Antibody immobilisation on fibre optic TIRF sensors

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.     

Minerals solubilizing and mobilizing microbiomes: A sustainable approach for managing minerals’ deficiency in agricultural soil

Agriculture faces challenges to fulfil the rising food demand due to shortage of arable land and various environmental stressors. Traditional farming technologies help in fulfilling food demand but they are harmful to humans and environmental sustainability. The food production along with agro-environmental sustainability could be achieved by encouraging farmers to use agro-environmental sustainable products such as biofertilizers and biopesticides consisting of live microbes or plant extract instead of chemical-based inputs. The eco-friendly formulations play a significant role in plant growth promotion, crop yield and repairing degraded soil texture and fertility sustainably. Mineral solubilizing microbes that provide vital nutrients like phosphorus, potassium, zinc and selenium are essential for plant growth and development and could be developed as biofertilizers. These microbes could be plant associated (rhizospheric, endophytic and phyllospheric) or inhabit the bulk soil and diverse extreme habitats. Mineral solubilizing microbes from soil, extreme environments, surface and internal parts of the plant belong to diverse phyla such as Ascomycota, Actinobacteria, Basidiomycota, Bacteroidetes, Chlorobi, Cyanobacteria, Chlorophyta, Euryarchaeota, Firmicutes, Gemmatimonadetes, Mucoromycota, Proteobacteria and Tenericutes. Mineral solubilizing microbes (MSMs) directly or indirectly stimulate plant growth and development either by releasing plant growth regulators; solubilizing phosphorus, potassium, zinc, selenium and silicon; biological nitrogen fixation and production of siderophores, ammonia, hydrogen cyanide, hydrolytic enzymes and bioactive compound/secondary metabolites. Biofertilizer developed using mineral solubilizing microbes is an eco-friendly solution to the sustainable food production system in many countries worldwide. The present review deals with the biodiversity of mineral solubilizing microbes, and potential roles in crop improvement and soil well-being for agricultural sustainability.     

Antitumor cytotoxic T-cell response induced by a survivin peptide mimic

Survivin is a tumor-associated antigen with significant potential as a cancer vaccine target. We have identified a survivin peptide mimic containing human MHC class I epitopes and a potential class II ligand that induces a potent antitumor response in C57BL/6 mice with GL261 cerebral gliomas. This peptide is able to elicit both CD8+ CTL and T helper cell responses in C57BL/6 mice. The corresponding region of the human survivin molecule represented by peptide SVN53-67 is 100% homologous to the murine protein, but SVN53-67 is weakly immunogenic in man. We evaluated several amino acid substitutions in putative human MHC I anchor positions in SVN53-67 to identify potential peptide mimics that could provide an enhanced antitumor immune response against human glioma and primary central nervous system lymphoma (PCNSL) cells in culture. We evaluated survivin peptides with predicted binding to human HLA-A*0201 antigen using peptide-loaded dendritic cells from PBMC of patients with these malignancies. One alteration (M57) led to binding to HLA-A*0201 with significantly higher affinity. We compared the ability of autologous dendritic cells loaded with SVN53-67 peptide and SVN53-67/M57 in CTL assays against allomatched and autologous, survivin-expressing, human malignant glioma and PCNSL cells. Both SVN53-67 and SVN53-67/M57 produced CTL-mediated killing of malignant target cells; however, SVN53-67/M57 was significantly more effective than SVN53-67. Thus, SVN53-67/M57 may act as a peptide mimic to induce an enhanced antitumor CTL response in tumor patients. The use of SVN53-67/M57 as a cancer vaccine might have application for cancer vaccine therapy.     

Sexual dimorphism of rat liver gene expression: regulatory role of growth hormone revealed by deoxyribonucleic Acid microarray analysis

GH has diverse physiological actions and regulates the tissue-specific expression of numerous genes involved in growth, metabolism, and differentiation. Several of the effects of GH on somatic growth and gene expression are sex dependent and are regulated by pituitary GH secretory patterns, which are sexually differentiated. The resultant sex differences in plasma GH profiles are particularly striking in rodents and are the major determinant of sex differences in pubertal body growth rates and the expression in liver of several cytochrome P450 (CYP) enzymes that metabolize steroids, drugs, and environmental chemicals of importance to endocrinology, pharmacology, and toxicology. DNA microarray analysis was used to identify rat liver-expressed genes that show sexual dimorphism, and to ascertain the role of GH as a regulator of their sexually dimorphic expression. Adult male and female rats were untreated or were treated with GH by 7-d continuous infusion using an Alzet osmotic minipump. Poly(A) RNA was purified from individual livers and Cy3- and Cy5-labeled cDNA probes cohybridized to Pan Rat Liver and 5K Rat Oligonucleotide microarrays representing 5889 unique rat genes. Analysis of differential gene expression profiles identified 37 liver-expressed, female-predominant genes; of these, 27 (73%) were induced by continuous GH treatment of male rats. Moreover, only three of 30 genes up-regulated in male rat liver by continuous GH treatment did not display female-dominant expression. Further analysis revealed that 44 of 49 male-predominant genes (90%) were down-regulated in the livers of continuous GH-treated male rats compared with untreated male rats, whereas only five of 49 genes that were down-regulated in male rats by continuous GH treatment were not male dominant in their expression. Real-time PCR analysis applied to a sampling of 10 of the sexually dimorphic genes identified in the microarray analysis verified their sex- and GH-dependent patterns of regulation. Taken together, these studies establish that GH-regulated gene expression is the major mechanistic determinant of sexually dimorphic gene expression in the rat liver model.     

