Unraveling the folding and dimerization properties of the human FoxP subfamily of transcription factors.

Human FoxP proteins share a highly conserved DNA-binding domain that dimerizes via three-dimensional domain swapping, although showing varying oligomerization propensities among its members. Here, we present an experimental and computational characterization of all human FoxP proteins to unravel how their amino acid substitutions impact their folding and dimerization mechanism. We solved the crystal structure of the forkhead domain of FoxP4 to then perform a comparison across all members, finding that their sequence changes impact not only the structural heterogeneity of their forkhead domains but also the protein-protein association energy barrier. Lastly, we demonstrate that the accumulation of a monomeric intermediate is an oligomerization-dependent feature rather than a common aspect of monomers and dimers in this protein subfamily.

Funcionalidad y personas mayores: ¿dónde estamos y hacia dónde ir?

[RESUMEN]. El proceso de envejecimiento poblacional llevará a un aumento en problemas de salud en personas mayores, principalmente relacionados a su funcionalidad. En esta línea, los países de la Región de las Américas deben comenzar a actuar para hacer frente a este desafío. Una de las tareas fundamentales en este reto tiene que ver con la capacidad de medir y monitorear la funcionalidad de la población. Los instrumentos utilizados para evaluarla están enfocados principalmente en las actividades básicas de la vida diaria, lo que limita su capacidad preventiva por ser las actividades instrumentales las que permiten un mejor pronóstico. Usando el caso de Chile identificamos desafíos para la Región, principalmente relacionados con las ventajas de incorporar nuevas metodologías de evaluación de la capacidad funcional que no solo permitan las estrategias actuales sino, también, detectar precozmente su deterioro y monitorear sus estadios a lo largo de la dependencia en las personas mayores. Ello permitirá, a la vez, evaluar las iniciativas de prevención y manejo de la pérdida de la funcionalidad.

[ABSTRACT]. The process of population aging will lead to an increase in health problems in older people, mainly related to their functionality. Accordingly, the countries of the Region of the Americas must begin to act to meet this challenge. One of the fundamental tasks involves the ability to measure and monitor functionality in the population. The tools used to evaluate functionality focus mainly on the basic activities of daily living, which limits their preventive capacity, since instrumental activities provide a better prognosis. Using the case of Chile, challenges for the Region were identified, mainly related to the advantages of adopting new methodologies that not only evaluate functional capacity, as current strategies do, but also detect its deterioration early and monitor its stages throughout the dependence stage in older people. This will also make it possible to evaluate initiatives for the prevention and management of loss of functionality.

[RESUMO]. O processo de envelhecimento populacional levará a um aumento de problemas de saúde nas pessoas idosas, principalmente relacionados à sua funcionalidade. Nesse sentido, os países da Região das Américas devem começar a agir para enfrentar esse desafio. Uma das tarefas fundamentais está relacionada à capacidade de medir e monitorar a funcionalidade da população. Os instrumentos utilizados para avaliá-la estão voltados principalmente para as atividades básicas da vida diária, o que limita sua capacidade preventiva, já que são as atividades instrumentais que permitem um melhor prognóstico. Utilizando o caso do Chile, identificamos desafios para a Região, principalmente relacionados às vantagens de incorporar novas metodologias de avaliação da capacidade funcional que não só habilitem as estratégias atuais, mas também detectem precocemente a deterioração da funcionalidade e monitorem suas fases ao longo da dependência das pessoas idosas. Isso permitirá, ao mesmo tempo, avaliar as iniciativas de prevenção e manejo da perda de funcionalidade.

Human FoxP Transcription Factors as Tractable Models of the Evolution and Functional Outcomes of Three-Dimensional Domain Swapping.

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function.

An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli.

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.

Author Correction: The protonation state of an evolutionarily conserved histidine modulates domain swapping stability of FoxP1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intrinsically Disordered Regions of the DNA-Binding Domain of Human FoxP1 Facilitate Domain Swapping.

Forkhead box P (FoxP) proteins are unique transcription factors that spatiotemporally regulate gene expression by tethering two chromosome loci together via functional domain-swapped dimers formed through their DNA-binding domains. Further, the differential kinetics on this dimerization mechanism underlie an intricate gene regulation network at physiological conditions. Nonetheless, poor understanding of the structural dynamics and steps of the association process impedes to link the functional domain swapping to human-associated diseases. Here, we have characterized the DNA-binding domain of human FoxP1 by integrating single-molecule Förster resonance energy transfer and hydrogen-deuterium exchange mass spectrometry data with molecular dynamics simulations. Our results confirm the formation of a previously postulated domain-swapped (DS) FoxP1 dimer in solution and reveal the presence of highly populated, heterogeneous, and locally disordered dimeric intermediates along the dimer dissociation pathway. The unique features of FoxP1 provide a glimpse of how intrinsically disordered regions can facilitate domain swapping oligomerization and other tightly regulated association mechanisms relevant in biological processes.

