A haplotype at the UCP1 gene locus contributes to genetic risk for type 2 diabetes in Asian Indians (CURES-72)

BACKGROUND: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. METHODS: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms, namely -3826A-->G, an A-->C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A-->G-5'UTR A-->C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). CONCLUSIONS: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

Association of the PPARGC1A gene polymorphism with diabetic nephropathy in an Asian Indian population (CURES-41)

BACKGROUND: The aim of this study was to evaluate the association of polymorphisms of the peroxisome proliferator-activated receptor gamma (PPARG) gene and peroxisome proliferators-activated receptor gamma co-activator 1 alpha (PPARGC1A) gene with diabetic nephropathy (DN) in Asian Indians. METHODS: Six common polymorphisms, 3 of the PPARG gene [-1279G/A, Pro12Ala, and His478His (C/T)] and 3 of the PPARGC1A gene (Thr394Thr, Gly482Ser, and +A2962G) were studied in 571 normal glucose-tolerant (NGT) subjects, 255 type 2 diabetic (T2D) subjects without nephropathy, and 141 DN subjects. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing. Logistic regression analysis was performed to assess the covariables associated with DN. RESULTS: Among the 6 polymorphisms examined, only the Gly482Ser of the PPARGC1A gene was significantly associated with DN. The genotype frequency of Ser/Ser genotype of the PPARGC1A gene was 8.8% (50/571) in NGT subjects, 7.8% (20/255) in T2D subjects, and 29.8% (42/141) in DN subjects. The odds ratios (ORs) for DN for the susceptible Gly/Ser and Ser/Ser genotype after adjusting for age, sex, body mass index, and duration of diabetes were 2.14 [95% confidence interval (CI), 1.23-3.72; P = 0.007] and 8.01 (95% CI, 3.89-16.47; P < 0.001), respectively. The unadjusted OR for DN for the XA genotype of the Thr394Thr polymorphism was 1.87 (95% CI, 1.20-2.92; P = 0.006) compared to T2D subjects. However, the significance was lost (P = 0.061) when adjusted for age, sex, BMI, and duration of diabetes. The +A2962G of PPARGC1A and the 3 polymorphisms of PPARG were not associated with DN. CONCLUSION: The Gly482Ser polymorphism of the PPARGC1A gene is associated with DN in Asian Indians.

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (-11377C-->G and -11282T-->C), one intronic SNP (+10211T-->G) and one exonic SNP (+45T-->G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T-->G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T-->G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07-1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3-1.8; P < 10(-7)) and that for GG genotype, 2.10 (95% CI 1.3-3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T-->G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.

Association of lipoprotein lipase gene polymorphisms with obesity and type 2 diabetes in an Asian Indian population

AIMS: Lipoprotein lipase (LPL), a pivotal enzyme in lipoprotein metabolism, catalyzes the hydrolysis of triglycerides of very low-density lipoproteins and chylomicrons. Assuming that the variants in the promoter of the LPL gene may be associated with changes in lipid metabolism leading to obesity and type 2 diabetes, we examined the role of promoter variants (-T93G and -G53C) in the LPL gene in an urban South Indian population. METHODS: The study subjects (619 type 2 diabetic and 731 normal glucose-tolerant (NGT) subjects) were chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The polymorphisms were genotyped using polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The two polymorphisms studied were not in LD. The -T93G was not associated with type 2 diabetes but was associated with obesity. 11.5% of the obese subjects (62/541) had the XG(TG+GG) genotype compared with 6.4% of the nonobese subjects (52/809; P=0.001). The odds ratio for obesity for the XG genotype was 1.766 (95% CI: 1.19-2.63, P=0.005). Subjects with XG genotype also had higher body mass index and waist circumference compared with those with TT genotype. With respect to G53C, subjects with the XC(GC+CC) genotype had 0.527 and 0.531 times lower risk for developing type 2 diabetes and obesity, respectively. CONCLUSIONS: Among Asian Indians, the -T93G SNP of the LPL gene is associated with obesity but not type 2 diabetes, whereas the -G53C SNP appears to be protective against both obesity and type 2 diabetes.

