Proteomic analysis of the organic matrix of the abalone <Emphasis Type="Italic">Haliotis asinina</Emphasis> calcified shell

Background  

The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.     

Cleavage Specificity of Enterococcus faecalis EnpA (EF1473), a Peptidoglycan Endopeptidase Related to the LytM/Lysostaphin Family of Metallopeptidases

Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl-glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpA_C, comprising residues 1374-1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpA_C cleaves the d-Ala-l-Ala bond formed by the d,d-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic d was identified as the minimum substrate of EnpA_C indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpA_C to assess enzyme specificity. EnpA_C did not cleave direct cross-links, but tolerated extensive variation in cross-bridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpA_C, which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family.     

Social organization in the Chinese water deer, <Emphasis Type="Italic">Hydropotes inermis</Emphasis>

The social organization of Chinese water deer was studied in a zoological park. Most adults lived together in the mixed zone, although other habitats were available. The overlap between individual areas was largest in females. In the mating season alone, less than half of the males established territories which overlapped in small spots where most encounters occurred. Females travelled freely throughout the entire available area, but non-territorial males stayed between the territories. Except in males during mating season, very few physical contacts other than sniffing took place between individuals regardless of age, sex or status. Each animal lived alone and did not show attraction or aggressiveness towards congeners. Grouping was temporary with no durable link between individuals, not even between mothers and daughters older than 5 months. The solitary life of individuals on a common ground and the establishment of seasonal territories make the water deer unique among ruminants. The species appears to be no more social than the water chevrotain, an “ancient” species. This fits well with other characteristics of the species.     

Simultaneous high-performance liquid chromatographic assay of droperidol and flunitrazepam in human plasma: Application to haemodilution blood samples collected during clinical anaesthesia

A simultaneous assay for droperidol and flunitrazepam by high-performance liquid chromatography has been developed and applied to blood samples collected during an acute normovolemic haemodilution under general anaesthesia. Haemodilution blood samples were stored at +4°C to be transfused, if required, to a patient during the post-surgical phase. A C₁₈ Supelclean cartridge was used for solid-phase extraction, and the recoveries were 74% and 89%, respectively, for droperidol and flunitrazepam. Compounds were chromatographed on a C₁₈ Novapak column at 250 nm, with a mobile phase of acetonitrile—10 mM ammonium acetate buffer (pH 6.7) (45:55, v/v). Nitrazepam was used as the internal standard. For both drugs, the assay was linear up to 500 μg/l, and the detection limits were 20 and 10 μg/l for droperidol and flunitrazepam, respectively, and their observed levels in haemodilution samples were 93 ± 82 μg/l and 76 ± 107 μg/l, respectively. Some of the values for flunitrazepam were higher than the minimal efficient concentration, defined as the plasma level observed at the time of the patient wakening from anaesthesia (12 ± 4 μg/l). According to our results, haemodilution sampling can be performed before induction of anaesthesia. When the blood is collected after the anaesthetic induction, it seems necessary to determine levels of the two drugs in haemodilution samples to avoid side-effects.     

Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.     

Which and How Many Patients Should Be Included in Randomised Controlled Trials to Demonstrate the Efficacy of Biologics in Primary Sj?gren’s Syndrome?

Objective

The goal of this study was to determine how the choice of the primary endpoint influenced sample size estimates in randomised controlled trials (RCTs) of treatments for primary Sjögren’s syndrome (pSS).

Methods

We reviewed all studies evaluating biotechnological therapies in pSS to identify their inclusion criteria and primary endpoints. Then, in a large cohort (ASSESS), we determined the proportion of patients who would be included in RCTs using various inclusion criteria sets. Finally, we used the population of a large randomised therapeutic trial in pSS (TEARS) to assess the impact of various primary objectives and endpoints on estimated sample sizes. These analyses were performed only for the endpoints indicating greater efficacy of rituximab compared to the placebo.

Results

We identified 18 studies. The most common inclusion criteria were short disease duration; systemic involvement; high mean visual analogue scale (VAS) scores for dryness, pain, and fatigue; and biological evidence of activity. In the ASSESS cohort, 35 percent of patients had recent-onset disease (lower than 4 years), 68 percent systemic manifestations, 68 percent high scores on two of three VASs, and 52 percent biological evidence of activity. The primary endpoints associated with the smallest sample sizes (nlower than 200) were a VAS dryness score improvement higher to 20 mm by week 24 or variable improvements (10, 20, or 30 mm) in fatigue VAS by week 6 or 16. For patients with systemic manifestations, the ESSDAI change may be the most logical endpoint, as it reflects all domains of disease activity. However, the ESSDAI did not improve significantly with rituximab therapy in the TEARS study. Ultrasound score improvement produced the smallest sample size estimate in the TEARS study.

Conclusion

This study provides valuable information for designing future RCTs on the basis of previously published studies. Previous RCTs used inclusion criteria that selected a small part of the entire pSS population. The endpoint was usually based on VASs assessing patient complaints. In contrast to VAS dryness cut-offs, VAS fatigue cut-offs did not affect estimated sample sizes. SGUS improvement produced the smallest estimated sample size. Further studies are required to validate standardised SGUS modalities and assessment criteria. Thus, researchers should strive to develop a composite primary endpoint and to determine its best cut-off and assessment time point.     

Sesquiterpenoids from Teucrium ramosissimum

An antiplasmodial bioguided investigation of the EtOAc extract of the aerial parts of Teucrium ramosissimum led to isolation and identification of three sesquiterpenoids, teucmosin, 4α-hydroxy-homalomenol C, 1β,4β,7α-trihydroxy-8,9-eudesmene and two trinorsesquiterpenoids, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one together with five known sesquiterpenoids, oplopanone, homalomenol C, oxo-T-cadinol, 1β,4β,6β-trihydroxyeudesmane, 1β,4β,7α-trihydroxyeudesmane and four flavonoids, 5-hydroxy-7,4′-dimethoxyflavone, salvigenin, genkwanin and cirsimaritin. The structures and the relative stereochemistry were elucidated by extensive spectroscopic studies including 1D and 2D NMR and mass spectrometry (MS). Homalomenol C, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione, oxo-T-cadinol and 1β,4β,6β-trihydroxyeudesmane displayed a significant in vitro antiplasmodial activity against Plasmodium falciparum with IC₅₀ values ranging from 1.2 to 5.0 μg/ml. Furthermore, no cytotoxicity was observed upon the human diploid lung cell line MRC-5 for these compounds.     

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74-84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a beta-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74-84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl(3). By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.