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RNA quadruplexes are non-canonical nucleic acid structures involved in several human disease states and are regulated by a specific subset of RNA helicases. Given the difficulty in identifying RNA quadruplex helicases due to the multifunctionality of these enzymes, we sought to provide a comprehensive in silico analysis of features found in validated RNA quadruplex helicases to predict novel human RNA quadruplex helicases. Using the 64 human RNA helicases, we correlated their amino acid compositions with subsets of RNA quadruplex helicases categorized by varying levels of evidence of RNA quadruplex interaction. Utilizing phylogenetic and synonymous/non-synonymous substitution analyses, we identified an evolutionarily conserved pattern involving predicted intrinsic disorder and a previously identified motif. We analyzed available next-generation sequencing data to determine which RNA helicases directly interacted with predicted RNA quadruplex regions intracellularly and elucidated the relationship with miRNA binding sites adjacent to RNA quadruplexes. Finally, we performed a phylogenetic analysis of all 64 human RNA helicases to establish how RNA quadruplex detection and unwinding activity may be conserved among helicase subfamilies. This work furthers the understanding of commonalities between RNA quadruplex helicases and provides support for the future validation of several human RNA helicases. Key words: RNA quadruplex, helicase, phylogeny, NIQI motif, intrinsic disorder. Les quadruplexes d'ARN sont des structures d'acides nucléiques non canoniques impliqués dans plusieurs états pathologiques chez l'humain et qui sont régulés par un sous-ensemble spécifique d'hélicases d'ARN. Etant donné la difficulté d'identifier les hélicases des quadruplexes d'ARN en raison de la multifonctionnalité de ces enzymes, les auteurs ont cherché à fournir une analyse in silico complète des caractéristiques des hélicases de quadruplexes d'ARN validées afin de prédire de nouvelles hélicases de quadruplexes d'ARN humain. En utilisant les 64 hélicases d'ARN humain, ils ont corrélé leurs compositions en acides aminés avec des sous-ensembles d'hélicases de quadruplexes d'ARN, classées en fonction du niveau de preuve d'interaction des quadruplexes d'ARN. En utilisant des analyses phylogénétiques et de substitutions synonymes/non-synonymes, ils ont identifié un motif conservé au cours de l'évolution impliquant un désordre intrinsèque prédit et un motif préalablement identifié. Ils ont analysé les données disponibles de séquençage de prochaine génération pour déterminer quelles hélicases d'ARN interagissaient directement avec les régions de quadruplexes d'ARN prédites à l'intérieur des cellules et ont élucidé une relation avec les sites de liaison de miARN adjacents aux quadruplexes d'ARN. Enfin, ils ont utilisé une analyse phylogénétique des 64 hélicases d'ARN humain pour établir comment l'activité de détection et de déroulement des quadruplexes d'ARN peut être conservée parmi les sous-familles d'hélicases. Ce travail permet de mieux comprendre les points communs entre les hélicases de quadruplexes d'ARN et d'étayer la future validation de plusieurs hélicases d'ARN humain. [Traduit par la Rédaction] Mots-clés: quadruplex d'ARN, hélicase, phylogénie, motif NIQI, désordre intrinsèque., Introduction Guanine quadruplexes (G4) are non-canonical four-stranded nucleic acid secondary structures composed of stacked guanine tetrads prevalent in both DNA and RNA (Guschlbauer et al. 1990; Fay et al. 2017b). [...]

Human 2'--5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'--5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond. Key words: oligoadenylate synthetase, oligoadenylate, viral RNA, RNA. Les 2'--5' oligoadenylate synthases (OAS) humaines forment une famille de proteines inductibles par l'interferon qui, a la suite d'une activation par de l'ARN double-brin, polymerisent l'ATP en oligoadenylates lies en 2'--5'. Dans cette etude, les auteurs ont sonde laspecificite pour l'ARN cofacteur des deux plus petites isozymes, OAS1et OAS2. Ils ont d'abord developpe une strategie permettant d'exprimer et de purifier l'OAS2 recombinante humaine a partir de cellules eucaryotes, et quantifie l'activite de l'enzyme par rapport a l'OAS1 in vitro. Ils ont ensuite confirme que les deux domaines d'OAS2, contrairement au domaine comportant la triade canonique d'acides aspartiques catalytiques seulement, sont requis a l'activite enzymatique. La cinetique enzymatique d'OAS1 et OAS2 en presence d'une variete d'ARN partenaires de liaison a permis de caracteriser la velocite maximale de la reaction et l'affinite apparente ARN-proteine d'ARN activateurs. Alors que dans cette etude, l'OAS1 peut etre activee catalytiquement par un ARN db de n'importe quelle longueur superieure a 19 pb, l'OAS2 montrait un accroissement marque de son activite en fonction de la longueur de l'ARN db, avec une longueur requise minimale de 35 pb. Il est interessant de noter que l'activation d'OAS2 etait aussi plus efficace lorsque l'ARN db comportait des extremites saillantes en 3' malgre l'absence d'impact significatif sur l'affinite de liaison. Les ARN viraux hautement structures qui sont etablis en tant qu'activateurs de l'OAS1 etaient incapables d'activer l'OAS2 sur la base de l'absence de segments etendus d'ARN plus longs que 35 pb. Dans l'ensemble, ces resultats pourront mettre en evidence des sous-categories distinctes d'ARN auxquelles les differentes isozymes d'OAS repondent. [Traduit par la Redaction] Mots-cles : oligoadenylate synthase, oligoadenylate, ARN viral, ARN., Introduction 2'--5'-Oligoadenylate synthetases (OAS) are a family of interferoninducible enzymes that act as pattern recognition receptors (PRR) to bind double-stranded RNA (dsRNA) during viral infection (Hovanessian 2007). Upon interaction with [...]

