18 research outputs found
Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes
The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity
Ethical and methodological considerations on conducting clinical research in poor and low-income countries: Viewpoint of the authors of the BEST TRIP ICP randomized trial in Latin America
Ethical and methodological considerations on conducting clinical research in poor and low-income countries: Viewpoint of the authors of the BEST TRIP ICP randomized trial in Latin America.
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Ethical and methodological considerations on conducting clinical research in poor and low-income countries: Viewpoint of the authors of the BEST TRIP ICP randomized trial in Latin America.
Development of a Severe Traumatic Brain Injury Consensus-Based Treatment Protocol Conference in Latin America
Antecedentes La lesión cerebral traumática grave (TCE) es un importante problema de salud mundial que afecta de manera desproporcionada a los países de ingresos bajos y medios (PIBM). El manejo de la hipertensión intracraneal en la sTBI es crucial para la supervivencia y la recuperación óptima. Los médicos de los países de ingresos altos utilizan habitualmente monitores de presión intracraneal (PIC), aunque se ha cuestionado su utilidad. Los monitores ICP generalmente no están disponibles en los países de ingresos bajos y medianos. No existen protocolos o literatura probados / basados en consenso para el tratamiento de la sTBI sin monitoreo de la PIC. Métodos Los investigadores desarrollaron encuestas en serie de SurveyMonkey para neurointensivistas y neurocirujanos latinoamericanos para determinar la práctica actual. Estos médicos tenían una amplia experiencia continua de rutina en sTBI sin monitorización de la PIC. Las encuestas se administraron y analizaron antes / durante / después de una conferencia de consenso en Buenos Aires de 2015. Los investigadores identificaron áreas de convergencia cegadas a las respuestas de los colegas. Un grupo de trabajo de 47 médicos, que representa a 15 países, que trata de forma rutinaria a pacientes con TCE sin monitores, desarrolló pautas de tratamiento basadas en consenso durante una conferencia facilitada de 3 días. Resultados Se agregaron elementos al protocolo a un umbral de acuerdo del 80%. Las encuestas de seguimiento resolvieron los elementos restantes con un 97% de acuerdo. El protocolo aborda tanto la disminución gradual (en la mejora) como el empeoramiento neurológico. Se identificaron opciones de tratamiento por etapas, además de problemas únicos de práctica clínica. Este proceso introdujo un método de investigación a un gran grupo multidisciplinario de médicos de LMIC. Este informe describe el proceso utilizado para desarrollar un protocolo específico de LMIC que es transferible a otras enfermedades / lesiones. El protocolo se está probando en 5 LMIC. Conclusiones Derivamos pautas consensuadas para el tratamiento de la sTBI sin monitorización de la PIC, e introdujimos un método de investigación a un gran grupo multidisciplinario de médicos de LMIC sin experiencia en tales métodos.Background Severe traumatic brain injury (sTBI) is a significant global health problem disproportionately affecting low- and middle-income countries (LMICs). Management of intracranial hypertension in sTBI is crucial to survival and optimal recovery. Practitioners in high-income countries routinely use intracranial pressure (ICP) monitors although their usefulness has been questioned. ICP monitors are usually unavailable in LMICs. No consensus-based/tested protocols or literature exists for sTBI treatment without ICP monitoring. Methods Investigators developed serial SurveyMonkey surveys for Latin American neurointensivists and neurosurgeons to determine current practice. These clinicians had extensive routine ongoing experience in sTBI without ICP monitoring. Surveys were administered and analyzed before/during/after a 2015 Buenos Aires consensus conference. Investigators identified areas of convergence blinded from colleagues’ responses. A 47-clinician task force, representing 15 countries, who routinely manage patients with sTBI without monitors developed consensus-based treatment guidelines during a 3-day facilitated conference. Results Elements were added to the protocol at an 80% agreement threshold. Follow-on surveys resolved remaining elements to 97% agreement. The protocol addresses both tapering (on improvement) and neuroworsening. Staged treatment options were identified, plus unique clinical practice issues. This process introduced a research method to a large multidisciplinary group of LMIC clinicians. This report describes the process used to develop an LMIC-specific protocol that is transferable to other diseases/injuries. The protocol is being tested in 5 LMICs. Conclusions We derived consensus-based guidelines for sTBI treatment without ICP monitoring, and introduced a research method to a large multidisciplinary group of LMIC clinicians naive to such method
