Low-frequency inherited complement receptor variants are associated with purpura fulminans.

ABSTRACT: Extreme disease phenotypes can provide key insights into the pathophysiology of common conditions, but studying such cases is challenging due to their rarity and the limited statistical power of existing methods. Herein, we used a novel approach to pathway-based mutational burden testing, the rare variant trend test (RVTT), to investigate genetic risk factors for an extreme form of sepsis-induced coagulopathy, infectious purpura fulminans (PF). In addition to prospective patient sample collection, we electronically screened over 10.4 million medical records from 4 large hospital systems and identified historical cases of PF for which archived specimens were available to perform germline whole-exome sequencing. We found a significantly increased burden of low-frequency, putatively function-altering variants in the complement system in patients with PF compared with unselected patients with sepsis (P = .01). A multivariable logistic regression analysis found that the number of complement system variants per patient was independently associated with PF after controlling for age, sex, and disease acuity (P = .01). Functional characterization of PF-associated variants in the immunomodulatory complement receptors CR3 and CR4 revealed that they result in partial or complete loss of anti-inflammatory CR3 function and/or gain of proinflammatory CR4 function. Taken together, these findings suggest that inherited defects in CR3 and CR4 predispose to the maladaptive hyperinflammation that characterizes severe sepsis with coagulopathy.

SingleCAnalyzer: Interactive Analysis of Single Cell RNA-Seq Data on the Cloud.

Single-cell RNA sequencing (scRNA-Seq) enables researchers to quantify the transcriptomes of individual cells. The capacity of researchers to perform this type of analysis has allowed researchers to undertake new scientific goals. The usefulness of scRNA-Seq has depended on the development of new computational biology methods, which have been designed to meeting challenges associated with scRNA-Seq analysis. However, the proper application of these computational methods requires extensive bioinformatics expertise. Otherwise, it is often difficult to obtain reliable and reproducible results. We have developed SingleCAnalyzer, a cloud platform that provides a means to perform full scRNA-Seq analysis from FASTQ within an easy-to-use and self-exploratory web interface. Its analysis pipeline includes the demultiplexing and alignment of FASTQ files, read trimming, sample quality control, feature selection, empty droplets detection, dimensional reduction, cellular type prediction, unsupervised clustering of cells, pseudotime/trajectory analysis, expression comparisons between groups, functional enrichment of differentially expressed genes and gene set expression analysis. Results are presented with interactive graphs, which provide exploratory and analytical features. SingleCAnalyzer is freely available at https://singleCAnalyzer.eu.

Bioorthogonal Chemistry Enables Single-Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase.

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs' structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

Características clínicas de la automedicación en adultos de tres Unidades de Salud Familiar de Paraguay en 2019

RESUMEN Introducción: la automedicación se define como la ingestión de medicamentos por iniciativa propia y sin receta médica. Esta práctica, que suele alcanzar cifras de prevalencia importantes, puede acarrear graves consecuencias a la salud de la población. Objetivos: describir las características de la automedicación en adultos de tres Unidades de Salud Familiar del Paraguay en el período agosto - septiembre del 2019. Metodología: estudio observacional, descriptivo y prospectivo en población adulta utilizando una entrevista estructurada previo consentimiento informado. Se midieron datos demográficos y variables relacionadas al consumo de medicamentos sin prescripción médica. Resultados: fueron estudiados 153 sujetos, de los cuales la edad media fue 43±17 años, con predominio del sexo femenino (77,2%). La situación laboral predominante fue quehaceres domésticos (39,2%). La mayoría (40%) tenía un nivel de escolaridad secundaria. En 101 casos (66%) se detectó algún grado de automedicación pues muchos admitieron ingerir más de un fármaco a la vez. La principal fuente de adquisición de los medicamentos fueron las farmacias (50%). La fuente de información del medicamento requerido para las afecciones de los sujetos estudiados fue principalmente el farmacéutico (37%). Los fármacos ingeridos con mayor frecuencia fueron los analgésicos (84%). El principal síntoma que motivo la automedicación fue el dolor (75%). Conclusión: la frecuencia de automedicación fue 66% predominando la ingesta de analgésicos y antipiréticos. Los síntomas que mayormente motivaron esta conducta fueron la fiebre y el dolor y la principal fuente de adquisición de los medicamentos fueron las farmacias.

