Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 invivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25α can be expressed in soluble form, whereas the other two, LKB1 and STRADα are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25α-STRADα complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25α and STRADα exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADα, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the α-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the α-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulatio

Estimating the uptake of traffic-derived NO2 from 15N abundance in Norway spruce needles

The 15N ratio of nitrogen oxides (NOx) emitted from vehicles, measured in the air adjacent to a highway in the Swiss Middle Land, was very high [δ15N(NO2) = +5.7‰]. This high 15N abundance was used to estimate long-term NO2 dry deposition into a forest ecosystem by measuring δ15N in the needles and the soil of potted and autochthonous spruce trees [Picea abies (L.) Karst] exposed to NO2 in a transect orthogonal to the highway. δ15N in the current-year needles of potted trees was 2.0‰ higher than that of the control after 4 months of exposure close to the highway, suggesting a 25% contribution to the N-nutrition of these needles. Needle fall into the pots was prevented by grids placed above the soil, while the continuous decomposition of needle litter below the autochthonous trees over previous years has increased δ15N values in the soil, resulting in parallel gradients of δ15N in soil and needles with distance from the highway. Estimates of NO2 uptake into needles obtained from the δ15N data were significantly correlated with the inputs calculated with a shoot gas exchange model based on a parameterisation widely used in deposition modelling. Therefore, we provide an indication of estimated N inputs to forest ecosystems via dry deposition of NO2 at the receptor level under field conditions

How Technical Innovations May Help to Prevent Drug Shortages in Switzerland

In this work, we investigated the technical feasibility of 'on-demand' production of selected drugs to cover their demand for a time window of 90 days. We focused on two sub-processes 'automated chemical synthesis' and 'formulation in micropellets'  to enable personalized dosing. The production of drugs 'on-demand' is challenging, important, but also attractive. Switzerland could thus gain access to an additional instrument for increasing resilience for supply-critical drugs. The biggest challenge in the case study presented here is the scalability of automated chemical synthesis and the application range of micropellet formulations

Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5′‐phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d‐sorbitol or d‐mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O‐acetyl‐l‐serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N‐deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]‐sulphate feeding experiments showed that the addition of glucose to dark‐treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N‐deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co‐ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergisticall

Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles

The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH4+, while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradatio

AtPTR4 and AtPTR6 are differentially expressed, tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectivel

A tradition and an epidemic: determinants of the campylobacteriosis winter peak in Switzerland

Campylobacteriosis is the most frequently reported food borne infection in Switzerland. We investigated determinants of infections and illness experience in wintertime. A case-control study was conducted in Switzerland between December 2012 and February 2013. Cases were recruited among laboratory-confirmed campylobacteriosis patients. Population-based controls were matched according to age group, sex and canton of residence. We determined risk factors associated with campylobacteriosis, and help seeking behaviour and illness perception. The multivariable analysis identified two factors associated with an increased risk for campylobacteriosis: consumption of meat fondue (matched odds ratio [mOR] 4.0, 95% confidence interval [CI] 2.3-7.1) and travelling abroad (mOR 2.7, 95% CI 1.1-6.4). Univariable analysis among meat fondue consumers revealed chicken as the type of meat with the highest risk of disease (mOR 3.8, 95% CI 1.1-13.5). Most frequently reported signs and symptoms among patients were diarrhoea (98%), abdominal pain (81%), fever (66%), nausea (44%) and vomiting (34%). The median perceived disease severity was 8 on a 1-to-10 rating scale. Patients reported a median duration of illness of 7days and 14% were hospitalised. Meat fondues, mostly "Fondue chinoise”, traditionally consumed during the festive season in Switzerland, are the major driver of the epidemic campylobacteriosis peak in wintertime. At these meals, individual handling and consumption of chicken meat may play an important role in disease transmission. Laboratory-confirmed patients are severely ill and hospitalisation rate is considerable. Public health measures such as decontamination of chicken meat and improved food handling behaviour at the individual level are urgently needed

2022 ESC Guidelines on cardio-oncology developed in collaboration with the European Hematology Association (EHA), the European Society for Therapeutic Radiology and Oncology (ESTRO) and the International Cardio-Oncology Society (IC-OS)

info:eu-repo/semantics/publishe

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference