Complete genome sequence of Pseudomonas aeruginosa lytic bacteriophage PA1O which resembles temperate bacteriophage D3112

A novel Pseudomonas aeruginosa lytic bacteriophage (phage), PA1Ø, was isolated, and its genome was sequenced completely. This phage is able to lyse not only P. aeruginosa but also Staphylococcus aureus. Genome analysis of PA1Ø showed that it is similar to a P. aeruginosa temperate phage, D3112, with the exception of the absence of a c repressor-encoding gene, which is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa.     

Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14

Background

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.

Principal Findings

In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.

Conclusions

We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.     

Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion

The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD) from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.     

Connexin 43 and metabolic effect of fatty acids in stressed endothelial cells

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.     

Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.     

Combined analysis of genome-wide association studies for Crohn disease and psoriasis identifies seven shared susceptibility loci

Psoriasis (PS) and Crohn disease (CD) have been shown to be epidemiologically, pathologically, and therapeutically connected, but little is known about their shared genetic causes. We performed meta-analyses of five published genome-wide association studies on PS (2,529 cases and 4,955 controls) and CD (2,142 cases and 5,505 controls), followed up 20 loci that showed strongest evidence for shared disease association and, furthermore, tested cross-disease associations for previously reported PS and CD risk alleles in additional 6,115 PS cases, 4,073 CD cases, and 10,100 controls. We identified seven susceptibility loci outside the human leukocyte antigen region (9p24 near JAK2, 10q22 at ZMIZ1, 11q13 near PRDX5, 16p13 near SOCS1, 17q21 at STAT3, 19p13 near FUT2, and 22q11 at YDJC) shared between PS and CD with genome-wide significance (p < 5 × 10⁻⁸) and confirmed four already established PS and CD risk loci (IL23R, IL12B, REL, and TYK2). Three of the shared loci are also genome-wide significantly associated with PS alone (10q22 at ZMIZ1, p_rs1250544 = 3.53 × 10⁻⁸, 11q13 near PRDX5, p_rs694739 = 3.71 × 10⁻⁰⁹, 22q11 at YDJC, p_rs181359 = 8.02 × 10⁻¹⁰). In addition, we identified one susceptibility locus for CD (16p13 near SOCS1, p_rs4780355 = 4.99 × 10⁻⁸). Refinement of association signals identified shared genome-wide significant associations for exonic SNPs at 10q22 (ZMIZ1) and in silico expression quantitative trait locus analyses revealed that the associations at ZMIZ1 and near SOCS1 have a potential functional effect on gene expression. Our results show the usefulness of joint analyses of clinically distinct immune-mediated diseases and enlarge the map of shared genetic risk loci.     

Abnormal pulmonary artery stiffness in pulmonary arterial hypertension: in vivo study with intravascular ultrasound

Background

There is increasing recognition that pulmonary artery stiffness is an important determinant of right ventricular (RV) afterload in pulmonary arterial hypertension (PAH). We used intravascular ultrasound (IVUS) to evaluate the mechanical properties of the elastic pulmonary arteries (PA) in subjects with PAH, and assessed the effects of PAH-specific therapy on indices of arterial stiffness.

Method

Using IVUS and simultaneous right heart catheterisation, 20 pulmonary segments in 8 PAH subjects and 12 pulmonary segments in 8 controls were studied to determine their compliance, distensibility, elastic modulus and stiffness index β. PAH subjects underwent repeat IVUS examinations after 6-months of bosentan therapy.

Results

At baseline, PAH subjects demonstrated greater stiffness in all measured indices compared to controls: compliance (1.50±0.11×10^–2 mm^2/mmHg vs 4.49±0.43×10^–2 mm^2/mmHg, p<0.0001), distensibility (0.32±0.03%/mmHg vs 1.18±0.13%/mmHg, p<0.0001), elastic modulus (720±64 mmHg vs 198±19 mmHg, p<0.0001), and stiffness index β (15.0±1.4 vs 11.0±0.7, p = 0.046). Strong inverse exponential associations existed between mean pulmonary artery pressure and compliance (r² = 0.82, p<0.0001), and also between mean PAP and distensibility (r² = 0.79, p = 0.002). Bosentan therapy, for 6-months, was not associated with any significant changes in all indices of PA stiffness.

Conclusion

Increased stiffness occurs in the proximal elastic PA in patients with PAH and contributes to the pathogenesis RV failure. Bosentan therapy may not be effective at improving PA stiffness.     

