Awake prone positioning in nonintubated spontaneous breathing ICU patients with acute hypoxemic respiratory failure (PRONELIFE)-protocol for a randomized clinical trial.

BACKGROUND: It is uncertain whether awake prone positioning can prevent intubation for invasive ventilation in spontaneous breathing critically ill patients with acute hypoxemic respiratory failure. Awake prone positioning could benefit these patients for various reasons, including a reduction in direct harm to lung tissue, and prevention of tracheal intubation-related complications. DESIGN AND METHODS: The PRONELIFE study is an investigator-initiated, international, multicenter, randomized clinical trial in patients who may need invasive ventilation because of acute hypoxemic respiratory failure. Consecutive patients admitted to participating ICUs are randomly assigned to standard care with awake prone positioning, versus standard care without awake prone positioning. The primary endpoint is a composite of tracheal intubation and all-cause mortality in the first 14 days after enrolment. Secondary endpoints include time to tracheal intubation and effects of awake prone positioning on oxygenation parameters, dyspnea sensation, and complications. Other endpoints are the number of days free from ventilation and alive at 28 days, total duration of use of noninvasive respiratory support, total duration of invasive ventilation, length of stay in ICU and hospital, and mortality in ICU and hospital, and at 28, 60, and 90 days. We will also collect data regarding the tolerance of prone positioning. DISCUSSION: The PRONELIFE study is among the first randomized clinical trials investigating the effect of awake prone positioning on intubation rate in ICU patients with acute hypoxemic failure from any cause. The PRONELIFE study is sufficiently sized to determine the effect of awake prone positioning on intubation for invasive ventilation-patients are eligible in case of acute hypoxemic respiratory failure without restrictions regarding etiology. The PRONELIFE study is a pragmatic trial in which blinding is impossible-however, as around 35 ICUs worldwide will participate in this study, its findings will be highly generalizable. The findings of the PRONELIFE study have the potential to change clinical management of patients who may need invasive ventilation because of acute hypoxemic respiratory failure. TRIAL REGISTRATION: ISRCTN ISRCTN11536318 . Registered on 17 September 2021. The PRONELIFE study is registered at clinicaltrials.gov with reference number NCT04142736 (October, 2019).

[Renal infarction and kidney rupture: complication of a massive cocaine intoxication in an intestinal carrier]. / Infarto y rotura renales: complicación de intoxicación masiva por cocaína en un portador intestinal.

Major complications derived from the use of cocaine have been described, alter nasal or intravenous administration of the drug. These complications are related to vascular spasm and secondary organ damage. We present the case of an intestinal cocaine packer--in slang, "mule"--, who suffered massive absorption of the drug, resulting n bowel, liver and renal ischemia. This situation, previously undescribe in the literature, ended in kidney rupture. An attempt of embolization, was unsatisfactory, and nephrectomy was finally required. The patient recovered uneventfully, with progressive renal functional improvement. This case, albeit quite exceptional, is illustrative of several of the renal actions of cocaine, and reveals the effects of absorption of cocaine at the intestinal level.

Activity of linezolid and tedizolid against clinical isolates of methicillin-resistant and methicillin and linezolid resistant Staphylococcus aureus: an in vitro comparison.

OBJECTIVE: The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Spain is approximately 20-30%. However, resistance to linezolid is rare, and the main reports are from nosocomial outbreaks. The objective of the present study was to compare the in vitro susceptibility of linezolid with that of tedizolid against MRSA isolates and methicillin-and linezolid-resistant isolates (MLRSA) mediated by the cfr gene. METHODS: The in vitro susceptibility of linezolid and tedizolid was determined using the E-test with 18 MRSA strains and 18 cfr-mediated MLRSA strains obtained from clinical isolates in the microbiology service of a tertiary university hospital. RESULTS: All MRSA strains were susceptible to both antibiotics. Analysis of the MRSA isolates revealed that the MIC50 and MIC90 of linezolid were 1.5 and 2 mg/L, respectively; those of tedizolid were 0.25 and 0.4 mg/L. The MIC50 and MIC90 of tedizolid remained at 0.75 and 1 mg/L against the MLRSA strains (MIC90 ≥ 8 mg/L). CONCLUSIONS: Both for MRSA and for MLRSA, the MICs obtained for tedizolid were at least 2 dilutions lower than those of linezolid, thus demonstrating between 2 and 4 times greater activity in vitro than linezolid.

