Structural diversity of human class II histocompatibility molecules induced by peptide ligands

SDS-PAGE analyses of stable HLA-DR1 complexes indicate that the binding of T cell epitopes can lead to multiple conformational variants. Whereas short T epitopes (<14-mer) induce complexes with apparent MW ranging from 47 to 57 kDa, longer peptides form generally high mobility complexes (44-45 kDa). The generation of HLA-DR1 conformational variants appears dependent on core peptide residues fitting inside the groove but can additionally be attributed to the presence of N- and C-terminal flanking residues (PFRs) acting as a complementary mechanism. These PFRs can jointly affect major histocompatibility complex class II conformation and stability, supporting the existence of alternative contacts at a distance from the classical binding site.     

Oestrone Sulphate Measurements for the Prediction of Small or Large Litters in Pigs

Serum from 88 pregnant sows and gilts was sampled 24 and 28 days after their first insemination or mating day. The oestrone sulphate (E1S) concentration in the samples was assessed with a commercially available radioimmunoassay kit modified for use with swine serum. The first aim was to test whether it was possible to predict litters of total number <10 piglets at term. The second aim was to compare the use of day 24 or day 28 samples, or of both, in this prediction.     

Evolutionary insight from whole-genome sequencing of experimentally evolved microbes

Experimental evolution (EE) combined with whole‐genome sequencing (WGS) has become a compelling approach to study the fundamental mechanisms and processes that drive evolution. Most EE‐WGS studies published to date have used microbes, owing to their ease of propagation and manipulation in the laboratory and relatively small genome sizes. These experiments are particularly suited to answer long‐standing questions such as: How many mutations underlie adaptive evolution, and how are they distributed across the genome and through time? Are there general rules or principles governing which genes contribute to adaptation, and are certain kinds of genes more likely to be targets than others? How common is epistasis among adaptive mutations, and what does this reveal about the variety of genetic routes to adaptation? How common is parallel evolution, where the same mutations evolve repeatedly and independently in response to similar selective pressures? Here, we summarize the significant findings of this body of work, identify important emerging trends and propose promising directions for future research. We also outline an example of a computational pipeline for use in EE‐WGS studies, based on freely available bioinformatics tools.     

Inference of genetic relatedness between viral quasispecies from sequencing data

Background

RNA viruses such as HCV and HIV mutate at extremely high rates, and as a result, they exist in infected hosts as populations of genetically related variants. Recent advances in sequencing technologies make possible to identify such populations at great depth. In particular, these technologies provide new opportunities for inference of relatedness between viral samples, identification of transmission clusters and sources of infection, which are crucial tasks for viral outbreaks investigations.

Results

We present (i) an evolutionary simulation algorithm Viral Outbreak InferenCE (VOICE) inferring genetic relatedness, (ii) an algorithm MinDistB detecting possible transmission using minimal distances between intra-host viral populations and sizes of their relative borders, and (iii) a non-parametric recursive clustering algorithm Relatedness Depth (ReD) analyzing clusters’ structure to infer possible transmissions and their directions. All proposed algorithms were validated using real sequencing data from HCV outbreaks.

Conclusions

All algorithms are applicable to the analysis of outbreaks of highly heterogeneous RNA viruses. Our experimental validation shows that they can successfully identify genetic relatedness between viral populations, as well as infer transmission clusters and outbreak sources.

     

Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation

The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and high-yielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.     

Reactivity of Lys(NH2)-containing peptides toward endopeptidases.

Lys(NH2)-containing peptides were subjected to various proteolytic enzymes which were selected for their well-documented specificity for arginyl and/or lysyl peptide bonds. Lys(NH2)-containing peptides were cleaved more rapidly by clostripain than the corresponding lysyl peptides. On the other hand, they proved to be resistant to Achromobacter protease I hydrolysis. The modified peptides synthesized in this study were more stable than the arginyl and lysyl analogues when incubated with trypsin or thrombin. The same tendency was observed when Lys(NH2)-containing peptides were incubated in diluted human serum, suggesting that the replacement of Arg or Lys by Lys(NH2) could be used to increase the stability of peptides in vivo.     

Shedding-Generated Met Receptor Fragments can be Routed to Either the Proteasomal or the Lysosomal Degradation Pathway

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.     

Novel technique for biliary reconstruction using an isolated gastric tube with a vascularized pedicle: a live animal experimental study and the first clinical case

Background

Biliary tract reconstruction continues to be a challenging surgical problem. Multiple experimental attempts have been reported to reconstruct biliary defects with different materials and variable outcome. Our aim was to evaluate a new method for biliary reconstruction using an isolated pedicled gastric tube in a live animal trial and also to present the first clinical case.

Methods

Seven mongrel dogs underwent biliary reconstruction using gastric tube harvested, completely separated from the greater curvature, and based on a vascularized pedicle with the right gastroepiploic vessels. The tube was interposed between the common bile duct (CBD) and the duodenum. Postoperative mortality, morbidity, liver functions, gross and microscopic histological picture were assessed. The first clinical case was also presented where, in a patient with post-cholecystectomy biliary injury, an isolated pedicled gastric tube was interposed between the proximal and distal ends of the CBD.

Results

One dog did not recover from anesthesia and another one died postoperatively from septic peritonitis. Five dogs survived the procedure and showed uneventful course and no cholestasis. The mean anastomotic circumference was 4.8 mm (range 4-6) for CBD anastomosis and 6.2 mm (range 5-7) for duodenal anastomosis. Histologically, anastomotic sites showed good evidence of healing. In the first clinical case, the patient showed clinical and biochemical improvement. Endoscopic retrograde cholangiography was feasible and assured patent biliary anastomoses.

Conclusion

In mongrel dogs, biliary reconstruction using pedicled gastric tube interposition between CBD and duodenum is feasible with satisfactory clinical results, anastomotic circumference and histological evidence of healing. The technique is also feasible in human and seems to be promising.     

Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Selective cleavage of an azaGly peptide bond by copper(II). Long‐range effect of histidine residue

Several reports have highlighted the interest of replacing Gly, a frequent amino acid within bioactive peptides, by azaGly (Agly) to improve their stability, activity or for the design of prodrugs. Because metal catalysis is increasingly used for tailoring peptide molecules, we have studied the stability of Agly peptides in the presence of metal ions. In this study, we show that Cu(II), unlike other metal ions such as Fe(II), Fe(III), Pd(II), or Pt(II), induces the cleavage of Agly peptides at room temperature and pH 7.3. The cleavage occurred in the absence of an anchoring His residue within the peptide but it was accelerated when this amino acid was present in the sequence. The influence of His residue on the cleavage rate was minimal when His and Agly were adjacent, whereas large effects were observed for distant His residues. The reaction between Cu(II) and Agly peptides induced the formation of Cu(I) species, which could be detected using bicinchoninic acid as a probe. The nature of products formed in this reaction allowed suggesting a mechanism for the Cu(II)‐induced cleavage of Agly peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.