Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Subtelomeric structure of Plasmodium falciparum chromosomes

Previous studies of subtelomeric regions in Plasmodium berghei led to the identification of subtelomeric repeats (2.3kb long) present in a variable number at many chromosomal ends. Both loss and increase in 2.3kb-repeat copy number are involved in chromosome-size polymorphisms. Subtelomeric losses leading to chromosome-size polymorphisms have been described by several authors in P.falciparum where the structure of subtelomeric regions is not known in detail. We therefore undertook their characterisation, by means of chromosome walking and jumping techniques, starting from the telomere-flanking sequence present in pPftel.1, the P.falciparum telomeric clone described by Vernick and McCutchan (1988). The results indicate that at least 20 (out of 28) chromosomal ends in P.falciparum 3D7 chromosomes share a subtelomeric region, about 40kb long, covering (but not limited to) the Rep20 region. Non repetitive, AT-rich portions flanking the Rep20 region on both sides are also conserved at most chromosomal ends.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Coronary heart disease in primary care: accuracy of medical history and physical findings in patients with chest pain - a study protocol for a systematic review with individual patient data.

BACKGROUND: Chest pain is a common complaint in primary care, with coronary heart disease (CHD) being the most concerning of many potential causes. Systematic reviews on the sensitivity and specificity of symptoms and signs summarize the evidence about which of them are most useful in making a diagnosis. Previous meta-analyses are dominated by studies of patients referred to specialists. Moreover, as the analysis is typically based on study-level data, the statistical analyses in these reviews are limited while meta-analyses based on individual patient data can provide additional information. Our patient-level meta-analysis has three unique aims. First, we strive to determine the diagnostic accuracy of symptoms and signs for myocardial ischemia in primary care. Second, we investigate associations between study- or patient-level characteristics and measures of diagnostic accuracy. Third, we aim to validate existing clinical prediction rules for diagnosing myocardial ischemia in primary care. This article describes the methods of our study and six prospective studies of primary care patients with chest pain. Later articles will describe the main results. METHODS/DESIGN: We will conduct a systematic review and IPD meta-analysis of studies evaluating the diagnostic accuracy of symptoms and signs for diagnosing coronary heart disease in primary care. We will perform bivariate analyses to determine the sensitivity, specificity and likelihood ratios of individual symptoms and signs and multivariate analyses to explore the diagnostic value of an optimal combination of all symptoms and signs based on all data of all studies. We will validate existing clinical prediction rules from each of the included studies by calculating measures of diagnostic accuracy separately by study. DISCUSSION: Our study will face several methodological challenges. First, the number of studies will be limited. Second, the investigators of original studies defined some outcomes and predictors differently. Third, the studies did not collect the same standard clinical data set. Fourth, missing data, varying from partly missing to fully missing, will have to be dealt with.Despite these limitations, we aim to summarize the available evidence regarding the diagnostic accuracy of symptoms and signs for diagnosing CHD in patients presenting with chest pain in primary care. REVIEW REGISTRATION: Centre for Reviews and Dissemination (University of York): CRD42011001170.

Desenvolupament en Network Simulator 2: protocol de migració per simular agents mòbils

Avui en dia, estem assistint a una expansió de la tecnologia d’agents mòbils i noves aplicacions basades en aquesta s’estan obrint pas constantment. Les aplicacions han de demostrar la seva viabilitat sobretot en entorns heterogenis i complexos com les xarxes MANET. En aquest projecte es desenvolupa un sistema per simular el comportament dels agents mòbils, ampliant l’actual simulador de xarxa NS2, i també es comprova la viabilitat de l’implementació de l’ETTMA pel triatge de víctimes en situacions d’emergència.

Agents mòbils i DTN: disseny i implementació d'un protocol d'encaminament en l'entorn PROSES

En aquest projecte s’ha treballat en l’entorn PROSES, on aeroports i avions de l’espai aeri són mules de transport sobre una xarxa DTN. L’objectiu principal és estudiar i simular dos escenaris concrets: l’enviament de notícies des de les torres de control als avions, i l’enviament de canvis de rutes de vol dels avions a un aeroport en qüestió. S’ha simulat el comportament de dos protocols d’encaminament diferents sobre els escenaris creats. Per a realitzar les proves s’ha utilitzat el simulador The ONE, s’ha implementat un nou protocol d’encaminament, s’ha creat un Generador de Mapes i Rutes, i s’han realitzat amb èxit les simulacions.

A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Inexperienced sonographers can successfully visualize and assess a three-dimensional image of the fetal face using a standardized ultrasound protocol

Introduction: A standardized three-dimensional ultrasonographic (3DUS) protocol is described that allows fetal face reconstruction. Ability to identify cleft lip with 3DUS using this protocol was assessed by operators with minimal 3DUS experience. Material and Methods: 260 stored volumes of fetal face were analyzed using a standardized protocol by operators with different levels of competence in 3DUS. The outcomes studied were: (1) the performance of post-processing 3D face volumes for the detection of facial clefts; (2) the ability of a resident with minimal 3DUS experience to reconstruct the acquired facial volumes, and (3) the time needed to reconstruct each plane to allow proper diagnosis of a cleft. Results: The three orthogonal planes of the fetal face (axial, sagittal and coronal) were adequately reconstructed with similar performance when acquired by a maternal-fetal medicine specialist or by residents with minimal experience (72 vs. 76%, p = 0.629). The learning curve for manipulation of 3DUS volumes of the fetal face corresponds to 30 cases and is independent of the operator's level of experience. Discussion: The learning curve for the standardized protocol we describe is short, even for inexperienced sonographers. This technique might decrease the length of anatomy ultrasounds and improve the ability to visualize fetal face anomalies.

