Predictors of mortality and neurological function in ICU patients recovering from cardiac arrest: A Spanish nationwide prospective cohort study.

OBJECTIVE: To identify predictors of mortality and neurological function in adult ICU patients recovering from cardiac arrest. DESIGN: A prospective cohort multicenter study was carried out. SETTING: Forty-six polyvalent ICUs. PATIENTS: A total of 595 patients recovering from out-of-hospital cardiac arrest (OHCA, n=285) or in-hospital cardiac arrest (IHCA, n=310). MAIN OUTCOME VARIABLES: Survival and recovery of neurological function. RESULTS: The mean cardiopulmonary resuscitation time was 18min (range 10-30). Moderate hypothermia was used in 197 patients, and 150 underwent percutaneous coronary intervention (PCI). Return of spontaneous circulation (ROSC) was achieved within 20min in 370 patients. Variables associated to mortality (ICU and in-hospital) were age (odds ratio [OR]=1.0, 95%CI 1.0-1.0 per year), non-cardiac origin of cardiac arrest (OR=2.16, 95%CI 1.38-3.38; P=0.001) and ROSC >20min (OR=3.07, 95%CI 1.97-4.78; P<0.001), whereas PCI and the presence of shockable rhythm exhibited a protective effect. Favorable neurological outcome was associated to shockable rhythm, ROSC <20min, and cardiac origin of arrest. Hypothermia did not affect survival or neurological outcome in the multivariate analysis. CONCLUSIONS: Age, non-cardiac origin of cardiac arrest and ROSC >20min were predictors of mortality. In contrast, cardiac arrest of cardiac origin, ROSC <20min, and defibrillable rhythms were associated to unfavorable neurological outcomes.

Combining electrokinetic transport and bioremediation for enhanced removal of crude oil from contaminated marine sediments: Results of a long-term, mesocosm-scale experiment.

Marine sediments represent an important sink of harmful petroleum hydrocarbons after an accidental oil spill. Electrobioremediation techniques, which combine electrokinetic transport and biodegradation processes, represent an emerging technological platform for a sustainable remediation of contaminated sediments. Here, we describe the results of a long-term mesocosm-scale electrobioremediation experiment for the treatment of marine sediments contaminated by crude oil. A dimensionally stable anode and a stainless-steel mesh cathode were employed to drive seawater electrolysis at a fixed current density of 11 A/m2. This approach allowed establishing conditions conducive to contaminants biodegradation, as confirmed by the enrichment of Alcanivorax borkumensis cells harboring the alkB-gene and other aerobic hydrocarbonoclastic bacteria. Oil chemistry analyses indicated that aromatic hydrocarbons were primarily removed from the sediment via electroosmosis and low molecular weight alkanes (nC6 to nC10) via biodegradation.

Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts.

Transforming growth factor-beta (TGF-beta) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF-beta stimulates this process remains poorly understood. We examined whether mitogen-activated protein kinase (MAPK) routes were involved in TGF-beta1-induced collagen expression in L(6)E(9) myoblasts. TGF-beta1 induced p38 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N-terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF-beta1-induced alpha(2)(I) collagen mRNA expression and accumulation. These data indicate that TGF-beta1-induced p38 activation is involved in TGF-beta1-stimulated collagen synthesis.

Role of the crc gene in catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway.

