A Systematic Mapping Approach of 16q12.2/FTO and BMI in More Than 20,000 African Americans Narrows in on the Underlying Functional Variation: Results from the Population Architecture using Genomics and Epidemiology (PAGE) Study

Genetic variants in intron 1 of the fat mass- and obesity-associated (FTO) gene have been consistently associated with body mass index (BMI) in Europeans. However, follow-up studies in African Americans (AA) have shown no support for some of the most consistently BMI-associated FTO index single nucleotide polymorphisms (SNPs). This is most likely explained by different race-specific linkage disequilibrium (LD) patterns and lower correlation overall in AA, which provides the opportunity to fine-map this region and narrow in on the functional variant. To comprehensively explore the 16q12.2/FTO locus and to search for second independent signals in the broader region, we fine-mapped a 646-kb region, encompassing the large FTO gene and the flanking gene RPGRIP1L by investigating a total of 3,756 variants (1,529 genotyped and 2,227 imputed variants) in 20,488 AAs across five studies. We observed associations between BMI and variants in the known FTO intron 1 locus: the SNP with the most significant p-value, rs56137030 (8.3×10-6) had not been highlighted in previous studies. While rs56137030was correlated at r2>0.5 with 103 SNPs in Europeans (including the GWAS index SNPs), this number was reduced to 28 SNPs in AA. Among rs56137030 and the 28 correlated SNPs, six were located within candidate intronic regulatory elements, including rs1421085, for which we predicted allele-specific binding affinity for the transcription factor CUX1, which has recently been implicated in the regulation of FTO. We did not find strong evidence for a second independent signal in the broader region. In summary, this large fine-mapping study in AA has substantially reduced the number of common alleles that are likely to be functional candidates of the known FTO locus. Importantly our study demonstrated that comprehensive fine-mapping in AA provides a powerful approach to narrow in on the functional candidate(s) underlying the initial GWAS findings in European populations

Evidence for an increase in cosmogenic 10Be during a geomagnetic reversal

Reversals in the geomagnetic field, which occur every few hundred thousand years, represent a dramatic change in the Earth's environment. Although there is no satisfactory theory for such reversals, it is generally accepted that the dipole field intensity decreases to <20% of its 'normal' value for a few thousand years during the change in direction. Because the galactic and solar cosmic rays which impinge on the Earth's atmosphere are charged, a significant fraction (about half) of them are deflected by the geomagnetic field. At the time of a reversal, this magnetic shielding is greatly reduced, and it has been suggested that the increased flux of high-energy particles could have effects on evolutionary or climatic processes. For example, the statistically significant coincidence in levels of some marine faunal extinctions and reversal boundaries in ocean sediments could be caused, directly or indirectly, by the decreased geomagnetic intensity during the reversal. We report here evidence in marine sediments for an increase in cosmogenic 10Be production in the Earth's atmosphere during the Brunhes-Matuyama reversal 730,000 yr ago. In addition to confirming an increase in cosmogenic isotope production, the results provide information on the magnitude and duration of the geomagnetic intensity decrease during such an event, and the depth at which remanent magnetism is acquired in marine sediments

Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P &lt; 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

Influence of emphysema distribution on pulmonary function parameters in COPD patients

