New ECCO model documents for Material Deposit and Transfer Agreements in compliance with the Nagoya Protocol.

The European Culture Collections' Organisation presents two new model documents for Material Deposit Agreement (MDA) and Material Transfer Agreement (MTA) designed to enable microbial culture collection leaders to draft appropriate agreement documents for, respectively, deposit and supply of materials from a public collection. These tools provide guidance to collections seeking to draft an MDA and MTA, and are available in open access to be used, modified, and shared. The MDA model consists of a set of core fields typically included in a 'deposit form' to collect relevant information to facilitate assessment of the status of the material under access and benefit sharing (ABS) legislation. It also includes a set of exemplary clauses to be included in 'terms and conditions of use' for culture collection management and third parties. The MTA model addresses key issues including intellectual property rights, quality, safety, security and traceability. Reference is made to other important tools such as best practices and code of conduct related to ABS issues. Besides public collections, the MDA and MTA model documents can also be useful for individual researchers and microbial laboratories that collect or receive microbial cultures, keep a working collection, and wish to share their material with others.

Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.

Msb2 is a Ste11 membrane concentrator required for full activation of the HOG pathway.

The high osmolarity glycerol (HOG) pathway, composed of membrane-associated osmosensors, adaptor proteins and core signaling kinases, is essential for the survival of yeast cells under hyper-osmotic stress. Here, we studied how the MAPKKK Ste11 might change its protein interaction profile during acute stress exposure, with an emphasis on the sensory system of the so-called Sho1/Msb2 signaling branch. To characterize the transience of protein-protein interactions we utilized a recently described enzymatic in vivo protein proximity assay (M-track). Accordingly, interaction signals between Ste11 and many of its signaling partners can already be detected even under basal conditions. In most cases these signals increase after stress induction. All the interactions are completely dependent on the function of the Ste11-adaptor protein Ste50. Moreover, the presence of either Msb2 or Hkr1 is necessary for observing the interaction between Ste11 and scaffolding factors such as Sho1 and Pbs2. Additional assays suggest that Msb2 is not only in close proximity to Ste11 but might function as an individual Ste11 concentrator at the plasma membrane. Our results confirm the existence of negative feedback systems targeting the protein levels of Ste11 and Msb2 and also hint at changes in the dissociation rates of intermediate signaling complexes.

M-Track: detecting short-lived protein-protein interactions in vivo.

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

Transcriptomic and proteomic approach for understanding the molecular basis of adaptation of Saccharomyces cerevisiae to wine fermentation.

Throughout alcoholic fermentation, Saccharomyces cerevisiae cells have to cope with several stress conditions that could affect their growth and viability. In addition, the metabolic activity of yeast cells during this process leads to the production of secondary compounds that contribute to the organoleptic properties of the resulting wine. Commercial strains have been selected during the last decades for inoculation into the must to carry out the alcoholic fermentation on the basis of physiological traits, but little is known about the molecular basis of the fermentative behavior of these strains. In this work, we present the first transcriptomic and proteomic comparison between two commercial strains with different fermentative behaviors. Our results indicate that some physiological differences between the fermentative behaviors of these two strains could be related to differences in the mRNA and protein profiles. In this sense, at the level of gene expression, we have found differences related to carbohydrate metabolism, nitrogen catabolite repression, and response to stimuli, among other factors. In addition, we have detected a relative increase in the abundance of proteins involved in stress responses (the heat shock protein Hsp26p, for instance) and in fermentation (in particular, the major cytosolic aldehyde dehydrogenase Ald6p) in the strain with better behavior during vinification. Moreover, in the case of the other strain, higher levels of enzymes required for sulfur metabolism (Cys4p, Hom6p, and Met22p) are observed, which could be related to the production of particular organoleptic compounds or to detoxification processes.

Expression of stress response genes in wine strains with different fermentative behavior.

The response to adverse growth conditions in yeast depends on the activation of signal transduction pathways which result in transcriptional changes and synthesis of protective molecules. During wine production, yeast cells are affected by a plethora of stress situations. In this work we have analyzed the fermentative behavior in synthetic must for six different wine yeast strains. In addition, we followed the expression of several stress response genes during the first half of the vinification. Our results indicate that common patterns of stress response are found among all the strains, but also that a subset of genes are differentially expressed according to the fermentative behavior of the various strains. Particularly, in the strains with the most severe fermentative problems, higher (and in some cases maintained) mRNA levels of many genes were found. The relevance of an equilibrium between stress response and growth efficiency during wine fermentation is discussed.

Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection.

During wine production, yeast cells are affected by several conditions that are adverse to growth (oxidative, osmotic and ethanol stress among others) and they should detect and respond to these conditions, otherwise alcoholic fermentation can be negatively affected. In this work we have analyzed the fermentative behaviour of 14 commercial and non-commercial strains in several synthetic musts. According to the data obtained these strains have been classified into three groups depending on whether or not vinification was completed (and on the amount of residual sugar remaining in the must if it was not). Moreover, we have determined the resistance of these strains to several stress situations under laboratory growth conditions. We have been able to establish a correlation between the groups based on fermentative behaviour and resistance to several stress conditions (especially oxidative and ethanol stress), by applying discriminant analysis to the data obtained in these experiments. Our results indicate a clear relationship between stress resistance and fermentative behaviour and this opens up the possibility of using this information as a criterion for the future selection of wine yeasts.