Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes

The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The optimization of the PCR protocol involved a gradient of annealing temperatures, as well as the application of seven PCR enhancers: formamide, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, BSA and Tween-20. The best results for AtCKX1 amplification were obtained at 55.1ºC, with the addition of 5% DMSO. Glycerol and trehalose also improved the sensitivity of amplification, while formamide, ethylene glycol and BSA enhanced only the amplification of control purified targets, but not the transcripts. Tween-20 inhibited PCR. DMSO enhanced AtCKX1 PCR amplification and improved the specificity of qPCR amplification, as well as the assay reproducibility. This work emphasizes the usefulness of additives, which are rarely used for PCR optimization in real-time experiments.Projekat ministarstva br. ON173015 i br. ON17302

Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes

The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The optimization of the PCR protocol involved a gradient of annealing temperatures, as well as the application of seven PCR enhancers: formamide, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, BSA and Tween-20. The best results for AtCKX1 amplification were obtained at 55.1ºC, with the addition of 5% DMSO. Glycerol and trehalose also improved the sensitivity of amplification, while formamide, ethylene glycol and BSA enhanced only the amplification of control purified targets, but not the transcripts. Tween-20 inhibited PCR. DMSO enhanced AtCKX1 PCR amplification and improved the specificity of qPCR amplification, as well as the assay reproducibility. This work emphasizes the usefulness of additives, which are rarely used for PCR optimization in real-time experiments.Projekat ministarstva br. ON173015 i br. ON17302

Kloniranje gena za ugljeni hidrat oksidazu iz biljke Lactuca sativa u kvasce saccharomyces cerevisiae i Pichia pastoris

We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene Department for Plant Physiology, Institute for Biological Research 'Siniša Stanković', drate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5α strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae stran InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme the further studies on purification and refolding of expressed protein are necessary.Ugljeni hidrat-oksidaza (CHO) iz zelene salate (Lactuca sativa) je enzim koji je do danas nedovoljno ispitan. Vrlo se malo zna o njegovoj strukturi i funkciji. CHO pripada velikoj familiji ugljenihidrat-oksidaza, koje oksiduju šećere. Svaki od članova ove velike familije dobio je ime po supstratu koji oksiduje. Oksidaze iz ove familije enzima imaju kako sličnu ulogu tako i sličnu strukutru. Sve ili većina ovih enzima su monomeri, čiji se polipeptidni lanac uvija u dva domena. Jedan od domena vezuje flavinski kofaktor, a drugi domen je supstrat vezujući. Većina njih oksidaciju supstrata vrši po takozvanom ping-pong mehanizmu. Sve oksidaze iz karbohidrat-oksidaza familije, pa među njima i enzim koji je predmet ove studije (CHO), danas su našle veliku primenu u industriji. CHO se može primenjivati kako u medicinskoj djagnostici, konkretno u biosenzorima za određivanje glukoze u krvi, u prehrambenoj industriji, poljoprivredi, proizvodnji hleba, deterdženata i u raznim drugim industrijskim oblastima. Problem sa ovim enzimom, kao i sa ostalim članovima ove familije, jeste niska koncentracija u prirodnim izvorima. Zato su danas razvijene različite metode rekombinantne tehnologije, kojima se dobijaju ovi enzimi. U ovom radu opisano je kloniranje gena za CHO iz zelene salate u dve vrste kvasaca (Saccharomyces cerevisiae i Pichia pastoris). Sintetički gen za CHO (1821 bp) iz zelene salate kloniran je u vektor pUC57. Escherichia coli soj DH5α korišćen je za kloniranje gena i održavanje plazmida. P. pastoris soj X-33 i S. cerevisiae soj InvSC1 korišćeni su za ekstracelularnu ekspresiju CHO. Aktivnost CHO određena je ABST esejom, a promena absorbance merena je na 405 nm. Potvrda prisustva enzima rađena je na DNK agaroznoj elektroforezi i SDS-PAGE. Posle transformacije P. pastoris X-33, nijedan od klonova nije pokazivao aktivnost CHO. Posle prve fermentacije, kolonije su testirane na SDS-PAGE. Kako su dva uzorka pokazala trake, koje ne postoje na praznom vektoru, ove trake bi mogle odgovarati željenom enzimu, CHO. Traka se nalazi na molekulskoj masi koja je veća od teoretske (više od 120 kDa). Enzim bi mogao biti glikolizovan i zbog toga pokazivati ovako velike vrednosti za molekulsku masu. S. cerevisiae soj InvSC1 transformisan je konstruktom CHO-pZES. Posle 24 sata, tri kolonije su porasle na ploči na kojoj su bile ćelije transformisane pomenutim konstruktom. Uzorci su testirani na SDS-PAGE. Jedan uzorak je pokazao traku na oko 110 kDa, ali aktivnost CHO nije potvrđena takođe. Cilj ove studije je bio kloniranje CHO u kvascima S. cerevisiae i P. pastoris, kao i njena ekspresija u ovim, danas široko primenjivanim ekspresionim sistemima