Objective: To evaluate the impact that the distribution of emphysema has on clinical and functional severity in patients with COPD. Methods: The distribution of the emphysema was analyzed in COPD patients, who were classified according to a 5-point visual classification system of lung CT findings. We assessed the influence of emphysema distribution type on the clinical and functional presentation of COPD. We also evaluated hypoxemia after the six-minute walk test (6MWT) and determined the six-minute walk distance (6MWD). Results: Eighty-six patients were included. The mean age was 65.2 ± 12.2 years, 91.9% were male, and all but one were smokers (mean smoking history, 62.7 ± 38.4 pack-years). The emphysema distribution was categorized as obviously upper lung-predominant (type 1), in 36.0% of the patients; slightly upper lung-predominant (type 2), in 25.6%; homogeneous between the upper and lower lung (type 3), in 16.3%; and slightly lower lung-predominant (type 4), in 22.1%. Type 2 emphysema distribution was associated with lower FEV1 , FVC, FEV1 /FVC ratio, and DLCO. In comparison with the type 1 patients, the type 4 patients were more likely to have an FEV1 < 65% of the predicted value (OR = 6.91, 95% CI: 1.43-33.45; p = 0.016), a 6MWD < 350 m (OR = 6.36, 95% CI: 1.26-32.18; p = 0.025), and post-6MWT hypoxemia (OR = 32.66, 95% CI: 3.26-326.84; p = 0.003). The type 3 patients had a higher RV/TLC ratio, although the difference was not significant. Conclusions: The severity of COPD appears to be greater in type 4 patients, and type 3 patients tend to have greater hyperinflation. The distribution of emphysema could have a major impact on functional parameters and should be considered in the evaluation of COPD patients

Understanding emotionally relevant situations in primary care dental practice: 1. Clinical situations and emotional responses

Background and aims. The stressful nature of dental practice is well established. Much less information is available on the coping strategies used by dentists and the emotions which underlie the stressful experience. Previous research has been almost exclusively questionnaire-based, limiting the range of emotions explored. This study used qualitative methods to explore the full extent of emotions and coping strategies associated with stressful events in primary dental practice. Method. Semi-structured interviews were conducted with 20 dentists in Lincoln and the surrounding area. Verbatim transcriptions were analysed using thematic analysis. Results. Participants reported a wide variety of stressful situations, consistent with the existing literature, which were associated with a diverse range of negative emotional responses including anxiety, anger and sadness. Dentists tended to have more difficulty identifying positive events and emotions. The designation of a situation as stressful or otherwise was dependent on the dentist's personal interpretation of the event. Data relating to the effects of stressors and the coping strategies used by dentists will be presented in subsequent papers. Conclusion. The situations which dentists find difficult are accompanied by a diverse set of emotions, rather than omnipresent 'stress.' This has implications for stress management programmes for those in dental practic

Immunization with Cocktail of HIV-Derived Peptides in Montanide ISA-51 Is Immunogenic, but Causes Sterile Abscesses and Unacceptable Reactogenicity

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886

Polymorphisms in PTK2 are associated with skeletal muscle specific force: an independent replication study

Purpose The aim of the study was to investigate two single nucleotide polymorphisms (SNP) in PTK2 for associations with human muscle strength phenotypes in healthy men. Methods Measurement of maximal isometric voluntary knee extension (MVCKE) torque, net MVCKE torque and vastus lateralis (VL) specific force, using established techniques, was completed on 120 Caucasian men (age = 20.6 ± 2.3 year; height = 1.79 ± 0.06 m; mass = 75.0 ± 10.0 kg; mean ± SD). All participants provided either a blood (n = 96) or buccal cell sample, from which DNA was isolated and genotyped for the PTK2 rs7843014 A/C and rs7460 A/T SNPs using real-time polymerase chain reaction. Results Genotype frequencies for both SNPs were in Hardy–Weinberg equilibrium (X 2 ≤ 1.661, P ≥ 0.436). VL specific force was 8.3% higher in rs7843014 AA homozygotes than C-allele carriers (P = 0.017) and 5.4% higher in rs7460 AA homozygotes than T-allele carriers (P = 0.029). No associations between either SNP and net MVCKE torque (P ≥ 0.094) or peak MVCKE torque (P ≥ 0.107) were observed. Conclusions These findings identify a genetic contribution to the inter-individual variability within muscle specific force and provides the first independent replication, in a larger Caucasian cohort, of an association between these PTK2 SNPs and muscle specific force, thus extending our understanding of the influence of genetic variation on the intrinsic strength of muscle.Published versio

Comparison of Influenza and SIV Specific CD8 T Cell Responses in Macaques

Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8+ T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8+ T-cells in 39 pigtail macaques expressing the common Mane-A*10+ (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8+ T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8+ T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8+ effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8+ T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8+ T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8+ T-cell response, profile may assist in controlling HIV disease