ABSTRACT Introduction: Self-medication is defined as the ingestion of medications on their own initiative and without a prescription. This practice, which usually reaches significant prevalence figures, can have serious consequences for the health of the population. Objectives: To describe the characteristics of self-medication in adults of three Family Health Units of Paraguay in the period August - September 2019. Methodology: Observational, descriptive and prospective study in the adult population using a structured interview with prior informed consent. Demographic data and variables related to the consumption of non-prescription medications were measured. Results: One hundred and fifty-three subjects were studied, with a mean age of 43±17 years and female predominance (77.2%). The predominant employment situation was household chores (39.2%). The majority (40%) had high school level. In 101 cases (66%) some degree of self-medication was detected as many admitted to ingest more than one drug at a time. The main source of acquisition of drugs were pharmacies (50%). The source of information on the medication required for the conditions of the subjects studied was mainly the pharmacist (37%). The drugs most frequently ingested were analgesics (84%). The main symptom that caused self-medication was pain (75%). Conclusion: The frequency of self-medication was 66% predominating the intake of analgesics and antipyretics. The symptoms that mostly motivated this behavior were fever and pain and the main source of medication acquisition was pharmacies.

Inversion of two-flux and four-flux radiative transfer models for determining scattering and absorption coefficients for a suspended particle device.

Intrinsic and extrinsic scattering and absorption coefficients of a suspended particle device (SPD) smart window sample at dark and clear appearance states-without and with applied electrical voltage, respectively-were determined by means of the Maheu, Letoulouzan, and Gouesbet four-flux (intrinsic) and Kubelka-Munk two-flux (extrinsic) radiative transfer models, respectively. Extrinsic values were obtained from fitting to the two-flux model taking into account the predominantly forward scattering of the SPD. As an approximation, the Fresnel reflection coefficients were integrated out to the critical angle of total internal reflection in order to compute diffuse interface reflectances. Intrinsic coefficients were retrieved by adding a new proposed approximation for the average crossing parameter based on the collimated and diffuse light intensities at each interface. This approximation, although an improvement of previous approaches, is not entirely consistent with the two-flux model results. However, it paves the way for further development of methods to solve the inverse problem of the four-flux model.

RaNA-Seq: Interactive RNA-Seq analysis from FASTQ files to functional analysis.

SUMMARY: RaNA-Seq is a cloud platform for the rapid analysis and visualization of RNA-Seq data. It performs a full analysis in minutes by quantifying FASTQ files, calculating quality control metrics, running differential expression analyses and enabling the explanation of results with functional analyses. Our analysis pipeline applies generally accepted and reproducible protocols that can be applied with two simple steps in its web interface. Analysis results are presented as interactive graphics and reports, ready for their interpretation and publication. AVAILABILITY: RaNA-Seq web service is freely available online at https://ranaseq.eu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RJSplot: Interactive Graphs with R.

Data visualization techniques provide new methods for the generation of interactive graphs. These graphs allow a better exploration and interpretation of data but their creation requires advanced knowledge of graphical libraries. Recent packages have enabled the integration of interactive graphs in R. However, R provides limited graphical packages that allow the generation of interactive graphs for computational biology applications. The present project has joined the analytical power of R with the interactive graphical features of JavaScript in a new R package (RJSplot). It enables the easy generation of interactive graphs in R, provides new visualization capabilities, and contributes to the advance of computational biology analytical methods. At present, 16 interactive graphics are available in RJSplot, such as the genome viewer, Manhattan plots, 3D plots, heatmaps, dendrograms, networks, and so on. The RJSplot package is freely available online at http://rjsplot.net.

Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops.

Upregulation of the procoagulant activity of monocytes by antibodies to beta2- glycoprotein I (ß2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by ß2GPI/anti-ß2GPI complexes and their binding sites on ß2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)- differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-ß2GPI were compared to cells treated with dimerized domain V of ß2GPI (ß2GPI-DV) or point mutants of ß2GPI-DV. We demonstrated that dimerized ß2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on ß2GPI is larger than previously thought and includes Lys308 in ß2GPI-DV. Intact residues in two phospholipid-binding loops of ß2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of ß2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on ß2GPI inducing procoagulant activity in monocytes can now be narrowed down to ß2GPI-DV. The ability of ß2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity.

D3GB: An Interactive Genome Browser for R, Python, and WordPress.

Genome browsers are useful not only for showing final results but also for improving analysis protocols, testing data quality, and generating result drafts. Its integration in analysis pipelines allows the optimization of parameters, which leads to better results. New developments that facilitate the creation and utilization of genome browsers could contribute to improving analysis results and supporting the quick visualization of genomic data. D3 Genome Browser is an interactive genome browser that can be easily integrated in analysis protocols and shared on the Web. It is distributed as an R package, a Python module, and a WordPress plugin to facilitate its integration in pipelines and the utilization of platform capabilities. It is compatible with popular data formats such as GenBank, GFF, BED, FASTA, and VCF, and enables the exploration of genomic data with a Web browser.

