Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.

Detection of antibodies against the huntingtin protein in human plasma.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder resulting from a CAG expansion in the huntingtin (HTT) gene, which leads to the production and accumulation of mutant huntingtin (mHTT). While primarily considered a disorder of the central nervous system, multiple changes have been described to occur throughout the body, including activation of the immune system. In other neurodegenerative disorders, activation of the immune system has been shown to include the production of antibodies against disease-associated pathological proteins. However, the existence of mHTT-targeted antibodies has never been reported. In this study, we assessed the presence and titer of antibodies recognizing HTT/mHTT in patients with HD (n = 66) and age- and gender-matched healthy controls (n = 66) using a combination of Western blotting and ELISA. Together, these analyses revealed that antibodies capable of recognizing HTT/mHTT were detectable in the plasma samples of all participants, including healthy controls. When antibody levels were monitored at different disease stages, it was observed that antibodies against full-length mHTT were highest in patients with severe disease while antibodies against HTTExon1 were elevated in patients with mild disease. Combined, these results suggest that antibodies detecting different forms of mHTT peak at different disease stages.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Costo-efectividad de la reanimación cardiopulmonar con el uso del desfibrilador externo automático, comparado con reanimación cardiopulmonar básica, para personas con pérdida de conciencia en espacios de afluencia masiva de público / Cost-effectiveness of cardiopulmonary resuscitation using an automated external defibrillator compared with a basic defibrillator in unconscious patients in crowded public spaces in Colombia

Resumen Objetivo: Estimar la razón de costo-efectividad de la reanimación cardiopulmonar con el uso del desfibrilador externo automático (DEA), comparado con la reanimación cardiopulmonar básica, para la reanimación de personas con pérdida de conciencia en espacios de afluencia masiva de público en Colombia. Métodos: Para estimar los costos y desenlaces de las dos alternativas de comparación, se diseñó un árbol de decisiones en el cual se reflejan los principales desenlaces posterior a la pérdida de conciencia y la reanimación con cualquiera de las dos alternativas. Se asumió la perspectiva del sistema de salud colombiano en un horizonte temporal comprendido entre el momento de la pérdida de conciencia de la persona hasta el ingreso al hospital. Las probabilidades de los eventos se obtuvieron de un meta-análisis de ensayos clínicos y la información de costos de fuentes oficiales y consulta directa a proveedores de DEA en Colombia. Los costos fueron expresados en pesos colombianos de 2016 y la efectividad en muertes evitadas. Se realizaron análisis de sensibilidad determinísticos y probabilísticos para estimar el impacto de la incertidumbre sobre las conclusiones. Resultados: La razón de costo-efectividad de la reanimación cardiopulmonar con DEA fue de $3.267.777 por muerte evitada. La probabilidad de que esta intervención sea costo-efectiva es superior al 90% para un umbral de costo-efectividad superior a 10 millones de pesos. Conclusión: Un programa de reanimación cardiopulmonar con desfibrilación temprana mediante el uso de DEA, en espacios de afluencia masiva de público, es una alternativa costo-efectiva para el sistema de salud colombiano.

Abstract Objective: To estimate the cost-effectiveness of cardiopulmonary resuscitation using an automated external defibrillator (AED) compared with basic cardiopulmonary resuscitation, for the resuscitation of unconscious patients in crowded public spaces in Colombia. Methods: A decision tree was designed in order to estimate the costs and outcomes of the two alternatives. This included the main outcomes after the loss of consciousness and resuscitation by any of the two alternatives. The perspective of the Colombian Health System was adopted in a time scale consisting of the time of loss of consciousness until hospital admission. The probabilities of the events were obtained from a meta-analysis of clinical trials, and the information on costs from official sources and direct consultations with AED providers in Colombia. The costs were expressed in Colombian pesos of 2016, and the effectiveness in deaths prevented. Deterministic and probabilistic sensitivity analyses were performed to estimate the impact of uncertainty on the conclusions. Results: The cost-effectiveness of cardiopulmonary resuscitation with AED was COP $3,267,777 per death avoided. The probability that this intervention would be cost-effective is greater than 90% for cost-effectiveness threshold greater than 10 million Colombian pesos. Conclusion: A cardiopulmonary resuscitation program with early defibrillation using an AED in crowded public spaces is a cost-effective alternative for the Colombian Health System.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1.

The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K+ (KATP) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening. Pancreatic KATP channels play an important role in insulin secretion. SUR1 mutations that result in increased levels of channel opening ultimately inhibit insulin secretion and lead to neonatal diabetes. In contrast, SUR1 mutations that disrupt trafficking and/or decrease gating of KATP channels cause congenital hyperinsulinism, where oversecretion of insulin occurs even in the presence of low glucose levels. Here, we present data on the effects of specific congenital hyperinsulinism-causing mutations (G716V, R842G, and K890T) located in different regions of the first nucleotide binding domain (NBD1). Nuclear magnetic resonance (NMR) and fluorescence data indicate that the K890T mutation affects residues throughout NBD1, including residues that bind MgATP, NBD2, and coupling helices. The mutations also decrease the MgATP binding affinity of NBD1. Size exclusion and NMR data indicate that the G716V and R842G mutations cause aggregation of NBD1 in vitro, possibly because of destabilization of the domain. These data describe structural characterization of SUR1 NBD1 and shed light on the underlying molecular basis of mutations that cause congenital hyperinsulinism.

Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

Cardiac biomakers for the evaluation of acute coronary syndrome.

The traditional diagnosis of myocardial infarction relies primarily within the exhibited patient's clinical presentation, electrocardiographic changes, and elevation in cardiac markers. Since the clinical presentation can be highly variable and EKG changes are not readily present for all patients, the use of markers of cardiac injury to support the diagnosis of myocardial infarction has become a fundamental part of the evaluation of a patient with suspected acute coronary syndrome. In 2007 the Joint European Society of Cardiology/ American College of Cardiology Committee for the Redefinition of Myocardial Infarction concluded that the main criteria for myocardial infarction should be a rise or fall of cardiac biomarkers (namely cardiac troponins and CK-MB) along with: (1) ischemic symptoms, (2) ischemic changes in EKG, (3) Q waves in EKG, or (4) imaging evidence of loss of myocardial viability or (5) wall motion abnormalities. These changes have increased search interests for more sensitive and specific markers of acute myocardial injury; furthermore, dedicated research has commenced in order to specifically allocate markers that could even predict myocardial ischemia. Therefore this article will review traditional employment of cardiac markers, providing current insight, information and experimental data with respect to emerging markers of myocardial ischemia.

Factores individuales (genéticos), clínicos, ambientales y de cuidado asociados al desarrollo de demencias, movimientos anormales y depresión en población de adultos mayores de 50 años, en Bogotá / Individual (genetic), clinical, environmental and care factors associated with the development of dementia, abnormal movements and depression among adults over 50, in Bogotá

Estudio analítico longitudinal, diseñado para definir prevalencia y factores de riesgo para los trastornos de la memoria, afectivos y movimientos anormales en 206 adultos mayores de la localidad cuarta de Bogotá en los barrios Quindío, La Victoria y San Blas durante 2005-2006. Los resultados revelan que el 65,5 por ciento de las personas participantes presentan algunas de las enfermedades estudiadas, es decir que dos de tres personas están afectadas. De los tres grupos de enfermedades las más frecuentes fueron las que alteran el estado de ánimo, después las que afectan las funciones intelectuales y en menor proporción las alteraciones de los movimientos. De acuerdo con el género, las mujeres presentan mayormente alteraciones del estado de ánimo, mientras que los hombres son más afectados por alteraciones de los movimientos. El 70 por ciento de los adultos mayores entrevistados tienen familiares que padecen los trastornos mencionados. Se encontró una asociación significativa de las enfermedades estudiadas con alguna de las variables analizadas, como la edad, el analfabetismo y la ocupación, lo cual constituye un importante avance en el conocimiento de los factores de riesgo de dichas patologías y permite desprender aplicaciones directas sobre estrategias de prevención, tratamiento y cuidado.

This longitudinal analytical study was designed to define prevalence and risk factors in memory loss alterations, mood disorders and abnormal movements among 206 senior adults from the Fourth Locality of Bogotá in the neighborhoods: Quindío, La Victoria and San Blas from 2005 to 2006. Results show that 65.5 per cent of the participants reported some the disorders studied. That is to say, two out of three people were affected. Mood disorders were the most frequent, followed by alterations of the intellectual functions, and, to a lesser degree, by abnormal movements. The gender analysis showed that women report mainly alterations in mood, while men are more affected...