PCR múltiplex para el diagnóstico de microorganismos atípicos transmitidos sexualmente / Multiplex PCR testing for the atypical microorganisms diagnosis sexually transmitted

Resumen Antecedentes: Las infecciones de transmisión sexual son un problema de salud pública mundial. El análisis rutinario incluye solo pruebas microbiológicas y serológicas para el diagnóstico de patógenos. Los microorganismos atípicos como Chlamydia trachomatis y micoplasmas no son identificados debido a los requerimientos. Además, no es incluida Gardnerella vaginalis, aunque se asocia a la vaginosis bacteriana. Objetivo: Desarrollar una PCR múltiplex para el diagnóstico de C. trachomatis, micoplasmas y G. vaginalis. Método: Se estandarizó la PCR múltiplex utilizando oligonucleótidos para C. trachomatis (gen ompA, orf6 plasmídico), Mycoplasma/Ureaplasma y G. vaginalis (genes rRNA16s). Resultados: Se estandarizaron pruebas de PCR múltiplex para los microorganismos estudiados, optimizándose las concentraciones y condiciones de las reacciones múltiplex. Se obtuvieron PCR dúplex para C. trachomatis (ompA, orf6), Chlamydia/Gardnerella y Chlamydia/micoplasmas y tríplex para Chlamydia/Mycoplasma/Ureaplasma. También un cuádruplex para Chlamydia/Mycoplasma/Ureaplasma/Gardnerella. Los resultados fueron verificados por PCR e hibridación automática (HybriSpot 12) y análisis in silico. Conclusión: Se desarrollaron pruebas de PCR múltiplex con una alta sensibilidad y especificidad para la identificación de C. trachomatis, micoplasmas y G. vaginalis.

Abstract Background: Sexually transmitted infections are a global public health problem. Routine analysis includes microbiological and serological tests for the diagnosis of pathogens. Atypical microorganisms such as Chlamydia trachomatis and mycoplasmas are not determined due to the requirements for their identification. Furthermore, Gardnerella vaginalis is not included despite being associated with bacterial vaginosis. Objective: To develop a multiplex PCR to diagnose Chlamydia, mycoplasmas, and Gardnerella. Method: Standardization of multiplex PCR tests was carried out using oligonucleotides for the identification of Chlamydia (ompA gene, plasmid orf6), Mycoplasma/Ureaplasma and Gardnerella (rRNA16s genes). Results: Multiplex PCR tests were standardized for the microorganisms studied, optimizing the concentrations and conditions of the multiplex reactions. Duplex PCR was obtained for Chlamydia (ompA, orf6), Chlamydia/Gardnerella, and Chlamydia/mycoplasmas, and triplex PCR for Chlamydia/mycoplasmas. Also, a quadruplex for Chlamydia, Mycoplasma/Ureaplasma and Gardnerella. PCR and automatic hybridization verified the results obtained (HybriSpot 12) and in silico analysis. Conclusion: Multiplex PCR tests with high sensitivity and specificity were developed to identify C. trachomatis, mycoplasmas, and G. vaginalis.

Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:ßheterodimer. For human αsubunits it has been shown that they depend on theirßsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same ßsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsßsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its ßsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Hit Compounds and Associated Targets in Intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. Chemical-genetic characterization through in vitro evolution combined with whole genome sequencing analysis was used identify novel drug targets and drug resistance genes in Mtb associated with its intracellular growth in human macrophages. We performed a genome analysis of 53 Mtb mutants resistant to 15 different hit compounds. We found nonsynonymous mutations/indels in 30 genes that may be associated with drug resistance acquisitions. Beyond confirming previously identified drug resistance mechanisms such as rpoB and lead targets reported in novel anti-tuberculosis drug screenings such as mmpL3, ethA, and mbtA, we have discovered several unrecognized candidate drug targets including prrB. The exploration of the Mtb chemical mutant genomes could help novel drug discovery and the structural biology of compounds and associated mechanisms of action relevant to tuberculosis treatment.

