Surface confined self-assembly of polyampholytes generated from charge-shifting polymers.

Polyampholyte-based films can be efficiently self-assembled onto a surface in a one-pot manner. By using a gradient of protons, morphogens, generated at an electrode surface, a charge-shifting polyelectrolyte present in solution can be transformed into a polyampholyte, leading to the continuous buildup of a film based on polyelectrolyte complexation.

Enzymatic Catalysis Combining the Breath Figures and Layer-by-Layer Techniques: Toward the Design of Microreactors.

Herein, we report the fabrication of microstructured porous surfaces with controlled enzymatic activity by combining the breath figures and the layer-by-layer techniques. Two different types of porous surfaces were designed based on fluorinated and carboxylated copolymers in combination with PS, using poly(2,3,4,5,6-pentafluorostyrene)-b-polystyrene (PS5F31-b-PS21) and polystyrene-b-poly(acrylic acid) (PS19-b-PAA10) block copolymers, respectively. For comparative purposes, flat surfaces having similar chemistry were obtained by spin-coating. Poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers incorporating alkaline phosphatase (ALP) were built on these porous surfaces to localize the enzyme both inside and outside of the pores using PS/PS5F31-b-PS21 surfaces and only inside the pores on PS/PS19-b-PAA10 surfaces. A higher catalytic activity of ALP (about three times) was obtained with porous surfaces compared to the flat ones. The catalysis happens specifically inside the holes of PS/PS19-b-PAA10surfaces, where ALP is located. This opens the route for applications in microreactors.

[Fast-tracking and regional anaesthesia: preliminary feasibility study]. / Fast-tracking en anesthésie locorégionale périphérique: étude préliminaire de faisabilité.

OBJECTIVE: The monitoring in the post-anaesthesia care unit (PACU) improves the safety, the comfort and the analgesia of patients. At present, studies suggest the possibility to bypass the PACU according to the principle of fast-tracking (FT). The aim of this study was to evaluate the feasibility and the safety of a simulated protocol of FT after a regional anaesthesia. PATIENTS AND METHODS: Seven hundred patients were prospectively included in this study over a period of 6 months. METHODS: The Withes' scoring system was used for determining when patients could be safely discharged from PACU. We added a variable concerning the monitoring of surgical site. A minimum score of 14 was required on arrival to the PACU to consider a FT. The success rate of blocks, the use of sedation or general anaesthesia were noted. Adverse events were recorded. RESULTS: The success rate of blocks was 93 %. The score was higher than 14 in 98 % of case on arrival to the PACU. Thirteen adverse events were reported before surgery and/or operating room. No adverse events were reported during the stay in the PACU. CONCLUSION: Regional anaesthesia seems to be an appropriate principle to fast-track the PACU. It could be a way to reduce health care costs, and can offer solution for the PACU congestion problem. In France, the fast-tracking is a marginal concept without any support regulatory. An evolution to such a practice could be considered.

The learning process of the hydrolocalization technique performed during ultrasound-guided regional anesthesia.

BACKGROUND: Because poor echogenicity of the needle remains a safety issue, we decided to analyze the learning process of the hydrolocalization technique (Hloc) performed to continuously identify needle-tip anatomical position during many ultrasound-guided regional anesthesia procedures. METHODS: Ten senior anesthesiologists naïve to the Hloc agreed to participate in the study. They were requested to perform 40 out-of-plane (OOP) approach ultrasound-guided axillary blocks (AB) each using the Hloc. The Hloc, which is a needle-tip localization principle, was performed by means of repetitive injections of a small amount of a local anesthetic solution (0.5-1 ml) under an ultrasound beam. Details of the learning process and skill acquisition of the Hloc were derived from the following parameters: the duration of block placement, a measure of the perceived difficulty of needle-tip visualization, a measure of block placement difficulty, and the amount of local anesthetics solution required for the technique. RESULTS: Four hundred ABs were performed. The success rate of an ultrasound-guided AB was 98%. The Hloc was successful in all patients. Skill acquisition over time of the Hloc was associated with a significant reduction of both the duration and the perceived difficulty of ABs placement. Apprenticeship data revealed that 20 blocks were required to successfully place AB within 5 min in most cases using the Hloc. CONCLUSION: The Hloc performed during the OOP approach of ultrasound-guided regional anesthesia is a simple technique with a relatively short learning process feasible for efficient placement of ABs.