Effect of polymorphisms in the PPARGC1A gene on body fat in Asian Indians

OBJECTIVE: To evaluate whether polymorphisms in the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A) gene were related to body fat in Asian Indians. METHODS: Three polymorphisms of PPARGC1A gene, the Thr394Thr, Gly482Ser and +A2962G, were genotyped on 82 type 2 diabetic and 82 normal glucose tolerant (NGT) subjects randomly chosen from the Chennai Urban Rural Epidemiology Study using PCR-RFLP, and the nature of the variants were confirmed using direct sequencing. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies using an expectation-maximization algorithm. Visceral, subcutaneous and total abdominal fat were measured using computed tomography, whereas dual X-ray absorptiometry was used to measure central abdominal and total body fat. RESULTS: None of the three polymorphisms studied were in LD. The genotype (0.59 vs 0.32, P=0.001) and allele (0.30 vs 0.17, P=0.007) frequencies of Thr394Thr polymorphism were significantly higher in type 2 diabetic subjects compared to those in NGT subjects. The odds ratio for diabetes (adjusted for age, sex and body mass index) for the susceptible genotype, XA (GA+AA) of Thr394Thr polymorphism, was 2.53 (95% confidence intervals: 1.30-5.04, P=0.009). Visceral and subcutaneous fat were significantly higher in NGT subjects with XA genotype of the Thr394Thr polymorphism compared to those with GG genotype (visceral fat: XA 148.2+/-46.9 vs GG 106.5+/-51.9 cm(2), P=0.001; subcutaneous fat: XA 271.8+/-167.1 vs GG 181.5+/-78.5 cm(2), P=0.001). Abdominal (XA 4521.9+/-1749.6 vs GG 3445.2+/-1443.4 g, P=0.004), central abdominal (XA 1689.0+/-524.0 vs GG 1228.5+/-438.7 g, P<0.0001) and non-abdominal fat (XA 18763.8+/-8789.4 vs GG 13160.4+/-4255.3 g, P<0.0001) were also significantly higher in the NGT subjects with XA genotype compared to those with GG genotype. The Gly482Ser and +A2962G polymorphisms were not associated with any of the body fat measures. CONCLUSION: Among Asian Indians, the Thr394Thr (G --> A) polymorphism is associated with increased total, visceral and subcutaneous body fat.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians

AIMS: The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-gamma co-activator-1 alpha (PGC-1alpha) gene with Type 2 diabetes in Asian Indians. METHODS: The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype frequencies were estimated using an expectation-maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264-2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. CONCLUSIONS: The A allele of Thr394Thr (G --> A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population.

Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK

This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

Understanding climate change projections for precipitation over western Europe with a weather typing approach

Precipitation over western Europe (WE) is projected to increase (decrease) roughly northward (equatorward) of 50°N during the 21st century. These changes are generally attributed to alterations in the regional large-scale circulation, e.g., jet stream, cyclone activity, and blocking frequencies. A novel weather typing within the sector (30°W–10°E, 25–70°N) is used for a more comprehensive dynamical interpretation of precipitation changes. A k-means clustering on daily mean sea level pressure was undertaken for ERA-Interim reanalysis (1979–2014). Eight weather types are identified: S1, S2, S3 (summertime types), W1, W2, W3 (wintertime types), B1, and B2 (blocking-like types). Their distinctive dynamical characteristics allow identifying the main large-scale precipitation-driving mechanisms. Simulations with 22 Coupled Model Intercomparison Project 5 models for recent climate conditions show biases in reproducing the observed seasonality of weather types. In particular, an overestimation of weather type frequencies associated with zonal airflow is identified. Considering projections following the (Representative Concentration Pathways) RCP8.5 scenario over 2071–2100, the frequencies of the three driest types (S1, B2, and W3) are projected to increase (mainly S1, +4%) in detriment of the rainiest types, particularly W1 (−3%). These changes explain most of the precipitation projections over WE. However, a weather type-independent background signal is identified (increase/decrease in precipitation over northern/southern WE), suggesting modifications in precipitation-generating processes and/or model inability to accurately simulate these processes. Despite these caveats in the precipitation scenarios for WE, which must be duly taken into account, our approach permits a better understanding of the projected trends for precipitation over WE.

