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2.  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.
doi:10.1371/journal.pone.0084427
PMCID: PMC3904830  PMID: 24489650
3.  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.
doi:10.1371/journal.ppat.1003390
PMCID: PMC3662668  PMID: 23717209
4.  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.
doi:10.1093/qjmed/hct061
PMCID: PMC3665909  PMID: 23538643
5.  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.
doi:10.1128/JB.00664-12
PMCID: PMC3416566  PMID: 22815452
6.  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.
doi:10.1091/mbc.E12-03-0251
PMCID: PMC3442409  PMID: 22833563
7.  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.
doi:10.7448/IAS.15.6.18078
PMCID: PMC3512455
8.  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.
doi:10.1083/jcb.201110031
PMCID: PMC3341166  PMID: 22547407
9.  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.
Background:
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.
Methods:
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).
Results:
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.
Conclusion:
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.
doi:10.1038/bjc.2011.132
PMCID: PMC3111153  PMID: 21505451
protein kinase B; Akt; biomarker; tumour; phosphorylation
10.  Genome Sequences of Mycoplasma alligatoris A21JP2T and Mycoplasma crocodyli MP145T▿ 
Journal of Bacteriology  2011;193(11):2892-2893.
Mycoplasma alligatoris and Mycoplasma crocodyli are closely related siblings, one being highly virulent and the other relatively attenuated. We compared their genomes to better understand the mechanisms and origins of M. alligatoris' remarkable virulence amid a clade of harmless or much less virulent species. Although its chromosome was refractory to closure, M. alligatoris differed most notably by its complement of sialidases and other genes of the N-acetylneuraminate scavenging and catabolism pathway.
doi:10.1128/JB.00309-11
PMCID: PMC3133112  PMID: 21460083
11.  Sulfasalazine and Mesalamine Modulate Beryllium-Specific Lymphocyte Proliferation and Inflammatory Cytokine Production 
Occupational exposure to beryllium (Be) results in Be sensitization (BeS) that can progress to pulmonary granulomatous inflammation associated with chronic Be disease (CBD). Be-specific lymphocytes are present in the blood of patients with BeS and in the blood and lungs of patients with CBD. Sulfasalazine and its active metabolite, mesalamine, are clinically used to ameliorate chronic inflammation associated with inflammatory bowel disease. We tested whether sulfasalazine or mesalamine could decrease Be-stimulated peripheral blood mononuclear cell (PBMC) proliferation in subjects with CBD and BeS and Be-induced cytokine production in CBD bronchoalveolar lavage (BAL) cells. CBD (n = 25), BeS (n = 12) and healthy normal control (n = 6) subjects were enrolled and ex vivo proliferation and cytokine production were assessed in the presence of Be and sulfasalazine or mesalamine. Be-stimulated PBMC proliferation was inhibited by treatment with either sulfasalazine or mesalamine. Be-stimulated CBD BAL cell IFN-γ and TNF-α cytokine production was decreased by treatment with sulfasalazine or mesalamine. Our data suggest that both sulfasalazine and mesalamine interfere with Be-stimulated PBMC proliferation in CBD and BeS and dampens Be-stimulated CBD BAL cell proinflammatory cytokine production. These studies demonstrate that sulfasalazine and mesalamine can disrupt inflammatory pathways critical to the pathogenesis of chronic granulomatous inflammation in CBD, and may serve as novel therapy for human granulomatous lung diseases.
doi:10.1165/rcmb.2009-0150OC
PMCID: PMC2951876  PMID: 19901345
granulomatous inflammation; IFN-γ; TNF-α; lymphocyte proliferation; berylliosis
13.  Nerve Sheath Myxoma (Neurothekeoma) of the Gingiva, A Case Report and Review of the Literature 
Head and Neck Pathology  2010;4(3):242-245.
Nerve sheath myxoma (NSM) is a benign peripheral nerve sheath tumor that rarely occurs in the oral cavity. Among the 23 reported intraoral cases, no lesion has previously been reported on the gingiva. In this report, we describe the first gingival case of oral neurothekeoma with histopathologic and immunohistochemical characteristics. The patient, a 32 year old female presented with a slowly growing gingival mass diagnosed clinically as an epulis. The lesion was surgically excised. Histopathologically, the lesion presented as a submucosal multinodular mass composed of spindle and stellate-shaped cells with a myxoid background. Immunohistochemically, the tumor cells were sporadically positive for S-100 and NSE and negative for GFAP, EMA, SMA, CD68 and HMB45. The immunoprofile of this lesion confirmed a Schwann cell origin. The lesion was followed up for 10 months with no reports of recurrence.
doi:10.1007/s12105-010-0183-5
PMCID: PMC2923317  PMID: 20502996
Nerve sheath myxoma; Neurothekeoma; Gingiva; Oral cavity; Benign tumor
14.  Towards a combined prognostic index for survival in HIV infection: the role of ‘non-HIV’ biomarkers 
HIV medicine  2009;11(2):143-151.
