Primer caso de tratamiento ex utero intraparto realizado por vía vaginal en un feto con síndrome de obstrucción congénita de la vía aérea superior / The first case of exutero intrapartum treatment performed using the vaginal route in a fetus with congenital high airway obstruction syndrome

El síndrome de obstrucción congénita de la vía aérea superior (CHAOS), es una condición infrecuente que causa asfixia o muerte perinatal inmediata, de no mediar una estrategia terapéutica que permita permeabilizar la vía aérea del paciente durante el nacimiento. El diagnóstico prenatal, es fundamental para delinear estrategias de tratamiento perinatal con el fin de minimizar la morbimortalidad de niños con anomalías congénitas. El tratamiento ex-útero intraparto (EXIT) es el procedimiento de elección. Clásicamente se realiza mediante una cesárea programada, manteniendo el soporte fetal a través de la circulación útero-placentaria. Se requiere un equipo altamente calificado y un trabajo coordinado para concretar el procedimiento en estas condiciones. Objetivo: El objetivo es reportar un caso de Síndrome de CHAOS, en el que se realizó un procedimiento EXIT en un niño nacido por parto vaginal, con la participación de un equipo multidisciplinario de profesionales de dos Instituciones Públicas de la Ciudad de Buenos Aires, en el marco de un Programa Conjunto de Diagnóstico y Tratamiento Fetal (AU)

Congenital high airway obstruction syndrome (CHAOS) is a rare entity causing perinatal asphyxia or immediate death if no therapeutic strategy is undertaken to correct airway patency at birth. Prenatal diagnosis is essential to plan perinatal strategies to decrease morbidity and mortality in children with congenital anomalies. The exutero intrapartum treatment (EXIT) is the procedure of choice. Classically, a programmed cesarean section is performed while the fetus is maintained on uteroplacental circulation. A highly trained team is required in the coordinated effort to perform the procedure. Aim: The aim of this study was to report on a case of CHAOS managed with an EXIT procedure in a child born through vaginal delivery performed by a multidisciplinary team of professionals belonging to two public institutions of the city of Buenos Aires in the framework of the Joint Program of Fetal Diagnosis and Treatment (AU)

Characterization of a computed tomography iterative reconstruction algorithm by image quality evaluations with an anthropomorphic phantom.

OBJECTIVE: This study aims to investigate the consequences on dose and image quality of the choices of different combinations of NI and adaptive statistical iterative reconstruction (ASIR) percentage, the image quality parameters of GE CT equipment. METHODS: An anthropomorphic phantom was used to simulate the chest and upper abdomen of a standard weight patient. Images were acquired with tube current modulation and different values of noise index, in the range 10-22 for a slice thickness of 5mm and a tube voltage of 120 kV. For each selected noise index, several image series were reconstructed using different percentages of ASIR (0, 40, 50, 60, 70, 100). Quantitative noise was assessed at different phantom locations. Computed tomography dose index (CTDI) and dose length products (DLP) were recorded. Three radiologists reviewed the images in a blinded and randomized manner and assessed the subjective image quality by comparing the image series with the one acquired with the reference protocol (noise index 14, ASIR 40%). The perceived noise, contrast, edge sharpness and overall quality were graded on a scale from -2 (much worse) to +2 (much better). RESULTS: A repeatable trend of noise reduction versus the percentage of ASIR was observed for different noise levels and phantom locations. The different combinations of noise index and percentage of ASIR to obtain a desired dose reduction were assessed. The subjective image quality evaluation evidenced a possible dose reduction between 24 and 40% as a consequence of an increment of ASIR percentage to 50 or 70%, respectively. CONCLUSION: These results highlighted that the same patient dose reduction can be obtained with several combinations of noise index and percentages of ASIR, providing a model with which to choose these acquisition parameters in future optimization studies, with the aim of reducing patient dose by maintaining image quality in diagnostic levels.

A suite of objective biomechanical measurement tools for personal load carriage system assessment.