Cloning of mini-mu bacteriophage in cosmids: in vivo packaging into phage lambda heads.

A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage lambda heads. This procedure has several advantages over packaging into Mu helper capsids: the amounts of DNA to be packaged can be increased, the packaging efficiency is improved, and the stability of transducing lysates is high.

A cos-mini-Mu vector for in vivo DNA cloning.

Construction of a mini-Mu plasmid vector containing a cosmid replicon is described. Upon derepression of mini-Mu transposition, bacterial DNA sequences can be flanked by the integrated mini-Mu. These sequences can then be packaged into lambda heads by superinfection with a lambda helper phage. Cosmid clones carrying particular bacterial genes can be recovered by selection after infection of appropriate strains with the cosmid transducing lambda lysate. We report here the successful in vivo cloning of several Escherichia coli genes using the transposoncosmid vector.

Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes.

A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

Expression of cloned genes by in vivo insertion of tac promoter using a mini-Mu bacteriophage.

The strong trp-lac(tac) promoter has been incorporated into the mini-Mu bacteriophage genome to form a mini-Mu-tac (mMu-tac) expression transposon. This mMu-tac element can transpose efficiently in Escherichia coli cells when derepressed and occasionally insert into a recombinant plasmid. When mMu-tac integration occurs upstream of a cloned gene in the orientation of its transcription, the tac promoter can direct the expression of the gene insert. mMu-tac contains translation stop codons downstream of the tac promoter. Thus, mMu-tac should be useful to express only those genes containing their own translational information. We report here the successful expression in E. coli of several prokaryotic genes using the transposon expression system.

Characterization of an insertion sequence-like element identified in plasmid pCIT264 from Lactococcus lactis subsp. lactis biovar diacetylactis.

Plasmid pCIT264 from Lactococcus lactis subsp. lactis biovar diacetylactis (L. diacetylactis) contains an insertion sequence (IS)-like element located in the citrate utilization (citQRP) cluster. This 967-nucleotide long element is bounded by 17 bp perfect inverted repeats and contains an open reading frame (ORF1) composed of 296 codons, which could encode a transposase. Expression of the IS from pCIT264 generates two mRNAs of 2900 and 1900 nucleotides. The transcription is driven by the P3 promoter, composed of a -10 region located at the right end of the IS and of a -35 region positioned downstream of this element. The IS-like element (IS982) is present in seven copies in the L.diacetylactis genome. The copy present in pCIT264 is highly stable and does not promote rearrangements of the cit cluster. We suggest that the stable maintenance of the IS-like element in pCIT264 could be due to a translational control of the putative transposase by an antisense RNA.

Cloning and molecular characterization of the citrate utilization citMCDEFGRP cluster of Leuconostoc paramesenteroides.

The citMCDEFGRP cluster from Leuconostoc paramesenteroides involved in citrate utilization was cloned and its nucleotide sequence determined. Homology of the inferred gene products with characterized enzymes reveals that citP encodes the citrate permease P, citC the citrate ligase and citDEF the subunits of the citrate lyase of Leuconostoc. Moreover, it suggests that citM encodes a Leuconostoc malic enzyme. Analysis of citrate consumption by and citrate lyase activity of Lc. paramesenteroides J1[pCITJ1] showed that its citrate permease and its citrate lyase are induced by the presence of citrate in the growth medium. Southern blot analysis demonstrated that the citMCDEFGRP cluster is located in a plasmid.

Peripheral venous blood gases and pulse-oximetry in acute cardiogenic pulmonary oedema.

