First case report of a human sepsis involving a recently identified anaerobic agent: Bacteroides faecis.

Up until now, Bacteroides faecis, a Gram-negative, anaerobic, non-motile, nonsporeforming rod has been principally described as a commensal microbe isolated from the feces of healthy adults. We report the first case of human Bacteroides faecis sepsis after removal of suspected post-colonic ischemia colonized epicardic electrodes. Electrodes and blood cultures both grew Gram-negative anaerobic rods but usual phenotypic methods and 16S rARN gene sequencing failed to ensure its species identification. B. faecis was finally identified using hsp60 gene sequencing. Because this species is not well-known and is difficult to identify, it may have been overlooked or misidentified in previous studies.

A novel peptide derived from vascular endothelial growth factor prevents amyloid beta aggregation and toxicity.

Amyloid-ß oligomers (Aßo) are the most pathologically relevant Aß species in Alzheimer's disease (AD), because they induce early synaptic dysfunction that leads to learning and memory impairments. In contrast, increasing VEGF (Vascular Endothelial Growth Factor) brain levels have been shown to improve learning and memory processes, and to alleviate Aß-mediated synapse dysfunction. Here, we designed a new peptide, the blocking peptide (BP), which is derived from an Aßo-targeted domain of the VEGF protein, and investigated its effect on Aß-associated toxicity. Using a combination of biochemical, 3D and ultrastructural imaging, and electrophysiological approaches, we demonstrated that BP strongly interacts with Aßo and blocks Aß fibrillar aggregation process, leading to the formation of Aß amorphous aggregates. BP further impedes the formation of structured Aßo and prevents their pathogenic binding to synapses. Importantly, acute BP treatment successfully rescues long-term potentiation (LTP) in the APP/PS1 mouse model of AD, at an age when LTP is highly impaired in hippocampal slices. Moreover, BP is also able to block the interaction between Aßo and VEGF, which suggests a dual mechanism aimed at both trapping Aßo and releasing VEGF to alleviate Aßo-induced synaptic damage. Our findings provide evidence for a neutralizing effect of the BP on Aß aggregation process and pathogenic action, highlighting a potential new therapeutic strategy.

Mobile robotic platforms for the acoustic tracking of deep-sea demersal fishery resources.

Knowing the displacement capacity and mobility patterns of industrially exploited (i.e., fished) marine resources is key to establishing effective conservation management strategies in human-impacted marine ecosystems. Acquiring accurate behavioral information of deep-sea fished ecosystems is necessary to establish the sizes of marine protected areas within the framework of large international societal programs (e.g., European Community H2020, as part of the Blue Growth economic strategy). However, such information is currently scarce, and high-frequency and prolonged data collection is rarely available. Here, we report the implementation of autonomous underwater vehicles and remotely operated vehicles as an aid for acoustic long-baseline localization systems for autonomous tracking of Norway lobster (Nephrops norvegicus), one of the key living resources exploited in European waters. In combination with seafloor moored acoustic receivers, we detected and tracked the movements of 33 tagged lobsters at 400-m depth for more than 3 months. We also identified the best procedures to localize both the acoustic receivers and the tagged lobsters, based on algorithms designed for off-the-shelf acoustic tags identification. Autonomous mobile platforms that deliver data on animal behavior beyond traditional fixed platform capabilities represent an advance for prolonged, in situ monitoring of deep-sea benthic animal behavior at meter spatial scales.

Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.

Correction: Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

Following the publication of this article the authors noted that two images were duplicated in Figure 2B. The corrected figure 2B is below. The authors wish to apologize for any inconvenience caused.

Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.

BACKGROUND: Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS: We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS: We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.

Degradation of a developmentally regulated mRNA in Xenopus embryos is controlled by the 3' region and requires the translation of another maternal mRNA.

By injecting the appropriately constructed plasmids into one-cell Xenopus embryos, we determined that the 3' region of the maternal Xenopus Eg2 mRNA confers instability on the chimeric mRNA transcribed from these plasmids. This instability, like that of the maternal Eg2 transcript, was abolished by treatment of the embryos with cycloheximide. Analysis of the polysome distribution of the maternal Eg2 mRNA in cycloheximide-treated and untreated embryos showed that Eg2 mRNA was released from polysomes after fertilization and that the stabilization caused by cycloheximide treatment was not due to a reloading of ribosomes onto the mRNA. Insertion of a stable hairpin loop (delta G = -50 kcal/mol) 5' to the reporter gene in the injected plasmid caused a 10- to 20-fold decrease in translation from the transcribed mRNAs. This decrease in translation did not abolish the instability conferred by the 3' Eg2 region. Therefore, the degradation of these chimeric mRNAs in Xenopus embryos requires the translation of another maternal mRNA coding for a trans-acting factor involved in mRNA degradation. Further restriction of the 3' Eg2 region, placed 3' to the reporter gene, showed that a cis-acting instability-conferring sequence is contained in a 497-nucleotide fragment.

The deadenylation conferred by the 3' untranslated region of a developmentally controlled mRNA in Xenopus embryos is switched to polyadenylation by deletion of a short sequence element.