For application to military and civilian needs, Defence Research and Development Canada--Toronto contracted Queen's University, Kingston to develop a suite of biomechanical assessment and analytical tools to supplement human-based load carriage system assessment methods. This suite of tools permitted efficient objective evaluation of biomechanical aspects of load-bearing webbing, vests, packs and their components, and therefore contributed to early system assessment and a rapid iterative design process. This paper is a summary of five assessment and analytical tools. A dynamic load carriage simulator was developed to simulate cadence of walking, jogging and running. The simulator comprised a computer-controlled pneumatic platform that oscillated anthropometrically weighted mannequins of varying dimensions from which measures of skin contact pressure, hip reaction forces and moments and relative pack-person displacements were taken. A stiffness tester for range of motion provided force-displacement data on pack suspension systems. A biomechanical model was used to determine forces and moments on the shoulders and hips, and validated using a static load distribution mannequin. Subjective perceptual rating systems were used gather soldier feedback during a standardized mobility circuit. Objective outcome measures were validated by means of other objective measures (e.g., Optotrak, video, Instron, etc.) and then compared to subjective ratings. This approach led to development of objective performance criteria for load carriage systems and to improvements in load carriage designs that could be used both in the military and in general.

Use of the Nd:YAG laser in the treatment of Early Childhood Caries.

AIM: This was to demonstrate that traditional therapy of Early Childhood Caries (ECC) can be improved with the use of the Nd:YAG laser. METHODS: This investigation was conducted on three 3 year-old children in which the four maxillary primary incisors were affected by ECC. The teeth were treated with the Nd:YAG laser and one unrestorable tooth for each child was extracted for investigation by scanning electron microscopy. RESULTS: The laser therapy provided several clinical advantages (reduction of dentine permeability and hypersensitivity, sterilization of the lased surface, and fluoride penetration within the tooth). Good patient compliance was also achieved due to the type of appliance and materials used, absence of noise and vibration, a lack of need for local analgesia, and the reduced number of brief appointments. CONCLUSION: This approach to treatment of ECC permits teeth to be retained in the dental arch and delays the use of traditional methods to a later time when the child has reached an age sufficient for cooperation with the practitioner.

Epitope tagging of chromosomal genes in Salmonella.

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640-6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage lambda red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

Growth-dependent DNA breakage and cell death in a gyrase mutant of Salmonella.

A class of gyrase mutants of Salmonella enterica mimics the properties of bacteria exposed to quinolones. These mutants suffer spontaneous DNA breakage during normal growth and depend on recombinational repair for viability. Unlike quinolone-treated bacteria, however, they do not show accumulation of cleavable gyrase-DNA complexes. In recA or recB mutant backgrounds, the temperature-sensitive (ts) allele gyrA208 causes rapid cell death at 43 degrees. Here, we isolated "suppressor-of-death" mutations, that is, secondary changes that allow a gyrA208 recB double mutant to survive a prolonged exposure to 43 degrees and subsequently to form colonies at 28 degrees. In most isolates, the secondary change was itself a ts mutation. Three ts alleles were mapped in genes coding for amino acyl tRNA synthetases (alaS, glnS, and lysS). Allele alaS216 completely abolished DNA breakage in a gyrA208 recA double mutant. Likewise, treating this mutant with chloramphenicol prevented death and DNA damage at 43 degrees. Additional suppressors of gyrA208 lethality include rpoB mutations and, surprisingly, icd mutations inactivating isocitrate dehydrogenase. We postulate that the primary effect of the gyrase alteration is to hamper replication fork movement. Inhibiting DNA replication under conditions of continuing macromolecular synthesis ("unbalanced growth") activates a mechanism that causes DNA breakage and cell death, reminiscent of "thymineless" lethality.

Helmet accommodation analysis using 3D laser scanning.

A method used to determine the probable population accommodation of a helmet sizing system is described. The method involves the use of 3D laser scanning, as a means of measuring helmet standoff distance (distance between the inside of the helmet and the skull), and the selection of a representative sample of test subjects. The laser scanner and the software developed to calculate standoff distance proved to be an excellent tool for the assessment of helmet fit. The main advantages include ease of use and visualization of problem areas. This 3D-analysis method gives designers objective evidence of the need for design changes as well as an idea of what these changes should be. A comparison was made between standoff distance results obtained from the scanner and those obtained using a physical measurement method (a probe). Although discrepancies were found between the two, sources of errors intrinsic to both methods make it difficult to determine which of the two methods yielded the truest standoff distance. Analysis of the comparison data shows laser scanning to be slightly more conservative than the probe method for standoff distance purposes, i.e. erring on the side of safety.

Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome.

