A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds' activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan

Megavejiga fetal. Presentación de un caso clínico

Introducción: La incidencia estimada de la megavejiga entre las semanas 10 y 14 de gestación está entre 1/900 a 1/1800 casos. Esta puede resolverse de forma espontánea en el 40-60% de los casos sin dejar secuelas o ser la manifestación de una patología progresiva obstructiva o un trastorno heterogéneo más complejo. El riesgo de aneuploidía es mayor en el caso de megavejiga moderada, entre 7 y 15 mm. Mediciones mayores se asocian más frecuentemente a causa obstructiva. Presentamos a continuación el caso de una megavejiga fetal, su diagnóstico y finalización. Caso clínico: Paciente de 42 años de raza blanca. Con antecedentes personales de depresión y urticaria colinérgica. Intervenida de cesárea segmentaria transversa en 1991. G5P2C1A1. Essure en 2012. Gestación producida por FIV con transferencia de 2 embriones congelados el día 02/02/2016 (óvulos propios y espermatozoides de la pareja). Gestación única con fecha probable de parto por ecografía el 21/10/2016. Acude a consulta a las 12 semanas para control ecográfico y cribado de cromosomopatías. Se realiza ecografía, observándose megavejiga de 11 mm, riñones hiperecogénicos con pelvis renales dilatadas. Resto de la exploración normal. Resultado de cribado combinado de alto riesgo. Se recomienda estudio de cariotipo fetal y arrays. Acude en la semana 14 para control ecográfico, se observa una megavejiga de 50 mm de diámetro con aumento de la ecogenicidad renal bilateral, dilatación de ambas pelvis renales y líquido amniótico escaso. Se le informa de mal pronóstico. Se presenta a las 15 semanas para la realización de amniocentesis, con el hallazgo de exitus fetal. El estudio anatomopatológico informó megavejiga de probable causa obstructiva, sin otros hallazgos. No se obtuvo cariotipo fetal. Conclusión La enfermedad renal obstructiva es un cuadro que incluye la hidronefrosis, el hidroureter, la megavejiga y la megauretra. La mortalidad debida a alteraciones del tracto urinario es alta, aproximadamente del 60 %. Existen distintas estrategias a seguir: control ecográfico, finalización de la gestación, inducción del parto y cirugía intraútero. La detección de estas alteraciones mediante la ecografía del primer trimestre del embarazo, afianzan la importancia que tiene la realización del ultrasonido en este período del embarazo para el asesoramiento de la pareja en cuanto a que conducta seguir.Introdución: A incidencia estimada da megavexiga entre as semanas 10 e 14 de xestación está entre 1/900 a 1/1800 casos. Esta pode resolverse de forma espontánea no 40-60% dos casos sen deixar secuelas ou ser a manifestación dunha patoloxía progresiva obstrutiva ou un trastorno heteroxéneo máis complexo. O risco de aneuploidía é maior no caso de megavexiga moderada, entre 7 e 15 mm. Medicións maiores asócianse máis frecuentemente a causa obstrutiva. Presentamos a continuación o caso dunha megavexiga fetal, o seu diagnóstico e finalización. Caso clínico: Paciente de 42 anos de raza branca. Con antecedentes persoais de depresión e urticaria colinérxica. Intervida de cesárea segmentaria transversa en 1991. G5 P2 C1A1. Essure en 2012. Xestación producida por FIV con transferencia de 2 embrións conxelados o día 02/02/2016 (óvulos propios e espermatozoides da parella). Xestación única con data probable de parto por ecografía o 21/10/2016. Acode a consulta ás 12 semanas para control ecográfico e rastrexo de cromosomopatías. Realízase ecografía, observándose megavexiga de 11 mm, riles hiperecoxenicos con pelvis renais dilatadas. Resto da exploración normal. Resultado de cribado combinado de alto risco. Recoméndase estudo de cariotipo fetal e arrays. Acode na semana 14 para control ecográfico, obsérvase unha megavexiga de 50 mm de diámetro con aumento da ecoxenicidade renal bilateral, dilatación de ambas as pelvis renais e líquido amniótico escaso. Infórmaselle de mal prognóstico. Preséntase ás 15 semanas para a realización de amniocentese, co achado de exitus fetal. O estudo anatomopatolóxico informou megavexiga de probable causa obstrutiva, sen outros achados. Non se obtivo cariotipo fetal. Conclusión A enfermidade renal  obstrutiva é un cadro que inclúe a  hidronefrose, o  hidroureter, a megavexiga e a  megauretra. A mortalidade debida a alteracións do tracto urinario é alta, aproximadamente do 60 %. Existen distintas estratexias a seguir: control ecográfico, finalización da xestación, indución do parto e cirurxía intraútero. A detección destas alteracións mediante a ecografía do primeiro trimestre do embarazo, afianzan a importancia que ten a realización do ultrasón neste período do embarazo para o asesoramento da parella en canto a que conduta seguir.Comunicación-póster presentada en la 34 Edición Nacional Formación S.E.G.O. celebrada en Oviedo del 12 al 16 de junio de 201

Editorial : Women in science : Genetics

No abstract available.https://www.frontiersin.org/journals/geneticsam2023Obstetrics and Gynaecolog

A non-canonical RNA silencing pathway promotes mRNA degradation in basal fungi

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing

Antifungal drug resistance evoked via RNAi-dependent epimutations

Microorganisms evolve via mechanisms spanning sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance (FK506R) and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the FKBP12 fkbA gene, giving rise to drug-resistant epimutants. FK506R epimutants readily reverted to the drug-sensitive wild-type (WT) phenotype when grown without drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNA (sRNA) and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves generation of a double-stranded RNA (dsRNA) trigger intermediate from the fkbA mature mRNA to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes

Polimerasas de RNA y su papel en el silenciamiento génico por RNA en Mucor circinelloides / Silvia Calo Valera; directores, Rosa María Ruiz Vázquez, Santiago Torres Martínez.

Tesis-Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TM 4076

Kinin B-1 and B-2 receptors in pig vessels: characterization of two monoreceptor systems

The coronary artery and renal vein of the adult pig are sensitive and reliable monoreceptor systems for studying kinin receptors. The pig coronary artery with intact endothelium is highly sensitive to bradykinin (BK, pEC50 8.6), while being insensitive to the B1 receptor agonist, LysdesArg9BK. The tissue responds to BK with concentration-dependent relaxation, which is prevented by B2 receptor antagonists, particularly DArg[Hyp3, Thi5, DTic7, Oic8]BK (HOE 140, pKB 9.3), (E)-3-(6-acetoamido-3-pyridyl)-N-(N-{2, 4-dichloro-3-[(2-methyl-8-quinolinyl)oxy-methyl]phenyl}-N- methylaminocarbonyl-methyl)acrylamide (FR 173657), a new non peptide compound (pKB 9.3), while B1 receptor antagonists (e.g. Lys[Leu8]desArg9BK) are inactive. The order of potency of kinin-related peptides in this vessel is: LysBK > or = BK > [Hyp3]BK > [Aib7]BK, a sequence typical of a B2 receptor system. Antagonists such as HOE 140 and FR 173657, at high concentrations reduce the maximum effect of BK and thus behave as noncompetitive antagonists. The kinin B1 receptor was studied in the pig renal vein without endothelium and incubated for several hours in order to allow for the de novo formation of this functional site. After 7-8 h in vitro incubation, the vessel shows high sensitivity to LysdesArg9BK (pEC50 8.3) and is insensitive to BK. The pig renal vein responds to B1 receptor agonists with concentration-dependent contraction which maintains a stable plateau and is prevented by selective B1 receptor antagonists such as Lys[Leu8]desArg9BK (pKB 6.7). The most active antagonist has been found to be desArg9HOE 140 (pA2 7.6) which acts as competitive antagonist in this preparation. Some B2 antagonists (e.g. HOE 140) show weak (pKB 6.1) anti-B1 receptor activity while the non-peptide compound FR 173657 is inactive on the B1 receptor and therefore acts as a potent and selective kinin B2 receptor antagonist in the pig. The data obtained in this study allow us to compare the porcine B2 and B1 receptors with those of other species including man, and underline some interesting features that are unique to the porcine functional sites

Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO₂ conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen

Generators of phenotypic diversity illustrated as a game of chance.

<p>Evolution is a multifactorial process that depends on numerous complex factors, including routes to diversity that can be deleterious, neutral, or advantageous depending on the environment. Here we depict these evolutionary trajectories using the analogy of a roulette wheel in which one possible path yields a winner that develops a new trait that allows the microorganism to survive in novel conditions, such as a host treated with antimicrobial agents. However, as in roulette, the odds are against the gambler and the other routes and outcomes are deleterious.</p