Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Total calcaneal allograft reconstruction of an Ewing's sarcoma in a child: Outcome and review of the literature.

BACKGROUND: Ewing's sarcoma rarely presents in bones of the feet. Surgical management usually includes amputation. Limb sparing surgery is anecdotal. CASE: We report the case of a 13-year-old boy with an Ewing sarcoma in his calcaneus who had a calcaneal reconstruction with total calcaneus allograft after induction chemotherapy. CONCLUSIONS: At 42 months of follow-up our patient remains disease free and functionally intact. A review of the exceptional limb salvage procedure options for malignant calcaneus tumor was performed.

Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them.

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in fish, seafood products and fish oil in Spain.

A total of 84 samples of wild and farmed fish, cephalopods and fish oils for animal feeding, traded in Spain, were analysed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in 2009-2012, by gas chromatography-ion trap tandem mass spectrometry (GC-MS-MS). The method was optimised for screening at moderate costs, allowing PCDD/Fs determination at 1 pg World Health Organization-toxic equivalent quantities (WHO-TEQ) g⁻¹ wet weight (w w) and dl-PCBs at 0.02 pg WHO-TEQ g⁻¹ w w. Concentrations in fish and cephalopods ranged from values below the limit of detection to 1.7 pg g⁻¹ WHO-TEQ sum PCDD/Fs and dl-PCBs, considered as safe with regard to EU legislation. Higher levels were found in cod livers (5.4-54.2) and fish oils (3.3-30.7), with one noncompliant sample in each group.

Composites para piezas posteriores: técnica clínica / Composites for posterior teeth: clinical technique

Clinical considerations to make successful posterior composite resins are an important hint for the professional to know. A new technique that may prevent leakage, enhancing marginal seal and different ways to handle polimerization shrinking are described

Rendimiento y composición química de las partes vegetativas del amaranto (Amaranthus hypochondriacus, L.) en diferentes etapas fisiológicas / Yield and chemical composition of the vegetal parts of the amaranth (Amaranthus hypochondriacus, L.) at different physiological stages

El genero Amaranthus comprende las especies conocidas comúmente como bledos, que al igual que otros vegetables de consumo tradicional, son fuente de nutrientes esenciales para el hombre. Los bledos o amarantos tienen buena aceptación como parte dela dieta del guatemalteco, lo que debe aprovecharse para incrementar su cultivo. Fundados en lo expuesto, se llevó a cabo la presente investigación, buscando el estado de madurez fisiológica más adecuado de corte para el consumo de las hojas de amaranto. En el trabajo de campo se utilizó un diseño de bloques al azar con tres tratamientos y ocho repeticiones. Dichos tratamientos consistieron en realizar la cosecha a los 25, 40 y 60 días despues de la emergencia de las plántulas. Se evaluaron las variables: altura de planta, número de hojas, área foliar, peso bruto (peso de hojas y tallos), peso neto (peso de hojas), y rendimientos en materia verde, materia seca y proteína. Se determinó, asimismo, la composición química del material cosechado, que incluyó el análisis del contenido de materia seca, humedaden fresco, humedad residual, proteína, carbohidratos, extracto etéreo, fibra cruda, cenizas, calcio, fósforo, hierro, beta-carotenos y oxalatos. Los resultados obtenidos en el estudio agronómico fueron sometidos a análisis de varianza para el diseño respectivo, encontrandose una diferencia significativa entre tratamientos para todas las variables en estudio. Los resultados del análisis bromatológico fueron analizados a su vez, con un diseño por completo al azar, encontrándose diferencias significativas para la mayoría de las variables estudiadas, excepto en cuanto ea extracto etéreo, calcio, hierro y oxalatos...

Evaluación de diferentes niveles de harina de amaranto (partes vegetativas), en sustitución de harina de alfalfa para conejos en crecimiento / Evaluation of different levels of amaranth flour (vegetative parts), in substitution for alfalfa flour for growing rabbits

Las partes vegetativas del amaranto pueden ser un recurso muy útil para la alimentación de las especies animales debido a sus característicias químicas y al alto rendimiento que se obtiene cuando se cosecha de 45 a 60 días después de la siembra. Con el propósito de determinar el valor alimenticio del amaranto (Amaranthus hypochondriacus) para conejos en crecimiento, se evaluaron seis dietas que contenían harina de hojas y tallos de amaranto deshidratado en niveles de 0, 15, 30, 45 y 60% en sustitución de harina de alfalfa. Ajeno a ello, se elaboró una dieta con hojas y tallos de amaranto cocido al vapor durante 5 min previo a su desecación y molido, y se agregó a un nivel de 60%. De acuerdo a los hallazgos, la harina de amaranto contenía 17.8% de proteína y 12.4% de fibra cruda en comparación con la harina de alfalfa, cuyo contenido proteínico era de 22.0%, y el de fibra cruda, de 23.3%. Los resultados indicaron que la harina de amaranto puede sustituir eficientemente a la harina de alfalfa hasta en un 15% del total de la dieta. No obstante, mayores niveles inducen retardo en el crecimiento y un cuadro patológico caracterizado por nefrosis intersticial y edema, más fácilmente observado al nivel de 60% en la dieta. Los resultados revelaron, asimismo, que un tratamiento con vapor mejora la calidad nutricional de la harina de la planta del amaranto