Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. RESULTS: The sensitivity of the assay was approximately 1.2 × 10(9 )(2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. CONCLUSIONS: The here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions

Transcriptional monitoring of steady state and effects of anaerobic phases in chemostat cultures of the filamentous fungus Trichoderma reesei

BACKGROUND: Chemostat cultures are commonly used in production of cellular material for systems-wide biological studies. We have used the novel TRAC (transcript analysis with aid of affinity capture) method to study expression stability of approximately 30 process relevant marker genes in chemostat cultures of the filamentous fungus Trichoderma reesei and its transformant expressing laccase from Melanocarpus albomyces. Transcriptional responses caused by transient oxygen deprivations and production of foreign protein were also studied in T. reesei by TRAC. RESULTS: In cultures with good steady states, the expression of the marker genes varied less than 20% on average between sequential samples for at least 5 or 6 residence times. However, in a number of T. reesei cultures continuous flow did not result in a good steady state. Perturbations to the steady state were always evident at the transcriptional level, even when they were not measurable as changes in biomass or product concentrations. Both unintentional and intentional perturbations of the steady state demonstrated that a number of genes involved in growth, protein production and secretion are sensitive markers for culture disturbances. Exposure to anaerobic conditions caused strong responses at the level of gene expression, but surprisingly the cultures could regain their previous steady state quickly, even after 3 h O(2 )depletion. The main effect of producing M. albomyces laccase was down-regulation of the native cellulases compared with the host strain. CONCLUSION: This study demonstrates the usefulness of transcriptional analysis by TRAC in ensuring the quality of chemostat cultures prior to costly and laborious genome-wide analysis. In addition TRAC was shown to be an efficient tool in studying gene expression dynamics in transient conditions

Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis

Peer reviewe

Response of ground macrolichens to site factors, co‐existing plants and forestry in boreal forests

Aim Ground lichens have declined in boreal and temperate Europe. The potential causes of the decline were explored by analysing the response of lichen cover to forest management, site and stand variables as well as co-existing plants in different boreal vegetation subzones. Location Finland. Methods Understorey vegetation was surveyed on a systematic network of 1721 sample plots in forests on mineral soil. The response of macrolichen cover to explanatory variables was analysed by generalised linear mixed models (GLMMs). Results Ground lichens favoured old Pinus sylvestris forests on xeric sites with sufficient light conditions. Intensive forest management regimes, such as regeneration cutting and soil ploughing decreased lichen cover, while lighter scarification methods had less effect on lichens. Lichens benefitted from intermediate cutting on sites with low bryophyte cover. Lichens responded similarly to site and stand variables in all boreal subzones, showing that despite heavy reindeer grazing these response patterns apply also in the northern boreal subzone. Lichens showed both negative (Vaccinium myrtillus, Empetrum nigrum and Calluna vulgaris) and positive (Vaccinium vitis-idaea) responses to increasing dwarf shrub cover. Generally, lichens negatively responded to increasing bryophyte cover, indicating either that bryophytes benefit from a decline of lichens or that there is real competition between these groups. The negative relationship between bryophytes and lichens strengthened as the tree canopy becomes denser. Conclusions Our study provides new quantitative insights into the effects of forest management and changes in forest structure as underlying factors for lichen decline both inside and outside the reindeer-herding area (i.e., the northern boreal subzone). These factors include increased canopy cover with increased shading as well as disturbance caused by regeneration cutting and soil preparation. The possible effects of the long-term legacy of nitrogen deposition, lack of forest fires and climate change are also discussed

Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase

AbstractCytochrome P450 2A6 (CYP2A6) is a polymorphic enzyme responsible for the oxidation of certain precarcinogens and drugs and is the major nicotine C-oxidase. The role of CYP2A6 for nicotine elimination was emphasised recently by the finding that smokers carrying defective CYP2A6 alleles consumed fewer cigarettes [Pianezza et al. (1998) Nature 393, 750]. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe reaction for the CYP2A6 enzyme. The present study describes an allele-specific PCR genotyping method that identifies the major defective CYP2A6 allele and accurately predicts the phenotype. An allele frequency of 1–3% was observed in Finnish, Spanish, and Swedish populations, much lower than described previously