The maternal Xenopus Eg mRNAs are adenylated and translated in the mature oocyte and then, after fertilization, are deadenylated and released from polysomes. Therefore, after fertilization, a change occurs in the cellular mechanisms that control mRNA adenylation. In the study reported here, we show that the 3' untranslated region of Eg2 mRNA contains a cis-acting element that is required for the deadenylation of chimeric RNAs after fertilization. This cis-acting element is contained within a single 17-nucleotide portion of the Eg2 mRNA. Disruption of this deadenylation element allows adenylation of the chimeric transcripts in the embryo. Therefore, this cis-acting element is part of the sequence information required for the developmental switch from adenylation to deadenylation of the maternal Eg2 mRNA in Xenopus embryos.

Stability of maternal mRNA in Xenopus embryos: role of transcription and translation.

The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.

RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.

Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.

A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to ß-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the ß-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target.

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.

Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein, which plays an essential, but largely unknown role in ribosome biogenesis. Nucleolin contains four consensus RNA-binding domains (CS-RBD), the presence of which suggests that the molecular function of this protein is likely reflected by its RNA-binding properties. Indeed, by immunocytological analysis performed on ribosomal transcription units, we have found several nucleolin molecules associated with nascent pre-rRNA. In mouse, two high-affinity binding sites with an apparent dissociation constant (Kd) of 50 to 100 nM have been mapped in the 5' ETS upstream from the early pre-rRNA processing site. Interestingly, nucleolin of mouse origin has recognized analogous sequences in the 5' ETS of human pre-rRNA. In parallel, selection-amplification (SELEX) experiments have identified an 18-nucleotide long RNA sequence that binds nucleolin with high affinity (Kd 5 to 20 nM) and shares a common UCCCGA motif with the characterized pre-rRNA binding sites. By mutagenesis and a structural analysis, we have characterized the nucleolin RNA binding site and found that it is constituted by a minimal 18-nucleotide long stem-loop structure. The sequence UCCCGA that is found within the hairpin loop is necessary for the specific interaction. Mutation of any of the C or G residues within this motif abolishes nucleolin interaction. Furthermore, point mutation in the stem that completely disrupt the hairpin structure also prevents nucleolin binding. By determining the minimal 5' and 3' ends of the RNA that is bound to the protein we concluded that nucleolin binding site is constituted by a short four to five-base-pair stem and an eight-nucleotide loop. This structural motif is very similar to hairpins recognized by two other CS-RBD-containing proteins (U1 snRNP A and U2 snRNP B"). Possible functional implications of our findings are discussed.

Recognition of pre-formed and flexible elements of an RNA stem-loop by nucleolin.

Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed.

Localization of nucleolin binding sites on human and mouse pre-ribosomal RNA.

Nucleolin, a major RNA binding protein of the nucleolus is found associated mainly to the pre-ribosomal particles and is absent from the cytoplasmic mature ribosomes. The role of this protein in ribosome biogenesis remains largely unknown, and is likely to be reflected by its RNA binding properties. Nucleolin contains in its central domain four RNA recognition motifs (RRM, also called RBD for RNA binding domain) which are conserved among different species. RNA binding studies have revealed that nucleolin interacts specifically with a short stem loop structure called NRE (nucleolin recognition element). We show that nucleolin extracted from human, hamster and mouse cells interacts with the same specificity and affinity to a mouse 5'ETS (external transcribed spacer) RNA fragment which contains a NRE motif. A similar structure within the human 5'ETS is also efficiently recognized by mouse nucleolin. We identified putative NRE not only in the 5'ETS but also in the 3'ETS, ITS (internal transcribed spacer) and in the 18S and 28S RNA sequences. This is in agreement with in vivo cross-linking data and a previous immunocytological analysis of ribosomal transcription units. Interestingly, we found that all the NRE localized in the 28S region are within the variable domains. Despite considerable sequence divergence of these domains, several of the NRE have sequences perfectly conserved between these two species. This suggests that these nucleolin binding sites might be functionally important, in particular for ribosome biogenesis.

Glucose dehydrogenase activity in Acinetobacter species.

A study of D-glucose oxidation by Acinetobacter species was carried out. Glucose-oxidizing strains were found distributed among almost all Acinetobacter species. 14C-glucose oxidation kinetics by non-proliferating cells with separation of oxidation products (14C-gluconate) by DEAE-cellulose paper chromatography was studied. Inhibition of glucose dehydrogenase (GDH) activity by 11 carbohydrates (mono- and disaccharides) and determination of the kinetic parameters showed that glucose oxidation was due to the action of membrane-bound GDH (inactive in vivo on disaccharides). On the basis of GDH inhibition patterns obtained, two groups were individualized. The first group of strains (identified as A. calcoaceticus, A. baumannii, A. lwoffii, A. johnsonii and Acinetobacter species 3, 9, 10 and 11) showed a greater affinity for glucose than the second group (A. haemolyticus, A. junii and Acinetobacter species 6 and 12). Restoration of GDH activity after addition of pyrroloquinoline quinone (PQQ) was studied in 187 strains previously found unable to oxidize glucose. GDH activity of 150 out of 166 strains identified as A. baumannii, A. johnsonii, A. lwoffii and Acinetobacter species 11 and 12 was restored. Eighteen of 21 strains identified as A. haemolyticus and Acinetobacter species 6 were unable to produce acid from glucose after addition of PQQ. Our results confirm that the former taxonomic scheme for the genus Acinetobacter (2 species differing only by glucose oxidation) is untenable and that, accordingly, identification of Acinetobacter strains at the species level must be performed using more modern methods, i.e. carbon source utilization tests.

