RESUMEN
Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Proteínas de la Membrana/genética , Oligodesoxirribonucleótidos/genética , Ovinos/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Masculino , Microinyecciones , Mutación , Reparación del ADN por Recombinación , Ovinos/embriologíaRESUMEN
Paracetamol overdose is common and microRNA (miR)-122 expression is increased with liver injury. We aimed to measure miR-122 in the setting of an abbreviated paracetamol overdose treatment regimen. We compared miRNA expression in patients treated for paracetamol poisoning with an abbreviated 12-h intravenous acetylcysteine regimen (200 mg/kg over 4 h, 50 mg/kg over 8 h) or a 20-h regimen (200 mg/kg over 4 h, 100 mg/kg over 16 h) (NACSTOP trial). miR-122 expression is increased (decreased cycle threshold (Ct) values) with paracetamol liver injury. We assessed miR-122 expression in patients receiving the two acetylcysteine regimens and in a separate group with acute liver injury (ALI). We examined 121 blood samples in 38 patients. After 20 h of acetylcysteine, median alanine transaminase (ALT) was 12 U/L (18, 14) versus 16 U/L (11, 21) ( p = 0.17) and median miR-122 Ct was 30.1 (interquartile range (IQR): 28.9, 33.3) versus 31.4 (28.9, 33.9) ( p = 0.7) in the NACSTOP abbreviated and control groups, respectively. Median normalized miR-122 Ct after 20 h of acetylcysteine was 2.2 (IQR 1.9, 6.4), 1.1 (0.7, 2.9), 63.9 (2.5, 168), 123.2 (40.9, 207.8) in the NACSTOP-abbreviated, NACSTOP-control, ALI and hepatotoxicity groups, respectively. There was no significant difference in ALT or miRNA between NACSTOP treatment groups and no signal of increased liver injury from an abbreviated 12-h acetylcysteine regimen. These findings suggest that an abbreviated acetylcysteine regimen in low-risk patients who have overdosed on paracetamol is safe. Further study is required to validate this finding utilizing miRNA as a comparative biomarker.
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Acetaminofén/envenenamiento , Acetilcisteína/administración & dosificación , Analgésicos no Narcóticos/envenenamiento , Sobredosis de Droga/tratamiento farmacológico , MicroARNs , Adolescente , Adulto , Sobredosis de Droga/genética , Femenino , Humanos , Infusiones Intravenosas , Masculino , Adulto JovenRESUMEN
PURPOSE: This work proposes a comparative evaluation of two of our patient-specific quality assurance processes involving ArcCHECK® (Sun Nuclear) and Gafchromic® EBT3 films (Ashland) in order to determine which detector is able to most effectively detect an anomaly in a deliberately biased tomotherapy plan. MATERIAL AND METHODS: A complex clinical head and neck tomotherapy plan was deliberately biased by introducing six errors: multileaf collimator leaf positional errors by leaving one and two central leafs closed during the whole treatment, initial radiation angle errors (+0.5° and +1.0°) and multileaf collimator leafs opening time errors (+0.5% and +1.0%). For each error-induced plan, comparison of ArcCHECK® with Gafchromic® EBT3 films (20.3×25.4cm2) was performed through two methods: a dose matrices subtraction study and a gamma index analysis. RESULTS: The dose matrices subtraction study shows that our ArcCHECK® processing is able to detect all the six induced errors contrary to the one using films, which are only able to detect the two biases involving multileaf collimator leaf positional errors. The gamma index analysis confirms the previous method, since it shows all six errors induced in the reference plan seem to be widely detected with ArcCHECK® with the more restrictive 1%/1mm gamma criterion, whereas films may only be able to detect biases in relation to multileaf collimator leaf positional errors. It also shows the common 3%/3mm gamma criterion does not allow deciding between both detectors in the detection of the six induced biases. CONCLUSION: Both comparative methods showed ArcCHECK® processing is more suitable to detect the six errors introduced in the reference treatment plan.
Asunto(s)
Garantía de la Calidad de Atención de Salud , Planificación de la Radioterapia Asistida por Computador , Radioterapia de Intensidad Modulada/normas , Humanos , Errores Médicos , Programas InformáticosRESUMEN
INTRODUCTION: Diagnostic features of myelodysplastic syndromes (MDS) are often polymorphic and nonspecific including anemia in most cases. Standard parameters provided by an automated analyzer seldom bring any argument for this diagnosis. The aim of this study was to investigate whether some structural parameters, not routinely provided by Sysmex™ XE 2100 analyzer, could help diagnose MDS in a simple way, adapted to routine practice. METHODS: Blood samples from 184 MDS fully annotated cases and 3545 normal blood count controls were performed with XE 2100 Sysmex™ analyzer. Quantitative and structural parameters were considered. RESULTS: We found that the structural neutrophil parameter, NEUT-X, converted into a semi-quantitative parameter, the granularity index (GI), could be used as a flag for MDS in front of anemia. Negative GI and anemia were able to make otherwise unrecognized MDS stand out in routine practice, increasing the number of slides addressed to review from 67% to 96%, without leading to a large excess of unfounded slide review among non-MDS. CONCLUSION: Including the GI index in the routine parameters provided by the Sysmex analyzer could be of major help for nonspecialized routine laboratories in detecting MDS.