genetically modified herbicide-tolerant cropping systems on weed seedbanks in two years of following crops 
Biology Letters  2005;2(1):140-143.
The Farm Scale Evaluations (FSEs) showed that genetically modified herbicide-tolerant (GMHT) cropping systems could influence farmland biodiversity because of their effects on weed biomass and seed production. Recently published results for winter oilseed rape showed that a switch to GMHT crops significantly affected weed seedbanks for at least 2 years after the crops were sown, potentially causing longer-term effects on other taxa. Here, we seek evidence for similar medium-term effects on weed seedbanks following spring-sown GMHT crops, using newly available data from the FSEs.
Weed seedbanks following GMHT maize were significantly higher than following conventional varieties for both the first and second years, while by contrast, seedbanks following GMHT spring oilseed rape were significantly lower over this period. Seedbanks following GMHT beet were smaller than following conventional crops in the first year after the crops had been sown, but this difference was much reduced by the second year for reasons that are not clear. These new data provide important empirical evidence for longer-term effects of GMHT cropping on farmland biodiversity.
PMCID: PMC1617187  PMID: 17148348
Farm Scale Evaluations; arable weeds; farmland biodiversity
24.  Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization†  
Molecular and Cellular Biology  2006;26(8):3231-3242.
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for the coagulant protease thrombin, is irreversibly activated by proteolysis. Unactivated PAR1 cycles constitutively between the plasma membrane and intracellular stores, thereby providing a protected receptor pool that replenishes the cell surface after thrombin exposure and leads to rapid resensitization to thrombin signaling independent of de novo receptor synthesis. Here, we show that AP2, a clathrin adaptor, binds directly to a tyrosine-based motif in the cytoplasmic tail of PAR1 and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PAR1 tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PAR1 constitutive internalization, loss of intracellular uncleaved PAR1, and failure of endothelial cells and other cell types to regain thrombin responsiveness. Our findings establish a novel role for AP2 in direct regulation of PAR1 trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.
PMCID: PMC1446942  PMID: 16581796
25.  On the rationale and interpretation of the Farm Scale Evaluations of genetically modified herbicide-tolerant crops. 
Farmland biodiversity and food webs were compared in conventional and genetically modified herbicide-tolerant (GMHT) crops of beet (Beta vulgaris L.), maize (Zea mays L.) and both spring and winter oilseed rape (Brassica napus L.). GMHT and conventional varieties were sown in a split-field experimental design, at 60-70 sites for each crop, spread over three starting years beginning in 2000. This paper provides a background to the study and the rationale for its design and interpretation. It shows how data on environment, field management and the biota are used to assess the current state of the ecosystem, to define the typical arable field and to devise criteria for selecting, sampling and auditing experimental sites in the Farm Scale Evaluations. The main functional and taxonomic groups in the habitat are ranked according to their likely sensitivity to GMHT cropping, and the most responsive target organisms are defined. The value of the seedbank as a baseline and as an indicator of historical trends is proposed. Evidence from experiments during the twentieth century is analysed to show that large changes in field management have affected sensitive groups in the biota by ca. 50% during a year or short run of years--a figure against which to assess any positive or negative effects of GMHT cropping. The analysis leads to a summary of factors that were, and were not, examined in the first 3 years of the study and points to where modelling can be used to extrapolate the effects to the landscape and the agricultural region.
PMCID: PMC1693276  PMID: 14561314

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