Early exposure to social disadvantages and later life body mass index beyond genetic predisposition in three generations of Finnish birth cohorts

BackgroundThe study aimed to explore the association between early life and life-course exposure to social disadvantage and later life body mass index (BMI) accounting for genetic predisposition and maternal BMI.MethodsWe studied participants of Helsinki Birth Cohort Study born in 1934-1944 (HBCS1934-1944, n=1277) and Northern Finland Birth Cohorts born in 1966 and 1986 (NFBC1966, n=5807, NFBC1986, n=6717). Factor analysis produced scores of social disadvantage based on social and economic elements in early life and adulthood/over the life course, and was categorized as high, intermediate and low. BMI was measured at 62years in HBCS1934-1944, at 46years in NFBC1966 and at 16years in NFBC1986. Multivariable linear regression analysis was used to explore associations between social disadvantages and BMI after adjustments for polygenic risk score for BMI (PRS BMI), maternal BMI and sex.ResultsThe association between exposure to high early social disadvantage and increased later life BMI persisted after adjustments (beta =0.79, 95% CI, 0.33, 1.25, p 0.22, 95% CI -0.91,1.35, p=0.700). In HBCS1934-1944 and NFBC1966, participants who had reduced their exposure to social disadvantage during the life-course had lower later life BMI than those who had increased their exposure (beta -1.34, [-2.37,-0.31], p=0.011; beta -0.46, [-0.89,-0.03], p=0.038, respectively).ConclusionsHigh social disadvantage in early life appears to be associated with higher BMI in later life. Reducing exposure to social disadvantage during the life-course may be a potential pathway for obesity reduction.Peer reviewe

Brain structural alterations in autism and criminal psychopathy

Publisher Copyright: © 2022 The AuthorsThe goal of this study was to elucidate the anatomical brain basis of social cognition through two disorders with distinctively different phenotypes of social interaction. We compared structural MR images of 20 individuals with autism spectrum disorder (ASD), 19 violent offenders with high psychopathic traits, and 19 control participants using voxel-based morphometry (VBM). Our earlier study showed lower grey matter volume (GMV) values in the insula, frontal cortex, and sensorimotor cortex of the offender group compared to controls. In the present study, the images of the ASD group revealed lower GMV in the left precuneus, right cerebellum, and right precentral gyrus in comparison with controls. The comparison between the offender and ASD groups showed lower GMV values for the right temporal pole and left inferior frontal gyrus in the offender group. There was also an overlap of both disorders in the right pre-central cortex, showing lower GMV compared to controls. Our findings suggest structural differences between violent offenders with high psychopathy traits and ASD individuals in the frontotemporal social brain network areas, previously associated with empathy. We also provide evidence of similar abnormal structures in the motor cortex for both of these disorders, possibly related to uniting issues of social cognition.Peer reviewe

Aspectos psicológicos associados à disfunção temporomandibular: uma revisão sistemática da literatura

A disfunção temporomandibular se configura como uma síndrome que afeta a região orofacial causando prejuízo à qualidade de vida. Atualmente, é explicada através do modelo etiológico multifatorial com vários fatores determinantes. Apesar de existir um consenso em relação à multifatoriedade, há pouca concordância em relação à importância dos fatores etiológicos envolvidos, e ainda não se sabe até que ponto estes fatores podem ser considerados predisponentes, desencadeantes ou perpetuantes. Diante disso, este estudo objetivou investigar, por meio de uma revisão sistemática de literatura, quais aspectos psicológicos são mais frequentemente associados à disfunção temporomandibular. Os artigos com datas entre 2000 e 2015 foram pesquisados nas bases de dados nacionais: SciELO, LILACS e BBO. Foram selecionados 21 artigos. Os resultados apontaram a depressão, o estresse, a ansiedade e características da personalidade como aspectos psicológicos mais associados à disfunção temporomandibular