Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895–1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

The surface temperatures of Earth: steps towards integrated understanding of variability and change

Surface temperature is a key aspect of weather and climate, but the term may refer to different quantities that play interconnected roles and are observed by different means. In a community-based activity in June 2012, the EarthTemp Network brought together 55 researchers from five continents to improve the interaction between scientific communities who focus on surface temperature in particular domains, to exploit the strengths of different observing systems and to better meet the needs of different communities. The workshop identified key needs for progress towards meeting scientific and societal requirements for surface temperature understanding and information, which are presented in this community paper. A "whole-Earth" perspective is required with more integrated, collaborative approaches to observing and understanding Earth's various surface temperatures. It is necessary to build understanding of the relationships between different surface temperatures, where presently inadequate, and undertake large-scale systematic intercomparisons. Datasets need to be easier to obtain and exploit for a wide constituency of users, with the differences and complementarities communicated in readily understood terms, and realistic and consistent uncertainty information provided. Steps were also recommended to curate and make available data that are presently inaccessible, develop new observing systems and build capacities to accelerate progress in the accuracy and usability of surface temperature datasets.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Targeted disruption of the intracellular domain of receptor FgfrL1 in mice.

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.

Contemporary spinal cord protection during thoracic and thoracoabdominal aortic surgery and endovascular aortic repair: a position paper of the vascular domain of the European Association for Cardio-Thoracic Surgery†.

Ischaemic spinal cord injury (SCI) remains the Achilles heel of open and endovascular descending thoracic and thoracoabdominal repair. Neurological outcomes have improved coincidentially with the introduction of neuroprotective measures. However, SCI (paraplegia and paraparesis) remains the most devastating complication. The aim of this position paper is to provide physicians with broad information regarding spinal cord blood supply, to share strategies for shortening intraprocedural spinal cord ischaemia and to increase spinal cord tolerance to transitory ischaemia through detection of ischaemia and augmentation of spinal cord blood perfusion. This study is meant to support physicians caring for patients in need of any kind of thoracic or thoracoabdominal aortic repair in decision-making algorithms in order to understand, prevent or reverse ischaemic SCI. Information has been extracted from focused publications available in the PubMed database, which are cohort studies, experimental research reports, case reports, reviews, short series and meta-analyses. Individual chapters of this position paper were assigned and after delivery harmonized by Christian D. Etz, Ernst Weigang and Martin Czerny. Consequently, further writing assignments were distributed within the group and delivered in August 2014. The final version was submitted to the EJCTS for review in September 2014.

Current status and recommendations for use of the frozen elephant trunk technique: a position paper by the Vascular Domain of EACTS.

The implementation of new surgical techniques offers chances but carries risks. Usually, several years pass before a critical appraisal and a balanced opinion of a new treatment method are available and rely on the evidence from the literature and expert's opinion. The frozen elephant trunk (FET) technique has been increasingly used to treat complex pathologies of the aortic arch and the descending aorta, but there still is an ongoing discussion within the surgical community about the optimal indications. This paper represents a common effort of the Vascular Domain of EACTS together with several surgeons with particular expertise in aortic surgery, and summarizes the current knowledge and the state of the art about the FET technique. The majority of the information about the FET technique has been extracted from 97 focused publications already available in the PubMed database (cohort studies, case reports, reviews, small series, meta-analyses and best evidence topics) published in English.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.