Laccase wiring on free-standing electrospun carbon nanofibres using a mediator plug.

Electrospun carbon nanofibres (CNFs) containing CNTs were produced by electrospinning and subsequent thermal treatment. This material was evaluated as a bioelectrode for biofuel cell applications after covalent grafting of laccase. Bis-pyrene-modified ABTS was used as a plug to wire laccase to the nanofibres leading to a maximum current density of 100 µA cm(-2).

Non-covalent double functionalization of carbon nanotubes with a NADH oxidation Ru(II)-based molecular catalyst and a NAD-dependent glucose dehydrogenase.

We report the double functionalization of multiwalled carbon nanotube electrodes by two functional pyrene molecules. In combination, an immobilized Ru(II)-based NADH oxidation catalyst and glucose dehydrogenase achieve highly efficient glucose oxidation with low overpotential of -0.10 V and high current densities of 6 mA cm(-2).

Interest of rehabilitation in healing and preventing recurrence of ankle sprains.

UNLABELLED: To assess the impact of rehabilitation on healing and recurrence rate of ankle sprain, 1year apart, 111 patients, who suffered an ankle sprain (67 men and 44 women; 17 mild sprains, 67 medium and 27 severe), were included by emergency physicians of four emergency rooms (ER) of Finistère. The physician was free to prescribe, or not, further investigations. He prescribed systematically to patients RICE (rest, ice, compression, elevation) protocol, put an ankle brace, and gave a prescription of standardized rehabilitation. The prescription was the same for the four ER. All patients were recalled to 1year. Of the 111 patients initially included, 21 patients were excluded for lack of response after three phone calls. In the end, 90 patients were assessable (56 men and 34 women), mean age 31.4±12.6years (range 15-55) at the time of initial trauma. Emergency physicians had diagnosed, initially, 16 mild sprains (17.8%), 56 medium sprains (62.2%) and 18 severe sprains (20%). Of the 90 patients, 73 patients have been rehabilitated (81.1%). Of the 44 accidents of everyday life, 31 were rehabilitated (70.5%). Of the 27 sports accidents, 25 were rehabilitated (92.6%). Of the 19 work-related injuries, 17 were rehabilitated (89.5%). There is no significant relationship between rehabilitation and no recurrence (P=0.45) nor between rehabilitation and full recovery of the ankle (P=0.59). CONCLUSION: We find no association between rehabilitation and prevention of recurrence, nor between rehabilitation and healing of patients. However, our study is limited by the small size of the non-rehabilitated group.

Single glucose biofuel cells implanted in rats power electronic devices.

We describe the first implanted glucose biofuel cell (GBFC) that is capable of generating sufficient power from a mammal's body fluids to act as the sole power source for electronic devices. This GBFC is based on carbon nanotube/enzyme electrodes, which utilize glucose oxidase for glucose oxidation and laccase for dioxygen reduction. The GBFC, implanted in the abdominal cavity of a rat, produces an average open-circuit voltage of 0.57 V. This implanted GBFC delivered a power output of 38.7 µW, which corresponded to a power density of 193.5 µW cm(-2) and a volumetric power of 161 µW mL(-1). We demonstrate that one single implanted enzymatic GBFC can power a light-emitting diode (LED), or a digital thermometer. In addition, no signs of rejection or inflammation were observed after 110 days implantation in the rat.

Adherence and effectiveness of rehabilitation in acute ankle sprain.

OBJECTIVE: To estimate adherence to and effectiveness of rehabilitation after acute ankle sprain. METHOD: Patients with acute ankle sprain attending four emergency departments were recruited between February and July 2009. After the initial examination (classification of the severity of the sprain), each patient received an Aircast(®) ankle brace and the same, standardized rehabilitation program. Between two and three months later; the patient was contacted by phone (always by the same investigator) in order to find out whether he/she had performed the prescribed rehabilitation, establish whether the physiotherapist had complied with the prescribed rehabilitation programme and assess subjective recovery. If a patient failed to respond to three phone calls, he/she was excluded from the study. RESULTS: Of the 245 patients initially included, 111 (67 men and 44 women; 17 mild sprains, 67 moderate sprains and 27 severe sprains) answered the "phone questionnaire". In terms of treatment adherence by the patient, 92 patients (82.9%) performed their rehabilitation (beginning an average of 13.8 days after the injury). In terms of prescription compliance by the physiotherapist, 88 patients (95.6%) received massage, 71 (77.2%) underwent physiotherapy, 83 (90.2%) performed weight training and 87 (94.5%) received proprioceptive training. Eighty-two patients said that they had received manipulative therapy that was not part of the prescribed programme. Impact on recovery: 61 patients (55%) considered that their injury had healed (10 mild, 42 medium and nine severe sprains), whereas 50 had not healed (seven mild, 25 medium and 18 severe sprains). There was no statistically significant association between recovery and compliance with rehabilitation. However, the application of massage (p=0.004) and proprioceptive training (p=0.017) were significantly associated with recovery, while physiotherapy, weight training and manipulative therapy were not. CONCLUSION: In acute ankle sprain, adherence with rehabilitation is good and the treating physiotherapists comply with the prescription. However, there was no statistically significant link between rehabilitation compliance and subjective recovery at 3 months. Revaluation of these patients at one year may be necessary for estimating the impact of rehabilitation on ankle function and the rate of injury recurrence.