Evaluación de las vulnerabilidades y potencialidades del paisaje en el municipio de Sacapulas, Quiché, Guatemala / The assessment of vulnerabilities and landscape potentials in the municipality of Sacapulas, Quiche, Guatemala

El departamento de El Quiché forma parte de un corredor seco, el cual limita sus capacidades agrícolas y de desarrollo, al mismo tiempo que posee importantes remanentes de arquitectura residencial tradicional rural de adobe y teja asociados al paisaje del altiplano con elementos representativos del bosque seco y especies endémicas. Estos remanentes han ido desapareciendo y ya son escasos en el país. El presente estudio determinó la vulnerabilidad paisajística desde un enfoque multidisciplinario, por medio de una investigación práctica explicativa. El resultado obtenido fue conocer la vulnerabilidad paisajística del municipio de Sacapulas recabada en cartografía georreferenciada, como una herramienta relevante para la toma de decisiones para proteger y/o potenciar el uso del paisaje con un enfoque de valoración patrimonial y de desarrollo sostenible. Se utilizó para análisis de paisaje la determinación de unidades de características homogéneas y áreas de visibilidad asociada al relieve desde un eje ubicado en la carretera principal, el método de análisis se organizó en tres niveles: (1) elementos primarios de la percepción, (2) de la percepción elaborados para el diagnóstico y (3) de la percepción para la toma de decisión. Dicho proceso utiliza como base cartografía digital georreferenciada. Se obtuvo un mapeo de vulnerabilidad visual y paisajística. El principal resultado es la cartografía que indica las áreas con mayor vulnerabilidad paisajística la cual permite a instituciones y tomadores de decisiones tomar en cuenta el valor paisajístico que esta puede tener en cuanto a ser intervenida en cualquier momento.

The department of El Quiché is part of a dry corridor, which limits its agricultural and development capabilities, at the same time it has important remnants of traditional rural residential architecture of adobe and tile associated with the highland landscape with representative elements of the dry forest and endemic species. These remnants have been disappearing and are already scarce in the country. The present study determined the landscape vulnerability from a multidisciplinary approach, through an explanatory practical investigation. The result obtained was to know the landscape vulnerability of the municipality of Sacapulas collected in geo-referenced cartography, as a relevant tool for making decisions to protect and / or enhance the use of the landscape with a focus on patrimonial valuation and sustainable development. The determination of units with homogeneous characteristics and visibility areas associated with the survey from the main location was used for landscape analysis, the analysis method was organized into three levels: (1) primary elements of perception, (2) perception elaborated for the diagnosis and (3) of the perception for the decision making. This process uses georeferenced digital cartography as a base. A mapping of visual and landscape vulnerability was obtained. The main result is the cartography that indicates the areas with greater landscape vulnerability that allows institutions and decision makers to take into account the landscape value that it can have as soon as it is intervened at any time.

Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from Amphitrite ornata.

The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H2O2, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.

Peroxygenase and oxidase activities of dehaloperoxidase-hemoglobin from Amphitrite ornata.

The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5'-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5'-Br2-indigo, and together suggest a newly identified oxidase activity for DHP.

Frequency and Temperature Dependence of Fabrication Parameters in Polymer Dispersed Liquid Crystal Devices.

A series of polymer dispersed liquid crystal devices using glass substrates have been fabricated and investigated focusing on their electrical properties. The devices have been studied in terms of impedance as a function of frequency. An electric equivalent circuit has been proposed, including the influence of the temperature on the elements into it. In addition, a relevant effect of temperature on electrical measurements has been observed.

Covalent Anchor Positions Play an Important Role in Tuning Catalytic Properties of a Rationally Designed MnSalen-containing Metalloenzyme.

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para- substituted substrates. The 1•Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min(-1)) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Noncovalent modulation of pH-dependent reactivity of a Mn-salen cofactor in myoglobin with hydrogen peroxide.

To demonstrate protein modulation of metal-cofactor reactivity through noncovalent interactions, pH-dependent sulfoxidation and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non-native Mn-salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn-salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen-bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen-bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen-bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the Mn-salen complex. Nonlinear least-squares fitting of rate versus pH plots demonstrates that 1.Mb(H64X) (X=H, R and F) and the control Mn-salen 1 exhibit pKa values varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1.Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1.Mb displays another pKa at low pH, with pKa of 5.0+/-0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen-bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds.