Impregnation of the Bacterial Cellulose Membrane with Biologically Produced Silver Nanoparticles

Different wound dressings with antibacterial property have been surveyed and one among them is bacterial cellulose (BC). Since the BC does not have antibacterial property, the biologically produced silver nanoparticles (SNPs) were impregnated into the BC. For the BC production, Hestrin–Schramm broth was used. Formation of the BC was proven by enzymatic hydrolysis. For SNPs production, the bacterial supernatant was treated with AgNO₃ and formation of SNPs was monitored through spectrophotometer, TEM and XRD. For impregnation of SNPs into the BC, the cleaned membrane was placed in the bacterial supernatant that contained 1 mmol of AgNO₃. The antibacterial assay was done for the BC/SNPs. Enzymatic hydrolysis proved the presence of the BC. Spectrophotometer and XRD results showed the formation of SNPs. TEM analysis revealed the presence of SNPs with sizes around 5–100 nm. SEM micrographs showed the impregnation of SNPs into the BC. Antibacterial test exhibited the antibacterial activity of the BC/SNPs.     

Effect of polymorphisms in the CSN3 (κ-casein) gene on milk production traits in Chinese Holstein Cattle

This study was designed to evaluate significant associations between single nucleotide polymorphisms (SNPs) and milk composition and milk production traits in Chinese Holstein cows. Six SNPs were identified in the κ-casein gene using pooled DNA sequencing. The identified SNPs were genotyped by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) methods from 507 individuals. Out of six, we identified three non-synonymous SNPs (g.10888T>C, g.10924C>A and g.10944A>G) that changed in the protein product. SIFT (Sorting_Intolerant_From_Tolerant) prediction score (0.01) demonstrated that protein changed Isoleucine > Threonine (g.10888T>C) will affect the phenotypes. Significant associations between identified SNPs and three yield traits (milk, protein and fat) and two composition traits (fat and protein percentages) were found whereas it did not reach significance for fat percentage in haplotypes association. Importantly, the significant SNPs in our results showed a large proportion of the phenotypic variation of milk protein yield and concentration. Our results suggest that CSN3 is an important candidate gene that influences milk production traits, and identified polymorphisms and haplotypes could be used as a genetic marker in programs of marker-assisted selection for the genetic improvement of milk production traits in dairy cattle.     

SNP characteristics predict replication success in association studies

Successful independent replication is the most direct approach for distinguishing real genotype–disease associations from false discoveries in genome-wide association studies (GWAS). Selecting SNPs for replication has been primarily based on P values from the discovery stage, although additional characteristics of SNPs may be used to improve replication success. We used disease-associated SNPs from more than 2,000 published GWASs to identify predictors of SNP reproducibility. SNP reproducibility was defined as a proportion of successful replications among all replication attempts. The study reporting association for the first time was considered to be discovery and all consequent studies targeting the same phenotype replications. We found that ?Log(P), where P is a P value from the discovery study, is the strongest predictor of the SNP reproducibility. Other significant predictors include type of the SNP (e.g., missense vs intronic SNPs) and minor allele frequency. Features of the genes linked to the disease-associated SNP also predict SNP reproducibility. Based on empirically defined rules, we developed a reproducibility score (RS) to predict SNP reproducibility independently of ?Log(P). We used data from two lung cancer GWAS studies as well as recently reported disease-associated SNPs to validate RS. Minus Log(P) outperforms RS when the very top SNPs are selected, while RS works better with relaxed selection criteria. In conclusion, we propose an empirical model to predict SNP reproducibility, which can be used to select SNPs for validation and prioritization.     

Eight SNPs of the Myf5 gene and diplotypes associated with growth and reproductive traits in Jinghai yellow chicken

The objective of this study was to analyze possible associations between single nucleotide polymorphisms (SNPs) in the Myf5 gene with chicken growth and reproductive traits. SNPs in Myf5 of the Jinghai yellow chicken were detected by the polymerase chain reaction single-strand conformation polymorphism method and the haplotypes were analyzed. Eight SNPs were identified in the exons of Myf5. Nine haplotypes were established in a group of 379 Jinghai yellow chickens. In terms of growth traits, least square analysis showed that haplotype H1H5 had significant effects on weight at weeks 8 and 12 (P < 0.05). Haplotype H2H6 had significant effects on weight at weeks 12 and 14 (P < 0.05). For reproductive traits, H1H5 had higher body weight for the first egg than H1H4 and H2H4 (P < 0.05), and H1H3 (P < 0.01). H1H3 had a poor performance in average egg weight at 300 days. On the other hand, H1H3 had an advantage in egg number at 300 days. The results showed that SNPs of Myf5 have certain effects on growth and reproductive traits in Jinghai yellow chickens, which can be used in marker-assisted selection to accelerate chicken genetic progress.     