Multiplex measurement of proinflammatory cytokines around miniscrews during orthodontic treatment / Medición múltiple de citocinas proinflamatorias alrededor de mini-implantes durante el tratamiento ortodóntico

ABSTRACT Objective : To determine the levels of proinflammatory cytokines around miniscrews with and without loading, used during orthodontic treatment. Material and Methods : A descriptive longitudinal study was executed with a sample of ten miniscrews inserted in patients that attended a dental clinic. Saliva and peri-implant crevicular fluid samples were taken: at baseline (T0), 24 hours after insertion (T1), 1 week after insertion (T2), 24 hours after loading (T3). The samples obtained were processed by immunoassay and read with a flow cytometer. Results : Data analysis was performed using the SPSS program version 21.0. To verify normal distribution, Kolmogorov-Smirnov test was used, followed by ANOVA to compare and determine statistical significance. Levels of inflammatory mediators in the peri-implant crevicular fluid at T1 had the following order of average values: IL-8>IL-1β >IL-6>TNF-α>IL-10>IL-12p70. Higher values were found 24 hours post-insertion. Salivary levels found were lower but the previously mentioned order was maintained. Statistically significant intergroup differences were found for IL-1β and IL-8 in the peri-implant crevicular fluid. The Scheffe post hoc test showed that there were no statistically significant differences when making an intragroup pair comparison of each mediator level in a given evaluation time. No statistically significant differences were found in saliva inter and intra-group for all the evaluated inflammatory mediators. Conclusions : The miniscrew loading did not generate an increase of the concentrations of the cytokines greater than the effect caused by the insertion per se. The highest levels of inflammatory mediators were found 24 hours post-insertion of the miniscrew.

RESUMEN Objetivo : Determinar los niveles de citocinas inflamatorias alrededor de mini-implantes con y sin carga, durante el tratamiento ortodóntico. Material y métodos : Se realizó un estudio descriptivo longitudinal con una muestra de diez mini-implantes. La toma de muestras de saliva y líquido crevicular peri-implantario fue realizada en cuatro tiempos: basal (T0), 24 horas post-inserción (T1), 1 semana post-inserción (T2), 24 horas post-carga (T3). Fueron procesadas mediante una técnica de inmunoensayo y citometría de flujo. Resultados : El análisis de los datos se realizó con el programa SPSS versión 21.0. Para verificar la distribución normal se utilizó la prueba de Kolmogorov-Smirnov, seguida de la prueba ANOVA para comparar y determinar la significancia estadística. Los niveles de mediadores inflamatorios en el líquido crevicular peri-implantario en T1 tuvieron el siguiente orden de valores promedio: IL-8> IL-1β> IL-6> TNF-α> IL-10> IL-12p70. Los valores más altos se encontraron 24 horas post-inserción. Los niveles en saliva fueron menores pero se mantuvo el orden mencionado. Se hallaron diferencias estadísticamente significativas entre grupos para IL-1β e IL-8 en el líquido crevicular peri-implantario. Posteriormente, la prueba post hoc de Scheffe demostró la ausencia de diferencias estadísticamente significativas en la comparación de pares intragrupo de cada mediador. No se encontraron diferencias estadísticamente significativas inter e intragrupo para todos los mediadores inflamatorios evaluados en saliva. Conclusiones : La carga del mini-implante no generó un aumento en la concentración de citocinas mayor que el provocado por la inserción per se. Los niveles más altos se encontraron 24 horas después de la inserción.

Corrosión en los mini-implantes de ortodoncia: una revisión de la literatura / Corrosion in orthodontic mini-implants: a literature review

RESUMEN Los mini-implantes son dispositivos metálicos que son temporalmente fijados al hueso y son utilizados para lograr una gran variedad de movimientos ortodónticos. Actualmente, se convirtieron en un importante dispositivo que ayuda al ortodoncista en todas las etapas del tratamiento. Los mini-implantes son hechos con uniones de diferentes metales, por lo que están expuestos al proceso de corrosión en la cavidad bucal. La liberación de iones puede causar efectos fisiológicos adversos, incluyendo citotoxicidad, genotoxicidad, carcinogenicidad y efectos alergénicos. El presente artículo busca hacer una revisión acerca de la corrosión de los mini-implantes de ortodoncia. La literatura reporta que la corrosión de mini-implantes contribuye a la inflamación, que es a su vez un factor que influye en la pérdida de estos dispositivos y la liberación de los iones metálicos es una de sus principales causas. Adicionalmente la corrosión puede producir perforaciones y pérdida de espesor del metal, lo que disminuye la resistencia mecánica y aumenta el riesgo de fractura.