Background
As those with HIV infection live longer, ‘non-AIDS’ condition associated with immunodeficiency and chronic inflammation are more common. We ask whether ‘non-HIV’ biomarkers improve differentiation of mortality risk among individuals initiating combination antiretroviral therapy (cART).
Methods
Using Poisson models, we analysed data from the Veterans Aging Cohort Study (VACS) on HIV-infected veterans initiating cART between 1 January 1997 and 1 August 2002. Measurements included: HIV biomarkers (CD4 cell count, HIV RNA and AIDS-defining conditions); ‘non-HIV’ biomarkers (haemoglobin, transaminases, platelets, creatinine, and hepatitis B and C serology); substance abuse or dependence (alcohol or drug); and age. Outcome was all cause mortality. We tested the discrimination (C statistics) of each biomarker group alone and in combination in development and validation data sets, over a range of survival intervals, and adjusting for missing data.
Results
Of veterans initiating cART, 9784 (72%) had complete data. Of these, 2566 died. Subjects were middle-aged (median age 45 years), mainly male (98%) and predominantly black (51%). HIV and ‘non-HIV’ markers were associated with each other (P<0.0001) and discriminated mortality (C statistics 0.68–0.73); when combined, discrimination improved (P<0.0001). Discrimination for the VACS Index was greater for shorter survival intervals [30-day C statistic 0.86, 95% confidence interval (CI) 0.80–0.91], but good for intervals of up to 8 years (C statistic 0.73, 95% CI 0.72–0.74). Results were robust to adjustment for missing data.
Conclusions
When added to HIV biomarkers, ‘non-HIV’ biomarkers improve differentiation of mortality. When evaluated over similar intervals, the VACS Index discriminates as well as other established indices. After further validation, the VACS Index may provide a useful, integrated risk assessment for management and research.
doi:10.1111/j.1468-1293.2009.00757.x
PMCID: PMC3077949  PMID: 19751364
anaemia; CD4 cell count; hepatitis C coinfection; hepatology; injecting drug use; outcomes; renal/kidney; risk groups; viral load
15.  Cell penetrating peptide inhibitors of Nuclear Factor-kappa B 
The nuclear factor kappa B (NF-κB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-κB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-κB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-κB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-κB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-κB signaling. Remarkably, several of these NF-κB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting.
doi:10.1007/s00018-008-8222-z
PMCID: PMC2975941  PMID: 18668204
Nuclear Factor kappa B; I kappa B Kinase; cell penetrating peptide; peptide transduction; signal transduction
16.  Is the Drugstore Safe? Counterfeit Diabetes Products on the Shelves 
It is no longer possible to identify counterfeit medical products, including medications and devices, by simply checking packaging and labeling. Improvements in technology have made it cheaper and easier to produce fake packaging and labels, making it nearly impossible for consumers and authorities to detect counterfeits without conducting tests on the products themselves, as illustrated by the sale of over one million counterfeit blood glucose test strips sold to unsuspecting U.S. consumers at drugstores in more than 35 states and in other countries around the world in the fall of 2006. The pricier the drugs, the more counterfeiters seek to mimic them to maximize returns, victimizing those patients at highest risk who rely on life-saving medications.
PMCID: PMC2787054  PMID: 20144408
anticounterfeiting; counterfeit drugs; fake pharmaceuticals; Internet pharmacies
17.  Maintenance treatment with quetiapine versus discontinuation after one year of treatment in patients with remitted first episode psychosis: randomised controlled trial  
Objective To study rates of relapse in remitted patients with first episode psychosis who either continued or discontinued antipsychotic drugs after at least one year of maintenance treatment.
Design 12 month randomised, double blind, placebo controlled trial.
Setting Early psychosis outpatient clinics in Hong Kong.
Participants 178 patients with first episode psychosis who had received at least one year of antipsychotic drug treatment between September 2003 and July 2006 and had no positive symptoms of psychosis.
Interventions Patients received either maintenance treatment with quetiapine (400 mg/day) or placebo and were followed up for the next 12 months or until a relapse occurred.
Main outcome measure Relapse assessed monthly and defined as re-emergence of psychotic symptoms (delusions, conceptual disorganisation, hallucinations, suspiciousness, and unusual thought content) according to predefined thresholds.
Results 178 patients were randomised (89 to quetiapine and 89 to placebo). The Kaplan-Meier estimate of the risk of relapse at 12 months was 41% (95% confidence interval 29% to 53%) for the quetiapine group and 79% (68% to 90%) for the placebo group (P<0.001). Although quetiapine was generally well tolerated, the rate of discontinuation due to adverse or serious adverse events was greater in the quetiapine group (18%; 16/89) than in the placebo group (8%; 7/89) (relative risk 2.29, 95% confidence interval 0.99 to 5.28; χ2=3.20, df=1; P=0.07).
Conclusion In a group of asymptomatic patients with first episode psychosis and at least one year of previous antipsychotic drug treatment, maintenance treatment with quetiapine compared with placebo resulted in a substantially lower rate of relapse during the following year.