The protonation state of an evolutionarily conserved histidine modulates domainswapping stability of FoxP1.

Forkhead box P (FoxP) proteins are members of the versatile Fox transcription factors, which control the timing and expression of multiple genes for eukaryotic cell homeostasis. Compared to other Fox proteins, they can form domain-swapped dimers through their DNA-binding -forkhead- domains, enabling spatial reorganization of distant chromosome elements by tethering two DNA molecules together. Yet, domain swapping stability and DNA binding affinity varies between different FoxP proteins. Experimental evidence suggests that the protonation state of a histidine residue conserved in all Fox proteins is responsible for pH-dependent modulation of these interactions. Here, we explore the consequences of the protonation state of another histidine (H59), only conserved within FoxM/O/P subfamilies, on folding and dimerization of the forkhead domain of human FoxP1. Dimer dissociation kinetics and equilibrium unfolding experiments demonstrate that protonation of H59 leads to destabilization of the domain-swapped dimer due to an increase in free energy difference between the monomeric and transition states. This pH-dependence is abolished when H59 is mutated to alanine. Furthermore, anisotropy measurements and molecular dynamics evidence that H59 has a direct impact in the local stability of helix H3. Altogether, our results highlight the relevance of H59 in domain swapping and folding stability of FoxP1.

Alargamiento óseo con minitutor externo como tratamiento de braquimetatarsianos / Bone lengthening with external mini-fixator as treatment for brachymetatarsals

Introducción: La braquimetatarsia es un acortamiento de algún metatarsiano, debido principalmente a una detención prematura del crecimiento de la fisis. Es más frecuente en mujeres, y afecta, sobre todo, al cuarto y al primer metatarsiano. El objetivo de este estudio fue evaluar los resultados del tratamiento mediante alargamiento óseo progresivo con minitutor externo. Materiales y Métodos: Estudio descriptivo retrospectivo. Serie de casos. Se analizaron los resultados de los pacientes operados con esta técnica en nuestro Centro. Se analizaron 6 casos, dos con braquimetatarsia bilateral. Todas eran mujeres y tenían braquimetatarsia del 4.° metatarsiano. El promedio de edad era de 14 años. Se evaluaron los siguientes aspectos: alargamiento en milímetros y complicaciones, como retraso de la consolidación, infección, luxación metatarsofalángica, articulación metatarsofalángica en flexión y satisfacción del paciente. Resultados: Se logró un alargamiento promedio de 15 mm. Dos pacientes tuvieron una infección superficial de las agujas. Una paciente evolucionó con luxación de la articulación metatarsofalángica; otra, con flexión de la articulación metatarsofalángica. No hubo complicaciones neurovasculares. Conclusiones: El alargamiento óseo guiado con tutor externo es un tratamiento seguro y eficaz, además lograría un largo mayor en milímetros que el alargamiento en un tiempo, con un mejor control del alargamiento de partes blandas y estructuras neurovasculares. Todas las complicaciones se resolvieron de manera simple, no afectaron el resultado final del tratamiento. Las complicaciones concuerdan con las descritas en la literatura internacional. Nivel de Evidencia: IV

Introduction: Brachymetatarsia is a shortening of any metatarsal bone, mainly due to premature growth arrest of the growth plate. It is more frequent in women, the fourth and the first metatarsal being the most affected bones. The aim of this study was to evaluate the outcomes of progressive bone lengthening with external mini-fixator. Methods: This is a retrospective, descriptive, case series study in which we analyzed the outcomes of all patients operated on with this technique in our Center. Six cases were evaluated, two of them presented bilateral brachymetatarsia. All patients were women, and had brachymetatarsia of the 4th metatarsal. The average age was 14 years. The following aspects were evaluated: lengthening in millimeters and presence of complications, such as delayed consolidation, infection, metatarsophalangeal joint dislocation, metatarsophalangeal joint in flexion and patient satisfaction. Results: On average, an increase of 15 mm was achieved. Two cases presented superficial infection at the needle insertion site. One patient progressed to metatarsophalangeal joint dislocation, and another one to metatarsophalangeal joint flexion. No neurovascular complications were observed. Conclusions: Bone lengthening with external fixator is a safe and effective treatment. A greater length in millimeters can be achieved in comparison to one-stage lengthening, as well as an improved soft-tissue neurovascular stretching. All complications resolved smoothly and did not affect final treatment results. Complications are similar to those described in the international literature. Level of Evidence: IV