ABSTRACT Mini-implants are metallic devices that are temporarily fixed to the bone and are used to achieve a wide variety of orthodontic movements. Today, they have become an important device that helps the orthodontist in all stages of treatment. The mini-implants are made with joints of different metals, so they are exposed to the corrosion process in the oral cavity. The release of ions can cause adverse physiological effects, including cytotoxicity, genotoxicity, carcinogenicity, and allergenic effects. This article seeks to review the corrosion of orthodontic mini-implants. The literature reports that the corrosion of mini-implants contributes to inflammation, which is a factor that influences the loss of these devices and the release of metal ions is one of its main causes. Additionally, corrosion can produce perforations and loss of thickness of the metal, which reduces the mechanical resistance and increases the risk of fracture.

Factores clínicos relevantes en el diagnóstico de un colapso posterior de mordida. Una revisión sistemática de reportes de caso 2021 / Relevant clinical factors in the diagnosis of a posterior bite collapse. a systematic review 2021

RESUMEN La presente investigación surge de una inquietud ante la falta de consenso sobre el concepto del colapso posterior de mordida, sobre el cual en la actualidad existe limitada literatura. El Objetivo fue Identificar los factores clínicos relevantes en el diagnóstico de colapso posterior de mordida. Es una revisión sistemática basada en reportes y series de caso. Se consideraron como dimensiones al tejido dentario, oclusión dentaria, tejido muscular, articulación temporomandibular, tejido periodontal y examen radiográfico. De 82 artículos seleccionados inicialmente, se realizó la revisión con 6 artículos, Con los artículos se ordenó la información describiendo cada uno de ellos, siendo las publicaciones entre 1987 y 2020. Podemos concluir que se identificaron los factores clínicos relevantes: pérdida de piezas dentarias, alteración de la dimensión vertical, periodontitis, desviación en la apertura y cierre del ATM, y reabsorción ósea como los más predominantes según las dimensiones de diagnóstico.

ABSTRACT The present research arises from a concern regarding the lack of consensus on the concept of posterior bite collapse on which there is currently very little literature. The Objective was identify the relevant clinical factors in the diagnosis of a posterior bite collapse. Is a systematic review based on reports and case series. Was considered as dimensions: dental occlusion, muscle tissue, temporomandibular joint, periodontal tissue and radiographic examination. Of 82 initially selected articles, the review was carried out with 6 articles. The information was ordered describing each one of them, being the publications between 1987 and 2020. We can Conclude that Relevant clinical factors for the correct diagnosis of a posterior bite collapse were identified according to the study dimensions: loss of teeth, alteration of the vertical dimension, periodontitis, deviation in the opening and closing of the TMJ, and bone resorption, as the most predominant according to the diagnostic dimensions.

Detection of oral biomarkers as a diagnostic tool for orthodontically induced inflammatory root resorption: a literature review / Detección de biomarcadores orales como herramienta de diagnóstico para la reabsorción radicular inflamatoria inducida ortodónticamente: una revisión de la literatura

ABSTRACT Orthodontically induced inflammatory root resorption (OIIRR) is one of the most common complications of orthodontic treatment. Historically, only radiographic methods were available, while offering ease of use and accessibility, many limitations exist. Problems of technique, standardization, limited points of view and radiation exposure remain. Resorption can only be detected after a significant portion of the root has been lost (60% to 70% mineralized tissue). Moreover, these methods are static and cannot indicate if the process of root resorption has arrested or is ongoing. Recently, oral fluids have been used as a tool for the diagnosis and monitoring of oral and systemic diseases through the detection of biomarkers. The purpose of this review is to describe the scientific evidence related to the prevalence, etiology, the traditional diagnostic methods and the advances in the detection of oral biomarkers through molecular biology for this pathology.

RESUMEN La reabsorción radicular inflamatoria inducida ortodónticamente (RRIIO) es una de las complicaciones más comunes del tratamiento de Ortodoncia. Históricamente, solo estaban disponibles los medios radiográficos para detectarla, sin embargo, requieren de exposición a radiaciones ionizantes potencialmente dañinas, la técnica radiográfica es sensible, detecta la reabsorción después de que se ha perdido una porción significativa de la raíz (60% al 70% del tejido mineralizado) y no proporciona información sobre su actividad. Recientemente, los fluidos orales vienen siendo utilizados como herramienta para el diagnóstico y monitoreo de enfermedades orales y sistémicas mediante la detección de biomarcadores. El propósito de esta revisión es describir la evidencia científica relacionada con la prevalencia y etiología, los métodos diagnósticos tradicionales y los avances en la detección de biomarcadores orales mediante biología molecular.

Herramientas digitales en el proceso de aprendizaje semipresencial en la Educación Dental Peruana durante la Pandemia COVID-19 / Digital tools in the blended learning process in Peruvian Dental Education during the COVID-19 Pandemic

RESUMEN La pandemia del COVID-19 ha generado un vacío pedagógico en la educación dental, que por la naturaleza clínica, práctica y laboratorial de las asignaturas obligó a los docentes a incorporar recursos didácticos digitales novedosos en los que se simulan entornos de aprendizaje que ayudan a fomentar el desarrollo de habilidades y conocimientos en busca de la mejora continua. No obstante, los estudiantes se han visto también en la necesidad de recurrir a plataformas virtuales que le permitan una mayor adaptación a este complejo sistema híbrido de aprendizaje semipresencial. Los hallazgos de la presente revisión, es producto de un estudio exploratorio que tuvo como propósito identificar las principales Tecnologías de la Información y Comunicación y organizarlas según herramientas de interpretación, modelado dinámico, comunicación, colaboración y organización. De esta manera proporcionamos a la comunidad académica una visión del impacto de ellas sobre el proceso de aprendizaje y autoaprendizaje, sus experiencias de uso y la percepción de los beneficios y limitaciones de estas nuevas tecnologías en un contexto actual en estudiantes de Odontología. En la primera parte abordamos el estado de arte sobre los componentes pedagógicos de las TIC, el papel que desempeñan en la educación superior, las características y los estándares que deben cumplir para que se constituyan en herramientas cognitivas aliadas de la educación formal, de manera que permitan la interacción y la gestión del conocimiento de los estudiantes. Seguidamente compartimos los resultados de una encuesta abierta de los estudiantes de pregrado de una Facultad de Odontología Peruana que se realizó como una fase preliminar diagnóstica que se constituyó el punto de partida para la validación de instrumentos de un trabajo de investigación sobre experiencias del uso de las TIC como herramientas digitales y cognitivas de enseñanza y aprendizaje en la educación dental en línea que cuenta con la autorización del Comité de Ética en Investigación de la Facultad de Medicina de la Universidad Nacional Mayor de San Marcos N° 0209; Las respuestas obtenidas muestran los recursos al servicio de su educación formal y científica relacionadas con habilidades propias de usuarios denominados "nativos digitales" motivándolos a ser los protagonistas de su aprendizaje y desarrollo de competencias para un adecuado desempeño profesional.

ABSTRACT The COVID-19 pandemic has generated a pedagogical vacuum in dental education, which, due to the clinical, practical and laboratory nature of the subjects, forced teachers to incorporate innovative digital teaching resources in which learning environments are simulated that help promote the development of skills and knowledge in search of continuous improvement. However, students have also seen the need to resort to virtual platforms that allow them to better adapt to this complex hybrid blended learning system. The findings of this review are the product of an exploratory study whose purpose was to identify the main Information and Communication Technologies and organize them according to interpretation tools, dynamic modeling; communication, collaboration and organization. In this way, we provide the academic community with a vision of their impact on the learning and self-learning process, their experiences of use and the perception of the benefits and limitations of these new technologies in a current context in Dentistry students. In the first part we address the state of the art on the pedagogical components of ICT, the role they play in higher education, the characteristics and standards they must meet to become cognitive tools allied to formal education, so that Allow the interaction and knowledge management of the students. Next, we share the results of an open survey of undergraduate students of a Peruvian School of Dentistry that was carried out as a preliminary diagnostic phase that became the starting point for the validation of instruments of a research work on experiences of the use of the ICT as digital and cognitive tools for teaching and learning in online dental education that has study code No. 0209 of the Research Ethics Committee of the Faculty of Medicine of the Universidad Nacional Mayor de San Marcos; The responses obtained show the resources at the service of their formal and scientific skills related to users' own skills called "digital natives" motivating them to be the protagonists of their learning and development of skills for adequate professional performance.