Association of JAG1 with Bone Mineral Density and Osteoporotic Fractures: A Genome-wide Association Study and Follow-up Replication Studies

Bone mineral density (BMD), a diagnostic parameter for osteoporosis and a clinical predictor of fracture, is a polygenic trait with high heritability. To identify genetic variants that influence BMD in different ethnic groups, we performed a genome-wide association study (GWAS) on 800 unrelated Southern Chinese women with extreme BMD and carried out follow-up replication studies in six independent study populations of European descent and Asian populations including 18,098 subjects. In the meta-analysis, rs2273061 of the Jagged1 (JAG1) gene was associated with high BMD (p = 5.27 × 10⁻⁸ for lumbar spine [LS] and p = 4.15 × 10⁻⁵ for femoral neck [FN], n = 18,898). This SNP was further found to be associated with the low risk of osteoporotic fracture (p = 0.009, OR = 0.7, 95% CI 0.57–0.93, n = 1881). Region-wide and haplotype analysis showed that the strongest association evidence was from the linkage disequilibrium block 5, which included rs2273061 of the JAG1 gene (p = 8.52 × 10⁻⁹ for LS and 3.47 × 10⁻⁵ at FN). To assess the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of c-Myc to the “G” but not “A” allele of rs2273061. A mRNA expression study in both human bone-derived cells and peripheral blood mononuclear cells confirmed association of the high BMD-related allele G of rs2273061 with higher JAG1 expression. Our results identify the JAG1 gene as a candidate for BMD regulation in different ethnic groups, and it is a potential key factor for fracture pathogenesis.     

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p_meta-Euro = 2.08 × 10⁻¹⁰), transmembrane protein 39A (TMEM39A; rs1132200; p_meta-all = 8.62 × 10⁻⁹), and 17q21 (rs1453560; p_meta-all = 3.48 × 10⁻¹⁰) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10⁻⁸ < p_meta-Euro < 9.99 × 10⁻⁵) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.     

Common genetic determinants of intraocular pressure and primary open-angle glaucoma

Intraocular pressure (IOP) is a highly heritable risk factor for primary open-angle glaucoma and is the only target for current glaucoma therapy. The genetic factors which determine IOP are largely unknown. We performed a genome-wide association study for IOP in 11,972 participants from 4 independent population-based studies in The Netherlands. We replicated our findings in 7,482 participants from 4 additional cohorts from the UK, Australia, Canada, and the Wellcome Trust Case-Control Consortium 2/Blue Mountains Eye Study. IOP was significantly associated with rs11656696, located in GAS7 at 17p13.1 (p = 1.4×10⁻⁸), and with rs7555523, located in TMCO1 at 1q24.1 (p = 1.6×10⁻⁸). In a meta-analysis of 4 case-control studies (total N = 1,432 glaucoma cases), both variants also showed evidence for association with glaucoma (p = 2.4×10⁻² for rs11656696 and p = 9.1×10⁻⁴ for rs7555523). GAS7 and TMCO1 are highly expressed in the ciliary body and trabecular meshwork as well as in the lamina cribrosa, optic nerve, and retina. Both genes functionally interact with known glaucoma disease genes. These data suggest that we have identified two clinically relevant genes involved in IOP regulation.     

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (10⁵ cells mL^-1) at densities of 10⁷, 10⁵, and 10³ PA14 cells mL^-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10?days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30?days; total MCs: 91.0%, 86.9%, and 61.6% at 30?days, respectively). However, the activity of PA14 diminished when its density decreased to less than 10⁶ cells mL^-1. At three initial algal densities (10⁶, 10⁵, and 10³ cells mL^-1), PA14 at a density of 10⁵ cells mL^-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30?days, respectively). Host range assays showed that at lower initial PA14 inoculation (10⁵ cells mL^-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (10⁷ cells mL^-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 10⁵ Pseudomonas aeruginosa PA14 cells mL^-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.     

Identification of novel genetic markers associated with clinical phenotypes of systemic sclerosis through a genome-wide association strategy

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10⁻¹², OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10⁻⁶, OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10⁻⁷, OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10⁻⁶¹, OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10⁻⁷⁶, OR = 8.84), and in NOTCH4 with ACA P = 8.84×10⁻²¹, OR = 0.55) and ATA (P = 1.14×10⁻⁸, OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.     