Cytochrome c oxidase deficiency (COXD) is an inherited disorder characterized by the absence or mutation in the genes encoding for the cytochrome c oxidase protein (COX). COX deficiency results in severe muscle weakness, heart, liver, and kidney disorders, as well as brain damage in infants and adolescents, leading to death in many cases. With no cure for this disorder, finding an efficient, inexpensive, and early means of diagnosis is essential to minimize symptoms and long-term disabilities. Furthermore, muscle biopsy, the traditional detection method, is invasive, expensive, and time-consuming. This study demonstrates the applicability of scanning electrochemical microscopy to quantify COX activity in living human fibroblast cells. Taking advantage of the interaction between the redox mediator N, N, N', N'-tetramethyl-para-phenylene-diamine, and COX, the enzymatic activity was successfully quantified by monitoring current changes using a platinum microelectrode and determining the apparent heterogeneous rate constant k0 using numerical modeling. This study provides a foundation for developing a diagnostic method for detecting COXD in infants, which has the potential to increase treatment effectiveness and improve the quality of life of affected individuals. [ABSTRACT FROM AUTHOR]

Sexual and gender minority (SGM) individuals experience high rates of homelessness and criminal justice system involvement, underscoring the need for supportive housing services. To explore the service needs of this population, we interviewed providers (n = 11) and clients (n = 10) from eight supportive housing organizations working with SGM populations in Los Angeles County, California, USA. We used the Consolidated Framework for Implementation Research to synthesize interview responses into themes (by domain and cross-cutting). Takeaways included the need for investment in systems of care for vulnerable SGM populations; the particular marginalization of Trans individuals and providers that serve them; the roles of supportive housing staff, residents, and leadership in cultivating an affirming environment; the prevalence of discrimination and stigma within supportive housing programs and broader society; and the complex interrelationships among SGM identity, homelessness, and criminal justice system involvement. These findings have important implications for supportive housing services and related policy. [ABSTRACT FROM AUTHOR]

Assembly of off-road bicycle handlebars with a stem that clamps the handlebar around its circumference would be expected to affect fatigue performance by introducing both assembly stresses and stress concentration. Because the effect of clamping on fatigue performance is unknown and because of the need to insure structural reliability in the stem-handlebar assembly to prevent serious injury, the objectives of the work reported by the present article were fourfold. One was to determine the stresses due to assembly and the stress concentration induced in a handlebar for two different clamp designs (i.e. 1-bolt and 2-bolt), a second was to determine experimentally the high cycle constant amplitude load fatigue lives of the two stem-handlebar assemblies, a third was to determine experimentally the variable amplitude load fatigue lives, and the fourth was to predict the variable amplitude load fatigue life with constant amplitude load fatigue test results. The handlebar was instrumented with strain gages and the assembly strains were measured when the stem clamps were tightened. The handlebar was also loaded as a cantilever beam while the applied strains were measured for each assembly. Stresses were computed and the maximum stresses induced by clamping exceeded 200 MPa for both assemblies. A method unique to this study was devised to determine the stress concentration at an arbitrary angular location around the circumference of the handlebar and for an arbitrary loading direction in the plane of the bicycle. For a load directed along an angle of -38degrees (clockwise rotation from horizontal viewed from the right), both stems created similar stress concentration; the location of maximum applied stress was shifted by 30degrees from the point that would be expected in the absence of assembly and the stress was increased by 40% at this location. The measured fatigue lifetimes for constant amplitude loading were similar for the two stem designs but the variable amplitude load fatigue lifetime for the 1-bolt stem assembly was shorter than that for the 2-bolt stem assembly by 19%. The fatigue lifetimes for variable amplitude loading based on constant amplitude load fatigue test results were predicted to within 3% and 33% forthe 1-bolt and 2-bolt stems, respectively. Thus, constant amplitude load fatigue test results can be used to approximate the variable amplitude load fatigue life. However, the ranking of different assemblies may not be accurately indicated by constant amplitude load fatigue data.