Extended conformational states dominate the Hsp90 chaperone dynamics.

The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a ß-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.

Mandibular angle morphology in open bite subjects according to the skeletal relationship: a cbct short report / Morfología del ángulo mandibular en sujetos con mordida abierta de acuerdo a la relación esquelética: un informe breve en cbct

Aim: The purpose of this research was to three-dimensionally evaluate the mandibular angle morphology in open bite subjects with different sagittal skeletal relationships. Material and Methods: Cone beam computed tomography (CBCT) images of 26 subjects (12 men and 14 women) with anterior open bite were evaluated. The sample included 3 groups categorized by their sagittal skeletal relationship (based on ANB angle and anteroposterior dysplasia indicator (APDI)): Class I (n=9), Class II (n=6) and Class III (n=11). The total gonial angle, upper gonial angle, lower gonial angle, intergonial width, interantegonial width and antegonial notch depth were measured. ANOVA and Tukey tests were used for intergroup comparison. The Kruskal Wallis test was also used when necessary. In addition, the Pearson correlation coefficient was calculated to evaluate significant correlations between overbite and antegonial notch depth with gonial angle, Frankfurt mandibular plane angle (FMA) and the palatal plane-mandibular plane (PP-MP). Results: A significant difference was only found on the upper gonial angle between Class II and Class III (p=0.047). The upper gonial angle showed greater values (48°±3°) with the mandibular branch toward backward in Class III subjects and lower values (42.42°±4.39°) with the mandibular ramus leaning forward in subjects with Class II skeletal relationship. Besides, only a statistically significant correlation was found between overbite and the lower gonial angle (r=-0.418, p=0.034). Conclusion: Mandibular angle morphology is similar in anterior open bite subjects with different sagittal skeletal relationships, except for the upper gonial angle which is increased in Class III and decreased in Class II subjects with open bite. Lower gonial angle is negatively correlated with overbite. This difference should be considered by orthodontists when planning their treatments.

Objetivo: El propósito de esta investigación fue evaluar tridimensionalmente la morfología del ángulo mandibular en sujetos de mordida abierta con diferentes relaciones esqueléticas sagitales. Material y Métodos: Se evaluaron imágenes de tomografía computarizada de haz cónico (CBCT) de 26 sujetos (12 hombres y 14 mujeres) con mordida abierta anterior. La muestra incluyó 3 grupos categorizados por su relación esquelética sagital (según el ángulo ANB y el indicador de displasia anteroposterior (APDI)): Clase I (n=9), Clase II (n=6) y Clase III (n=11). Se midieron el ángulo goniaco total, el ángulo goniaco superior, el ángulo goniaco inferior, el ancho intergonial, el ancho interantegonial y la profundidad de la entalladura antegonial. Se utilizaron las pruebas ANOVA y Tukey para la comparación intergrupal. La prueba de Kruskal Wallis también se utilizó cuando fue necesario. Además, se calculó el coeficiente de correlación de Pearson para evaluar correlaciones significativas entre la sobremordida y la profundidad de la entalladura antegonial con el ángulo goniaco, el ángulo del plano mandibular de Frankfurt (FMA) y el plano palatino-plano mandibular (PP-MP). Resultados: Solo se encontró una diferencia significativa en el ángulo goniaco superior entre la Clase II y la Clase III (p=0.047). El ángulo gonial superior mostró valores mayores (48°±3°) con la rama mandibular hacia atrás en sujetos Clase III y valores más bajos (42,42°±4,39°) con la rama mandibular inclinada hacia adelante en sujetos con Clase II esquelética relación. Además, solo se encontró una correlación estadísticamente significativa entre la sobremordida y el ángulo goniaco inferior (r= -0,418, p= 0,034). Conclusión: La morfología del ángulo mandibular es similar en sujetos con mordida abierta anterior con diferentes relaciones esqueléticas sagitales, excepto por el ángulo goniaco superior que aumenta en la Clase III y disminuye en sujetos de Clase II con mordida abierta. El ángulo gonial inferior se correlaciona negativamente con la sobremordida. Los ortodoncistas deben considerar esta diferencia al planificar sus tratamientos.

Client binding shifts the populations of dynamic Hsp90 conformations through an allosteric network.

Hsp90 is a molecular chaperone that interacts with a specific set of client proteins and assists their folding. The underlying molecular mechanisms, involving dynamic transitions between open and closed conformations, are still enigmatic. Combining nuclear magnetic resonance, small-angle x-ray scattering, and biochemical experiments, we have identified a key intermediate state of Hsp90 induced by adenosine triphosphate (ATP) binding, in which rotation of the Hsp90 N-terminal domain (NTD) yields a domain arrangement poised for closing. This ATP-stabilized NTD rotation is allosterically communicated across the full Hsp90 dimer, affecting distant client sites. By analyzing the interactions of four distinct clients, i.e., steroid hormone receptors (glucocorticoid receptor and mineralocorticoid receptor), p53, and Tau, we show that client-specific interactions with Hsp90 select and enhance the NTD-rotated state and promote closing of the full-length Hsp90 dimer. The p23 co-chaperone shifts the population of Hsp90 toward the closed state, thereby enhancing client interaction and processing.

New subtype of PCH1C caused by novel EXOSC8 variants in a 16-year-old Spanish patient.

We report the case of a 16-year-old Spanish boy with cerebellar and spinal muscular atrophy, spasticity, psychomotor retardation, nystagmus, ophthalmoparesis, epilepsy, and mitochondrial respiratory chain (MRC) deficiency. Whole exome sequencing (WES) uncovered three variants (two of them novel) in a compound heterozygous in EXOSC8 gene (NM_181503.3:c.[390+1delG];[628C>T;815G>C]) that encodes the exosome complex component RRP43 protein (EXOSC8). In order to assess the pathogenicity of these variants, expression experiments of RNA and protein for EXOSC8 were carried out. The c.[390+1delG] variant produces the elimination of exon 7 (r.[345_390del]; p.[Ser116LysfsTer27]) and a decrease of the RNA expression in relation to the other allele (p.[Pro210Ser;Ser272Thr]). Furthermore, total mRNA expression is reduced by 30% and the protein level by 65%. EXOSC8 is an essential protein of the exosome core, a ubiquitously expressed complex responsible for RNA processing and degradation. Recessive mutations in EXOSC8 cause pontocerebellar hypoplasia type 1C (PCH1C), and currently, only two homozygous variants in this gene have been described. However, unlike PCH1C-affected individuals with EXOSC8 variants, our patient presents a normal supratentorial cerebral tissue (neither corpus callosum hypoplasia nor hypomyelination) with a less severe phenotype and longer survival. In conclusion, our data expand both genetic and phenotypic spectrum associated with EXOSC8 variants.

The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit.

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

Characterization of a complex phenotype (fever-dependent recurrent acute liver failure and osteogenesis imperfecta) due to NBAS and P4HB variants.

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants.

Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle.

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.

The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90.

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP-driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N-terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD-MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand ß8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD-MD arrangement with the accessibility of a client binding site on the NTD.

Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90.

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

The molecular basis of chaperone-mediated interleukin 23 assembly control.

The functionality of most secreted proteins depends on their assembly into a defined quaternary structure. Despite this, it remains unclear how cells discriminate unassembled proteins en route to the native state from misfolded ones that need to be degraded. Here we show how chaperones can regulate and control assembly of heterodimeric proteins, using interleukin 23 (IL-23) as a model. We find that the IL-23 α-subunit remains partially unstructured until assembly with its ß-subunit occurs and identify a major site of incomplete folding. Incomplete folding is recognized by different chaperones along the secretory pathway, realizing reliable assembly control by sequential checkpoints. Structural optimization of the chaperone recognition site allows it to bypass quality control checkpoints and provides a secretion-competent IL-23α subunit, which can still form functional heterodimeric IL-23. Thus, locally-restricted incomplete folding within single-domain proteins can be used to regulate and control their assembly.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.