Development of a severe traumatic brain injury consensus-based treatment protocol conference in Latin America
Antecedentes La lesión cerebral traumática grave (TCE) es un importante problema de salud mundial que afecta de manera desproporcionada a los países de ingresos bajos y medios (PIBM). El manejo de la hipertensión intracraneal en la sTBI es crucial para la supervivencia y la recuperación óptima. Los médicos de los países de ingresos altos utilizan habitualmente monitores de presión intracraneal (PIC), aunque se ha cuestionado su utilidad. Los monitores ICP generalmente no están disponibles en los países de ingresos bajos y medianos. No existen protocolos o literatura probados / basados en consenso para el tratamiento de la sTBI sin monitoreo de la PIC. Métodos Los investigadores desarrollaron encuestas en serie de SurveyMonkey para neurointensivistas y neurocirujanos latinoamericanos para determinar la práctica actual. Estos médicos tenían una amplia experiencia continua de rutina en sTBI sin monitorización de la PIC. Las encuestas se administraron y analizaron antes / durante / después de una conferencia de consenso en Buenos Aires de 2015. Los investigadores identificaron áreas de convergencia cegadas a las respuestas de los colegas. Un grupo de trabajo de 47 médicos, que representa a 15 países, que trata de forma rutinaria a pacientes con TCE sin monitores, desarrolló pautas de tratamiento basadas en consenso durante una conferencia facilitada de 3 días. Resultados Se agregaron elementos al protocolo a un umbral de acuerdo del 80%. Las encuestas de seguimiento resolvieron los elementos restantes con un 97% de acuerdo. El protocolo aborda tanto la disminución gradual (en la mejora) como el empeoramiento neurológico. Se identificaron opciones de tratamiento por etapas, además de problemas únicos de práctica clínica. Este proceso introdujo un método de investigación a un gran grupo multidisciplinario de médicos de LMIC. Este informe describe el proceso utilizado para desarrollar un protocolo específico de LMIC que es transferible a otras enfermedades / lesiones. El protocolo se está probando en 5 LMIC. Conclusiones Derivamos pautas consensuadas para el tratamiento de la sTBI sin monitorización de la PIC, e introdujimos un método de investigación a un gran grupo multidisciplinario de médicos de LMIC sin experiencia en tales métodos.Background Severe traumatic brain injury (sTBI) is a significant global health problem disproportionately affecting low- and middle-income countries (LMICs). Management of intracranial hypertension in sTBI is crucial to survival and optimal recovery. Practitioners in high-income countries routinely use intracranial pressure (ICP) monitors although their usefulness has been questioned. ICP monitors are usually unavailable in LMICs. No consensus-based/tested protocols or literature exists for sTBI treatment without ICP monitoring. Methods Investigators developed serial SurveyMonkey surveys for Latin American neurointensivists and neurosurgeons to determine current practice. These clinicians had extensive routine ongoing experience in sTBI without ICP monitoring. Surveys were administered and analyzed before/during/after a 2015 Buenos Aires consensus conference. Investigators identified areas of convergence blinded from colleagues’ responses. A 47-clinician task force, representing 15 countries, who routinely manage patients with sTBI without monitors developed consensus-based treatment guidelines during a 3-day facilitated conference. Results Elements were added to the protocol at an 80% agreement threshold. Follow-on surveys resolved remaining elements to 97% agreement. The protocol addresses both tapering (on improvement) and neuroworsening. Staged treatment options were identified, plus unique clinical practice issues. This process introduced a research method to a large multidisciplinary group of LMIC clinicians. This report describes the process used to develop an LMIC-specific protocol that is transferable to other diseases/injuries. The protocol is being tested in 5 LMICs. Conclusions We derived consensus-based guidelines for sTBI treatment without ICP monitoring, and introduced a research method to a large multidisciplinary group of LMIC clinicians naive to such method