A constitutive expression system for high‐throughput screening

To reduce the amount of consumables and number of pipetting steps in high‐throughput screening, a constitutive expression system was developed that comprises four different promoters of varying strength. The system was validated by the expression of different sucrose phosphorylase enzymes from Leuconostoc mesenteroides, Lactobacillus acidophilus and Bifidobacterium adolescentis in 96‐deep‐ and low‐well plates at three temperatures. Drastically improved soluble expression in mini‐cultures was observed for the enzymes from L. mesenteroides strains by reducing the promoter strength from strong to intermediate and by expressing the proteins at lower temperatures. In contrast, the enzymes from B. adolescentis and L. acidophilus were expressed most efficiently with a strong promoter. The constitutive expression of sucrose phosphorylases in low‐well plates resulted in a level of activity that is equal or even better than what was achieved by inducible expression. Therefore, our plasmid set with varying constitutive promoters will be an indispensable tool to optimize enzyme expression for high‐throughput screening.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

A global study of relationships between leaf traits, climate and soil measures of nutrient fertility

Aim This first global quantification of the relationship between leaf traits and soil nutrient fertility reflects the trade‐off between growth and nutrient conservation. The power of soils versus climate in predicting leaf trait values is assessed in bivariate and multivariate analyses and is compared with the distribution of growth forms (as a discrete classification of vegetation) across gradients of soil fertility and climate. Location All continents except for Antarctica. Methods Data on specific leaf area (SLA), leaf N concentration (LNC), leaf P concentration (LPC) and leaf N:P were collected for 474 species distributed across 99 sites (809 records), together with abiotic information from each study site. Individual and combined effects of soils and climate on leaf traits were quantified using maximum likelihood methods. Differences in occurrence of growth form across soil fertility and climate were determined by one‐way ANOVA. Results There was a consistent increase in SLA, LNC and LPC with increasing soil fertility. SLA was related to proxies of N supply, LNC to both soil total N and P and LPC was only related to proxies of P supply. Soil nutrient measures explained more variance in leaf traits among sites than climate in bivariate analysis. Multivariate analysis showed that climate interacted with soil nutrients for SLA and area‐based LNC. Mass‐based LNC and LPC were determined mostly by soil fertility, but soil P was highly correlated to precipitation. Relationships of leaf traits to soil nutrients were stronger than those of growth form versus soil nutrients. In contrast, climate determined distribution of growth form more strongly than it did leaf traits. Main conclusions We provide the first global quantification of the trade‐off between traits associated with growth and resource conservation ‘strategies’ in relation to soil fertility. Precipitation but not temperature affected this trade‐off. Continuous leaf traits might be better predictors of plant responses to nutrient supply than growth form, but growth forms reflect important aspects of plant species distribution with climate.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system

Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrogen supply effects on leaf dynamics and nutrient input into the soil of plant species in a sub-arctic tundra ecosystem

Global warming will lead to increased nitrogen supply in tundra ecosystems. How increased N supply affected leaf production, leaf turnover and dead leaf N input into the soil of Empetrum nigrum and Andromeda polifolia (evergreens), Eriophorum vaginatum (graminoid) and Betula nana (deciduous) in a sub-arctic tundra in northern Sweden between 2003 and 2007 was experimentally investigated. There was considerable interspecific variation in the response of leaf production to N addition, varying from negative, no response to a positive response. Nitrogen addition effects on leaf turnover also showed considerable variation among species, varying from no effect to increased leaf turnover (up to 27% in Eriophorum). Nitrogen addition resulted in a four to fivefold increase in N content in the dead leaves of both evergreens and a 65% increase in Eriophorum. Surprisingly, there was no increase in Betula. The response of dead leaf P contents to N addition was rather species specific. There was no response in Empetrum, whereas there were significant increases in Andromeda (+214%) and Eriophorum (+32%), and a decrease of 47% in Betula. As an overall result of the changes in leaf production, leaf turnover and dead leaf N and P contents, nitrogen addition increased in all species except Betula the amount of N and, for Andromeda and Eriophorum the amount of P transferred to the soil due to leaf litter inputs. However, the way in which this was achieved differed substantially among species due to interspecific differences in the response of the component processes (leaf production, leaf turnover, dead leaf nutrient content).     

Mitochondrial dysfunction leads to reduced chronological lifespan and increased apoptosis in yeast

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.