Valoració de la introducció d’un protocol diagnòstic-terapèutic de la pancreatitis aguda fent especial èmfasi en els marcadors precoços de gravetat

Del 10 al 20% de les pancreatitis agudes(PA) són greus. L’objetiu del treball és la valoració dels marcadors precoços de gravetat. Es presenta un estudi prospectiu de 130 pacients amb PA analitzant les classificacions de Ranson, Apache II, Índex de Severitat del TAC(IST), hematòcrit i PCR en les primeres 24h comparant-los amb els criteris d’Atlanta. La proporció de necrosi, d’ingrés a UCI i de mortalitat obtingudes en la sèrie estudiada en el nostre entorn són similars als de la literatura. Segons els nostres resultats l’hematòcrit en les primeres 24hores és predictor de mortalitat i l’Apache II d’ingrés a UCI.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenothypical gentamicin resistance amplification (frequencies of 10-3 to 10-5, compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromossomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

The population structure of Trypanosoma cruzi: expanded analysis of 54 strains using eight polymorphic CA-repeat microsatellites

Recently we cloned and sequenced the first eight Trypanosoma cruzi polymorphic microsatellite loci and studied 31 clones and strains to obtain valuable information about the population structure of the parasite. We have now studied 23 further strains, increasing from 11 to 31 the number of strains obtained from patients with chronic Chagas disease. This expanded set of 54 strains and clones analyzed with the eight microsatellites markers confirmed the previously observed diploidy, clonal population organization and very high polymorphism of T. cruzi. Moreover, this new study disclosed two new features of the population genetic structure of T. cruzi. The first was the discovery that, similarly to what we had previously shown for strains isolated from insect vectors, mammals and humans with acute disease, isolates from patients in the chronic phase of Chagas disease could also be multiclonal, albeit at a reduced proportion. Second, when we used parsimony to display the genetic relationship among the clonal lineages in an unrooted Wagner network we observed, like before, a good correlation of the tree topography with the classification in three clusters on the basis of single locus analysis of the ribosomal RNA genes. However, a significant new finding was that now the strains belonging to cluster 2 split in two distant sub-clusters. This observation suggests that the evolutionary history of T. cruzi may be more complex than we previously thought.

Repeat Gamma Knife surgery for recurrent trigeminal neuralgia: long-term outcomes and systematic review.

Object The purpose of this study was to establish the safety and efficacy of repeat Gamma Knife surgery (GKS) for recurrent trigeminal neuralgia (TN). Methods Using the prospective database of TN patients treated with GKS in Timone University Hospital (Marseille, France), data were analyzed for 737 patients undergoing GKS for TN Type 1 from July 1992 to November 2010. Among the 497 patients with initial pain cessation, 34.4% (157/456 with ≥ 1-year follow-up) experienced at least 1 recurrence. Thirteen patients (1.8%) were considered for a second GKS, proposed only if the patients had good and prolonged initial pain cessation after the first GKS, with no other treatment alternative at the moment of recurrence. As for the first GKS, a single 4-mm isocenter was positioned in the cisternal portion of the trigeminal nerve at a median distance of 7.6 mm (range 4-14 mm) anterior to the emergence of the nerve (retrogasserian target). A median maximum dose of 90 Gy (range 70-90 Gy) was delivered. Data for 9 patients with at least 1-year followup were analyzed. A systematic review of literature was also performed, and results are compared with those of the Marseille study. Results The median time to retreatment in the Marseille study was 72 months (range 12-125 months) and in the literature it was 17 months (range 3-146 months). In the Marseille study, the median follow-up period was 33.9 months (range 12-96 months), and 8 of 9 patients (88.9%) had initial pain cessation with a median of 6.5 days (range 1-180 days). The actuarial rate for new hypesthesia was 33.3% at 6 months and 50% at 1 year, which remained stable for 7 years. The actuarial probabilities of maintaining pain relief without medication at 6 months and 1 year were 100% and 75%, respectively, and remained stable for 7 years. The systematic review analyzed 20 peer-reviewed studies reporting outcomes for repeat GKS for recurrent TN, with a total of 626 patients. Both the selection of the cases for retreatment and the way of reporting outcomes vary widely among studies, with a median rate for initial pain cessation of 88% (range 60%-100%) and for new hypesthesia of 33% (range 11%-80%). Conclusions Results from the Marseille study raise the question of surgical alternatives after failed GKS for TN. The rates of initial pain cessation and recurrence seem comparable to, or even better than, those of the first GKS, according to different studies, but toxicity is much higher, both in the Marseille study and in the published data. Neither the Marseille study data nor literature data answer the 3 cardinal questions regarding repeat radiosurgery in recurrent TN: which patients to retreat, which target is optimal, and which dose to use.