Common vitamin D pathway gene variants reveal contrasting effects on serum vitamin D levels in African Americans and European Americans

Vitamin D deficiency is more common among African Americans (AAs) than among European Americans (EAs), and epidemiologic evidence links vitamin D status to many health outcomes. Two genome-wide association studies (GWAS) in European populations identified vitamin D pathway gene single-nucleotide polymorphisms (SNPs) associated with serum vitamin D [25(OH)D] levels, but a few of these SNPs have been replicated in AAs. Here, we investigated the associations of 39 SNPs in vitamin D pathway genes, including 19 GWAS-identified SNPs, with serum 25(OH)D concentrations in 652 AAs and 405 EAs. Linear and logistic regression analyses were performed adjusting for relevant environmental and biological factors. The pattern of SNP associations was distinct between AAs and EAs. In AAs, six GWAS-identified SNPs in GC, CYP2R1, and DHCR7/NADSYN1 were replicated, while nine GWAS SNPs in GC and CYP2R1 were replicated in EAs. A CYP2R1 SNP, rs12794714, exhibited the strongest signal of association in AAs. In EAs, however, a different CYP2R1 SNP, rs1993116, was the most strongly associated. Our models, which take into account genetic and environmental variables, accounted for 20 and 28 % of the variance in serum vitamin D levels in AAs and EAs, respectively.     

Genetic polymorphisms associated with rubella virus-specific cellular immunity following MMR vaccination

Rubella virus causes a relatively benign disease in most cases, although infection during pregnancy can result in serious birth defects. An effective vaccine has been available since the early 1970s and outbreaks typically do not occur among highly vaccinated (≥2 doses) populations. Nevertheless, considerable inter-individual variation in immune response to rubella immunization does exist, with single-dose seroconversion rates ~95 %. Understanding the mechanisms behind this variability may provide important insights into rubella immunity. In the current study, we examined associations between single nucleotide polymorphisms (SNPs) in selected cytokine, cytokine receptor, and innate/antiviral genes and immune responses following rubella vaccination in order to understand genetic influences on vaccine response. Our approach consisted of a discovery cohort of 887 subjects aged 11–22 at the time of enrollment and a replication cohort of 542 older adolescents and young adults (age 18–40). Our data indicate that SNPs near the butyrophilin genes (BTN3A3/BTN2A1) and cytokine receptors (IL10RB/IFNAR1) are associated with variations in IFNγ secretion and that multiple SNPs in the PVR gene, as well as SNPs located in the ADAR gene, exhibit significant associations with rubella virus-specific IL-6 secretion. This information may be useful, not only in furthering our understanding immune responses to rubella vaccine, but also in identifying key pathways for targeted adjuvant use to boost immunity in those with weak or absent immunity following vaccination.     

Natural genetic variation in MIR172 isolated from Brassica species

The present study reports a natural variation in microRNA172 (MIR172) family members isolated from six species of genus Brassica. The analysis of nucleotide polymorphism across 44 Brassica MIR172 homologs revealed a higher conservation in the predicted precursors relative to flanking regions. Single nucleotide polymorphisms (SNPs) were detected in miRNA and miRNA*. The 21-nt miRNA sequence was conserved in all MIR172 members except MIR172a. However, the miRNA* sequence was conserved only in MIR172a compared to A. thaliana. Non-canonical Brassica variants of precursor miR172a were detected wherein SNP at 5′ terminal in mature miR172a resulted in a sequence identical to mature miR172e. SNPs and indels in precursors resulted in varied stem-loop structures of differing stabilities (ΔG) implying a differential efficiency of miRNA biogenesis. A sequence based phylogram revealed ortholog specific groupings of MIR172 irrespective of genetic background. A Northern analysis in Brassica juncea displayed the cumulative expression of miR172 isoforms in all tissues representing different developmental stages with levels gradually increasing from vegetative to reproductive stages. Detection of high content of miR172 in roots indicates the possibility of additional roles of Brassica miR172 in root development.     

Melanocortin-4 receptor (MC4R) polymorphisms are associated with growth and meat quality traits in sheep

The involvement of melanocortin 4 receptor gene (MC4R) in food intake and body weight regulation is well characterized. MC4R mutations are the most frequent monogenic cause of human obesity. Significant associations have been revealed between MC4R mutations and productive traits in pigs, cattle and poultry. Herein, fluorescence-based conformation sensitive gel electrophoresis was used to identify two single nucleotide polymorphisms (SNPs) in the coding region (93G>A and 292G>A) and two SNPs in the 3′-UTR area (1016G>A and 1240T>C) of MC4R gene in 132 German Merino sheep. We found that the 1016G>A mutation in the 3′-UTR was significantly associated with body weight at 120 and 180 days, average daily gain, back fat thickness and loin-eye area. Allele A located at the 292th position of MC4R gene representing Arg98 was associated with significantly higher loin-eye area in sheep. For the synonymous 93G>A mutation, A allele carrier animals had higher back fat thickness. Our results provide evidence that the MC4R gene may be a candidate gene for growth and meat quality traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in German Merino sheep.     