Expression of the alkane degradation pathway encoded in the OCT plasmid of Pseudomonas putida GPo1 is induced in the presence of alkanes by the AlkS regulator, and it is down-regulated by catabolic repression. The catabolic repression effect reduces the expression of the two AlkS-activated promoters of the pathway, named PalkB and PalkS2. The P. putida Crc protein participates in catabolic repression of some metabolic pathways for sugars and nitrogenated compounds. Here, we show that Crc has an important role in the catabolic repression exerted on the P. putida GPo1 alkane degradation pathway when cells grow exponentially in a rich medium. Interestingly, Crc plays little or no role on the catabolic repression exerted by some organic acids in a defined medium, which shows that these two types of catabolic repression can be genetically distinguished. Disruption of the crc gene led to a six- to sevenfold increase in the levels of the mRNAs arising from the AlkS-activated PalkB and PalkS2 promoters in cells growing exponentially in rich medium. This was not due to an increase in the half-lives of these mRNAs. Since AlkS activates the expression of its own gene and seems to be present in limiting amounts, the higher mRNA levels observed in the absence of Crc could arise from an increase in either transcription initiation or in the translation efficiency of the alkS mRNA. Both alternatives would lead to increased AlkS levels and hence to elevated expression of PalkB and PalkS2. High expression of alkS from a heterologous promoter eliminated catabolic repression. Our results indicate that catabolic repression in rich medium is directed to down-regulate the levels of the AlkS activator. Crc would thus modulate, directly or indirectly, the levels of AlkS.

Differential shedding of transmembrane neuregulin isoforms by the tumor necrosis factor-alpha-converting enzyme.

The neuregulins (NRGs) are a family of EGF-like factors that activate receptor tyrosine kinases of the ErbB/HER type. Some NRGs are membrane anchored and are released upon cleavage of the ectodomain. Here we have investigated the characteristics of the cleavage of different transmembrane NRG isoforms (proNRG) that diverge in domains that have been implicated in the regulation of the cleavage of other membrane-anchored growth factors. We show that cleavage of proNRGs is complex and generates several cell-bound truncated fragments. Comparison of the resting generation of these truncated fragments between proNRG forms that diverge in the linker region that connects the EGF-like module to the transmembrane domain revealed that proNRG beta 2a was relatively resistant to processing compared to proNRG beta 4a which was processed more efficiently than proNRG alpha 2a. An important role for this linker in proNRG cleavage was supported by deletion analysis of this region that prevented NRG solubilization. Studies aimed at the identification of the proteolytic machinery responsible for proNRG processing indicated that metalloproteases were involved in proNRG processing. This was further supported by the fact that cleavage of proNRG alpha 2c was defective in fibroblasts derived from TACE(-/-) animals that express an inactive form of the metalloprotease TACE.

Characterization of bacterial strains able to grow on high molecular mass residues from crude oil processing.

Oil residues containing high molecular mass hydrocarbons, rich in polyaromatic compounds, are frequent end-products of crude oil processing and are poorly biodegradable. Their disposal poses an environmental problem. Through batch-enrichments from contaminated soils we have isolated and characterized seven bacterial strains that can use a residue from crude oil processing as a source of carbon and energy. The residue was a complex mixture of high molecular mass compounds, including saturated, aromatic and polycyclic aromatic hydrocarbons (PAHs). Analysis of the metabolic profiles of the strains isolated showed that they could all metabolize long-chain-length alkanes efficiently, but not PAHs. Strains degrading naphthalene, a simple PAH, did exist in the soil inocula used, but could be isolated only when enrichments were performed using pure naphthalene as the sole carbon source. All strains tested emulsified the oil residue and their ability to produce surfactants was studied.

Stimulation of cleavage of membrane proteins by calmodulin inhibitors.

The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca(2+). Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-alpha and pro-neuregulin-alpha2c, the receptor tyrosine kinase, TrkA, and the beta-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca(2+) increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction.

A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway.