Epidemiological profiles between equol producers and nonproducers: a genomewide association study of the equol-producing phenotype

Equol is a daidzein (a phytoestrogen isoflavone) metabolite of gut bacteria, and the ability to produce equol varies between individuals and reduces the risks of several diseases. We tested the effects of equol production on health in Koreans and identified the genetic factors that determine the equol-producing phenotype. In 1391 subjects, the equol-producing phenotype was determined, based on measurements of serum equol concentrations. The anthropometric and blood biochemical measurements between equol producers and nonproducers were analyzed by LC-MS/MS. Genetic factors were identified in a genomewide association study (GWAS), and the interaction between genetic factors and the equol-producing phenotype was examined. We observed that 70.1 % of the study population produced equol. Blood pressure was significantly lower in equol producers (beta ± SE = −1.35 ± 0.67, p = 0.045). In our genomewide association study, we identified 5 single-nucleotide polymorphisms (p < 1 × 10⁻⁵) in HACE1. The most significant SNP was rs6927608, and individuals with a minor allele of rs6927608 did not produce equol (odds ratio = 0.57 (95 % CI 0.45–0.72), p value = 2.5 × 10⁻⁶). Notably, the interaction between equol production and the rs6927608 HACE1 SNP was significantly associated with systolic blood pressure (p value = 1.3 × 10⁴). Equol production is linked to blood pressure, and HACE1, identified in our (GWAS), might be a determinant of the equol-producing phenotype.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0292-8) contains supplementary material, which is available to authorized users.     

Genetic loci associated with plasma phospholipid n-3 fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium

Long-chain n-3 polyunsaturated fatty acids (PUFAs) can derive from diet or from α-linolenic acid (ALA) by elongation and desaturation. We investigated the association of common genetic variation with plasma phospholipid levels of the four major n-3 PUFAs by performing genome-wide association studies in five population-based cohorts comprising 8,866 subjects of European ancestry. Minor alleles of SNPs in FADS1 and FADS2 (desaturases) were associated with higher levels of ALA (p = 3×10⁻⁶⁴) and lower levels of eicosapentaenoic acid (EPA, p = 5×10⁻⁵⁸) and docosapentaenoic acid (DPA, p = 4×10⁻¹⁵⁴). Minor alleles of SNPs in ELOVL2 (elongase) were associated with higher EPA (p = 2×10⁻¹²) and DPA (p = 1×10⁻⁴³) and lower docosahexaenoic acid (DHA, p = 1×10⁻¹⁵). In addition to genes in the n-3 pathway, we identified a novel association of DPA with several SNPs in GCKR (glucokinase regulator, p = 1×10⁻⁸). We observed a weaker association between ALA and EPA among carriers of the minor allele of a representative SNP in FADS2 (rs1535), suggesting a lower rate of ALA-to-EPA conversion in these subjects. In samples of African, Chinese, and Hispanic ancestry, associations of n-3 PUFAs were similar with a representative SNP in FADS1 but less consistent with a representative SNP in ELOVL2. Our findings show that common variation in n-3 metabolic pathway genes and in GCKR influences plasma phospholipid levels of n-3 PUFAs in populations of European ancestry and, for FADS1, in other ancestries.     

Pressure-accelerated dissociation of amyloid fibrils in wild-type hen lysozyme

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.     

A genome-wide association study of nephrolithiasis in the Japanese population identifies novel susceptible Loci at 5q35.3, 7p14.3, and 13q14.1

Nephrolithiasis is a common nephrologic disorder with complex etiology. To identify the genetic factor(s) for nephrolithiasis, we conducted a three-stage genome-wide association study (GWAS) using a total of 5,892 nephrolithiasis cases and 17,809 controls of Japanese origin. Here we found three novel loci for nephrolithiasis: RGS14-SLC34A1-PFN3-F12 on 5q35.3 (rs11746443; P = 8.51×10⁻¹², odds ratio (OR) = 1.19), INMT-FAM188B-AQP1 on 7p14.3 (rs1000597; P = 2.16×10⁻¹⁴, OR = 1.22), and DGKH on 13q14.1 (rs4142110; P = 4.62×10⁻⁹, OR = 1.14). Subsequent analyses in 21,842 Japanese subjects revealed the association of SNP rs11746443 with the reduction of estimated glomerular filtration rate (eGFR) (P = 6.54×10⁻⁸), suggesting a crucial role for this variation in renal function. Our findings elucidated the significance of genetic variations for the pathogenesis of nephrolithiasis.