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium? array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.     

Mutations in microRNA Binding Sites of CEP Genes Involved in Cancer

The CEP genes play a pivotal role in the replication of the cell. CEP family proteins form the major constituents of the centrosome and play a prominent role in centriole biogenesis and in cell replication. Alteration in CEP genes will result in disruption of cell cycle that may in turn cause cancer. In our study, we found that 16 of the CEP genes are a potential target to miRNA that binds to complementary sequences in 3′untranslated regions (UTR) of mRNA and stop them from translation. Single nucleotide polymorphisms (SNPs) occurring naturally in such miRNA binding site can alter the miRNA: mRNA interaction and can significantly alter gene expression. We developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 44 high-confidence SNPs that may impair miRNA target sites in the 3′UTR of 16 genes. Further we performed expression analysis to shed light on the potential tissues that might be affected by mutation, enrichment analysis to find the metabolic functions of the gene, and network analysis to highlight the important interactions of CEP genes with other genes to provide insight that complex network will be disturbed upon mutation. In this study, we explored and prioritised the SNPs in CEP gene which could act as a potential target in centrosome-associated human disease. Our analysis would provide a thoughtful insight to wet lab researches to understand the expression pattern of CEP genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.     

Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case–control studies of type 2 diabetes in developing countries

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.     

A comparison of type 2 diabetes risk allele load between African Americans and European Americans

The prevalence of type 2 diabetes (T2D) is greater in populations of African descent compared to European-descent populations. Genetic risk factors may underlie the disparity in disease prevalence. Genome-wide association studies (GWAS) have identified >60 common genetic variants that contribute to T2D risk in populations of European, Asian, African and Hispanic descent. These studies have not comprehensively examined population differences in cumulative risk allele load. To investigate the relationship between risk allele load and T2D risk, 46 T2D single nucleotide polymorphisms (SNPs) in 43 loci from GWAS in European, Asian, and African-derived populations were genotyped in 1,990 African Americans (n = 963 T2D cases, n = 1,027 controls) and 1,644 European Americans (n = 719 T2D cases, n = 925 controls) ascertained and recruited using a common protocol in the southeast United States. A genetic risk score (GRS) was constructed from the cumulative risk alleles for each individual. In African American subjects, risk allele frequencies ranged from 0.024 to 0.964. Risk alleles from 26 SNPs demonstrated directional consistency with previous studies, and 3 SNPs from ADAMTS9, TCF7L2, and ZFAND6 showed nominal evidence of association (p < 0.05). African American individuals carried 38–67 (53.7 ± 4.0, mean ± SD) risk alleles. In European American subjects, risk allele frequencies ranged from 0.084 to 0.996. Risk alleles from 36 SNPs demonstrated directional consistency, and 10 SNPs from BCL11A, PSMD6, ADAMTS9, ZFAND3, ANK1, CDKN2A/B, TCF7L2, PRC1, FTO, and BCAR1 showed evidence of association (p < 0.05). European American individuals carried 38–65 (50.9 ± 4.4) risk alleles. African Americans have a significantly greater burden of 2.8 risk alleles (p = 3.97 × 10^?89) compared to European Americans. However, GRS modeling showed that cumulative risk allele load was associated with risk of T2D in European Americans, but only marginally in African Americans. This result suggests that there are ethnic-specific differences in genetic architecture underlying T2D, and that these differences complicate our understanding of how risk allele load impacts disease susceptibility.     

Single-nucleotide polymorphism associations in common with immune responses to measles and rubella vaccines

Single-nucleotide polymorphisms (SNPs) in candidate immune response genes were evaluated for associations with measles- and rubella-specific neutralizing antibodies, interferon (IFN)-γ, and interleukin (IL)-6 secretion in two separate association analyses in a cohort of healthy immunized subjects. We identified six SNP associations shared between the measles-specific and rubella-specific immune responses, specifically neutralizing antibody titers (DDX58), secreted IL-6 (IL10RB, IL12B), and secreted IFN-γ (IFNAR2, TLR4). An intronic SNP (rs669260) in the antiviral innate immune receptor gene, DDX58, was significantly associated with increased neutralizing antibody titers for both measles and rubella viral antigens post-MMR vaccination (p values 0.02 and 0.0002, respectively). Significant associations were also found between IL10RB (rs2284552; measles study p value 0.006, rubella study p value 0.00008) and IL12B (rs2546893; measles study p value 0.005, rubella study p value 0.03) gene polymorphisms and variations in both measles- and rubella virus-specific IL-6 responses. We also identified associations between individual SNPs in the IFNAR2 and TLR4 genes that were associated with IFN-γ secretion for both measles and rubella vaccine-specific immune responses. These results are the first to indicate that there are SNP associations in common across measles and rubella vaccine immune responses and that SNPs from multiple genes involved in innate and adaptive immune response regulation may contribute to the overall human antiviral response.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.