Differentiation of Acinetobacter calcoaceticus sensu stricto from related Acinetobacter species by electrophoretic polymorphism of malate dehydrogenase, glutamate dehydrogenase and catalase.

Acinetobacter baumannii, unnamed Acinetobacter species 3 (studied by P.J.M. Bouvet and P.A.D. Grimont) and unnamed DNA group 13 (studied by I. Tjernberg and J. Ursing) are the most prevalent Acinetobacter species in hospitals. Using the identification scheme of Bouvet and Grimont, it is sometimes difficult to differentiate these species from A. calcoaceticus sensu stricto, a species of the natural environment that has seldom been found associated with human infection. Genetically identified Acinetobacter isolates belonging to A. calcoaceticus sensu stricto (n = 12), A. baumannii (n = 22), Acinetobacter species 3 (n = 15) and DNA group 13 of Tjernberg and Ursing (n = 26), Acinetobacter species 10 (n = 2), Acinetobacter species 11 (n = 2) and 3 strains ungrouped by DNA-DNA hybridization were investigated for electrophoretic separations of L-malate dehydrogenase (MDH), glutamate dehydrogenase (GDH) and catalase (CAT). All A. calcoaceticus sensu stricto isolates were easily differentiated from those of other species investigated by their high MDH values (relative mobility (Rf) = 78), their low GDH values (Rf range: 24-28) and CAT values (Rf range: 34-42). Acinetobacter species 3 was differentiated from A. baumannii and DNA group 13 of Tjernberg and Ursing by high CAT values. A. baumannii could not be differentiated from Tjernberg and Ursing DNA group 13. Acinetobacter species 10 was clearly differentiated from Acinetobacter species 11. Once an Acinetobacter is phenotypically identified with the four closely related species investigated here, electrophoretic analysis of MDH, GDH and CAT might be a useful complement to the identification scheme of Bouvet and Grimont for accurately identifying A. calcoaceticus sensu stricto.

Delineation of new proteolytic genomic species in the genus Acinetobacter.

Twenty-seven proteolytic Acinetobacter strains differing phenotypically from the 12 previously described Acinetobacter species were studied by DNA/DNA hybridization using the S1 nuclease method to assess their relatedness. Five DNA groups (genomic species 13 to 17) containing 20 strains were delineated. Seven strains remained ungrouped. Within species, the level of DNA relatedness to the reference strains ranged from 64 to 99%, with delta Tm values below 3.5 degrees C. DNA group 13 was 31 to 42% related to group 14. DNA group 15 was 59 to 69% related to group 16, with delta Tm values between 4.5 and 6 degrees C. DNA group 17 was 51 to 61% related to DNA groups 15 and 16 with delta Tm values between 5.5 and 7.5 degrees C. The seven ungrouped strains were 28 to 60% related to the five newly delineated genomic species with delta Tm between 6.5 and 13.5 degrees C. Reference strains of the five genomic species were 5 to 22% related to the type or reference strains of the 12 Acinetobacter genomic species previously described. Biochemically, DNA groups 13 to 17 and ungrouped strains could not be separated unambiguously and therefore are not named.

Cell surface hydrophobicity of 88 clinical strains of Acinetobacter baumannii.

We studied the surface hydrophobicity of 88 Acinetobacter baumannii strains of clinical origin, using both salt aggregation and adherence to paraxylene tests. Strains were divided into 2 groups: the first included 65 strains isolated from various clinical samples (infected catheters, tracheal and bladder devices); the second included 23 strains isolated from skin obtained from healthy controls. High surface hydrophobicity was observed in 92% of the first group of strains and in only 5% of the second.

Nosocomial outbreaks due to amikacin-resistant tobramycin-sensitive Acinetobacter species: correlation with amikacin usage.

Fifty-seven patients in the Val-de-Grâce hospital were infected or colonized with amikacin-resistant, tobramycin-sensitive Acinetobacter spp. between January 1985 and December 1987. This resistance phenotype was attributed to the recently described 3'-O-aminoglycoside phosphotransferase (APH(3')-VI), on the basis of substrate profile and DNA-DNA hybridization, and was mainly encountered in various biotypes of A. baumannii isolated from patients. It was also encountered in saprophytic A. johnsonii isolates from the hands of 11 healthy workers among the medical staff, which provided evidence for the dissemination of an epidemic gene among different biotypes and species of Acinetobacter. A retrospective epidemiological survey showed a significant correlation between amikacin consumption and case incidence in the wards where cross-infection had occurred.