Detection of tobacco mosaic tobamovirus in cigarettes through RT-PCR

TMV was tried to recover from a variety of branded cigarettes and cigars. Tobacco from six different brands of cigarettes and cigar were processed and reverse transcriptase polymerase chain reaction was employed for the detection of TMV. RTPCR confirmed the presence of TMV in tobacco from one brand of cigarette and one brand of cigar. Bean plants (Phaseolus vulgaris) were inoculated manually with tobacco sap of cigarettes resulting in the production of localized disease lesions. Together, these results showed that tobacco used to make cigarettes and cigars can function as an effective disease vector, potentially aiding the movement of infectious TMV between countries. This is an important finding prompting a need to test smoking tobacco for other virus particles that infect tobacco plants and survive processing as well as considering biosecurity measures to limit virus transmission

Species-specific identification of Leishmania in naturally infected sand flies captured in Mato Grosso do Sul State, Brazil

An increase in cutaneous and visceral leishmaniasis cases has been reported in recent years in the state of Mato Grosso do Sul, Brazil, and little is known to date about their etiological agents. An investigation into natural Leishmania infection of sand flies captured in this state between December 2003 and August 2004 was carried out. Mini-exon sequences were used as targets to identify Leishmania, and an RFLP technique was employed for those identified as belonging to the Viannia subgenus. Calculation of the minimal infection rate (MR) revealed that 1.6% of sand flies captured in the forest, peridomicile and intradomicile were positive. Six species were found to be infected by Leishmania (V.) braziliensis. Interestingly, two of the six species. Lutzomyia longipalpis and Nyssomyia whitmani, were captured in anthropic environments. The findings of this study constitute a useful tool for planning control measures against this disease in the State of Mato Grosso do Sul. (C) 2010 Elsevier B.V. All rights reserved.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

CYP3A5*3A allele is associated with reduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia

Background: The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of xenobiotics. CYP3A5 SNPs have been associated with variations on enzyme activity that can modify the metabolism of several drugs. Methods: In order to evaluate the influence of CYP3A5 variants on response to lowering-cholesterol drugs, 139 individuals with hypercholesterolemia were selected. After a wash-out period of 4 weeks, individuals were treated with atorvastatin (10 mg/day/4 weeks). Genomic DNA was extracted by a salting-out procedure. CYP3A5*3C, CYP3A5*6 and CYP3A5*1D were analyzed by PCR-RFLP and DNA sequencing. Results: >Frequencies of the CYP3A5*3C and CYP3A5*1D alleles were lower in individuals of African descent (*3C: 47.8% and *1D: 55.2%) than in non-Africans (*3C: 84.9% and *1D 84.8%, p<0.01). Non-Africans carrying *3A allele (*3C and *1D combined alleles) had lower total and LDL-cholesterol response to atorvastatin than non-*3A allele carriers (p<0.05). Conclusion: CYP3A5*3A allele is associated with reduced cholesterol-lowering response to atorvastatin in non-African individuals. (C) 2008 Elsevier B.V. All rights reserved.

Population structure of the migratory fish Prochilodus lineatus (Characiformes) from rio Grande basin (Brazil), an area fragmented by dams

1. Prochilodus lineatus (Prochilodontidae, Characiformes) is a migratory species of great economic importance both in fisheries and aquaculture that is found throughout the Jacui, Paraiba do Sul, Parana, Paraguay and Uruguay river basins in South America. Earlier population studies of P. lineatus in the rio Grande basin (Parana basin) indicated the existence of a single population; however, the range of this species has been fragmented by the construction of several dams. Such dams modified the environmental conditions and could have constrained the reproductive migration of P. lineatus, possibly leading to changes in the population genetic structure. 2. In order to evaluate how genetic diversity is allocated in the rio Grande basin, 141 specimens of P. lineatus from eight collection sites were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with 15 restriction enzymes. 3. Forty-six haplotypes were detected, and 70% of them are restricted. The mean genetic variability indexes (h = 0.7721 and pi = 1.6%) were similar to those found in natural populations with a large effective size. Fst and Exact Test values indicated a lack of structuring among the samples, and the model of isolation by distance was tested and rejected. 4. The haplotype network indicated that this population of P. lineatus has been maintained as a single variable stock with some differences in the genetic composition (haplotypes) between samples. Indications of population expansion were detected, and this finding was supported by neutrality tests and mismatch distribution analyses. 5. The present study focused on regions between dams to serve as a parameter for further evaluations of genetic variability and the putative impact of dams and repopulation programmes in natural populations of P. lineatus. Copyright (C) 2011 John Wiley & Sons, Ltd.