An attempt to validate the modification of the American-European consensus definition of acute lung injury/acute respiratory distress syndrome by the Berlin definition in a university hospital.

PURPOSE: The Berlin definition for acute respiratory distress syndrome (ARDS) is a new proposal for changing the American-European consensus definition but has not been assessed prospectively as yet. In the present study, we aimed to determine (1) the prevalence and incidence of ARDS with both definitions, and (2) the initial characteristics of patients with ARDS and 28-day mortality with the Berlin definition. METHODS: We performed a 6-month prospective observational study in the ten adult ICUs affiliated to the Public University Hospital in Lyon, France, from March to September 2012. Patients under invasive or noninvasive mechanical ventilation, with PaO2/FiO2 <300 mmHg regardless of the positive end-expiratory pressure (PEEP) level, and acute onset of new or increased bilateral infiltrates or opacities on chest X-ray were screened from ICU admission up to discharge. Patients with cardiogenic pulmonary edema were excluded. Patients were further classified into specific categories by using the American-European Consensus Conference and the Berlin definition criteria. The complete data set was measured at the time of inclusion. Patient outcome was measured at day 28 after inclusion. RESULTS: During the study period 3,504 patients were admitted and 278 fulfilled the American-European Consensus Conference criteria. Among them, 18 (6.5 %) did not comply with the Berlin criterion PEEP ≥ 5 cmH2O and 20 (7.2 %) had PaO2/FiO2 ratio ≤200 while on noninvasive ventilation. By using the Berlin definition in the remaining 240 patients (n = 42 mild, n = 123 moderate, n = 75 severe), the overall prevalence was 6.85 % and it was 1.20, 3.51, and 2.14 % for mild, moderate, and severe ARDS, respectively (P > 0.05 between the three groups). The incidence of ARDS amounted to 32 per 100,000 population per year, with values for mild, moderate, and severe ARDS of 5.6, 16.3, and 10 per 100,000 population per year, respectively (P < 0.05 between the three groups). The 28-day mortality was 35.0 %. It amounted to 30.9 % in mild, 27.9 % in moderate, and 49.3 % in severe categories (P < 0.01 between mild or moderate and severe, P = 0.70 between mild and moderate). In the Cox proportional hazard regression analysis ARDS stage was not significantly associated with patient death at day 28. CONCLUSIONS: The present study did not validate the Berlin definition of ARDS. Neither the stratification by severity nor the PaO2/FiO2 at study entry was independently associated with mortality.

[Peripartum period and hemophilia carriers]. / Gestion du péri-partum de la patiente conductrice d'une hémophilie.

Women who are carriers for hemophilia are usually considered as safe carriers. However, they can present hemorragic symptoms associated with low factor VIII or IX levels. During pregancy, factor VIII increases whereas factor IX does not. The peripartum period is at risk of increased bleeding in these women. Here are presented reports of clinical data concerning two hemophilia carriers with low factor VIII or IX (30-40%) during the peripartum period. They received remifentanil and ketamine for labor pain management because of contraindication of epidural and spinal analgesia. Delivery occured quickly but they presented immediate moderate postpartum haemorrage. They did not necessitate blood transfusion. The one with hemophilia A received desmopressin just after delivery and the other one received factor IX when she arrived in delivery room. Blood factor VIII or IX has to be assessed in these women with familial history of hemophilia and bleeding. During pregnancy, factor VIII increases and can be assessed many times during pregnancy expecting a level over 50%. Factor IX does not really increase during pregancy and hemorrage can occur. Epidural and spinal anesthesia seem to be contraindicated as far as recommandations are concerned. Coagulation factor substitution is a mean of increasing factor level before these anaesthesias and can be discussed for each case.

Delivery of tidal volume from four anaesthesia ventilators during volume-controlled ventilation: a bench study.

BACKGROUND: Tidal volume (V(T)) must be accurately delivered by anaesthesia ventilators in the volume-controlled ventilation mode in order for lung protective ventilation to be effective. However, the impact of fresh gas flow (FGF) and lung mechanics on delivery of V(T) by the newest anaesthesia ventilators has not been reported. METHODS: We measured delivered V(T) (V(TI)) from four anaesthesia ventilators (Aisys™, Flow-i™, Primus™, and Zeus™) on a pneumatic test lung set with three combinations of lung compliance (C, ml cm H2O(-1)) and resistance (R, cm H2O litre(-1) s(-2)): C60R5, C30R5, C60R20. For each CR, three FGF rates (0.5, 3, 10 litre min(-1)) were investigated at three set V(T)s (300, 500, 800 ml) and two values of PEEP (0 and 10 cm H2O). The volume error = [(V(TI) - V(Tset))/V(Tset)] ×100 was computed in body temperature and pressure-saturated conditions and compared using analysis of variance. RESULTS: For each CR and each set V(T), the absolute value of the volume error significantly declined from Aisys™ to Flow-i™, Zeus™, and Primus™. For C60R5, these values were 12.5% for Aisys™, 5% for Flow-i™ and Zeus™, and 0% for Primus™. With an increase in FGF, absolute values of the volume error increased only for Aisys™ and Zeus™. However, in C30R5, the volume error was minimal at mid-FGF for Aisys™. The results were similar at PEEP 10 cm H2O. CONCLUSIONS: Under experimental conditions, the volume error differed significantly between the four new anaesthesia ventilators tested and was influenced by FGF, although this effect may not be clinically relevant.

Bacterial resistance in French acne patients.

BACKGROUND: The percentage of strains of Propionibacterium acnes resistant to antibiotics in acne is increasing in many countries, raising the question of the risks associated with bacterial resistance. Numerous series of cases have been published on European populations of acne patients, but at the moment we still have very few data regarding France. OBJECTIVES: The aim of this study was to evaluate the prevalence of P. acnes resistant strains to erythromycin, tetracycline, and doxycycline in a population of French acne patients. METHODS: Specimens were collected from 273 patients in 43 centers located throughout France (12 hospitals and 31 private office practices). RESULTS: 75.1% of the patients were carriers of P. acnes strains resistant to erythromycin and 9.5% to tetracycline. One hundred percent of strains resistant to tetracycline were also resistant to doxycycline. There was no significant difference among the regions and between patients followed in hospital or private office. CONCLUSIONS: It clearly appears that the percentage of patients with P. acnes strains resistant to erythromycin is similar to that in other countries, but lower for tetracycline or doxycycline in comparison to other European countries.

Model for stathmin/OP18 binding to tubulin.

Stathmin/OP18 is a regulatory phosphoprotein that controls microtubule (MT) dynamics. The protein does not have a defined three-dimensional structure, although it contains three distinct regions (an unstructured N-terminus, N: 1-44; a region with high helix propensity, H 1: 44-89; and a region with low helix propensity, H 2: 90-142). The full protein and a combination of H 1 and H 2 inhibits tubulin polymerization, while the combination of H 1 and the N-terminus is less efficient. None of the individual three regions alone are functional in this respect. However, all of them cross-link to alpha-tubulin, but only full-length stathmin produces high-molecular-weight products. Mass spectrometry analysis of alpha-tubulin-stathmin/OP18 and its truncation products shows that full-length stathmin/OP18 binds to the region around helix 10 of alpha-tubulin, a region that is involved in longitudinal interactions in the MT, sequestering the dimer and possibly linking two tubulin heterodimers. In the absence of the N-terminus, stathmin/OP18 binds to only one molecule of alpha-tubulin, at the top of the free tubulin heterodimer, preventing polymerization.

A tale of two secondary structure elements: when a beta-hairpin becomes an alpha-helix.

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the GB1 domain by a sequence with strong beta-hairpin propensity in isolation. In particular, we have chosen the sequence of the second beta-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated beta-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.

Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability.

