RESUMEN
OBJECTIVEThe majority of children with myelomeningocele undergo implantation of CSF shunts. The efficacy of adding surveillance imaging to clinical evaluation during routine follow-up as a means to minimize the hazard associated with future shunt failure has not been thoroughly studied.METHODSA total of 300 spina bifida clinic visits during the calendar years between 2012 and 2016 were selected for this study (defined as the index clinic visit). Each index visit was preceded by a 6-month period during which no shunt evaluation of any kind was performed. At the index clinic visit, all patients were evaluated by a neurosurgeon. Seventy-four patients underwent previously scheduled surveillance CT or shunt series scans in addition to clinical evaluation (surveillance imaging group), and 226 patients did not undergo surveillance imaging (clinical evaluation group). Subsequent unexpected events, defined as emergency department visits, caregiver-requested clinic visits, and shunt revision surgeries were reviewed. The timing and likelihood of an unexpected event in each of the 2 groups were compared using Cox proportional hazard survival analysis. The rate of shunt revision surgery in the follow-up period as well as the associated outcomes and rate of complications were analyzed.RESULTSThe clinical characteristics of the 2 groups were similar. In the clinical evaluation group, 4 of 226 (1.8%) patients underwent shunt revision based on clinical findings during the index visit, compared to 8 of 74 (10.8%) patients in the surveillance imaging group who underwent shunt revision based on clinical and imaging findings at that visit (p < 0.05). In the subsequent follow-up period, there were 74 unexpected events resulting in 10 shunt revisions in the clinical evaluation group, for an event rate of 33% and operation rate of 13.5%. In the surveillance imaging group there were 23 unexpected events resulting in 2 shunt revisions, for an event rate of 34.8% and an operation rate of 8.7%; neither difference was statistically significant. The complication rate for shunt revision surgery was also not different between the groups.CONCLUSIONSObtaining predecided, routine surveillance imaging in children with myelomeningocele and shunted hydrocephalus resulted in more shunt revisions in asymptomatic patients. For patients who had negative results on surveillance imaging, the rate of shunt revision in the follow-up period was not significantly decreased compared to patients who underwent clinical examination only at the index visit.
Asunto(s)
Derivaciones del Líquido Cefalorraquídeo/estadística & datos numéricos , Hidrocefalia/diagnóstico , Meningomielocele/diagnóstico , Reoperación/estadística & datos numéricos , Adolescente , Derivaciones del Líquido Cefalorraquídeo/efectos adversos , Niño , Preescolar , Servicio de Urgencia en Hospital/estadística & datos numéricos , Falla de Equipo/estadística & datos numéricos , Estudios de Seguimiento , Humanos , Hidrocefalia/diagnóstico por imagen , Hidrocefalia/cirugía , Lactante , Meningomielocele/diagnóstico por imagen , Meningomielocele/cirugía , Selección de Paciente , Vigilancia de la Población/métodos , Tomografía Computarizada por Rayos X/estadística & datos numéricosRESUMEN
Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), which is a molecule secreted by Streptomyces scabies, the plant pathogen responsible for potato scab. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis in vitro, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but is readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expand our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates.
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Aminohidrolasas/metabolismo , Nitrocompuestos/farmacología , Compuestos Organometálicos/farmacología , Elementos de Transición/farmacología , Aminohidrolasas/química , Barbitúricos/química , Barbitúricos/metabolismo , Catálisis , Hidrólisis/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Nitrocompuestos/química , Compuestos Organometálicos/química , Salicilatos/química , Salicilatos/metabolismo , Elementos de Transición/químicaRESUMEN
Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0â Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the ß-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.
Asunto(s)
Epítopos/química , Fragmentos Fab de Inmunoglobulinas/química , Proteínas de la Membrana/química , Anticuerpos de Cadena Única/química , Animales , Cristalización , Cristalografía por Rayos X , Ratones , Modelos Moleculares , Conformación ProteicaRESUMEN
Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.