Extremidad superior gravemente lesionada: Reporte de un caso clínico / Mangled upper extremity. Case report

Se presenta un paciente con una extremidad superior gravemente lesionada por arma de fuego. Inicialmente fue tratado con múltiples limpiezas quirúrgicas y colocación de tutor externo AO. El tratamiento definitivo consistió en tutor externo monolateral, además de injerto estructural de cresta ilíaca. Durante el manejo inicial, se detecta lesión del nervio radial, con nervio en continuidad. Dado el proceso cicatricial y la infección de partes blandas, no se logra manejar en la etapa aguda. Posteriormente se realizan transferencias tendinosas para nervio radial. La evolución fue favorable y la función de la extremidad superior resultó satisfactoria. Se comunica este caso debido a su complejo manejo a causa de las lesiones óseas, neurovasculares, cutáneas y de partes blandas. Nivel de Evidencia: IV

We present a patient with a severely injured upper extremity due to a shotgun. Patient was initially managed with multiple surgical debridements and an external AO fixator. Final treatment was performed with a monolateral external fixator in addition to a iliac crest structural graft. During initial management, a radial nerve injury was observed; the nerve was intact, though devitalized. Given the scarring process and soft tissue infection, treatment was performed in the acute phase. Subsequently, tendon transfers were made to treat a radial nerve injury. Patient evolved favorably and regained adequate function of the upper extremity. This case is presented due to its complex management due to bone, neurovascular, skin, and soft tissue involvement. Level of Evidence: IV

Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers.

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1.

The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize by three-dimensional domain swapping, whereby structural elements are exchanged between adjacent subunits, leading to an intertwined dimer. Recent evidence has largely stressed the biological relevance of domain swapping in FoxP, as several disease-causing mutations have been related to impairment of this process. Here, we explore the equilibrium folding and binding mechanism of the forkhead domain of wild-type FoxP1, and of two mutants that hinder DNA-binding (R53H) and domain swapping (A39P), using size-exclusion chromatography, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. Our results show that domain swapping of FoxP1 occurs at micromolar protein concentrations within hours of incubation and is energetically favored, in contrast to classical domain-swapping proteins. Also, DNA-binding mutations do not significantly affect domain swapping. Remarkably, equilibrium unfolding of dimeric FoxP1 follows a three-state N2 ↔ 2I ↔ 2U folding mechanism in which dimer dissociation into a monomeric intermediate precedes protein unfolding, in contrast to the typical two-state model described for most domain-swapping proteins, whereas the A39P mutant follows a two-state N ↔ U folding mechanism consistent with the second transition observed for dimeric FoxP1. Also, the free-energy change of the N ↔ U in A39P FoxP1 is âˆ¼2 kcal⋅mol(-1) larger than the I ↔ U transition of both wild-type and R53H FoxP1. Finally, hydrogen-deuterium exchange mass spectrometry reveals that the intermediate strongly resembles the native state. Our results suggest that domain swapping in FoxP1 is at least partially linked to monomer folding stability and follows an unusual three-state folding mechanism, which might proceed via transient structural changes rather than requiring complete protein unfolding as do most domain-swapping proteins.

Carbon footprint of premium quality export bananas: case study in Ecuador, the world's largest exporter.

Nowadays, the new international market demands challenge the food producing countries to include the measurement of the environmental impact generated along the production process for their products. In order to comply with the environmentally responsible market requests the measurement of the greenhouse gas emissions of Ecuadorian agricultural goods has been promoted employing the carbon footprint concept. Ecuador is the largest exporter of bananas in the world. Within this context, this study is a first assessment of the carbon footprint of the Ecuadorian premium export banana (Musa AAA) using a considerable amount of field data. The system boundaries considered from agricultural production to delivery in a European destination port. The data collected over three years permitted identifying the hot spot stages. For the calculation, the CCaLC V3.0 software developed by the University of Manchester is used. The carbon footprint of the Ecuadorian export banana ranged from 0.45 to 1.04 kg CO2-equivalent/kg banana depending on the international overseas transport employed. The principal contributors to the carbon footprint are the on farm production and overseas transport stages. Mitigation and reduction strategies were suggested for the main emission sources in order to achieve sustainable banana production.

A ribokinase family conserved monovalent cation binding site enhances the MgATP-induced inhibition in E. coli phosphofructokinase-2.