Trends from 2002 to 2010 in Daily Breakfast Consumption and its Socio-Demographic Correlates in Adolescents across 31 Countries Participating in the HBSC Study

Breakfast is often considered the most important meal of the day and children and adolescents can benefit from breakfast consumption in several ways. The purpose of the present study was to describe trends in daily breakfast consumption (DBC) among adolescents across 31 countries participating in the HBSC survey between 2002 to 2010 and to identify socio-demographic (gender, family affluence and family structure) correlates of DBC. Cross-sectional surveys including nationally representative samples of 11–15 year olds (n = 455,391). Multilevel logistic regression analyses modeled DBC over time after adjusting for family affluence, family structure and year of survey. In all countries, children in two-parent families were more likely to report DBC compared to single parent families. In most countries (n = 19), DBC was associated with family affluence. Six countries showed an increase in DBC (Canada, Netherland, Macedonia, Scotland, Wales, England) from 2002. A significant decrease in DBC from 2002 was found in 11 countries (Belgium Fr, France, Germany, Croatia, Spain, Poland, Russian Federation, Ukraine, Latvia, Lithuania and Norway), while in 5 countries (Portugal, Denmark, Finland, Ireland, Sweden) no significant changes were seen. Frequency of DBC among adolescents in European countries and North America showed a more uniform pattern in 2010 as compared to patterns in 2002. DBC increased significantly in only six out of 19 countries from 2002 to 2010. There is need for continued education and campaigns to motivate adolescents to consume DBC. Comparing patterns across HBSC countries can make an important contribution to understanding regional /global trends and to monitoring strategies and development of health promotion programs.     

Correlates of dental visits among community‐residing Latino elders: a public health alert

doi:10.1111/j.1741‐2358.2009.00335.x
Correlates of dental visits among community‐residing Latino elders: a public health alert Objectives: To examine oral service utilisation in a probability sample of community‐residing Latino elders. Background: Older Latinos are at a potential increased risk of oral diseases, given their higher prevalence of co‐morbidities and lower rate of dental service utilisation. Methods: A prevalence survey was conducted among a random sample of Latino (largely Puerto Rican) elders (n = 205; mean age = 75.8; SD ± 5.3) in New York City during 2001–2002. A systematic random sample was drawn from the Centers for Medicare and Medicaid Services Beneficiary tape files. Current use of oral health services and self‐reported health conditions was obtained. Functional and cognitive impairment were assessed. Results: Less than half of the sample reported a dental visit in the previous year. The average time since the last dental visit was 54 months (SD ± 84.5). Last year dental visit compliers were more likely to be unmarried, living alone, with higher levels of education, fewer health conditions and less impairment with activities of daily living. In multivariate analyses, problem‐oriented behaviour, Medicaid beneficiary, education, living alone, chronic health conditions and mobility impairment explained 14% of the ‘time since last dental visit’ variance. Conclusions: Given that socio‐demographic and level of functioning determinants appear to influence the frequency of dental visits, a multilevel approach to oral health promotion is imperative.     

Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, including diabetic nephropathy. Endothelial-derived nitric oxide synthase (eNOS) gene polymorphisms affect eNOS activity and are associated with endothelial dysfunction. We evaluated the association of the constitutive endothelial nitric oxide synthase gene (eNOS) polymorphisms with type 2 diabetic nephropathy. We genotyped three polymorphisms of eNOS (Two SNPs: -786T > C, 894G > T and one 27-bp repeat polymorphism in Intron 4 (27VNTR)) in type 2 diabetic nephropathy patients (cases: n = 195) and type 2 diabetic without nephropathy (controls: n = 255), using validated PCR-RFLP assays. We measured serum NO levels in these subjects and examined its correlation with diabetic nephropathy and eNOS genotypes. The frequency of CC (-786T > C), TT (894G > T) and aa genotypes (27VNTR) were significantly higher in diabetic nephropathy patients as compared to the diabetes without nephropathy group (CC: P = 0.003, TT: P = 0.03, aa: P < 0.0001). These mutant genotypes were found to be associated with higher risk of nephropathy (-786T > C: OR: 5.5, 95%CI: 1.53-19.79; 894G > T: OR: 1.8, 95%CI: 1.03-3.16; Intron 4: OR: 6.23, 95%CI: 2.23-16.31). Haplotype with all the wild alleles (T-b-G) was found to be associated with a decreased risk of nephropathy (OR: 0.68, P = 0.005) and haplotype with all mutant alleles (C-a-T) was associated with higher risk of diabetic nephropathy as compared to diabetes without nephropathy group (OR: 2.6, P = 0.14). No significant linkage disequilibria were observed among the variants in this case-control study. The serum NO levels were observed to be significantly (P < 0.05) lower in mutant allele carriers ('C' allele of T-786C SNP and/or 'T' allele of G894T SNP) as compared with the wild-type allele carriers (-786T and/or 894G) within each of the subject groups (with and without nephropathy). These results suggest that the eNOS gene locus is associated with diabetic nephropathy and the functional polymorphisms (-786T > C & 894G > T) might lead to a decreased expression of eNOS gene.     

Cellular differentiation and nitrogenase activity in the cyanobacteriumAnabaena

Nitrogenase activity at periods of differentiation of heterocysts and akinetes was assayed by the acetylene reduction technique. There was no nitrogenase activity in ammoniumgrown, non-heterocystousAnabaena sp.; the activity appeared only after a lag-phase of about 17 – 21 h after the ammonium-grown culture had been transferred to medium free of combined nitrogen. This activity started appearing as the proheterocysts were developing to mature heterocysts. Maximum nitrogenase activity was attained with exponential phase of culture and mature heterocysts. This activity gradually decreased with the differentiation of akinetes. Only insignificant nitrogenase activity was observed in old cultures in which most cells had matured into akinetes.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.