The leuB gene from the psychrotrophic strain Vibrio sp. I5 has been cloned and sequenced. The gene codes for 3-isopropylmalate dehydrogenase, a 360-residue, dimeric enzyme involved in the biosynthesis of leucine. Three recently solved homologous isopropylmalate dehydrogenase (IPMDH) crystal structures from thermophilic and mesophilic organisms have been used to build a homology model for the psychrotrophic IPMDH and to deduce the possible structural reasons for its decreased thermostability. According to our model the psychrotrophic IPMDH contains fewer stabilizing interactions than its mesophilic and thermophilic counterparts. Elements that have been identified as destabilizing in the comparison of the psychrotrophic, mesophilic and thermophilic IPMDHs are a smaller number of salt-bridges, a reduction in aromatic-aromatic interactions, fewer proline residues and longer surface loops. In addition, there are a number of substitutions of otherwise strictly conserved residues that can be linked to thermostability.

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.

Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli.

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40 degrees C the K(m) of E. coli IPMDH was 105 microM for IPM and 321 microM for NAD, the kcat was 69 s-1. The half denaturation temperature was 64 degrees C, which was 20 degrees C lower than that of the thermophile enzyme.

Revision of the amino-acid sequence of 3-isopropylmalate dehydrogenase from Salmonella typhimurium by means of X-ray crystallography.

The amino acid (aa) sequence of the leuB gene product of Salmonella typhimurium, 3-isopropylmalate dehydrogenase (IPMDH), has been revised using electron density maps from X-ray structure determination. The nucleotide (nt) sequence of both strands of leuB has been redetermined to confirm the crystallographic findings. It does not agree with the previously reported S. typhimurium leuB nucleotide sequence [Andreadis and Rosenthal, Biochim. Biophys. Acta 1129 (1992) 228-230].

The reversible replacement of internal potassium by caesium in isolated turtle heart.

1. By perfusing the isolated turtle heart with Cs solution, most of the intracellular K can be replaced by Cs. After 3--4 hr, Cs approaches a steady-state distribution with a concentration slightly below initial K concentration. 2. During the initial stages of perfusion, the heart accumulated Cs and lost K, the exchange ratio of K for Cs was estimated to be about 0.6. Subsequently perfusion yielded an equi-ionic substitution of K by Cs. 3. In presence of DNP (2 x 10(-4) M), K efflux and Cs accumulation increased but the low initial K--Cs exchange was abolished. Then, the replacement of K by Cs took place at a ratio of K:Cs of about 0.8. Ouabain (10(-5) M) suppressed uptake of Cs whereas K loss was the same as with DNP. 4. These results confirm that permeability of the cardiac sarcolemma to Cs is low, and suggest that the movement of Cs must be mainly attributed to its active transport into cells by the ionic exchange which normally transports K+ and is coupled to the extrusion of Na. The initial low net K efflux could be explained by an accumulation process which facilitates retention of K by the heart. A mechanism of this kind would be described as active reabsorption of some K present in extracellular space and would consequently reduce the uptake of Cs. 5. After loading the heart with Cs, perfusion with K solution showed the exchange of Cs for K at a ratio of Cs:K of about 0.5. K reaccumulation is reduced by ouabain (10(-5) M) and comes back to a normal steady-state distribution after 5 hr. At this time, K concentration was slightly below normal K value, but only half Cs content was eliminated. After 15 hr of perfusion, intracellular K remained constant whereas 15% of the original Cs remained.

[Effect of 2,4 dinitrophenol and ouabain on the ability of cesium ions to substitute for intracellular potassium ions in isolated and perfused turtle heart (author's transl)]. / Effet du DNP et de l'ouabaïne sur les conditions de substitution du césium au potassium intracellulaire dans le cas du coeur isolé et perfusé de Tortue

1. It has been shown that most of the intracellular potassium of turtle heart can be replaced by cesium. 2. The uptake of cesium is reduced by the presence of either DNP 5.10(-5) M or ouabain 10(-6) M in the external medium. The presence of 10(-5) M ouabain markedly inhibits the uptake of cesium. 3. These results lead to the conclusion that the intracellular accumulation of cesium occurs via an active inward transport system wich operates with the simultaneous efflux of sodium.