Functional brain network centrality is related to APOE genotype in cognitively normal elderly

INTRODUCTION: Amyloid plaque deposition in the brain is an early pathological change in Alzheimer's disease (AD), causing disrupted synaptic connections. Brain network disruptions in AD have been demonstrated with eigenvector centrality (EC), a measure that identifies central regions within networks. Carrying an apolipoprotein (APOE)-ε4 allele is a genetic risk for AD, associated with increased amyloid deposition. We studied whether APOE-ε4 carriership is associated with EC disruptions in cognitively normal individuals. METHODS: A total of 261 healthy middle-aged to older adults (mean age 56.6 years) were divided into high-risk (APOE-ε4 carriers) and low-risk (noncarriers) groups. EC was computed from resting-state functional MRI data. Clusters of between-group differences were assessed with a permutation-based method. Correlations between cluster mean EC with brain volume, CSF biomarkers, and psychological test scores were assessed. RESULTS: Decreased EC in the visual cortex was associated with APOE-ε4 carriership, a genetic risk factor for AD. EC differences were correlated with age, CSF amyloid levels, and scores on the trail-making and 15-object recognition tests. CONCLUSION: Our findings suggest that the APOE-ε4 genotype affects brain connectivity in regions previously found to be abnormal in AD as a sign of very early disease-related pathology. These differences were too subtle in healthy elderly to use EC for single-subject prediction of APOE genotype

Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population

<p>Abstract</p> <p>Background</p> <p>Animal identification is pivotal in governmental agricultural policy, enabling the management of subsidy payments, movement of livestock, test scheduling and control of disease. Advances in bovine genomics have made it possible to utilise inherent genetic variability to uniquely identify individual animals by DNA profiling, much as has been achieved with humans over the past 20 years. A DNA profiling test based on bi-allelic single nucleotide polymorphism (SNP) markers would offer considerable advantages over current short tandem repeat (STR) based industry standard tests, in that it would be easier to analyse and interpret. In this study, a panel of 51 genome-wide SNPs were genotyped across panels of semen DNA from 6 common breeds for the purposes of ascertaining allelic frequency. For SNPs on the same chromosome, the extent of linkage disequilbrium was determined from genotype data by Expectation Maximization (EM) algorithm. Minimum probabilities of unique identification were determined for each breed panel. The usefulness of this SNP panel was ascertained by comparison to the current bovine STR Stockmarks II assay. A statistically representative random sampling of bovine animals from across Northern Ireland was assembled for the purposes of determining the population allele frequency for these STR loci and subsequently, the minimal probability of unique identification they conferred in sampled bovine animals from Northern Ireland.</p> <p>Results</p> <p>6 SNPs exhibiting a minor allele frequency of less than 0.2 in more than 3 of the breed panels were excluded. 2 Further SNPs were found to reside in coding areas of the cattle genome and were excluded from the final panel. The remaining 43 SNPs exhibited genotype frequencies which were in Hardy Weinberg Equilibrium. SNPs on the same chromosome were observed to have no significant linkage disequilibrium/allelic association. Minimal probabilities of uniquely identifying individual animals from each of the breeds were obtained and were observed to be superior to those conferred by the industry standard STR assay.</p> <p>Conclusions</p> <p>The 43 SNPs characterised herein may constitute a starting point for the development of a SNP based DNA identification test for European cattle.</p

Electrochemical methods for speciation of trace elements in marine waters. Dynamic aspects

The contribution of electrochemical methods to the knowledge of dynamic speciation of toxic trace elements in marine waters is critically reviewed. Due to the importance of dynamic considerations in the interpretation of the electrochemical signal, the principles and recent developments of kinetic features in the interconversion of metal complex species will be presented. As dynamic electrochemical methods, only stripping techniques (anodic stripping voltammetry and stripping chronopotentiometry) will be used because they are the most important for the determination of trace elements. Competitive ligand ex- change-adsorptive cathodic stripping voltammetry, which should be considered an equilibrium technique rather than a dynamic method, will be also discussed because the complexing parameters may be affected by some kinetic limitations if equilibrium before analysis is not attained and/or the flux of the adsorbed complex is in fluenced by the lability of the natural complexes in the water sample. For a correct data interpretation and system characterization the comparison of results obtained from different techniques seems essential in the articulation of a serious discussion of their meaning

Yeast biodiversity in vineyard environments is increased by human intervention

One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio