EPIDEMIOLOGÍA DE LAS QUEMADURAS PEDIÁTRICAS: SEIS AÑOS DE EXPERIENCIA EN UNA UNIDAD ESPECIALIZADA DE ALTA COMPLEJIDAD / EPIDEMIOLOGY OF BURNS PEDIATRIC: SIX YEARS OF EXPERIENCE IN A SPECIALIZED UNIT HIGH COMPLEXITY

ntroducción: Las lesiones por quemaduras son una patología grave, que pueden conducir a una gran morbilidad y una mortalidad significativa, pero también tienen un impacto sanitario-económico considerable. El objetivo de este estudio fue describir epidemiológicamente la población hospitalizada en la Unidad de Quemados del Hospital de Pediatría "Prof. Dr. Juan P. Garrahan" entre los años 2015 y 2020. Material y métodos: Estudio observacional, descriptivo-analítico, transversal, con evaluación y análisis de datos registrados en base de datos de historias clínicas digitalizadas. Resultados: La serie incluyó 214 pacientes, 60,3% sexo masculino, mediana de edad 4.6 años (0-16,6), 63% provenientes de la provincia de Buenos Aires, 78% de traslados se hicieron por vía terrestre con tiempo promedio de 55,6 minutos (DS 81,9), 52,8% ingresaron en los meses de otoño-invierno, 80% carecían de cobertura social. La etiología lesional fue fuego y variantes (69,2%) y escaldaduras (25,7%). El 49% reunieron criterios de lesión inhalatoria. La mediana de superficie corporal quemada (SCQ) fue 30% (0-100%), lesiones tipo B (profundas) 16,2% (0-100%) y gravedad crítica (37,4%) y grave (19,2%), requiriendo una mediana de 5 actos quirúrgicos (0-55). El 87,3% de los ingresos fue en Cuidados Intensivos, con mediana de estancia hospitalaria de 33 días (1-243) y relación promedio %SCQ/días internación 1,9 (DS 2,1). El uso de Asistencia Respiratoria Mecánica (ARM) fue 68,7% con una mediana de 7.5 días (1-100). La mortalidad de la serie fue 9,8% y estuvo asociada estadísticamente a lesión inhalatoria (p=0,0001), profundidad lesional B (p=0,00001) y uso de ARM (p=0,0011). Conclusion: Los resultados de este estudio concluyen que el sexo masculino, la franja etaria < 5 años, los ingresos en otoño-invierno, las lesiones por fuego, el grupo de gravedad crítico y la utilización de ARM son datos epidemiológicos predominantes correspondientes a una Unidad de Quemados de Alta Complejidad y deben ser tenidos en cuenta para la planificación y adecuación de los recursos asistenciales

Burn injuries are a serious pathology, which can lead to high morbidity and significant mortality, but also have a considerable health-economic impact. The objective of this study was to epidemiologically describe the population hospitalized in the Burn Unit of the Pediatric Hospital "Prof.Dr. Juan P. Garrahan" between 2015 and 2020.Material and method: Observational, descriptive-analytical, cross-sectional study, with evaluation and analysis of data recorded in a database of digitized medical records. Results: The series included 214 patients, 60,3% male, median age 4,6 years (0-16,6), 63% from the province of Buenos Aires, 78% of transfers were made by land with an average time of 55,6 minutes (DS 81,9), 52,8% entered in the fall-winter months, 80% lacked social coverage. The lesional etiology was fire and variants (69,2%) and scalds (25,7%). 49% met criteria for inhalation injury. The median body surface area burned (SCQ) was 30% (0-100%), type B (deep) injuries 16,2% (0-100%) and critical (37,4%) and severe (19,2%) severity,requiring a median of 5 surgical acts (0-55). 87,3% of the admissions were in Intensive Care, with a median hospital stay of 33 days (1-243) and average ratio %SCQ/days hospitalization 1,9 (DS 2,1). The use of Mechanical Respiratory Assistance (MRA) was 68,7% with a median of 7,5 days (1-100). Mortality in the series was 9,8% and was statistically associated with inhalation injury (p=0,0001), injury depth B (p=0,00001) and use of MRA (p=0,0011). Conclusion: The results of this study conclude that male sex, the age group <5 years, admissions in autumn-winter, fire injuries, the critical severity group and the use of MRA are predominant epidemiological data corresponding to a Unit of High Complexity Burns and must be taken into account for the planning and adaptation of care resources

Isoforms of the DHTKD1-Encoded 2-Oxoadipate Dehydrogenase, Identified in Animal Tissues, Are not Observed upon the Human DHTKD1 Expression in Bacterial or Yeast Systems.

Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.

Analysis of gene expression in penicillin G induced persistence of Chlamydia pneumoniae.

Chlamydia pneumoniae is responsible for respiratory tract infections and has been associated to chronic diseases such as atherosclerosis. The involvement of C. pneumoniae in chronic diseases may be correlated to its ability to induce persistent forms in which Chlamydiae remain viable but are not cultivable. The aim of our study is to investigate C. pneumoniae specific gene activities associated with the development of Chlamydial persistence in a cell culture system in the presence of penicillin G. Chlamydia-infected HEp 2 cells were incubated with or without penicillin G for up to 72 hours. The relative mRNA expression levels of early and late genes in treated and untreated cell cultures were determined by Real-time RT-PCR. Our results revealed a consistent down-regulation of Chlamydial hctA and hctB genes (p=0.012 and p=0.003 respectively) in association with up-regulation of htrA gene (p=0.002) during penicillin G-induced persistence suggesting these gene sets as leading candidate for in vivo investigation of the development of persistent Chlamydial infection. In conclusion, the Chlamydial expression pattern of hctA, hctB, and htrA genes may be helpful to identify target molecules to diagnose and treat Chlamydia-associated chronic diseases.

Stability and oligomerization of recombinant GadX, a transcriptional activator of the Escherichia coli glutamate decarboxylase system.

One of the most important strategies that enteric bacteria adopt for maintaining the cytoplasmic pH neutral under acid stress involves the glutamate decarboxylase (Gad) system. The system works by the concerted action of a cytoplasmic, pyridoxal 5'-phosphate-dependent glutamate decarboxylase and a transmembrane antiporter, which imports glutamate and exports gamma-aminobutyrate (GABA), the decarboxylation product, thereby providing local buffering of the extracellular environment. Herein, we provide a preliminary biochemical characterization of GadX, an activator of the Gad system belonging to the AraC/XylS family of bacterial transcriptional regulators. The GadX protein has been purified as a chimeric MalE-GadX with a yield of 15-20 mg/l of bacterial culture. The fusion protein is fairly stable, although a conformational change occurs upon storage, which reduces the binding affinity by a factor of 2, without affecting the binding pattern. Partial removal of the MalE moiety from the fusion protein triggers the formation of a species which is likely to be a heterodimer, or a higher oligomer, of the type GadX/MalE-GadX. This experimental evidence is in line with the well-known tendency of AraC/XylS-like proteins to dimerize via their N-terminal domain.

Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant.

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.

The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system.

Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid-dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of gamma-aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co-transcribed with gadB and that the functional glutamic acid-dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone-like protein H-NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H-NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H-NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H-NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.

The roles of His-167 and His-275 in the reaction catalyzed by glutamate decarboxylase from Escherichia coli.

Two histidine residues in glutamate decarboxylase from Escherichia coli, potential participants in catalysis because they are conserved among amino acid decarboxylases and because they are at the active site in the homologous enzyme ornithine decarboxylase, were mutated. His-275 is shown to bind the cofactor pyridoxal 5'-phosphate but not to contribute directly to catalysis. The H275N enzyme was unable to bind the cofactor whereas the H275Q mutant contained 50% of the normal complement of cofactor and its specific activity (expressed per mole of cofactor) was 70% of that of the wild-type enzyme. The H167N mutant bound the cofactor tightly, its specific activity was approximately half that of the wild-type enzyme and experiments in D2O showed that it catalyzed replacement of the carboxyl group with retention of configuration as does the wild-type enzyme. Comparison of reaction profiles by observing changes in the absorbance of the cofactor after stopped-flow mixing, revealed that a slow reaction, in which approximately one-third of the wild-type enzyme is converted to an unreactive complex during catalysis, does not occur with the H167N mutant enzyme. This reaction is attributed to a substrate-induced conformational change, a proposal that is supported by differential scanning calorimetry.

Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli.

Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2. The E. coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci. Their protein products differ in only five amino acid residues, four of which are located in the N-terminal region (Smith et al., 1992, J. Bacteriol. 174, 5820-5826). Herein, we report the sequences of the two gad genes, including their regulatory regions. Both genes were separately cloned into the vector pQE60, for overexpression under the control of the lac promoter. In this way, we have succeded in separately expression large quantities of each pure isoform. The two isoforms were characterized biochemically and all evidence, including that from analysis of the complex pre-steady-state kinetic behavior of the enzymes, indicates that the functional properties of the two isoenzymes are identical.