The AlkS regulator, encoded by the alkS gene of the Pseudomonas oleovorans OCT plasmid, activates the expression of a set of enzymes that allow assimilation of alkanes. We show that the AlkS protein regulates, both negatively and positively, the expression of its own gene. In the absence of alkanes, alkS is expressed from promoter PalkS1, which is recognized by sigmaS-RNA polymerase, and whose activity is very low in the exponential phase of growth and considerably higher in stationary phase. AlkS was found to downregulate this promoter, limiting expression of alkS in stationary phase when alkanes were absent. In the presence of alkanes, AlkS repressed PalkS1 more strongly and simultaneously activated a second promoter for alkS, named PalkS2, located 38 bp downstream from PalkS1. Activation of PalkS2 allowed efficient transcription of alkS when alkanes were present. Transcription from PalkS2 was modulated by catabolite repression when cells were provided with a preferred carbon source. We propose that the expression of alkS is regulated by a positive feedback mechanism, which leads to a rapid increase in alkS transcription when alkanes are present. This mechanism should allow a rapid induction of the pathway, as well as a fast switch-off when alkanes are depleted. An improved model for the regulation of the pathway is proposed.

Role of the alternative sigma factor sigmaS in expression of the AlkS regulator of the Pseudomonas oleovorans alkane degradation pathway.

The AlkS protein activates transcription from the PalkB promoter, allowing the expression of a number of genes required for the assimilation of alkanes in Pseudomonas oleovorans. We have identified the promoter from which the alkS gene is transcribed, PalkS, and analyzed its expression under different conditions and genetic backgrounds. Transcription from PalkS was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. The PalkS -10 region was similar to the consensus described for promoters recognized by Escherichia coli RNA polymerase bound to the alternative sigma factor sigmaS, which directs the expression of many stationary-phase genes. Reporter strains containing PalkS-lacZ transcriptional fusions showed that PalkS promoter is very weakly expressed in a Pseudomonas putida strain bearing an inactivated allele of the gene coding for sigmaS, rpoS. When PalkS was transferred to E. coli, transcription started at the same site and expression was higher in stationary phase only if sigmaS-RNA polymerase was present. The low levels of AlkS protein generated in the absence of sigmaS were enough to support a partial induction of the PalkB promoter. The -10 and -35 regions of PalkS promoter also show some similarity to the consensus recognized by sigmaD-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase PalkS is probably recognized both by sigmaD-RNA polymerase (inefficiently) and by sigmaS-RNA polymerase (present at low levels), leading to low-level expression of the alkS gene. sigmaS-RNA polymerase would be responsible for the high level of activity of PalkS observed in stationary phase.

Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway.

Pseudomonas oleovorans GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein. In the presence of alkanes, AlkS activates the expression of promoter PalkB, from which most of the genes of the pathway are transcribed. In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the alk genes with the metabolic status of the cell. Reporter strains bearing PalkB-lacZ transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the PalkB promoter by a nonmetabolizable inducer. Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate. When cells were grown in Luria-Bertani rich medium, PalkB was strongly down-regulated. This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to Escherichia coli, a heterologous genetic background. Possible mechanisms responsible for PalkB regulation are discussed.

Analysis of the complete nucleotide sequence of African swine fever virus.

We present an analysis of the complete genome of African swine fever virus (ASFV) strain BA71V, including 80 kbp of novel sequence and 90 kbp previously reported by several authors. The viral DNA is 170,101 nucleotides long and contains 151 open reading frames. Structural and/or functional information is available on 113 viral proteins. ASFV encodes five multigene families, putative membrane and secreted proteins, and enzymes involved in nucleotide and nucleic acid metabolism (including DNA repair) and protein modification. Database comparisons have provided clues about genes that may modulate the virus-host interaction, thus, possibly controlling ASFV virulence and persistence. The virus possesses genes similar to CD2, IkappaB, C-type lectins, MyD116/gadd34/gamma, 34.5, bcl-2/bax, iap, NifS, and ERV1, which may allow a viral regulation of cell adhesion, apoptosis, and redox metabolism, as well as of the host immune response against ASFV infection. The proteins encoded by different ASFV isolates are highly similar, the most variable ones being those belonging to multigene families, some membrane proteins, and those containing tandem repeats. DNA sequence data confirm the intermediate characteristics of ASFV between poxviruses and iridoviruses, supporting the notion that ASFV belongs to an independent virus family.

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.