[Assessment of the contribution of the immunochromatographic pneumococcal urinary antigen test to the etiological diagnosis of pneumonia in hospitalized adults]. / Bilan de la contribution de l'antigénurie pneumocoque par test immunochromatographique au diagnostic étiologique des pneumonies chez l'adulte hospitalisé.

AIM OF THE STUDY: To assess the usefulness and prescription practices of the Binax Now Streptococcus pneumoniae urinary antigen test in hospitalized adults. PATIENTS AND METHODS: The results of the pneumococcal urinary antigen tests (UAT) performed from January 2002 to September 2004 were related to that of microbiological cultures, and in positive patients to radiographic findings and C-reactive protein (CRP) levels. The evolution of the number of prescriptions and positivity rate in 2007 versus 2002-2004 was analyzed. RESULTS: The pneumococcal UAT was positive in 32 of the 278 patients included from 2002 to 2004 (11.5%). Results were concordant with that of microbiological cultures in 90% of the 247 documented cases. Pneumococcal etiology was considered to be definite in 19 patients (isolation of S. pneumoniae from blood, 17 patients; or pleural fluid, two patients), of whom 15 had a positive UAT (sensitivity: 79%); to be probable in 22 patients (positive UAT, 17 patients and/or isolation of S. pneumoniae from respiratory samples, six patients), and was retained in 39 of the 41 patients (positive predictive value: 93.7%). CRP was greater than 100mg/L in 34 of 39 documented patients and lobar alveolar radiographic opacities observed in 25 of 28 documented patients. In 2007, the dramatic increase in the number of UAT prescriptions and the diversification of prescribing units were associated to a decreased positivity rate (8.1%). CONCLUSION: Whereas the pneumococcal UAT clearly increases etiological diagnosis, pneumococcal pneumonia cannot be ruled out if negative. Indications for its use need to be refined to improve the cost-effectiveness of this test.

Hydrolysis of pectins with different degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme.

The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme.

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme.

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Addition of a glycophosphatidylinositol to acetylcholinesterase. Processing, degradation, and secretion.

We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet omega/omega + 1/omega + 2, particularly omega - 1. 5) Glypiation was not affected by the closeness of the omega site to the alpha(10) helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an omega site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of omega. 7) Glypiation was not affected by the presence of an N-glycosylation site at omega or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region.

The glial and the neuronal glycine transporters differ in their reactivity to sulfhydryl reagents.

The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na(+)/Cl(-)-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na(+) coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na(+)-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.

Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors.

NMDA receptors are allosterically inhibited by Zn2+ ions in a voltage-independent manner. The apparent affinity for Zn2+ of the heteromeric NMDA receptors is determined by the subtype of NR2 subunit expressed, with NR2A-containing receptors being the most sensitive (IC50, approximately 20 nM) and NR2C-containing receptors being the least sensitive (IC50, approximately 30 microM). Using chimeras constructed from these two NR2 subtypes, we show that the N-terminal LIVBP-like domain of the NR2A subunit controls the high-affinity Zn2+ inhibition. Mutations at four residues in this domain markedly reduce Zn2+ affinity (by up to >500-fold) without affecting either receptor activation by glutamate and glycine or inhibition by extracellular protons and Ni2+ ions, indicating that these residues most likely participate in high-affinity Zn2+ binding.

Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit.

Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure. Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP. In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit. Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft. Upon Zn binding the two lobes close tightly around the divalent cation. Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand. Such a functional organization may apply to other eukaryotic iGluRs.

Evaluation of the cytocompatibility of three endodontic materials.

The goal of this in vitro study was to evaluate the relative cytocompatibility of three endodontic materials: calcium hydroxide, a calcium oxide-based compound, and a zinc oxide-eugenol-based sealer. The evaluation was conducted 24, 72, and 168 h after contact with the compounds and involved three complementary techniques: a colorimetric cytotoxicity test, scanning electron microscopy, and flow cytometry. The results we obtained confirmed the initial cytotoxicity of the zinc oxide-eugenol-based sealer and showed that the calcium oxide-based compound had the same relative cytocompatibility as calcium hydroxide.

The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative "back door".

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

An evaluation of the CO2 laser for endodontic disinfection.

The goal of this study was to evaluate the bactericidal action of the CO2 laser on animal teeth infected with an endodontic bacterial species. After instrumentation, 24 freshly extracted incisors were inoculated with a known concentration of Actinomyces odontolyticus and incubated anaerobically for 18 h. The incisors were separated into three groups: group 1--untreated control teeth; group 2--teeth treated with 3% NaOCl; and group 3--teeth lased with a CO2 laser at 5 W using three successive 9.9 s irradiation periods with 10 s between treatments. For each of the three groups, 60 microliters samples were removed using gel loading capillary pipette tips, and the diluted samples were plated in triplicate on Columbia agar plates. After a 5- to 6-day incubation, the colony-forming units were counted, and the quantitative results were subjected to an analysis of variance. The results of this analysis indicated an average 85% decrease in the colony-forming units in the laser-treated group, compared with the control group. According to Fisher and Scheffé tests, the differences in the averages between the control and laser groups were statistically significant (p < 0.05). The NaOCl treatment was statistically superior to the CO2 laser treatment.

Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp.

This study aimed to evaluate the microbiota of necrotic pulp in teeth without carious lesions where the crown and root were intact and to test the sensitivity of this microbiota to antibiotics in order to improve treatment. The necrotic pulp was sampled from 26 single-rooted teeth in intact pulp chambers. A total of 84 strains were isolated. The number of species isolated per tooth varied from 2 to 8, with a strong component (81%) of anaerobic bacteria. The most commonly represented species were Bacteroides gracilis, Propionibacterium acnes, Fusobacterium nucleatum, Prevotella buccae and Eubacterium lentum. The sensitivity of these organisms to amoxicillin, amoxicillin combined with clavulanate and tetracycline was evaluated by Etest on 38 isolates. For all strains tested, the minimum inhibitory concentration values obtained were low and substantially below effective serum concentrations for these antibiotics. These data enable us to devise suitable treatments for acute development of apical lesions and to prevent dissemination of this source of infection to the rest of the host.

Craniofacial anomalies and malformations in respiratory chain deficiency.

We report on facial anomalies including round face, high forehead, flat philtrum, apparently low-set ears, and short neck in 4 unrelated patients with mitochondrial respiratory enzyme deficiency. Pre- and postnatal growth retardation with microcephaly, brachydactyly, and hypoplasia of distal and middle phalanges was present in all 4 cases. The diagnosis of respiratory chain deficiency was confirmed by enzymatic and molecular studies. The combination of facial anomalies, prenatal growth failure, and malformations is suggestive of antenatal expression of the disease, and raises the question of the part that respiratory chain deficiencies play in human malformations.

Evaluation of carbohydrate-deficient transferrin compared with Tf index and other markers of alcohol abuse.

It has been known for some years that a partial deglycosylation of transferrin occurs in the sera of alcohol abusers. Different methods have been proposed in order to evaluate this carbohydrate-deficient fraction of serum transferrin. Chromatofocusing or isoelectric focusing followed by direct immunofixation have been used until now. Recently, a new method called the carbohydrate-deficient transferrin (CDT) test based on ion-exchange chromatography has been developed by Stibler et al. (Alcohol Clin Exp Res 10:535-544, 1986). Here we compare this new method with results obtained using our Tf index determination method. The upper limit of normal values was set to the 90th percentile of the values observed in a reference population. The population under investigation consisted of 50 healthy volunteers and 160 alcohol abusers whose ethanol consumption was evaluated through a questionnaire. Sensitivity and specificity of the CDT test have been found higher than 0.76 and 0.90, respectively. The correlation between both methods was 0.794, a satisfactory result considering that the CDT test and the Tf index do not exactly measure the same part of the carbohydrate-deficient transferrin. In a population of 23 patients with liver diseases not related to alcohol abuse, no abnormal CDT value was observed. We can conclude from these results that the CDT test now seems to be the best test to detect alcohol abusers.

Kinetics of serum glutamate dehydrogenase during short-term alcohol withdrawal.

The behavior of serum glutamate dehydrogenase after alcohol withdrawal was studied in 64 alcoholics admitted to a detoxification center. The enzyme activity decreased in 57 patients by a median of -35.8 percent in 24 h. Thus, the decrease of serum glutamate dehydrogenase after 24 h of ethanol abstinence can serve as a test of alcohol abuse.

The decrease of low serum gamma glutamyl transferase during short-term abstinence.

We present a study of 107 in-patients of a detoxification center. As expected, 81 percent of them showed upon admittance an increased serum gamma glutamyl transferase (GGT). After alcohol withdrawal, GGT decreased in all but one of these patients. More surprisingly, even among the rest of the patients exhibiting upon admittance a GGT result within the reference range, there still occurred a significant decrease in 50 percent of the cases. Thus, whether the initial GGT is high or normal, we observed a decrease in 96 patients out of 107, i.e., a sensitivity of 0.90. The decrease test consists in asking a subject to refrain from any alcohol intake during a short period, e.g., seven days. If any significant diminution of serum GGT occurs, the possibility of alcohol abuse should at least be given serious consideration before being rejected. This test was used up to now only when GGT was initially high. The present results show that it can be attempted even when GGT is initially within the normal range, with a sensitivity of 0.90.