Oral and Anal Vaccination Confers Full Protection against Enteric Redmouth Disease (ERM) in Rainbow Trout

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3×10⁸ CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.     

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Two affinity states of M1 muscarine receptors

1. The binding of oxotremorine-M to M1 muscarine receptors was examined by measuring competition between the agonist and 3H-pirenzepine, using rabbit hippocampal membranes suspended in 20 mM Tris buffer containing 1 mM Mn2+. 2. Both ligands interacted with a single class of receptors. The receptors could assume two affinity states for oxotremorine-M, with equal numbers of high-affinity (KH) and low-affinity (KL) sites. 3. KH interconverted reversibly to KL in the absence of divalent cations and interconverted reversibly to a state similar to KL in the presence of guanyl 5'-yl imidodiphosphate. 4. The results are compatible with a model in which a pair of receptor molecules can be stabilized by a guanine nucleotide-binding "G protein" and have one site each of KH and KL affinity.     

Correlation Between Degradation of Bacteriophage T2 Deoxyribonucleic Acid and the Resistance of Escherichia coli to Infection

The ability of certain strains of Escherichia coli to degrade T2 deoxyribonucleic acid to acid-soluble fragments is correlated with their high capacity to survive T2 infection.     

Vegetation demographics in Earth System Models: A review of progress and priorities

Numerous current efforts seek to improve the representation of ecosystem ecology and vegetation demographic processes within Earth System Models (ESMs). These developments are widely viewed as an important step in developing greater realism in predictions of future ecosystem states and fluxes. Increased realism, however, leads to increased model complexity, with new features raising a suite of ecological questions that require empirical constraints. Here, we review the developments that permit the representation of plant demographics in ESMs, and identify issues raised by these developments that highlight important gaps in ecological understanding. These issues inevitably translate into uncertainty in model projections but also allow models to be applied to new processes and questions concerning the dynamics of real‐world ecosystems. We argue that stronger and more innovative connections to data, across the range of scales considered, are required to address these gaps in understanding. The development of first‐generation land surface models as a unifying framework for ecophysiological understanding stimulated much research into plant physiological traits and gas exchange. Constraining predictions at ecologically relevant spatial and temporal scales will require a similar investment of effort and intensified inter‐disciplinary communication.     

Role of Phosphorylation in Moesin Interactions with PIP2-Containing Biomimetic Membranes

Moesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological processes, including viral budding and infection. Its interaction with the plasma membrane occurs via a key phosphoinositide, the phosphatidyl(4,5)inositol-bisphosphate (PIP₂), and phosphorylation of residue T558, which has been shown to contribute, in cellulo, to a conformationally open protein. We study the impact of a double phosphomimetic mutation of moesin (T235D, T558D), which mimics the phosphorylation state of the protein, on protein/PIP₂/microtubule interactions. Analytical ultracentrifugation in the micromolar range showed moesin in the monomer and dimer forms, with wild-type (WT) moesin containing a slightly larger fraction (～30%) of dimers than DD moesin (10–20%). Only DD moesin was responsive to PIP₂ in its micellar form. Quantitative cosedimentation assays using large unilamellar vesicles and quartz crystal microbalance on supported lipid bilayers containing PIP₂ reveal a specific cooperative interaction for DD moesin with an ability to bind two PIP₂ molecules simultaneously, whereas WT moesin was able to bind only one. In addition, DD moesin could subsequently interact with microtubules, whereas WT moesin was unable to do so. Altogether, our results point to an important role of these two phosphorylation sites in the opening of moesin: since DD moesin is intrinsically in a more open conformation than WT moesin, this intermolecular interaction is reinforced by its binding to PIP₂. We also highlight important differences between moesin and ezrin, which appear to be finely regulated and to exhibit distinct molecular behaviors.