A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine β -Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria

A mathematical model of tissue-engineered cartilage development under cyclic compressive loading

In this work a coupled model of solute transport and uptake, cell proliferation, extracellular matrix synthesis and remodeling of mechanical properties accounting for the impact of mechanical loading is presented as an advancement of a previously validated coupled model for free-swelling tissue-engineered cartilage cultures. Tissue-engineering con- structs were modeled as biphasic with a linear elastic solid, and relevant intrinsic mechanical stimuli in the constructs were determined by numerical simulation for use as inputs of the coupled model. The mechanical dependent formulations were derived from a calibration and parametrization dataset and validated by comparison of normalized ratios of cell counts, total glycosaminoglycans and collagen after 24h continuous cyclic unconfined compression from another dataset. The model successfully fit the calibration dataset and predicted the results from the validation dataset with good agreement, with average relative errors up to 3.1 and 4.3%, respectively. Temporal and spatial patterns determined for other model outputs were consistent with reported studies. The results suggest that the model describes the interaction between the simultaneous factors involved in in vitro tissue-engineered cartilage culture under dynamic loading. This approach could also be attractive for optimization of culture protocols, namely through the application to longer culture times and other types of mechanical stimul

Extract2Chip—Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance

Funding Information: This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 by the authors.Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein–protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein–protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.publishersversionpublishe

Micromechanical study of the load transfer in a polycaprolactone-collagen hybrid scaffold when subjected to unconfined and confined compression

Scaffolds are used in diverse tissue engineering applications as hosts for cell proliferation and extracellular matrix formation. One of the most used tissue engineering materials is collagen, which is well known to be a natural biomaterial, also frequently used as cell substrate, given its natural abundance and intrinsic biocompatibility. This study aims to evaluate how the macroscopic biomechanical stimuli applied on a construct made of polycaprolactone scaffold embedded in a collagen substrate translate into microscopic stimuli at the cell level. Eight poro-hyperelastic finite element models of 3D printed hybrid scaffolds from the same batch were created, along with an equivalent model of the idealized geometry of that scaffold. When applying an 8% confined compression at the macroscopic level, local fluid flow of up to 20 [Formula: see text]m/s and octahedral strain levels mostly under 20% were calculated in the collagen substrate. Conversely unconfined compression induced fluid flow of up to 10 [Formula: see text]m/s and octahedral strain from 10 to 35%. No relevant differences were found amongst the scaffold-specific models. Following the mechanoregulation theory based on Prendergast et al. (J Biomech 30:539-548, 1997. https://doi.org/10.1016/S0021-9290(96)00140-6 ), those results suggest that mainly cartilage or fibrous tissue formation would be expected to occur under unconfined or confined compression, respectively. This in silico study helps to quantify the microscopic stimuli that are present within the collagen substrate and that will affect cell response under in vitro bioreactor mechanical stimulation or even after implantation

Impacto de un programa intervención em alunos del segundo ciclo

O objetivo do presente estudo consistiuem avaliar um programa de intervenção junto a alunos do 2º ciclo de escolaridade nas seguintes dimensões: tomada de decisão, conhecimentos sobre sexualidade, competências sociais, assertividade e autoconceito. Metodologia: Participaram 145 alunos, distribuídos pelos grupos controle e experimental. Os instrumentos utilizados foram: TCU Decision-Making; Questionário de Conhecimentos sobre Sexualidade; Assertion Self-Statement Test- Revised; Questionário de Competências Sociais; Piers-Harris Children’s Self-Concept Scale. Os resultados revelaram diferenças no pós-teste entre os grupos ao nível da sexualidade. Verificaram-se diferenças do pré-teste para o pós-teste no grupo experimental nos níveis da sexualidade, da assertividade e das competências sociais. No grupo experimental encontraram-se associações positivas entre tomada de decisão, competências sociais e assertividade, bem como entre sexualidade, competências sociais e autoconceito, no pós-teste. Os preditores da assertividade no pós-teste foram tomada de decisão, sexualidade e competências sociais. Como conclusão, os resultados enfatizam a importância de intervenção junto a adolescentes, particularmente na tomada de decisão, na sexualidade e nas competências sociais. Palavras-chave: Habilidades sociais, sexualidade, autoconceito.In this study we evaluate an intervention program in the following dimensions: Decision Making, Knowledge on Sexuality, Social Skills, Assertiveness and Self-Concept with students in 5th and 6th grade. Methodology: 145 students participated in the study divided by control and experimental group. The instruments used were: Decision-Making TCU, Knowledge on Sexuality Questionnaire; Assertion Self-Statement Test-Revised;Social Skills Questionnaire, and Piers-Harris Children's Self- Concept Scale. The results indicate differences at post-test between the groups on knowledge regarding sexuality. There were also differences from pre-test to post-test in the experimental group on knowledge on sexuality, assertiveness and social skills. Positive associations among decision making, social skills and assertiveness were found as well as among knowledge on sexuality, social skills and self-concept, in the experimental group, in the pos-test. Finally, the predictors of assertiveness regarding health behaviors, in the pos-test were: decision making, knowledge regarding sexuality and social skills. The results emphasize the importance of intervention for adolescents in terms of health promotion particularly in decision making, sexuality and social skills.El objetivo del presente estudio fue evaluar un programa de intervención con alumnos del 2º ciclo de escolaridad en las siguientes dimensiones: Toma de Decisión, Conocimientos sobre Sexualidad, Habilidades Sociales, Asertividad y Autoconcepto. Metodología: Participaron 145 alumnos, distribuidos en grupo control y experimental. Los instrumentos utilizados fueron: TCU Decision-Making; Cuestionario de Conocimientos sobre Sexualidad; AssertionSelf-Statement Test-Revised; Cuestionario de Habilidades Sociais; Piers-Harris Children'sSelf-Concept Scale. Los resultados mostraron diferencias en el post-test entre los grupos en cuanto a la sexualidad. Se verificaron diferencias del pre-test para el post-teste en el grupo experimental, cuanto a sexualidad, asertividad y habilidades sociales. Se encontraron asociaciones positivas entre toma de decisión, habilidades sociales y asertividad, así como entre sexualidad,habilidades sociales yautoconcepto, en el post-test en el grupo experimental. Los predictores de la asertividad en el post-test fueron toma de decisión, sexualidad y habilidades sociales. Los resultados destacan la importancia de la intervención con adolescentes particularmente en la toma de decisiones, sexualidad y habilidades sociales.(undefined

The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra 1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86 degrees C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H-2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A(0)A(1)-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea

Structure of NADH: quinone oxidoreductase from<i>Acidianus ambivalens</i>: electron entry point of aerobic respiratory chain

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X-ray structure of full-length human RuvB-Like 2 – mechanistic insights into coupling between ATP binding and mechanical action

Abstract RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling

Innovative sea surface monitoring with GNSS-REflectometry aboard ISS: Overview and recent results from GEROS-ISS

GEROS-ISS (GEROS hereafter) stands for GNSS REflectometry, Radio Occultation and Scatterometry onboard the International Space Station. It is a scientific experiment, proposed to the European Space Agency (ESA) in 2011 for installation aboard the ISS. The main focus of GEROS is the dedicated use of signals from the currently available Global Navigation Satellite Systems (GNSS) for remote sensing of the System Earth with focus to Climate Change characterisation. The GEROS mission idea and the current status are briefly reviewed