Effects of avoidance or use of neuromuscular blocking agents on outcomes in tracheal intubation: a Cochrane systematic review.

Cohort studies have indicated that avoidance of neuromuscular blocking agents (NMBA) is a risk factor for difficult tracheal intubation. However, the impact of avoiding NMBA on tracheal intubation, possible adverse effects, and postoperative discomfort has not been evaluated in a systematic review of randomised trials. We searched several databases for trials published until January 2017. We included randomised controlled trials comparing the effect of avoiding vs using NMBA. Two independent authors assessed risk of bias and extracted data. The risk of random errors was assessed by trial sequential analysis (TSA). We included 34 trials (3565 participants). In the four trials judged to have low risk of bias, there was an increased risk of difficult tracheal intubation with no use of NMBA [random-effects model, risk ratio (RR) 13.27, 95% confidence interval (CI) 8.19-21.49, P<0.00001, TSA-adjusted CI 1.85-95.04]. The result was confirmed when including all trials, (RR 5.00, 95% CI 3.49-7.15, P<0.00001, TSA-adjusted CI 1.20-20.77). There was a significant risk of upper airway discomfort or injury by avoiding NMBA (RR=1.37, 95% CI 1.09-1.74, P=0.008, TSA-adjusted CI 1.00-1.86). None of the trials reported mortality. Avoiding NMBA was significantly associated with difficult laryngoscopy, (RR 2.54, 95% CI 1.53-4.21, P=0.0003, TSA-adjusted CI 0.27-21.75). In a clinical context, one must balance arguments for using NMBA when performing tracheal intubation.

Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes.

BACKGROUND: Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone complexes and the levels of total free-plasma DNA were elevated in post-cardiac arrest patients compared with healthy individuals, and to examine if these biomarkers were capable of predicting mortality. METHODS: We included 42 comatose out-of-hospital cardiac arrest patients and collected blood samples after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus . The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. RESULTS: We found no difference in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P < 0.01) and the area under the ROC curve was 0.78 (95% confidence interval: 0.59;0.96). The level of total free-plasma DNA was increased in the 22-h sample compared with healthy individuals (P < 0.001) but no significant difference was found between non-survivors and survivors 30 days after the cardiac arrest (all P ≥ 0.06). CONCLUSION: An increased level of DNA-histone complexes was associated with increased mortality and that the level of total free-plasma DNA was elevated post-cardiac arrest.

Natural killer cells accumulate in lung-draining lymph nodes and regulate airway eosinophilia in a murine model of asthma.

Increasing evidence suggests a key role for the innate immune system in asthma development. Although the role of Natural Killer (NK) cells in allergic asthma is poorly known, modifications of the blood NK cell populations have been found in asthmatic and/or allergic patients. Their repartition and activation status in the inflammatory (lungs) and the regulatory (draining lymph nodes) sites of the allergic reaction is unknown. The aim of our study was to monitor NK cell migration pattern and activation status and to investigate the consequences of NK cell depletion during allergic airway reaction in a mouse model. Ovalbumin sensitization and challenges of BALB/cByJ mice had no effect on the total number of lung NK cells but significantly decreased the number of most mature NK cells and increased the level of the activation marker CD86. In the lung-draining mediastinal lymph nodes, ovalbumin sensitization and challenges led to increased number of NK cells, and more precisely, immature NK cells and increased expression of CD86. Ovalbumin-sensitized mice also exhibited increased percentage of proliferating NK cells in lung-draining mediastinal lymph nodes. Anti-ASGM1 antibody treatment depleted most NK cells and decreased bronchoalveolar lavage eosinophilia but did not modify airway responsiveness. Altogether, our study shows that pulmonary allergic sensitization induces modification in the NK cell compartment at the inflammatory and regulatory sites and suggests that NK cells may participate in the regulation of the asthmatic response and, more particularly, to the allergic airway eosinophilia.

On the origin of bacterial resistance to penicillin: comparison of a beta-lactamase and a penicillin target.

Structural data are now available for comparing a penicillin target enzyme, the D-alanyl-D-alanine-peptidase from Streptomyces R61, with a penicillin-hydrolyzing enzyme, the beta-lactamase from Bacillus licheniformis 749/C. Although the two enzymes have distinct catalytic properties and lack relatedness in their overall amino acid sequences except near the active-site serine, the significant similarity found by x-ray crystallography in the spatial arrangement of the elements of secondary structure provides strong support for earlier hypotheses that beta-lactamases arose from penicillin-sensitive D-alanyl-D-alanine-peptidases involved in bacterial wall peptidoglycan metabolism.

[Implementation of hygiene procedures, microbial and physical controls and environmental monitoring in radiopharmacy department]. / Mise en oeuvre de procédures d'hygiène et de contrôles physiques et microbiologiques d'environnement au sein d'une unité de radiopharmacie.

Preparation of radiopharmaceuticals for injection involves compliance with the regulations for pharmaceutical drugs and radionuclides. The microbiological quality must be ensured, radiation exposure limited, and radioactive contamination of personnel and the environment prevented. Based on work concerning compliance and in accordance with changes in recent regulations, the facilities of the radiopharmacy department of the Louis Mourier Hospital have been optimized. Physical and microbiological controls of equipment and facilities have been implemented to monitor workstations and their environment with respect to microbiological quality. Three hygiene guidelines have also been implemented: improving hygiene practices, personal clothing, practical training on hygiene and its evaluation.

The late, but not early, asthmatic response is dependent on IL-5 and correlates with eosinophil infiltration.

Early-phase reactions (EPRs) and late-phase reactions (LPRs) are characteristic features of bronchial asthma, although the pathogenetic mechanisms responsible for each of the responses are not fully defined. A murine model of EPRs and LPRs was developed to investigate the role of IL-5 and eosinophils in development of both responses. After initial intraperitoneal sensitization and airway challenge to ovalbumin (OVA), mice were provoked by additional exposure to OVA. An EPR, characterized by a transient increase in airway responsiveness, was observed 5-30 minutes after antigen provocation. This response was followed by an LPR that reached its maximum at 6 hours after challenge and was characterized by increased airway responsiveness and significant lung eosinophilia. The EPR was blocked by cromoglycate and albuterol, whereas the LPR was abolished by cromoglycate and hydrocortisone. Before provocation with allergen, administration of anti-IL-5 antibody prevented the influx of eosinophils into the lung tissue and abolished the LPR but not EPR. These results suggest that IL-5 and eosinophils are essential for development of the LPR, but not EPR, in this model.

Purification and properties of the exocellular beta-lactamase of Actinomadura strain R39.

The exocellular beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) of Actinomadura R39 consists of one single polypeptide chain of molecular weight about 15 200. It exhibits a highly asymmetrical shape, has a low isoelectric point (at pH 5.0) and contains about 9.3% (w/w) of a polydeoxyribonucleotide with which it forms a rather stable complex. Removal of a substantial amount of this deoxyribonucleotide by treatment with DNAase I has no effect on the enzyme activity. The beta-lactamase has a wide spectrum of activity. Penicillins and delta 3-cephalosporins can be either good or poor substrates. Oxacillin, which is a poor substrate of most beta-lactamases from Gram-positive bacteria, is a good substrate of the beta-lactamase of Actinomadura R39. Its best substrate, however, is nitrocefin (kcat/Km: 2300 000 M-1.s-1; catalytic centre activity: 210 s-1). The kcat/Km values observed with some penicillins and delta 3-cephalosporins are similar to the values of the bimolecular rate constants that govern the formation of the acyl-enzyme intermediates between these antibiotics and the serine D-alanyl-D-alanine peptidase that is also secreted by the same strain Actinomadura R39. Such a relationship, however, is not observed with all the beta-lactam compounds tested.

Studies on the primary structures of the exocellular D-alanyl-D-alanine peptidases of Streptomyces strain R61 and Actinomadura strain R39.

The Mr 37 000 D-alanyl-D-alanine peptidase excreted by Streptomyces R61 and the Mr 53 000 D-alanyl-D-alanine peptidase excreted by Actinomadura R39 are both characterized by a very uneven distribution of the basic (Arg + Lys) amino acid residues. Trypsin degradation of the heat-denatured enzymes generates (1) thirteen soluble peptides which contain from 2 to 28 residues in the case of the R61 enzyme and nineteen soluble peptides which contain 2 to 39 residues in the case of the R39 enzyme; and (2) three large segments or core peptides which, irrespective of the enzymes from which they originate, consist of 50-60, 70-80 and 110-120 residues. About 90% of the basic (Arg + Lys) amino acid residues are recovered in the soluble tryptic peptides. The core peptides represent 62% (Mr approximately 23 000) and 45% (Mr approximately 24 000) of the untreated R61 and R39 enzymes, respectively. One 28-residue soluble peptide isolated from the R61 enzyme represents the N-terminal portion of the protein whose sequence has been established. The penicillin attachment site of the R61 enzyme has been located in one of the core peptides. For the R39 enzyme, indirect evidence shows that the penicillin binding site is probably within one of the soluble peptides.

Molecular characterisation of a versatile peroxidase from a Bjerkandera strain.

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)--putatively involved in manganese binding and H83 and W172--potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both LiP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain.

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G.

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.

Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters.

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

Penicillin and beyond: evolution, protein fold, multimodular polypeptides, and multiprotein complexes.

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and beta-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes.

Cloning, nucleotide sequence and amplified expression of the gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G.

The gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G has been cloned in Escherichia coli DH5 alpha MCR via pBR322 or 325, and then transferred into Streptomyces lividans TK24 via pIJ486, with substantial amplification of the expressed DD-peptidase. The gene has the information for the synthesis of a 255 amino acid precursor, the amino terminal region of which has the characteristic features of a signal peptide. The primary structure as deduced from nucleotide sequencing confirms that previously determined by chemical methods except for the occurrence of an Asp instead of Asn at position 1 and an additional Ala immediately downstream of Pro67.

Nucleotide sequence of the gene encoding the active-site serine beta-lactamase from Actinomadura R39.

The gene encoding the extracellular, active-site serine beta-lactamase of Actinomadura R39, previously cloned into Streptomyces lividans, has the information for the synthesis of a 304 amino acid protein, the amino terminal region of which has the characteristic features of a signal peptide. The Actinomadura R39 beta-lactamase is another member of the class A beta-lactamases. In particular, it shows high homology with the beta-lactamase of Bacillus licheniformis.

Overproduction and properties of the mannuronate alginate lyase AlxMB.

In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 micrograms per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.

Cloning and nucleotide sequencing of the gene encoding the beta-lactamase from Citrobacter diversus.

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.

Sequence of a gene encoding a (poly ManA) alginate lyase active on Pseudomonas aeruginosa alginate.

The recombinant plasmid pAL-A3 bears a (poly ManA) alginate lyase-encoding gene that originates from the marine bacterium ATCC 433367 (Brown et al., Appl. Environ. Microbiol. (1991) 57, 1870-1872). The alginate lyase produced by Escherichia coli TC4 harbouring pAL-A3 was purified to protein homogeneity and the corresponding gene sequenced, giving access to the first known primary structure of an alginate lyase. The 265-amino acid residue alginate lyase showed lytic activity on a Pseudomonas aeruginosa alginate isolated from a cystic fibrosis patient. Unexpectedly, the alginate lyase thus characterized differed from that isolated from the culture medium of the bacterium ATCC 433367 (Romeo and Preston, Biochemistry (1986) 25, 8385-8391).

Cloning and nucleotide sequence of the gene encoding the gamma-D-glutamyl-L-diamino acid endopeptidase II of Bacillus sphaericus.

The gene encoding the Bacillus sphaericus gamma-D-glutamyl-L-diamino acid endopeptidase II, a cytoplasmic enzyme involved in cell sporulation [1], contains the information for a 271-amino acid protein devoid of a signal peptide. The endopeptidase lacks sequence relatedness with other proteins of known primary structure except that its C-terminal region has significant similarity with the C-terminal region of the 54-kDa P54 protein of Enterococcus faecium, of unknown function [2].

Modulation of the allergen-induced human IgE response in Hu-SCID mice: inhibitory effect of human recombinant IFN-gamma and allergen-derived lipopeptide.

We have previously established a model to study the in vivo human IgE response using humanized SCID mice. Allergic SCID mice were obtained following intraperitoneal injection with mononuclear cells from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, and sensitization by Dpt allergen intraperitoneal injection (immunization) or Dpt aerosol (inhalation). Human serum IgE was measured in allergic SCID mice after administration of human recombinant IFN-gamma or the lipopeptide LP 52-71 (derived from peptide p52-71 from Der p 1, Dpt major allergen, coupled to a lipophilic moiety), during the immunization or the inhalation phase. IFN-gamma inhibited human IgE production when given at the time of immunization, but not during inhalation. This effect was long-lasting as Dpt aerosol, given one month after immunization and IFN-gamma administration, failed to increase IgE levels. Unlike Dpt or p52-71, LP 52-71 failed to induce human IgE production at day 14 and 21 after its injection, but did inhibit the development of the IgE response after a secondary Dpt-challenge. Moreover, LP 52-71 administration 14 days after Dpt inhalation decreased IgE levels, in contrast to peptide 52-71, which increased IgE levels. Thus, taken together these results indicate that the development of the human IgE response in allergic SCID mice can be modulated by modified allergen and a Th1 cytokine.

The division and cell wall gene cluster of Enterococcus hirae S185.

A chromosomal 10355-bp segment of Enterococcus hirae S185 contains nine orfs which occur in the same order as the MraW-, FtsL-, PBP3-, MraY-, MurD-, MurG-, FtsQ-, FtsA- and FtsZ-encoding genes of the division and cell wall clusters of Escherichia coli and Bacillus subtilis. The E. hirae DNA segment lacks the genes which in E. coli encode the ligases Ddl, MurC, MurE and MurF and the integral membrane protein FtsW. The encoded E. hirae and E. coli proteins share 25% to 50% identity except FtsL and FtsQ (approximately = 14% identity).