Trial registration Clinical trials NCT00334035.
doi:10.1136/bmj.c4024
PMCID: PMC2924475  PMID: 20724402
18.  Gene Variants Associated With Ischemic Stroke 
Background and Purpose
The purpose of this study was to determine whether 74 single nucleotide polymorphisms (SNPs), which had been associated with coronary heart disease, are associated with incident ischemic stroke.
Methods
Based on antecedent studies of coronary heart disease, we prespecified the risk allele for each of the 74 SNPs. We used Cox proportional hazards models that adjusted for traditional risk factors to estimate the associations of these SNPs with incident ischemic stroke during 14 years of follow-up in a population-based study of older adults: the Cardiovascular Health Study (CHS).
Results
In white CHS participants, the prespecified risk alleles of 7 of the 74 SNPs (in HPS1, ITGAE, ABCG2, MYH15, FSTL4, CALM1, and BAT2) were nominally associated with increased risk of stroke (one-sided P<0.05, false discovery rate=0.42). In black participants, the prespecified risk alleles of 5 SNPs (in KRT4, LY6G5B, EDG1, DMXL2, and ABCG2) were nominally associated with stroke (one-sided P<0.05, false discovery rate=0.55). The Val12Met SNP in ABCG2 was associated with stroke in both white (hazard ratio, 1.46; 90% CI, 1.05 to 2.03) and black (hazard ratio, 3.59; 90% CI, 1.11 to 11.6) participants of CHS. Kaplan-Meier estimates of the 10-year cumulative incidence of stroke were greater among Val allele homozygotes than among Met allele carriers in both white (10% versus 6%) and black (12% versus 3%) participants of CHS.
Conclusions
The Val12Met SNP in ABCG2 (encoding a transporter of sterols and xenobiotics) was associated with incident ischemic stroke in white and black participants of CHS.
doi:10.1161/STROKEAHA.108.521328
PMCID: PMC2881155  PMID: 19023099
brain infarction; cerebrovascular accident; epidemiology; genetics; prevention; risk factors
19.  G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes 
The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.
doi:10.1146/annurev.pharmtox.48.113006.094646
PMCID: PMC2869288  PMID: 17995450
GPCR; arrestin; ubiquitin; trafficking; clathrin; PDZ; bioinformatic
20.  Polymorphism in the Apolipoprotein(a) Gene, Plasma Lipoprotein(a), Cardiovascular Disease, and Low-dose Aspirin Therapy 
Atherosclerosis  2008;203(2):371-376.
Objective
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).
Conclusions
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.
doi:10.1016/j.atherosclerosis.2008.07.019
PMCID: PMC2678922  PMID: 18775538
cardiovascular disease; aspirin; lipoproteins; genetics; Lp(a)
21.  Primary prevention of cardiovascular disease: a web‐based risk score for seven British black and minority ethnic groups 
Heart  2006;92(11):1595-1602.
Objective
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.
Design
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.
Setting
Two community‐based surveys.
Participants
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.
Results
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.
Conclusions
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.
doi:10.1136/hrt.2006.092346
PMCID: PMC1861244  PMID: 16762981
22.  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.
Objectives
To develop a cardiovascular risk assessment tool that is feasible and easy to use in primary care (general practice (GP) model).
Design
Prospective cohort study.
Setting
23 towns in the United Kingdom.
Participants
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).
Results
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.
Conclusions
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.
doi:10.1136/hrt.2005.085381
PMCID: PMC1861043  PMID: 16547204
23.  Single Nucleotide Polymorphisms Associated with Coronary Heart Disease Predict Incident Ischemic Stroke in the Atherosclerosis Risk in Communities 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 multiethnic 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.
doi:10.1159/000155637
PMCID: PMC2662496  PMID: 18799872
Genetic factor; Coronary heart disease; Ischemic stroke; Myocardial infarction; Single nucleotide polymorphisms
24.  Polymorphisms Associated with Both Noncardioembolic Stroke and Coronary Heart Disease: Vienna Stroke Registry 
Noncardioembolic stroke and coronary heart disease (CHD) may share genetic predispositions. We tested the hypothesis that genetic variants which are associated with risk of CHD would also be associated with risk of noncardioembolic stroke in 562 cases from the Vienna Stroke Registry and 815 controls. We selected 6 gene variants that had been consistently associated with risk of CHD in 3 studies, including the Atherosclerosis Risk in Communities study, and found that 4 of these gene variants were also associated with risk of noncardioembolic stroke. The odds ratios for noncardioembolic stroke were 1.31 (90% CI 1.07–1.60) for rs3900940 in MYH15, 1.24 (90% CI 1.01–1.5) for rs20455 in KIF6, 1.21 (90% CI 0.99–1.49) for rs1010 in VAMP8, and 1.20 (90% CI 0.95–1.50) for rs10757274 on chromosome 9p21.
doi:10.1159/000236914
PMCID: PMC2818395  PMID: 19752551
Single nucleotide polymorphisms; Coronary heart disease; Stroke; Risk factors; Genetics

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