Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography.

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy.

During replication, expression, and repair of the eukaryotic genome, cellular machinery must access the DNA wrapped around histone proteins forming nucleosomes. These octameric protein·DNA complexes are modular, dynamic, and flexible and unwrap or disassemble either spontaneously or by the action of molecular motors. Thus, the mechanism of formation and regulation of subnucleosomal intermediates has gained attention genome-wide because it controls DNA accessibility. Here, we imaged nucleosomes and their more compacted structure with the linker histone H1 (chromatosomes) using high-speed atomic force microscopy to visualize simultaneously the changes in the DNA and the histone core during their disassembly when deposited on mica. Furthermore, we trained a neural network and developed an automatic algorithm to track molecular structural changes in real time. Our results show that nucleosome disassembly is a sequential process involving asymmetrical stepwise dimer ejection events. The presence of H1 restricts DNA unwrapping, significantly increases the nucleosomal lifetime, and affects the pathway in which heterodimer asymmetrical dissociation occurs. We observe that tetrasomes are resilient to disassembly and that the tetramer core (H3·H4)2 can diffuse along the nucleosome positioning sequence. Tetrasome mobility might be critical to the proper assembly of nucleosomes and can be relevant during nucleosomal transcription, as tetrasomes survive RNA polymerase passage. These findings are relevant to understanding nucleosome intrinsic dynamics and their modification by DNA-processing enzymes.

Plataforma TI de vigilancia entomológica del Aedes aegypti con colaboración colectiva, Web GIS y mHealth / IT platform for entomological surveillance of Aedes aegypti with crowdsourcing, Web GIS and mHealth / Plataforma de TI para vigilância entomològica de Aedes aegypti com colaboração coletiva, Web GIS e mHealth

RESUMEN El mosquito Aedes aegypti es una especie antropoflica que se ha adaptado a entornos urbanos y es el principal vector de enfermedades como el dengue, la fiebre de Zika, la enfermedad del Chikungunya y la fiebre amarilla, lo que representa una importante carga al sistema de salud, en especial en países tropicales donde es endèmico. Ejercer apropiadamente la vigilancia en salud pública es fundamental para la prevención de estas enfermedades mediante sistemas de información. El propósito de este trabajo es proporcionar una plataforma de tecnologias de la información (TI), integrando tecnologias abiertas Web GIS y mHealth para la vigilancia entomológica del vector a partir de colaboración abierta distribuida para la generación de mapas de infestación. Se realizó un piloto con un grupo focal de 23 estudiantes del curso de epidemiologia, que permitió registrar l20 elementos en 55 reportes en la Universidad de los Llanos para la generación automática de 21 mapas de calor de sintomas, zancudos y criaderos, y un mapa global de infestación. Este trabajo sugiere una perspectiva novedosa de interacción y participación colaborativa de la comunidad con las autoridades de salud soportado por las TI.

ABSTRACT The Aedes aegypti mosquito is an anthropophilic species that has adapted to urban environments and it is the main vector of diseases such as dengue, Zika fever; Chikungunya disease and yellow fever; which represents a significant burden on the health system, especially in tropical countries where it is endemic. Properly exercising public health surveillance is essential for the prevention of these diseases through information systems. The purpose of this work is to provide an information technology (IT) platform, integrating open technologies Web GIS and mHealth for the entomological surveillance ofthe vector; based on crowdsourcing for the generation of infestation maps. A pilot was carried out with a focus group of 23 students from the epidemiology course, which allowed the registration of l20 elements in 55 reports at the Universidad de los Llanos for the automatic generation of 2l heatmaps of symptoms, mosquitoes and breeding sites, and a global infestation map. This work suggests a novel perspective of interaction and collaborative participation of the community with health authorities supported by IT

RESUMO O mosquito Aedes aegypti è uma espècie antropofílica que se adaptou aos ambientes urbanos e è o principal vetor de doenças como dengue, febre Zika, doença Chikungunya e febre amarela, o que representa uma carga significativa para o sistema de saúde, especialmente em países tropicais onde é endêmica. O exercício adequado da vigilância em saúde pública è essencial para a prevenção dessas doenças por meio de sistemas de informação. O objetivo deste trabalho è fornecer uma plataforma de tecnologia da informação (TI), integrando tecnologias abertas Web GIS e mHealth para a vigilância entomológica do vetor com base em uma colaboração aberta distribuída para a geração de mapas de infestação. Um piloto foi realizado com um grupo focal de 23 estudantes do curso de epidemiologia, que permitiu o registro de l20 elementos em 55 relatórios na Universidad de los Llanos para a geração automática de 2l mapas de calor de sintomas, mosquitos e criadouros, e um mapa de infestação global. Este trabalho sugere uma nova perspectiva de interação e participação colaborativa da comunidade com autoridades de saúde apoiadas por TI.

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography.

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity.

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Riesgo de transmisión de SARS-CoV-2: evaluación espacial multicriterio en un municipio de Colombia, 2020 / Risk of transmission of SARS-CoV-2: multi-criteria spatial evaluation in a municipality of Colombia, 2020

RESUMEN Objetivo Zonificar el riesgo de transmisión de SARS-CoV-2 en Villavicencio, Colombia, mediante una evaluación espacial multicriterio. Materiales y Métodos Se implementó un modelo de evaluación multicriterio, a través de un proceso de análisis jerárquico integrado a un sistema de información geográfica. Como criterios fueron incluidos atributos descriptores de las amenazas y vulnerabilidades de transmisión viral identificados mediante un modelo epidemiológico en una misma escala numérica adimensional y proporcional a la probabilidad de contagio. Las alternativas evaluadas corresponden a entidades espaciales representadas por pixeles. Los criterios fueron ponderados de acuerdo con el juicio experto de los evaluadores, con los cuales se realizó el cálculo de una matriz de prioridades relativas normalizada, que permitió la estimación de un vector de pesos, cuyo grado de inconsistencia fue admisible. La magnitud del riesgo se calculó con una sumatoria ponderada de la valoración de los criterios, de acuerdo con un geoproceso de álgebra de mapas. Resultados La heterogeneidad espacial del riesgo de transmisión de SARS-CoV-2 fue descrita en Villavicencio, lo que permitió identificar las áreas con mayor probabilidad de transmisión localizadas en barrios caracterizados por una alta vulnerabilidad socioeconómica. Conclusiones La representación cartográfica derivada de la implementación de un modelo multicriterio, integrado a un Sistema de Información Geográfica, en el análisis de riesgo de transmisión de SARS-CoV-2 constituye un aporte metodológico relevante para la toma de decisiones que definan estrategias de mitigación a escala local y que faciliten la localización y optimización de recursos por parte de las autoridades sanitarias.(AU)

ABSTRACT Objective To zoning the risk of SARS-CoV-2 transmission in Villavicencio, Colombia, through a multi-criteria spatial evaluation. Materials and Methods A multi-criteria evaluation model was implemented, through a hierarchical analysis process, integrated into a Geographic Information System. As criteria, descriptive attributes of the threats and vulnerabilities of viral transmission identified by means of an epidemiological model were included, on the same dimensionless numerical scale and proportional to the probability of contagion; the alternatives evaluated correspond to spatial entities represented by pixels. The criteria were weighted according to the expert judgment of the evaluators, with whom the calculation of a normalized matrix of relative priorities was performed, which allowed the estimation of a vector of weights, the degree of inconsistency of which was admissible. The magnitude of the risk was calculated with a weighted summation of the evaluation of the criteria, according to a map algebra geoprocessing. Results The spatial heterogeneity of the risk of SARS-CoV-2 transmission was described in Villavicencio, allowing the identification of the areas with the highest probability of transmission, located in neighborhoods characterized by high socioeconomic vulnerability. Conclusions The cartographic representation derived from the implementation of a multicriteria model, integrated to a Geographical Information System, in the SARS-CoV-2 transmission risk analysis, constitutes a relevant methodological contribution for decision-making defining strategies of mitigation at the local level, facilitating the location and optimization of resources by the health authorities.(AU)

[Risk of transmission of SARS-CoV-2: multi-criteria spatial evaluation in a municipality of Colombia, 2020]. / Riesgo de transmisión de SARS-CoV-2: evaluación espacial multicriterio en un municipio de Colombia, 2020.

OBJECTIVE: To zoning the risk of SARS-CoV-2 transmission in Villavicencio, Colombia, through a multi-criteria spatial evaluation. MATERIALS AND METHODS: A multi-criteria evaluation model was implemented, through a hierarchical analysis process, integrated into a Geographic Information System. As criteria, descriptive attributes of the threats and vulnerabilities of viral transmission identified by means of an epidemiological model were included, on the same dimensionless numerical scale and proportional to the probability of contagion; the alternatives evaluated correspond to spatial entities represented by pixels. The criteria were weighted according to the expert judgment of the evaluators, with whom the calculation of a normalized matrix of relative priorities was performed, which allowed the estimation of a vector of weights, the degree of inconsistency of which was admissible. The magnitude of the risk was calculated with a weighted summation of the evaluation of the criteria, according to a map algebra geoprocessing. RESULTS: The spatial heterogeneity of the risk of SARS-CoV-2 transmission was described in Villavicencio, allowing the identification of the areas with the highest probability of transmission, located in neighborhoods characterized by high socioeconomic vulnerability. CONCLUSIONS: The cartographic representation derived from the implementation of a multicriteria model, integrated to a Geographical Information System, in the SARS-CoV-2 transmission risk analysis, constitutes a relevant methodological contribution for decision-making defining strategies of mitigation at the local level, facilitating the location and optimization of resources by the health authorities.

High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier.

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis.

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and ß-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

Methylation of histone H3 lysine 9 occurs during translation.

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum.

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membrane fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.