[Ultrasound guided regional anaesthesia: an effective method for cleaning the probes]. / Anesthésie locorégionale périphérique sous échographie: efficacité de stratégie de décontamination de la sonde.

OBJECTIVES: The objective of this study was to determine if the ultrasound probe can act as a vector for cross-infection and to compare two cleaning methods for ultrasound probes in order to limit or control the transmission risk. STUDY DESIGN: Prospective study. PATIENTS AND METHODS: The first part of the study (P1) was conducted to evaluate the possibility of the ultrasound probe to serve as a source of cross-contamination. Thirty blocks were placed under ultrasound guidance in elective outpatients. After each procedure (Proc), the ultrasound probe was decontaminated/cleaned using either an antiseptic solution spray (AS: n=15) or just wiped with two dry paper sheets (DP: n=15), in a randomly assigned order. Bacteriological samples were collected before and after each decontamination/cleaning methods and inoculated on a chocolate agar plates. The second part of the study (P2) was conducted to compare the effectiveness of two cleaning methods for ultrasound probes. The ultrasound probes were exposed to a large inoculum of three bacteria (Inoc). They were then cleaned/decontaminated using either DP (n=10) or AS (n=10), in a randomly assigned order. Bacteriological samples were collected before and after each cleaning/decontamination methods and inoculated on a chocolate agar plates. RESULTS: During P1, after Pro, all probes were found to be sterile before and after both AS and DP. During P2, after Inoc, all probes were found infected (CFU>150) but were considered sterile (CFU<10) after both DP and AS. CONCLUSION: The results of this study suggest that the risk of cross-infection during ultrasound guidance in locoregional anaesthesia is really low. Our data suggest that wiping ultrasound probe with two dry paper sheets is an adequate cleaning method to prevent cross-contamination risk.

[Evaluation of synthetic glue for the fixation of continuous peripheral nerve catheters]. / Evaluation d'une colle liquide pour la fixation des cathéters périnerveux périphériques.

Accidental dislodgement of continuous peripheral nerve catheters remains a frequent problem that causes failure of postoperative analgesia. We have assessed the use of new synthetic glue (Mastisol) to secure and maintain catheters in the correct position among 60 patients. This method allowed securing an effective fixation in 94% of cases,resulting in efficient ambulatory orthopedic surgery postoperative analgesia. No nervous or infectious complications were observed. This technique offers a simple, complementary method to secure peripheral nerve catheters.

[Ultrasound-guided infraclavicular block: a preliminary study of feasibility]. / Bloc infraclaviculaire sous contrôle échographique: une étude préliminaire de faisabilité.

OBJECTIVES: To assess the feasibility of neurostimulation and ultrasound guidance combination for infraclavicular brachial plexus block (ICB) technique. STUDY DESIGN: Prospective study. PATIENTS AND METHODS: Fifty consecutive patients scheduled for hand, forearm or elbow surgery were included. METHODS: A single stimulation lateral approach technique of ICB was performed. During the procedure, neurostimulation and ultrasound guidance were combined. The feasibility of ICB was assessed using a visual analogue scale (VAS, 0: impossible, 100: very simple) for ultrasound anatomical structures identification (VAS(Anat)) and for block placement (VAS (Block)). The success rate of ICB block was noted. RESULTS: No patient required general anaesthesia conversion. Median VAS+/-SD of VAS(Anat) and VAS(Block) were of 84+/-15 and 96+/-7, respectively. Success rate of ICB was 96%. No specific complication of ICB technique was noted. CONCLUSION: Combination of neurostimulation and ultrasound guidance is feasible. Combination of neurostimulation and ultrasound guidance secured ICB. Ultrasound-evidenced spread of local anaesthetics increased the success rate of ICB.

[Efficiency of secondary posterior trunk single stimulation, low volume infraclavicular plexus block for upper limb surgery]. / Bloc plexique infraclaviculaire en monostimulation: l'injection au contact du faisceau postérieur permet de réduire le volume d'anesthésique local.

OBJECTIVES: To assess the efficiency of a posterior secondary trunk single stimulation, low volume (30 ml 1.5% mepivacaine) infraclavicular brachial plexus block (ICB) technique. STUDY DESIGN: Prospective study. PATIENTS AND METHODS: One hundred consecutive patients scheduled for hand, forearm or elbow surgery were included. ICB was placed using a single stimulation technique. 30 ml 1.5% mepivacaine was injected when an evoked distal radial motor type response was elicited for 0.3-0.6 mA intensity current. Based upon both sensory and motor distribution ICB, characteristics and performance were assessed. RESULTS: No patient required general anesthesia conversion. Success rate was 92%. 8 patients required a total amount of 10 complementary distal troncular blocks. No specific complication of ICB technique was accoutered. All patients completed full neurological recovery from ICB 24 hours after surgery. CONCLUSION: 30 ml mepivacaine 1.5% ICB is suitable for upper limb surgery.

A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

Massive gene decay in the leprosy bacillus.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.

The decaying genome of Mycobacterium leprae.

Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.

The integrated genome map of Mycobacterium leprae.

The integrated map of the Mycobacterium leprae genome unveiled for the first time the genomic organization of this obligate intracellular parasite. Selected cosmid clones, isolated from a genomic library created in the cosmid vector Lorist6, were identified as representing nearly the complete genome and were subsequently used in the M. leprae genome sequencing project. Now a new version of the integrated map of M. leprae can be presented, combining the mapping results from the Lorist6 cosmids with data obtained from a second genomic library constructed in an Escherichia coli-mycobacterium shuttle cosmid, pYUB18. More than 98% of the M. leprae genome is now covered by overlapping large insert genomic clones representing a renewable source of well defined DNA segments and a powerful tool for functional genomics.

Comparative genomics of the mycobacteria.

The genus mycobacteria includes two important human pathogens Mycobacterium tuberculosis and Mycobacterium lepra. The former is reputed to have the highest annual global mortality of all pathogens. Their slow growth, virulence for humans and particular physiology makes these organisms extremely difficult to work with. However the rapid development of mycobacterial genomics following the completion of the Mycobacterium tuberculosis genome sequence provides the basis for a powerful new approach for the understanding of these organisms. Five further genome sequencing projects of closely related mycobacterial species with differing host range, virulence for humans and physiology are underway. A comparative genomic analysis of these species has the potential to define the genetic basis of these phenotypes which will be invaluable for the development of urgently needed new vaccines and drugs. This minireview summarises the different techniques that have been employed to compare these genomes and gives an overview of the wealth of data that has already been generated by mycobacterial comparative genomics.

Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur.

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.

Comparative genomics of the leprosy and tubercle bacilli.

To achieve the quantum leap in understanding required to overcome two major human diseases, leprosy and tuberculosis, systematic and comparative genome analysis has been undertaken. New insight into the biology of their causative agents has been obtained and the principle findings are reported here.

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays.

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.

Analysis of the proteome of Mycobacterium tuberculosis in silico.

Novel bioinformatics routines have been used to provide a more detailed definition of the proteome of Mycobacterium tuberculosis H37Rv. Over half of the current proteins result from gene duplication or domain shuffling events while one-sixth show no similarity to polypeptides described in other organisms. Prominent among the genes that appear to have been duplicated on numerous occasions are those involved in fatty acid metabolism, regulation of gene expression, and the unusually glycine-rich PE and PPE proteins. Protein similarity analysis, coupled with inspection of the genetic neighbourhood, was used to explore possible functional relatedness. This uncovered four large mce operons whose proteins may mediate initial interactions between the tubercle bacillus and host cells, together with a cluster of genes that might encode components of a structure required for secretion of ESAT-6 like proteins. Close linkage of the mmpL genes, encoding large membrane proteins, with those required for fatty acid metabolism suggests involvement in lipid transport. Compared to free-living bacteria, M. tuberculosis has a significantly smaller transport protein repertoire and this may reflect its intracellular lifestyle.