The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K(+) or Cs(+). Kinetic characterization of the enzyme shows that K(+) and Na(+) alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K(+) (but not Na(+)) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K(+) (but not Na(+)) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs(+) or K(+). These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.

El primer equipo interdisciplinario de Costa Rica / The first team of interdisciplinay of Costa Rica

El abuso sexual es un conjunto de actitudes de un sujeto que realiza en detrimento de otro, generalmente menor, para su autosatisfacción. Las estadísticas de los diferentes países apuntan a que se está convirtiendo en una epidemia que nos está asfixiando. La prevención de la revictimización, una política establecida en el Poder Judicial de Costa Rica desde hace más de diez años, dio como fruto la conformación de los equipos interdisciplinarios. Al inicio, estos abarcaron una serie de disciplinas tan disímiles entre sí, que comprendían tanto el derecho, como la medicina, la psicología, el trabajo social y la psiquiatría. Con el paso de los años los equipos se fueron reduciendo hasta quedar conformados por el psicólogo, el trabajador social y el médico forense, que asiste a una reunión semanal con ellos y en donde se discuten las diferentes conclusiones de las tres disciplinas. Hoy en día son equipos establecidos prácticamente en todas las cabeceras de provincia de nuestro país con funciones periciales muy específicas en el tratamiento legal de las causas de los delitos sexuales.

Sexual Abuse is series of actitudes of a subject, Which he realize in detriment of other, usually younger, for his self satisfaction. The statistics of differents countries shows that this issue is torning into epidemics amounts ant its suffocating us. Prevention in revictimation is a policy established in Costa Rica for over a decade and for this we has had form interdisciplinary teams. In early times those temas had a lot of uncompatible disciplines soch as team is formed by only three members: the forensic doctor, the psychologist and the social worker, ehom attent to a weekly metting to analize the different conclusions of each especiallist. Actually there are teams established in almost all head of province in our country with percial function well established in the legal treatment of the causes of these crimes.

El daño corporal: reflexiones médico legales sobre la reparación del Daño Corporal / Corporal damage legal: reflections medical on the repair of corporal damage

Cerca del 10 por ciento de la población costarricense padece de alguna minusvalía, producto del riesgo que conlleva el trabajo en sí mismo. Desde 1865 se han hecho acciones legislativas para atender la reparación del daño corporal de origen laboral, hasta que se llega a establecer la Ley de Riesgos del Trabajo en 1982. Dicha ley esta inspirada en los baremos de amplio uso. En el presente artículo se hace una exposición de amplio uso. En el presente artículo se hace una exposición de la aplicación de dichos instrumento de valoración del daño corporal, en las diferentes instancias médico legales que operan en Costa Rica.

Fujometría Doppler en la enfermedad hipertensiva severa del embarazo con retardo del crecimiento intrauterino / Doppler velocimetry in sever hypertensive disease of pregnancy with intrauterine growth retardation

Objetivo: Evaluar el rol del uso de la ultrasonografía Doppler de las arterias umbilical, uterina y cerebral media en el manejo de la enfermedad hipertensiva con retardo de crecimiento intrauterino y su asociación con resultados perinatales adversos. Material y métodos: En este estudio retrospectivo realizado en el centro de diagnóstico ecográfico Ecomed, se seleccionó 32 pacientes por presentar embarazo de riesgo alto, de un total de 158. A las pacientes se les realizó evaluación del bienestar fetal y Doppler de la arteria uterina, umbilical y cerebral, diagnosticándose RCIU y alteraciones del Doppler materno y fetal, por la presencia de notch en la arteria uterina uni o bilateral y por la disminución de la relación entre los índices de resistencia de la arteria cerebral/arteria umbilical, que normalmente es mayor que 1. Un Doppler de arteria umbilical con ausencia de diástole y un trazo con flujo en reversa fueron considerados signos ominosos para el feto. Se hizo estudios seriados para determinar la conducta obstétrica. Se hizo seguimiento de las pacientes hasta la interrupción del embarazo y posteriormente la evolución de los neonatos en la UCI de recién nacidos, para la determinación de la morbimortalidad neonatal. Conclusiones: La enfermedad hipertensiva del embarazo con retardo de crecimiento intrauterino es causa de morbimortalidad perinatal, cuando se asocia a velocimetría Doppler fetal anormal. La presencia de notch en la arteria uterina podría ser un elemento de diagnóstico para predecir toxemia y retardo de crecimiento intrauterino. La prueba de bienestar fetal modificado podría implementarse para obtener mejores resultados perinatales.