BACKGROUND: The role of venous blood gases as an alternative to arterial blood gases in patients with severe acute heart failure has not been established. OBJECTIVE: To assess the correlation between arterial and peripheral venous blood gases together with pulse-oximetry (SpO2), as well as to estimate arterial values from venous samples in the first hours upon admission of patients with acute cardiogenic pulmonary oedema. METHODS: Simultaneous venous and arterial blood samples were extracted on admission and over the next 1, 2, 3, 4, and 10 hours. SpO2 was also registered at the same intervals. RESULTS: A total of 178 pairs of samples were obtained from 34 consecutive patients with acute cardiogenic pulmonary oedema. Arterial and venous blood gases followed a parallel course in the first hours, showing high correlation rates at all time intervals. Venous samples underestimated pH (mean difference -0.028) and overestimated CO2 (+5.1 mmHg) and bicarbonate (+1 mEq/l). Conversely, SpO2 tended to underestimate SaO2 (mean±SD: 93.1±9.1 vs. 94.2±8.4). Applying simple mathematical formulae based on these differences, arterial values were empirically calculated from venous samples, showing acceptable agreement in the Bland-Altman test. Likewise, a venous pH <7.32, pCO2 >51.3 mmHg, and bicarbonate <22.8 mEq/l could fairly identify arterial acidosis, either respiratory or metabolic, with a test accuracy of 92, 68, and 91%, respectively. CONCLUSIONS: In patients with cardiogenic pulmonary oedema, arterial blood gas disturbances may be estimated from peripheral venous samples. By monitoring SpO2 simultaneously, arterial punctures could often be avoided.

Thermal regulation of membrane lipid fluidity by a two-component system in Bacillus subtilis.

This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of Bacillus subtilis, composed of a Δ5-desaturase (encoded by the des gene) and the canonical two-component system DesK-DesR, to study the transcriptional regulation of des during cold shock. Students analyze the expression of a reporter transcriptional fusion between the des promoter and the bacterial lacZ gene in a wild-type B. subtilis strain and in des or desK-desR mutants grown under different culture conditions. Measurements of ß-galactosidase activity allow them to investigate how the Des pathway works and to assess the role of each component of this regulatory system.

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis.

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37 degrees C. When cultures were transferred from 37 degrees C to 20 degrees C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20 degrees C, reaching levels of 20% of the total fatty acid content. [14C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20 degrees C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20 degrees C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.

L-cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase.

Random Tn917 mutagenesis of Bacillus subtilis followed by selection of lipoic acid auxotrophs led to the isolation of the cysH gene. The gene was sequenced and found to encode a phosphoadenylylsulfate sulfotransferase with a molecular mass of 27 kDa. Expression of lacZ fused to the cysH promoter was repressed by cysteine and sulfide and induced by sulfur limitation, indicating that cysH is controlled at the level of transcription.

Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids, in Bacillus subtilis.

The Bacillus subtilis gpsA gene was cloned by complementation of an Escherichia coli gpsA strain auxotrophic for sn-glycerol 3-phosphate. The gene was sequenced and found to encode an NAD(P)H-dependent dihydroxyacetone phosphate reductase with a deduced molecular mass of 39.5 kDa. The deduced amino acid sequence showed strong conservation with that of the E. coli homolog and to other procaryotic and eucaryotic dihydroxyacetone phosphate reductases. The physical location of gpsA on the B. subtilis chromosome was at about 200 degrees. Disruption of the chromosomal gpsA gene yielded B. subtilis strains auxotrophic for glycerol, indicating that the gpsA gene product is responsible for synthesis of the sn-glycerol 3-phosphate required for phospholipid synthesis. We also found that transformation of the classical B. subtilis glycerol auxotrophs with a gpsA-containing genomic fragment yielded transformants that grew in the absence of glycerol. In agreement with prior work, our attempts to determine the reductase activity in B. subtilis extracts were unsuccessful. However, expression of the B. subtilis gpsA gene in E. coli gave reductase activity that was only slightly inhibited by sn-glycerol 3-phosphate. Since the E. coli GpsA dihydroxyacetone phosphate reductase is very sensitive to allosteric inhibition by sn-glycerol 3-phosphate, these results indicate that the B. subtilis gpsA-encoded reductase differs from that of E. coli. It seems that B. subtilis regulates sn-glycerol 3-phosphate synthesis at the level of gene expression rather than through the E. coli mechanism of strong allosteric inhibition of an enzyme produced in excess.

In vivo cloning of DNA into multicopy cosmids by mini-Mu-cosduction.

A general in vivo procedure for cloning Escherichia coli genes into cosmids has been developed. The method we describe here uses a deleted Mu phage (a mini-Mu) to transpose E. coli genes into cosmids during mini-Mu replication. The resulting cosmids clones are packaged in vivo into lambda phage particles. Plasmids carrying a particular DNA sequence can be selectively recovered after infection of a new host with the in vivo constructed genomic cosmid library. This system was used successfully to clone several E. coli genes.