The notion that transcription can generate supercoils in the DNA template largely stems from work with small circular plasmids. In the present work, we tested this model in the bacterial chromosome using a supercoiling-sensitive promoter as a functional sensor of superhelicity changes. The leu-500 promoter of Salmonella typhimurium is a mutant and inactive variant of the leucine operon promoter that regains activity if negative DNA supercoiling rises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants). Activation of the leu-500 promoter was analysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon. Some insertions inhibited leu-500 promoter activation in the absence of inducer. This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the mechanism responsible for activation. Superimposed on these effects, transcription of the inserted gene stimulated or inhibited leu-500 promoter activity depending on whether this gene was oriented divergently from the leu operon or in the same direction respectively. Interestingly, transcription-mediated inhibition of leu-500 promoter was observed with inserts as far as 5 kb from the leu operon, and it could be relieved by the introduction of a strong gyrase site between the inserted element and the leu-500 promoter. These results are consistent with the idea that transcriptionally generated positive and negative supercoils can diffuse along chromosomal DNA and, depending on their topological sign, elicit opposite responses from the leu-500 promoter.

Inducible prophages contribute to Salmonella virulence in mice.

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.

Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci.

Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37 degrees C. This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of the hisR locus, which encodes the tRNA(His). We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close to oriC, and the his operon, far from oriC. Some of the conclusions drawn here may apply to other operons as well.

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

Unsuspected prophage-like elements in Salmonella typhimurium.

We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).

Long-distance effect of downstream transcription on activity of the supercoiling-sensitive leu-500 promoter in a topA mutant of Salmonella typhimurium.

Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.

Hyper-negative template DNA supercoiling during transcription of the tetracycline-resistance gene in topA mutants is largely constrained in vivo.

The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is thought to enhance twin-domain partitioning. Thus, it is often assumed that the 'hyper-negative' linking difference of plasmid DNA from topA mutants reflects unconstrained, hyper-negative DNA supercoiling inside the cell. We tested the validity of this assumption in the present study. A DNA sequence that undergoes a gradual B to Z transition under increasing negative superhelical tension was used as a sensor of unconstrained negative supercoiling. Z-DNA formation was probed at a site upstream from the inducible pTac promoter fused either to the tet gene or to the gene for cytosolic chloramphenicol acetyl transferase (cat). Although plasmid DNA linking deficit increased more extensively in topA mutants following tet activation than following cat activation, no significant differences were observed in the extents to which the B to Z DNA transition is stimulated in the two cases. We infer that the excess linking deficit of the tet-containing plasmid DNA reflects constrained negative DNA supercoiling inside the cell.

A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require rec functions for viability.

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37 degrees C. Selection of suppressors that concomitantly restore growth at 37 degrees C and SOS induction in a recB- background yielded mutations that relleve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone-treated gyr+ recB- strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37 degrees C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of "cleavable' gyrase-DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA-cells at restrictive temperatures. Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.

RNA polymerase (rpoB) mutants selected for increased resistance to gyrase inhibitors in Salmonella typhimurium.

Some rifampicin-resistance (RifR) mutations make bacteria slightly resistant to the gyrase inhibitors novobiocin (Nov) and nalidixic acid (Nal). This suggested that it might be possible to isolate rpoB mutants using either drug for positive selection. In an initial test, we confirmed the presence of Rif-resistant isolates among clones selected for Nov resistance. These mutants are also more resistant to Nal. In a subsequent experiment, we found that mutants selected for low-level resistance to Nal include isolates harboring mutations genetically linked to the rpoB locus; of two such mutants studied, one is temperature-sensitive for growth. These two mutants, which are only marginally affected in their response to Nov, are normally sensitive to Rif and thus might be representative of a new class of rpoB alleles. The Rif-resistant and Rif-sensitive rpoB alleles that increase resistance to gyrase inhibitors have one property in common: they all suppress, to varying degrees, the defect in his operon regulation (transcriptional deattenuation) caused by a gyrase defect or inhibition by novobiocin. To further analyse the transcription-supercoiling relationships in these mutants, we examined the ability of RNA polymerase to recruit gyrase activity during transcription. This was done by two independent approaches: (i) observing transcription-induced accumulation of hyper-negatively supercoiled plasmid DNA in a topA mutant background and (ii) measuring transcription-induced plasmid DNA cleavage in the presence of oxolinic acid. Results indicate that the rpoB alleles described in this study diminish the recruitment of gyrase activity by the transcription process. This property correlates with a decrease in the rate of transcription initiation.(ABSTRACT TRUNCATED AT 250 WORDS)